Campylobacter

Campylobacter Third Edition

Editors

Irving Nachamkin Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Christine M. Szymanski Institute for Biological Sciences National Research Council Ottawa, Ontario Canada and

Martin J. Blaser Departments of Medicine and Microbiology New York University School o f Medicine and New York Harbor Veterans Affairs Medical Center New York, New York

ASM

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PRESS

Washington, DC

CONTENTS

Contributors Preface

I.

ix xv

The Organism

1. Taxonomy of the Family Campylobacteraceae 3 Lies Debruyne, Dirk Gevers, and Peter Vandamme 2. Population Biology of Campylobacter jejuni and Related Organisms 27 Martin C. J. Maiden and Kate E. Dingle 3. Complexity and Versatility in the Physiology and Metabolism of Campylobacter 41 jejuni David J. Kelly 4. Comparative Genomics of Campylobacter 63 jejuni Olivia L. Champion, Suaad Al-Jaberi, Richard A. Stabler, and Brendan W. Wren

5. Comparative Genomics of Campylobacter Species Other Than Campylobacter 73 jejuni William G. Miller

7. Clinical Significance of Carnpylobacter and Related Species Other Than Campylobacter jejuni and Campylobacter coli 123 Albert J. Lastovica and Ban Mishu Allos

8. Burden of Illness of Campylobacteriosis and Sequelae 151 Kdre M d b a k and Arie Havelaar 9. Epidemiology of Carnpylobacter jejuni Infections in Industrialized Nations 163 Christine K. Olson, Steen Ethelberg, Wilfrid van Pelt, and Robert V. Tauxe 10. Molecular Epidemiology of Campylobacter Species 191 Stephen L. W. On, Noel McCarthy, William G. Miller, and Brent J. Gilpin 11. Isolation, Identification, Subspecies Differentiation, and Typing of Campylobacter 213 fetus Marcel A. P. van Bergen, Jos P. M. van Putten, Kate E. Dingle, Martin J. Blaser, and Jaap A. Wagenaar

12. Diagnosis and Antimicrobial Susceptibility of Campylobacter Species 227 Collette Fitzgerald, Jean Whichard, and Irving Nachamkin

Clinical and Epidemiologic Aspects of 11. Campylobacter Infections

13. Guillain-BarrC Syndrome and Campylobacter Infection 245 Bart C. Jacobs, Alex van Belkum, and Hubert P. Endtz

6. Clinical Aspects of Carnpylobacter jejuni and Campylobacter coli Infections 99 Martin J. Blaser and Jargen Engberg

14. Mechanisms of Antibiotic Resistance in Campylobacter 263 Qijing Zhang and Paul J. Plummer

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CONTENTS

15. National Molecular Subtyping Network for Food-Borne Bacterial Disease Surveillance in the United States 277 Peter Gerner-Smidt, Steven G. Stroika, and Collette Fitzgerald

111.

IV.

Glycobiology

25. N-Linked Protein Glycosylation in Campylobacter 447 Harald Nothaft, Saba Amber, Markus Aebi, and Christine M. Szymanski

Pathogenesis and Immunity 26. 0-Linked Flagellar Glycosylation in Campylobacter 471 Susan M. Logan, Ian C. Schoenhofen, and Patricia Guerry 9

16. Interaction of Campylobacter jejuni with Host Cells 289 Robert 0. Watson andlorge E. G a l h

Cell Biology of Human Host Cell Entry by 17. Campylobacter jejuni 297 Lan Hu and Dennis]. Kopecko 8

Campylobacter jejuni Secretes Proteins via 18. the Flagellar Type I11 Secretion System That Contribute to Host Cell Invasion and Gastroenteritis 3 15 Charles L. Larson, Jeffrey E. Christensen, Sophia A. Pacheco, Scott A. Minnich, and Michael E. Konkel

27. Campylobacter jejuni Lipooligosaccharides: Structures and Biosynthesis 483 Michel Gilbert, Craig T. Parker, and Anthony P. Moran 28. Campylobacter jejuni Capsular Polysaccharide 505 Andrey V. Karlyshev, Brendan W. Wren, and Anthony P. Moran 29. Campylobacter Metabolomics 523 Evelyn C. Soo, David J. McNally, lean-Robert Brisson, and Christopher W. Reid

19. Innate Immunity in Campylobacter Infections 333 Nicole M. Iovine

V.

20. Chemosensory Signal Transduction Pathway of Campylobacter jejuni 351 Victoria Korolik and Julian Ketley

Regulation of Flagellar Gene Expression 30. and Assembly 545 David R. Hendrixson

21. Animal Models of Campylobacter jejuni Infections 367 Linda S. Mansfield, David B. Schauer, andJames G. Fox

Natural Competence and Transformation in 3 1. Campylobacter 559 Rebecca S. Wiesner and Victor]. DiRita

Genes and Gene Expression

22. Rabbit Model of Guillain-BarrC 381 Syndrome Nobuhiro Yuki

Survival Strategies of Campylobacter jejuni: 32. Stress Responses, the Viable but Nonculturable State, and Biofilms 571 Sarah L. Svensson, Emilisa Frirdich, and Erin C. Gaynor

23. Pathogenesis of Campylobacter fetus 401 Martin 1.Blaser, Diane G. Newell, Stuart A. Thompson, and Ellen L. Zechner

33. Iron Metabolism, Transport, and Regulation 59 1 Alain Stintzi, Arnoud H. M. van Vliet, andlulian M. Ketley

24. Development of a Human 429 Vaccine David R. Tribble, Shahida Baqar, and Stuart A. Thompson

Regulation of Genes in Campylobacter 34. jejuni 611 Marc M. S. M. Wosten, Andries van Mourik, and 10s P. M. van Putten

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CONTENTS

VI.

Food Safety and Intervention

35. Campylobacter in the Food 627 Supply Wilma Jacobs-Reitsma, Ulrike Lyhs, and Jaap Wagenaar Transmission of Antibiotic Resistance from 3 6. Food Animals to Humans 645 Frank M. Aarestrup, Patrick F. McDermott, and Henrik C. Wegener

37. Poultry Colonization with Campylobacter and Its Control at the Primary Production Level 667 Jaap A. Wagenaar, Wilma Jacobs-Reitsma, Merete Hofshagen, and Diane Newell 38. Bacteriophage Therapy and Campylobacter 679 Ian F. Connerton, Phillippa L. Connerton, Paul Barrow, Bruce S. Seal, and Robert J. Atterbuy Index

695

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CONTRIBUTORS

Frank M . Aarestrup National Food Institute, DK-1790 Copenhagen V, Denmark

Martin J. Blaser Department of Medicine, New York University School of Medicine, New York, NY 10016

Suaad Al-Jaberi Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, United Kingdom

Jean-Robert Brisson NRC-Institute for Biological Sciences, Ottawa, Ontario K1A OR6, Canada Olivia L. Champion School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom

Ban Mishu Allos Division of Infectious Diseases, Vanderbilt University School of Medicine, Nashville, TN 37232

Jeffrey E. Christensen School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234

Markus Aebi Department of Biology, Institute of Microbiology, Eidgenossische Technische Hochschule (ETH) Zurich, CH-8093 Zurich, Switzerland

Ian F. Connerton School of Biosciences, Division of Food Sciences, University of Nottingham, Loughborough, Leics LE12 SRD, United l n g d o m

Saba Amber Department of Biology, Institute of Microbiology, Eidgenossische Technische Hochschule (ETH) Zurich, CH-8093 Zurich, Switzerland

Phillippa L. Connerton School of Biosciences, Division of Food Sciences, University of Nottingham, Loughborough, Leics LE12 5RD, United Kingdom

Robert J. Atterbury Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 ~ D U Y United Kingdom

Lies Debruyne Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Shahida Baqar Enteric Diseases Program, Naval Medical Research Center, Silver Spring, MD 20910-7500

Kate E. Dingle Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom

Paul Barrow School of Veterinary Medicine and Science, Division of Food Sciences, University of Nottingham, Loughborough, Leics LE12 SRD, United Kingdom

Victor J. DiRita Department of Microbiology and Immunology and Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI 48 103 ix

x

CONTRIBUTORS

Hubert P. Endtz Laboratory Sciences Division, International Centre for Diarrheal Disease Research Bangladesh, Dhaka 1212, Bangladesh

Patricia Guerry Naval Medical Research Center, Silver Spring, MD 20910

Jargen Engberg Department of Clinical Microbiology, Hvidovre Hospital, DK-2650 Hvidovre, Denmark

Arie Havelaar Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and the Environment, Bilthoven, The Netherlands

Steen Ethelberg Department of Epidemiology and Department of Bacteriology, Statens Serum Institut, DK-2300 Copenhagen S, Denmark

David R. Hendrixson Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Collette Fitzgerald Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333

Merete Hofshagen National Veterinary Institute, Oslo, Norway

James G. Fox Division of Comparative Medicine and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Lan Hu Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892

Emilisa Frirdicb Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada

Nicole M . Iovine Department of Medicine, New York University School of Medicine, Veteran’s Administration Medical Center, New York, NY 10010

Jorge E. Gal& Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536

Bart C . Jacobs Departments of Neurology and Immunology, Erasmus University Medical Center, Rotterdam 3015 CE, The Netherlands

Erin C. Gaynor Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada Peter Gerner-Smidt Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333 Dirk Gevers Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium Michel Gilbert Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada Brent J. Gilpin Water Management Programme, Institute of Environmental Science and Research Ltd., Christchurch, New Zealand

Wilma Jacobs-Reitsma RIKILT Institute of Food Safety, 6708 PD Wageningen, The Netherlands Andrey V. Karlysbev London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom David J. Kelly Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom Julian Ketley Department of Genetics, University of Leicester, Leicester LE1 7RH, United Kingdom Michael E. Konkel School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234

CONTRIBUTORS

Dennis J. Kopeck0 Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892 Victoria Korolik Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia Charles L. Larson School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234 Albert J. Lastouica Department of Biotechnology, University of the Western Cape, Bellville 7535, South Africa

xi

Scott A. Minnich Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, MOSCOW, ID 83844-3 052 a r e Mslbak Department of Epidemiology, Statens Serum Institute, Copenhagen, Denmark Anthony P. Moran Department of Microbiology, National University of Ireland, Galway, Ireland, and Institute for Glycomics, Griffith University, Queensland, Australia Irving Nachamkin Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283

Susan M . Logan Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

Diane G. Newel1 Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom

Ulrike Lyhs Seinajoki Unit, Ruralia Institute, University of Helsinki, FI-60320 Seinajoki, Finland

Harald Nothaft Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

Martin C . J. Maiden Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom

Christine K. Olson Enteric Diseases Epidemiology Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333

Linda S. Mansfield Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Stephen L. W . On Food Safety Programme, Institute of Environmental Science and Research Ltd., Christchurch, New Zealand

Noel McCarthy Department of Zoology, University of Oxford, Oxford OX1 3SY, United Kingdom

Sophia A. Pacheco School of Molecular Biosciences, Washington State University, Pullman, WA 99 164-4234

Patrick F. McDermott National Antimicrobial Resistance Monitoring System, Center for Veterinary Medicine, U.S. Food & Drug Administration, Laurel, MD 20708

Craig T. Parker Produce Safety and Microbiology Research, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710

David J. McNally NRC-Institute for Biological Sciences, Ottawa, Ontario K1A OR6, Canada

Paul J. Plummer Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, JA 50011

William G. Miller Produce Safety and Microbiology Research Unit, USDA, ARS, Albany, CA 94710

Christopher W. Reid NRC-Institute for Biological Sciences, Ottawa, Ontario K1A OR6, Canada

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CONTRIBUTORS

David B. Schauer Division of Comparative Medicine and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

David R. Tribble Preventive Medicine and Biometrics Department, Uniformed Services University of the Health Sciences, Bethesda, MD 208 14-5119

Ian C . Schoenhofen Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada

Alex van Belkum Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam 3015 CE, The Netherlands

Bruce S. Seal Poultry Microbiological Safety Research Unit, Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30605 Evelyn C . So0 NRC-Institute for Marine Biosciences, Halifax, Nova Scotia B3H 321, Canada Richard A. Stabler Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, United Kingdom Alain Stintzi Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada Steven G. Stroika Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333 Sarah L. Svensson Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada Christine M . Szymanski Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada Robert V. Tauxe Division of Foodborne, Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333 Stuart A. Thompson Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912

Marcel A. P. van Bergen Division of Infectious Diseases, Animal Sciences Group, OIE Reference Laboratory for Campylobacteriosis, 8200 AB Lelystad, The Netherlands Peter Vandamme Laboratory of Microbiology, Ghent University, B9000 Gent, Belgium Andries van Mourik Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands Wilfrid van Pelt Department of Epidemiology and Surveillance, Rijksinsituut voor Volksgezondheid en Milieu, 3721 MA Bilthoven, The Netherlands Jos P. M . van Putten Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, 3508 TD Utrecht, The Netherlands

Arnoud H . M . van Vliet Institute of Food Research, Norwich NR4 7UA, United Kingdom Jaap A . Wagenaar Division of Infectious Diseases, Animal Sciences Group , OIE Reference Laboratory for Campylobacteriosis and WHO Collaboration Centre for Campylobacter, 8200 AB Lelystad, and Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, 3.508 TD Utrecht, The Netherlands Robert 0. Watson Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536 Henrik C . Wegener National Food Institute, DK-1790 Copenhagen V, Denmark

CONTRIBUTORS

xiii

Jean Whichard Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333

Nobuhiro Yuki GBS Laboratory, 3-9-5 Kamiikedai, Ota-ku, Tokyo 145-0064, Japan

Rebecca S. Wiesner University of Michigan, Ann Arbor, MI 48103

Ellen L. Zechner Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria

Marc M . S. M . Wosten Department of Infectious Diseases and Immunology, Utrecht University, 3584 CL Utrecht, The Netherlands Brendan W. Wren Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WClE 7HT, United Kingdom

Qijing Zhang Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011

PREFACE

late Gerald Aspinall, has provided a structural basis for important polysaccharides and glycolipids from the organism and great insight into glycosylation systems, which also are present in other prokaryotes and eukaryotes. This work, combined with an increased understanding of the molecular biology of gene expression in Campylobacter, is helping to form a more complete picture about the organism and its interaction with the host and environment. Ultimately, research will help improve human health through understanding the immunology of campylobacter infection and the pertinent host defenses. These will lead to the development of strategies to reduce infection, through either effective vaccines or improved food safety. The ecology of food safety also has seen a dramatic increase in research, with emphasis on understanding the extent of campylobacter in the food supply, transmission of antibiotic resistant campylobacters from food animals to humans, and control of campylobacter at the food source. The above advances have helped to form the nucleus for the 3'd edition of Campylobacter. We are grateful to the scientific groups from around the world that have contributed to the preparation of these outstanding chapters. These represent the generous sharing of our growing knowledge of these important zoonotic pathogens of humans. Finally, we especially thank Greg Payne at ASM Press for his help and guidance throughout the process of developing this volume. Irving Nachamkin Christine M. Szymanski Martin J. Blaser February 2008

Our understanding of the clinical aspects, epidemiology, and pathogenesis of Campylobacter infection has increased dramatically since publication of the second edition of Campylobacter in 2000. As the number of species within the family has expanded, so has our knowledge of this group of organisms in terms of their physiology, population biology, and diversity. The ability to understand genomic diversity in Campylobacter is due in great part to improvements in technology that have advanced comparative analyses. The sequencing of the first C. jejuni strain in 2000 was a milestone in Campylobacter genetics and brought to light aspects of biology that previously could not have been identified. Since then, additional complete Campylobacter genome sequences have been published and have provided new insights about the genomic diversity of these organisms, which now is covered extensively in this new edition. Campylobacter infections and their complications, such as Guillain-BarrC syndrome, cause significant morbidity in specific populations. There is growing recognition of Campylobacter among clinicians as well as by the lay public. Antimicrobial resistance also continues to increase and poses new issues regarding therapies. Of particular note are the impact that researchers in our field have had on government regulation of antimicrobial agent use in food animals and how effecting change will ultimately improve human health. Our understanding of the pathogenesis of Campylobacter infections has advanced greatly, especially in the area of signal transduction pathways and cell biology of the organism. The emerging field of Campylobacter glycobiology, which was pioneered by the

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I. THE ORGANISM

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 1

Taxonomy of the Family Campylobacteraceae LIESDEBRUYNE, DIRKGEVERS,AND PETER VANDAMME

fer, and species-specific core genomes and strainspecific accessory genomes have been described for several bacterial species (Dagan and Martin, 2006; Kunin et al., 2007). It would be unrealistic to expect that in the near future bacterial species will be described on the basis of whole-genome sequences only. Several recent studies (Goris et al., 2007; Konstantinidis et al., 2006), however, demonstrated that multiple parameters in whole-genome sequences reflect the traditional delineation of species at the 70% DNA-DNA hybridization level. In particular, multilocus sequence analysis (MLSA), a technique based on sequence analysis of fragments of multiple household genes present in the core genome, has been put forward as a novel approach for the classification of bacteria (Gevers et al., 2005). Konstantinidis et al. (2006) demonstrated that phylogenetic trees based on MLSA data can bear the same phylogenetic signal as trees that are based on sequence analysis of the entire common gene pool, implying that one does not need entire genome sequences for all strains under comparison to design taxonomic frameworks equivalent to frameworks that are based on whole-genome content. MLSA schemes have been developed for a growing number of bacteria, including some members of the family Campylobacteraceae (see below). Additional studies are needed to provide pragmatic criteria for determining how we can use MLSA data to define species and subdivisions within species for poorly studied or newly discovered taxa.

This chapter will present a traditional overview of the biological diversity of Campylobucter and Arcobacter but will also address the taxonomic information that has become available through whole-genome sequence analysis. Starting in the 1990s, genomic sequencing drastically changed our understanding of the relationships between microorganisms. Many new papers have been evaluating diversity within and among species by exploiting whole-genome information content, and the bacterial species concept emerged from obscurity into the spotlight (Doolittle and Papke, 2006; Gevers et al., 2005; Konstantinidis et al., 2006). Since the 1960s, the level of whole-genome DNA-DNA hybridization is the accepted standard for species delineation, with 70% DNA-DNA reassociation being the recommended species cutoff. Genomic DNA-DNA hybridization has been used as the key technology for the phylogenetic delineation of bacterial species because whole-genome sequences were assumed to provide a rational and objective basis for the description and delineation of bacterial species. While waiting for easy access to whole-genome sequences to emerge, the genomic DNA-DNA hybridizations were the best approach to comparing wholegenome sequences. Meanwhile, sequencing technology improved so dramatically that several hundreds of bacterial genomes have been-or are in the process of beingsequenced and annotated. It has become apparent that apart from the traditional nucleotide changes, other selective forces such as gene deletion, gene duplication, horizontal gene transfer, and chromosomal rearrangements shape the bacterial genome and that considerable fractions of the genome can be strain specific. Numerous studies, however, have stressed the dominance of vertical over horizontal gene trans-

TAXONOMIC HISTORY The early history of the genus Campylobacter, starting with the first description by Theodor Escher-

Lies Debruyne, Dirk Gevers, and Peter Vandamme Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Bioinformatics and Evolutionary Genomics, Ghent University/VIB, Technologiepark 927, B-9052 Gent, Belgium. Dirk Gevers Gent, Belgium.

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4

DEBRUYNE ET AL.

ich in 1886, the first isolation of a Vibrio-like organism from aborted ovine fetuses by McFadyean and Stockman in 1913, the emergence of several novel “Vibrio” species in a range of animals with and without clinical symptoms, and the creation of the genus Campylobacter in 1963, has been outlined in detail in several previous reviews (Butzler, 2004; Skirrow, 2006). In 1963, Sebald and VCron transferred two of these Vibrio species, Vibrio fetus and Vibrio bubulus, into the new genus Campylobacter as Campylobacter fetus and Campylobacter bubulus, respectively. The former was the bacterium first isolated by McFadyean and Stockman (1913), which was subsequently also isolated from aborted bovine fetuses (Smith and Taylor, 1919) and from blood cultures of women who aborted (Vinzent et al., 1947); the latter was an organism isolated from the bovine vagina and semen (Florent, 1953). The reasons for transferring these two species into the novel genus Campylobacter included their low DNA base composition, their microaerophilic growth requirements, and their nonfermentative metabolism. Ten years later, VCron and Chatelain (1973) published a more comprehensive study on the taxonomy of the microaerophilic Vibrio-like organisms and considered four distinct species in the genus Campylobacter: C. fetus (the type species), Campylobacter coli (a vibrio isolated from feces of pigs with diarrhea [Doyle, 1948]), Campylobacter jejuni (a vibrio isolated from the feces of cattle with diarrhea Uones et al., 19311, blood cultures of humans with gastroenteritis [King, 1957; Levy, 19461 and aborted sheep fetuses [Bryans et al., 1960]), and Campylobacter sputorum. The latter comprised two subspecies, C. sputorum subsp. sputorum (a vibrio isolated from sputum of a patient with bronchitis [Prihot, 1940; Tunicliff, 19141) and C. sputorum subsp. bubulus (a vibrio isolated from the bovine vagina and semen [Florent, 19531). The application of a filtration technique used in veterinary microbiology allowed Butzler and coworkers in the early 1970s to exploit the small cell dimensions and the vigorous motility of Campylobacter cells to selectively isolate them from stools of humans with diarrhea (Butzler et al., 1973; Dekeyser et al., 1972). The main breakthrough, however, was provided a few years later by Skirrow, who described a selective supplement comprising a mixture of vancomycin, polymyxin B, and trimethoprim that was added to a basal medium (Skirrow, 1977). This simple isolation procedure thus enabled routine diagnostic microbiology laboratories to isolate campylobacters and to evaluate their clinical role. The availability of adequate isolation procedures led to a renewed interest in Campylobacter research

during the early 1980s. As a consequence, manifold Campylobacter-like organisms were isolated from a variety of human, animal, and environmental sources, and gradually, new species were described (Benjamin et al., 1983; Fox et al., 1989; Gebhart et al., 1985; Lawson et al., 1981; Marshall et al., 1984; McClung et al., 1983; Neil1 et al., 1985; Tanner et al., 1981). Classical biochemical tests routinely used for the identification of clinical bacteria often yielded negative or variable results within Campylobacter species. This poor biochemical reactivity and lack of clear-cut differential characters led to the wide application of vernacular names for many groups of Campylobacter-like organisms, and the term CLO (Campylobacter-like organism) became widely used. Some of these CLO groups were later classified as novel species, but several were identified as biochemical variants of well-known species (Vandamme, 2000). Still others remained unnamed: for example, the CLO-3 strain that was isolated in the early 1980s was not formally classified pending the isolation and characterization of additional strains belonging to the same taxon (Totten et al., 1985). An overview of these empirical names with their final classification has been given previously (Vandamme, 2000). Also in the 1980s, the study of bacterial phylogeny as imprinted in the degree of rRNA cistron similarity became more popular and was increasingly used to evaluate and revise bacterial classification schemes. Long-standing genera like Pseudomonas, Flavobacterium, Bacteroides, and many others were found to be extremely heterogeneous, and their classification was gradually revised to conform to the new phylogenetic (or “natural”) insights. The first rRNA-based phylogenetic studies of Campylobacter species were published in the late 1980s and demonstrated that Campylobacter too was heterogeneous, as it comprised three distinct phylogenetic lineages that also included members of the genus Wolinella and two Bacteroides species, Bacteroides gracilis and Bacteroides ureolyticus, which appeared phylogenetically distinct from the true Bacteroides species (Vandamme, 2000). In 1989, Goodwin et al. summarized genotypic and phenotypic arguments to exclude the gastric species Campylobacter pylori (humans) and Campylobacter mustelae (ferrets) from the genera Campylobacter and Wolinella and proposed a novel genus, Helicobacter, to accomodate both. Two years later, a complete revision of the taxonomy and nomenclature of the genus Campylobacter and related bacteria was proposed by Vandamme et al. (1991a). The results of an extensive DNA-rRNA hybridization study of over 60 strains representing all known Campylobacter species, CLO groups, and putative relatives such

CHAPTER 1

as Wolinella, Bacteroides, and “Flexispira” species corroborated and extended the 16s rRNA sequencing data. Carnpylobacter, together with Wolinella and “Flexispira” and B. gracilis and B. ureolyticus, was found to represent a separate, sixth rRNA superfamily sensu De Ley within the group of the gramnegative bacteria. This phylogenetic lineage is now known as the epsilon subdivision of the proteobacteria. Within this lineage, C. fetus, Campylobacter hyointestinalis (Gebhart et al., 1985), Campylobacter concisus (Tanner et al., 1981), Campylobacter mucosalis (Lawson et al., 1981; Roop et al., 1985a), C. sputorum, C. jejuni, C. coli, Campylobacter lari (Benjamin et al., 1983), Campylobacter upsaliensis (Sandstedt and Ursing, 1991), Wolinella curva (Tanner et al., 1984), Wolinella recta (Tanner et al., 1981), B. gracilis, and B. ureolyticus constituted the first rRNA homology cluster. Campylobacter nitrofigilis (McClung et al., 1983), Campylobacter cryaerophila (Neil1 et al., 1985), and several other aerotolerant CLO strains (which were later classified as Arcobacter butzleri and Arcobacter skirrowii vandamme et al., 1992]), constituted the second rRNA homology cluster. Helicobacter pylori (Goodwin et al., 1989; Marshall et al., 1984), H. mustelae (Fox et al., 1989; Goodwin et al., 1989), Campylobacter cinaedi (Totten et al., 1985), Campylobacter fennelliae (Totten et al., 1985), W. succinogenes (Wolin et al., 1961), “Flexispira rappini“ (Bryner et al., 1986), and the CLO-3 strain (Totten et al., 1985) constituted a third rRNA homology cluster. In addition, the free-living saprophytic Campylobacter strains of Laanbroek et al. (1977) and Wolfe and Pfennig (1977) (later reclassified as Sulfurospirillum species) belonged to the same phylogenetic lineage but did not belong to one of the three major rRNA homology clusters. A revised classification was proposed that took into account the phylogenetic results derived from the DNA-rRNA hybridization experiments and that published data on the genotype and phenotype of these organisms. The genus Carnpylobacter was restricted to those species belonging to the rRNA homology cluster containing C. fetus, the type species of the genus Campylobacter. The generically misnamed W. curva and W. recta, and in a subsequent study, B. gracilis (Vandamme et al., 1995), were included in the emended genus Campylobacter as Campylobacter curvus, Campylobacter rectus, and Campylobacter gracilis, respectively. B. ureolyticus was obviously a close relative of the emended genus Campylobacter, but its taxonomic status was not changed pending further study of this taxon and the isolation of similar bacteria (Vandamme et al., 1991a, 1995). The name Arcobacter (with Arcobacter nitrofigilis as the type species) was proposed for the organisms be-

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TAXONOMY OF THE CAMPYLOBACTERACEAE

5

longing to the second rRNA homology cluster. Finally, C. cinaedi and C. fennelliae were transferred into the genus Helicobacter as H. cinaedi and H. fennelliae, respectively, and an emended genus description was proposed. Wolinella succinogenes remained the only species of the genus Wolinella. Several new Campylobacter, Arcobacter, and Helicobacter species have been reported since then. Campylobacter and Arcobacter species will be discussed below. A detailed overview of Helicobacter species is beyond the scope of the present chapter. Finally, the genera Campylobacter and Arcobacter were accomodated in the new bacterial family, the Campylobacteraceae (Vandamme and De Ley, 199l), because they shared several genotypic and phenotypic characteristics. The family Campylobacteraceae currently also comprises the generically misclassified species B. ureolyticus and the genus Sulfurospirillum (Stolz et al., 2005). In addition, a growing number of free-living Epsilonproteobacteria occupy phylogenetic positions among the Campylobacter, Arcobacter, Sulfurospirillum, and Wolinella-Helicobacter clades. The allocation of these bacteria to a bacterial family such as the Campylobacteraceae or others needs further study. Figure 1 represents a traditional phylogenetic tree of the family Campylobacteraceae that is based on percentage of 16s rRNA gene sequence similarity.

WHOLE-GENOME TAXONOMY OF CAMPYLOBACTER AND RELATED BACTERIA Species Tree of Campylobacter and Related Bacteria The progress in the whole-genome studies of Campylobacter and related bacteria presents an opportunity to produce a more robust species tree. Data sets consisting of a single gene or a small number of concatenated genes have a significant probability of supporting conflicting topologies. By contrast, a fully resolved species tree with maximum support can come from the concatenation of a large number of carefully selected genes, which whole-genome sequences provide in abundance. A total of 25 complete and incomplete genome sequences have been obtained for 15 species belonging to the Campylobacteraceae and related bacteria (A. butzleri, C. coli, C. concisus, C. curvus, C. fetus, C. jejuni, C. lari, C. upsaliensis, Helicobacter acinonychis, H. hepaticus, H. mustelae, H. pylori, Sulfurimonas denitrificans, Thiornicrospira crunogena, and W. succinogenes; http: //www.genomesonline.org/). These genomes were subjected to a comparative genome analysis to

6

DEBRUYNE ET AL.

-

-Sulfurospirillum cavolei Phe91T(AB246781) -Sulfurospirillum sp. DSM 806 (AF144694) Sulfurospirillum barnesii SES-3' (AF038843) ulfurospirillum deleyianum Spirillum 5 17.5' (Y13671) Sulfurospirillum multivorans K' (X82931) Sulfurospirillum halorespirans PCE-M2' (AF218076) "CandidatusArcobacter sulfidicus" (AY035822) Arcobacter butzleri ATCC 49616T(AY621116) obacter cibarius LMG 21996T(AJ607391) Arcobacter skirrowii CCUG 10374T(L14625) Arcobacter cryaerophilus CCUG 17801' (L14624) Arcobacter nitrofigilis CCUG 15893T(L14627) Arcobacter halophilus LA3 lBT(AF513455) Campylobacter hyointestinalis subsp. lawsonii CHY 5' (AF097685) -CCampylobacier lanienae NCTC 13004T(AF043425) Campylobacter hyointestinalis subsp. hyointestinalis NCTC 11608' (AF097689) mpylobacterfetus subsp.fetus ATCC 27374' (LO4314) Campylobacterfetus subsp. venerealis ATCC 19438T(L14633) Campylobacter canadensis L266T(EF621894) Campylobacter upsaliensis CCUG 14913' (L14628) -CCampylobacter helveticus NCTC 12470T(U03022) Campylobacter coli CCUG 11283' (LO4312) Campylobacter lari CCUG 23947' (LO4316) Campylobacter insulaenigrae NCTC 12927T(AJ620504) Campylobacterjejuni subsp. doylei CCUG 24567T(L14630)

: L

-

-I

-

c

Figure 1. Phylogenetic tree of the family Campylobacteruceae and related bacteria, based on percentage of 16s rRNA gene sequence similarity.

screen for protein families that together might reveal a major vertical component of species phylogeny (D. Gevers, unpublished data). Orthologous gene families were selected as phylogenetic markers if they met the following criteria: (i) had exactly one copy in each of

the 25 strains, (ii) had a minimum length of 100 amino acids after ambiguous positions were marked out, and (iii) showed no noisy phylogenetic signal from recombination or horizontal gene transfer. This resulted in a carefully selected set of 60 core genes

CHAPTER 1

that could be used to estimate the phylogeny of these bacteria by maximum likelihood (Fig. 2). In general, this species tree correlates nicely with the phylogenetic tree that is based on 16s rRNA gene sequences, but compared with the latter, it has a superior resolution to distinguish closely related bacteria such as C. jejuni subsp. jejuni, C. jejuni subsp. doylei, and C. coli. This tree also confirms and widens previous insights in the evolutionary relationships based on 12 concatenated protein data sets including only seven species (Fouts et al., 2005). By including more C. jejuni genomes, it is not only confirmed that C. jejuni RM1221 is more closely related to the C. jejuni subsp. jejuni strains than to C. coli, but it is also shown that it is closer to any C. jejuni subsp. jejuni than to C. jejuni subsp. doylei 269.97. Further, it confirms that W. succinogenes DSM 1740Tis more closely related to Helicobacter spp. than to any Campylobacter species. In addition, it was found that S. denitrificans ATCC 3 3 889= (reclassification from Thiomicrospira denitrificans) is more closely related to A. butzleri and Campylobacter than to T. crunogena.

0.2

I -

I

I

TAXONOMY OF T H E CAMPYLOBACTERACEAE

7

Whole-Genome Comparisons

As in other groups of bacteria, bioinformatic analyses of the growing number of whole-genome sequences of Campylobacter strains is explaining to which extent strains within species and strains representing different species are different. Goris et al. (2007) demonstrated that the recommended cutoff of 70% DNA-DNA reassociation for species delineation corresponds to 69% conserved DNA and 95% average nucleotide identity (ANI), a measure for evolutionary relatedness that is based on sequence similarity between orthologous genes. As shown in Fig. 3 , the AN1 values among C. jejuni strains do fall above this cutoff and range between 95.24 and 99.68%, with a significant partition between its two subspecies: intrasubspecies values for C. jejuni subsp. jejuni are on average 97.85% ( _ t 0.62%), and intersubspecies values for C. jejuni subsp. doylei versus C. jejuni subsp. jejuni are on average 95.80% (k0.28%). In comparison, Helicobacter pylori comprises a more heterogenous set of strains, with lower AN1 values ranging between 94.05 and 95.57%. The AN1 values

H . pylori 26695 H. p ylori H PAG 1 H . pylori399 H. acinon ychis str. Sheeba H. mustelae ATCC 43772 H . hepaticus ATCC 51449 Wolinella succinogenes DSM 1740 Sulfurimonas denitrificans ATCC 33889 Arcobacter butzleri 40 18 C. fetus subsp. fetus 82-40

HB93-13 81-176 260.94 C. j e j u n i subsp. j e j u n i CG8486 .C. j e j u n i RM1221 C. j e j u n i subsp. j e j u n i NCTC 11168 C. jejuni subsp. jejuni 84-25 CF93-6 Thiomicrospira crunogena X C L- 2

Figure 2. Whole-genome-based phylogeny of Campylobacter and related bacteria. Out of 365 Campylobacter core gene families, a concatenation of 60 carefully selected genes was used to estimate the phylogeny of these bacteria by maximum likelihood. Orthologous protein families were determined by OrthoMCL (Li et al., 2003). Protein sequence alignments were generated by Muscle (Edgar, 2004), and poorly conserved regions were trimmed by Gblocks (Castresana, 2000). Phylogenetic trees with 100 bootstrap replicates were. obtained by the PhyML algorithm (Guindon and Gascuel, 2003) with a JIT amino acid substitution matrix and a discrete gamma model. All nodes had 100% bootstrap support.

8

DEBRUYNE ET AL.

pylori-acinonychis 0

u 1

s!

60

0 5

0

80

90

100 100 I

95

90

85

80

75

70

Average Nucleotide Identity (ANI)

Figure 3. Genetic diversity among genomes of Cumpylobacter and related bacteria. Every data point represents a wholegenome comparison between two genomes (both intra- and interspecies) and shows the percentage AN1 on the x axes plotted against the percentage of gene content similarity on the y axes. Only comparisons with AN1 values above 70% are shown because this is the range in which AN1 is considered to be a robust and sensitive method for measuring evolutionary relatedness (Konstantinidis and Tiedje, 2005). AN1 values were calculated as described previously (Konstantinidis and Tiedje, 2005), and gene content similarity values were calculated on the basis of reciprocal best BLASTp hits.

are useful for quantifying evolutionary relatedness between different species and correspond to wholegenome-based phylogenies. The closest related species of c. jejuni among the included genomes is c. coli (ANIs of 83.23 -t 0.29%), and the second closest is C. upsaliensis (ANIs of 72.89 0.10%). In comparison, the evolutionary distance between H. pylori and H. acinonychis is smaller than any two species of Campylobacter included in our analyses (ANIs of 89.25 ? 0.18). The gene content similarity values, shown in Fig. 3, were determined to reveal the effect of ecology on this group of organisms. It has been reported before that the environment is one of the main factors in shaping the evolution of the gene content of an organism (Konstantinidis et al., 2006). Larger intraspecific genomic differences could be the outcome of a differential evolution of those strains in different environments. The intraspecific values of the C. jejuni

*

strains (excluding the C. jejuni subsp. jejuni CG8486 strain) go as low as 74%, indicative of an ecologically versatile organism. These differences are likely to be determined by the broad host range of C. jejuni, which includes human, cattle, and poultry as well as a large variety of wild bird species (Schouls et al., 2003). Similar large genomic differences within a species have been reported for Escherichia coli (Konstantinidis et al., 2006). The unfinished C. jejuni subsp. jejuni CG8486 genome sequence has significantly fewer annotated genes in comparison with other strains of this species (1,421 versus 1,750 2 100, respectively), which explains why comparisons with this strain result in more extreme gene content similarity values, and thus its gene content similarity values should be considered with caution. The AN1 values do not require the same reservation because they are not affected by large genome size differences in the same way. In contrast to the intraspecific values

CHAPTER 1

of the C. jejuni strains, the H. pylori strains show substantially smaller genomic differences, ranging between 83 and 90%, even though their AN1 values indicate a larger evolutionary distance. This is a strong indication of its narrower ecological nichethat is, it is limited to the human host. It is well established that the phenotypes and genotypes of C. jejuni and C. coli are remarkably similar, resulting in an important taxonomic and diagnostic problem. Figure 3 provides us with a quantitative measure of this similarity. Although the AN1 values reflect a clear distinction between these two species, c. coli seems to have a large set of genes in common with some of the C. jejuni strains, likely explaining the confusing phenotypic test results. Which and How Many Genes Are Needed To Predict the Whole-Genome-Based Phylogeny? Because of the limitations of 16s rRNA gene analysis for accurate species-level identification of campylobacters (see below), several other genes were examined for taxonomic and identification purposes. Karenlampi et al. (2004) demonstrated that partial groEL gene sequences could be used to construct a phylogeny similar to that obtained by 16s rRNA gene sequences, but with lower interspecies and high intraspecies similarities. Korczak et al. (2006) used rpoB sequences and also obtained a good congruence between rpoB and 16s rRNA gene sequence-based trees. Again, resolution for the rpoB gene was much higher than for the 16s rRNA gene, enabling differentiation of even closely related Campylobacter species, as well as of most subspecies. In 2001, Dingle and coworkers (2001) reported a first multilocus sequence typing scheme that is based on seven housekeeping loci to study population biology in C. jejuni. Since then, the analysis of polymorphisms in multiple coding gene sequences has been used in several other Campylobacter species for typing purposes (Dingle et al., 2005; Litrup et al., 2007; van Bergen et al., 2005a). Miller et al. (2005) reported the first multispecies, multilocus sequence-based scheme that allowed analysis of multiple Campylobacter species (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus) but used it for strain typing and not for studying the phylogeny and identification of these bacteria. Yet the analysis of multilocus sequences currently represents the most realistic approach for studying species diversity and evolution at large (Gevers et al., 2005). But how do a few genes correlate with whole-genome-based phylogeny in the case of the Campylobacteraceae, and what genes should preferentially be used to improve phylogenetic accuracy in a cost-effective way? Genes currently used in multi-

TAXONOMY OF THE CAMPYLOBACTERACEAE

9

locus sequencing approaches are often selected primarily on the basis of being used previously in other taxa, being essential, being unlinked in the genome, and having a successful primer design. However, their phylogenetic robustness is overlooked, and a selection based on their predictive power for the whole-genome-based phylogeny would be better. Taking such an approach, it has been shown that a more accurate phylogeny for the Escherichia coli group can be obtained from three best-performing genes compared with the concatenated alignment of eight commonly used markers (Konstantinidis et al., 2006). The availability of 17 Campylobacteraceae genomic sequences allows a similar analysis. We evaluated the correlation between all the 548 Campylobacteraceae core genes separately, and we assessed the whole-genome-based phylogeny by performing comparisons both in terms of tree topology (Robinson-Foulds symmetric difference distance) and branch length (branch score distance of Kuhner and Felsenstein) with the FTREEDIST (http: //emboss. sourceforge.net/) and TOPD (Puigbo et al., 2007) software tools. This methodology guided the selection toward a total of 60 best-performing genes with tree topologies and branch lengths highly similar to the whole-genome-based tree, i.e., the species tree. However, single-gene-based trees correlate poorly with the species tree at the shorter evolutionary scales, i.e., among the very closely related strains of C. jejuni, where only the concatenation of multiple loci provides enough resolving power. This can be explained by the finding that C. jejuni has a weakly clonal population structure as a result of high levels of intraspecific recombination (Suerbaum et al., 2001). Among the genes that are chosen as good phylogenetic markers, there appears to be no functional bias (Table 1). By use of the general functional classification from the COG (Cluster of Orthologous Genes) database (Tatusov et al., 1997), it was found that the genes are evenly distributed over the categories of information storage and processing (25%), cellular processes and signaling (28%), and metabolism (32%), with the remainder classified in the “poorly characterized” category or not classified in any COG at all. For a gene to be practically useful as a phylogenetic marker in an MLSA approach, it should allow the development of a set of conserved primers of limited degeneracy. To identify regions corresponding to the most highly conserved segments of each of the 60 best-performing genes, amino acid alignments were submitted to the BLOCKS Multiple Alignment Processor (Henikoff et al., 1995; http://

Table 1. Functional classification of 60 selected phylogenetic markers for the Campylobacteraceae" Gene name

Product description

P7fA miaA" miaB dusB

Peptide chain release factor 1 tRNA delta(2)-isopentenylpyrophosphatetransferase tRNA-i(6)A37 thiotransferase enzyme tRNA-dihydrouridine synthase B Ribosomal protein L25 GTP-binding protein Glycyl-tRNA synthetase beta subunit Methionyl-tRNA formyltransferase DNA gyrase, B subunit Exodeoxyribonuclease VII, large subunit Excinuclease ABC subunit C DNA polymerase type I Holliday junction DNA helicase RuvB DNA gyrase, A subunit DNA processing protein A PP-loop family protein Chromosome partitioning protein, ParA family Mur ligase family protein Membrane-associated zinc metalloprotease, putative Methyltransferase GidB Outer-membrane lipoprotein carrier protein precursor Permease, putative Peptidase, M23 /M37 family Lipopolysaccharide heptosyltransferase I Penicillin-binding protein DNA repair protein ATPase subunit of heat shock protein H s l W Heat shock protein 90 Mg chelatase-related protein Trigger factor Preprotein translocase, SecY subunit Signal recognition particle-docking protein Homoserine dehydrogenase Aspartate kinase Carbamoyl-phosphate synthase small chain Carbamoyl-phosphate synthase, large subunit CTP synthetase HIT family protein Phosphatase, Ppx/ GppA family Fructose-1,6-bisphosphatase ATP-NAD kinase, putative Dephospho-CoA kinase 4-Hydroxybenzoate octaprenyltransferase Glutamyl-tRNA reductase Dihydropteroate synthase Bifunctional 3,4-dihydroxy-2-butanone 4-phosphate Synthase/ GTP cyclohydrolase I1 protein Oxygen-independent coproporphyrinogen 111 oxidase Riboflavin synthase subunit alpha D-3-phosphoglycerate dehydrogenase Acetyl-CoA carboxylase beta subunit Phosphatidylserine decarboxylase Peptidase, putative GTP-binding protein, GTPl/ Obg family tRNA modification GTPase Conserved hypothetical protein Porphobilinogen deaminase Translation elongation factor Ts Multidrug resistance efflux transporter, putative Delta-aminolevulinic acid dehydratase Peptidase, U32 family

7p"'

ychF dYS fmt" gY7B

xseA" uvrc polA ruvB gY7A

dprA" b b

murF

gidB' lolA* z.

waaC radA hslU htpG

*

tig secY ftsY* hom lysC carA carB PYrG

*

fbP b

coaE* ubiA hemA folP ribB hemN ribE* serA accD psdb b

obg tmzE hemC tsf hemB

COG

J J J J J J J J

L L L L

L L LU D D M M M M M M M M

0 0 0 0 0 U U E E EF EF F FGR FP G G H H

H H H

H H HE I I R R R S

X X X X X

Genes for which automatic primer design was not successful are indicated with an asterisk. hFunctional annotation as the Cluster of Orthologous Genes (COG) database (Tatusov et al., 1997; http://www. ncbi.nlm.nih.gov/ COG).

a

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Next Page CHAPTER 1

bioinformatics.weizmann.ac.il/blocks/).The output from BLOCKS was subsequently subjected to automatic PCR primer design by CODEHOP with parameters maintained at their default settings (Rose et al., 1998). For a total of 41 of 60 genes, a primer combination could be generated whose predicted products ranged from 400 to 1,500 bp (Table 1). The strength of an MLSA approach lies in the concatenation of multiple genes buffering against the distorting effects of recombination at a single locus and resulting in an increased resolving power. Knowing a list of best-performing genes raises the question of how many genes should be used to come up with a cost-effective consensus phylogeny. Figure 4 shows the results of an in silico MLSA analysis that is based on a random combination of 2 to 15 genes selected from the best-performing gene set. When we look at the averages of 100 repetitions per evaluated loci count, we find that each additional gene used improves the correlation with the whole-genome-based phylogeny. However, it does this in a more prominent way, by comparing the lower loci count, whereas the gain in the use of higher loci count is less pronounced. These results also show that combinations of two genes exist that are more congruent to the whole-genome-based phylogeny than some combinations of seven or more genes. However, when only a few genes are used, the buffering capac-

I

I

I

I

I

1

2

3

5

7

I

1

0

I

1

5

Number of genes

Figure 4. In silico MLSA analysis with variable loci count. For a variable number (n = 1-15) of concatenated loci (x axis) randomly selected from the set of best-performing genes (Table l), the differences in tree topology and branch lengths with the wholegenome-based phylogeny was determined and expressed as a tree distance value (y axis). For each number of loci, the procedure was repeated 100 times, and the distributions of the distances between the maximum likelihood trees are plotted as box and whisker plots.

TAXONOMY OF THE CAMPYLOBACTERACEAE

11

ity against unexpected conflicting signals in even a few lineages is too limited. Moreover, in interpreting this analysis, it should be kept in mind that this set of genes was defined by using the same set of strains whose phylogeny we are trying to estimate. Therefore, the analysis does not guarantee similar performance for an independent set of strains. Modern statistical approaches have the potential to make the choice of genes less critical by using sequence information to extract a phylogenetic signal while simultaneously identifying the genes that show discordant signals for particular stains and incorporating this information in the analysis (Didelot and Falush, 2007).

GENERAL DESCRIPTION The Family Campylobacteraceae The genera Campylobacter, Arcobacter, and Sulfurospirillum and the generically misclassified B. ureolyticus form a family of gram-negative, nonsaccharolytic bacteria with microaerobic growth requirements and a low G+C content. This definition is in complete agreement with the original criteria used by Sebald and V&on (1963) to separate several Vibrio species from the genuine vibrios, and to include the former into the new genus Campylobacter. Members of the family Campylobacteraceae occur as commensals or parasites in humans and domestic animals (Campylobacter and Arcobacter) or as free-living environmental bacteria (Arcobacter and Sulfurospirillum). The type genus is the genus Campylobacter Sebald and Vkron 1963. Members of the family Campylobacteraceae have following general characteristics. Cells are curved, S-shaped, or spiral rods that are 0.2 to 0.8 p m wide and 0.5 to 5 p m long. They are gram negative and do not form spores. Cells in old cultures may form spherical or coccoid bodies. They are typically motile, with a characteristic corkscrew-like motion performed by means of a single polar unsheathed flagellum at one or both ends of the cell. Campylobacter and Arcobacter cells grow under microaerobic conditions and have a respiratory and chemoorganotrophic type of metabolism. Some grow under aerobic or anaerobic conditions. Energy is obtained from amino acids or tricarboxylic acid cycle intermediates, not carbohydrates. Carbohydrates are neither fermented nor oxidized. The optimum growth temperature is 30 to 37°C. Typical biochemical characteristics are reduction of fumarate to succinate; negative methyl red reaction and acetoin and indole production; and for most species, reduction of nitrate, absence of hippurate hydrolysis, and presence of oxidase activity.

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DEBRUYNE ET AL.

Menaquinones are the only respiratory quinones detected, with menaquinone-6 (three different structural types) and menaquinone-5 as major components. rRNA genes (16s and 23s) of some strains comprise internal transcribed spacers or intervening sequences. The presence of these internal transcribed spacers is strain specific, not species specific. The DNA base ratio ranges between 27 and 47 mol%. The Genus Campylobacter General characteristics

Campylobacter cells are mostly slender, spirally curved rods, 0.2 to 0.8 p m wide and 0.5 to 5 p m long. Cells of some species are predominantly curved or straight rods. Cells in old cultures may form coccoid bodies; these are considered degenerative forms rather than a dormant stage of the organism. Cells of most species are motile, with a characteristic corkscrew-like motion performed by means of a single polar unsheathed flagellum at one or both ends of the cell. Cells of some species are nonmotile (C. gracilis) or have multiple flagella (Campylobacter showae). In general, biochemical characteristics are as described for the family Campylobacteraceae. Several species grow in anaerobic conditions with fumarate or nitrate as electron acceptor; these species grow only in microaerobic conditions if hydrogen, formate, or succinate is supplemented as electron source. Gelatin, casein, starch, and tyrosine are not hydrolyzed. Oxidase activity is present in all species except C. gracilis. No lipase or lecithinase activity is present. Some species are pathogenic for humans and animals. This organism is found in the reproductive organs, intestinal tract, and oral cavity of humans and animals. The G + C of the DNA ranges from 29 to 47 mol%. At present, there are 17 validly named Campylobacter species, with Campylobacter fetus Sebald and VCron 1963 as the type species. There is no simple gold standard for the routine isolation of all Campylobacter species. Simultaneous application of a microaerobic atmosphere containing hydrogen with a filtration method and a selective base is methodologically the optimal solution. However, the predominant species in human infection can be readily grown in a microaerobic atmosphere on selective media without the necessity to use hydrogen. To evaluate the presence of less common species, appropriate culture conditions need to be applied. Menaquinone-6 (2-methyl-3-farnesyl-farnesyl1.4-naphthoquinone) and a methyl-substituted menaquinone-6 (2,[5 or 81-dimethyl-3-farnesyl-far-

nesyl-1.4-naphthoquinone)have been reported as major respiratory quinones in Campylobacter species. List of the species of the genus Campylobacter C . fetus. The first isolated Campylobacter was almost certainly C. fetus (McFadyean and Stockman, 1913). The species comprises two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis, both of which are considered primary pathogens. C. fetus subsp. fetus has been associated with abortion in sheep and sporadic abortion in cattle, and it has been isolated from a wide range of sources, including ungulates, fowl, reptiles, and humans. It is considered an uncommon human pathogen. It causes systemic infection in immunodeficient patients, or, rarely, neonatal sepsis and septic abortion. C. fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, an infectious venereal disease that can lead to infertility, abortion, and embryo death. It is mainly isolated from the bovine genital tract. Differentiation between the two subspecies is not straightforward. Biochemical differentiation is based on a single phenotypic test, i.e., tolerance to 1% glycine. It was suggested that, additionally, tolerance to 0.1% potassium permanganate, 0.005% basic fuchsin, and 64 mg literp1 cefoperazone may be useful for discrimination between the two subspecies (On and Harrington, 2001). Whole-cell protein electrophoresis, cellular fatty acid analysis, serotyping, and ribotyping do not separate the two taxa. Molecular methods used for discrimination between the two subspecies include numerical analysis of pulsedfield gel electrophoresis (PFGE) DNA profiles (On and Harrington, 2001), amplified fragment-length polymorphism (AFLP) (Duim et al., 2001), randomly amplified polymorphic DNA (RAPD) analysis (Tu et al., 2005), and several PCR tests (e.g., van Bergen et al., 2005b). C. fetus strains of reptile origin are divergent from C. fetus strains from mammalian origin, based on 16s rRNA, recA and sapD gene sequences, RAPD, PFGE, and DNA-DNA hybridization experiments. This suggests that they might represent a distinct taxon (Tu et al., 2005).

C. hyointestinalis. C. hyointestinalis was originally isolated from pigs with intestinal disorders and includes two subspecies: C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii. The former has been isolated from the intestines of pigs and hamsters, fecal samples from cattle, deer, and humans, and occasionally from the blood of patients with bacteremia. C. hyointestinalis subsp. hyointestinalis is associated with porcine proliferative

CHAPTER 1

enteritis and diarrhea in animals and humans, but its pathogenic role is unclear. C. hyointestinalis subsp. lawsonii has been isolated from the stomach of pigs. Its clinical significance is unknown. When grown on common blood agar bases, only some C. hyointestinalis strains grow microaerobically without hydrogen (all strains grow microaerobically in the presence of hydrogen). Both whole-cell protein and macrorestriction profiles, and AFLP analysis can be used for high-resolution identification and for determination of epidemiological relationships in this species (Duim et al., 2001; On and Harrington, 2000).

Campylobacter lanienae. During a routine screen of healthy abattoir workers, Campylobacterlike organisms were isolated from fecal samples of two individuals. Cells were slender, slightly curved rods, with single, bipolar unsheathed flagella (Logan et al., 2000). C. lanienae was also isolated from fecal samples from healthy pigs and has been detected by means of conventional and quantitative PCR in bovine fecal samples. C. sputorum. Bacteria currently classified in the species C. sputorum (On et al., 1998), were originally described as multiple Vibrio species. In 1914, Tunicliff observed a vibrio, later classified as Vibrio sputorum (PrCvot, 1940), in sputum of a patient with bronchitis. Similar bacteria isolated from the bovine vagina and semen were classified as Vibrio bubulus (Florent, 1953). These two species were thought to be related at the subspecies level on the basis of their extensive biochemical similarities and were renamed C. sputorum subsp. sputorum and C. sputorum subsp. bubulus (VCron and Chatelain, 1973). In addition, Firehammer (1965) described a fecal-type vibrio isolated from sheep that differed mainly from V. bubulus by its strong catalase activity, for which the name Vibrio fecalis was proposed. Subsequent DNA-DNA hybridization studies (Roop et al., 1985b) revealed a high level of DNA-DNA relatedness between C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and V. fecalis, and also indicated that C. sputorum subsp. mucosalis (see below) represented a distinct species. Limited biochemical variation between these taxa had been noted (catalase production, and growth on 3.5% NaCl and 1.0% ox-bile media), and therefore Roop et al. (1985b) proposed that these taxa be referred to as source-specific biovars of C. sputorum (C. sputorum bv. sputorum, C. sputorum bv. bubulus, and C. sputorum bv. fecalis). Subsequent studies by On et al. (1998) questioned the legitimacy of C. sputorurn bv. bubulus as a distinct taxon be-

TAXONOMY OF THE CAMPYLOBACTERACEAE

13

cause the tests used to distinguish this taxon from other biovars were found to be poorly reproducible. The identification of urease-positive variants of C. sputorum isolated from cattle feces (Atabay et al., 1997) resulted in the proposal of a new biovar structure, defined by reactions in two simple and reproducible tests: presence of catalase and urease activity. Strains of C. sputorum bv. sputorum produce neither catalase nor urease. They are found in the oral cavity, feces (normal and diarrheic), and abscesses and other skin lesions of humans; the genital tract of bulls; abortions in sheep; and the feces of sheep and pigs. Their pathogenicity is unknown. Strains of C. sputorum bv. faecalis produce catalase, but not urease. They have been isolated from the feces of sheep and cattle, and their pathogenicity is unknown. Finally, strains of C. sputorum bv. paraureolyticus produce urease, but not catalase. Strains are isolated from the feces of cattle and from human diarrhea; their pathogenicity is unknown.

C. mucosalis. The name C. sputorum subsp. mucosalis was originally proposed for a group of strains thought to be a causal agent of proliferative enteritis in pigs (Lawson et al., 1981). However, subsequent studies have identified Lawsonia intracellularis as the principal pathogen in this disease, and DNA-DNA hybridization demonstrated that this organism represented a distinct Campylobacter species (Roop et al., 1985a). C. mucosalis strains do not grow microaerobically on common agar bases in an atmosphere without hydrogen. Three distinct serovars, A, B, and C, have been described (Lawson et al., 1981), which were shown to have strikingly distinct whole-cell protein patterns (Costas et al., 1987; Vandamme et al., 1990). C. mucosalis strains have been isolated from the intestinal mucosa of pigs with porcine intestinal adenamatosis, necrotic enteritis, regional ileitis, and proliferative hemorrhagic enteropathy; and from the porcine oral cavity. Their clinical significance is unknown. Human infections supposedly caused by this organism were shown to be misidentified C. concisus infections (On, 1994). More recently, Tyrrell et al. (2003) reported the isolation of C. rnucosalis, identified by biochemical means, from the tongue dorsum of patients with oral malodor. C . concisus and C:curvus. Tanner et al. (1981, 1984) described two groups of strains isolated from gingival crevices of humans with gingivitis, periodontitis, and periodontosis, which were named C. concisus and C. curvus (the latter was originally described as w. cuwa; see above). Subsequent studies identified C. concisus in stool and blood samples of

14

DEBRUYNE ET AL.

children and adults with and without diarrhea, and more recently also in patients with Barrett’s esophagus, a complication of chronic gastroesophageal reflux disease. The high prevalence of C. concisus in clinical settings has been demonstrated when appropriate isolation conditions are used. Whole-cell protein electrophoresis and DNA-DNA hybridization experiments (Van Etterijck et al., 1996; Vandamme et al., 1989) revealed that C. concisus is a heterogeneous species, which has been confirmed by a range of molecular techniques, including RAPD (Engberg et al., 2005; Matsheka et al., 2006; Van Etterijck et al., 1996), ribotyping (Engberg et al., 2005), and PFGE (Matsheka et al., 2002). AFLP analysis revealed at least four distinct genomospecies within C. concisus (Aabenhus et al., 2005). At present, no clear differences have been observed between isolates from patients with diarrhea and isolates from healthy carriers; therefore, the role of C. concisus as a human enteric pathogen remains unclear. C. cuwus has been rarely isolated from human fecal samples, and it has been implicated in hepatic and bronchial abscesses. Strains of both species require hydrogen for growth in microaerobic conditions.

C. rectus, C . gracilis, and C. showae. C. rectus, C. gracilis, and C. showae are hydrogen-requiring campylobacters for which optimal growth is obtained in anaerobic conditions. Their cell morphology is most unusual. C. rectus (Tanner et al., 1981) is a rather plump, straight rod; its cellular surface is covered with a distinctive array of hexagonal, packed, macromolecular subunits. Strains have been isolated from gingival crevices of humans, in cases of appendicitis, in patients with Barrett’s esophagus, and from extraoral abscesses. It is considered a putative periodontal pathogen. It has been suggested that periodontal disease may contribute to preterm low birth weight, but because many confounding factors may influence preterm delivery, there is no conclusive evidence that periodontal pathogens like C. rectus are actually involved (Buduneli et al., 2005). C. gracilis cells are straight and nonmotile rods (Tanner et al., 1981). C. gracilis is the only oxidasenegative Campylobacter species, although the pattern of cytochromes found in C. gracilis resembled that reported for other campylobacters. Strains have been isolated from gingival crevices; from visceral, head, and neck infections; in soft tissue abscesses; in pneumonia and empyema; and in an ischial wound in humans. The association of C. gracilis with serious deep tissue infection, coupled with a high frequency of antibiotic resistance, suggests that it is an underesti-

mated pathogen, even though conclusive evidence for pathogenicity is lacking. C. showae cells are straight rods and have polar bundles of two to five unsheathed flagella (Etoh et al., 1993). Strains have been isolated from human dental plaque, from infected root canals, and from periodontitis lesions. Pathogenicity is unknown.

Campylobacter hominis. C. hominis was initially described as “Candidatus Campylobacter hominis,” an uncultivated species found in the gastrointestinal tract of healthy human individuals (Lawson et al., 1998). After modifications of the isolation methodology, the organism was subsequently isolated by anaerobic culture. Similar to C. gracilis, C. hominis cells are nonmotile straight rods without flagella (Lawson et al., 2001). C. horninis has been detected by speciesspecific PCR in both healthy and diarrheic human fecal samples. C. jejuni and C. coli. In 1927, Smith and Orcutt described a group of Vibrio-like bacteria from the feces of cattle with diarrhea. Jones and coworkers (193 1) showed a causal relationship between these microaerophilic vibrios and bovine dysentery and subsequently named the organism Vibrio jejuni. Similar organisms were detected in blood cultures of humans with gastroenteritis and aborted sheep fetuses. In 1944, Doyle isolated yet another vibrio from feces of pigs with diarrhea and classified it as Vibrio coli (Doyle, 1948). At present, C. jejuni and C. coli are by far the most important human enteropathogens among the campylobacters. C. jejuni infection is also a known antecedent of Guillain-BarrC syndrome. Within C. jejuni, two subspecies are recognized: C. jejuni subsp. jejuni and C. jejuni subsp. doylei. Strains of the latter species differ biochemically from the former by the absence of nitrate reduction, cephalothin susceptibility, and often catalase activity (a weak reaction may also be observed). Many common molecular-based methods are not suitable for subspeciation, but recently a multiplex PCR based on the nap locus was proposed for C. jejuni subspecies differentiation (Miller et al., 2007). The pathogenic role of C. jejuni subsp. doylei is unknown. Although it is isolated infrequently, it is obtained primarily from human clinical samples, and it is often associated with bacteremia, notably in infants. Biochemically, C. jejuni subsp. jejuni and C. coli differ only in their ability to hydrolyze hippurate, for which C. coli is negative. However, hippuratenegative C. jejuni subsp. jejuni strains are well recognized. Because of the limitations of biochemical differentiation, a wide range of alternative, mainly

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genotypic methods has been developed for differentiation between C. coli and C. jejuni subsp. jejuni (e.g., Jensen et al., 2005; Mandrel1 et al., 2005; Nayak et al., 2005; Sails et al., 2001). The complexity of this taxonomic area has been illustrated by data showing that strains first described as a distinct species, c. hyoilei, are in fact c. coli (Vandamme et al., 1997), despite a higher 16s rRNA sequence similarity to C. jejuni (Alderton et al., 1995). A multilocus sequence typing study of C. coli demonstrated that C. hyoilei strains belonged to the sequence type 1017 complex, which comprised the majority of the C. coli sequence types and included strains from Australia, Belgium, Canada, Denmark, Japan, and the United States and which were isolated from humans and a range of animals (Miller et al., 2005).

C. lari. C. luri was originally referred to as the group of nalidixic acid-resistant thermophilic campylobacters (the NARTC group). These strains were primarily differentiated from C. jejuni and C. coli by their resistance to nalidixic acid, their anaerobic growth in the presence of trimethylamine N-oxide hydrochloride, and later also by the absence of indoxy1 acetate hydrolysis. However, nalidixic acidsusceptible strains (NASC strains), urease-producing strains (UPTC strains), and urease-producing nalidixic acid-susceptible strains (Endtz et al., 1997) were also identified as C. luri variants by onedimensional whole-cell protein electrophoresis and semiquantitative DNA-DNA hybridization (Vandamme et al., 1991b). Endtz et al. (1997) reported a striking heterogeneity among and within the different groups of C. luri variants, which was confirmed by Duim and coworkers (2004), who clearly distinguished four subgroups by means of numerical analysis of AFLP profiles and of partial protein profiles. Preliminary DNA-DNA hybridization experiments revealed that some of these groups may represent distinct species, but at present, the taxonomic status of these groups remains unclear. C. luri strains have been isolated from intestinal contents of seagulls and other animals, river water, and shellfish. In humans, C. luri has been occasionally isolated from diarrheic feces and from cases of bacteremia and other extraintestinal infections in both immunocompetent and immunodeficient patients. Its pathogenicity is unknown. Campylobacter insulaenigae. Four Cumpylobacter-like strains were isolated from rectal swabs taken from common seals and the intestinal contents of a porpoise in northern Scotland and represented a distinct Campylobacter species, named C. insulaenigrae (Foster et al., 2004). C. insuluenigrue

TAXONOMY OF THE CAMPYLOBACTERACEAE

15

has also been isolated from free-living and stranded northern elephant seals in California. Although initially reported as nonthermotolerant, several of the Californian C. insuluenigrue strains were able to grow at 42"C, leading to an initial misidentification of some of these strains as C. lari (Stoddard et al., 2007). The pathogenic potential of these bacteria in marine mammals is unknown.

Campylobacter canadensis. During a study of the bacterial flora of the cloacae of whooping cranes (Grus umericuna), 10 atypical Campylobacter isolates were recovered on two separate occasions and were classified as C. canadensis. The cells were polymorphic in shape (sigmoid to coccoid) and possessed a single polar flagellum. The prevalence of C. cunudensis and its role are at present unknown (Inglis et al., 2007). C. upsaliensis and Campylobacter helveticus. Both C. upsaliensis and C. helveticus are catalasenegative or weakly positive thermotolerant campylobacters. C. upsuliensis strains have been isolated from feces of enteric and asymptomatic humans, dogs, cats, and meercats as well as from human blood samples. Strains associated with a human abortion, a breast abscess, hemolytic-uremic syndrome, and a prosthetic knee infection have been described. C. helveticus has been isolated from feline and canine fecal samples, but at present, no strains have been isolated from human sources. Reports have emerged worldwide implicating C. upsuliensis as a human enteric pathogen, and in some regions, it is preceded only by C. jejuni in being the most frequently isolated Cumpylobucter species in human stool samples. Accumulated clinical and epidemiological data support the importance of C. upsuliensis as a primary human pathogen, even though direct evidence is at present still lacking. Differentiation of Campylobacter species Apart from the recent single-locus, multilocus, or whole-genome-based analyses discussed above, a variety of approaches have been used to identify campylobacters to the species level. Some of these are discussed below. Classical phenotypic characteristics. Most routine laboratories use basic biochemical tests to identify campylobacters. The lack of application of highly standardized procedures and the well-known biochemical inertness of campylobacters often make biochemical identification of these bacteria difficult be-

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cause the discrimination among species or subspecies may rely on one differential character, such as presence of hippuricase or urease activity (On, 1996). A computerized scheme of nearly 70 biochemical tests readily differentiated virtually all Campylobacter species (On et al., 1996; On and Holmes, 1995). Traditional taxonomic analyses. In taxonomic studies, the reference method for the delineation of bacterial species is determination of the level of DNA-DNA hybridization (Vandamme et al., 1996). A variety of DNA-DNA hybridization studies have been performed before the phylogenetic relationships of campylobacters were established by means of rRNA-directed studies and focused on the species known since the early 1980s (detailed overviews are given by Vandamme and Goossens [1992] and On [1996]). Chemotaxonomic techniques such as wholecell protein and fatty acid analysis have been used in a range of taxonomic studies for the identification of Campylobacter species. In particular, electrophoresis of whole-cell proteins by highly standardized sodium dodecyl sulfate-polyacrylamide gel electrophoresis has proven to be extremely reliable to identify Campylobacter and other strains at the species level because numerous studies have revealed a correlation between high similarity in whole-cell protein content and level of DNA-DNA hybridization (On, 1996; Vandamme et al., 1996). rRNA-based approaches. Sequence analysis of the rRNA genes became the ultimate tool to study bacterial phylogeny during the 1980s, and international databases have been constructed to collect sequences and to make them publicly available. Nowadays, it is common to identify unknown organisms that are of particular interest by means of 16s rRNA sequence analysis and comparison with rRNA sequences present in international databases (Gorkiewicz et al., 2003). Several important points should be addressed here. Although unsurpassed in its capacity to reveal the phylogenetic neighborhood of an unknown bacterium, comparison of entire 16s rRNA sequences is generally not adequate for the identification of strains to the species level. It has been reported that strains belonging to different species may have identical 16s rRNA sequences and that strains of one species may have 16s rRNA genes that differ up to 3% (Stackebrandt and Goebel, 1994). Harrington and On (1999) demonstrated differences of up to 4.3% of the total 16s rRNA sequence between C. hyointestinalis strains. There is definitely a lack of knowledge, not only of the strain-to-strain variation within a species, but also of the interoperon variation within a single

strain as reported by Clayton et al. (1995). Therefore, concluding that an unknown organism belongs to a particular species because it shares a high percentage of its 16s rRNA gene sequence with another strain of that species, or concluding that an unknown represents a novel species because it shares only 97% of its 16s rRNA gene sequence with its closest neighbor is premature in the absence of other data. Alternatively, sequence information derived from rRNA cistrons has been used successfully to design and validate many species-, group-, or genusspecific primers and probes (Liibeck et al., 2003; On, 1996). Applied in a PCR or a hybridization assay, these primers and probes offer valuable alternatives for identification of Campylobacter taxa. Finally, broad-spectrum molecular identification schemata that are based on restriction fragment analysis of PCR amplicons derived from 16s (Cardarelli-Leite et al., 1996; Marshall et al., 1999) and 23s (Gonzalez et al., 2006; Hurtado and Owen, 1997) rRNA genes have also been described. AFLP analysis. AFLP analysis is based on selective amplification of restriction fragments of chromosomal DNA and has been widely used in a variety of bacterial genera, including Campylobacter, both for identification and typing purposes. It is a reproducible and discriminatory technique, particularly well suited for computer-assisted data analysis. In Campylobacter, AFLP schemes were initially developed for typing purposes, incorporating only C. jejuni subsp. jejuni and C. coli, and two studies revealed clear differences in AFLP profiles between these two species (Duim et al., 1999; Kokotovic and On, 1999). These observations were extended to other Campylobacter species (Duim et al., 2001; On and Harrington, 2000) and demonstrated that numerical analysis of AFLP profiles is well suited for identification at the species, subspecies, and strain levels. Numerous subsequent studies have applied AFLP to explore intraspecies diversity (e.g., Aabenhus et al., 2005; Duim et al., 2004) and epidemiological relationships (e.g., Keller et al., 2007; Siemer et al., 2005) of Campylobacter strains. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In

MALDI-TOF MS, biological samples are mixed with a matrix that is chosen such that it specifically absorbs the energy of a laser beam. The high-energy impact is followed by the formation of ions that are extracted through an electric field; these are subsequently focused and detected as a m / z (mass/charge) spectrum. In a preliminary study, Winkler et al. (1999) analyzed a set of C. jejuni, C. coli, C. fetus,

CHAPTER 1

H. pylori, and H. mustelae strains by means of both whole cells and partially purified extracts from lysed cells, and they reported the detection of discriminatory biomarker ions in the 10- to 20-kDa range. Further research into the potential of MALDI-TOF MS for identification purposes was performed by Mandrell and coworkers (2005). They analyzed whole bacterial cells from a large collection of well-characterized reference strains from C. coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis, and they observed species-identifying biomarker ions in the 9- to 14-kDa range for each of the six species. Most difficulty was observed for differentiating the closely related C. upsaliensis and C. helveticus. The Genus Arcobacter General characteristics

Arcobacter cells are slender, spirally curved rods 0.2 to 0.9 pm wide and 0.5 to 3 p m long; S-shaped or helical cells are often present. Cells in old cultures may form spherical or coccoid bodies and loose spiral filaments up to 20 p m long. Arcobacter is motile, with a characteristic corkscrew-like motion performed by means of a single polar unsheathed flagellum at one or both ends of the cell. In general, biochemical characteristics are as described for the family Campylobacteraceae. In addition, arcobacters are able to grow in aerobic and anaerobic conditions and at 15°C. Hydrogen is not required. Indoxyl acetate is hydrolyzed. No hydrolysis of gelatin, casein, starch, hippurate, and tyrosine is present. Habitat is extremely diverse. Arcobacter is unique among the Epsilonproteobacteria in that it combines plant and animal associated species and free-living environmental species within one genus. Some species are associated with humans and animals and are found in the reproductive organs and abortions of various animals and in the intestinal tract of humans and animals. Other sources include water reservoirs, river and surface water, sewage, oil field communities, marine and hypersaline environments, and deep-sea hydrothermal vents; one species is a plant-associated nitrogen fixer. The G+C of the DNA ranges from 27 to 35 mol%. Menaquinone-6 (2 -methyl-3 -farnesyl-farnesyl- lY4-naphthoquinone) and a second menaquinone-6, the detailed structure of which has not been determined, were reported as major respiratory quinones. The genus Arcobacter presently comprises six validly named species, with Arcobacter nitrofigilis Vandamme, Falsen, Rossau, Hoste, Segers, Tytgat, and De Ley 1991 as the type species.

TAXONOMY OF THE CAMPYLOBACTERACEAE

17

Isolation procedures for Arcobacter spp. are often based on filtration methods or selective media developed for Campylobacter, Yersinia, or even Leptospira species (Vandamme, 2000). In addition, several isolation methods were developed specifically for Arcobacter spp. (deBoer et al., 1996; Houf et al., 2001a; Johnson and Murano, 1999a, 1999b) by using a variety of selective media, in some cases preceded by an enrichment step. However, several of these methods are suboptimal because arcobacters may be overgrown by campylobacters present in the same specimens, and they may be susceptible to some of the antimicrobial agents used in the selective media (Houf et al., 2001b). In 2001, an Arcobacter enrichment broth and Arcobacter selective agar were developed for the isolation of arcobacters from poultry products, which allowed the recovery of A. butzleri, Arcobacter cryaerophilus, and A. skirrowii and successfully inhibited the contaminating flora (Houf et al., 2001a). In recent years, this procedure was validated for several other matrices, including animal feces (Van Driessche et al., 2003), human feces (Houf and Stephan, 2007), and poultry intestinal content (Van Driessche and Houf, 2007). Species of the genus Arcobacter A. nitrofigilis. A. nitrofigilis strains occur in the roots and rhizosphere of the salt marsh plant Spartina alternifiora, where they fix nitrogen (McClung et al., 1983). They differ from most other members of the family Carnpylobacteraceae by their preference for a high salt concentration (optimum growth is in the presence of 10 to 40 g/liter NaCl), and by the presence of nitrogenase and urease activity. A. cryaerophilus. In 1985, Neil1 and coworkers performed an extensive phenotypic characterization of aerotolerant Campylobacter strains isolated from various animal sources. They concluded that the aerotolerant strains were only distantly related to strains of the other Campylobacter species examined and emphasized that they formed a heterogeneous group. Their findings were confirmed by integrated phenotypic, genotypic, and chemotaxonomic studies of, in part, the same isolates (Kiehlbauch et al., 1991; Vandamme et al., 1991a, 1992), which entailed in the description of a novel genus Arcobacter, with three separate animal-associated species: A. cryaerophilus, A. butzleri, and A. skirrowii. Within A. cryaerophilus, two subgroups, referred to as subgroup 1 or group 1A and subgroup 2 or group lB, have been described (Kiehlbauch et al., 1991; Vandamme et al., 1992). Strains of these subgroups differ in their whole-cell protein and fatty acid

18

DEBRUYNE ET AL.

patterns, and in the restriction fragment length polymorphisms of the rRNA genes. Moderate levels of DNA-DNA hybridization were detected between strains of the different subgroups, suggesting that they could be given a distinct taxonomic rank. However, at present, no formal nomenclatural modifications have been proposed. A. cryaerophilus strains have been isolated from cases of human bacteremia and diarrhea; from chicken, pork, and duck carcasses; from bovine, ovine, and porcine abortions; from fecal samples from cattle, pigs, and horses; and from mastitis in cattle. One study reported its isolation from a naturally infected rainbow trout. Their pathogenicity is unknown. A. butzleri. A. butzleri strains have been isolated from human blood and diarrheic feces; from feces of various animals with diarrhea including nonhuman primates, pigs, horses, cattle, an ostrich and a tortoise; from bovine and porcine abortions; from various food products including ground pork, chicken, and turkey; from surface and drinking water reservoirs and canal waters; from seawater; and from plankton. This species is associated with enteritis, abdominal cramps, bacteremia, and appendicitis in humans and with enteritis and abortion in animals. It was identified as the fourth most isolated Campylobacter-like organism from human diarrheal stool in Belgium and France (Prouzet-Mauleon et al., 2006; Vandenberg et al., 2004). Screening for arcobacters in asymptomatic humans yielded no positive results for A. butzleri (Houf and Stephan, 2007).

A. skirrowii. A. skirrowii strains have been isolated from preputial fluids of bulls; from bovine, ovine, and porcine abortions; from diarrheic feces of various animals including sheep and cattle; and from chicken carcasses. At present, there is one report on the isolation of A. skirrowii from a patient with chronic diarrhea, but it was unclear whether the strain was the etiological agent (Wybo et al., 2004). Their pathogenicity remains unknown. Arcobacter cibarius. A. cibarius strains were first isolated during a long-term study of Arcobacter contamination on poultry carcasses. Twenty isolates, recovered from 13 unrelated broiler carcasses, yielded no Arcobacter species-specific PCR amplicon, but they did yield a genus-specific PCR amplicon (Houf et al., 2005). Additional data demonstrated that the 20 isolates represented a novel Arcobacter species. The organism has also been isolated from porcine and ovine feces (K. Houf, unpublished data) and pig-

gery effluent (Chinivasagam et al., 2007). At present, their pathogenic potential is unknown.

Arcobacter halophilus. A. halophilus is an obligately halophilic Arcobacter species isolated from a hypersaline lagoon on Laysan Atoll in the northwestern Hawaiian Islands, and currently comprises a single strain. Phenotypic and genotypic data placed the type strain LA3 lBT within the genus Arcobacter, but it can be distinguished from all recognized arcobacters by its obligate halophily and fatty acid composition (Donachie et al., 2005). “Candidatus Arcobacter sulfidicus.” “Candidatus A. sulfidicus” is a highly motile sulfide-oxidizing bacterium, unique among prokaryotes in that it excretes elemental sulfur in the form of hydrophilic rigid filaments, as opposed to globular or amorphous sulfur found with other sulfur oxidizers (Wirsen et al., 2002). The organism currently remains a candidate species that is difficult to cultivate, not because of the nutritional aspects of cultivation, but because of the required growth apparatus and conditions (Wirsen et al., 2002). The formation of filamentous sulfur appears to be a specific adaptation permitting these organisms to colonize the specialized niche, characterized by dynamic fluid movement and high sulfide and low oxygen concentrations, required for their survival. The worldwide occurrence of microbial filamentous sulfur formation in marine ecosystems indicates that it is likely to play an important yet undefined role in global sulfur and carbon cycles (Sievert et al., 2007). As opposed to the chemoorganotrophic growth found in other Arcobacter spp., this organism has a chemoautotrophic mode of nutrition. Experiments demonstrated that it has a microaerobic growth preference and that it aggregates within the oxic-anoxic interface by exhibiting a chemotactic response (Sievert et al., 2007). Other Organisms On and coworkers (2002, 2003) reported the isolation of five strains associated with pig abortions and one from duck feces, which were believed to represent a novel Arcobacter species, as assessed by AFLP profiling, phenotype, and 16s rRNA gene sequence analysis. However, formal description was deferred until the identification of at least 10 isolates, in accordance with recommendations for minimal taxonomic standards for this group of bacteria (Ursing et al., 1994). Also, a number of culture-independent studies have documented the existence of additional

CHAPTER 1

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TAXONOMY OF THE CAMPYLOBACTERACEAE

19

Arcobacter taxa, as assessed by 16s rRNA gene phylogeny, in environmental samples including oil field communities (Gevertz et al., 2000; Sette et al., 2007; Voordouw et al., 1996), cyanobacterial mats (Teske et al., 1996), activated sludge of a large municipal wastewater treatment plant (Snaidr et al., 1997), and deep-sea hydrothermal vents (Moussard et al., 2006). Other potentially novel species have been detected in the tunic matrix of the colonial ascidian Diplosoma migrans (Schuett et al., 2005) and as part of the Chilean oyster (Tiostrea chilensis) microbiota (Romero et al., 2002). Because none of these novel taxa was cultivated, they were not formally named.

PCR for identification of A. butzleri and A. cryaerophilus (Brightwell et al., 2007). As for the aforementioned PCR assays, A. cibarius was not included in the evaluation of these DNA-based identification methods. The nucleotide sequences of the gyrA gene for A. butzleri, A. cryaerophilus, A. cibarius, and A. skirrowii have been determined. Phylogenetic analysis of the generated gyrA sequences provided a result similar to 16s rRNA gene phylogeny. Phylogenetic analysis allowed a good separation of these species from each other and from related Campylobacter and Helicobacter species (Abdelbaqi et al., 2007).

Differentiation of Arcobacter Species

The Genus Sulfurospirillum

Basic biochemical tests are still routinely used to identify arcobacters to the species level, and, like for campylobacters, this is hampered by their biochemical inertness and intraspecies variability. In particular, the separation of A. cryaerophilus subgroup 2 and A. butzleri is tedious (Vandamme et al., 1992). As mentioned for Campylobacter, numerical analysis of a large number of phenotypic characteristics (On et al., 1996; On and Holmes, 1995) and whole-cell protein electrophoresis can also be used for Arcobacter identification. Whole-cell fatty acid analysis did not allow discrimination between A. butzleri from A. cryaerophilus subgroup 2, but this technique did permit differentiation of all other Arcobacter taxa (Vandamme and Goossens, 1992). Several PCR assays for the discrimination of animal-associated Arcobacter species have been developed in the last decade, often in the form of a multiplex reaction (Brightwell et al., 2007; Houf et al., 2000; Kabeya et al., 2003). At present, no PCR assay has been developed that included A. cibarius. Restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16s rRNA (Cardarelli-Leite et al., 1996) or 23s rRNA (Hurtado and Owen, 1997) differentiated A. butzleri from other arcobacters but did not differentiate A. cryaerophilus from A. skirrowii. A PCR-restriction fragment length polymorphism analysis that used either a 16s or 23s rRNA amplicon in combination with only one restriction enzyme permitted genuslevel discrimination between Arcobacter, Campylobacter, and Helicobacter (Gonzalez et al., 2006). Numerical analysis of AFLP profiles allowed discrimination of four Arcobacter species (i.e., A. butzleri, A. cryaerophilus, A. skirrowii and A. nitrofigilis) but did not include A. cibarius or A. halophilus (On et al., 2003). Other DNA-based methods include 16s rRNA-based DNA probes (Wesley et al., 1995), ribotyping (Kiehlbauch et al., 1994), and real-time

The genus Sulfurospirillum was established by Schumacher et al. (1992) to accommodate two freeliving Campylobacter-like strains (Spirillum 5 17.ST and Campylobacter sp. “Veldkamp”), which formed a distinct clade within the rRNA superfamily VI (Vandamme et al., 1991a). At present, there are seven named Sulfurospirillum species, with Sulfurospirillum deleyianum (Wolfe and Pfennig 1977) Schumacher, Kroneck, and Pfennig 1993 as the type species. Strain Spirillum 5 175T, isolated from anoxic mud from a forest pond near Heiningen, Germany, was assigned as the type strain of the species Sulfurospirillum deleyianum. Sulfurospirillum arcachonense was isolated from oxidized surface sediment in an intertidal mud flat near Arcachon, France (Finster et al., 1997). Stolz et al. (1999) described Sulfurospirillum barnesii, formerly referred to as “Geospirillum barnesii,” and Sulfurospirillum arsenophilum. The type strain of S. barnesii, SES3T, was isolated from a selenate-contaminated freshwater lake in western Nevada, USA (Oremland et al., 1994), and is able to use selenate and arsenate as terminal electron acceptors. The type strain for S. arsenophilum, MIT-13T, was isolated from arsenic-contaminated watershed sediments in eastern Massachusetts, USA (Ahmann et al., 1994), and is able to reduce arsenate. Sulfurospirillum halorespirans PCE-M2T, isolated from a polluted anaerobic soil, and Sulfurospirillum multivorans KT (formerly Dehalospirillum multivorans), isolated from activated sludge, both have the ability to reduce the pollutant tetrachloroethylene (Luijten et al., 2004; Scholzmuramatsu et al., 1995). Sulfurospirillum cavolei Phe9 lTwas isolated from petroleum-contaminated groundwater in an underground crude oil storage cavity in Japan (Kodama et al., 2007). Sulfurospirilla are extremely fastidious, and very few strains have been isolated and studied. Speciation is primarily based on differences in 16s rRNA gene

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DEBRUYNE ET AL.

sequences, although there are obvious differences in the metabolism of these organisms too. Cells of all of these species are slender, spirally curved rods 0.1 to 0.5 p m wide and 1.0 to 3 p m in length. They may form helical chains of two or more cells. As far as the data available indicate, their general biochemical profiles conform to that of the family Campylobacteraceae. All species show oxidase activity. Growth occurs between 8 and 36°C. The G+C of the DNA ranges from 32 to 42 mol%. Menaquinone-6, a methyl-substituted menaquinone-6 (referred to as thermoplasmaquinon-6), and menaquinone-5 have been reported as respiratory quinones in S. deleyianum; menaquinone-6 and thermoplasmaquinon-6 were found in S. arcachonense. Members of this genus are all sulfur reducers but exhibit metabolic versatility. Different isolation procedure have been described, all of which involve enrichment procedures (Finster et al., 1997; Oremland et al., 1994; Wolfe and Pfennig, 1977). Several other presently unnamed organisms also belong to the genus Sulfurospirillum. Coleman et al. (1993) isolated a marine sulfur-reducing bacterium, designated strain SM-5, implicated in the reduction of Fe(II1). A nitrate-reducing Sulfurospirillum sp., strain KW, was isolated from an oil field (Hubert and Voordouw, 2007). Strain “Veldkamp” (DSM 806), isolated from activated sludge (Laanbroek et al., 1977), was originally classified as a Sulfurospirillum sp. (Schumacher et al., 1992) and was later reclassified as S. deleyianum, but phylogenetic analysis of the 16s rRNA gene sequence of this strain indicates that it may represent a separate species (Fig. 1). Strain NP4, isolated from groundwater with high arsenic concentrations, is classified as a Sulfurospirillum species on the basis of 16s rRNA gene sequence analysis, and it differs from the other Sulfurospirillurn isolates in its carbon source and electron acceptor usage profiles (MacRae et al., 2007). This organism may adversely affect water quality by reducing arsenate to the more toxic arsenite.

Bacteroides ureolyticus B. ureolyticus (Jackson and Goodman, 1978) cells are nonmotile rods that grow microaerobically when hydrogen is provided. Representative strains of this species were included in a polyphasic taxonomic study to elucidate their taxonomic status (Vandamme et al., 1995). B. ureolyticus resembles campylobacters in respiratory quinone content, DNA base ratio, and most of its phenotypic characteristics, but differs from campylobacters in fatty acid composition and proteolytic metabolism. This organism was, however, not formally reclassified pending a thorough taxo-

nomic characterization of additional B. ureolyticuslike bacteria (Vandamme et al., 1995). Strains have been isolated from superficial ulcers and soft tissue infections, urethritis, vaginosis, and periodontal disease. A pathogenic role is suggested by its predominance in mixed infections and its strong proteolytic activity, which may enable tissue destruction. Acknowledgments. We are indebted to the Fund for Scientific Research-Flanders (Belgium) for research grants (P.V.) and for a position as a postdoctoral fellow (D.G.).

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oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26:1628-1635. Sails, A. D., A. J. Fox, F. J. Bolton, D. R. Wareing, D. L. Greenway, and R. Borrow. 2001. Development of a PCR ELISA assay for the identification of Campylobacter jejuni and Campylobacter coli. Mol. Cell. Probes 15:291-300. Sandstedt, K., and J. Ursing. 1991. Description of Campylobacter upsaliensis sp. nov. previously known as the CNW group. Syst. Appl. Microbiol. 14:39-45. Scholzmuramatsu, H., A. Neumann, M. Messmer, E. Moore, and G. Diekert. 1995. Isolation and characterization of Dehalospirillum multivorans gen. nov., sp. nov., a tetrachloroetheneutilizing, strictly anaerobic bacterium. Arch. Microbiol. 163:4856. Schouls, L. M., S. Reulen, B. Duim, J. A. Wagenaar, R. J. Willems, K. E. Dingle, F. M. Colles, and J. D. Van Embden. 2003. Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J. Clin. Microbiol. 41:15-26. Schuett, C., H. Doepke, W. Groepler, and A. Wichels. 2005. Diversity of intratunical bacteria in the tunic matrix of the colonial ascidian Diplosoma migrans. Helgoland Mar. Res. 59:136-140. Schumacher, W., P. M. H. Kroneck, and N. Pfennig. 1992. Comparative systematic study on Spirillum 5 175, Campylobacter and Wolinella species: description of Spirillum 5 175 as Sulfurospirillum deleyianum gen. nov., spec. nov. Arch. Microbiol. 158:287293. Sebald, M., and M. V6ron. 1963. Teneur en bases de I’ADN et classification des vibrions. Ann. lnst. Pasteur 105:897-910. Sette, L. D., K. C. Simioni, S. P. Vasconcellos, L. J. Dussan, E. V. Neto, and V. M. Oliveira. 2007. Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin. Antonie Leeuwenhoek 91:253-266. Siemer, B. L., E. M. Nielsen, and S. L. On. 2005. Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping. Appl. Environ. Microbiol. 71:1953-1958. Sievert, S. M., E. B. Wieringa, C. 0. Wirsen, and C. D. Taylor. 2007. Growth and mechanism of filamentous-sulfur formation by “Candidatus Arcobacter sulfidicus” in opposing oxygensulfide gradients. Environ. Microbiol. 9:271-276. Skirrow, M. B. 1977. Campylobacter enteritis: a “new” disease. Br. Med. J. 2:9-11. Skirrow, M. B. 2006. John McFadyean and the centenary of the first isolation of Campylobacter species. Clin. Infect. Dis. 43: 1213-1217. Smith, T., and M. Orcutt. 1927. Vibrios from calves and their serological relation to Vibrio fetus. J. Exp. Med. 45:391-397. Smith, T., and M. S. Taylor. 1919. Some morphological and biochemical characters of the spirilla (Vibrio fetus n. sp.) associated with disease of the fetal membranes in cattle. J. Exp. Med. 310: 299-312. Snaidr, J., R. Amann, I. Huber, W. Ludwig, and K. H. Schleifer. 1997. Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl. Environ. Microbiol. 63:2884-2896. Stackebrandt, E., and B. M. Goebel. 1994. A place for DNA-DNA reassociation and 16s ribosomal RNA sequence analysis in the present species definition in bacteriology. lnt. J. Syst. Bacteriol. 44~846-849. Stoddard, R. A., W. G. Miller, J. E. Foley, J. Lawrence, F. M. Gulland, P. A. Conrad, and B. A. Byrne. 2007. Campylobacter insulaenigrae isolated from Northern Elephant Seals (Mirounga angustirostris) in California. Appl. Environ. Microbiol. 73:17291735.

Stolz, J. F., D. J. Ellis, J. S. Blum, D. Ahmann, D. R. Lovley, and R. S. Oremland. 1999. Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the epsilon Proteobacteria. lnt. J. Syst. Bacteriol. 49:1177-1180. Stolz, J. F., R. S. Oremland, B. J. Paster, F. E. Dewhirst, and P. Vandamme. 2005. Genus 111. Sulfurospirillum Schumacher, Kroneck, and Pfennig 1993, 188VP (Effective publication: Schumacher, Kroneck, and Pfennig 1992, 291), emend. Fienster, Liesack and Tindall 1997d, 1216, p. 1165-1168. In D. J. Brenner, N. R. Krieg, J. T. Staley, and G. M. Garrity (ed.), BergeySManual of Systematic Bacteriology, vol. 2. Springer-Verlag, New York. Suerbaum, S., M. Lohrengel, A. Sonnevend, F. Ruberg, and M. Kist. 2001. Allelic diversity and recombination in Campylobacter jejuni. J. Bacteriol. 183:2553-2559. Tanner, A. C. R., S. Badger, C. H. Lai, M. A. Listgarten, R. A. Visconti, and S. S. Socransky. 1981. Wolinella gen. nov., Wolinella succinogenes (Vibriosuccinogenes Wolin et. al.) comb. nov., and description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from humans with periodontal disease. lnt. J. Syst. Bacteriol. 31:432-445. Tanner, A. C. R., M. A. Listgarten, and J. L. Ebersole. 1984. Wolinella curva sp. nov.: “Vibriosuccinogenes” of human origin. Int. J. Syst. Bacteriol. 34:275-282. Tatusov, R. L., E. V. Koonin, and D. J. Lipman. 1997. A genomic perspective on protein families. Science 278:63 1-637. Teske, A., P. Sigalevich, Y. Cohen, and G. Muyzer. 1996. Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16s ribosomal DNA fragments as a tool for isolation in pure cultures. Appl. Environ. Microbiol. 62:4210-4215. Totten, P. A., C. L. Fennell, F. C. Tenover, J. M. Wezenberg, P. L. Perine, W. E. Stamm, and K. K. Holmes. 1985. Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men. J. Infect. Dis. 151:131-139. Tu, Z. C., W. Eisner, B. N. Kreiswirth, and M. J. Blaser. 2005. Genetic divergence of Campylobacter fetus strains of mammal and reptile origins. J. Clin. Microbiol. 43:3334-3340. Tunicliff, R. 1914. An anaerobic vibrio isolated from a case of acute bronchitis. J. Infect. Dis. 15:350-351. Tyrrell, K. L., D. M. Citron, Y. A. Warren, S. Nachnani, and E. J. Goldstein. 2003. Anaerobic bacteria cultured from the tongue dorsum of subjects with oral malodor. Anaerobe 9:243-246. Ursing, J. B., H. Lior, and R. J. Owen. 1994. Proposal of minimal standards for describing new species of the family Campylobacteraceae. Int. J. Syst. Bacteriol. 445342445. van Bergen, M. A., K. E. Dingle, M. C. Maiden, D. G. Newell, L. van der Graaf-Van Bloois, J. P. van Putten, and J. A. Wagenaar. 2005a. Clonal nature of Campylobacter fetus as defined by multilocus sequence typing. J. Clin. Microbiol. 435888-5898. van Bergen, M. A., G. Simons, L. van der Graaf-van Bloois, J. P. van Putten, J. Rombout, I. Wesley, and J. A. Wagenaar. 2005b. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. J. Med. Microbiol. 54: 1217-1224. Vandamme, P. 2000. Taxonomy of the family Campylobacteraceae, p. 3-26. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Vandamme, P., M. I. Daneshvar, F. E. Dewhirst, B. J. Paster, K. Kersters, H. Goossens, and C. W. Moss. 1995. Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov. lnt. J. Syst. Bacteriol. 45:145-152.

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Vandamme, P., and J. De Ley. 1991. Proposal for a new family, Campylobacteraceae.Int. J. Syst. Bacteriol. 41:45 1-455. Vandamme, P., E. Falsen, B. Pot, B. Hoste, K. Kersters, and J. De Ley. 1989. Identification of EF group 22 campylobacters from gastroenteritis cases as Campylobacter concisus. J. Clin. Microbiol. 27: 1775-1781. Vandamme, P., E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley. 1991a. Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int. J , Syst. Bacteriol. 41~88-103. Vandamme, P., and H. Goossens. 1992. Taxonomy of Campylobacter, Arcobacter, and Helicobacter: a review. Zentralbl. Bakteriol. 276:447-472. Vandamme, P., B. Pot, E. Falsen, K. Kersters, and J. De Ley. 1990. Intra- and interspecific relationships of veterinary campylobacters revealed by numerical analysis of electrophoretic protein profiles and DNA:DNA hybridizations. Syst. Appl. Microbiol. 13~295-303. Vandamme, P., B. Pot, M. Gillis, P. de Vos, K. Kersters, and J. Swings. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60:407-438. Vandamme, P., B. Pot, and K. Kersters. 1991b. Differentiation of campylobacters and Campylobacter-like organisms by numerical analysis of one-dimensional electrophoretic protein patterns. Syst. Appl. Microbiol. 1457-66. Vandamme, P., L. J. Van Doorn, S. T. a1 Rashid, W. G. Quint, J. van der Plas, V. L. Chan, and S. L. On. 1997. Campylobacter hyoilei Alderton et al. 1995 and Campylobacter coli Veron and Chatelain 1973 are subjective synonyms. Int. J. Syst. Bacteriol. 47~1055-1060. Vandamme, P., M. Vancanneyt, B. Pot, L. Mels, B. Hoste, D. Dewettinck, L. Vlaes, C. van den Borre, R. Higgins, and J. Hommez. 1992. Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. Int. J. Syst. Bacteriol. 42:344-356. Van Driessche, E., and K. Houf. 2007. Discrepancy between the occurrence of Arcobacter in chickens and broiler carcass contamination. Poult. Sci. 86:744-751. Van Driessche, E., K. Houf, J. Van Hoof, L. De Zutter, and P. Vandamme. 2003. Isolation of Arcobacter species from animal feces. FEMS Microbiol. Lett. 229:243-248.

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Van Etterijck, R., J. Breynaert, H. Revets, T. Devreker, Y. Vandenplas, P. Vandamme, and s. Lauwers. 1996. Isolation of Campylobacter concisus from feces of children with and without diarrhea. J. Clin. Microbiol. 34:2304-2306. Vandenberg, O., A. Dediste, K. Houf, S. Ibekwem, H. Souayah, S. Cadranel, N. Douat, G. Zissis, J. P. Butzler, and P. Vandamme. 2004. Arcobacter species in humans. Emerg. Infect. Dis. 10:1863-1867. Viron, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and V6ron and designation of the neotype strain for the type species Campylobacter fetus (Smith and Taylor) Sebald and VCron. lnt. J. Syst. Bacteriol. 23:122-134. Vinzent, R., J. Dumas, and N. Picard. 1947. Septicemie grave eu cours de la grossesse, due iun vibrion. Avortement consecutif. C. R Acad. Med. 131:90. Voordouw, G., S. M. Armstrong, M. F. Reimer, B. Fouts, A. J. Telang, Y. Shen, and D. Gevertz. 1996. Characterization of 16s rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide-oxidizing bacteria. Appl. Environ. Microbiol. 62: 16231629. Wesley, I. V., L. Schroeder-Tucker, A. L. Baetz, F. E. Dewhirst, and B. J. Paster. 1995. Arcobacter-specific and Arcobacter butzleri-specific 16s rRNA-based DNA probes. J. Clin. Microbiol. 33:1691-1698. Winkler, M. A., J. Uher, and S. Cepa. 1999. Direct analysis and identification of Helicobacter and Campylobacter species by MALDI-TOF mass spectrometry. Anal. Chem. 71:3416-3419. Wirsen, C. O., S. M. Sievert, C . M. Cavanaugh, S. J. Molyneaux, A. Ahmad, L. T. Taylor, E. F. DeLong, and C. D. Taylor. 2002. Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur. Appl. Environ. Microbiol. 68:3 16-325. Wolfe, R. S., and N. Pfennig. 1977. Reduction of sulfur by Spirillum 5175 and syntrophism with Chlorobium. Appl. Environ. Microbiol. 33 :427-43 3. Wolin, M. J., E. A. Wolin, and N. J. Jacobs. 1961. Cytochromeproducing anaerobic vibrio, Vibrio succinogenes, sp. n. J. Bacteriol. 81:911-917. Wybo, I., J. Breynaert, S. Lauwers, F. Lindenburg, and K. Houf. 2004. Isolation of Arcobacter skirrowii from a patient with chronic diarrhea. J. Clin. Microbiol. 42:1851-1852.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Biaser 6 2008 ASM Press, Washington, DC

Chapter 2

Population Biology of Campylobacter jejuni and Related Organisms MARTIN

c. J. MAIDEN AND KATE E. DINGLE

must take account of population biology and evolution. Population studies of bacteria comprise three elements: (i) sampling the population, (ii) measuring variation in the population, and (iii) interpreting the variation. Sampling the natural world is a nontrivial problem, and it is impossible to achieve the ideal of a truly random or even wholly representative population sample. In microbiology there are additional technical questions about the reliability of culture methods-or indeed other methods-for accurately capturing all of the variation present in a given sample. For widely distributed multihost bacteria, such as the members of the genus Campylobacter, these issues are particularly relevant (Bolton and Robertson, 1982) and have to be mitigated by the adoption of appropriately designed structured sampling schemes that are driven by the particular question being investigated. Molecular methods can go some way to mitigating problems with culture (Abulreesh et al., 2006), but even these can be subject to sensitivity issues, and the lack of bacterial isolates limits the extent to which biological variation can be accurately characterized. Once bacterial isolates are available, exhaustively measuring the variation present among genomes is possible by the appropriate application of highthroughput nucleotide sequence determination technology (Maiden, 2000). Two approaches can be used. The existence of a complete genome from one example of the population being analyzed enables the nucleotide sequence of genes in many isolates to be determined with PCR-based direct nucleotide sequence determination; this has the advantage that it can also be done on specimens containing noncultur-

Studies of the ecology and evolution of bacterial populations, which require measurement of variation, theory development, and testing, are often carried out independently from the investigation of microbial function that are usually conducted within a reductionist framework by means of controlled experiments. There is a potentially creative tension between these two approaches: population studies require the collection and analysis of large numbers of bacterial isolates, while on the other hand, functional studies commonly involve the detailed examination of one, or at most a few, example isolates, with little or no reference to population variation. The integration of these approaches is necessary if recent technical advances in data collection are to be fully exploited. The availability of whole-genome sequences for many bacteria, including the principal human and animal pathogens (Bentley and Parkhill, 2004), has highlighted the need for better interaction of these approaches. A whole-genome sequence is in many ways the apotheosis of a single organism study, and in principle, the determination of the complete genetic information for any organism enables all aspects of its biology to be deduced. In practice, however, current and anticipated knowledge of biochemistry and genetics is too limited to permit such deductions from genome sequences alone. Biochemistry is still not a high-throughput science in the sense that genome sequence determination is, and detailed investigations of all cistrons from all genomes are impractical. Consequently, most of the information in annotated genomes comes from comparative studies, the quality of which is dependent on the quality of the comparisons (Brenner, 1999). Once a comparative element is introduced into a study, it perforce

Martin C. J. Maiden Department of Zoology, University of Oxford, South Parks Rd., Oxford OX1 3PS, United Kingdom. Kate E. Dingle Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.

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able organisms. Conversely, the complete genome of many samples can be compared with microarray, or better, rapid whole-genome resequencing approaches, although this generally requires the availability of isolates to prepare genomic DNA. The former approach has the advantage of producing defined data rapidly, whereas the latter provides the intriguing prospect of comprehensive comparative analysis. At the present time, the whole-genome sequence determination approach remains impractical as a result of cost constraints. Even if such data were to become available, new analysis algorithms would be required to cope with the quantity of data generated. The use of an evolutionary framework is essential to the interpretation of biological variation, and a number of analytical approaches have been developed. These allow the variation present in a given population to be explored with a view to establishing the biological forces that have generated the variation present. In the case of the bacteria, which are fast growing, have small genomes, and live in intimate contact with their environment, genetic variation can be especially informative in understanding their ecology. The most effective analysis methods are parametric and model based, and in this respect, recently developed analytical tools that are based on coalescent theory are especially valuable (Balding, 2003; Didelot and Falush, 2007; Falush et al., 2003). Informal techniques can also be valuable in the primary analysis of data (Feil et al., 2004). Here we explore the contribution that population studies have made to our understanding of the biology of the Campylobacter, and we argue that such studies have a central role to play in understanding the epidemiology and pathogenesis of this important group of gramnegative bacteria.

ANALYSIS OF POPULATIONS OF CAMPYLOBACTER The factors that make members of the genus Campylobacter difficult to study with conventional single-isolate approaches-in particular their ubiquity and diversity-also make them tractable to, and indeed attractive subjects of, population studies. The biology underlying the diversity of members of the genus Campylobacter, including factors such as host range and specialization, as well as being interesting biological problems in their own right, are also relevant to the main human interaction with these bacteria, namely their propensity to cause gastroenteritis. Carnpylobacter jejuni and Campylobacter coli cause the majority of human cases of Campylobacter-associated gastroenteritis; these two organisms are asso-

ciated with approximately 90 and 10% of cases, respectively (Gillespie et al., 2002). Campylobacter upsaliensis is frequently identified in domestic cats and dogs, and it causes occasional cases of gastroenteritis in humans. Campylobacter helveticus is also found in domestic animals, but it appears not to cause human disease. Campylobacter lari can cause gastroenteritis in humans. It has been isolated from shellfish (Endtz et al., 1997), but it is most commonly found among wild birds. Campylobacter fetus is primarily associated with infections in livestock, which result in abortion and infertility, particularly in sheep and cattle. C. fetus can also be isolated from goats, pigs, horses, fowl, and reptiles and causes occasional human infections. Transmission to humans generally occurs orally through contaminated food or water (Garcia et al., 1983). A range of techniques have been used to study variation within the genus Campylobacter. Indeed, the plethora of isolate characterization techniques available could be said to have obscured rather than illuminated research into populations of these organisms (Wassenaar and Newell, 2000). In this respect, it is necessary to distinguish those bacterial characterization schemes that can be sensibly used for population studies from those that cannot. It is relatively trivial to develop a typing scheme that can classify isolates as distinct or indistinguishable (Achtman, 1996), but it is much more problematic to develop schemes that generate reproducible data that can be analyzed to investigate the biology of the organism in question. Because virtually all variation in bacterial populations is genetic or ultimately genetically determined, the availability of high-throughput nucleotide sequence determination has provided influential advances in isolate characterization. Thanks to the combination of the availability of complete genome sequences of C. jejuni, for example that of isolate NCTC 11168 (Parkhill et al., 2000), and PCR amplification technology, it is now possible to establish definitively the genetic variation present at any part of the genome by PCR-direct nucleotide sequence determination. Other methods, such as multilocus enzyme electrophoresis (Aeschbacher and Piffaretti, 1989), pulsed-field gel electrophoresis analysis (Yan et al., 1991), and amplified fragment-length polymorphism (Duim et al., 1999), are at best proxies for nucleotide sequence diversity and therefore generally provide less information (Sails et al., 2003a, 2003b). In addition, the data produced by these techniques are not as tractable to phylogenetic and statistical analysis as nucleotide sequences themselves. The application of high-throughput nucleotide sequence-based approaches (Maiden, 2000) to large

CHAPTER 2

isolate collections and the analysis of these data have enabled the diversity of C. jejuni populations to be defined (Dingle et al., 2002) and the relationship of this population to other members of the genus compared (Dingle et al., 2005; Miller et al., 2005; van Bergen et al., 2005). At present, the major technique used is multilocus sequence typing (MLST) (Maiden et al., 1998). MLST is a generic approach for the characterization of bacterial isolates that aims to identify lineages in bacterial populations by indexing the variation present in several (commonly seven) housekeeping genes located at various parts of the chromosome (Maiden, 2006). The advantages of using housekeeping genes is their presence in all, or nearly all, isolates, combined with the fact that although they contain measurable variation, they are subject to stabilizing selection for conservation of metabolic function. Given that horizontal genetic exchange is common in the microbial world (Narra and Ochman, 2006), the use of multiple loci is important because single loci can be misleading indicators of relationship by descent. MLST is in effect a nucleotide sequence-based version of multilocus enzyme electrophoresis (Selander et al., 1986) in that it indexes of genetic variation at housekeeping loci with nucleotide sequencing. This approach has a number of advantages in addition to generating definitive, reproducible, and transportable data. For MLST, a viable isolate is not required, and the process is scalable from one isolate to many hundreds or thousands by the exploitation of liquid handling technology and automated DNA analyzers. For a group of organisms as diverse as those making up the genus Campylobacter, this is a particular advantage because it facilitates the analysis of very large numbers of isolates. MLST also introduced the innovation of sharing data via the Internet (Chan et al., 2001; Jolley et al., 2004) and promoted the development of novel analysis tools (Didelot and Falush, 2007; Feil et al., 2004, 1999; Jolley et al., 2001). MLST approaches have the advantage that they can simultaneously address large-scale or more local population questions, especially in combination with other sequence-based typing approaches (Maiden, 2006). In the Campylobacter MLST scheme, the sequences of seven allele fragments are determined after amplification from genomic DNA or a killed cell suspension. In common with other schemes, short gene fragments, generally 400 to 550 bp, are used because these are easily sequenced on both DNA strands by using only one extension primer pair, ensuring rapidity, cost effectiveness, and accuracy. Each unique sequence of an allele fragment is assigned an arbitrary number in order of discovery. These unique

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sequences are stored electronically in the “profiles database,” which is essentially a dictionary that catalogs variation and that is used to assign typing data as they are collected (Jolley et al., 2004). The allele numbers are combined into allelic profiles, or sequence types (STs), which are also arbitrarily assigned. Allele numbers and STs are in effect shorthand codes for bacterial variation, with each ST representing a unique combination of nucleotide sequence of, in the case of C. jejuni, 3,309 bp in length and including the variation present at seven separate loci. STs can be grouped into “clonal complexes,” surrogates of genetic lineages that are described in detail for C. jejuni below. The definitions of these clonal complexes are also stored in the profiles database. Analysis can be conducted either at the level of clonal complex, ST, allele, or nucleotide sequences, including all or a subset of the MLST data, depending on the questions being addressed (Maiden, 2006).

VARIATION WITHIN THE GENUS CAMPYLOBACTER MLST schemes are now available for many members of the genus Campylobacter. The first to be developed was for C. jejuni (Dingle et al., 2001a); schemes are also available for C. coli (Dingle et al., 2005), C. lari, C. upsaliensis, C. helveticus (Miller et al., 2005), and C. fetus (van Bergen et al., 2005). These schemes have established the basic population structure of the members of the genus and are beginning to demonstrate associations of genotypes with particular animal hosts (Dingle et al., 2002; French et al., 2005). The relationships of these different Campylobacter microbiological species with human and veterinary disease are being more precisely defined on the basis of the insights available from MLST data. The level of genetic divergence among Campylobacter housekeeping genes is as high as 80.9% (Miller et al., 2005). Consequently, only C. jejuni can be MLST typed reliably by using the oligonucleotide primers originally described (Dingle et al., 2001b), which were designed by using the first draft of the C. jejuni genome sequence (Parkhill et al., 2000). Two approaches have been used to adapt this MLST scheme to other Campylobacter species. First, the scheme was extended to C. coli and C. fetus by using a large collection of different C. jejuni oligonucleotide primers in PCR amplifications of DNA from C. coli and C. fetus (Dingle et al., 2005; van Bergen et al., 2005). The amplification reactions were performed under conditions of reduced stringency to

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permit the amplification of corresponding housekeeping gene sequences from heterologous species. The sequences of these amplicons were determined, and new primers were designed that were specific to C. coli or C. fetus. The same seven housekeeping genes were used as the C. jejuni scheme to allow comparisons of diversity, population structure, and epidemiology among the different species. The C. coli MLST scheme was validated by using 53 isolates from humans, chickens, and pigs, together with 15 Penner serotype reference isolates (Dingle et al., 2005). The C. fetus MLST scheme was validated by using a diverse collection of 140 geographically diverse C. fetus isolates from a variety of animal hosts and humans (van Bergen et al., 2005). A second approach to extending the C. jejuni MLST scheme to other species was made possible by the availability of the draft genome sequences of C. coli, C. upsaliensis, and C. lari (Fouts et al., ZOOS), enabling the design of degenerate oligonucleotide primers, so that individual primer pairs could be used to amplify MLST loci from up to five different Campylobacter species (Miller et al., 2005). This had the advantage of easily detecting interspecies genetic recombinants, genetic hybrids, within the genus Campylobacter and detecting mixed samples. In addition to C. jejuni, the species targeted were C. coli and C. helveticus, for which the same loci are used as the C. jejuni scheme. Schemes have also been developed for C. lari, for which adk and pgi were substituted for aspA and gltA, and C. upsaliensis, for which adk and pgi were substituted for gltA and pgm. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed, demonstrating the utility of the approach for distinguishing among these bacteria. More than 80% of the isolates had unique STs. Genetic recombinants have since been described between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus (D’lima et al., 2007; Kinana et al., 2007; Miller et al., 2005). The MLST data for all of the above schemes are available at the pubMLST website (http: //pubmlst. org/campylobacter/), stored in web-accessible customized databases (Fig. 1)that permit online analysis and comparison (Jolley et al., 2004; Jolley and Maiden, 2006). The MLST scheme used for C. jejuni and C. coli is represented by a single PubMLST isolate database, which is integrated with typing data from the flaA, flaB, and porA genes used for antigen gene sequencing by AgdbNet software (Jolley and Maiden, 2006) (Fig. lb). Data from the other MLST schemes are also available at pubMLST. Although MLST was originally developed with a view to identifying clones or lineages within bacterial

populations, especially those of pathogens (Maiden et al., 1998), it has many other applications, including addressing the vexed question of bacterial “speciation” (Stackebrandt et al., 2002). Where the same genes have been chosen for different members of the genus, their interrelationships can be investigated in a number of ways, the simplest being a phylogeny based on the nucleotide sequences. A simple phylogeny drawn for those members of the genus Campylobacter for which MLST data are available shows that C. fetus is the most divergent member of the group, with C. jejuni and C. coli sharing a common ancestor, as do C. lari and C. insulaenigrae, and C. upsaliensis and C. helveticus (Fig. 2). Phylogenic trees represent genealogies, with the branches illustrating pathways of descent. Conventional algorithms use genetic distances, calculated in various ways, to reconstruct these phylogenies, by comparing nucleotide sequences on the assumption that the sequence changes occur according to a more or less constant clock rate. Such a representation is unproblematic if there is no, or at least very little, genetic exchange among the taxa being analyzed, as in the case of the named Campylobacter species. However, as the frequency of gene transfer increases among the organisms placed on divergent branches of the tree, the reliability of a conventionally generated treelike phylogeny decreases. This is because a single genetic transfer event, which is common in some bacterial populations, may introduce a large number of sequence changes, resulting in a long branch, while a single mutation, a comparatively rare event, would generate a short branch. The generation of mosaic genes by intragenic recombination further complicates the interpretation of conventional phylogenies (Maiden, 1993). It is now known that genetic exchange (sometimes referred to as lateral gene transfer or “localized sex” [Maynard Smith et al., 19911) is quite common in the bacteria and needs to be taken into account when reconstructing bacterial genealogies (Maynard Smith et al., 1993). In the case of the relationships among the different members of the genus, a conventional tree, such as that shown in Fig. 2, is more or less reliable, as there is relatively little genetic exchange among the bacteria classified as microbiological species. Indeed, bacterial “species” are probably best defined as populations of bacteria that recombine infrequently, rather than the more traditional definitions that are based on DNA homology (Stackebrandt et al., 2002). The problem of such a definition is that it is difficult to define the levels of gene flow that permit the emergence of bacterial species, and in any case, estimating these parameters is complex and can be sensitive to the sampling frame used. Further, the different levels

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31

a

N

N

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% 0

0 0 0

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tn

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Figure 1. Carnpylobacter MLST databases. (a) The growth of the Carnpylobacterjejuni and Campylobacter coli database since its inception in 2001. Open squares represent isolates submitted; closed squares, sequence types. (b) The structure of the pubMLST database network, which is used for all of the Campylobacter databases. It allows the integration of data from various sources in a variety of public or private databases, ensuring that a common typing nomenclature is used.

of horizontal genetic exchange may have different effects on diverse bacterial populations. Wholly asexual populations, i.e., those in which genetic exchange is negligible, will conform to the clonal model of population structure. Clonality is an inevitable consequence of reproduction by binary fission because any mutations that occur in the population are limited to the descendants of the mother cell in which they arose. Diversity reduction events such as bottlenecking (Achtman, 1997) or periodic selection (Levin, 1981) purge the population of diversity, resulting in a population that is dominated by lineages with distinct genotypes and therefore phenotypes. Clonal populations will conform to a num-

ber of predictions: (i) alleles at different loci will exhibit linkage disequilibrium; that is, they will occur in nonrandom combinations; (ii) they will exhibit relationships that conform to a treelike phylogeny, with branches generated by mutation events; and (iii) they will exhibit congruence; that is, the same phylogeny will be recorded at different loci. The population structure of wholly sexual populations is characterized by a lack of lineage structure, with genetic variation that exhibits (i) linkage equilibrium, (ii) a netlike phylogeny, and (iii) incongruence, with each locus recording a unique evolutionary history (Maynard Smith, 1989). In practice, few bacteria occupy the extreme of an entirely clonal or

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C.insulaenigrae \

C. lari \

,

C. jejuni C. upsaliensis

I

w

C. helveticus

C. fetus

Figure 2. Relationships among the different Campylobacter microbiological species. The phylogeny was reconstructed with concatenated sequences from the alleles common to all of the schemes (gln, gly, and tkt).

wholly nonclonal population, and the process of binary fission imposes some element of clonality on all bacterial populations (Feil et al., 1999; Spratt and Maiden, 1999). Understanding bacterial population structure therefore becomes an issue of determining the rate of gene flow and applying an appropriate model, i.e. clonal, partially clonal, or nonclonal. Within the genus Campylobacter, different types of population structure have been observed within bacteria currently assigned to different microbiological species. By far the most studied to date are C. jejuni and C. coli, reflecting their importance in human disease.

Campylobacter jejuni The MLST data collected to date show that C. jejuni is highly diverse with a total of 2,243 distinct STs from some 3,704 isolates deposited in the pubMLST database (http://pubmlst.org/ campylobacter/); many more isolates, perhaps in excess of 10,000, have been characterized by MLST in a variety of studies. In addition to the most widely used scheme, two other C. jejuni MLST schemes have been developed. As expected, given the nature of MLST systems, all three schemes provide similar perspectives on C. jejuni biology (Dingle et al., 2001a; Manning et al., 2003; Suerbaum et al., 2001). Analyses of subsets of the MLST data by maximum likelihood methods show that the phylogenies generated from the different loci are incongruent,

violating the predictions of clonality (Dingle and Maiden, 2005). In addition, there is little evidence for treelike structure in nucleotide sequences of the individual alleles (Dingle et al., 2001a; Suerbaum et al., 2001). There is, however, some evidence of linkage disequilibrium in these data sets (Dingle and Maiden, 2005; Schouls et al., 2003). These features, which are seen in a number of other bacteria analyzed by MLST (Maiden, 2006), are consistent with a partially clonal population, that is, one that contains distinct clusters of related isolates that do not share clonal relationships with each other. In other words, although each member of each cluster shares a common ancestor with other members of the same cluster, cluster recombination has reassorted genetic variation wholesale among clusters. Clusters of this type are recognized in MLST data as clonal complexes, which are defined informally within the MLST databases on the basis of a number of biological properties (Maiden, 2006). Examination of data sets from partially clonal organisms reveals differences in the frequency distribution of STs in that some STs are much more frequent in population samples than others. Typically these STs are also recovered in isolates obtained over prolonged periods of time and from diverse geographic locations. In addition, such STs are often characterized by having many low-frequency relatives that vary from them at one, two, or three loci. These STs correspond to “central genotypes” (sometimes referred to as “founders”) that define and give their name to the clonal complex. The clonal complex then includes all isolates with the central ST plus those with STs sharing up to four identical loci. Thus, the ST45 clonal complex includes ST-45 itself and all of its single, double, and triple locus variants (Fig. 3). In most cases, the variants are generated by recombination rather than point mutation. The influence of recombination on the population structure of C. jejuni is further indicated by the relationship of alleles to STs in the database. Although there were a total of 2,243 distinct STs, organized into 42 clonal complexes at the time of writing, the number of alleles at each locus was much smaller, ranging from 134 (for aspA) to 256 (for pgm). This relationship shows that most genotypes are assembled by reassortment of preexisting alleles, not by the generation of novel alleles by mutation or intra-allele recombination. This in turn implies that the recombination fragment sizes are rather larger than the average size of the MLST alleles (402 to 507 bp in C. jejuni) and, given that these are substantial fractions of the average size of C. jejuni cistrons (948 bp; Parkhill et al., 2000), the genes themselves. This observation is somewhat at odds with some estimates

I

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of average recombination fragment sizes of 225 to 750 bp obtained by approximate likelihood methods (Fearnhead et al., 2005) but consistent with the estimate of 3.3 kbp obtained with apparently less precise methods (Schouls et al., 2003). However, these observations further underline the importance of genetic exchange in the population biology and evolution of Campylobacter. Clonal complexes are surrogates for lineages, and the utility of the clonal complex as a unit of analysis presumably reflects the strength of the lineage structure within populations of C. jejuni. The recent development of the ClonalFrame algorithm (Didelot and Falush, 2007) permits the formal analysis of these lineages for the first time. This program identifies clonal lineages while estimating the likelihood that particular changes have occurred by either recombination or single base-pair mutation, avoiding the problems associated with distance-based methods. The principal disadvantage of this approach is that it is computationally intensive to the extent that it is impractical to apply to all samples in the MLST databases and has to be rerun each time a new ST is added. It is, however, invaluable for testing the robustness of the informal clonal complex designations. Analysis of sample data sets with ClonalFrame shows that C. jejuni clonal complexes correspond to genetic lineages, as predicted. It also confirms the observation that particular clonal complexes are associated with different isolation sources. This probably reflects the fact that clonal complexes represent lineages that are adapted to particular niches that, at least in some cases, correspond to different host species. For example, ST-45 complex C. jejuni are isolated primarily from chickens, environmental sources, or human disease, ST-61 complex mostly from bovine or ovine sources, the environment, or human disease, and ST-42 complex mostly from bovine or ovine sources and human disease (Dingle et al., 2002) (Fig. 4). Some relationships among clonal complexes are apparent from the ClonalFrame analysis; for example, ST-283 complex and ST-45 complex appear to be related, as do ST-48 complex, ST-353 complex, and ST-682 complex (Fig. 4). Most branches, however, converge in a “star phylogeny,” showing that at least for data from seven loci, ClonalFrame analysis cannot resolve deeper phylogenies. This may be a consequence of extensive horizontal genetic exchange disrupting all clonal signal in the population, or it may be a limitation of the numbers of loci used in the analysis. The ST-21 complex is also poorly resolved in this analysis, also appearing as a star phylogeny; this also might be resolved by the inclusion of more loci in future studies. Because the

ST-21 complex is found in a variety of host sources, there may be sublineages within this clonal complex adapted to particular niches. Further support for these ideas comes from the assignment of host types on the basis of MLST data. By use of the structure algorithm (Falush et al., 2003) and a reference set of isolates, the origins of variant alleles in a set of ST-21 complex isolates were assigned and compared with the host origin of the isolate itself. In 18 of 22 cases, the assignment of the variant alleles corresponded with the isolation source, in terms of host animal of the variant strain, indicating that the strain (i) had been resident in the host, and (ii) had preferentially acquired alleles associated with that particular host (McCarthy et al., 2007). Clonal complex, a surrogate for lineage, is therefore a fundamental unit of C. jejuni biology, and much of the confusion present in the field was a consequence of the fact that earlier typing schemes, especially serotyping, did not accurately reflect the clonal complexes (Dingle and Maiden, 2005). The lineages are sufficiently stable to be recovered over periods of time and to be associated with particular hosts. Given that most sampling effort is focused on human disease cases and food animals, it is likely that clonal complexes associated by nonfarm animal sources are poorly represented in current isolate collections, and it is likely that many additional lineages associated with wild animal sources exist (French et al., 2005). In one case a lineage had been identified and named as a discrete subspecies. Isolates characterized as C. jejuni subspecies doylei have been classified as distinct from other C. jejuni isolates on the basis that they are primarily isolated from humans and have a propensity to cause bacteremia. Analysis of MLST data with conventional phylogenetic techniques and microarray-based comparative genome indexing suggested that C. jejuni doylei was distinct from “C. jejuni jejuni” (Parker et al., 2007). This division into two clades is not supported by ClonalFrame analysis, which suggests that the isolates belonging to C. jejuni doylei are just another lineage. This analysis also provides the following explanation for the discrepancy. The C. jejuni doylei isolates are located in a sparsely sampled part of the C. jejuni tree generated by ClonalFrame, which contains isolates mostly of environmental origin. Moreover, the well-sampled part of the tree, which contains mostly isolates derived from humans or food animals, contains a number of major lineages that are related to each other as well as to the ST-21 complex. Thus, a collection of reference isolates that reflected current isolate collections would have very few of the samples of nonhuman and nonhuman food origin, and very many of those from humans and their food animals. There-

CHAPTER 2

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\ C.doylei

Figure 4. Correspondence of clonal complexes with genealogies. Clonal complex designations were used to annotate a phylogeny generated by ClonalFrame. The size of the pie charts represents the number of isolates in each clonal complex, with the color indicating the relative contribution of each source: black, human disease; white, chickens and chicken meat; dark gray, ovines and ovine meat; light gray, environmental sources. Data from Dingle et al. (2002) and Parker et al. (2007).

fore, analyses comprising C. jejuni doylei and other human disease isolates, however performed, will show C. jejuni doylei to be distinct because other isolates with a similar relationship to the humancentered isolates will be absent. An interpretation that is consistent with both sets of data is that C. jejuni doylei isolates represent a lineage associated with an environmental source that very rarely comes into contact with humans, but when it does, it tends to cause more severe disease. The observations on the potentially misleading characterization of the C. jejuni doylei isolates as a subspecies distinct from all other C. jejuni lineages introduces another implication of the population

structure of C. jejuni. Comparisons of the genomes of multiple isolates, especially by microarray methods, are popular and considered to be important (Champion et al., 2005). However, the inferences that can be gained from such studies are dependent on the genes included in the microarrays used and the isolates included in the analysis. It is known that not all C. jejuni isolates contain all of the genes present in the population, and it is likely that different lineages typically have distinct gene complements. A consequence of this is that any comparison of gene complement that is based on a microarray assembled with only one genome sequence can only provide resolution of the C. jejuni population into two groups:

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“like the reference isolate” and “unlike the reference isolate.” Thus, microarray-based “genomotyping” with microarrays that are based on C. jejuni 11168 can only divide C. jejuni collections into two groups: like NTCC1168 (which happens to be an ST-21 complex isolate) and unlike NTCC1168 (Champion et al., 2005). Adding data from more genomes will help in this process, but the MLST data assembled so far indicate that data from a minimum of 42 isolates (one from each clonal complex identified to date) should be used to generate the microarrays if the association of particular clonal complexes with host sources are to be informatively defined. In general, clonal complex structure should be taken into account when choosing isolates for inclusion in functional analyses or multiple genome sequencing. Although human infection is one of the most important practical applications of studies of Campylobacter populations, in terms of Campylobacter population dynamics and evolution, infection is probably irrelevant. Human infection is almost certainly incidental, and little if any importance should be attached in terms of onward evolutionary spread of C. jejuni. Understanding the population biology of Carnpylobacter is, however, crucial in understanding the transmission to humans and developing means for its control. MLST has permitted direct comparisons to be made among studies of C. jejuni epidemiology (Gillespie et al., 2002). The identification of outbreaks and potential sources of infection relies on the precise characterization of isolates and knowledge of their interrelationships. The clonal complex is a valuable unit of analysis for epidemiological investigation, allowing the comparison of data from diverse sources worldwide. This has demonstrated that different clonal complexes are important in different geographical regions of the world, probably reflecting different food animal sources of human infection (Dingle et al., 2002; Duim et al., 2003; Mickan et al., 2007; Sopwith et al., 2006) (Fig. 4). MLST has also been of value in investigating the link of C. jejuni genotypes with specific disease syndromes (Dingle et al., 2001b). Antigen sequence typing generates high levels of discrimination among bacterial isolates by measuring the variation present in antigen genes that are subject to diversifying selection, presumably by virtue of their exposure to the host immune system. The inclusion of these data are therefore useful in identifying very closely related bacterial isolates, for example in an outbreak situation, and in studies of the immune response to bacteria, but their high rates of change make them less useful in unambiguously identifying lineages. Combining MLST and antigen gene sequence data can be a highly effective means of

achieving high-resolution characterization data that also allow long-range comparisons. For the identification of disease outbreaks, clonal complex designation alone is insufficiently precise to reliably detect outbreaks (Clark et al., 2003; Fitzgerald et al., 2001; Sails et al., 2003b). Sequence typing data also provide the resolution and reproducibility necessary to investigate the diversity of Campylobacter isolates obtained from farms and the environment (Colles et al., 2003; French et al., 2005; Manning et al., 2003). These studies have shown that genotypes most like those from human disease are recovered from farm animals (Colles et al., 2003; Manning et al., 2003), while the genotypes from environmental sources seem to be distinct (Fig. 4) (French et al., 2005). Certain clonal complexes are associated with particular food animal sources, with the ST-61 complex especially associated with cattle (Dingle et al., 2002; French et al., 2005) and the ST-403 with pigs (Manning et al., 2003). Large-scale studies of isolates from a wide variety of sources provide the prospect that in the near future, it will be possible to trace the movement of Campylobacter genotypes through the food chain (Allen et al., 2007) and attribute cases of human infection to particular host sources with reasonable precision (McCarthy et al., 2007). MLST has also been used to examine antibiotic-resistant isolates obtained from poultry (Kinana et al., 2006, 2007).

Campylobacter coli C. coli has a similar host range to C. jejuni and is associated with up to 10% of human Campylobacter infections. At the time of writing, the MLST allelic profile database contained 708 C. coli STs. Preliminary analyses of MLST data from C. coli (Miller et al., 2005) identified two putative clonal complexes among a small collection of 37 STs. The first was designated ST-1017 complex (31STs) and the second ST-1052 complex (4 STs). Now that more data are available, ST-1017 has been shown to be part of the ST-828 complex, and ST-1052 is currently unassigned. Two large C. coli clonal complexes have now been confirmed, the largest being ST-828 complex, with 425 STs, and the other ST-1150 complex, with 25 STs (http: //pubmlst.org/campylobacter/). This indicates the difficulty of identifying the central genotype of a clonal complex when only few data are available. Of the 708 STs currently in the PubMLST database, 258 are unassigned to a clonal complex. These STs were analyzed for the presence of clonal complexes by the BURST algorithm (Feil et al., 2004), and a new large lineage was identified, ST-

CHAPTER 2

1172 complex, containing 130 STs. Two smaller possible clonal complexes, ST-2013 (eight STs) and ST2461 (seven STs), were also identified. Five groups of 4 isolates, two groups of 3 isolates, and 10 paired isolates were identified, with 67 singletons remaining. This suggests that the C. coli population structure is similar to but not identical to C. jejuni, C. coli apparently comprising a smaller number of genetic lineages. The two large clonal complexes identified so far appear to resemble ST-21 complex in C. jejuni in that they are multihost and could potentially be better understood by the sequencing of additional loci. Five studies performed in four different countries have used MLST to study C. coli isolated from food animals and human disease (D’lima et al., 2007; Kinana et al., 2007; Litrup et al., 2007; Thakur and Gebreyes, 2005; Thakur et al., 2006). Pigs are considered a common reservoir for C. coli infections in humans; therefore, several studies have used MLST to examine the genotypes associated with pigs. A MLST-based comparison of 151 C. coli isolates from pigs on the farm and at slaughter demonstrated a high level of genetic diversity, with a total of 74 STs identified among the isolates. Most STs found on the farm were not found at slaughter, but different STs were, suggesting an important role for crosscontamination at slaughter (Thakur and Gebreyes, 2005; Thakur et al., 2006). They also compared isolates from animals reared with and without the use of antibiotics. ST-828 complex was described as containing isolates from both the production systems examined. A study of 36 C. coli isolates from chickens in Senegal described a low level of genetic diversity, with 13 STs, and no clear genetic lineages, probably due to the small size of the data set (Kinana et al., 2007); however, 10 of the STs identified were members of ST-828 complex. There was no apparent link between quinolone resistance and ST. In a study of 160 Danish C. coli isolates from humans, broilers, pigs, and cattle, high genetic diversity was observed, with 84 STs being identified. Ten percent of the isolates possessed STs that were found in human, poultry, and pig isolates. Only 10% of the isolates from pigs shared STs with isolates from humans, and these shared STs were found in poultry isolates as well. Therefore, C. coli isolates from pigs were presumed not to be a significant source of human infections in Denmark (Litrup et al., 2007). A study of 59 multidrug-resistant C. coli strains from turkeys demonstrated that only 3 of the 14 STs accounted for 69% of the isolates, and these STs were turkey specific, implying that multidrug-resistant C. coli strains from turkeys in North Carolina, USA, are characterized by a relatively small number of clonal groupings (D’Lima et al., 2007).

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37

The studies by Litrup et al. and D’Lima et al. illustrate the potential of C. coli MLST for tracking the origins of human infection. They also illustrate the requirement for large data sets before host associations start to become apparent. Miller et al. (2006) have made major advances in this respect by typing 488 C. coli strains from four different food animal sources (cattle, chickens, swine, and turkeys) collected at different times over a 6-year period from different geographical locations in the United States. A total of 149 STs were identified. The 185 swine strains were the most diverse, possessing 82 STs. The cattle strains were the most clonal; 52 (83%) of 63 strains possessed a single ST (ST-1068). Most STs and alleles were host associated, i.e., found primarily in strains from a single food-animal source. Only 12 (8%) of 149 STs were found in multiple sources, indicating the potential for MLST to help trace human infections (Miller et al., 2006). C . fetus Population Structure and Molecular Epidemiology

A large and diverse collection of 140 C. fetus isolates, including a wide range of hosts and geographic locations, was used to validate the C. fetus MLST scheme (van Bergen et al., 2005). Fourteen different STs were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. The C. fetus MLST data indicated that this species is genetically homogeneous compared with the heterogeneity of other Campylobacter species for which MLST data are available (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus). A total of 91% of C. fetus isolates was assigned to only four STs. C. fetus is divided into two subspecies: C. fetus subspecies fetus (associated with abortions most often in sheep, and a wide range of other hosts) and C. fetus subspecies venerealis (isolated mainly from cattle). The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis, which appears to represent a bovine clone. Congruence was observed among C. fetus subspecies, sap types, and ST. MLST therefore confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche. No associations among the STs and host or geographic region were identified. Other Species The extended MLST method (Miller et al., 2005) was used to type 20 C. lari isolates, 78 C. upsaliensis, and nine C. helveticus. Each species was ge-

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netically diverse, the majority of the STs being unique (http://pubmlst.org/campylobacter/).A total of 15 C. lari STs, 66 C. upsaliensis STs, and 8 C. helveticus STs were identified.

CONCLUSIONS Since the development of MLST 10 years ago (Maiden et al., 1998), major progress has been made in understanding the population biology of C. jejuni and its relatives. The application of high-throughput nucleotide sequence-based methods, and particularly MLST, has not only improved isolate characterization but also illuminated areas of the biology of these highly diverse organisms that are relevant to understanding and controlling disease. It is instructive to reflect that the first study of C. jejuni and C. coli population structure by multilocus enzyme electrophoresis (Aeschbacher and Piffaretti, 1989) provided many insights that have proved to be correct and that have been extended and deepened by MLST studies. The clonal complex has proved to be a powerful and adaptable unit for analysis of population structure that also enables long-term epidemiological monitoring. Including data from antigen-encoding genes provides a highly discriminatory system for tracking genotypes through the food chain and in epidemiological monitoring. The association of particular genotypes with particular host species provides the prospect of accurately determining the relative importance of different sources of human infection. Ongoing nucleotide sequence-based studies involving large numbers of isolates and improved genealogical analysis tools provide the highly attractive prospect that well within the next 10 years, the population biology of these organisms, at least insofar as it relates to human infection, will be effectively resolved. Acknowledgments. M.C.J.M. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. The United Kingdom Department of Environment, Food and Rural Affairs supported our work (contract numbers 020604 and 020611). We thank Samuel Sheppard for assistance in the preparation of the figures.

REFERENCES Abulreesh, H. H., T. A. Paget, and R. Goulder. 2006. Campylobacter in waterfowl and aquatic environments: incidence and methods of detection. Enuiron. Sci. Technol. 40:7122-7131. Achtman, M. 1996. A surfeit of YATMs? J. Clin. Microbiol. 34: 1870. Achtman, M. 1997. Microevolution and epidemic spread of serogroup A Neisseria meningitidis-a review. Gene 192:135-140. Aeschbacher, M., and J.-C. Piffaretti. 1989. Population genetics of human and animal enteric Campylobacter strains. Infect. lmmun. 57~1432-1437.

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juni clones in Curacao, Netherlands Antilles. 1.Clin. Microbiol. 415593-5597. Duim, B., T. M. Wassenaar, A. Rigter, and J. Wagenaar. 1999. High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting. Appl. Environ. Microbiol. 65:236923 75. Endtz, H. P., J. S. Vliegenthart, P. Vandamme, H. W. Weverink, N. P. van den Braak, H. A. Verbrugh, and A. van Belkum. 1997. Genotypic diversity of Campylobacter lari isolated from mussels and oysters in The Netherlands. Int. J. Food Microbiol. 34:7988. Falush, D., M. Stephens, and J. K. Pritchard. 2003. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164: 1567-1587. Fearnhead, P., N. G. Smith, M. Barrigas, A. Fox, and N. French. 2005. Analysis of recombination in Campylobacter jejuni from MLST population data. J. Mol. Evol. 61:333-340. Feil, E. J., B. C. Li, D. M. Aanensen, W. P. Hanage, and B. G. Spratt. 2004. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol. 186:1518-1530. Feil, E. J., M. C. J. Maiden, M. Achtman, and B. G. Spratt. 1999. The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis. Mol. Biol. Evol. 16:1496-1502. Fitzgerald, C., L. 0. Helsel, M. A. Nicholson, S . J. Olsen, D. L. Swerdlow, R. Flahart, J. Sexton, and P. I. Fields. 2001. Evaluation of methods for subtyping Campylobacter jejuni during an outbreak involving a food handler. J. Clin. Microbiol. 39:23862390. Fouts, D. E., E. F. Mongodin, R. E. Mandrell, W. G. Miller, D. A. Rasko, J. Ravel, L. M. Brinkac, R. T. DeBoy, C. T. Parker, S . C. Daugherty, R. J. Dodson, A. S . Durkin, R. Madupu, S. A. Sullivan, J. U. Shetty, M. A. Ayodeji, A. Shvartsbeyn, M. C. Schatz, J. H. Badger, C. M. Fraser, and K. E. Nelson. 2005. Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species. PLoS Biol. 3:e15. French, N., M. Barrigas, P. Brown, P. Ribiero, N. Williams, H. Leatherbarrow, R. Birtles, E. Bolton, P. Fearnhead, and A. Fox. 2005. Spatial epidemiology and natural population structure of Campylobacter jejuni colonizing a farmland ecosystem. Environ. Microbiol. 7:1116-1126. Garcia, M. M., G. M. Ruckerbauer, M. D. Eaglesome, and W. E. Boisclair. 1983. Detection of Campylobacter fetus in artificial insemination bulls with a transport enrichment medium. Can. J. Comp. Med. 47:336-340. Gillespie, I. A., S . J. O’Brien, J. A. Frost, G. K. Adak, P. Horby, A. V. Swan, M. J. Painter, K. R. Neal, and C. S. S. S. Collaborators. 2002. A case-case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses. Emerg. Infect. Dis. 8:937-942. Joky, K. A., M. S . Chan, and M. C. Maiden. 2004. mlstdbNetdistributed multi-locus sequence typing (MLST) databases. BMC Bioinfomzatics 5:86. Joky, K. A., E. J. Feil, M. S . Chan, and M. C. Maiden. 2001. Sequence type analysis and recombinational tests (START). Bioinfomzatics 17: 1230-123 1. Joky, K. A., and M. C. Maiden. 2006. AgdbNet-antigen sequence database software for bacterial typing. BMC Bioinformatics 7:314. Kinana, A. D., E. Cardinale, I. Bahsoun, F. Tall, J. M. Sire, S. Breurec, B. Garin, C. Saad-Bouh Boye, and J. D. Perrier-GrosClaude. 2007. Campylobacter coli isolates derived from chickens

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in Senegal: diversity, genetic exchange with Campylobacter jejuni and quinolone resistance. Res. Microbiol. 158:138-142. Kinana, A. D., E. Cardinale, F. Tall, I. Bahsoun, J. M. Sire, B. Garin, S. Breurec, C. S . Boye, and J. D. Perrier-Gros-Claude. 2006. Genetic diversity and quinolone resistance in Campylobacter jejuni isolates from poultry in Senegal. Appl. Environ. Microbiol. 72:3309-3313. Levin, B. R. 1981. Periodic selection, infectious gene exchange and the genetic structure of E. coli populations. Genetics 99:l-23. Litrup, E., M. Torpdahl, and E. M. Nielsen. 2007. Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark.1. Appl. Microbiol. 103:210-218. Maiden, M. C. 2006. Multilocus sequence typing of bacteria. Annu. Rev. Microbiol. 60561-588. Maiden, M. C. J. 1993. Population genetics of a transformable bacterium: the influence of horizontal genetical exchange on the biology of Neisseria meningitidis. FEMS Microbiol. Lett. 112: 243-250. Maiden, M. C. J. 2000. High-throughput sequencing in the population analysis of bacterial pathogens. Int. J. Med. Microbiol. 290~183-190. Maiden, M. C. J., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95:3140-3145. Manning, G., C. G . Dowson, M. C. Bagnall, I. H. Ahmed, M. West, and D. G. Newell. 2003. Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni. Appl. Environ. Microbiol. 69:6370-6379. Maynard Smith, J. 1989. Trees, bundles or nets. Trends Ecol. Evol. 4 :3 02-3 04. Maynard Smith, J., C. G. Dowson, and B. G. Spratt. 1991. Localized sex in bacteria. Nature 349:29-31. Maynard Smith, J., N. H. Smith, M. O’Rourke, and B. G. Spratt. 1993. How clonal are bacteria? Proc. Natl. Acad. Sci. USA 90: 4384-4388. McCarthy, N. D., F. M. Colles, K. E. Dingle, M. C. Bagnall, G. Manning, M. C. Maiden, and D. Falush. 2007. Host-associated genetic import in Campylobacter jejuni. Emerg. Infect. Dis. 13: 267-272. Mickan, L., R. Doyle, M. Valcanis, K. E. Dingle, L. Unicomb, and J. Lanser. 2007. Multilocus sequence typing of Campylobacter jejuni isolates from New South Wales, Australia. J. Appl. Microbiol. 102:144-152. Miller, W. G., M. D. Englen, S . Kathariou, I. V. Wesley, G . Wang, L. Pittenger-Alley, R. M. Siletz, W. Muraoka, P. J. FedorkaCray, and R. E. Mandrell. 2006. Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals. Microbiology 152:(Pt. 1):245-255. Miller, W. G., S . L. On, G. Wang, S . Fontanoz, A. J. Lastovica, and R. E. Mandrell. 2005. Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus. J. Clin. Microbiol. 43:2315-2329. Narra, H. P., and H. Ochman. 2006. Of what use is sex to bacteria? Cum Biol. 16:R705-R710. Parker, C. T., W. G. Miller, S . T. Horn, and A. J. Lastovica. 2007. Common genomic features of Campylobacter jejuni subsp. doylei strains distinguish them from C. jejuni subsp. jejuni. BMC Microbiol. 7 5 0 . Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S . Holroyd, K. Jagels, A. V. Karlyshev, S . Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H.

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van Vliet, S . Whitehead, and B. G. Barrel]. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668. Sails, A. D., B. Swaminathan, and P. I. Fields. 2003a. Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strain associations identified by multilocus enzyme electrophoresis. J. Clin. Microbiol. 41:4058-4067. Sails, A. D., B. Swaminathan, and P. I. Fields. 2003b. Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni. J. Clin. Microbiol. 41:4733-4739. Schouls, L. M., S . Reulen, B. Duim, J. A. Wagenaar, R. J. Willems, K. E. Dingle, F. M. Colles, and J. D. Van Embden. 2003. Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J. Clin. Microbiol. 41:15-26. Selander, R. K., D. A. Caugant, H. Ochman, J. M. Musser, M. N. Gilmour, and T. S . Whittam. 1986. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 515337-884. Sopwith, W., A. Birtles, M. Matthews, A. Fox, S . Gee, M. Painter, M. Regan, Q. Syed, and E. Bolton. 2006. Campylobacter jejuni multilocus sequence types in humans, northwest England, 20032004. Emerg. Infect. Dis. 12:1500-1507. Spratt, B. G., and M. C. J. Maiden. 1999. Bacterial population genetics, evolution and epidemiology. Proc. R. SOC. Lond. B Biol. Sci. 354:701-710.

Stackebrandt, E., W. Frederiksen, G. M. Garrity, P. A. Grimont, P. Kampfer, M. C. Maiden, X. Nesme, R. Rossello-Mora, J. Swings, H. G. Truper, L. Vauterin, A. C. Ward, and W. B. Whitman. 2002. Report of the ad hoc committee for the reevaluation of the species definition in bacteriology. Int. /. Syst. Bacteriol. 52(Pt. 3):1043-1047. Suerbaum, S., M. Lohrengel, A. Sonneveld, F. Ruberg, and M. Kist. 2001. Allelic diversity and recombination in Campylobacter jejuni. J. Bacteriol. 183:2553-2559. Thakur, S., and W. A. Gebreyes. 2005. Campylobacter coli in swine production: antimicrobial resistance mechanisms and molecular epidemiology. J. Clin. Microbiol. 435705-5714. Thakur, S., W. E. Morrow, J. A. Funk, P. B. Bahnson, and W. A. Gebreyes. 2006. Molecular epidemiologic investigation of Campylobacter coli in swine production systems, using multilocus sequence typing. Appl. Environ. Microbiol. 725666-5669. van Bergen, M. A., K. E. Dingle, M. C. Maiden, D. G. Newell, L. van der Graaf-Van Bloois, J. P. van Putten, and J. A. Wagenaar. 2005. Clonal nature of Campylobacter fetus as defined by multilocus sequence typing. J. Clin. Microbiol. 435888-5898. Wassenaar, T. M., and D. G. Newell. 2000. Genotyping of Campylobacter species. Appl. Environ. Microbiol. 66: 1-9. Yan, W., N. Chang, and D. E. Taylor. 1991. Pulsed-field gel electrophoresis of Campylobacter jejuni and Campylobacter coli genomic DNA and its epidemiologic application. J. Infect. Dis. 163:1068-1072.

Cumpylobucter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 3

Complexity and Versatility in the Physiology and Metabolism of

Campylobacter jejuni DAVIDJ. KELLY

UNDERSTANDING PHYSIOLOGY AND METABOLISM HELPS TO UNDERSTAND THE COLONIZATION AND DISEASE PROCESS

comparative genomics. The challenge now is to use this information to discover which metabolic pathways or enzyme activities are essential in the different environments that C. jejuni is capable of surviving or growing in. This will give us insights into the strategies it uses for successful colonization, and it will suggest how C. jejuni can be a commensal in the avian gastrointestinal tract, a pathogen in the human gastrointestinal tract, and a persistent survivor in the environment and the food chain. In these different situations, the metabolism of the bacteria may be significantly altered, yet these crucial changes are poorly understood at present. In this chapter, the current knowledge on some of the metabolic aspects of C. jejuni physiology, with emphasis on those features of carbon, nitrogen, and electron flow that are likely to be of importance in understanding growth in the environment and in vivo, will be reviewed. The chapter will focus on catabolic pathways, i.e., those involved in the breakdown of extracellular solutes, yielding energy and key intracellular intermediates that are the building blocks for new cell growth. Since I last reviewed this area (Kelly, 2005), a number of important papers have been published. I have endeavored to include most of these, but because of space limitations, the material is necessarily selective.

The recent availability of the genome sequences of several campylobacters, particularly Campylobacter jejuni, has refocused attention on the physiological and metabolic properties of this medically and economically important group of the Epsilonproteobacteria. For many years, such studies have taken second place to work on virulence and colonization, but it is now becoming clear that the particular metabolic properties of any pathogen are intimately related to its ability to cause disease, and a greater understanding of these relationships is crucial to gain a full picture of pathogenicity. The campylobacters can be difficult organisms to culture, often have complex nutritional requirements, and are microaerophilic, and this combination of features has undoubtedly hindered progress in elucidating many aspects of their physiology and metabolism. Nevertheless, such studies are central to understanding growth in animal and human hosts, survival in the environment and the food chain, and mechanisms of pathogenicity, as well as what carbon sources are being used in vivo. Physiological studies may also provide leads in identifying potential targets for controlling or preventing the growth of campylobacters. The determination of the complete genome sequence of C. jejzkni NCTC 11168 (Parkhill et al., 2000) provided many new insights into its biology, not least in terms of metabolism. Complete genome sequences are now available for additional diverse strains of C. jejuni, for example 81-176 (Hofreuter et al., 2006), RM1221 (Fouts et al., 2005), and CG8486, a recent clinical isolate from Thailand (Poly et al., 2007), which provide a wealth of material for

TRANSPORTING SOLUTES IN AND OUT OF CAMPYLOBACTER JEJUNI Overview

Solute transport is a fundamental prerequisite for metabolism in the cytoplasm of hydrophilic and polar growth substrates. Active solute transport sys-

David J. Kelly Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.

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tems can be broadly classified into two major groups on the basis of their energy-coupling mechanisms. Primary systems are often multicomponent and depend on direct hydrolysis of chemical bonds to release free energy for solute transport, often ATP hydrolysis. In contrast, secondary systems are usually simpler (sometimes a single transport protein) and use a preexisting electrochemical ion-gradient across the cytoplasmic membrane to power solute uptake, often the proton motive force or a sodium ion gradient. C. jejuni contains many types of both classes of transport systems. As an example, the NCTC 11168 genome contains about 92 distinct transport proteins, 37% of which form primary systems and 57% of which form secondary systems. Most primary systems are accounted for by ABC-type transporters (about 30 complete systems, both importers and exporters), with the remaining primary systems comprising three P-type ATPases (the cation transporters Cjll5.S and Cj1161, and the Kdp potassium transporter) and one F-type ATPase (the proton-translocating ATP synthase). Of the secondary transporters, most fall into the major facilitator superfamily group, consisting of a single transport protein, but there are one or more representatives of about 20 other distinct families of secondary transporters, each of which will have specialized roles in solute movements. It is often difficult to accurately predict the substrates used by transporters from sequence homologies. As a general observation, we can conclude that C. jejuni NCTC 11168, for example, has few carbohydrate transporters, but many systems for amino acid and organic acid transport. This reflects the asaccharolytic nature of its metabolism and its reliance on amino acids as major carbon sources (discussed below). Here, there is only space to describe a selection of the transport systems present that relate to the major metabolic pathways in C. jejuni for which some functional data are available. Amino Acid Transport As described below, amino acids are thought to be the most important carbon and energy sources for C. jejuni, and data are now accumulating on the transport of these substrates, which suggests a number of interesting connections between pathogenicity and metabolism. This is particularly evident in the dual roles of several amino acid binding proteins, which are also immunogenic surface protein antigens and adhesins. PEBla and the PEBl transport system: aspartate and glutamate transport In a study designed to identify proteins suitable for use in serology and as vaccine candidates, Pei et

al. (1991) identified four proteins of 28, 29, 30, and 31 kDa in acidic glycine extracts of whole cells of strain 81-176. These were termed PEBl to PEB4, respectively. The structural gene for PEBl (pebla)was cloned by Pei and Blaser (1993) by using polyclonal antibody raised against purified PEBla protein in a screen of a hgtl 1 library of genomic DNA from strain 81-176. The gene was mutated, and the null mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture (Pei et al., 1998). Moreover, mouse challenge studies indicated that the rate and duration of intestinal colonization was significantly lower and shorter, respectively, compared with the wild-type strain. These data have established the PEBla protein as an important colonization and virulence factor in C. jejuni. Western blot analysis showed that PEBla is present in all C. jejuni and C. coli strains examined, but it appears to be absent from C. fetus, C. lari, and Helicobacter pylori (Pei et al., 1991). From database searching that used the deduced amino acid sequence of PEBla, Pei and Blaser (1993) showed that the protein is homologous to the periplasmic-binding protein component of amino acid ABC transporters, for example GlnH (27.8% identity) and HisJ (28.9% identity). A linked open reading frame encodes a protein with homology with the membrane proteins GlnQ and HisP, and the genome sequence of strain NCTC 11168 clearly reveals that pebla in this strain (CjO921c) is part of a four-gene operon encoding a typical ABC transporter. A potential dual role for PEBla as both a surface-exposed adhesin and a periplasmic solute binding protein can be rationalized because of the unusual existence of two predicted processing sites in the signal sequence for signal peptidase I and 11, which may be responsible for the localization of the protein both in the periplasm and on the cell surface (Pei and Blaser, 1993). PEBla is encoded by CjO921c in the C. jejuni NCTC 11168 genome (Parkhill et al., 2000). Overexpression and purification of the recombinant form of Cj092 1 followed by steady-state fluorescence titrations with all 20 amino acids (Leon-Kempis et al., 2006) showed that the protein specifically bound Laspartate and L-glutamate with low (micromolar) Kd values but had a low affinity for L-asparagine and Lglutamine. The other amino acids did not bind. Significantly, a CjO921c mutant was completely deficient in glutamate transport, showed a -20-fold reduction in aspartate transport compared with the parent strain, and was unable to grow on either aspartate or glutamate as a carbon source in minimal media (Leon-Kempis et al., 2006). It is thus clear that PEBla is the periplasmic component of an aspartate/gluta-

CHAPTER 3

mate ABC transporter, essential for optimal microaerobic growth on these dicarboxylic amino acids. The vast majority of PEBla in C. jejuni was found to be periplasmic (Leon-Kempis et al., 2006), consistent with its solute binding function, but some must be surface exposed in order for the protein to act as an adhesin. The presence of a signal peptidase I1 recognition site might suggest that the protein could be anchored in the outer membrane, but no evidence for this was found from fractionation and localization studies (Leon-Kempis et al., 2006). Rather, the presence of PEBla in acidic glycine extracts and in concentrated culture supernatant samples supports the suggestion of Pei and Blaser (1993) that the protein can be exported across the outer membrane. Although the mechanism for this is unclear, the two-component signal peptide of PEBla is extremely similar to that of endoglucanase (Egl) from Pseudomonas solanacearum (Huang and Schell, 1992; Pei and Blaser, 1993), the two-step export of which has been studied in detail. Egl is first directed across the inner membrane to the periplasm by the lipoprotein signal peptide, then is further cleaved at the signal peptidase I site during export across the outer membrane (Huang and Schell, 1992). A similar process may apply to PEBla, although in this case, most of the fully mature protein appears to remain in the periplasm. Recently, the crystal structure of PEBla at 1.5 8, resolution has been obtained (Miiller et al., 2007), allowing insights into the mechanism of solute binding. The protein has a two-domain a / p structure, characteristic of periplasmic extracytoplasmic solute receptors and a chain topology related to the type I1 subfamily. An aspartate ligand, clearly defined by electron density in the interdomain cleft, forms extensive polar interactions with the protein, the majority of which are made with the larger domain. Arg89 and Asp174 form ion-pairing interactions with the main chain a-carboxyl and a-amino groups, respectively, of the ligand, while Arg67, Thr82, Lysl9, and Tyrl56 coordinate the ligand side chain carboxyl. Lysl9 and Arg67 line a positively charged groove, which favors binding of Asp over the neutral Asn. The ligand-binding cleft is also of sufficient depth to accommodate a glutamate. This is the first structure to be determined of an ABC-type aspartate binding protein and explains the high affinity of the protein for aspartate and glutamate and its much weaker binding of asparagine and glutamine. Stopped-flow fluorescence spectroscopy indicates a simple bimolecular mechanism of ligand binding, with high association-rate constants (Miiller et al., 2007). Interestingly, sequence alignments and phylogenetic analyses revealed PEBla homologues to be

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43

present in some gram-positive bacteria. The alignments also suggested a more distant homology with GltI from Escherichia coli, a known glutamate and aspartate binding protein, but Lysl9 and Tyrl56 are not conserved in GltI. These results provide a structural basis for understanding both the solute transport and adhesin/virulence functions of PEBla.

CjaA: cysteine transport Interestingly, PEBla is not the only differentially localized solute binding protein in C. jejuni with an unusual signal peptide. Another C. jejuni immunodominant surface antigen and vaccine candidate, CjaA (Cj0982), has been shown to be a cysteine binding protein component of an ABC system, and the three-dimensional structure of this protein has been determined (Miiller et al., 2005). The signal peptide of CjaA contains a strongly predicted recognition sequence for signal peptidase I1 but a much less convincing atypical signal peptidase I site, so the sorting mechanism may not be the same as for PEBla. Indeed, preliminary data suggest that CjaA is mainly located on the periplasmic side of the inner membrane (Godlewska et al., 2005). CjaA is localized in an operon with CjaB, which encodes a predicted solute:proton symporter (Wyszynska et al., 2006), but there is no evidence that CjaB has a functional role in cysteine transport. Instead, the predicted membrane permease and ABC protein subunits that could make up a functional ABC-type cysteine transporter along with CjaA appear to be localized in an unlinked region of the chromosome, and they have been detected by bioinformatic comparisons with other bacteria (Miiller et al., 2005). Serine transport As described below, serine is a good carbon source for the growth of C. jejuni. The genome sequence of NCTC 11168 reveals a two-gene operon (Cj1624c and Cj1625c) encoding homologues of the E. coli serine dqhydratase (SdaA) and serine transporter (SdaC) for L-serine transport and catabolism. The hydropathy profile of SdaC shows that it contains 10 or 11 potential membrane-spanning segments, and its overall topology is similar to the E. coli SdaC protein and other members of the hydroxyl aromatic amino acid permease family. Biochemical evidence suggests that serine transport in C. jejuni is actually carried out by at least two systems, a lowaffinity L-serine-specific transporter encoded by sdaC, and a high-affinity transporter, which was revealed by analysis of the residual serine transport kinetics in an sdaC mutant (Velayudhan et al., 2004). The iden-

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KELLY

tity of the high-affinity transporter is unknown, but competition studies with other amino acids suggest it could be a dual serine-threonine transporter. This type of system is known in other bacteria-for example the Na+-coupled serine-threonine uptake system in E. coli (Hama et al., 1987). Organic acid transport systems There appears to be a multiplicity of transporters for organic acids encoded in the C. jejuni genome; at least nine distinct secondary systems can be deduced in strain NCTC 11168. The physiological roles for many of these are not clear, but various strains of C. jejuni have been shown to grow on pyruvate (Velayudhan and Kelly, 2002), citrate, malate, fumarate, succinate, and lactate (Hinton, 2006), all of which will require active transport across the cytoplasmic membrane. C4-dicarboxylate transport (malate, succinate, fumarate, and possibly also aspartate) seems to be particularly important in C. jejuni. These substrates can feed directly into the citric acid cycle (CAC), malate and succinate can act as direct electron donors for aerobic respiration, and fumarate is an alternative electron acceptor under oxygen-limiting conditions. There are three genes (CjOO25c, Cj1097, and Cj 1192) that encode members of the dicarboxylate/ amino acid: cation symporter family. Cj1192 is a likely DctA homologue, well known in other bacteria to transport malate/succinate/fumarate for catabolism under aerobic conditions. DcuA (Cj088) and DcuB (Cj0671) are paralogous and partially redundant C4-dicarboxylate secondary uptake systems that are known to function in fumarate:succinate antiport during anaerobic fumarate respiration in enteric bacteria like E. coli (Unden and Kleefeld, 2004). Because C. jejuni is known to carry out fumarate respiration (see below) and the active site of fumarate reduction by the fumarate reductase catalytic subunit FrdA is on the cytoplasmic side of the membrane, the Dcu proteins are essential for this process. Moreover, by acting as electroneutral antiporters, by using the outwardly directed succinate concentration gradient to power the uptake of fumarate, they provide a bioenergetically efficient solution to the need to deliver fumarate to the inside of the cell. Significantly, microarray and reverse transcriptase PCR studies have shown that both dcuA and dcuB are upregulated in expression in C. jejuni grown in the chick gut compared with laboratory media (Woodall et al., ZOOS), suggesting that fumarate respiration may be of increased importance in this oxygen-limited in vivo environment (see below). E. coli also contains another Dcu protein, DcuC. The role of this is thought to be

as a dedicated succinate efflux system during fermentative growth (Unden and Kleefeld, 2004). Interestingly, strains NCTC 11168 and RM1221 each contain a dcuC pseudogene, while in strain 81-176, there are apparently two functional full-length dcuC genes in addition to dcuA and dcuB homologues (Hofreuter et al., 2006). This suggests an increased role for dicarboxylate metabolism in 8 1-176. Whether this might relate to the increased pathogenicity of this strain is not yet clear.

CENTRAL CARBON METABOLISM IN C. JEJUNI Gluconeogenesis and Anaplerotic Reactions Genes encoding enzymes of the Embden-Meyerhof (EM) and pentose phosphate pathways are present in the genome of the currently sequenced strains of C. jejuni, but these strains lack the key enzymes of the Entner-Doudoroff pathway. Most of the reactions of the EM pathway are reversible in vivo, with the notable exceptions of the 6-phosphofructokinase and pyruvate kinase (PYK) reactions, both of which constitute key control points in many organisms. The C. jejuni strains sequenced thus far lack a gene that could encode a 6-phosphofructokinase, which would account for the well-known inability of this species to catabolize glucose (Velayudhan and Kelly, 2002; Kelly, 2005). However, a gene encoding fructose-1,6bisphosphatase is present, which suggests that the EM pathway functions solely in gluconeogenesis. Surprisingly, a homologue of PYK (Cj0392 in strain NCTC 11168) is present in the C. jejuni genome. This is unexpected because PYK catalyzes the physiologically irreversible conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP at the final stage of the glycolytic pathway (Fig. 1).PYK is thus a catabolic enzyme that should not be required if the only function of the EM pathway in C. jejuni is in gluconeogenesis. A p y k mutant displayed a slightly decreased growth rate in complex media but was able to grow with pyruvate, lactate, and malate as carbon sources in defined media (Velayudhan and Kelly, 2002). PYK is present in cell extracts at a high specific activity (>800 nmol min-' mg-' protein), is activated by fructose-l,6-bisphosphate,and displays other regulatory properties strongly indicative of a catabolic role. Therefore, PYK may function in the catabolism of unidentified substrates, which are metabolized through PEP. Thus, the lower part of the EM pathway in C. jejuni may have a catabolic role in some situations (e.g., during in vivo growth) even though the absence of 6-phosphofructokinase does not allow the full catabolism of hexose sugars. In this

CHAPTER 3

PHYSIOLOGY AND METABOLISM OF C. 7E7UNI

45

Serine I

1

+Acetate A

proline

PutA

\ glutama&e

cytoplasm

i GSSG --GG-T Glu

Proline

i

Glutamate Aspartate

Glutathione (GSSG) Figure 1. Major pathways of central carbon metabolism and amino acid utilization in C. jejuni. The cell is represented with an outer membrane (OM) and inner membrane (IM), enclosing the periplasm, and a cytoplasm within which the major pathways of carbon metabolism are shown. Key amino acid transport systems are shown as black rectangles in the inner membrane. The black circle emphasizes the transamination reaction that converts glutamate to aspartate. The major enzymes are shown next to the reactions catalyzed. SdaC, serine transporter; SdaA, serine dehydratase; Pyk, pyruvate kinase; Pyc, pyruvate carboxylase; Pck, PEP carboxykinase; Por, pyruvate:acceptor oxidoreductase; Acs, acetyl-CoA synthetase; Pta, phosphotransacetylase; AckA, acetate kinase; GltA, citrate synthase; Acn, aconitase; Icd, isocitrate dehydrogenase; Oor, 2oxog1utarate:acceptor oxidoreductase; SUC,succinyl-Cod synthetase; Sdh, succinate dehydrogenase; Fum, fumarase; Mqo, ma1ate:quinone oxidoreductase; Mdh, malate dehydrogenase (NAD linked); AspA, aspartase; Aat, aspartate:glutamate aminotransferase; GlnA, gluramine synthase; GltBD, glutamate synthase; PutA, proline dehydrogenase; PutP, proline transporter; GGT, y-glutarnyl transpeptidase. The conversion of glutathione to glutamate ocurrs in the periplasm of some strains only (dotted arrow). The Pebl system is an ABC transporter containing the periplasmic aspartateightamate binding protein Pebla. Fld, flavodoxin; Fd, ferredoxin. OAA, oxaloacetate; PEP, phosphoenol pyruvate; AcP, acetyl-phosphate.

context, it is interesting to note that although a pseudogene in NCTC 11168, strain 81-176 contains a glpT gene encoding a glycerol-3-phosphate transporter homologue (Hofreuter et al., 2006), which may allow the latter strain to grow on this substrate, which can be catabolized by the lower part of the glycolytic pathway. Net synthesis of oxaloacetate (OM) from pyruvate or PEP is required to permit the CAC to fulfill

both biosynthetic and energy-conserving roles. C. jejuni possesses homologues of anaplerotic enzymes that may function in this capacity (Fig. l),the activities of some of which have been detected by nuclear magnetic resonance (Mendz et al., 1997) and direct assay (Velayudhan and Kelly, 2002). Pyruvate carboxylase (PYC) is a key anaplerotic enzyme encoded by pycA (Cj1037c) and pycB (CjO933c) homologues, which correspond to the biotin carboxylase and bi-

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KELLY

otin carboxyl carrier subunits, respectively, of the two-subunit type of carboxylase (Goss et al., 1981). PYC fulfills an important function in many organisms by catalyzing the ATP-dependent carboxylation of pyruvate to yield OAA. Phosphoenolpyruvate carboxykinase (PCK) provides the PEP required for gluconeogenesis and catalyzes the ATP-dependent decarboxylation of OAA to PEP. C. jejuni has a pckA gene (CjO932c), and interestingly, it is contiguous with the pycB homologue. In addition to PYC and PCK, another predicted decarboxylating/carboxylating enzyme located at the metabolic node around pyruvate is malate oxidoreductase (malic enzyme [MEZ]). MEZ catalyzes the oxidative decarboxylation of malate to pyruvate coupled with NAD(P) reduction. Cj 1287c encodes a probable malate oxidoreductase homologous to that of Bacillus stearothermophilus (Parkhill et al., 2000). Insertion mutants in pycA, pycB, and mez have been generated (Velayudhan and Kelly, 2002). However, this could not be achieved for pckA, indicating that PCK could be an essential enzyme in C. jejuni. The lack of PEP synthase and pyruvate orthophosphate dikinase activities in cell-free extracts suggest a unique role for PCK in PEP synthesis. A pycA mutant was able to grow normally in complex media but was unable to grow in defined media with pyruvate or lactate as major carbon sources, thus confirming an important role for PYC in anaplerosis. In view of the high K, of MEZ for malate (-9 mM) and the lack of a growth phenotype of the rnez mutant, MEZ seems to have only a minor anaplerotic role in C. jejuni (Velayudhan and Kelly, 2002). A Complete CAC with Some Characteristics of Anaerobes The genome sequences of the strains of C. jejuni so far sequenced indicate the presence of a complete oxidative CAC (Fig. l),with all of the key enzymes present including homologues of succinate dehydrogenase or a type B fumarate reductase (which could also act in reverse as a succinate dehydrogenase), the a and /3 subunits of succinyl-CoA synthetase (SCS; sucAB genes) and an NAD-linked malate dehydrogenase and a ma1ate:quinone oxidoreductase. Importantly however, genes for the pyruvate and 2-oxoglutarate dehydrogenase complexes usually found in respiratory aerobes are absent. In fact, C. jejuni uses unrelated enzymes more commonly found in obligately anaerobic bacteria to perform the same reactions. This has consequences for understanding the bioenergetics of campylobacters and so will be discussed in some detail.

The entry of carbon from pyruvate into the cycle requires its oxidative decarboxylation to acetyl-CoA, which, in most aerobic bacteria, is carried out by the pyruvate dehydrogenase multienzyme complex, with NAD used as an electron acceptor. However, in many obligate anaerobes, a flavodoxin- or ferredoxin-dependent pyruvate:acceptor oxidoreductase (POR) catalyzes this reaction. Daucher and Kreig (1995) showed that many species and strains of Campylobacter possess this latter type of enzyme, which can be detected by using benzyl or methyl viologen as an artificial electron acceptor in cell-free extracts. The significance of this is that such acceptor: oxidoreductases are iron-sulfur-cluster-containing enzymes, which are usually sensitive to inactivation by molecular oxygen or reactive oxygen species. This has been clearly shown in the related H. pylori by the oxygen lability of the purified pyruvate:flavodoxin oxidoreductase (Hughes et al., 1995). In addition, both H. pylori and C. jejuni contain a related oxygen labile CAC enzyme, 2-oxog1utarate:acceptor oxidoreductase (OOR; Hughes et al., 1998; Kelly, 2001), which replaces the function of the 2-oxoglutarate dehydrogenase multienzyme complex found in conventional aerobes. Campylobacter jejuni is a true microaerophile that exhibits oxygen-dependent growth but is unable to grow at normal atmospheric oxygen tensions, and it has been proposed that the presence of these proteins might contribute to the microaerophilic phenotype of both Helicobacter and Campylobacter (Kelly, 1998, 2001, 2005). However, there is an interesting difference between the POR of C. jejuni and H. pylori in that the former enzyme is a large single-subunit protein containing four separate functional domains that are present as separate gene products in H. pylori. In contrast, the OORs of both bacteria are similar four-subunit enzymes. The situation in C. jejuni and the fact that there is only a rather low overall sequence similarity between the POR and OOR subunit sequences in H. pylori indicate that these two enzymes have evolved independently. An important question is how the reduced flavodoxin or ferredoxin produced in the POR/OOR reactions is reoxidized and thus how the substrate derived electrons are used by the bacteria for energy conservation. In H. pylori, it was shown that in cellfree extracts in the presence of CoA and flavodoxin, pyruvate could support the reduction of NADP, even though NADP is not a substrate of the purified POR enzyme (Hughes et al., 1998). This suggested that an additional redox protein might mediate the transfer of electrons from POR-reduced flavodoxin to NADP, which might then be used as a respiratory substrate. A likely candidate for this enzyme has recently been identified in H. pylori as HP1164, a flavodoxin:qui-

CHAPTER 3

none reductase that can catalyze flavodoxin-dependent NADP reduction in vitro and that appears to be unique to and highly conserved in many Epsilonproteobacteria, including C. jejuni (St Maurice et al., 2007). In NCTC 11168, the flavodoxin:quinone reductase homologue is Cj0559 (St Maurice et al., 2007), but it has not yet been shown whether this might mediate the same reaction. An alternative route for the electrons derived from pyruvate or 2-0x0glutarate via ferredoxin/ flavodoxin is a more direct interaction with the respiratory chain. As discussed below, an interesting possibility is via the unusual NDH-1 complex, which does not contain NAD(P)Hbinding subunits in C. jejuni and related bacteria.

AMINO ACID CATABOLISM AND NITROGEN ASSIMILATION The main carbon sources used by C. jejuni in vivo are likely to be amino acids because the bacterium is unable to metabolize exogenous sugars but does have the transport and enzymatic capacity for amino acid catabolism. There are essentially four enzymatic amino acid catabolic mechanisms that might be operative, depending on the particular chemistry of the amino acid side chain: (i) deamination (oxidative or nonoxidative), (ii) transamination via an oxo-acid acceptor like 2-oxoglutarate or OAA, (iii) p elimination of ammonia via dehydratases or amino acid:ammonia lyases, and (iv) decarboxylation. These four mechanisms must provide intermediates that can readily feed into central metabolic pathways, and some amino acids might require a combination of enzymes to satisfy this requirement. In C. jejuni NCTC 11168, on the basis of the available genome sequence and a consideration of the most likely catabolic pathways in which these enzymes participate, there are enzymes that would be predicted to allow the complete catabolism of only aspartate, asparagine, glutamate, glutamine, serine, and proline (Fig. 1). This is a narrow range of amino acids, but it accords exactly with the amino acid utilization data of Leach et al. (1997), and interestingly, aspartate, glutamate, proline, and serine are the most abundant amino acids in chicken excreta (Parsons et al., 1982). CjOO21c encodes a potential fumarylacetoacetate hydrolase that is the final enzyme in tyrosine catabolism in some bacteria, but the other enzymes of this complex pathway appear to be absent. No amino acid catabolic decarboxylases are present, but the three other principal mechanistic routes are represented (Fig. 1).Asparagine might be deaminated to aspartate by Cj0029 (a predicted asparaginase), and there are two possible aspartate/glutamate transaminases (CjOl50 and

PHYSIOLOGY AND METABOLISM OF C. TETUNI

47

Cj0762). A key aspartate catabolic enzyme is likely be encoded by Cj0087, a predicted aspartate:ammonia lyase (aspartase) that would directly form fumarate by p-elimination of ammonia. Proline could be oxidized to glutamate via a predicted bifunctional proline dehydrogenase PutA (Cj1503) with A'pyrroline-5-carboxylate as an intermediate. The electrons from this reaction may feed into the respiratory chain at the level of menaquinone (Fig. 2). Glutamate catabolism is likely to proceed via transamination to aspartate. Glutamate and glutamine metabolism is also dominated by the glutamine synthase and glutamine:2-oxoglutarate amino-transferase system, which is the major route by which nitrogen (in the form of ammonium ions) is incorporated into cellular amino acids. 2-Oxoglutarate is the primary amino group acceptor in the interconversion of glutamine and glutamate, and it is a crucial intermediate in C. jejuni amino acid metabolism. Some biochemical studies have been carried out on amino acid utilization in both batch and continuous cultures (Karmali et al., 1986; Westfall et al., 1986; Leach et al., 1997; Mendz et al., 1997). In complex media, the most heavily depleted amino acids were serine, aspartate, glutamate, and proline (Leach et al., 1997). In continuous culture, there was a strong growth rate effect on the utilization of different amino acids, with aspartate and serine being metabolized preferentially at high dilution (growth) rates, with a switch to glutamine and proline at lower dilution rates (Leach et al., 1997). L-Serine seems to be a particularly favored amino acid for catabolism. C. jejuni possesses an active serine dehydratase (deaminase) that converts serine to pyruvate and ammonia. The pyruvate can be rapidly and efficiently metabolized via POR and the CAC, which may explain why this amino acid is so well utilized (Fig. 1).Most serine dehydratases from aerobic bacteria are pyridoxal 5'-phosphate (PLP)dependent enzymes. Results (Velayudhan et al., 2004) have shown that the sdaA gene in C. jejuni encodes an L-serine dehydratase that is devoid of PLP; instead, it is an oxygen-labile iron-sulfur enzyme. The enzyme lacks the consensus sequence centered around a lysyl residue to which PLP binds via a Schiff base (Ogawa et al., 1989), in addition to lacking the glycine-rich region reported for all PLP-dependent enzymes (Marceau et al., 1988). Moreover, the PLP-reactive carbonyl reagents phenylhydrazine and hydroxylamine did not significantly affect enzyme activity. The absorption spectrum of the purified protein was, however, typical of that for an iron-sulfur protein, with broad peaks at 300 to 350 nm and at 420 nm (Velayudhan et al., 2004). Consistent with this, the C. jejuni SdaA se-

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KELLY

FldA/Fd

NO3-

NapAB

(Cj078 11782)

Cj0020 and Cj0358

NrfH --* (Cj1358)

NAD(P)H

SorAB Malate Proline

? e Gluconate

Tor/Dor Cj026

(CjO408-410)

f

Succinate 4

TMAO/DMSO

TMADMS

Figure 2. Major electron transport pathways in C. jejuni. Integral membrane oxidoreductases on the electron donor side of the menaquinone (MK) pool include an NDH-1-like complex (Cj1566-1579), the electron donor to which is unknown, hydrogenase, formate dehydrogenase, and succinate dehydrogenase. Peripherally associated oxidoreductases include (among several others) ma1ate:quinone oxidoreductase, proline dehydrogenase, and a lactate dehydrogenase. Reducing equivalents are transferred to menaquinone in the lipid bilayer of the inner membrane. Menaquinol reduces the cytochrome bc, complex, which in turn reduces periplasmic cytochrome c. Cytochrome c is reoxidized by one of the terminal oxidases, a cb-type cytochrome c oxidase. A separate quinol oxidase (CioAB) is also present. Cytochrome c may also be reoxidized by hydrogen peroxide in the periplasm through the activity of two separate CCPs. Several alternative reductases are present in C. jejuni. Fumarate reductase (FrdCAB) catalyzes electron transfer from menaquinol to fumarate as terminal acceptor. Periplasmic nitrate (Nap), nitrite (Nrf), and TMAOiDMSO reductases (ToriDor) are also present. An additional type of DMSO reductase (DmsABC) is present in strain 81-176 (not shown; see text and Fig. 3 for details). Cj0378/379 are homologues of the E. coli YedZ/YedY proteins, which are a b-type cytochrome and a molybdoprotein reductase, respectively, but the substrate reduced is unknown. Solid lines indicate experimentally established or highly likely routes of electron transport; dotted lines indicate uncertainty as to the exact route, possibly with the participation of unidentified additional redox proteins. Figure modified and updated from Kelly (2005).

quence contains four conserved cysteine residues, which could coordinate a [4Fe-4S] cluster. Moreover, both the spectral features and enzyme activity were lost on exposure to air, but activity could be restored by treatment with ferrous iron and a reducing agent (Velayudhan et al., 2004). These properties are similar to those exhibited by the L-serine dehydratases of many anaerobes, which are PLP-independent, iron-sulfur-containing enzymes (Grabowski et al., 1993). They form a family of enzymes that share mechanistic similarities with the dehydration of citrate by aconitase (Grabowski et al., 1993), but they are generally highly specific for L-serine deamination. The use of an oxygen-labile dehydratase for serine catabolism is another example of the use by C. jejuni of a type of enzyme more commonly found in anaerobic bacteria than aerobes.

Mutation of sduA resulted in a 10-fold reduction in the activity of serine dehydratase in cell-free extracts. The residual activity was found to be due to the PLP-dependent biosynthetic L-threonine dehydratase (IlvA; CjO828c), but this activity was unable to support growth of the mutant on L-serine in a minimal medium, and there was no evidence from 'H-nuclear magnetic resonance spectroscopy for significant utilization of L-serine by intact cells (Velayudhan et al., 2004). However, growth of the sduA mutant in a complex medium was indistinguishable from that of the parent strain. This indicates that an inability to deaminate serine does not result in a growth disadvantage when a larger array of carbon sources in addition to amino acids are available. Most significantly, however, it was found that there was a defect in colonization of 2-week-old chickens by the

CHAPTER 3

isogenic sdaA mutant compared with its wild-type colonization-proficient parent strain (Velayudhan et al., 2004). The data suggest that a source of serine is readily available to the bacterium and that its catabolism via SdaA is a determinant of its ability to grow in the chicken cecum. These results may also indicate a high degree of selectivity in the types of amino acid utilized by C. jejuni in this niche. Role for y-Glutamyl Transpeptidase in Amino Acid Catabolism and Colonization in C. j e j m i Although all of the key enzymes discussed above are present in each of the sequenced C. jejuni strains, there is an interesting situation regarding one enzyme, y-glutamyl transpeptidase (GGT), which is present in some strains, e.g., 81-176 (Hofreuter et al., 2006) and 81116 (Barnes et al., 2007), but not in NCTC 11168 or RM1221. Colonization studies with isogenic ggt mutants have clearly shown that this gene is required for persistent colonization of l-day-old chicks (Barnes et al., 2007) and also a mouse model of infection (Hofreuter et al., 2006). GGT catalyzes the transpeptidation of the y-glutamyl group of glutathione, and it is an essential enzyme in glutathione metabolism in most animal tissues. Its role in bacteria, in which it is also widespread, has been less clear because other enzymes of glutathione metabolism and the y-glutamyl cycle are often absent, and also because in gram-negative bacteria, GGT is a periplasmic enzyme. However, GGT also catalyzes the hydrolysis of glutamine to form glutamate and ammonia, and glutathione to form glutamate and cysteinylglycine. The hydrolysis reaction of GGT has been largely overlooked in assessing the function of the enzyme in prokaryotes, but a recent elegant study in H. pylori by Shibayama et al. (2007) has revealed that the kinetics of the hydrolysis reaction are far more favorable than the y-glutamyl transferase reaction, and that the likely role of GGT in H. pylori is to supply the bacteria with glutamate for catabolism by the hydrolysis of extracellular glutathione or glutamine. These substrates would be hydrolyzed in the periplasm and then the glutamate transported inside the cell (Shibayama et al., 2007). Given the importance of amino acid catabolism for the growth of C. jejuni, as emphasized above, and the known abundance of glutathione (and almost certainly glutamine) in avian and mammalian tissues, it is tempting to speculate that in those strains that possess a ggt gene, a similar physiological role in supplying glutamate applies (Fig. 1). Other Sources of Nitrogen Low concentrations of environmental ammonium ions and those produced as a result of amino

PHYSIOLOGY AND METABOLISM OF C. TETUNI

49

acid catabolism by deamination or @elimination reactions as described above can be assimilated via the glutamine synthase and glutamine:2-oxoglutarate amino-transferase system. Other sources of nitrogen are also likely to be available to C. jejuni in the host-for example, urea and uric acid in mammalian and avian hosts, respectively. Unlike H. pylori and some thermophilic campylobacters, C. jejuni does not possess a urease gene to allow the direct hydrolysis of urea to ammonia and carbon dioxide. An alternative route of urea catabolism is via urea carboxylase to allophanate (urea-l-carboxylate), which can then be hydrolyzed to yield ammonia (Kanamori et al., 2004). These reactions may be carried out by Cj1541 and Cj1542 in NCTC 11168, with the cognate genes also being conserved in RM1221 and 81176, although biochemical data are lacking. Degradation of the purine ring system of uric acid is more complex and requires a set of enzymes to oxidize and hydrolyze uric acid to allantoin, allantoate, and then on to urea. Clear homologues of uricase (urate oxidase) and allantoinase appear to be lacking in C. jejuni, but a possible enzyme to hydrolyze the product of the uricase reaction (5-hydroxyisourate) is present in all the genome-sequenced strains (Cj0715 in NCTC 11168). This is hydroxyisourate hydrolase, a protein containing a eukaryotic-like transthyretin domain (Lee et al., 2005).

PHYSIOLOGY OF MICROAEROBIC GROWTH Campylobacters and many related Epsilonproteobacteria are well known as microaerophiles. The term microaerophile refers to microorganisms that, although requiring oxygen for growth, are unable to grow at normal atmospheric oxygen tensions; these organisms are thought to be adapted to particular niches that contain low oxygen concentrations (Kreig and Hoffman, 1986; Kelly et al., 2001). Most strains of C. jejuni are routinely cultured in gas atmospheres containing 3 to 10% (vol/vol) oxygen and 5 to 10% (vol/vol) carbon dioxide. However, during its life cycle, C. jejuni must be exposed to highly variable oxygen concentrations, and it thus presents an interesting paradox; although it is oxygen sensitive, it nevertheless must be able to survive high environmental oxygen tensions, resist the oxidative stresses encountered in vivo, and adapt to the severe oxygen limitation of the gut. The relationship between C. jejuni and oxygen is therefore one of the major defining features of the biology of this food-borne pathogen, yet it is also one of the least studied and understood. Indeed, although many genera of freeliving and pathogenic microaerophilic bacteria are

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known, it is still the case that the molecular and physiological bases of microaerophily are not fully understood for any bacterium. A facile explanation would be a deficiency of oxidative stress defenses. All aerobic bacteria are at risk from damage by molecular oxygen, as the stepwise one-electron reduction of 0, results in the formation of the superoxide radical (02-) and hydrogen peroxide (H,O,), which can be removed by the well-characterized superoxide dismutase and catalaselperoxidase enzymes. Significantly, although some microaerophiles do lack these activities (Kreig and Hoffman, 1986) there are many species-including C. jejuni-that contain them, so additional, more complex explanations need to be invoked. As is apparent from the discussion above, C. jejuni, and indeed related microaerophiles, contain several key enzymes that are oxygen sensitive and that may be major targets for inactivation by both molecular oxygen and reactive oxygen species. This may be one reason for the oxygen sensitivity of growth of these bacteria. Sensing of oxygen is clearly important in microaerophiles both for oxygen avoidance at supraoptimal levels and for maximizing the use of alternative pathways of electron transport (and thus energy conservation) if oxygen concentrations become too limiting for aerobic growth. Mechanisms of oxygen sensing are unclear in C. jejuni; from the genome sequence, it is obvious that many of the global regulatory systems present in “model” aerobic bacteria are absent. There are few transcriptional regulators, only seven two-component systems and no RpoStype stationary-phase sigma factor. Significantly, the SoxRS and OxyR oxidative stress regulators are absent, and there are no Arc or Fnr-type systems (Parkhill et al., 2000). Nevertheless, previous microarray studies have provided evidence that C. jejuni upregulates several alternative electron transport pathways in response to oxygen limitation, both in vitro (Gaynor et al., 2004) and in the chick gut (Woodall et al., 2005). The data from the latter study suggest that there may be a coordinated response to limiting oxygen in the sense that metabolically related sets of genes show upregulation in vivo, i.e., the chick gut, compared with in vitro.

PATHWAYS OF ELECTRON TRANSPORT IN C. JEJUNI Organization and Assembly of Electron Transport Chains The complexity of electron transport systems are a key element in the ability of bacteria to grow under a variety of environmental conditions. This is because

the composition and degree of branching of the electron transport chain determines the metabolic flexibility a bacterium possesses in terms of the variety of electron donors and acceptors that can be used to support growth. The simplest types of electron transport chain consist of a primary dehydrogenase to extract electrons from a substrate electron donor, a membrane soluble quinone as a mobile carrier of the electrons (in the form of hydrogen atoms), and a terminal reductase to reduce an appropriate electron acceptor. Key properties are (i) the degree of branching at both “dehydrogenase” and “reductase” ends of the chain; (ii) the ability to use alternative electron acceptors to molecular oxygen, with suitably positive midpoint redox potentials (EM7);(iii) the presence of a variety of types of cytochromes as additional electron carriers and often more than one type of quinone; (iv) interactions between electron transport pathways, optimizing the possibility of each reductant being paired with a wide choice of oxidants; and (v) the degree to which each electron transport chain contributes to proton translocation and energy transduction. The early detailed studies of Hoffman and Goodman (1982) and Carlone and Lascelles (1982) indicated that the respiratory chain of C. jejuni was complex. This work can now be interpreted in terms of the available genome sequences of C. jejuni. Figure 2 shows a partial reconstruction of the major predicted electron transport pathways in strain NCTC 11168 as deduced from the genome sequence and biochemical evidence. A variety of primary dehydrogenases can be identified that feed electrons to a menaquinone pool. C. jejuni contains a proton-translocating cytochrome bc, complex feeding electrons to a periplasmic c-type cytochrome (probably Cjll53 in NCTC 11168) and then to a high-affinity cb-type oxidase, which allows efficient energy conservation when oxygen is used as electron acceptor. Some periplasmic dehydrogenases ( e g , for sulfite and gluconate; see below) probably feed electrons to the same periplasmic cytochrome c (Fig. 1).C. jejuni also contains a number of terminal reductases that allow respiration with a variety of alternative electron acceptors to oxygen. These are discussed in more detail below. The respiratory chain in C. jejuni is clearly highly branched and is significantly more complex than might be expected for such a small-genome pathogen. It should be noted, however, that this complexity is currently underestimated because although a large majority of the components of the respiratory chains can be fitted into diagrams such the one in Fig. 2, there are still a number of uncharacterized cytochromes and other redox proteins. For example, the

CHAPTER 3

molybdoenzyme encoded by Cj0379 is a homologue of the E. coli YedY protein, the three-dimensional structure of which is known (Loschi et al., 2004), but the physiological substrate has not been identified (Fig. 1).Strain 81-176 contains an additional c-type cytochrome and an associated biogenesis system not present in the other strains (Hofreuter et al., 2006). All the key electron transport enzymes contain redox components like heme, iron-sulfur clusters, or molybdenum, nickel, copper, or other cofactors, which have their own pathways of biosynthesis and incorporation into the relevant apoproteins. The biogenesis of the electron transport chains of C. jejuni is thus critically dependent on metal-ion transport and complex cofactor biosynthesis enzymes. In addition, many of the proteins involved in the various pathways of respiration described below must be exported to the periplasm for their function. In the case of the c-type cytochromes, this is achieved via the Sec translocation system, with covalent heme attachment to the apoprotein occurring posttranslationally in the periplasm. In the case of complex cofactor containing proteins like molybdoenzymes (see below), cofactor attachment occurs in the cytoplasm and the mature proteins are translocated across the cytoplasmic membrane via the “twin arginine” translocation (TAT) system. TAT signal peptides can be easily recognized in the N-terminal regions of such proteins in C. jejuni, which also contains homologues of the tatAIE (Cj1176c), tatB (CjOS79c), and tatC (CjOS78c) translocase genes. Electron Donors Hydrogen and formate may be key energy sources in vivo C. jejuni contains a set of structural genes for a NiFe-type “uptake” hydrogenase, as well as accessory genes for nickel incorporation, which could mediate the transfer of electrons from gaseous hydrogen to the quinone pool (Fig. 2). C. jejuni has been shown to possess hydrogenase activity in membrane fractions (Carlone and Lascelles, 1982; Hoffman and Goodman, 1982). Hydrogen could be an important source of electrons for the growth of campylobacters in the gut because many obligate anaerobes in the intestine produce hydrogen from redox reactions associated with fermentation. It would be of great interest to know whether hydrogenase activity is essential for or contributes to the colonization of the avian gut by C. jejuni, as studies with H. pylori have shown that hydrogenase mutants do not colonize a mouse model of infection, and microelectrode measurements have shown that hydrogen is present in the

*

PHYSIOLOGY AND METABOLISM OF C. TETUNI

51

mouse stomach at concentrations much greater than the K , value of the hydrogenase enzyme (Olson and Maier, 2002). In addition to hydrogen, formate is also a good electron donor because the formate/bicarbonate couple has a highly negative redox potential (EM, -420 mV). Formate is also produced by anaerobes in the gut from mixed-acid-type fermentation reactions, so it is available to an organism like C. jejuni, which has the enzymatic capacity for formate oxidation. Respiratory activities in membrane vesicles were SO to100 times greater with formate or hydrogen as substrates when compared with the rates achieved with other electron donors such as succinate, lactate, malate, or NADH, suggesting that the former are indeed excellent electron donors (Hoffman and Goodman, 1982) and indicating the presence of an active formate dehydrogenase. The genome sequence of C. jejuni 11168 reveals an operon encoding putative formate dehydrogenase subunits IfdhA-D/CjlSllc-CjlS08c).C. jejuni fdhA (Cjl51lc) encodes a large 104-kDa selenocysteine containing molybdoprotein equivalent to the E. coli 110 kDa FdnG (a)subunit. fdhB (Cj1510c) encodes a 24 kDa iron-sulfur subunit equivalent to the E. coli 32-kDa FdnH (p) subunit. fdhC (CjlS09c) encodes a 3.5-kDa cytochrome b subunit equivalent to the E. coli 20-kDa FdnI ( y ) subunit. In addition to these three subunits, both E. coli and C. jejuni encode an FdhD protein (29 kDa) required for activity of the formate dehydrogenase enzyme complex (Berg et al., 1991). Molecular and functional studies are lacking on the C. jejuni enzyme. In particular, it would be of great interest to know whether fdh mutants are deficient in colonization of chickens or other animal models because this would provide evidence for the utilization and importance of formate as an in vivo electron donor. The puzzle of NAD(P)H oxidation and the function of complex I In most organisms, NADH is the major electron donor for oxidative phosphorylation, via interaction with a proton-translocating quinone oxidoreductase (often referred to as complex I, Nuo, NQO, or NDH-l), whereas the usual role of NADPH is as a source of electrons for cytoplasmic biosynthetic reactions. NDH-1 is a complex enzyme with a multitude of redox centers and is most commonly made up of 14 different subunits in bacteria. Electrons are passed from NADH via one FMN and nine Fe centers in NDH-1, to ubiquinone/menaquinone in the respiratory chain. Four protons are translocated

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across the membrane for every two electrons transferred. The genome sequences of C. jejuni show that the bacterium contains a cluster of genes (Cj1566 to Cj1579 in NCTC 11168) encoding a potential NADH:quinone oxidoreductase of the NDH-1 type (Fig. 2). However, examination of the deduced proteins encoded by this gene cluster suggests that the complex may not actually oxidize NADH because of the lack of the N Q O l (NuoE) and N Q 0 2 (NuoF) subunits (Smith et al., 2000; Kelly, 2005). These subunits are thought to be essential components for the function of the NDH-1 complex. NuoF binds NADH and also possesses a bound FMN and an Fe-S center. NuoE, G, By and I have cysteine residues that coordinate Fe-S clusters. In place of the expected nuoE and nuoF genes, C. jejuni (and also H. pylori and related Epsilonproteobacteria) has open reading frames of unknown function. Significantly, these open reading frames encode unique small proteins that do not contain any obvious NAD(P)H-binding motif. There are two possibilities regarding the function of this complex in C. jejuni. The first is that electrons from NAD(P)H are transferred to it via an intermediate protein that interacts with the N Q O l and N Q 0 2 replacements (Finely 1998; Smith et al., 2000). Alternatively, coupling of NAD(P)H with the respiratory chain may not occur via the NDH-1 homologue at all, but through an alternative quinone reductase, which may or may not be proton translocating. This implies that an as yet unidentified electron donor interacts with the Cj1566-1579 complex, and that its function is different from most bacterial NDH-1 systems. The nature of the electron donor and whether this type of enzyme is proton translocating are important unresolved questions for understanding the bioenergetics of C. jejuni. One possibility, discussed above, is that flavodoxin/ ferredoxin reduced by the POR/OOR complexes in the CAC donates electrons directly to the NDH-1-like enzyme. Organic acids as electron donors Several types of organic acids (in addition to formate) appear to be able to act as direct electron donors to the respiratory chain in c. jejuni (Fig. 2). Hoffman and Goodman (1982) demonstrated the activity of a lactate dehydrogenase in oxygen-linked respiration, although analysis of the NCTC 11168 genome sequence suggests no obvious locus for a membrane associated lactate dehydrogenase enzyme in C. jejuni. An annotated L-lactate dehydrogenase (Cj1167) is probably a fermentative enzyme. Malate can act as a direct electron donor to the quinone pool

due to the presence of a flavoprotein-type malate oxidoreductase (Mqo). Analysis of the C. jejuni genome reveals a putative oxidoreductase (Cj0393) with sequence similarity to the E. coli ma1ate:quinone oxidoreductase and to the Mqo of H. pylori, indicating that these bacteria use a similar enzyme for malate oxidation (Kather et al., 2000). This is also consistent with the data of Hoffman and Goodman (1982), which showed that malate stimulated respiration in C. jejuni, producing a H'/O ratio of 2.0. Although Mqo and thus malate oxidation is predicted to be localized on the cytoplasmic side of the inner membrane, an interesting additional potential periplasmic electron donor in each of the sequenced C. jejuni strains is gluconate (Fig. 2), which is based on the prediction of a flavin-containing gluconate dehydrogenase encoded by the Cj0414 and Cj0415 genes in strain NCTC 11168, the former with a TAT signal sequence. It is likely that gluconate-derived electrons will enter the chain at the level of the periplasmic cytochrome c (Fig. 2). In enteric bacteria, gluconate has been shown to be a significant in vivo growth substrate (Chang et al., 2004) and is an abundant component of intestinal mucus. It is highly likely that this substrate will be encountered by C. jejuni in vivo, but it is unlikely to be used as a growth substrate and carbon source because C. jejuni lacks the Entner-Doudoroff pathway for its intracellular catabolism. The interconversion of fumarate and succinate can be carried out by two related enzymes: by succinate dehydrogenase, which is expressed under aerobic conditions, or by fumarate reductase, which is induced under anaerobiosis. Succinate dehydrogenase (succinate:quinone oxidoreductase) catalyzes electron transfer from succinate to quinone, where succinate is oxidized to fumarate as part of the tricarboxylic acid cycle. As noted above, C. jejuni appears to have a complete CAC with genes encoding clearly identifiable homologues of all of the conventional enzymes (Parkhill et al., 2000), including separate sdh and frd operons. Sulfite: an unexpected electron donor in C . jejuni The ability to use sulfite as a respiratory electron donor is usually associated with free-living chemolithotrophic sulfur-oxidizing bacteria. However, it has been shown that C. jejuni has the ability to respire sulfite, with oxygen as the electron acceptor (Myers and Kelly, 2005). The C. jejuni NCTC 11168 CjOOO4c and CjOOOSc genes encode a monoheme cytochrome c and molybdopterin oxidoreductase, respectively, homologous to the su1fite:cytochrome c

CHAPTER 3

oxidoreductase of Starkeya novella (Fig. 2). Western blot tests of C. jejuni periplasm probed with a SorA antibody demonstrated cross-reaction of a 45-kDa band, consistent with the size of Cj0005, and a CjOOO4c mutant was unable to respire with sulfite or metabisulfite as electron donors but showed wildtype rates of formate dependent respiration (Myers and Kelly, 2005). Evidence from inhibitor studies suggested that electrons from sulfite enter the respiratory chain after the bc, complex at the level of cytochrome c (Fig. 2), and a fractionation study confirmed that the majority of the su1fite:cytochrome c oxidoreductase activity is located in the periplasm (Myers and Kelly, 2005). Sulfite is an extremely widespread inorganic anion in many environments, particularly low-oxygen niches in soil or water, where it is more stable than in aerobic conditions. The possession of a sulfite respiration system may thus contribute to the survival of C. jejuni in such environments, particularly because this bacterium has a high-affinity cb-type cytochrome c oxidase, which could allow sulfite respiration at extremely low oxygen concentrations. Sulfite respiration may also provide a detoxification mechanism for this normally growth-inhibiting compound. The presence of a sulfite respiration system in C. jejuni is another example of the surprising diversity of the electron transport chain in this small genome pathogen. Oxygen-Dependent Respiration in C. jejuni Most bacteria possess at least two terminal oxidases, often a quinol oxidase and a cytochrome c oxidase (Poole and Cook, 2000), with distinct catalytic properties and regulation of expression. Whereas only one terminal oxidase, a cb-type cytochrome c oxidase, has been found to terminate the respiratory chain of Helicobacter pylori (Nagata et al., 1996), all sequenced strains of Campylobacter jejuni have two terminal oxidases (Parkhill et al., 2000; Fouts et al., 2005; Hofreuter et al., 2006). This confirms the earlier spectroscopic work of Carlone and Lascelles (1982) and Hoffman and Goodman (1982). One oxidase is a cb-type cytochrome c oxidase, similar to that described in H. pylori, and the other resembles a cytochrome bd-type quinol oxidase. Analysis of the NCTC 11168 genome sequence, for example, reveals the presence of a cydAB-like operon encoding subunits I and I1 of such a quinol oxidase. However, in a recent detailed study of the terminal oxidases of NCTC 11168 (Jackson et al., 2007), it is clear from the lack of optical spectral signals associated with the high-spin hemes b and d that this

. PHYSIOLOGY AND METABOLISMOF c. mmr

53

oxidase is not of the conventional cytochrome bd type. Mutation of the cydAB operon was without significant effect on growth, but it did reduce formatesupported respiratory activity. However, the cydABencoded oxidase enhanced survival of cells cultured in a gaseous environment containing 5% (vol/vol) oxygen and was relatively cyanide resistant and has now been renamed CioAB (cyanide-insensitive oxidase). The oxygen affinity of each oxidaie was determined by means of a highly sensitive assay that exploits globin deoxygenation during respirationcatalyzed oxygen uptake (Jackson et al., 2007). The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K, = 0.8 pM) and a V,, of >20 nmol/ mg/s. The alternative ccoNOQP-encoded cyanidesensitive oxidase, which encodes a cytochrome cbtype enzyme, plays a major role in the aerobic respiratory metabolism of C. jejuni because it appeared to be essential for viability, as judged by the failure of repeated attempts to construct a knockout mutation. The cytochrome cb-oxidase exhibited a high oxygen affinity, with a K,,, value of 40 nM and a V, of 6 to 9 nmol/mg/s. Therefore, C. jejuni has two oxidases with different structural and kinetic properties. The marked differences in affinity for oxygen probably reflect distinct physiological roles in response to, and survival under, various degrees of oxygen provision. In particular, the possession of a cb-type oxidase is likely to endow the bacterium with the ability to continue respiration at extremely low oxygen concentrations, as might be found in the niches that it occupies in the gut of animals or humans. Hydrogen Peroxide as an Electron Acceptor Reduction of oxygen to water is a four-electron reaction, but toxic oxygen intermediates formed by incomplete reduction include the superoxide anion (O,-), hydrogen peroxide (H,O,), and the hydroxyl radical (HO'). Hydrogen peroxide can be degraded to H,O and 0, by the cytoplasmic enzyme catalase, but in the periplasm, H,O, can be broken down to water alone by a periplasmic cytochrome c peroxidase (CCP), with the requirement of reduced cytochrome c as an electron donor (Atack and Kelly, 2007). Proton translocation and energy conservation may thus accompany electron transport from a primary electron donor to hydrogen peroxide via the electron transport chain. Although the periplasmic location of the peroxidase means that the enzyme itself is not energy conserving, the reduction of hydrogen peroxide could allow proton translocation to occur at the level of the primary dehydrogenases and also,

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as in C. jejuni, through the operation of the cytochrome bc, complex. The importance of the energyconserving (as opposed to the peroxide detoxification) function of CCPs is difficult to define, but Goodhew et al. (1988), working with the catalasenegative microaerophile Campylobacter mucosalis, were able to show that addition of hydrogen peroxide resulted in uncoupler-sensitive proton extrusion in classical proton-pulse type experiments, albeit with a low stoichiometry (about 0.6 H+/H202).It was suggested that this may be important in the bioenergetics of the bacterium during growth on formate where periplasmic hydrogen peroxide may be generated as a side reaction of the formate dehydrogenase. The true physiological role of bacterial CCPs is actually not well understood (Atack and Kelly, 2007). It is well known that exposure of bacterial cells to excess oxygen increases the rate of H,O, production (Seaver and Imlay, 2004). Paradoxically, however, bacterial ccp genes appear to be upregulated preferentially under low oxygen (microaerobic) or even anaerobic conditions. This may be relevant in pathogenic bacterialike C. jejuni because a drop in oxygen levels may be a signal of entry into the host and would allow the deployment of a periplasmic first line of defense against the attack of hydrogen peroxide-producing phagocytes, for example. In prokaryotes, CCPs are usually diheme proteins, containing two hemes C, one high potential and one low potential, although triheme CCPs have recently been recognized (Atack and Kelly, 2007). The high-potential heme is the source of the second electron for H,O, reduction, and the low-potential heme acts as a peroxidatic center. C. jejuni strains NCTC 11168, 81-176, and RM1221 each have two separate genes encoding putative CCP homologues. In NCTC strain 11168, Cj0020 and Cj03.58 are 34 kDa and 37 kDa, respectively, with both proteins containing two cytochrome c heme-binding site signatures. Cj0358 appears to be a typical CCP, which clusters phylogenetically with other similar enzymes from the bacteria in the epsilon group of the proteobacteria (e.g., H. pylori). These enzymes are all related to the “classical” Pseudomonas aeruginosa type of CCP, which has been studied in detail mechanistically (Atack and Kelly, 2007). However, Cj0020 appears not to be a closely related paralogue of Cj0358, and it is not contained within any of the four major groups that can be identified by phylogenetic analysis (Atack and Kelly, 2007). It does appear to be distantly related to the triheme enzymes from Zymononas mobilis and Gluconobacter oxydans, even though Cj0020 contains only two heme-binding mo-

tifs. Mutants in the CjOO2Oc gene show increased M. sensitivity to killing by hydrogen peroxide Atack and D. J. Kelly, unpublished data), and mutants in the 81-176 homologue are unable to effectively colonize chicks (Hendrixson and DiRita, 2004). It is not yet known whether this putative CCP also plays a role in virulence. The second CCP in C. jejuni, Cj0358, is also needed for resistance to hydrogen peroxide (J. M. Atack and D. J. Kelly, unpublished data), and its expression is increased significantly in the chick cecum (Woodall et al., 200.5), which is thought to be a low-oxygen niche. Cj0358 is also negatively regulated under iron-limited conditions by the iron response regulator Fur, by its homologue PerR (the peroxide response regulator [Holmes et al., 200.5]), and by the temperature-responsive regulator, RacR (Bras et al., 1999). Taken together, these studies suggest a complex regulatory pattern, which may well be related to colonization and possibly pathogenicity.

u.

Growth of C. jejuni under Oxygen Limitation: Respiration by Alternative Electron Acceptors In addition to oxygen-dependent electron transport, it is also clear that the genome of C. jejuni encodes a number of reductases that would be predicted to allow the bacterium to carry out respiration with several alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, trimethy1amine-Noxide (TMAO), and dimethyl sulfoxide (DMSO) (Kelly, 2001; Sellars et al., 2002; Myers and Kelly, 2005; Pittman et al., 2007). Nevertheless, Sellars et al. (2002) found that although growth with these alternative electron acceptors (see below) was insignificant under strictly anaerobic conditions, such growth was possible under severely oxygen-limited conditions. Their results indicated that some oxygen requiring metabolic reaction or reactions prevented anaerobic growth. Because C. jejuni only contains genes for an oxygen-requiring class I type of ribonucleotide reductase (RNR), it was suggested that the inability to synthesize DNA anaerobically is the most likely explanation. Consistent with this, cells incubated anaerobically with electron acceptors did not divide properly but formed filaments analogous to those seen after treatment of aerobic cells with the RNR inhibitor hydroxyurea (Sellars et al., 2002). Thus, C. jejuni can use alternative electron acceptors in energy-conserving reactions, but only if some oxygen is present to satisfy the requirement for deoxyribonucleotide production. This conclusion is directly relevant for understanding growth of C. jejuni in the gut because it implies that in the niches occupied by

CHAPTER 3

the bacterium, small amounts of oxygen must be present for continued viability. Whether in these severely oxygen-limited niches alternative electron acceptors are being used in addition to or even instead of molecular oxygen will require studies with the relevant mutants in animal models. Fumarate as electron acceptor In the absence of oxygen, fumarate (EM7+30 mV) can be used as an alternative terminal electron acceptor for an electron transport chain by using menaquinol (EM7-74 mV), and fumarate respiration is important in energy conservation in many anaerobic bacteria. C. jejuni strains have an frdCAB operon, encoding subunits highly similar to the thoroughly investigated fumarate reductase of Wolinella succinogenes (Lancaster, 2002). This enzyme is a type B quino1:fumarate reductase, which is reversible and has two b-type hemes bound to the quinol-oxidizing FrdC subunit, which spans the membrane. FrdB is an iron-sulfur cluster containing electron transfer protein, which passes electrons on to the peripheral FrdA subunit, which contains a flavin adenine dinucleotide (FAD) cofactor, the site of fumarate reduction. Because the site of fumarate reduction (which consumes 2H+ per mole of fumarate reduced to succinate) is on the cytoplasmic side of the membrane, and quinol oxidation releases protons into the periplasm, this type of fumarate reductase should be an electrogenic (and energy conserving) enzyme. However, experimental evidence from W. succinogenes has shown that the overall reaction is electroneutral (Kroger et al., 2002). To explain this, the E-pathway hypothesis has been advanced (Lancaster, 2002), which proposes a counterbalancing proton-translocating pathway in the enzyme from periplasm to cytoplasm via a conserved glutamate residue that is coupled to electron transfer between the two b hemes. The operation of this energy-dissipating pathway is essential for the function of the enzyme, even though the overall redox reaction of fumarate reduction by menaquinol is thermodynamically favorable. It is highly likely that similar considerations apply to the homologous C. jejuni Frd complex and that, like the remaining periplasmic reductases described below, net energy conservation occurs only at the level of the primary dehydrogenases. Nitrate and nitrite as electron acceptors

C. jejuni NCTC 11168 has both nitrate (Nap) and nitrite (Nrf) reductases located in the periplasm (Sellars et al., 2002; Pittman et al., 2007). The Nap type of nitrate reductase is a two-subunit enzyme consisting of NapA, a -90-kDa catalytic subunit that

PHYSIOLOGY AND METABOLISM OF C. 7E7UNI

55

binds a bis-molybdenum guanosine dinucleoside cofactor and a [4Fe-4S] cluster, and NapB, a -16-kDa diheme c-type cytochrome. NapA is a substrate for the TAT system and requires an additional protein, NapD, as a “proofreading” chaperone in the export process (Palmer et al., 2005). In C. jejuni, the electron transport pathway to nitrate via NapAB is not immediately obvious because nitrate reduction in other Nap systems is usually coupled to quinol oxidation by a membrane-anchored tetraheme cytochrome, NapC. However, a napAGHBLD operon is present in C. jejuni, which does not contain napC. Analysis of the NCTC strain 11168 genome shows that it does encode a napC homologue (Cj1358c), which is directly upstream of the nrfA nitrite reductase gene (Cj13.57~)~ implying that it is part of the nitrite reductase system. This is the case in Wolinella succinogenes, which encodes a NapC-like subunit (NrfH) similar to the product of Cj1358c (Simon et al., 2000). How, then, is the NapAB complex coupled to menaquinol oxidation in C. jejuni? Pittman et al. (2007) showed that the iron-sulfur protein NapG has a major role in electron transfer to the NapAB complex, and an additional protein, NapH, probably acts together with NapG as a quinol dehydrogenase, as in E. coli (Brondijk et al., 2004). It was found, however, that a napG mutant still showed slow nitratedependent growth under oxygen-limited conditions, which was abolished in a nrfH/napG double mutant, suggesting that a low rate of electron transfer from NrfH to NapAB can occur (Pittman et al., 2007). Interestingly, NapG/H are specifically involved in electron transfer from ubiquinol to nitrate in the E. coli Nap system (Brondijk et al., 2002) and are not required for menaquinol-dependent nitrate reduction (Brondijk et al., 2002), indicating a fundamentally different quinol specificity in C. jejuni. The epsilon group of proteobacteria (including W. succinogenes and C. jejuni) are unusual in containing an additional gene of unknown function, designated napL, within their nap operons (Simon et al., 2003). NapL is predicted to be a soluble periplasmic protein with no obvious cofactor binding motifs. A napL mutant possessed -50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor (Pittman et al., 2007). It may possibly be involved in the assembly or maturation of NapA. There is evidence that nitrate respiration may play a significant role in the growth of human and animal pathogens in vivo. In Mycobacterium bovis, membrane-bound nitrate reductase (Nar) activity has been shown to contribute to virulence (Weber et al., 2000). As yet, it is not known if the C. jejuni Nap has any in vivo role; it may also have other functions,

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such as in survival in foods or the environment where nitrate is present, or in nitrogen assimilation in combination with nitrite reductase, which produces ammonia. It should be noted that because of the periplasmic location of the Nap and Nrf enzymes, a proton-motive force can only be generated at the level of the primary dehydrogenases and not from quinol oxidation during electron transfer. A role for the nitrite reductase NrfA in nitric oxide detoxification Cj1357c in strain NCTC 111168 encodes the periplasmic pentaheme cytochrome c nitrite reductase (NrfA), which is the terminal enzyme in the sixelectron dissimilatory reduction of nitrite to ammonia (Simon, 2002; Pittman et al., 2007). High specific activities of nitrite reduction can be detected in intact cells of C. jejuni by using methyl viologen as the electron donor (Sellars et al., 2002); a nrfA mutant lacks this activity and shows no oxygen-limited growth with nitrite (Pittman et al., 2007). NrfA is a wellstudied enzyme, which in most bacteria contains an unusual CXXCK motif at the heme 1 binding site, instead of the CXXCH motif found in the vast majority of heme c-containing enzymes. This requires a dedicated lyase for covalent attachment of the active site heme (Pisa et al., 2002). The C. jejuni NrfA contains a novel ligation site at heme 1, with a “conventional” CXXCH motif instead of CXXCK. This would explain the absence of genes encoding Nrfspecific heme lyases that are found in other bacteria (Einsle et al., 2000), but a site-directed His-for-Lys substitution in the W. succinogenes NrfA results in low catalytic activity (Pisa et al., 2002), so how the enzyme achieves high activity in C. jejuni is unknown. Interestingly, the predicted NrfAs of the closely related C. coli, C. lari, and C. upsuliensis (Fouts et al., 2005) also contain the heme 1 CXXCH motif rather than CXXCK. Cytochrome c nitrite reductases can also carry out the five electron reduction of the nitric oxide radical ( N O ) to NH4+ (Costa et al., 1990). Work in E. coli has shown that this reaction may be physiologically relevant under microaerobic conditions, and it has been proposed that NrfA plays a significant role in nitric oxide detoxification in addition to flavohemoglobin (Hmp) and flavorubredoxin (NorV), the other major E. coli N O detoxification systems (Poock et al., 2002). In C. jejuni, Hmp and NorV are absent, but there is a single domain globin (Cgb), which has been shown to protect against nitrosative stress (Elvers et al., 2004). In keeping with this, Cgb is inducible by nitrosative stress via the regulatory protein NssR (Elvers et al., 2005). nrfA mutants were found

to be hypersensitive to various forms of nitrosative stress, suggesting that NrfA does indeed play a role in nitric oxide metabolism (Pittman et al., 2007). However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite itself causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of a nrfA mutant, in media containing nitrite (Pittman et al., 2007). Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defense against nitrosative stress.

S- or N-oxides as electron acceptors TMAO and DMSO are structurally similar compounds that are widely distributed in many environments, particularly water; TMAO is an excretory product of some fish, and DMSO is produced by some algae (McCrindle et al., 2005). It is therefore likely that campylobacters will come into contact with these electron acceptors in aquatic environments, soil, etc. In fact, a wide range of bacteria possess enzyme systems that catalyze the reduction of TMAO and DMSO (often by the same enzyme), which is a two electron transfer process, yielding trimethylamine or dimethyl sulfide, respectively: (CH,),SO

+ 2H+ + 2e-

-+ -+

(CH,),S (CH,),NO

+ 2H+ + 2e-

(CH,),N

H,O EM, = +160 mV

H,O EM, = +130 mV

As can be seen, the midpoint redox potentials of the DMSO/ dimethyl sulfide and TMAO/ trimethylamine couples are not highly positive, making them thermodynamically poorer electron acceptors compared with, say, oxygen or nitrate, but better than fumarate. Both TMAO and DMSO reductase activities have been demonstrated in intact cells of C. jejuni NCTC 11168 by using methyl viologen as an artificial electron donor, and TMAO- and DMSOdependent growth under oxygen-limited conditions has also been demonstrated (Sellars et al., 2002). Analysis of the C. jejuni NCTC 11168 genome reveals a gene encoding a 93-kDa molybdoprotein TMAO/DMSO reductase homologue (Cj0264) containing conserved residues for the binding of a molybdenum cofactor and an N-terminal “twin arginine” translocase recognition motif for transport to the periplasm. The CjO264c gene was mutated, and the mutant was found to be deficient in both TMAO and DMSO reductase activity, indicating that a single enzyme is responsible for both activities (Sellars et al.,

PHYSIOLOGY AND METABOLISM OF C. TETUNl

CHAPTER 3

2002). In addition, there is an upstream gene encoding a 22-kDa monoheme c type cytochrome (CjO265c) with similarity to the C terminus of the membrane-anchored pentaheme c type cytochrome TorC of E. coli, which transfers electrons to the catalytic TMAO reductase subunit, TorA (Gon et al., 2001; Sellars et al., 2002). An interesting unresolved issue here is how electrons get from the quinol pool to the CjO265/CjO264 complex (Fig. 3 ) . In other bacteria, a family of tetraheme cytochromes (the NapC family; see above) are often involved in connecting terminal reductases in the periplasm with the quinol pool. The additional four hemes of the E. coli TorC protein have this role, but the only NapC homologue in C. jejuni is NrfH (described above), the electron donor to the nitrite reductase. Growth studies with a nrfH mutant clearly showed that it was still capable of oxygen-limited growth with both TMAO and DMSO (Pittman et al., 2007). Because Cj0265 alone is unlikely to act as a quinol oxidase, there appears to be a novel route of electron transfer to these electron acceptors in strain NCTC 11168, which merits further investigation. Of the other genome-sequenced strains of C. jejuni, RM1221 and 81-176 contain a Cj0264 homologue and are thus likely to have a similar type

TMAOiDMSO + 2H+

HCOO - + H.0

He',-+

TMA/DMS

of TMAO/DMSO reductase to that found in NCTC 11168. Given the presence of TMAO and DMSO in water and soil, the physiological role of this enzyme might be assumed to aid survival outside the host. However, strain 81-176 contains, in addition to a Cj0264 homologue, a structurally distinct type of DMSO reductase, which has proven to be interesting with regard to a potential role in vivo (Hofreuter et al., 2006). This system is similar to the DmsABC DMSO reductase complex in E. coli (McCrindle et al., 2005) consisting of a large extrinsic membrane bound molybdoprotein (DmsA), a smaller electron transferring iron-sulfur subunit (DmsB), and an intrinsic membrane anchor and quinol binding subunit (DmsC). Electrons would be transferred from the quinol pool via DmsC then DmsB to DmsA in the periplasm, where the reduction of DMSO would take place (Fig. 3 ) . Like the periplasmic Cj0264/ Cj0265 system, the arrangement of this enzyme across the membrane means that it is not energy conserving, but electron flow through either system can allow energy to be conserved at the level of the primary dehydrogenases (Fig. 3 ) , as with the Nap and Nrf systems described above. Significantly, Hofreuter et al. (2006) showed that a dmsA mutant was less able to colonize a mouse

+ 2H20

TMAODMSO

TMA/DMS

2H+

'&

Cj026

membrane I

2H+

57

cytoplasm

I

2H+

Figure 3. Likely topological organization of two types of TMAO/DMSO reductases found in strains of C. jejuni. In each case, formate is depicted as a typical electron donor to the menaquinone pool (MK), through the action of formate dehydrogenase (Fdh). (a) The Tor/Dor type of reductase system that has been characterized in strain 11168 (Sellars et al., 2002) is illustrated, which is also present in strains RM1221 and 81-176. There is uncertainty over the mechanism of electron transfer from the MK to the monoheme Cj0265 cytochrome (dashed rectangular hypothetical quinol oxidoreductase). In (b), the additional DmsABC-type system shown in strain 81-176 to be needed for optimal colonization of a mouse infection model (Hofreuter et al., 2006) is illustrated. Note that because both of these TMAO/DMSO reductases are predicted to be periplasmic, reduction of the electron acceptor (which consumes protons-in bold) and quinol oxidation (which releases protons-in bold) occur on the same (periplasmic) side of the membrane. Thus, a proton-motive force is only generated at the level of the primary dehydrogenase (binding and releasing protons on opposite sides of the membrane). MGD, molybdenum guanosine dinucleoside cofactor.

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model of infection than the isogenic wild-type parent strain, implying that this enzyme has an important role in the colonization process. It seems unlikely that DMSO is the substrate of this enzyme in an animal host, and it is possible that in vivo, it is reducing a different S oxide-for example, methionine sulfoxide-because it is known that this class of enzyme often has a broad substrate specificity. It is intriguing that this type of DmsABC system has also been shown to be important for virulence in another bacterial pathogen, Actinobacillus pleuropneumoniae (Baltes et al., 2003). The fact that some strains of C. jejuni, a small-genome pathogen, can possess two distinct functional electron transport chains to TMAO/ DMSO underlines its surprising metabolic versatility and indicates a possible role for these electron acceptors in aiding survival outside and inside the host.

Leon-Kempis, John Atack, Marie Thomas, Jon Smart, Chat Phansopa, and Andy Hitchcock.

ADDENDUM IN PROOF Since this chapter was written, several papers have been published which clarify some aspects of the topics covered, particularly growth in vivo. D. Weerakoon and J. Olson (1.Bacteriol, 190: 915-925, 2008) have shown experimentally that reduced flavodoxin is indeed the electron donor to the unusual complex I in C. jejuni. Weingarten et al. (Appl. Environ. Microbiol., in press, 2008) have shown that mutants in napA and nrfA colonize chicks less well than the wild-type parent strain, while cydA and CjO264c mutants show wild-type levels of colonization. This indicates a possible role for nitrateinitrite reduction in growth of C. jejuni in chickens. A ccoN::cm (cb-oxidase) mutant showed no colonization of chickens, although this strain was very oxygen sensitive and had a significant microaerobic growth defect. Bingham-Ramos and Hendrixson (Infect. Immun., in press, 2008) have characterized the cytochrome c peroxidases in strain 81-176 and have shown a potential role in vivo for the Cj0020 homologue but not for the Cj0358 homologue.

CONCLUSIONS REFERENCES

Understanding the metabolism and physiology of C. jejuni and related bacteria has been greatly facilitated by the availability in recent years of several genome sequences and by better experimental techniques for their culture and biochemical analysis. One conclusion that can be drawn is that many campylobacters show an unexpected metabolic versatility, which is particularly reflected in the complexity of the electron transport chains in C. jejuni, as reviewed here. Despite a genome size of only 1.7 Mb, it is clear that this bacterium has retained complex enzymes with a variety of redox cofactors that enable it to be flexible with respect to the variety of electron donors and acceptors it can use. This is surely a function of its ability to occupy so many niches. Published and ongoing colonization studies with metabolic mutants are crucial and are beginning to answer questions about the nature of C. jejuni metabolism during growth in vivo. This information could be useful in identifying a means to control the growth of C. jejuni in animal reservoirs, for example. It is now also becoming increasingly clear that the application of both conventional biochemical techniques and postgenomic technologies like metabolomics and systems biology will be required to fully elucidate the metabolism of this important pathogen. Acknowledgments. Work in my laboratory on Campylobacter jejuni has been supported by grants and studentships from the United Kingdom Biotechnology and Biological Sciences Research Council and by Don Whitley Scientific Ltd., Shipley, West Yorkshire, United Kingdom. I gratefully acknowledge the hard work of past and current students and postdocs on Campylobacter in my laboratory: Jyoti Velayudhan, Mike Sellars, Mark Gidley, Stephen Hall, Marc Pittman, Jon Myers, Ed Guccione, Maria del Rocio

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Cumpylobucter, 3rd ed. Edited by I. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 4

Comparative Genomics of Campylobacter jejuni OLIVIA L. CHAMPION, SUAAD AL-JABERI,RICHARDA. STABLER,AND BRENDANW. WREN

The sequencing of the first Campylobacter jejuni strain in 2000 was a watershed in Campylobacter genetics and brought to light unanticipated aspects of biology of this important gastrointestinal pathogen. Since this milestone, three further complete C. jejuni genome sequences have been published that provide definitive glimpses of the genome diversity of this species. However, for the genome comparison of larger sets of C. jejuni strains, microarray-based technology has been widely applied and is currently the method of choice. Such studies have identified key genes that could be used to differentiate strains from different sources. The use of comparative genomics combined with robust methods for data analysis will continue and will form the basis for the development of rational intervention strategies to reduce C. jejuni in the food chain. In this chapter, we review the salient comparative features of the four fully sequenced genomes and reveal highlights from selected wholegenome microarray studies.

the basis of amino acid similarity to proteins of known function (Parkhill et al., 2000). After the completion of further C. jejuni genome sequences, these programs could be used for both inter- and intraspecies genomic comparisons. Furthermore, the availability of multiple C. jejuni genome sequences resulted in a shift away from the notion that any single strain could typify the entire species. More recently, the original annotation of NCTC 11168 has been updated in light of new studies on Campylobacter species and other microorganisms. The c. jejuni NCTC 11168 reannotation reduced the total number of coding sequences from 1,654 to 1,643, of which 90.0% have additional information regarding the motif identification and/or new relevant literature (Gundogdu et al., 2007). The reannotation led to 18.2% of coding sequence product functions being revised. This provides a useful framework for future C. jejuni studies and is a testament to the rapid pace of Campylobacter research in the postgenomic era.

CAMPYLOBACTER JEJUnrI GENOME SEQUENCES

GENOME DIVERSITY The relative genome diversity of bacterial species varies from clonal (genetically uniform) to genetically highly variable. The genomes of the three human clinical isolates share 1,474 core genes, with 35 genes unique to 81-176, 38 genes unique to CG8486, and 8 genes unique to NCTC 11168 (Fig. 1). All four sequenced C. jejuni genomes have revealed evidence of chromosomal loci that appear to be hot spots for intra- and intergenomic combination. For example, the genomes of NCTC 11168, RM1221, and 81-176 all have DNA unique to each strain in the loci cjO.564 and cj0.570. Similarly, identical homologues of cj1687 and cjl688 flank two

At the time of writing, four fully sequenced and annotated C. jejuni genomes have been published, including three human and one chicken isolate. Details of the strains and their basic genome statistics are summarized in Table 1. The publication of the first C. jejuni genome (Parkhill et al., 2000) paved the way for comparative genomics of this species. Initially the NCTC 11168 genome was compared with other publicly available sequences by computer programs such as Blast and the Artemis comparison tool. Such comparisons provide a means for attributing putative phenotypes to C. jejuni predicted coding sequences on

Olivia L. Champion School of Biosciences, University of Exeter, Geoffrey Pope Building, Exeter EX4 4QD, United KingSuaad Al-Jaberi, Richard A. Stabler, and Brendan W. Wren * Department of Infectious & Tropical Diseases, London School dom. of Hygiene & Tropical Medicine, Keppel St., London WClE 7HT, United Kingdom.

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Table 1. Summary of C. jejuni genomes sequenced to date and comparison to C. doylei Strain 11168" 81-176 CG8486 Fa41221 C. doylei

G+C content Size (Mb) No. of genes % coding 30.55 30.61 30.43 30.31 31.02

1.64 1.59 1.61 1.78 1.85

1,643 1,707 1,588 1,940 2,037

92 93 ? 90 82

No. of No. of structural RNAs pseudogenes 54 54 ?b

56 55

20 0 ?b

46 25 1

Source

Origin

Human diarrhea Human diarrhea Human diarrhea Chicken skin

United Kingdom United States Thailand United States

Reannotated genome (Gundogdu et al., 2007). *None reported.

a

unique 81-176 open reading frames (ORFs), although no DNA inserts were found between these two ORFs in the NCTC 11168 and RM1221 genomes. A PCR screening of C. jejuni clinical isolates revealed that 6 of 16 possessed inserts between cj1687 and cj1688 (Hofreuter et al., 2006). C. jejuni demonstrates diversity in the DNA restriction modification systems, which is thought to influence its ability to acquire genetic information by horizontal gene transfer (Miller et al., 2005). Strain 81-176 encodes a complete type I DNA restriction modification system absent in 11168 and RM1221. This restriction modification has a G+C content of 28%, varying from the genome average of 30.61% G+C, suggesting that it may have been acquired by horizontal gene transfer. Likewise, strain CG8486 encodes two complete type I restriction modification systems, the first of which is positioned between homologues of

n NCTC 11168

1

8

Figure 1. Venn diagram of the genome content of the three sequenced C. jejuni human isolates (NCTC 11168, CG8486, and 81-176) (adapted from Poly et al., 2007). The gene content of 81-176 is based on the results of Hofreuter et al. (2006). These estimations exclude the capsule, LOS, and flagellin posttranslational modification loci.

cj0759 and cj0760 (as is the novel restriction modification type I system of 81-176). The second novel type I restriction modification system is located between cj1448 and cjl555, replacing the corresponding NCTC 11168 ORFs that are absent from the CG8486 genome. Strain CG8486 also contains one novel type I11 restriction modification system. GENE GAZING AND CLUES T O SURVIVAL AND PATHOGENESIS Although isolated from a human with campylobacteriosis and thus clearly pathogenic, NCTC 11168 contains none of the usual suspects in terms of pathogenesis. The genome of strain NCTC 11168, like those of RM1221 and CG8486, does not contain plasmids. However, strain 81-176 possesses two previously sequenced plasmids: pVir and pTet (Bacon et al., 2000, 2002; Batchelor et al., 2004). This suggests that pVir may be important for pathogenesis of a subset of C. jejuni strains. However, no clear mechanism has yet been demonstrated. Conflicting reports have been published regarding the correlation of pVir in C. jejuni strains and the clinical presentation of bloody diarrhea. A study carried out in Canada revealed that pVir was present in 17 of 104 clinical isolates of C. jejuni and was significantly associated with bloody diarrhea, a marker for invasiveness (Tracz et al., 2005). However, a later study of 125 C. jejuni isolates carried out in Holland revealed no correlation between the presence of pVir and bloody diarrhea (Louwen et al., 2006). The 81-176 genome sequence also revealed a 6-kb element integrated at a leu tRNA with the characteristics of an integrated plasmid. This integrated plasmid encodes TraN and TraG homologues that function in type IV secretion systems. The integrated plasmid also contains a CJEll55 homologue contained on the CJIE3 genomic island found in the genome of RM1221. The integrated plasmid in strain 81-176 is positioned between homologues of cj0936 and cj0937. The integrated plasmids identified in all C. jejuni strains

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sequenced to date have not been linked with pathogenesis. In addition to lacking plasmids, NCTC 11168 revealed virtually no insertion sequences, no prophages or pathogenicity islands, and few repeat sequences. Similarly, C. jejuni CG8486 (Poly et al., 2007) also lacks these genetic elements. However, this is not true of all sequenced C. jejuni strains. Although the genome of RM1221 shows synteny with that of NCTC 11168, the genome of RM1221 is disrupted with four large integrated elements (Fouts et al., 2005). A Campylobacter Mu-like phage (CMLP1) encoding MuA and MuB transposase homologues is located upstream of argC (CjE0275). CMLP1 contains 5 '-TG-3' dinucleotides flanked by a 5-bp direct repeat (TATGC), consistent with those found in Mulike prophages (Morgan et al., 2002). Bacteriophages can be vehicles of horizontal gene transfer that can increase bacterial fitness (Desiere et al., 2001; Hendrix, 2002) and can influence many aspects of virulence, from adhesion and invasion to host evasion and toxin production (Wagner and Waldor, 2002). Although CMLPl does not encode any characterized virulence determinants, its presence may influence phenotypes by insertional inactivation (Fouts et al., 2005). Pathogenicity islands are often associated with tRNA loci, and unlike CMLP1, integrated elements 2 and 4 (CJIE2 and CJIE4) in the RM1221 genome have integrated into the 3' end of arginyl and methionyl tRNA genes. Phage-related endonucleases, methylases, or repressors are predicted to be encoded by ORFs found in these elements. However, RM1221 CJIE3 is located within an arginyl tRNA yet possesses no phage-related ORFs, suggesting that it is of plasmid origin or a genomic island. The majority of genes on CJIE3 are of unknown function, although 23% share homology with Helicobacter hepaticus ATCC 51449 genomic island (HHGI1) (Suerbaum et al., 2003). CMLPl is inducible with mitomycin C, but it is unknown whether the remaining three integrated elements in strain RM1221 can be excised. Because of the unknown function of the majority of genes sequenced in these integrated elements, putative roles cannot be predicted. However, functions affecting virulence or bacterial fitness cannot be ruled out. Although strain 81-176 exhibits high levels of invasion in vitro (Hu and Kopecko, 1999; Oelschlaeger et al., 1993) and is highly pathogenic in vivo (Black et al., 1988; Prendergast et al., 2004; Russell et al., 1989), the genome sequence lacks prophages. The genome of strain 81-176 is syntenic with both the NCTC 11168 and RM1221 genomes, with a few disruptions where genomic islands have been in-

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serted. The C. jejuni 81-176 genome contains 37 novel genes that are absent or pseudogenes in strains 11168 and RM1221. These are involved with electron pathways, energy metabolism, and respiratory pathways and include ORFs encoding predicted proteins with homology to DmsA, DmsB, DmsC, DmsD, and TorD, components of a Wollinella succinogenes putative anaerobic dimethyl sulfoxide reducatase (Hofreuter et al., 2006). Genome 81-176 also contains genes encoding proteins with homology to cytochrome c and several cytochrome c biosynthesis proteins. These findings suggest that strain 81-176 may have expanded respiratory pathways (Hofreuter et al., 2006). Similarly, although present as pseudogenes and thus redundant in both NCTC 11168 and RM1221, genes kdpA-C involved with potassium uptake are apparently functional in 8 1-176. Although all three genomes possess the KtrA/KtrB potassium uptake system (Tholema et al., 2005), the presence of an additional system in 81-176 may enhance its fitness in inside phagocytic vacuoles where potassium concentrations are low (Wagner et al., 2005). Strain 81-176 also contains an ORF, ggt, encoding a predicted protein with significant homology to a gammaglutamyl transpeptidase found in Helicobacter pylori. Although the mechanism is not characterized, this enzyme has been shown to have a role in H. pylori colonization in vivo (McGovern et al., 2001).

GENE SEQUENCING AND POTENTIAL PHASE VARIATION Scouring the genomes has revealed few definitive clues to genetic elements that may be important in virulence. However, scrutiny of raw nucleotide data can reveal repeat sequences or polymeric tracts (e.g., homopolymeric tracts of repeat As, Cs, Gs, and Ts) that can provide clues to genes that slip in and out of frame (Parkhill et al., 2000). Such phase-variable genes can provide a simple primordial genetic mechanism for switching key determinants on and off depending on the environment's selective pressures (van Belkum et al., 1998). The presence of hypervariable sequences in genes encoding the biosynthesis or modification of surface structures such as capsular polysaccharide, flagellum, and lipooligosaccharide (LOS) has been a common theme in each of the C. jejuni genomes sequenced to date. The combinatorial mathematics of phase variation means that a bacterium with just 20 phase-variable loci can potentially exist in over a million different states. Several dozen phase-variable genes in surface antigen biosynthesis loci have been identified in the strains sequenced to date. The rep-

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ertoire of envelope-associated structures provides C. jejuni with astonishing diversity when interacting with its environment, including immune evasion in the human host but also during its survival in nonhuman environmental reservoirs.

GENE SEQUENCING AND CELL SURFACE GLYCOSTRUCTURES Surface structures, particularly glycan-modified surface structures, often play important roles for bacterial pathogens in interacting with host cells and the host immune system. The most rapid advance in the biology of C. jejuni postgenomic era has been our basic understanding of cell surface glycoconjugates fueled by the importance of these structures in disease pathogenesis, including immune-mediated neuropathies. C. jejuni has been termed a “hyperglycemic bug” in that it has dedicated a considerable part of its relatively small genome to the biosynthesis of several glycostructures. These include LOS, capsular polysaccharide, and both 0- and N-linked glycosylation pathways (the only bacterium proven to have both pathways). The C. jejuni LOS, capsule, and 0linked glycosylation system of flagellin have been found to be highly variable between strains, with multiple genetic mechanisms used to affect the observed structural differences (Gilbert et al., 2002; Karlyshev et al., 2005). One of the key discoveries resulting from the NCTC 11168 genome sequence was the presence of a highly variable C. jejuni capsular polysaccharide (Karlyshev and Wren, 2001; Karlyshev et al., 2005). Genomic comparisons of C. jejuni strains of different Penner serotype by means of microarrays have revealed that capsule is the likely Penner serodeterminant (Dorrell et al., 2001). In addition, all C. jejuni strains sequenced to date contain highly variable LOS biosynthesis genes and flagellin modification loci. The C. jejuni cell surface polysaccharides are discussed in more detail elsewhere (chapters 25 to 29).

C . JEJUnrr GENOMICS TO PHENOMICS Genome sequence is not an end in its own right, but a means for hypothesis generation to be tested in the laboratory. Genomic comparisons must be coupled with assays in the laboratory to unravel gene function and understand differences in strain phenomes. In strain 8 1-176, loss-of-function mutations were constructed in dmsA, cytC, and ggt to assess their contribution to virulence. These genes are thought to be involved in respiratory pathways of

strain 81-176 and so were tested under low 0, conditions, as would be experienced by C. jejuni in the gut during colonization. Interestingly, in vitro, no growth defect was demonstrated for all dmsA, cytC, and ggt mutants in BHI 10% CO, or under anaerobic conditions. Furthermore, mutants showed no reduction in invasiveness and survival in intestinal epithelial cells compared with wild type. However, a significant colonization defect was observed for dmsA and cytC in vivo after 3 weeks (Hofreuter et al., 2006). The ggt knockout also showed a colonization defect compared with wild-type 81-176 during competitive index experiments. By week 7 after infection, shedding of ggt knockout strains was significantly reduced compared with wild type, and by week 8, significantly fewer ggt knockout strains were recovered from mouse tissue compared with wild-type 8 1-176. These differences led the authors to suggest that 81176 specific genes dmsA, cytC, and ggt may contribute to the increased virulence of strain 8 1-176 in vivo (ferret model) compared with strain 11168. However, during in vivo experiments in the ferret model, strain CG8486 was equally as pathogenic as strain 81-176 (five of seven animals developed diarrhea after infection with both CG8486 and 81-176). Strain 8 1-176 genes dmsA, cytC, and ggt were not found in strain CG8486, although the same level of invasiveness in vivo was observed. Previous studies that used strain 81-176 have indicated that invasiveness in vitro correlates with disease in the ferret model (Bacon et al., 2001; Goon et al., 2006; Yao et al., 1997). However, despite displaying equal levels of pathogenicity in vivo, during in vitro comparisons of invasiveness in INT-407 cells, CG8486 invaded 1,000-fold less than strain 81-176. The disparity between in vitro and in vivo phenotypes of loss-of-function mutants dmsA, cytC, and ggt in strain 81-176 is not understood. Multiple C. jejuni genome sequences have revealed that the presence of specific virulence genes in the genome does not appear to correlate with the level of pathogenicity in vitro and in vivo of a strain. Loci encoding C. jejuni surface antigens possess the highest levels of genetic variation. Understanding the variation in these loci may hold the key to understanding strain differences in pathogenicity. Further research underpinning environmental stimuli, including host-pathogen interactions, implicit in surface antigen variability is required.

COMPARATIVE GENOMICS BY MICROARRAY Traditional phylogenetic classification of bacteria to study evolutionary relatedness is based on the

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characterization of a limited number of genes and of rRNA or signature sequences. However, because of the acquisition of DNA through lateral gene transfer, the differences between closely related bacterial strains can be vast. By contrast, whole-genome sequencing comparisons allow a multitude of genes to be compared. Nevertheless, whole-scale genome sequencing remains an expensive endeavor, and such comparisons are limited to only a handful of strains. DNA microarrays represent an alternative technology for whole-genome comparisons, enabling a bird’s-eye view of all the genes absent or present in a given genome as compared with the reference genome on the microarray. Harnessing DNA microarray information through interrogative and robust algorithms has enabled a true “comparative phylogenomics” approach to be developed. Recent comparative phylogenomics studies have been undertaken on increasingly large collections of strains from defined origins. A common feature from many of these studies has been the unexpectedly large genetic diversity between strains within the same species, blurring our definition of species boundaries. Whole-genome comparisons typically identify sets of core genes shared by all strains in a species and accessory genes present in one of more strains in a species that often result from gene acquisition. It is these differences that can often be used to identify genes/genetic islands related to gainof-function traits in pathogenic strains. Uncovering the mechanisms behind this variability is fundamental in understanding and ultimately counteracting infection. Moreover, given the range of diseases associated with some bacterial pathogens and the diverse genotypic and phenotypic properties of clinical and environmental isolates, microarrays have proved to be particularly useful for determining correlates of pathogenicity (Hinchliffe et al., 2003; Howard et al., 2006) and in deciphering the epidemiology and ecological niches of the organism (Champion et al., 2005; Stabler et al., 2006).

CASE STUDIES OF C. JEjUNI GENOMIC COMPARISONS BY MICROARRAY The first C. jejuni microarray, or clone array, which used clone vectors as a by-product from the genome sequencing project, was constructed in 2001 (Dorrell et al., 2001). This clone array demonstrated a proof of principle for microarray technology for C. jejuni and provided a platform for comparison of multiple strains at a whole-genome level. Microarray comparisons revealed an unexpectedly large level

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of genetic diversity among strains of C. jejuni. A set of core genes shared by all strains compared to date have been identified. In addition, accessory genes have also been identified that are present in one or more strains in the species and are often the result of gene acquisition. These differences can be particularly useful for retrospective analysis of bacterial evolution and population genetics, both recent and in the long term. The initial observation on genetic variability in the gastrointestinal pathogen C. jejuni (Dorrell et al., 2001) has been expanded by several recent studies (Leonard et al., 2003; Pearson et al., 2003; Taboada et al., 2004), suggesting C. jejuni exhibits high levels of genome diversity and low levels of genome plasticity (Taboada et al., 2004). The original estimate of C. jejuni accessory genes of ~ 2 0 %(Dorrell et al., 2001) has now been increased to ~ 3 7 % (Taboada et al., 2004). However, many of these accessory genes mapped to previously defined variable loci, suggesting that large regions of the C. jejuni genome are genetically stable (Taboada et al., 2004). Unfortunately, it has proved difficult to use microarray data to identify genetic markers predictive of source or outcome of infection. A comparison of isolates from patients who developed Guillain-BarrC syndrome (GBS) with isolates from patients with uncomplicated gastrointestinal infection failed to identify specific GBS genes or regions (Leonard et al., 2004). A whole-genome microarray was used to determine the activities of cytolethal distending toxin (CDT) and hemolysin along with survival characteristics under aerobic conditions at room temperature (On et al., 2006). Numerical analysis of microarray data clearly delineated strains into two clusters, genotype cluster l and 2 (GC1 and GC2). CDT and hemolysin activities of GC1 strains were not statistically different from those of cluster GC2 strains. However, viability during aerobic incubation of cluster GC1 strains was statistically lower than corresponding estimates of cluster GC2 strains. The number of missing or highly divergent genes in cluster GC1 strains with respect to NCTC 11168 was also statistically significantly greater compared with those of cluster GC2 strains. Out of the genes present in NCTC 11168, 67 genes were characteristically missing or divergent among cluster GC1 strains. Of these, 53 genes were localized within 11 major gene clusters, of which 8 were associated with surface structures, including flagellar, lipooligosaccharide, and membrane transport proteins. This study indicates a correlation between C. jejuni genomic content, particularly in surface-coding regions, and its capacity for environmental survival, and it may explain why

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genetics, which plays an important role in the investigation of C. jejuni pathogenicity.

certain serotypes are more commonly reported in human diseases (On et al., 2006). Studies that use an ORF-specific C. jejuni DNA microarray to assess clinical isolates associated with five independent clusters of infection were compared with data from random amplified polymeric DNA and Penner serotyping (Leonard et al., 2003). Microarray analysis provided a method for comparison of isolates that is more gene specific than are other existing genotyping methods and that can reveal genomic differences between isolates that randomly amplified polymorphic DNA analysis may not identify (Leonard et al., 2003). By means of comparative genomic hybridization, many genes were found to be divergent across multiple strains and were uniquely variable in single strains. Despite the large increase in the collective number of variable genes, nearly half of them were mapped to previously defined variable loci, and it therefore appears that large regions of the C. jejuni genome are genetically stable (Taboada et al., 2004). On the whole bacterial chromosome, regions of variable genes often are present in large clusters, suggesting that they were acquired or lost from the genome in groups during evolution. The data generated by microarray technology are highly accurate and readily comparable among laboratories. Studies carried out by microarray technology promise a clearer understanding of the population biology of this important pathogen. This in turn will lead to a better understanding of population

COMPARATIVE PHYLOGENOMICS Although the whole-genome comparisons of C. jejuni by DNA microarrays have been useful in identifying candidate genes that may be involved in pathogenesis and survival, they do not provide any clue to the evolutionary origins and phylogeny of C. jejuni. Therefore, we developed comparative phylogenomics, whole-genome comparisons of microbes via DNA microarrays combined with Bayesian-based algorithms to model the phylogeny of a given microorganism (Fig. 2). We applied this initially to 111 C. jejuni isolates from a spectrum of sources, including humans (n = 70), chickens (n = 17), bovines (n = 13), ovines (n = 5), and the environment (n = 6). Remarkably, from this data, the Bayesian phylogeny of the isolates revealed two distinct clades unequivocally supported by Bayesian probabilities (P = 1) (Champion et al., 2005). On further analysis, these two clades appeared to correlate with the origin of the strains and included a livestock clade comprising 3 1 (88.6%) of 35 of the livestock isolates and a nonlivestock clade containing all environmental isolates. Several genes were identified as characteristic of strains in the livestock clade. The most prominent

MrBayes

C. jejuni Strain Collection

I

1

gDNA

Microarray

-

K '

Molecular Biology Analysis

A Genespring

Test Vs Common Reference (C. jejuni 11168) Quality Contrc

/

\

I-

Phylogenetic Tree production

I

GACK

Gene Calling

Figure 2. Comparative phylogenomics pipeline. Graphical representation of steps involved in production of a phylogenetic tree. Genomic DNA from each member of the C. jejuni strain collection was hybridized to a BpGS C. jejuni 11168 microarray with C. jejuni 11168 gDNA as control. Florescent intensities were calculated by BlueFuse. GeneSpring calculated intensity ratios and removed low-quality data points. GACK was used to convert ratio data to binary present/absent data. MrBayes used the binary data to construct a putative phylogeny by means of a Bayesian algorithm. The resulting tree was statistically tested for robustness. Further C. jejuni strains can be fed into the pipeline, and selection may be influenced by current phylogeny prediction.

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was a cluster of six genes (cj1321 to cj1326) within the genetically variable flagellin glycosylation locus. In subsequent studies, this locus has been confirmed as being present in approximately 50% of strains, and its precise role in these C. jejuni strains is the subject of intense study. Surprisingly, the initial comparative phylogenomics study showed that 39 (55.7%) of 70 C. jejuni human isolates were found in the nonlivestock clade, suggesting that many C. jejuni infections may be from nonlivestock (and possibly nonagricultural) sources. More recently, we have studied over 230 C. jejuni strains from diverse origin and have further demonstrated the split of strains into two clades. Approximately half of the human isolates from this study are not associated with the livestock clade (S. Al-Jaberi, and B. W. Wren, unpublished data). These studies may provide insight into a previously unidentified reservoir of C. jejuni infection that may have implications in disease control strategies. The comparative phylogenomics approach provides a robust methodological prototype and has been applied to other bacterial pathogens (Howard et al., 2006; Stabler et al., 2006).

CONCLUSION AND FUTURE PERSPECTIVES Until the year 2000, the genetics of C. jejuni was investigated in a piecemeal gene-by-gene approach, with no clear overview. The availability of whole-genome sequences has facilitated research into genotypes and phenotypes in their entirety, heralding a golden era in Campylobacter microbiology. Robust methods of data analysis are required to maximize the new information gained from both genome sequences and comparative genome analyses via microarrays. DNA microarrays represent a powerful enabling technology for the whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. Such studies have increased our ability to differentiate between C. jejuni, highlighting previously undetected genetic differences and population structures, and providing new insight into virulence and evolution of C. jejuni. Hypothesis trawling is possible through comparison of whole genomes of strains with different characteristics by both genome sequencing and DNA microarrays. Subsets of genes that differ between the strains can be identified and their roles hypothesized on the basis of strain phenotypes. Gene homology searches with other sequenced bacteria provide clues

*

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to the function of many of the genes in the genome. However, the function of many C. jejuni genes remains unknown, and learning their roles will aid understanding of the unique functioning of this problematic pathogen. Advances in genetic manipulation of C. jejuni, including the development of speciesspecific promoters and complementation techniques, have fueled research into gene function. However, the lack of a small, portable animal model to study C. jejuni pathogenesis is still a stumbling block for studying gene function in vivo. The next challenge for microbiologists in this postgenomic era is to correlate C. jejuni genome to phenome. This will provide a clear and more comprehensive understanding of the biology of C. jejuni.

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Gilbert, M., M. F. Karwaski, S . Bernatchez, N. M. Young, E. Taboada, J. Michniewicz, A. M. Cunningham and w. w. Wakarchuk. 2002. The genetic bases for the variation in the lipooligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 277:327-337. Goon, S., C. P. Ewing, M. Lorenzo, D. Pattarini, G. Majam, and P. Guerry. 2006. A sigma28-regulated nonflagella gene contributes to virulence of Campylobacter jejuni 81-176. Infect. lmmun. 74:769-772. Gundogdu, O., S . D. Bentley, M. T. Holden, J. Parkhill, N. Dorrell, and B. W. Wren. 2007. Re-annotation and re-analysis of the Campylobacter jejuni NCTCl1168 genome sequence. BMC Genomics 8~162. Hendrix, R. W. 2002. Bacteriophages: evolution of the majority. Theoretical Population Biol. 61:471-480. Hinchliffe, S . J., K. E. Isherwood, R. A. Stabler, M. B. Prentice, A. Rakin, R. A. Nichols, P. C. Oyston, J. Hinds, R. W. Titball, and B. W. Wren. 2003. Application of DNA microarrays to study the evolutionary genomics of Yersinia pestis and Yersinia pseudotuberculosis. Genome Res. 13:2018-2029. Hofreuter, D., J. Tsai, R. 0. Watson, V. Novik, B. Altman, M. Benitez, C. Clark, C. Perbost, T. Jarvie, L. Du, and J. E. Galan. 2006. Unique features of a highly pathogenic Campylobacter jejuni strain. Infect. lmmun. 74:4694-4707. Howard, S . L., W., Gaunt, J. Hinds, A. A. Witney, R Stabler, and B. W. Wren. 2006. Application of comparative phylogenomics to study the evolution of Yersinia enterocolitica and to identify genetic differences relating to pathogenicity. J. Bacteriol. 188: 3645-3653. Hu, L., and D. J. Kopecko. 1999. Campylobacter jejuni 81-176 associates with microtubules and dynein during invasion of human intestinal cells. Infect. lmmun. 67:4171-4182. Karlyshev, A. V., 0. L. Champion, C. Churcher, J. R. Brisson, H. C. Jarrell, M. Gilbert, D. Brochu, F. St Michael, J. Li, W. W. Wakarchuk, I. Goodhead, M. Sanders, K. Stevens, B. Stevens, J. Parkhill, B. W. Wren, and C. M. Szymanski. 2005. Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses. Mol. Microbiol. 55:90-103. Karlyshev, A. V., and B. W. Wren. 2001. Detection and initial characterization of novel capsular polysaccharide among diverse Carnpylobacterjejuni strains using Alcian blue dye. J. Clin. Microbiol. 39:279-284. Leonard, E. E., 11, T. Takata, M. J. Blaser, S. Falkow, L. S. Tompkins, and E. C. Gaynor. 2003. Use of an open-reading framespecific Campylobacter jejuni DNA microarray as a new genotyping tool for studying epidemiologically related isolates. J. Infect. Dis. 187:691-694. .eonard, E. E., 11, L. S . Tompkins, S . Falkow, and I. Nachamkin. 2004. Comparison of Campylobacter jejuni isolates implicated in Guillain-Barre syndrome and strains that cause enteritis by a DNA microarray. Infect. lmmun. 72:1199-1203. .ouwen, R. P., A. van Belkum, J. A. Wagenaar, Y. Doorduyn, R. Achterberg, and H. P. Endtz. 2006. Lack of association between the presence of the pVir plasmid and bloody diarrhea in Campylobacter jejuni enteritis. J. Clin. Microbiol. 44:18671868. McGovern, K. J., T. G. Blanchard, J. A. Gutierrez, S . J. Czinn, S . Krakowka, and P. Youngman. 2001. Gamma-glutamyltransferase is a Helicobacter pylori virulence factor but is not essential for colonization. Infect. Immun. 69:4168-4173. Miller, W. G., B. M. Pearson, J. M. Wells, C. T. Parker, V. V. Kapitonov, and R. E. Mandrell. 2005. Diversity within the Campylobacter jejuni type I restriction-modification loci. MicrobiolOD 151:337-351.

Morgan, G. J., G. F. Hatfull, S . Casjens, and R. W. Hendrix. 2002. Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus. J. Mol. Biol. 317:337-359. Oelschlaeger, T. A., P. Guerry, and D. J. Kopecko. 1993. Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii. Proc. Natl. Acad. Sci. USA 90:6884-6888. On, S. L., N. Dorrell, L. Petersen, D. D. Bang, S . Morris, S . J. Forsythe, and B. W. Wren. 2006. Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses. lnt. J. Med. Microbiol. 296:353-363. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S . Holroyd, K. Jagels, A. V. Karlyshev, S . Moule, M. J. Pallen, C. W. Penn, M. A. Quail, M. A. Rajandream, K. M. Rutherford, A. H. van Vliet, S . Whitehead, and B. G. Barrell. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668. Pearson, B. M., C. Pin, J. Wright, K. I'Anson, T. Humphrey, and J. M. Wells. 2003. Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays. FEBS. Lett. 554~224-230. Poly, F., T. Read, D. R. Tribble, S . Baqar, M. Lorenzo, and P. Guerry. 2007. Genome sequence of a clinical isolate of Campylobacter jejuni from Thailand. Infect. lmmun. 75:34253433. Prendergast, M. M., D. R Tribble, S . Baqar, D. A. Scott, J. A. Ferris, R. I. Walker, and A. P. Moran. 2004. In vivo phase variation and serologic response to lipooligosaccharide of Campylobacter jejuni in experimental human infection. Infect. lmmun. 72:916-922. Russell, R G., M. J. Blaser, J. I. Sarmiento, and J. Fox. 1989. Experimental Campylobacter jejuni infection in Macaca nemestrina. Infect. lmmun. 57:1438-1444. Stabler, R A., D. N. Gerding, J. G. Songer, D. Drudy, J. S . Brazier, H. T. Trinh, A. A. Witney, J. Hinds, and B. W. Wren. 2006. Comparative phylogenomics of Clostridium dificile reveals clade specificity and microevolution of hypervirulent strains. J. Bacteriol. 188:7297-7305. Suerbaum, S., C. Josenhans, T. Sterzenbach, B. Drescher, P. Brandt, M. Bell, M. Droge, B. Fartmann, H. P. Fischer, Z. Ge, A. Horster, R. Holland, K. Klein, J. Konig, L. Macko, G. L. Mendz, G. Nyakatura, D. B. Schauer, Z. Shen, J. Weber, M. Frosch, and J. G. Fox. 2003. The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus. Proc. Natl. Acad. Sci. USA 100:7901-7906. Taboada, E. N., R. R. Acedillo, C. D. Carrillo, W. A. Findlay, D. T. Medeiros, 0. L. Mykytczuk, M. J. Roberts, C. A. Valencia, J. M. Farber, and J. H. Nash. 2004. Large-scale comparative genomics meta-analysis of Campylobacter jejuni isolates reveals low level of genome plasticity. J. Clin. Microbiol. 42:45664576. Tholema, N., M. Vor der Bruggen, P. Maser, T. Nakamura, J. I. Schroeder, H. Kobayashi, N. Uozumi, and E. P. Bakker. 2005. All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus. J. Biol. Chem. 280:4114641154. Tracz, D. M., M. Keelan, J. Ahmed-Bentley, A. Gibreel, K. Kowalewska-Grochowska, and D. E. Taylor. 2005. pVir and bloody diarrhea in Campylobacter jejuni enteritis. Emerg. Infect. Dis. 11:838-843.

CHAPTER 4

van Belkum, A., S., Scherer, L. van Alphen, and H. Verbrugh. 1998. Short-sequence DNA repeats in prokaryotic genomes. Microbiol. Mol. Biol. Rev. 62:275-293. Wagner, D., J. Maser, B. Lai, Z. Cai, C. E. Barry 111, K. Honer Zu Bentrup, D. G. Russell, and L. E. Bermuda. 2005. Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes in-

COMPARATIVE GENOMICS OF c. rmuw

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dicates pathogen-induced microenvironments within the host cell's endosomal system. J. Immunol. 174:1491-1500. Wagner, P. L., and M. K. Waldor. 2002. Bacteriophage control of bacterial virulence. Infect. Immun. 70:3985-3993. Yao, R., D. H. Burr, and P. Guerry. 1997. CheY-mediated rnodulation of Campylobacter jejuni virulence. Mol. Microbiol. 23: 1021-1031.

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NJC genomes were limited; for some species, e.g., C. cuwus and C. hominis, the total genomic data available consisted of the 16s rDNA sequence and perhaps the sequence of a few genes. Thus, for the NJC species, the genetic basis of virulence, host preference and colonization could not be identified. The availability of eight new NJC genomes (for the purposes of this chapter, C. jejuni subsp. doylei is considered an NJC) permits genomic comparisons between the NJC group and between the NJC genomes and the C. jejuni subsp. jejuni genomes. Such comparisons will identify common virulence determinants, surface structures, metabolic pathways, signal transduction and chemotaxis proteins, and other features contained within all Campylobacter strains. Ideally, in addition, Campylobacter genome comparisons might identify genes and pathways specific to particular Campylobacter species, providing the genetic basis for phenotypes observed for that species and identifying also the genetic basis underlying differences in host range, colonization, and pathogenicity. Campylobacter genomic comparisons permit also analyses regarding evolution and the origin of particular Campylobacter species and/or subspecies. Additionally, genomic analysis of the genus Campylobacter is enhanced by the presence of multiple related Campylobacterales genomes. These include the ge)~ nome of Arcobacter butzleri (Miller et al., 2 0 0 7 ~the three Helicobacter pylori genomes (strains 26695 [Tomb et al., 19971, J99 [Alm, 19991, and HPAGl [GenBank accession number NC0008086.1]), and the genomes of Helicobacter hepaticus (Suerbaum et al., 2003), Helicobacter acinonychis (Eppinger et al., 2006), Wolinella succinogenes (Baar et al., 2003), and Sulfurimonas denitrificans (formerly Thiomicrospira denitrificans [Takai et al., 20061; GenBank accession number CP000153.1). Additionally, the genomes of more distantly related Epsilonproteobacteria, such as Sulfurovum and Nitratiruptor (Nakagawa et al., 2007), have been completed. This chapter will compare and contrast the genomes of Campylobacter species other than C. jejuni subsp. jejuni. Where relevant, the NJC genomes will be compared with related genomes, especially those of C. jejuni subsp. jejuni and A. butzleri. Elements important in overall genome structure, such as genomic islands, prophage, and insertion sequences (ISs) will be discussed. Individual topics, including restriction modification (R-M), metabolism, and virulence will be presented. Discussion of surface structures (e.g., lipooligosaccharide, capsule, and C. fetus subsp. fetus S-layer proteins) and signal transductiod chemotaxis will be presented elsewhere in this book (chapters 27, 28, 23, and 20, respectively). Finally, genomic data from the organisms C. jejuni subsp.

doylei 269.97, C. fetus subsp. fetus 82-40, C. curvus 525.92, C. concisus 13826, and C. hominis ATCC BAA-381 are from Fouts et al. (unpublished data).

GENERAL FEATURES OF THE NJC GENOMES The first Campylobacter genome to be sequenced, C. jejuni subsp. jejuni NCTC 11168 (Parkhill et al., 2000), was approximately 1.6 Mb in size with a G+C content of 30.6%. Analysis of the NJC genomes (Table 1) indicates that these genomes are similar in size (approximately 1.53 to 1.97 Mb). The G+C content of most of the NJC genomes is 29.7 to 34.5%, with the exception of C. curvus 525.92, which has the much higher G+C content of 44.5%. These numbers contrast with the genome sequence of the related organism A. butzleri RM4018, which has both a larger size (2.3 Mb) and lower G+C content (27%). The total number of coding sequences (CDSs) per Campylobacter genome is proportional to the size of the genome, with an average gene size range of 988 to 1,051 bp. Finally, the CDSs with assigned function are fairly consistent across the sequenced Campylobacter genomes, with an assigned function/ total CDSs average of 46%. Pairwise BLASTP comparisons identified 8 80 proteins conserved across all of the NJC proteomes. Of these 880 proteins, 753 were conserved further across the family Campylobacteraceae (i.e., Campylobacter + Arcobacter), and 569 were conserved still further across the order Campylobacterales (Campylobacteraceae + Helicobacteraceae). The identities of the majority of proteins within the core protein set are, predictably, proteins with housekeeping functions, e.g., those involved in protein synthesis, transcription, DNA replication, flagellar biosynthesis, and biosynthesis of purines and pyrimidines. However, 294 proteins in the core protein set have been classified as “conserved hypothetical” or have been assigned only a general function. The majority of proteins within the core protein set have homologs in other bacterial taxa: only 27 of the 880 proteins would be considered unique, within Campylobacterales, to Campylobacter. Only a few of these 27 proteins, e.g., SerC, have assigned function; the remainder are conserved hypothetical. The proteomes of C. concisus 13826 and C. hominis ATCC BAA-381 were not included in the BLASTP analysis. Because the exclusion of C. lari from the pairwise BLASTP analysis would lead to a larger core protein set as a result of the inclusion of multiple biosynthetic genes (see below), it is likely that the size of the core protein set will diminish as new Campylobacter proteomes are factored into the analysis.

Table 1. General features of the sequenced Cumpylobucteruceae genomes" Parameter Genome size (bp) Yo G+C CDS numbers" Pseudogenesd Assigned function Generallunknown function No. of prophagelgenomic islands1CRISPRs No. of rRNA operons Plasmids (kb) No. of IS elements No. of poly G:C tracts (>8 bp) Average tract size No. of restrictionlmodification systems Type I (hsd) Type I1 Type 111 No. of two component systems Response regulator Sensor histidine kinase

Cii

Cii

3 1;3;4;180 6 18 9.4

-1,660,000 34.5 1,670 ND 79 1 879 0/1/0 3 3;110 0 75 13.6

1,525,460 29.7 1,500 13 736 764 11010 3 46 0 15 9.7

29 2 1

1 2 0

49 69 29

10 6"

9 7

7 4

1,616,554 30.6 1,622 32 670 952 O/l/O 3 37;45 0 19 9.3

1,845,106 30.6 1,728 125 811 917

1,684,122 31.4 1,639 18 785 853

11.511

o/o/o

3 0 3 74 10.6

19 3 1

2 1 0

9 6

9 7

3 0 0 29 9.4

1,777,831 30.3 1,800 47 758 1042 21211 3 0 0 24 9.9

1 3 0

10 6

01011

RM2100

RM2228

81-176

1,641,481 30.6 1,597 27 833 764

cu RM319.5'

Cjd 269.97

Cii RM1221

11168

cc

Ab

Cur 525.92

RM4018

1,773,615 33.3 1,710 27 795 915 011/1 3 0 0 30 9

1,971,264 44.5 1,875 21 743 1,132 01312 3 0 0 9 10

2,341,251 27.1 2,259 5 1,011 1,248 1/3/0 5 0 0 0 NIAf

19 2 0

1 1 0

1 19 0

0 0 0

8 6

12 12

15 14

42 37

Cf 82-40

Cl

Cjj, Campylobacter jejuni subsp. jejuni: Cjd, C. jejuni subsp. doylei: Cc, C. coli: Cu, C. upsaliensis; Cl, C. lari; Ck C. fetus subsp. fetus; Cur, C. curvus; Ab, Arcobacter butzleri; CRISPRs, clustered regularly interspaced short palindromic repeats. bNumbers based on preliminary analysis of the RM3195 draft genome. "Total does not include pseudogenes, where applicable. dTotals include potential contingency genes. 'ND, not determined. fNA, not applicable. g Contains putative pseudogenes or contingency genes. Two sensor histidine kinases are putative pseudogenes. a

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By use of the core protein set, pairwise average amino acid identities between any two Campylobacter proteomes were calculated (Fig. 1).For example, the 880 core proteins of C. jejuni subsp. doylei 269.97 had an average amino acid identity per protein of 87.2% when compared by BLASTP to their homologs within the 880 core proteins of C. coli RM2228. Average amino acid identity values ranged from 97.6% for C. jejuni subsp. doylei 269.97 and the related C. jejuni subsp. jejuni genomes to 58.4 to 59.6% for C. curvus 525.92 and C. fetus subsp. fetus 82-40 when compared with any of the thermophilic Campylobacter genomes. The amino acid identity values between C. fetus subsp. fetus and C. curvus were 64.9%, indicating that although they demonstrate similar amino acid identity values when compared with the thermophilic campylobacters, they are similarly unrelated to each other. The most related genomes to C. jejuni subsp. jejuni were, in order: C. jejuni subsp. doylei, C. coli, C. upsaliensis, C. lari, and C. fetus subsp. fetuslC. curvus. Amino acid identities varied by 1.1% between the C. jejuni subsp. jejuni genomes; however, the level of strain-to-strain variation in the NJC genomes is presently unknown. Multilocus sequence typing (MLST) of the NJC genomes suggests that similar levels of strain-to-strain variation exist within these species (Miller et al., 2005), especially within C. fetus subsp. fetus, where interstrain variation is remarkably low (van Bergen et al., 2005). However, the seven genes utilized by MLST represent a very small fraction of the total genome, and MLST genes are generally housekeeping genes with intrinsically lower levels of variation. Strain-to-strain variation would also be expected to be influenced by lateral gene transfer. For example, the sequenced genomes of C. jejuni subsp. jejuni RM1221 and C. jejuni subsp. doylei 269.97 contain regions that originated in C. coli and C. upsaliensis, respectively. The C. coli region within strain

I

cii 98.9

I

I

RM1221 encodes core proteins and thus formed part of the 1.1% variation within C. jejuni subsp. jejuni. MLST analysis of other Campylobacter species has identified other instances of putative lateral gene transfer (data not shown). Unfortunately, a more thorough analysis of strain-to-strain variation at the sequence level within the NJC species will require sequencing additional genomes. However, this solution is not presently feasible because of the high cost of genomic sequencing. A more reasonable middle ground would entail sequencing of a much larger gene set than the seven sequenced by MLST. This gene set would have to be small enough to enable the analysis of multiple strains but large enough to present an accurate representation of intraspecific variation.

PLASMIDS Plasmids of a wide range of sizes have been observed in many NJC species, including C. coli (Taylor and Tracz, 2005), C. upsaliensis (Owen and Hernandez, 1990; Stanley et al., 1994) and other, nonthermophilic campylobacters (Boosinger et al., 1990; Waterman et al., 1993; Willoughby et al., 2005; data not shown). The primary phenotypic marker associated with Campylobacter plasmids is antibiotic resistance. Genes encoding resistance to tetracycline, kanamycin, and chloramphenicol have been identified on Cumpylobacter plasmids (Trieber and Taylor, 2000). Cumpylobacter plasmids fall into two basic groups: cryptic plasmids and megaplasmids. Although cryptic plasmids are small, generally <5 kb in size, and megaplasmids are much larger molecules, in Campylobacter as large as 180 kb, the primary distinction between the two types of plasmids is related to

Cid

cc

cu

c/

Cf

Cur

97.6 100

87.7 87.2 I00

77.6 77.2 77.4 100

71.9 71.6 71.7 69.8 I00

59.4 59.3 59.5 58.4 59.3

59.6 59.5 59.6 58.7 59.6

I

i

i I

i

I

i

I

Cjj cid Cc CU

c/

Figure 1. Average amino acid identities of the Cumpylobucter proteomes. Pairwise BLASTP comparisons of the Cumpylobacter proteomes were performed. For each protein from each proteome, the protein with the most significant homology from the other proteomes was identified. Significant homology was defined as >30% amino acid sequence identity with at least 75% sequence overlap. From these matches, a set of 880 core proteins, conserved across the Cumpylobacter proteomes, was identified. Values represent average amino acid identities between the core protein sets of any two given Cumpylobucter proteomes. See Table 1, footnote a, for abbreviations.

CHAPTER 5

COMPARATIVE GENOMICS OF NON-C. 7ETUNr CAMPYLOBACTER SPP.

the genes encoded by the plasmids. Cryptic plasmids are defined as plasmids that encode only one or two replication proteins and, usually but not always, one or two mobilization proteins. Megaplasmids are distinguished by the presence of several genes that encode proteins involved in plasmid conjugation. Cryptic Plasmids Multiple NJC cryptic plasmids have been identified and characterized (Kienesberger et al., 2007; Miller et al., 2007a; Waterman et al., 1993). In some strains, multiple plasmids are present (Kienesberger et al., 2007; Miller et al., 2007a). One such strain is C. coli RM2228, which contains three cryptic plasmids, pCC2228-1 (1.3 kb), pCC2228-2 (3.3 kb) and pCC2228-3 (4.3 kb), and the 180-kb megaplasmid pCC2228-4 (Miller et al., 2007a). The presence of three cryptic plasmids in strain RM2228 suggests that these three plasmids represent multiple incompatibility (Inc) groups (Miller et al., 2007a). The existence of multiple Inc groups within this strain is evidenced further by predicted differences in the replication origin and replication apparatus of each cryptic plasmid. pCC2228-1 is unusual in both its small size (1.3 kb) and the presence of only one CDS on the plasmid. Comparison of this CDS to the GenBank nr database suggests that this CDS encodes a replication protein of the Firmicutes RepL family. RepL-family plasmids have been shown to replicate via a rollingcircle mechanism (Hefford et al., 1997), although no data are available to indicate that pCC2228-1 replicates by a similar mechanism. Rolling circle plasmids are not unique in Campylobacterales. Such plasmids have been identified in Helicobacter (Kleanthous et al., 1991); however, the replication proteins from these plasmids belong to the unrelated Rep-2 family. pCC2228-2 is a typical theta-replicating, iteroncontaining plasmid related to the majority of characterized Campylobacter plasmids, including most of the Campylobacter shuttle plasmids. Iterons are binding sites for the cognate replication (Rep) protein, and binding of Rep to the iterons is necessary for replication and copy number control (Chattoraj, 2000). pCC2228-3 is also an iteron-containing plasmid. However, it differs from pCC2228-2 in that the replication protein, RepC, is unrelated to the RepAB proteins of pCC2228-2, and that the plasmid organization is repCliteronlmob instead of iteronlrepAB1 mob. Most of the characterized NJC cryptic plasmids resemble pCC2228-2 in both gene content and iteronlrepABlmob organization. One exception is the C. fetus subsp. venerealis plasmid pCFVlO8 (Kienes-

77

berger et al., 2007), which is similar in organization to pCC2228-3 and pCJ1170. Miller et al. (2007a) proposed that as many as nine Inc groups exist within the Campylobacter cryptic plasmids. This estimate was based on the presence of both unique iteron repeat sequences and phylogenetically distinct replication proteins. Plasmids from the same putative Inc group have been isolated from different species. For example, plasmids pCC2228-3 and pCJ1170, isolated from C. coli and C. jejuni subsp. jejuni, respectively, contain identical iteron repeats and Rep proteins (Miller et al., 2007a). The existence of similar plasmids in different Campylobacter species indicates that some cryptic plasmids have an expanded host range. For instance, the pIP1455-derived shuttle vectors have been used to transform C. jejuni subsp. jejuni, C. coli, C. lari, and C. fetus subsp. fetus (Kienesberger et al., 2007; Miller et al., 2000; data not shown). pIP1455-derived plasmids do not replicate in C. hyointestinalis, although plasmids derived from C. hyointesintalis replicate also in C. fetus (Waterman et al., 1993).Additionally, pIP1455-derived plasmids have not been used successfully to transform C. upsaliensis (data not shown); however, this may reflect enhanced restriction-modification in this species rather than host range. Other plasmids, such as pCFV108, have been demonstrated also to possess a narrower host range (Kienesberger et al., 2007). Megaplasmids Potentially, the most interesting of the Campylobacter plasmid types are the megaplasmids. This is due to their much greater size, with a concomitant larger number of CDSs, and their similarity to characterized Campylobacter genomic islands (Fouts et al., 2005). Unfortunately, sequence information exists for only four NJC megaplasmids: the 44.7-kb C. coli megaplasmid pCC3 1 (Batchelor et al., 2004) and the three megaplasmids described by Fouts et al. (2005): the 180-kb C. coli megaplasmid pCC22284, the 110-kb C. upsaliensis megaplasmid pCU3 1952, and the 46-kb C. lari megaplasmid pCL2100. All four megaplasmids encode homologs of the Tra type IV secretionlplasmid conjugation proteins, termed Cmg (Campylobacter mating genes) by Batchelor et al. (2004), or other proteins- involved in plasmid conjugation and/or DNA transfer. In addition, pCC2228-4 and pCC3 1 encode antibiotic resistance genes: both plasmids encode the tetracycline resistance gene tetO, and pCC2228-4 encodes putative kanamycin (kan) and hygromycin-B (hyg) resistance genes. The tetO gene in both plasmids resides in an area of high G+C content (39 to 40%), suggesting

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MILLER

lateral gene transfer (Batchelor et al., 2004). However, the TetO protein of pCC2228-4 is only 93% identical to the TetO protein of pCC3 1 and is almost identical (99% identity) to the TetO protein of C. jejuni subsp. jejuni 81-176 megaplasmid pTet. Intriguingly, the pCC2228-4 kan and hyg genes are contained within a putative mobile element similar to clostridial transposons (see below). Additionally, pCC2228-4 contains three identical copies of an IS, ISCcol. The remainder of the genes contained on the four Campylobacter megaplasmids encodes mainly proteins of unknown function. Nevertheless, several of these genes have homologs in Epsilonproteobacterial or other eubacterial taxa or, significantly, homologs within Campylobacter genomic islands (Fouts et al., 2005). pCC31 contains several genes homologous to genes from the Actinobacillus actinomycetemcomitans plasmid pVT745 (Batchelor et al., 2004), and pCC2228-4 contains genes homologous to genes from Vibrio spp. It is likely that the four NJC megaplasmids described here represent only a small subset of the total NJC megaplasmids. It is also likely that the gene content of NJC megaplasmids is modulated by lateral gene transfer and the presence of repeated sequences and mobile elements. Recombination between repeat sequences and mobile elements would result necessarily in a more modular megaplasmid structure, leading to new combinations of genes. This is especially true with pCC2228-4, which contains four mobile elements and where approximately 25% of the megaplasmid is composed of repeated sequences.

HOMOPOLYMERIC G:C TRACTS, CONTINGENCY GENES, PSEUDOGENES, AND MISMATCH REPAIR Homopolymeric G:C tracts have been identified in all of the NJC genomes (Fouts et al., 2005) (Table 1). The number of G:C tracts 8 bp or longer ranges from nine in C. cuwus 525.92 to at least 75 in C. upsaliensis RM3195. The average length of these tracts is similar to those reported in C. jejuni subsp. jejuni (approximately 9 to 10 bp) with the exception of C. upsaliensis; homopolymeric G:C tracts in C. upsaliensis RM3195 are as long as 19 bp and average nearly 14 bp in length. Neither the length nor number of homopolymeric G:C tracts in the NJC genomes is proportional to the G+C content of the genome; C. cuwus 525.92 has the fewest number of G:C tracts but has the highest G+C content (44.5%; Table 1).Similar to homopolymeric G:C tracts in C. jejuni subsp. jejuni (Gilbert et al., 2005; Miller et al., 2005b; Parkhill et al., 2000), homopolymeric G:C

tracts within the NJC genomes are located often within genes involved in synthesis of surface structures and restriction/modification genes. For example, 46 of 74 of the C. jejuni subsp. doylei 269.97 homopolymeric G:C tracts are located within genes related to surface structure/restriction-modification. Hypervariability at these loci has been demonstrated to lead to changes in surface structures (Guerry et al., 2002; Karlyshev et al., 2005; Linton et al., 2000). It is likely that hypervariable G:C tracts in the NJC genomes would lead to similar changes in phenotype. Contingency (phase-variable) genes, characterized by hypervariable G:C tracts, have been identified in the C. coli, C. lari, and C. upsaliensis genomes (Fouts et al., 2005); no data are available for the remaining NJC genomes. Although putative contingency genes within the thermophilic NJC genomes have been identified, the proportion of these genes with respect to total G:C tract-containing genes is small. Identification of contingency genes within the NJC genomes has relied on a sufficient level of sequencing coverage. Tracts with a low level of coverage (e.g., two to three reads) may appear stable simply because not enough data are available to detect hypervariability. Thus, additional experiments will be necessary to determine whether each homopolymeric tract is stable or hypervariable. Additionally, it is possible that some tracts may be hypervariable only under certain environmental conditions. Therefore, tracts that appear stable after growth on laboratory media may become hypervariable when the strain is inoculated into a host. Finally, stability of homopolymeric tracts can vary between strains. This was demonstrated in C. jejuni subsp. jejuni 30 and RM1516, where the homopolymeric G:C tracts within identical hsdS genes were stable in strain 30 and hypervariable in strain RM1516 (Miller et al., 2005b). Several genes within the NJC genomes are frameshifted or have other defects and are considered putative pseudogenes (Table 1).Many of these frameshifts are centered on a homopolymeric G:C tract; thus, it is possible that these G: C-tract-containing pseudogenes are phase variable. As before, additional investigations will be necessary to determine the nature of these putative contingency genes. Despite the presence of putative contingency genes within the population of pseudogenes, contained within each NJC genome are gene fragments or genes with a combination of authentic (nonhypervariable) frameshifts and point mutations. The C. jejuni subsp. doylei 269.97 genome is predicted to contain 125 pseudogenes. Twenty-nine of these pseudogenes in strain 269.97 contain homopolymeric G:C tracts and are potentially phase variable. The remainder are related

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COMPARATIVE GENOMICS OF NON-C. TETUNI CAMPYLOBACTER SPP.

to genes encoding proteins involved in signal transduction (e.g., methyl-accepting chemotaxis proteins), transport (e.g., feoB), restriction/modification (e.g., hsd), and metabolism (e.g., pro& sdhABC, and nap). In two instances, the origin of pseudogenes in strain 269.97 can be ascribed to insertional inactivation, either via a genomic island or an IS element. However, there are no definite data on why C. jejuni subsp. doylei 269.97 contains so many putative pseudogenes relative to the other NJC genomes. The genome of strain 269.97 does contain 74 homopolymeric G:C tracts. Indeed, there is a rough correlation between the number of G:C tracts and the number of pseudogenes. However, these tracts account only for 23% of the total pseudogenes in C. jejuni subsp. doylei 269.97. It is more likely that a root cause exists that accounts for the presence of both pseudogenes and contingency genes; this cause is possibly related to DNA repair. An unexpected feature of the A. butzleri RM4018 genome was the absence of homopolymeric G:C tracts (Table 1; Miller et al., 2 0 0 7 ~ a) ~feature shared also by the genome of the related S. denitrificans. Therefore, comparison of the A. butzleri and S. denitrificans genomes to other genomes of the epsilon subdivision would perhaps permit construction of a model describing the formation of homopolymeric G:C tracts. Analysis of these two genomes revealed the presence of two to four members of the DnaQ superfamily (Miller et al., 2 0 0 7 ~ genes ) ~ that were absent in the other Epsilonproteobacterial genomes. The DnaQ superfamily includes the 3 ’ 4 5 ’ exonuclease ExoX (Viswanathan and Lovett, 1999). The 3 ’ 4 5 ’ exonucleases are key components of the methyl-directed mismatch repair (MMR) system. MMR depends on three functions: the MutSLH endonuclease complex, chromosomal methylation (to identify parental/daughter strands), and multiple 5’-3 ’ and 3 ‘ 4 5 ’ single-strand DNA exonucleases (Iyer et al., 2006). Although genes encoding 5 ’ 4 3 ’ single-strand DNA exonucleases (e.g., recJ) have been identified in Campylobacter and Helicobacter, no protein with predicted 3 ’ 4 5 ’ exonuclease activity has been identified. Multiple mutations in the Escherichia coli 3 ‘ 4 5 ’ exonucleases resulted in a 32fold increase in +1 frameshifts and an 11-fold increase in -1 frameshifts (Viswanathan and Lovett, 1998). Therefore, it is possible that the presence of homopolymeric G:C tracts can be explained in part by the state of MMR in an organism: organisms such as A. butzleri RM4018, which are predicted to encode both 5 ’ 4 3 ’ and 3 ’ 4 . 5 ’ single-strand DNA exonucleases, would be predicted to contain a fully functional MMR system.

79

GENOME STRUCTURE Genomic Colinearity between the Campylobacter Genomes Total genomic colinearity between C. jejuni subsp. jejuni and the NJC genomes is quite low. The exceptions are the C. coli RM2228 and C. jejuni subsp. doylei 269.97 genomes. The RM2228 and C. jejuni subsp. jejuni NCTC 11168 genomes are remarkably colinear (Fig. 2A), with only a small number of inversions in the RM2228 genome relative to the NCTC 11168 genome. Only two of these are of any substantial size and are clustered near the predicted origin of replication (approximately bp 1).The C. jejuni subsp. doylei 269.97 genome contains a large genomic inversion, relative to the NCTC 11168 genome, bounded by IS elements (Fig. 2B); otherwise, the C. jejuni subsp. doylei and C. jejuni subsp. jejuni genomes would be largely colinear. For the remainder of the NJC genomes, pairwise comparisons of colinearity resemble Fig. 2C, in which the gene orders of C. lari RM2100 and C. jejuni subsp. jejuni NCTC 11168 were compared, where no obvious large syntenic regions exist. Although overall colinearity between the NJC genomes is generally low, pockets of localized synteny can be identified within the genomes. Such syntenic pockets represent clusters of genes that remain linked within all members of the genus. Analysis of the Campylobacter genomes identified 44 clusters of colinear genes with a minimum size of five CDSs (Table 2). These clusters range in size from 5 to 21 CDSs, with an average size of 8 CDSs. In some cases, these clusters contain genes that encode similar functions or components of a single pathway. For example, cluster 18 encodes tricarboxylic acid (TCA) cycle enzymes, and cluster 44 encodes SecY and 20 ribosomal proteins. However, for most of the clusters, no common biological function can be defined, and it is unclear why the genes in these clusters remain linked throughout the genus. The total number of genes present- in these clusters is 357, indicating that the majority of Campylobacter genes are not located in syntenic clusters and are not tightly linked. As with the core protein set, the C. concisus and C. hominis genomes were not analyzed for localized synteny; therefore, it is likely that some of these 44 clusters will be absent from future lists that include additional Campylobacter genomes. Bacteriophage Mu (CMLP1 and AMLP1) The bacteriophage Mu-like element CMLPl (Campylobacter Mu-like - _ phage) was identified first in jejuni subsp. jejuni R&i1221(Fouts et al., 2005).

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C. jejuni NCTC 11168

1800 b

1600

Q)

6

1400

(0 (v

1200

.-

2

3 1000

D

.-c 2 .%

'

800 600 400 200

0

C.jejuni NCTC 11168

C

C. jejuniNCTC 11168

Figure 2. Pairwise dot plot comparisons of selected Cumpylobucter genomes. Each protein in the C. jejuni subsp. jejuni NCTC 11168 proteome was compared with the proteomes of C. coli RM2228 (A), C. jejuni subsp. doylei 269.97 ( B ) , and C. luri RM2100 (C) by BLASTP analysis. Each dot represents a protein with significant homology, using the parameters from Fig. 1, to a C. jejuni subsp. jejuni NCTC 11168 protein. The x and y axis values represent gene numbers.

Amplification of other NJC strains with a MuB primer set has detected CMLP-related elements in other Campylobacter species, such as C. coli, C. lari, C. upsaliensis, C. helveticus, C. fetus subsp. fetus, C. mucosalis, C. hyointestinalis, C. concisus, and C. curvus

(data not shown), although none of the sequenced NJC genomes is predicted to contain a CMLP-related element. CMLP-related elements have also been identified in non-Campylobacter Epsilonproteobacterial species, e.g., Helicobacter bilis, A. butzleri, A. skirro-

CHAPTER 5

COMPARATIVE GENOMICS OF NON-C. TETUNI CAMPYLOBACTER SPP.

81

Table 2. Localized regions of synteny within Campylobacter Avg. aa identity (vs Cjj)" Region no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

Locus CjOO59c-CjOO68 CjOO98-CjOll8 CjOl23c-CjO12 7c CjOl29c-CjO138 CjO147c-CjO155c CjO156c-CjO166 CjOl86c-CjO193c CjO232c-CjO236c Cj0273-CjO2 77 Cj0281c-CjO288c CjO317-CjO322 CjO328c-CjO333c CjO365c-CjO369c CjO394c-CjO405 CjO472-CjO4 79 CjO495-CjO499 Cj0509c-CjO5 16 Cj0531-el0538 CjO539-CjO545 CjO580c-CjO590 Cj0595c-CjO600 CjO63Oc-CjO635 CjO639c-CjO652 CjO699c-CjO714 CjO795c-CjO8OOc Cj080l-Cj0807 Cj089 1c-CjO897c CjO925-CjO930 CjO954c-CjO96 1c CjO992c-CjO997 CjlO56c-CjlO62 CjlO9Oc-CjlO95 CjlO99-CjllO5 Cjl123c-Cjl127c Cjll75c-Cjll82c Cj1226c-Cj123 1 Cjl27Oc-Cjl282 Cjl366c-Cjl3 70 Cjl487c-Cjl495c Cjl592-Cj1596 Cjl606c-Cjl610 Cjl634c-Cjl638 Cjl639-Cjl645 Cjl688c-Cjl708c

No. of genes 10 21 5 10 9 10 8 5 5 8 6

6 5 12 8 5 8 8 7 11 6 6 14 16 6 7 7 6 8 6 7 6 7 5 8 6 12 5 9 5 5 5 7 21

Cjd

Cc

Cl

Cu

Cff

Cur

97 98 98 97 98 98 98 98 99 98 97 98 97 98 99 96 97 99 97 96 98 97 98 98 97 98 97 98 98 96 99 97 98 96 98 98 99 99 98 99 99 98 99 99

88 87 86 83 82 81 87 88 91 89 87 90 85 80 97 80 83 96 83 78 86 84 82 85 80 87 86 85 84 84 88 89 79 87 86 92 85 88 85 98 86 85 87 98

71 70 63 72 69 63 77 82 80 76 73 81 59 65 88 56 69 83 67 66 72 66 63 76 58 74 68 75 68 70 74 71 68 65 70 73 71 68 77 93 68 66 70 91

83 80 73 74 75 67 77 82 84 77 79 85 69 72 93 68 77 91 72 71 76 68 72 81 71 77 76 78 74 71 76 83 71 72 75 80 79 75 77 94 75 71 75 93

60 57 52 58 60 50 62 60 67 61 61 70 47 55 81 50 59 63 51 56 59 54 52 66 52 63 58 68 57 58 58 59 55 53 58 62 60 59 63 86 57 55 54 82

58 58 53 59 65 48 62 66 69 65 60 70 46 56 82 51 60 60 50 56 54 57 52 66 50 63 61 69 59 59 64 57 56 53 58 61 61 60 61 85 56 54 55 83

amino acid; Cjj, Campylobacter jejuni subsp. jejuni; Cjd, C. jejuni subsp. doylei; Cc, C. coli; Cl, C. lari; Cu, C. upsaliensis; Cfk C. fetus subsp. fetus; Cur, C. cuwus.

aaa,

wii, and A. cryaerophilus (data not shown). The genome of the A. butzleri RM4018 contains a Mu-like phage, termed AMLP1 (Arcobacter -Mu-like phage) (Miller et al., 2 0 0 7 ~ ) Al&ough . several of th; proteins encoded by AMLP1 have homologs within CMLP1, there are differences between the two phages, such as the absence of the CMLP1 extracellular DNase in AMLP1. DNA microarray analysis and preliminary sequence analysis of other CMLP elements also indicate differences in gene content be-

tween the CMLP elements (Parker et al., 2006; data not shown), suggesting that CMLPl and AMLP1 are just two members of a much larger family of Mu-like bacteriophage prevalent within the Epsilonproteobacteria. Other Prophage Bacteriophage Mu-like elements are identified in bacterial genomes as a result of the presence of sev-

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era1 well-conserved components, such as the genes that encode the Mu transposase. However, members of other less well-characterized prophage families can be difficult to identify, and confirmation of such prophage relies often on the presence of a few phagerelated genes and one or two putative capsid genes. Moreover, a protein with low similarity to a phagerelated protein does not always signify a bacteriophage origin. Thus, for several of the NJC integrated elements, additional investigations will be necessary ( e g , bacteriophage induction) before an element can be identified definitively as a prophage. Nevertheless, the integrated element CLIEl (Campylobacter -lari -integrated element) in C. lari m 2 l O O is almost certainly a limbdoid-type bacteriophage, similar to the element CJIE4 (Campylobacter jejuni integrated element) in C. j e j u s subsp. jejuni-RM1221. Additionally, one of the integrated elements in C. jejuni subsp. doylei 269.97 may be a prophage, although this label is more tenuous.

ulence functions were identified within these genome islands. In fact, no biological role has been ascribed to any of the nonprophage Campylobacter genomic islands. However, two NJC genomic islands are particularly noteworthy: the C. curuus 525.92 element CURIE2 (Curvus integrated element) and the C. jejuni subspxoylei 269.97 eiement DIE6. CURIE2 contains the sole Type I R-M locus in C. curvus 525.92, suggesting that this R-M locus was acquired through lateral transfer. This is similar to A. butzleri RM4018, in which the only DNA methyltransferase present in the genome is contained within a genomic island (Miller et al., 2 0 0 7 ~ )DIE6 . is a clear representation of lateral gene transfer between C. upsaliensis and C. jejuni subsp. doylei. DIE6 contains 26 CDSs, all encoding proteins with homologs of high similarity (? = 97.5% identity) to proteins of C. upsaliensis RM3195. The C. upsaliensis CDSs are colinear with the C. jejuni subsp. doylei CDSs and are predicted to encode mainly glycosyltransferases and other proteins involved in capsular biosynthesis.

Genomic Islands/Integrated Plasmids

CFUSPR Elements

As with identification of prophage, the distinction between genomic island and integrated plasmid can be subtle and not discerned readily. Certainly, the presence of plasmid-related genes, such as the plasmid-conjugation-related tra genes, implies a plasmid origin. One such integrated plasmid is the element DIE4 (Doylei integrated element), which contains at least Tl tra genes. Integrated plasmids can be identified also through homology to characterized megaplasmids, as was the case with the C. jejuni subsp. jejuni RM1221 element CJIE3 (Fouts et al., 2005). However, the remaining NJC genomic islands do not have significant homology to existing characterized plasmids, indicating either that these elements have a nonplasmid origin or that the ancestor plasmids of these islands have not yet been identified. In the NJC genomes, genomic islands have been identified in C. jejuni subsp. doylei, C. upsaliensis, C. fetus subsp. fetus, and C. curvus. Consistent with the integrated elements of C. jejuni subsp. jejuni RM1221 (Fouts et al., 2005), many NJC genomic islands contain a phage-related integrase/ recombinase-encoding gene on one end, insertion points in or in close proximity to tRNAs (or the tmRNA in DIE1) and terminal repeats of 15 to 32 bp. The size of the NJC genomic islands ranges from 7 CDSs in C. fetus subsp. fetus 82-40 to DIE4, which contains 52 CDSs. Unfortunately, the NJC genomic islands are predicted to mostly encode conserved hypothetical proteins of unknown or at best general function. No drug-resistance genes or genes encoding putative vir-

CRISPRs (clustered regularly interspaced short palindromic repeats) have been identified in -40% of the bacterial and archaeal genomes (Kunin et al., 2007). These repeats are generally 25 to 50 bp in length and are separated by nonrepetitive spacer sequences of similar length. The spacer sequences are often unique, and spacers in some CRISPRs have been demonstrated to be of extrachromosomal origin, i.e., phage and plasmids (Bolotin et al., 2005). CRISPR-associated (Cas) protein families are present only in CRISPR-containing strains and are always found adjacent to a CRISPR (Haft et al., 2005). CRISPR elements have been organized into at least eight different subtypes (Haft et al., 2005) on the basis of factors such as gene composition, repeat length, and Cas sequence similarity. In addition to the Cas proteins (Casl-7), other CRISPR-associated protein families unique to particular CRISPR subtypes, such as Csn, Csm, Cmr, and Csh, have also been identified (Haft et al., 2005). CRISPR elements of the Nmeni subtype (Haft et al., 2005) have been identified in C. jejuni subsp. jejuni (Fouts et al., 2005; Schouls et al., 2003). Similarly, C. jejuni subsp. doylei 269.97 contains also an Nmeni-subtype CRISPR element. These elements have repeat lengths of 36 bp and contain only Casl, Cas2, and the CRISPR-associated protein Csnl. Although the C. jejuni subsp. doylei CRISPR repeat sequences are identical to those of C. jejuni subsp. jejuni NCTC 11168 and RM1221, the spacer sequences are unique, and the C. jejuni subsp. doylei CRISPR contains six repeats instead of the five pres-

CHAPTER 5

COMPARATIVE GENOMICS OF NON-C. 7ElUNl CAMPYLOBACTER SPP.

ent in strain NCTC 11168 and the four present in strain RM1221. No CRISPR elements were identified in the genomes of C. coli RM2228, C. lari RM2100, and C. upsaliensis RM3195 (Fouts et al., 2005). However, CRISPRs are present in the genomes of C. curvus 525.92 and C. fetus subsp. fetus 82-40. In fact, C. cuwus 525.92 contains two CRISPRs, and the repeat sequences of the two CRISPRs are different. The subtype of the first cannot be identified because of the absence of associated Cas proteins; this CRISPR contains nine repeats of 30 bp with an average spacer length of 36.4 bp. The second CRISPR element is of the Hmari subtype and contains four repeats of 25 bp and an average spacer length of 40.7 bp. This subtype is defined by the presence of Cas proteins Casl6 and two additional CRISPR-associated proteins, Cshl and Csh2. The C. fetus subsp. fetus CRISPR element is highly unusual. This element is bounded by two CRISPR arrays with an additional repeat sequence two genes downstream. The left CRISPR array contains 22 repeats, and the right CRISPR array contains 27 repeats. All of the repeats, including the downstream singlet, have identical 3 0-bp sequences, and the spacers within the arrays are 34 to 37 bp, with an average length of 35.9 bp. Another unusual feature is the size (22 CDSs) of the element and the organization and identities of the CRISPR-associated proteins: Cas proteins Casl-6 are linked to the right CRISPR array; however, several phage-related proteins, including a putative capsid protein, are linked to the left CRISPR array. The C. fetus subsp. fetus CRISPR element subtype is undefined and perhaps novel; however, BLASTP analysis indicates that the Cas proteins are similar to clostridial Cas proteins (e.g., C. kluyveri, C. novyi, and C. tetani) and the Cas proteins of Syntrophomonas wolfei. The presence of phage-related genes within the C. fetus subsp. fetus 82-40 CRISPR element is intriguing, considering that CRISPR elements have been demonstrated to provide resistance to bacteriophage in Streptococcus therrnophilus (Barrangou et al., 2007). In S. therrnophilus, phage resistance was demonstrated to be conferred by the addition of one to four novel spacers (with concomitant addition of repeats) to the internal (Cas-linked) end of the CRISPR array (Barrangou et al., 2007). These novel spacers are identical to phage sequences, and the presence of a spacer sequence identical to a phage sequence provides resistance against bacteriophage that contain that spacer sequence (Barrangou et al., 2007). It is possible that the Cas proteins serve to degrade bacteriophage DNA and insert small fragments as novel CRISPR spacers. In this case, the phage proteins in the C. fetus subsp. fetus CRISPR

83

element may have resulted from an error in which a larger than normal bacteriophage fragment was inserted. Another possibility is that the phage sequences originated via recombination between the terminal spacer sequence and a bacteriophage, although the small size of the spacer makes this unlikely. BLASTN analysis of the C. fetus subsp. fetus spacer sequences did not identify any homologous sequences within the NCBI nr database. It should prove interesting to characterize both additional C. fetus subsp. fetus CRISPR elements and C. fetus subsp. fetus bacteriophage and investigate phage resistance in this organism. ISs and Transposons The genomes of H. acinonychis Sheeba, W. succinogenes DSM 1740, and S. denitrificans ATCC 33889, as well as all three sequenced H. pylori genomes, are predicted to contain several ISs. However, the genomes of C. jejuni subsp. jejuni NCTC 11168 and RM1221 do not contain any IS elements, outside of a degenerate IS606-like element adjacent to the 5 s rRNA gene of rrnC. The genomes of C. lari RM2100, C. upsaliensis RM3195, C. fetus subsp. fetus 82-40, and C. curvus 525.92 also do not contain IS elements, suggesting that IS elements, although common within the Epsilonproteobacteria, are rare in Campylobacter. Nevertheless, IS elements have been identified in two Carnpylobacter genomes: C. jejuni subsp. doylei 269.97 and C. coli RM2228. Three copies of an IS60.5-like element (Kersulyte et al., 1998), termed here ISCjdl, are present in the C. jejuni subsp. doylei 269.97 genome. All three copies of ISCjdl are identical at the nucleotide level and are predicted to encode two transposases: the 12.3kDa IS200-like TnpA and the 47.7-kDa IS1341-like TnpB. ISCjdl is 1,851 bp, and its right end is similar (12/14 bp) to the right end of IS60.5; also, the target sequence (5’-TTTAAA) is similar to that of IS605 (Kersulyte et al., 1998). The orientation of ISCjd1-1 is reversed relative to the orientation of the other two elements. C. jejuni subsp. doylei 269.97 contains a large chromosomal inversion whose breakpoints are bounded by elements ISCjdl-1 and ISCjd1-3 (Fig. 2). Because these two elements have opposite orientations, it is likely that this inversion arose via homologous recombination between these IS elements. Similar IS60.5-mediated genomic rearrangements have been observed in H. pylori (Kersulyte et al., 1998). The C. coli RM2228 contains six copies of a novel 1,870-bpY IS606-related IS termed ISCcol (Fouts et al., 2005). Three copies are located on the chromosome; the other three copies are located on the 180-kb megaplasmid pCC2228-4. All six copies

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are nearly identical at the nucleotide level; however, two of the genomic copies contain identical 344-bp deletions within tnpA. Although IS606 and ISCcol share a common target sequence (5’-TTTAT) and have nearly identical (12/13 nt) right ends, the two ISs differ in the orientation of the transposase genes: tnpA and tnpB are transcribed divergently in IS606 and transcribed in the same direction in ISCcol. In addition to the IS elements present in the C. coli RM2228 genome and the megaplasmid pCC2228-4, pCC2228-4 is predicted to contain a mobilizable transposon similar to the clostridial transposons Tn44.52 and Tn44.53 (Adams, 2002). Mobilizable transposons encode mobilization functions but are not conjugative, relying on conjugative machinery provided in trans to transfer into recipient strains. Mobilizable transposons contain a resolvase rather than a transposase and contain a transfer origin, oriT, within the transposon; transposition proceeds through a nonreplicative circular intermediate (Adams, 2002). The putative mobilizable transposon of pCC2228-4 contains homologs of the clostridial proteins TnpX (resolvase), TnpV (excisionase), and TnpY in addition to a MobA/MobL family mobilization protein. The mobilizable transposons of Clostridium often contain antibiotic resistance genes, e.g., Tn44.51 and Tn4453 confer chloramphenicol resistance (Adams, 2002). Similarly, the C. coli mobilizable transposon contains two antibiotic resistance genes: aphA-3, which confers resistance to kanamycin, and an aminoglycoside phosphotransferase gene similar to an E. coli hygromycin-B resistance gene. The transposon encodes also a putative pyroglutamyl peptidase. The presence of identical copies of ISCcol on the strain RM2228 chromosome and megaplasmid suggests that Hfr (high frequency of recombination) strain formation may be possible wGhin Campylobacter. In E. coli, the conjugative F episome cointegrates into the chromosome via homologous recombination between IS elements common to both the chromosome and the F plasmid, resulting in an Hfr strain. Because the F episome contains an oriT transfer origin, conjugation between the Hfr strain and a suitable recipient generally leads to transfer of chromosomal sequences in addition to plasmid sequences. Thus, analogous Hfr formation in Campylobacter would result necessarily in significant lateral gene transfer between Campylobacter strains and perhaps different Campylobacter species. Therefore, Campylobacter IS elements would be predicted to affect greatly genome structure through both IS-mediated inversions/deletions and Hfr-mediated large-scale transfer of genetic material.

EVOLUTION AND PHYLOGENY Comparisons of multiple related genomes within a taxon can lead to insights about the evolution of that taxon and the formation of specific lineages or insights about the origin of particular species or subspecies. An example of metabolic lineages identified from comparing the Campylobacter genomes are the three dihydroorotase (DHOase) lineages identified within the Epsilonproteobacteria. DHOase is encoded by pyrC and catalyzes the second step in pyrimidine biosynthesis. In mammalian cells, DHOase is one domain of the CAD multifunctional protein, along with carbamoyl phosphate synthetase and aspartate transcarbamoylase (ATCase) (McPhail and Shepherdson, 2006). In some bacterial taxa, DHOase is a homodimer, not associated with ATCase (Brichta et al., 2004). Bacteria often contain two pyrC genes, encoding the type I1 homodimeric DHOase and a defective type I DHOase related to the multifunctional complex enzyme and demonstrated to associate with ATCase (Schurr et al., 1995). The defective DHOases lack a histidine zinc-binding domain critical for enzymatic activity (Schurr et al., 1995). The Campylobacter genomes are predicted to encode both the type I1 and nonfunctional type I DHOases, with the exception of C. fetus, C. cuwus, and C. concisus, which encode only a type I DHOase. Unlike the DHOases of the other campylobacters, these type I DHOases contain both zinc-binding domains and are presumably functional. Also, consistent with the identification of a functional ATCaseassociated DHOase, pyrC is linked in these three genomes to pyrB, which encodes the ATCase. Thus, two separate DHOase lineages are present within Campylobacter; all of the sequenced Helicobacteraceae genomes are predicted to encode two pyrC genes and would be members of the lineage that includes the thermophilic campylobacters. A third DHOase lineage is exemplified by the A. butzleri RM4018 genome that encodes only the homodimeric type I1 DHOase. Members of this lineage include the deepsea-vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. The biological significance of these three lineages is unclear. It has been proposed that functional type I DHOases provide thermal protection for unstable intermediates and permit growth at higher temperatures (McPhail and Shepherdson, 2006). However, this appears unlikely in Campylobacter because members of the C. fetus DHOase lineage generally grow at lower temperatures than the thermophilic campylobacters. Genomic comparisons and analysis can sometimes lead to new perspectives regarding the origin of species or subspecies. One such example is the or-

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COMPARATIVE GENOMICS OF NON-C. 7E7UNI CAMPYLOBACTER SPP.

igin of C. jejuni subsp. doylei. The defining characteristic of C. jejuni subsp. doylei is the absence of nitrate reductase activity (Steele and Owen, 1988). DNA microarray analysis and genomic sequencing revealed defects in the nitrate reductase locus nap, specifically in the two genes encoding the large and small subunits of nitrate reductase, NapA and NapB, respectively (Miller et al., 2007b). In eight C. jejuni subsp. doylei strains tested, all napA loci contained a deletion, and the napB loci of seven strains contained either deletions or point mutations leading to truncated proteins. The napA loci of an additional 19 C. jejuni subsp. doylei isolates also contained deletions. Unexpectedly, the size of the napA deletions in all 27 C. jejuni subsp. doylei isolates was identical (2,761 bp) with identical endpoints. The presence of these identical napA deletions, identified in strains isolated across four continents, suggests that C. jejuni subsp. doylei arose from C. jejuni subsp. jejuni as a result of a single evolutionary event. Divergence at napB occurred subsequently as the constraint maintaining an active nitrate reductase was removed with the loss of NapA. The putative C. jejuni subsp. jejuni ancestor of C. jejuni subsp. doylei has not been identified.

METABOLISM AND RESPIRATION Embden-Meyerhof-Parnas and EntnerDoudoroff Pathways Members of the genus Campylobacter cannot utilize sugars as a sole carbon source. The inability to metabolize sugars can be traced to the absence of two key components of the Embden-Meyerhof-Parnas (EMP or glycolytic) pathway. The first component is the phosphoenolpyruvate-dependent phosphotransferase system, which simultaneously transports and phosphorylates sugars. The second component is phosphofructokinase, which catalyzes the conversion of fructose-6-phosphate to fructose-1,6 bisphosphate. Some members of the Helicobacteraceae, e.g., H. acinonychis and H. pylori (but not H. hepaticus), contain enzymes of the Entner-Doudoroff (ED) pathway. Like the EMP pathway, the ED pathway converts glucose-6-phosphate to glyceraldehyde-3-phosphate but utilizes a gluconate-6-phosphate intermediate rather than a fructose-6-phosphate intermediate. Thus, the ED pathway obviates the need for phosphofructokinase. Helicobacters are also missing the terminal step of the EMP pathway catalyzed by pyruvate kinase. However, the ED pathway produces also pyruvate in addition to glyceraldehyde-3-phosphate. Thus, the presence of the ED pathway in H. pylori, in addition to a predicted hexose transporter and glucokinase, would indicate that H. pylori can

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utilize glucose as a sole carbon source; glucose utilization in H. pylori was demonstrated by Mendz et al. (1993). In addition to the EMP enzymes conserved throughout Campylobacter, C. jejuni subsp. doylei 269.97 is predicted to encode all of the ED pathway enzymes. In addition, this strain is predicted to encode a putative glucose/hexose transporter and a glucokinase homologous to proteins from Helicobacter. Therefore, the presence of these latter two proteins plus the combined enzymes of the EMP and ED pathways would suggest that C. jejuni subsp. doylei 269.97, like H. pylori, can utilize at least glucose as a sole carbon source. However, no data are available to indicate that C. jejuni subsp. doylei can utilize glucose or other carbohydrates. A surprising feature of the ED pathway in C. jejuni subsp. doylei is that the pathway-encoding genes are organized in a cluster and located between the isoleucinyl-tRNA and 23s rRNA genes in the rrnB rRNA locus. The presence of these Helicobacter-related genes in C. jejuni subsp. doylei suggests that this cluster was acquired from a member of this genus. Helicobacter strains that possess the ED pathway also contain a phosphoglucose isomerase (PGI) distinct from those found in the other Epsilonproteobacteria. These PGI proteins are similar to those from multiple bacterial taxa, including the Gammaproteobacteria, whereas the PGI proteins from Campylobacter, Arcobacter, and H. hepaticus are similar to Thermotoga and archaeal taxa. c. jejuni subsp. doylei 269.97 is predicted to encode both PGI types. Another example of dichotomy in the EMP enzymes is represented by fructose bisphosphate aldolase (FBA). Curiously, however, the Helicobacter FBAs are members of the type IIB group, which includes FBAs from Thermotoga and archaeal taxa, whereas Campylobacter FBAs (including C. jejuni subsp. doylei FBA) are members of the type IIA group, which includes FBAs from the Gammaproteobacteria. TCA Cycle

A complete oxidative tricarboxylic acid (TCA) cycle is present in C. jejuni subsp. jejuni, as evidenced by the presence of all three subunits of succinate dehydrogenase (encoded by sdhABC). The genome of C. jejuni subsp. doylei 269.97 contains the sdhABC genes. However, these genes are heavily fragmented and are unlikely to be functional. Genes encoding succinate dehydrogenase are absent from the C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus genomes; however, succinate dehydrogenase is predicted to be encoded by both C. cuwus and C. concisus. Thus, complete oxidative TCA cycles would be

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predicted to be present in these latter two species and C. jejuni subsp. jejuni, whereas C. coli, C. upsaliensis, and C. fetus subsp. fetus would be predicted to contain a branched noncyclic pathway, similar to that present within H. pylori (Marais et al., 1999). Campylobacter lari RM2100 does not encode citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, or succinyl-CoA synthetase (W. G. Miller et al., unpublished data). Therefore, it would appear that the TCA cycle is missing from this organism. The absence of &A, which encodes citrate synthase, was noted first during the development of an MLST method for C. lari (Miller et al., 2005a) because gltA is a member of the C. jejuni subsp. jejuni MLST gene set. Also, the absence of gltA was noted during development of an MLST method for the related species C. insulaenigrae (Stoddard et al., 2007), suggesting that the TCA cycle may be absent in this species as well. Campylobacter lari RM2100 is predicted to encode pyruvate carboxylase, malate dehydrogenase, fumarase, and fumarate reductase. Therefore, in strain RM2100, pyruvate can be interconverted into oxaloacetate, malate, fumarate, and succinate, presumably to provide metabolic precursors. Formation of acetyl-CoA via oxidative decarboxylation of pyruvate is catalyzed in Campylobacter by a pyruvate: flavodoxin oxidoreductase (Daucher and Kreig, 1995). Pyruvate:flavodoxin oxidoreductases are usually oxygen labile, and it has been proposed that this enzyme contributes in part to the microaerophily of Campylobacter (Kelly, 2005). Instead of pyruvate:flavodoxin oxidoreductase, the genome of the aerobic microorganism A. butzleri RM4018 is predicted also to encode all three subunits of pyruvate dehydrogenase, an enzyme found often in aerobic bacteria. Additionally, strain RM4018 is predicted to encode two fumarases: fumarase C, encoded also by Campylobacter and Helicobacter, and fumarase A. Fumarase A is an iron-sulfur-clustercontaining enzyme that is unstable under aerobic conditions, whereas fumarase C is stable in the presence of oxygen. The presence of TCA cycle enzymes with different oxygen labilities suggests that A. butzleri RM4018 is able to grow at a much wider range of oxygen concentrations than either Campylobacter or Helicobacter. Indeed, A. butzleri RM4018 has been demonstrated to grow anaerobically, microaerobically, and aerobically (Miller et al., 2 0 0 7 ~ ) . Respiration On the basis of the different environmental niches inhabited by the NJC genomes, it might be predicted that the respiratory pathways present in

these strains would be substantially diverse. Indeed, comparative analysis of the NJC genomes reveals a large degree of variation in the electron transport pathways. Most of the respiratory proteins described in C. jejuni subsp. jejuni NCTC 11168 (Kelly, 2005) are present also in the NJC genomes. The respiratory pathways of C. jejuni subsp. doylei 269.97 are the most limited among the characterized NJC genomes, and the pathways of C. curvus 525.92 are the most extensive. Besides the aforementioned deletions at the nitrate reductase (nap) locus, C. jejuni subsp. doylei 269.97 is also not predicted to encode the trimethyl amine-N-oxide (TMAO) reductase TorA (homologous to CjO264c). Unlike the nap locus, loss of torA is not due to genomic deletions but rather due to multiple frameshifts, resulting in a presumably nonfunctional protein. Intriguingly, both cytochrome c peroxidases, homologs of Cj0020 and Cj0358, are also present in strain 269.97 as pseudogenes. Although C. jejuni subsp. doylei 269.97 does not reduce nitrate, it is predicted to encode the NrfAH nitrite reductase. The genome of C. curvus 525.92 is predicted to encode several electron acceptors novel to Campylobacter. Two proteins are predicted members of the hybrid-cluster protein/prismane family; members of this family have two Fe/S clusters: a [4Fe-4S] cubane cluster and a hybrid [4Fe-2S-20] cluster. One protein has similarity to carbon monoxide dehydrogenases, and the other, also encoded by C. fetus subsp. fetus 82-40, has similarity to hydroxylamine reductases. However, both types are similar and can be interconverted via a single amino acid change (He0 et al., 2002; Wolfe et al., 2002). Hybrid-cluster proteins are expressed optimally under anaerobic conditions in the presence of nitrate or nitrite (Wolfe et al., 2002). C. fetus subsp. fetus and C. curvus 525.92 are predicted also to encode a putative hydroxylamine oxidoreductase that catalyzes the conversion of hydroxylarnine to nitrite. C. fetus subsp. fetus 82-40 contains homologs of the C. jejuni subsp. jejuni nitrite reductase NrfAH; however, homologs of these proteins were not found in C. curvus. Campylobacter curvus 525.92, C. fetus subsp. fetus 82-40, and A. butzleri RM4018 contain the NorB nitric oxide reductase large subunit. The cognate NorC nitric oxide reductase small subunit is not predicted to be encoded by any of these three strains, and no data are available as to whether these NorB homologs retain full activity in the absence of NorC. Campylobacter curvus and C. fetus subsp. fetus contain also the NosZ nitrous oxide reductase that reduces N 2 0 to N2. The presence of the prismane family proteins along with the putative hydroxylamine oxidoreductase and the NorB and NosZ homologs suggests that

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COMPARATIVE GENOMICS OF NON-C. TETUNI CAMPYLOBACTER SPP.

nitrogen metabolism in C. fetus subsp. fetus and C. curvus may be more complex than in the other NJC species. An additional electron acceptor present in C. curvus 525.92 is the polysulfide reductase PsrABC. Polysulfide reductase was characterized first in the Helicobacteraceae species W. succinogenes (Krafft et al., 1995). In W. succinogenes, Psr uses electrons obtained from hydrogenases or formate dehydrogenase to reduce polysulfides (e.g., tetrasulfide S,2- and pentasulfide SS2-) via a methyl-menaquinone-6 electron carrier (Dietrich and Klimmek, 2002). Reduction of polysulfides generates hydrogen sulfide; thus, presence and activity of this enzyme in strain 525.92 is consistent with the H,S production phenotype observed in C. cuwus strains. Finally, in addition to five putative TMAO reductases contained in C. curvus 525.92, this strain is also predicted to contain a novel DmsABC anaerobic DMSO reductase. In addition to the HyaABCD Ni/Fe hydrogenase, C. curvus 525.92 and C. fetus subsp. fetus 8240 contain a second hydrogenase, the hydrogenase-4 encoded by hyfABCEFGHI. As in E. coli, the hyf cluster of C. curvus is linked to focB, which encodes a putative formate/nitrate transporter (Skibinski et al., 2002); focB is not linked to the hyf cluster in C. fetus subsp. fetus. Linked also to both clusters are putative hydrogenase maturation genes hycH and h y d . Two additional variable respiration-related loci are present within the NJC genomes, cydAB and the NADH:quinone oxidoreductase complex (NDH-1) encoded by the 14-gene nu0 cluster. CydAB is one of two terminal oxidases in C. jejuni subsp. jejuni, the other being the cb-type cytochrome c oxidase encoded by ccoNOPQ. All NJC genomes contain both the CcoNOPQ and CydAB terminal oxidases, with the exception of C. lari and C. fetus subsp. fetus, which do not contain the CydAB terminal oxidase. The CydAB terminal oxidase is found also only in certain Helicobacteraceae species: H. pylori and H. acinonychis do not contain this oxidase, but W. succinogenes and H. hepaticus are predicted to encode CydAB. The NJC genomes all contain similar NDH1 complexes with the exception of C. fetus subsp. fetus 82-40. The NDH-1 complex of this strain is similar to the NDH-1 complex of A. butzleri RM4018 and differs from the other complexes in one important aspect. The NDH-1 complex is composed of 14 subunits: two subunits, NuoE and NuoF, are critical for binding and oxidizing NADH (Smith et al., 2000). In C. jejuni subsp. jejuni and H. pylori, the nuoE and nuoF genes have been replaced by genes encoding proteins of unknown function. Thus, it is not certain whether the NDH-1 complexes in these two species are functional (Kelly, 2005). However, the A. butzleri and C. fetus subsp. fetus NDH-

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1 complexes contain NuoE and NuoF homologs, distinguishing these two complexes from the NDH-1 complexes of the other NJC genomes. The A. butzleri and C. fetus subsp. fetus Nu0 complexes are also distinct as a result of the fusion of the nuoC and nuoD CDSs, supporting further their similarity. Curiously, the A. butzleri RM4018 nuo complex has a second copy of gltA, which encodes citrate synthase, located between the nuoF and nuoG genes (Miller et al., 2 0 0 7 ~ ) .It is unknown at present whether this gltA encodes a functional citrate synthase, although the high similarity between the two enzymes would suggest that it is functional. It is also not known why this second gltA copy is located in the nuo cluster. The presence of anaerobic electron acceptors, e.g., fumarate reductase, would indicate that campylobacters such as C. jejuni subsp. jejuni should grow anaerobically. However, C. jejuni subsp. jejuni is incapable of anaerobic growth (Kelly, 2005). Sellars et al. (2002) proposed that the anaerobic growth of C. jejuni subsp. jejuni was restricted as a result of the presence of oxygen-requiring metabolic reactions in this organism, specifically reduction of ribonucleotides by the class I ribonucleoside-diphosphate reductase (RNR) encoded by the genes nrdA and nrdB. Class I enzymes contain a tyrosyl radical that is generated only in the presence of oxygen. However, the genomes of C. fetus subsp. fetus 82-40 and C. curvus 525.92 (as well as A. butzleri Rh44018) are predicted to encode the NrdD anaerobic-type class I11 RNR and anaerobic reductase activator protein NrdG, in addition to the NrdAB class I RNR. All three class I11 RNR-containing strains can grow anaerobically, supporting this hypothesis. Genomic Reduction In addition to the TCA cycle and respiratory genes absent from the C. lari RM2100 genome, several other genes common to Campylobacter are absent also from this genome (W. G. Miller et al., unpublished data). Many of these genes encode proteins involved in amino acid biosynthesis, including argCOBD, gltB, gltD, proA, proB, trpAFDE, trpC, metEF, metAB, and leuABCD. In addition to these amino acid biosynthetic genes, genes related to the biosynthesis of pantothenate, thiamine, tetrahydrofolate, and biotin are absent. Thus, strain RM2100 would be predicted to be multiply auxotrophic. In some cases, only particular branches or sections of biosynthetic pathways are missing. For example, the aromatic amino acid pathway from erythrose4-phopshate to chorismate is present in strain RM2100, as are the branches from chorismate to both phenylalanine and tyrosine; however, the path-

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way leading to synthesis of tryptophan is missing. Also, strain RM2100 is predicted to encode ArgG and ArgH; thus, theoretically, RM2100 could synthesize arginine if provided with an exogenous source of citrulline. Finally, genes encoding the hemin and enterochelin transporters ChuABCD and CeuBCDE are missing from strain RM2100, as is the gene encoding the autoinducer-2 production protein, LuxS. The mechanism leading to this genomic reduction in C. lari RM2100 is unknown. C. lari genes described in this and previous sections have been deleted and are not present within the RM2100 genome as fragmented or frameshifted pseudogenes. In fact, loss of these genes would require 21 separate deletions. C. jejuni subsp. doylei 269.97 is also missing the ceuBCDE gene cluster. However, in this strain, loss of the ceu cluster is linked to insertion of the DIE2 genomic island. With the exception of the prophage CLIE1, C. lari RM2100 contains no genomic islands or mobile elements. Thus, genomic rearrangements modulated by these elements are not the cause of the C. Zari genomic reduction. About half of the deletions do occur at syntenic breakpoints, suggesting that gene loss in strain RM2100 may be due to genome rearrangement. However, the other half of the deletions occur within localized syntenic regions. Notably, one such region in C. jejuni subsp. jejuni contains (in order) the oor gene cluster, sucCD, mdh, icd, and Cj0530. In C. Zari RM2100, sucCD and icd have been deleted as a result of two separate and presumably independent events. These multiple deletions within the C. lari RM2100 genome would be predicted to have a profound effect on metabolism, colonization, and the overall lifestyle of this strain. However, strain RM2100 is a human clinical isolate; therefore, it is unlikely that these deletions have a serious detrimental effect on pathogenicity in humans. The absence of gZtA from both C. lari and C. insulaenigrae might suggest that many of the genes absent from strain RM2100 are absent also from C. insulaenigrae. Absence of this gene from both species may imply a common ancestor and/or may reflect the similar environmental niches that both species occupy: C. lari in shorebirds and shellfish, and C. insulaenigrae in marine mammals (Stoddard et al., 2007). It is possible that genomic reduction in C. Zari is confined to strain RM2100. Additional investigations will be necessary to determine the extent of genome reduction in C. Zari and perhaps in the related species C. insuZaenigrae. An intriguing insight into genomic reduction in Campylobacter relates to motility in C. hominis. Preliminary analysis of the Campylobacter hominis ATCC BAA-381 genome indicates that this strain

does not encode any genes involved in motility: flagellar genes, flagellar-related genes (e.g., pflA), chemotaxis genes, or genes encoding methyl-accepting chemotaxis (MCP domain) proteins. Similar to the genes absent in C. Zari, these motility-related loci are present in the C. jejuni subsp. jejuni NCTC 11168 genome in at least 30 unlinked chromosomal locations. Therefore, from the C. jejuni subsp. jejuni perspective, multiple independent deletion events would have had to occur to result in the loss of motility genes in C. hominis. However, another explanation is likely. The motility genes in the deep-sea-vent Epsilonproteobacterial taxon Nitratiruptor are heavily clustered, with one cluster containing over 40 chemotaxis and flagellar genes. Additionally, in A. butxZeri RM4018, a flagellar cluster of 23 genes is present in the genome. It is interesting to note that the more evolutionarily “distant” the Epsilonproteobacterial taxon, the more the motility genes are clustered. Thus, it would be much more probable for loss of the multiple motility genes to result from deletion of one or a few clusters rather than 30 independent deletion events. A similar mechanism of clustered loci may explain the genomic reduction in C. Zari. Clustering of such related loci may also be indicative of an origin via lateral gene transfer (Nakagawa et al., 2007). Finally, an obvious consequence of loss of motility in C. horninis would be reduced virulence, perhaps contributing to the putative commensal nature of this organism.

RESTRICTION/MODIFICATION SYSTEMS Compared with H. pylori, Campylobacter species contain relatively few R-M systems. With the exception of C. upsaliensis RM3195, the NJC strains contain at most five R-M systems: generally one Type I system encoded by the hsd locus and a few Type I1 or I11 R-M systems, and a small number of DNA methyltransferases. In addition, like C. jejuni subsp. jejuni and H. pylori, several NJC R-M genes contain homopolymeric G: C tracts and are therefore putative contingency genes. Type I (hsd) R-M Systems The Type I R-M enzyme is a multisubunit complex composed of HsdR (endonuclease), HsdM (methyitransferase), and HsdS (specificity subunit) subunits in the stoichiometry R,S,M,. On the basis of several factors, including DNA sequence similarity and immunological cross-reactivity, Type I R-M systems have been divided into four families: IA, IB, IC, and ID (Titheradge et al., 2001; Fig. 3). HsdM pro-

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COMPARATIVE GENOMICS OF NON-C. TETUNI CAMPYLOBACTER SPP.

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W. succinogenesHsdM S. denitrificans HsdM H. pylon’HP1403 ” ‘ [ H . pyloriJHP1423

H.acinonychis HsdM H. py/oriJHP0415 H.pylori HP0463 H.pvlori HPAGI 0438

I

looL

C, upsaliensis HsdMl Ciistrain 81-176 HsdMl

Figure 3. Phylogenetic tree depicting the similarities among HsdM polypeptides from Cumpylobucterules and from selected bacterial taxa. Bootstrap values (>75%) are based on 500 replicates and are indicated as percentages. Cumpylobucter HsdM proteins are bold. Representatives of the IA (IA),IB (IB), IC (IC) and ID (ID) families are included for comparison. Type I families, identified previously (A, B, AB, C, D, and F), are shaded in gray. See Table 1, footnote a, for abbreviations.

teins within a family are well conserved, with HsdM proteins from different families having low amino acid sequence identity. An analysis of 73 C. jejuni subsp. jejuni strains demonstrated that the C. jejuni subsp. jejuni Type I systems are members of at least three families: IC and the novel “IAB” and “IF” families (Miller et al., 2005b). Extension of this analysis to the NJC strains (Fig. 3) emphasizes the familial organization of the Campylobacter Type I R-M systems. C. fetus subsp. fetus 82-40 HsdM is 80% identical to S. denitrificans ATCC 33889 HsdM but only 21 and 33% identical to the HsdM proteins of C. curvus 525.92 and C. concisus 13826, respectively. Similarly, C. curvus HsdM (ccIB’y)demonstrates greater homology to Streptococcus pneumoniae R6 HsdM (“IB”; 61.2%) than to the HsdM protein from the related C. concisus (“IC”; 20.6%). On the basis of the analysis presented in Fig. 3, at least five additional Type I families would be predicted to exist within the Epsilonproteobacteria.

Like H. pylori, C. upsaliensis RM3195 is predicted to contain three hsd loci: locus 1 is a member of a novel family that includes the C. jejuni subsp. jejuni 81-176 locus cjj812 76-0776-0780, and loci 2 and 3 are members of the IB and IC families, respectively. The hsdM gene of locus 3 contains a 15-bp G:C tract; therefore, hsdM3 may be a contingency gene. Homopolymeric G:C tracts have been identified within IC hsd genes. The IC hsdS genes of C. jejuni RM1516 and 30 also contain G:C tracts, and the homopolymeric tract of RM1516 hsdS was demonstrated to be hypervariable (Miller et al., 2005b). Similar to the C. upsaliensis RM3195 hsdM3 gene, other NJC hsd loci contain putative contingency geneslpseudogenes. The hsd locus of C. lari RM2100 contains a frameshift within hsdM. Campylobacter lari hsdM does not contain a homopolymeric G:C tract but does contain a homopolymeric A tract adjacent to the frameshift, suggesting that C. lari hsdM may be a contingency gene. Additionally,

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both hsd loci of C. jejuni subsp. doylei 269.97 contain potential defects: the Type IC locus hsdS gene contains a homopolymeric G:C tract, whereas the Type IAB locus hsdR and hsdM genes are degenerate.

mosomal methylation patterns, making the chromosome remarkably resistant to restriction. This is probably the explanation for the observation that many C. upsaliensis strains are refractory to analysis by pulsed-field gel electrophoresis (data not shown).

Type II/III R-M Systems Excluding C. upsaliensis RM3195, the NJC genomes encode few Type I1 or Type I11 restriction enzymes, ranging from one putative Type I1 pseudogene in C. curuus to three restriction enzymes in C. jejuni subsp. doylei 269.97. One Type I1 restriction enzyme, the C. jejuni subsp. jejuni enzyme CjeI, is encoded by C. coli, C. upsaliensis, C. fetus subsp. fetus, and C. curvus. However, the C. upsaliensis and C. curuus homologs are fragmented and presumably nonfunctional, and it is not known whether the other two homologs recognize the same target sequence as the C. jejuni subsp. jejuni enzyme. The Type 11s enzyme encoded by Cj0031/32 is also found in C. jejuni subsp. doylei, C. coli, and C. lari. As in C. jejuni subsp. jejuni NCTC 11168, the C. coli Cj0032/32 homolog is a contingency gene; however, the C. jejuni subsp. doylei and C. lari homologs do not contain poly G:C tracts. Eight Type I1 and Type I11 restriction enzymes are predicted to be encoded by the C. upsaliensis RM3195 genome (Fouts et al., 2005; data not shown). However, the genes encoding two Type I1 restriction enzymes (including CjeI) are fragmented and may be nonfunctional. Three of the eight R-M systems are homologous to R-M systems from H. pylori, and one Type I1 system is similar to the Haemophilus influenzae Hin4II system (Azarinskas et al., 2006), although in strain RM3195, the two Hin4II genes encoding the m5C and m6A DNA methyltransferases have been fused into one open reading frame.

C . upsaliensis Methyltransferases In addition to the Type I, 11, and I11 methyltransferases, the C. upsaliensis RM3 195 genome also contains at least 15 additional genes that encode adenineand cytosine-specific DNA methyltransferases (Fouts et al., 2005). The majority (9 of 15) of these methyltransferases have homologs within H. pylori, whereas 5 of the remaining methylases are similar to proteins from other taxa: e.g., Mod-7 is 40% identical to a methyltransferase from Thermoplasma volcanium, and Mod-17 is 51% identical to a methyltransferase from Bacillus stearothermophilus. It is unclear what biological role these multiple methylases play in C. upsaliensis. Undoubtedly, the combined action of the suite of methyltransferases within C. upsaliensis would lead to dense, overlapping chro-

VIRULENCE/PATHOGENICITY LOCI Cytolethal Distending Toxin One of the major proposed virulence factors in Campylobacter is the cytolethal distending toxin (CDT), an exotoxin encoded by the cdtA, cdtB, and cdtC genes. CDT has been demonstrated to irreversibly block eukaryotic cells in the G2 phase of the cell cycle (Whitehouse et al., 1998). The genomes of the NJC strains C. coli RM2228, C. lari RM2100, C. upsaliensis RM3195, and C. fetus subsp. fetus 82-40 all contain the complete cdt operon, consistent with previous studies investigating CDT production in these species (Asakura et al., 2007; Bang et al., 2001; Mooney et al., 2001; Ohya et al., 1993). On the basis of genomic analyses, C. concisus 13826, C. curvus 525.92, and C. jejuni subsp. doylei 269.97 are not predicted to encode CDT. A CDT-like toxin was observed in some strains of C. concisus (Engberg et al., 2005); therefore, either CDT is encoded only by a subset of C. concisus strains, or the CDT-like toxin is encoded by an as yet unidentified genomic determinant. The cdt operon in C. jejuni subsp. doylei 269.97 is represented only by a remnant of the cdtA gene. Deletions within the cdt operon were observed in all eight C. jejuni subsp. doylei strains characterized by Parker et al. (2007), suggesting that absence of CDT is characteristic of the C. jejuni subsp. doylei subspecies. Although the C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus genomes encode CDT, the CDT subunits demonstrate a substantial amount of diversity when compared with the average amino acid identities between the species. For example, the average amino acid identity between C. jejuni subsp. jejuni and C. coli proteins is 87.7% (Fig. l),yet the amino acid identities between the C. jejuni subsp. jejuni and C. coli CdtA, CdtB, and CdtC subunits are 47.4%, 67.2%, and 49.4%, respectively. Similar levels of diversity were reported by Asakura et al. (2007), who reported CdtABC identities of 47.6Y0, 67.3%, and 47.9%. Differences between these two sets of numbers probably reflect strain variation in cdt sequence. In some instances, variation between the subunits can lead to changes in activity. For example, Asakura et al. (2007) demonstrated that CDTs from different strains within a given species possessed vari-

COMPARATIVE GENOMICS OF NON-C. TETUNI CAMPYLOBACTER SPP.

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able levels of activity. In addition, CDTs from C. coli were generally inactive. Asakura et al. (2007) cloned and sequenced the cdt loci of C. coli and C. fetus; the positions of the cdt operons reported correspond to the positions of cdt operons in the C. coli RM2228 and C. fetus subsp. fetus 82-40 genomes. However, a second cdt cluster is present in the C. fetus subsp. fetus 82-40 genome. This locus (CFF8240-0951-0957) is unusual in that it contains five additional cdt genes in the order cdtC2-cdtA2-cdtC3-cdtB2-cdtA3;a putative frameshifted cdtB gene is present downstream of the first cdtC gene in the cluster. Thus, C. fetus subsp. fetus 82-40 contains three cdtA genes, at least two cdtB genes, and three cdtC genes. Even more surprising is the fact that very little similarity exists between the various C. fetus subsp. fetus CDT subunits; the greatest amount of sequence identity is between CdtB1 and CdtB2 (55.9%; Fig. 4). It is not known if the C. fetus subsp. fetus CDT subunits independently assort, i.e., whether CdtB1 can complex with any of the CdtA or CdtC subunits. If so, then C. fetus subsp.

fetus can potentially encode 18 unique exotoxins. It is likely that differences in CDT activity reflect changes in critical amino acids within the CDT subunits (Asakura et al., 2007). Therefore, it is possible that the C. fetus subsp. fetus CDTs, as a whole, possess a range of activities. It is not known whether other C. fetus subsp. fetus strains also possess multiple cdt operons. Other Virulence Factors The remainder of the virulence factors, identified initially in C. jejuni subsp. jejuni, are found in different subsets of the NJC genomes. For example, the fibronectin adhesin CadF is predicted to be encoded by all of the NJC genomes, as is the Campylobacter invasion antigen, CiaB, and the putative virulence determinant MviN. However, the JlpA lipoprotein is encoded only by C. coli RM2228 and C. upsaliensis RM3195 and not by the C. lari, C. fetus subsp. fetus, or C. cuwus genomes. Similarly, the PEBla adhesin subunit is not encoded by C. lari RM2100 or by C. curvus 525.92. Curiously, CiaB

A Cjlll68-CdtA Cjl221-CdtA Cc2228-CdtA Cl2100-CdtA Cu3195-CdtA Cff-CdtAl

C ff-CdtA2 Cff-CdtA3 Hhep-CdtA

Cjlll68-Cdtl Cjl221-CdtB Cc2228-CdtB C12100-CdtB Cu3195-CdtB Cff-CdtBl Cff-CdtB2 Hhep-CdtB

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67.2 66.5 71.7 59.3

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t

58.5

Figure 4. Pairwise comparisons of the Cumpylobucterules cytolethal distending toxin subunits. Pairwise comparisons of the CdtA (A; lower triangle), CdtB (B) and CdtC (A; upper triangle) subunits were performed by BLASTP. Values represent amino acid identities.

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and MviN are also encoded by the Nautiliales taxon Caminibacter and by the deep-sea-vent Epsilonproteobacterial taxa Sulfurovum and Nitratiruptor (Nakagawa et al., 2007). The presence of putative virulence determinants, like CiaB, in nonpathogenic taxa leads to several conclusions: (i) the deep-rooted Epsilonproteobacterial taxa (e.g., Nitratiruptor) are pathogenic, (ii) the function of CiaB in these taxa is not the same as the function in C. jejuni subsp. jejuni, (iii) critical amino acid differences between the CiaB proteins result in decreased or nonexistent activity in the nonpathogenic taxa, and (iv) CiaB may not be a true virulence determinant. It is highly unlikely that organisms such as Nitratiruptor are pathogenic; thus, it is probably safe to exclude the first option. Also, there is evidence indicating that CiaB is a virulence determinant (Raphael et al., 2005). This leaves options (ii) and (iii). It is entirely possible that the biological role of CiaB has evolved over time. Thus, the function that this protein had in an ancestral Epsilonproteobacterial organism may not resemble the function that it currently possesses in Campylobacter. The scenario in which the function of CiaB has evolved into its current state is more likely than the scenario in which the CiaB virulence determinant in taxa such as Caminibacter became nonfunctional as a result of the accumulation of point mutations (option iii). A fifth option is that the biological function of CiaB is identical in all taxa, but that the virulence phenotype only manifests in Campylobacter inside the human host. Additional experimental data will be necessary to define the role of these virulence determinants in the NJC genomes. Very few additional virulence determinants were observed after analysis of the NJC genomes. Two such determinants were identified in the genomes of C. fetus subsp. fetus 82-40 and C. jejuni subsp. doylei 269.97. C. fetus subsp. fetus 82-40 contains a putative filamentous hemagglutinin of the HecA family. The putative hecA gene in this strain is linked to the associated hecB gene. A hecAlhecB homolog is also in the genome of A. butzleri RM4018 (Miller et al., 2 0 0 7 ~ )Campylobacter . coli RM2228 also contains a hecA CDS, but this sequence is heavily fragmented and is not predicted to encode a presumably functional hemagglutinin; however, strain RM2228 does encode a presumably functional HecB protein. The genome of C. jejuni subsp. doylei 269.97 is predicted to encode an extracellular serine protease of the IgAl family. IgAl protease family proteins have been also demonstrated to cleave human coagulation factor V (Brunder et al., 1997), hemoglobin (Otto et al., 1998), human chorionic gonadotrophin hormone, granulocyte-macrophage colony stimulating factor,

the CD8 surface antigen of cytotoxic T lymphocytes, and lysosomal glycoproteins (Mistry and Stockley, 2006). It may not be coincidental that the two strains that contain hemagglutinid blood-factor-related virulence determinants are members of species isolated often from human blood cultures. Additional experiments will be necessary to determine whether these virulence factors are functional in vivo.

CONCLUSIONS Genomic data for the NJC species are incomplete: at the time of this writing, the annotation for many of these genomes ( e g , C. upsaliensis RM3195) has not been finalized. Therefore, many novel and interesting CDSs may still be hidden within the NJC genomes. Also, it is likely that additional Campylobacter genomes will soon be sequenced, further adding to the genomic data available. Analysis of the Campylobacter genomes is also enhanced by the existence of other Epsilonproteobacterial genomes; several NJC CDSs have homologs not within Campylobacter but in related taxa, such as A. butzleri, W. succinogenes, and H. pylori. Thus, knowledge of genomes such as those of Arcobacter sulfidicus, Helicobacter mustelae, and Caminibacter-all three nearing completion-will also prove beneficial. Nevertheless, despite the incomplete nature of the NJC genomes, these genomes have greatly expanded our knowledge of Campylobacter biology. Many of the features described in this chapter were identified after in silico analysis of the NJC genomes: thus, experimental evidence for many of these features is absent. Therefore, many of the pathways and systems described and hypotheses proposed will need to be supported by future investigations. However, one benefit of multiple, related sequenced genomes is the fact that CDSs are often conserved across multiple Campylobacter taxa. For example, 33% of the core Campylobacter proteins are conserved hypothetical proteins, or proteins assigned only a general function. Thus, experimental data related to one of these proteins are likely to apply to its homologs within the core protein set, potentially increasing the experimental database for multiple CDSs en bloc. Comparative analysis of the NJC genomes has also identified novel mobile elements, genomic islands, and prophage. Additionally, it has provided evidence of lateral gene transfer between Cumpylobacter species. Comparative genome analysis can therefore be utilized in investigations related to genome structure and evolution of the genus. Major contributors to strain variation in Campylobacter are the genomic islands and integrated elements. Al-

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though the current biological role of these genomic islands appears to be the synthesis of a plethora of conserved hypothetical proteins, as new experimental data are obtained, it is likely that the functions of the proteins encoded by these elements will be ascertained. Analysis of the genomic islands should also include a more thorough characterization of the family of plasmids and prophage that exist within Campylobacter; this is because the proposed origin of genomic islands, and for that matter CRISPR elements, is extrachromosomal. Finally, genomic data for the NJC genomes will lead to new typing methods and perhaps culture methods. Indeed, completion of the NJC C. coZi, C. Zuri, and C. upsuliensis genomes resulted in novel MLST methods for these three species (Miller et al., 2005a) and other sequence-based typing methods (Klena et al., 2004). Similar sequence data also led to novel MLST methods for Arcobacter spp. (W. G. Miller et al., unpublished data). Many of the NJC genomes are difficult to culture and are often overlooked in clinical samples, presumably as a result of differences in atmospheric, growth temperature, and media requirements or as a result of variable resistance to commonly used antibiotics. Comparative genomic analysis may improve culturing and detection techniques and provide a more accurate picture of the role that the NJC genomes play in human illnesses. REFERENCES Adams, V., D. Lyras, K. A. Farrow, and J. I. Rood. 2002. The clostridial mobilisable transposons. Cell. Mol. Life Sci. 59:20332043. A h , R. A., L. S. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. deJonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomicsequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180. Asakura, M., W. Samosornsuk, M. Taguchi, K. Kobayashi, N. Misawa, M. Kusumoto, K. Nishimura, A. Matsuhisa, and S. Yamasaki. 2007. Comparative analysis of cytolethal distending toxin (cdt) genes among Campylobacter jejuni, C. coli and C. fetus strains. Microb. Pathog. 42:174-183. Azarinskas, A., 2. Maneliene, and A. Jakubauskas. 2006. Hin411, a new prototype restriction endonuclease from Haemophilus influenzae RFL4: discovery, cloning and expression in Escherichia coli. 1.Biotechnol. 123:288-296. Baar, C., M. Eppinger, G. Raddatz, J. Simon, C. Lanz, 0. Klimmek, R. Nandakumar, R. Gross, A. Rosinus, H. Keller, P. Jagtap, B. Linke, F. Meyer, H. Lederer, and s. C. Schuster. 2003. Complete genome sequence and analysis of Wolinella succinogenes. Proc. Natl. Acad. Sci. USA 100:11690-11695. Bang, D. D., F. Scheutz, P. Ahrens, K. Pedersen, J. Blom, and M. Madsen. 2001. Prevalence of cytolethal distending toxin (cdt) genes and CDT production in Campylobacter spp. isolated from Danish broilers. J. Med. Microbiol. 50:1087-1094.

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11. CLINICAL AND EPIDEMIOLOGIC ASPECTS OF CAMPYLOBACTER INFECTIONS

Campylobacter, 3rd ed. Edited by I. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 6

Clinical Aspects of Campylobacter jejuni and Campylobacter coli Infections MARTIN J. BLASERAND JORGENENGBERG

Clinical Pathology

In this chapter, we describe the clinical aspects of infection with Campylobacter jejuni (C. jejuni subsp. jejuni) and C. coli, which are the main causes of Campylobacter enteritis in humans. Our account includes extraintestinal infections that may arise either as a complication of enteritis or as an apparently spontaneous event, but not infections due to species other than C. jejuni and C. coli, which are described in chapter 7 (which includes infection due to C. jejuni subsp. doylei). Campylobacter-associated GuillainBarr6 syndrome has taken on such importance that it also merits its own chapter (chapter 13). An overview of Campylobacter-associated illness is shown diagrammatically in Fig. l.

The essential lesion in Campylobacter enteritis is an acute inflammatory enteritis, which commonly extends down the intestine to affect the colon and rectum. Terminal ileitis and cecitis with mesenteric adenitis are prominent features. Because autopsy and surgical material are rare, nearly all our knowledge of the histological changes in the gut are derived from biopsy specimens obtained at proctosigmoidoscopy. These are described below (see “Colitis”). For infection to become established, campylobacters must first survive gastric acidity to colonize the jejunum and ileum. Lowering of gastric acidity, therefore, facilitates infection, which is well established in relation to Salmonella infections. A casecontrol study indicated that therapy with the proton pump inhibitor omeprazole roughly doubles the risk of acquiring Campylobacter enteritis; this effect was not seen with H, receptor antagonists, perhaps reflecting their lesser potency (Neal et al., 1996). Colonization of the gut mucosa depends on unimpaired bacterial motility and the ability of bacteria to attach to the surface of mucosal cells. The rapid motility and spiral shape of campylobacters enable them to move easily through the viscous mucus overlying the mucosa. These and other aspects of pathogenesis are discussed in section I11 of this book.

DESCRIPTION OF DISEASE

Campylobacter enteritis is an acute diarrheal disease with clinical manifestations like those of other acute bacterial gut infections of the intestinal tract, such as salmonellosis or shigellosis (Table 1).Clinically, Campylobacter enteritis cannot be distinguished from these infections, although the presence of a prodromal period of fever without diarrhea, intense abdominal pain, or prostration would favor a diagnosis of Campylobacter enteritis. A definitive diagnosis can only be made by detecting campylobacters in the feces. There does not seem to be any clear difference between infections caused by C. jejuni and C. coli when assessed by frequency of diarrhea, blood in stool, abdominal pain, fever, vomiting, mean duration of illness, or admission to hospitals.

Immune response Antibodies to Campylobacter antigens appear in the serum from about the fifth day of illness, peak within 2 to 4 weeks, and then decline over several

Martin J. Blaser Department of Medicine, New York University School of Medicine, 550 First Ave., OBV A-606, New York, NY 10016. Jsrgen Engberg Department of Clinical Microbiology, 445 Hvidovre Hospital, Ketteglrd All6 30, DK-2650 Hvidovre, Denmark.

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Immunodeficient

Healthy

Intestinal Complications Extra-Intestinal Infections Bacteremia Metastatic seeding infections

T

fl

I

Late Onset Complications * Reactive arthritis * Guillain-Bsrrk syndrome Postinfectious irritable bowel syndrome Figure 1. Diagram illustrating an overview of illnesses due to C. jejuni and C. coli.

months (Black et al., 1988; Blaser and Duncan, 1984; Strid et al., 2001). Intestinal antibodies are also produced (Black et al., 1992). Volunteer studies show that Campylobacter infection confers short-term immunity to the homologous strain (Black et al., 1988, 1992), but it is not known for how long it lasts, nor how broad the immunity is from a single infection. Sequential infections arising 6 to 18 months apart have been recorded, invariably with a strain that is different from the original one (Kell and Ellis, 1985; M. B. Skirrow, unpublished data). However, under

Table 1. Clinical features of Campylobacter enteritis derived from surveys of community outbreaks in which 50 or more people were affected and analyzed" Symptom

No. of Outbreaksb

Fever Diarrhea Headache Abdominal pain Myalgia Vomiting" Blood in feces

25 26 21 26 5

20 7

Frequency of symptom (%) Mean

Median

Range

50 84 41 79 42 15 15

52 85 47 80 37 11 13

6-75 52-100 6-69 56-99 28-59 1-42 0.5-32

"Compiled from the following references: Fahey et al. (1995); Hoskins and Davies (1983); Itoh et al. (1983); Jones et al. (1981); Matsuaki and Katayama (1984); McNaughton et al. (1982); Melby et al. (1990); Mentzing (1981); Porter and Reid (1980); Potter et al. (1983); Rogol et al. (1983); Sacks et al. (1986); Stalder et al. (1983); Stehr-Green et al. (1991); Taylor et al. (1982); Tettmar and Thornton (1981); Vogt et al. (1982); D. A. Robinson, unpublished data. bNumbers of outbreaks from which data are obtained. "Excluding waterborne outbreaks because the exceptionally high frequency of vomiting in several waterborne outbreaks suggested the additional presence of norovirus.

natural conditions, immunity is gained from relatively few infections, despite the heterogeneity of C. jejuni strains. In developing countries where repeated infection is common in early childhood, infection rates decline with age, fewer infections are associated with diarrhea, and the duration and magnitude of convalescent excretion of campylobacters are reduced. This is paralleled by a progressive increase in specific serum immunoglobulin (Ig) A antibody (Blaser et al., 1986). In addition, in developing countries, Campylobacter enteritis is virtually absent in older children and adults. However, this evidence does not prove conclusively that immunity depends on repeated reexposure to the organism; other factors have been observed to influence immunity as well. In a seroepidemiologic study among rural children in Wisconsin, increasing age and farm residence were associated with C. jejuni seropositivity, and the clinic visit rate for diarrhea was 46% lower among farm-resident children compared with those who did not reside on farms. The findings are consistent with repeated antigenic stimulation in a farm environment with subsequent development of clinical immunity (Belongia et al., 2003). The importance of the humoral response is indicated by the problems experienced by subjects with hypogammaglobulinemia, who are likely to develop prolonged and sometimes severe infections (see below). Campylobacter-specific IgA antibody is secreted in the breast milk of immune mothers and partially protects against infection in their infants (Nachamkin et al., 1994). Considerations of the immune response are clinically relevant

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in that illness gets increasingly milder with age among children in developing countries.

to have more severe illness than those whose illness starts with diarrhea.

Infective dose

Diarrheal stage

There are many influencing factors, but in general, the infective dose of C. jejuni is often low. Infection has been induced with doses as low as 500 bacteria in experimental human infection (Black et al., 1988; Robinson, 1981). This low figure is consistent with the large size of milk-borne and waterborne outbreaks of Campylobacter enteritis, which may further lower the infective dose by buffering action or rapid washthrough of stomach contents. Higher doses lead to higher attack rates.

The onset of diarrhea betrays the intestinal nature of the infection. It is commonly profuse, watery, and bile stained, and it is sometimes prostrating. Surveys have shown that at least 50% of patients attending emergency rooms have 10 or more bowel movements per day. After 1 or 2 days of diarrhea, frank blood appears in the stools in about 15% of patients (30% of hospitalized patients), indicating a progression of infection in the tissues of the colon and rectum (see section “Colitis” below). Large variations in invasion frequency have been observed among strains of C. jejuni, and the occurrence of the plasmid pVir has been associated with bloody stool, suggesting that clinically severe (bloody) diarrhea may in part be strain dependent (Tracz et al., 2005). However, a recent study could not support an association between the presence of the pVir plasmid and bloody diarrhea and suggests a role for other virulence determinants (Louwen et al., 2006). In a large waterborne outbreak, the frequency of bloody diarrhea was approximately one-third of culture-confirmed infections, compared with less than 3% of non-culture-confirmed infections, and it reflects the age and severity bias in surveillance activity that is exclusively based on passive case detection from samples received by the health care system (Engberg et al., 1998). In two studies, gross or occult blood in the stools was shown to be more frequent than in Salmonella or Shigella enteritis (Blaser et al., 1983; Rao and Fuller, 1992). Nausea is a frequent symptom, but only about 15% of patients vomit. A particular feature of Campylobacter enteritis is abdominal pain, which may become continuous and sufficiently intense so as to mimic acute appendicitis; this is the most frequent reason for admission of patients with Campylobacter enteritis to the hospital. Although the pain is usually central, it can radiate to the right iliac fossa and make diagnosis difficult (see below). Outbreaks of Campylobacter enteritis in schools have been reported in which 30% of children had abdominal pain without diarrhea (Matsuaki and Katayama, 1984; Wilson et al., 1983).

Incubation period The mean incubation period of Campylobacter enteritis, calculated from 17 point-source outbreaks, is 3.2 days, with a range of 18 h to 8 days. The extremes of the range are suspect because the outliers might not represent infection from the defined source. A range of 1to 7 days is a reasonable working estimate. We emphasize that the mean incubation period of 3 days is longer than that of most other intestinal infections, a point of significance when questioning patients with suspected food-borne infection. Onset and prodrome The clinical consequences depend in part on the virulence of the infecting strain, the challenge dose, and the susceptibility of the patient. As described above, people who have had previous Campylobacter infection, such as those who habitually drink raw milk, or children brought up in developing countries will probably experience no symptoms. Others are likely to become ill. The disease described below is that shown by patients sufficiently ill to seek medical attention, but unrecorded milder degrees of illness are undoubtedly common. The onset is often abrupt with cramping pains in the abdomen, quickly followed by diarrhea, but about 30% of patients experience a nonspecific influenza-like prodrome with one or more symptoms of fever, headache, dizziness, and myalgia. Rigors have been recorded in up to 22% of patients, and fever may be sufficiently high to cause convulsions in children (Jones et al., 1981) or delirium in adults. Meningismus also has been observed. The prodrome can be highly misleading in the absence of abdominal symptoms, which may not appear for two or even three days. Patients with prodromal symptoms tend

Recovery stage After a variable period, usually about 3 to 4 days into the illness, the diarrhea begins to ease and the patient’s condition improves, although abdominal pain may persist for several more days. The mistake many patients make at this stage is to eat too much

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of erythromycin (Paulet and Coffernils, 1990). The proportion of patients admitted to the hospital shows even greater variation between surveys (0.5 to 32%). Most surveys indicate a hospitalization range of 5 to 10%. A fatal outcome is rare and usually confined to elderly patients or those with another serious disease (Skirrow et al., 1993; Smith and Blaser, 1985). The burden to society of Campylobacter-associated illness is discussed in more detail in chapter 8.

too soon, which invites a prompt return of symptoms, especially abdominal pain. Minor relapses have been reported in 15 to 25% of patients who seek care from physicians (Blaser et al., 1979; Drake et al., 1981; Pitkanen et al., 1983). Weight loss of up to 5 kg is a common outcome. Patients continue to excrete campylobacters in their feces for several weeks after they have clinically recovered, unless the infection has been treated with antibiotics. Estimation of excretion period depends on the sensitivity of detection methods. With direct plating methods, 50% of samples are culture negative after 3 weeks, but in a study that used more sensitive methods, a mean excretion period of 37.6 days (maximum 69 days) was found (Kapperud et al., 1992). Long-term carriage has only been observed in patients with immune deficiency, notably hypogammaglobulinemia and AIDS (see below). The question of convalescent Campylobacter excretion is academic: it has never been proved that such individuals transmit infection. A diagram of the typical course of Campylobacter enteritis is provided in Fig. 2.

SPECIAL ASPECTS OF INFECTION

Campylobacter Enteritis in Children The pattern of infection in children, especially newborn infants, differs sufficiently from the above description to warrant special consideration. Fortunately, illness is seldom severe. Fever is often absent, but vomiting and the passage of blood in the stools are more frequent. In a hospital-based study, 92% of children under 1 year of age had frank blood in their stools (Karmali and Fleming, 1979). In the newborn, the passage of blood, often without obvious diarrhea, can mimic intussusception, with the risk of an unnecessary laparotomy being performed. In cases in which the infant shows more severe illness, the clinical picture can resemble necrotizing enterocolitis (Torphy and Bond, 1979). The high frequency of blood in the stools of infected children reflects the frequency of colitis, although few have performed sigmoidoscopic examinations in the young (Guandalini et al., 1983). Chronic colitis also has been described (Heyman et al., 1982). Pancolitis was recorded in a 14-year-old boy (Lambert et al., 1979) and a 4-year-old girl who apparently died from Campylobacter infection superimposed on Crohn’s disease (Coffin et al., 1982). In another case, a 7-year-old boy with suspected bowel

Morbidity The average duration of illness is difficult to measure because of a multiplicity of variables, such as the immune status of the host, the virulence of the infecting strain, and the criteria used to define illness. A survey of nine outbreaks affecting about 1,500 people showed a mean duration of illness of 4.6 days, but in one of the outbreaks, a third of the patients were ill for more than 7 days (Millson et al., 1991). A survey of sporadic cases in Norway recorded a mean figure of 3.8 days lost from work or school and 14.6 days for the presence of symptoms (Kapperud et al., 1992). There is a remarkable case of a young man who allegedly had Campylobacter enteritis for 17 years and who was ultimately cured by a course

-- a/

I *

Days

,

Incubation period

Ingest ion

1 I

1

2

3

4

5

6

7

Fever

Abdominal pain

-/

Diarrhoea

1-111

Blood in faeces

Campylobacters

in faeces

50%

Figure 2. Diagram illustrating the typical course of Cumpylobucter enteritis. Reprinted from D. Greenwood, R. Slack, and J. Peutherer (ed.), Medical Microbiology, 15th ed. (Churchill Livingstone, Edinburgh, 1997).

CHAPTER 6

CLINICAL ASPECTS OF c. r E r m I AND

obstruction was found by radiography to have colonic distension, fluid levels, and aphthous ulcers of his colon, all apparently due to Campylobacter infection (Bentley et al., 1985). Neonatal Infection Neonatal infection usually is acquired at birth from infected mothers. The mother may not be having diarrhea at the time, nor is there always a history of diarrhea (Anders et al., 1981; Karmali et al., 1984). At least five outbreaks in neonatal nurseries have been reported in which there has been nosocomial spread of infection from some communally used items, such as inadequately disinfected thermometers or baby baths (Goossens et al., 1986; Hershkowici et al., 1987; Rusu and Lucinescu, 1988; Terrier et al., 1985; van Dijk and van der Straaten, 1988). In one such case, C. jejuni was isolated from a rubber bath plug in the affected nursery (van Dijk and van der Straaten, 1988). In another outbreak, 11 infants developed C. jejuni meningitis (Goossens et al., 1986). There is a single report of a 3*/2-week-old boy with hemolytic anemia associated with C. jejuni gastroenteritis (Damani et al., 1993). Rashes Urticaria appearing late in Campylobacter enteritis has been described on several occasions (Bradshaw et al., 1980; Bretag et al., 1984; Hoskins and Davies, 1983; Lopez-Brea et al., 1984). Most remarkable was when 12 boys sought care for urticaria near the end of an outbreak of Campylobacter enteritis in a United Kingdom boarding school, in which some 400 boys had symptoms; not all of the 12 boys with urticaria had had diarrheal symptoms (Hoskins and Davies, 1983). Erythema nodosum appearing 1 to 2 weeks after Campylobacter enteritis has been reported in four patients, all women (Ashworth and Engblish, 1984; Eastmond et al., 1983; Ezpeleta et al., 1992; Lambert et al., 1979). A patient whose forearms, elbows, and shins were affected had terminal ileitis (Lambert et al., 1979). Immune complex vasculitis has been reported in association with Campylobacter enteritis, but the patients had underlying diseases that could have contributed to the skin pathology (Autenrieth et al., 1996; Nagaratnam et al., 1990; Schsnheyder et al., 1995). Infection in Immunodeficient Patients Hypogammaglobulinemia and AIDS are the conditions most often associated with more severe

c. COLI INFECTIONS

103

Campylobacter infection. Chronic carriage of campylobacters with recurrent enteritis, often with bacteremia, are typical problems. A study of Campylobacter infection in patients infected with human immunodeficiency virus (HIV), 76% of whom had AIDS, showed that 10% had bacteremia (Molina et al., 1995), a rate much higher than in immunocompetent hosts. Another study showed that bacteremia in HIV-infected patients was often a severe debilitating febrile illness requiring multiple and prolonged courses of antimicrobial therapy (Tee and Mijch, 1998). The incidence of Campylobacter infection in patients with AIDS has been calculated to be 40-fold higher compared with immunocompetent hosts (Sorvillo et al., 1991). In a survey of 41 hypogammaglobulinemic patients, 5 had experienced at least one episode of C. jejuni septicemia, 3 with an erysipelas-like cellulitis (Kerstens et al., 1992). Repeated courses of antimicrobial treatment are often needed in these patients, and this carries with it the risk that the infecting strain will become resistant, often compounding an intractable problem. Limited experience suggests that the best approach is to combine an antimicrobial agent with an immunoglobulin preparation. A commercial IgM (Pentaglobin) was used successfully with antibiotics to cure two hypogammaglobulinemic patients of their C. jejuni infections (Borleffs et al., 1993), and the bactericidal properties of the preparation were demonstrated in vitro against several serotypes of C. jejuni (Autenrieth et al., 1995). The anti-Campylobacter IgM antibodies present in this preparation appear essential because eight other immunoglobulins containing only IgG lacked a bactericidal effect (Autenrieth et al., 1995). Another report describes the successful combination of maternal plasma with ciprofloxacin in treating an intractable 2-year-long C. jejuni infection in a 7-year-old boy with X-linked agammaglobulinemia (Autenrieth et al., 1996).

INTESTINAL COMPLICATIONS Appendicitis Abdominal pain in Campylobacter enteritis may be intense and continuous, and it may radiate to the right iliac fossa. In most patients, the appendix is not affected (“pseudo-appendicitis”), and the pain is caused by terminal ileitis and mesenteric adenitis. Yet occasionally there is genuine appendicitis, and campylobacters can be isolated from the inflamed appendixes (Chan et al., 1983; Mtgraud et al., 1982; Morlet and Glancy, 1986; Pearson et al., 1982). True

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guarding and rebound tenderness demand surgical attention whether or not Campylobacter infection is thought to be present. Although appendicitis associated with Campylobacter enteritis seems to occur infrequently, in actuality, it is most likely often overlooked as a result of appropriate specimens not being routinely cultured. A survey of 25 l children admitted to a surgical unit with suspected appendicitis showed that 6 (2.4%) had Campylobacter infection, 3 of whom (1.2%) had genuine appendicitis, and 3 had mesenteric adenitis with normal appendixes (Pearson et al., 1982). In a larger survey of 533 older children and adults, the Campylobacter infection rate was almost the same (2.8%), but only one patient showed minor histological evidence of acute appendicitis (Puylaert et al., 1989). The other infected patients were shown by graded-compression ultrasound to have a characteristic picture of mural thickening of the terminal ileum and cecum with enlarged mesenteric lymph nodes, but no image of the appendix. As a result of these sonograms, 26 planned appendectomies were canceled (Puylaert et al., 1989). The proportion of patients with Campylobacter enteritis referred for suspected appendicitis is small. In a large milk-borne outbreak in the United Kingdom (Jones et al., 1981), the fraction was 0.1%, and among sporadic infections reported to the Communicable Disease Surveillance Centre, London, the figure was 0.27% (unpublished data). Nevertheless, even these small proportions (1to 400 to 1 to 1,000 infections) could account for hundreds of cases a year (or more) because Campylobacter infections in the population are so common.

Colitis Sigmoidoscopy and analysis of rectal biopsy specimens show that elements of colitis and proctitis are present in most patients with Campylobacter enteritis sufficiently severe to have these tests done (Blaser et al., 1980; Colgan et al., 1980; Lambert et al., 1979; McKendrick et al., 1982; Price et al., 1979). In some patients, colitis is a dominant feature, and then the problem is to distinguish the condition from acute nonspecific inflammatory bowel disease (IBD). Stool cultures should be obtained as soon as possible, and it is worth determining whether the local laboratory can undertake rapid diagnosis by direct microscopic examination of feces for campylobacters. Cultures that use filtration techniques to identify other Carnpylobacter and related species should also be done. There are conflicting views on how reliably the distinction between Campylobacter infection and

IBD can be made by sigmoidoscopy and analysis of rectal biopsy samples, with some workers finding little difficulty (Mee et al., 1985; Price et al., 1979) but others encountering problems in some patients (Lambert et al., 1979; McKendrick et al., 1982). First, the changes seen in Campylobacter enteritis are indistinguishable from those of other acute bacterial infections of the gut, such as those caused by Salmonella and Shigella infections. Endoscopic appearances range from mildly hyperemic intact epithelium to mucosal edema, granularity, friability, spontaneous bleeding, and patchy aphthous type ulceration (Colgan et al., 1980; Lambert et al., 1979; McKendrick et al., 1982; Mee et al., 1985). Active colitis may extend at least as far as the splenic flexure, and one patient has been described as having ulcers up to 3 X 5 cm and a “cobblestone” mucosa, but with “skip areas” of normal mucosa (Loss et al., 1980). Histology shows acute inflammation of the mucosa with edema, infiltration by polymorphonuclear leukocytes, and crypt abscess formation (Colgan et al., 1980; Lambert et al., 1979; McKendrick et al., 1982; Price et al., 1979; van Spreeuwel et al., 1985). In one study, the degree of histological abnormality was found to correlate with a history of blood in the stools, but not with fever, abdominal pain, or diarrhea (Colgan et al., 1980). In contrast to IBD, the lesions are focal, there is little or no distortion of the crypts or mucosa, and there is no striking depletion of mucus cells. However, the appearances overlap, especially in patients with Campylobacter enteritis who have had the infection for more than a week, in whom chronic inflammatory cells may be numerous (McKendrick et al., 1982). The main distinguishing histological features are listed in Table 2. It is important to remember that an apparent flare-up of IBD may be due to an intercurrent intestinal infection, such as Campylobacter enteritis (Goodman et al., 1980; Newman and Lambert, 1980). Pathogens must be looked for in such patients so that appropriate antimicrobial treatment can be started at the earliest opportunity. It is unlikely that Campylobacter infection plays any part in the cause of IBD; no excess of patients with antibodies to Campylobacter antigens were found in serological surveys of patients with IBD (Blaser et al., 1984a, 1984b; Melby and Kildebo, 1988).

Toxic Megacolon Toxic megacolon complicating Campylobacter enteritis has been reported in three patients, all adult women (Gould, 1985; McKinley et al., 1980; Stephenson and Cotton, 1985). Unless perforation of the bowel is suspected, it is best to avoid surgical

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Table 2 . Comparison of histological features that help to differentiate Cumpylobucter and other infective causes of proctocolitis from acute inflammatory bowel disease (IBD)" Feature

Infective colitis

Distribution of lesions Crypt architecture Mucus cell depletion Inflammatory cells Distribution of cells Epithelioid granulomata Basal lymphoid aggregates Isolated giant cells

IBD

Focal and segmental Preserved Absent or mild Mainly polymorphs in focal collections; crypt abscess formation Mainly mucosal Absent Absent In upper part of glandular epithelium

Diffuse and general Distorted, atrophic Chronic inflammatory cells (especially plasma cells) Submucosa may be involved Often present Present Basally located

aBased on data from Surawicz and Belic (1984).

treatment in such patients and to rely instead on vigorous antimicrobial treatment. Intestinal Hemorrhage Severe intestinal hemorrhage has been reported. One such patient was a previously healthy 24-yearold nurse who had to undergo emergency hemicolectomy for a massively bleeding ulcer in the terminal ileum (Michalak et al., 1980). Ileostomy Stoma Ulceration Patients with an ileostomy may develop partial strangulation of their stoma if they get Cumpylobucter enteritis, presumably through congestion and edema. In two patients with long-established ileostomies resulting from total colectomy for IBD, the stoma became extensively ulcerated but eventually healed without lasting damage (Meuwissen et al., 1981; Skirrow, 1981). Perirectal Abscess

A perirectal abscess in a 64-year-old woman yielded C. jejuni and Citrobucter freundii by culture 3 weeks after she had experienced diarrhea presumed to have been caused by the C. jejuni strain (Krajden et al., 1986). EXTRAINTESTINAL INFECTION Bacteremia Bacteremia in Cumpylobucter enteritis is seldom reported, but it probably occurs commonly as a transient event in the early stages of infection, especially in patients with rigors and high fever. There are three reasons why C. jejuni bacteremia is not detected more frequently: (i) blood cultures are rarely taken

early in the disease; (ii) C. jejuni is generally sensitive to the bactericidal properties of normal serum, unlike C. fetus, which is serum resistant; and (iii) not all methods of detecting bacteremia are equally sensitive for Cumpylobucter species (Wang and Blaser, 1986). A survey of laboratory reports made to the Communicable Disease Surveillance Centre, London, showed an average bacteremia rate of 1.5 per 1,000 intestinal infections, but there was wide variation with age (Skirrow et al., 1993). The highest rate (5.9 per 1,000) was in patients aged 65 years or more, and the lowest (0.3 per 1,000) was in children aged 1 to 4 years (Fig. 3 ) . Rates in male subjects were nearly twice as high as those in female subjects. Although some patients had immunodeficiency or other underlying disease, 71% were apparently normal (in contrast to patients with C. fetus infection, most of whom had some predisposing disorder). C. jejuni strains belonging to Penner serogroups HS:4 and HS: 18 were more frequent among blood than fecal isolates, but subsequent genotypic analysis of these and additional strains did not suggest that blood isolates were especially invasive or serum resistant (Jackson et al., 1997a). In a smaller, more recent study performed at two centers in Denmark, the bacteremia rate was found to be 8 per 1,000 intestinal infections (Schsnheyder et al., 1995). C. jejuni can cause serious septicemic infection in patients whose immune system is severely compromised (see above). A fatal outcome was reported in two patients who had undergone splenectomy for thalassemia, both of whom had iron overload (Jackson et al., 1997b; Meyrieux et al., 1996). Infection of the Hepatobiliary System Hepatitis

A mild degree of hepatic inflammation may be a regular feature of Cumpylobucter enteritis. Several re-

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4

I I

5

I

<1

1-4

5-14

15-24 25-34 35-44 45-54 55-64 265

Age in Years

Figure 3. Distribution of Cumpylobacter bacteremia cases in England and Wales, 1981 to 1991. Solid line indicates number of cases (n = 374); dashed line, cases per 1,000 intestinal infections. Reprinted from Skirrow et al. (1993).

ports have shown slightly increased serum transaminases in hospitalized patients with Campylobacter enteritis (Jackson et al., 1997b; McKendrick et al., 1982; Pitkanen et al., 1983). Some strains of C. jejuni have been shown to produce hepatotoxic factors in mice (Kita et al., 1992), and in experimentally infected rhesus monkeys, the liver and gallbladder were found to be the most consistently colonized organs after the intestines (Fitzgeorge et al., 1981). However, obvious clinical hepatitis is rare. Two young men were found to have enlarged livers and increased liver enzymes 1 week after the onset of Campylobacter enteritis (Humphrey, 1993). In three other patients with clinical hepatitis, analysis of liver biopsy samples showed evidence of hepatitis: a 52year-old man had mild mononuclear cell infiltration in portal areas, with variation in hepatocyte and nuclear size (Reddy and Thomas, 1982); a 48-year-old alcoholic man had cholestatic hepatitis with lymphohistiocytic infiltration of portal tracts and C. jejuni bacteremia (Nahum et al., 1982); and a previously healthy 50-year-old woman, also with bacteremia (with C. coli), had neutrophil infiltration of the portal tracts with adjacent areas of necrosis (Ampelas et al., 1982). More recently, an 82-year-old woman admitted to the hospital with rigors, fever, and watery diarrhea due to C. jejuni was found to have acute hepatitis (Korman et al., 1997). Significantly, the hepatitis resolved in all six of these patients with treatment directed against the infecting Campylobacter strain. Cholecystitis

C. jejuni is a bile-tolerant organism, so it is not surprising that it is sometimes found in the gallblad-

der in association with acute or acute-complicatingchronic cholecystitis (Darling et al., 1979; Drion et al., 1988; Gerritsen van der and Veringa, 1993; Mertens and De, 1979; Pereira et al., 1981). Patients may or may not give a history of recent diarrhea. Prospective searches for campylobacters in bile from several hundred cholecystectomy samples yielded none, so biliary infection appears to be uncommon (Darling et al., 1979).

Pancreatitis Acute pancreatitis has been reported in a few patients, all adults, during the acute stages of Campylobacter enteritis. Abdominal pain was a dominant symptom in each (Castilla et al., 1989; De Bois et al., 1989; Dutronc et al., 1995; Gallagher et al., 1981; Ponka and Kosunen, 1981). An 88-year-old woman had acute pancreatitis and C. jejuni bacteremia without diarrhea (Ezpeleta et al., 1992). The pancreatitis resolved satisfactorily in all of these patients after they had received appropriate antimicrobial treatment. The mechanism by which campylobacters cause pancreatitis is unknown. The possibility that a mild degree of pancreatitis is a regular feature of the disease was suggested by a Finnish study of 188 hospitalized patients with Campylobacter enteritis. In that study, 22% of patients were considered to have pancreatitis on the basis of increased serum amylase or lipase levels (Pitkanen et al., 1983). However, in a study of 22 other patients, none was found to have increased enzyme levels (Murphy et al., 1991).

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Campylobacter Abortion and Perinatal Infection

Renal and Urinary Tract Disease

C. fetus has been traditionally regarded as the cause of epizootic abortion of sheep and cattle, but it was only after the implication of C. jejuni and C. coli in human infections that the role of these organisms in veterinary medicine became apparent. A survey in the United Kingdom showed that C. jejuni and C. coli account for about 40 and 20% of ovine abortion cases, respectively; only 40% are caused by C. fetus. This predilection of campylobacters for uterine and fetal tissues in ruminants is fortunately uncommon in humans. Nevertheless, there have been nearly 30 reports of septic abortion or stillbirth due to Campylobacter infection. About half were caused by C. fetus, one by C. hyointestinalis, and the remaining half by C. jejuni or C. coli. The average stages of gestation for women infected with C. fetus or with C. jejuni are 28 and 19 weeks, respectively. However, five infants infected in utero with C. fetus were born sufficiently late in gestation to survive premature birth, whereas none of the C. jejuni-infected infants survived; only two were born late enough to have a chance of survival. The pathology in humans resembles that of Campylobacter abortion in sheep, namely an acute placentitis that is severe enough to cause fetal death by placental insufficiency, although infection eventually reaches the fetus via amniotic fluid. The campylobacters probably reach the placenta by hematogenous spread from the intestinal tract, although this theory is unproven. There is no evidence that infection ascends from the genital tract. Although most infected mothers have fever and about half have diarrhea shortly before or at the time of abortion, maternal illness is seldom pronounced, and there may be little to indicate the nature of the problem. Twenty patients with Campylobacter abortion were reviewed by Simor et al. (1986), and several more patients have been described since then (Denton and Clarke, 1992; Moscuna et al., 1989; Simor and Ferro, 1990). A 28-year-old woman with acquired agammaglobulinemia had recurrent abortions after a persistent attack of C. jejuni diarrhea (Pines et al., 1983). The factors that allow campylobacters to infect the human placenta are unknown. In a study in the United Kingdom, C. jejuni and C. coli were isolated from the rectal swabs of 0.2% of women in labor, most of whom had no symptoms relating to the organism (Youngs and Roberts, 1985). Thus, many thousands of pregnant women must develop the infection without any untoward effect. Some infections are almost certainly overlooked, but even allowing for this, Campylobacter-induced abortion remains a rare event.

There are reports of eight patients, five of them children under the age of 5 years, who developed hemolytic-uremic syndrome within a few days of developing Campylobacter enterocolitis (Chamovitz et al., 1983; Denneberg et al., 1982; Dickgiesser, 1983; May et al., 1986; Shulman and Moel, 1983). An exception is the case of an adult patient in whom the interval was 1 month (Delans et al., 1984). Most of the patients had blood in their stools, and all recovered except one, a 4-month-old Bangladeshi infant who died (Haq et al., 1985). A further report describes a 72-year-old woman who developed the closely related syndrome of thrombotic thrombocytopenic purpura 5 days after the onset of nonbloody C. jejuni diarrhea (Morton et al., 1985). Because only one of these reports states that enterohemorrhagic Escherichia coli were not isolated, it is possible that the isolation of campylobacters was coincidental to undiscovered enterohemorrhagic E. coli infection. Several of the incidents arose before the role of enterohemorrhagic E. coli in hemolytic-uremic syndrome was known or before routine measures for their isolation were implemented. Nephritis Rare instances of nephritis, mainly glomerulonephritis, complicating Campylobacter enteritis have been reported. An 18-year-old man developed mild acute glomerulonephritis during an attack of C. jejuni enteritis (Menck, 1981). A 33-year-old man was shown to have moderately severe diffuse proliferative endocapillary glomerulonephritis by analysis of renal biopsy specimens a few days into an attack of C. jejuni diarrhea; he required hemodialysis but recovered completely (Maidment et al., 1985). A 5-year-old girl developed glomerulonephritis with pulmonary hemorrhage and anemia (Goodpasture’s syndrome) 3 to 4 weeks after the onset of C. jejuni diarrhea. Analysis of renal biopsy samples showed immune complexmediated crescentic glomerulonephritis, and C. jejuni antigen was identified in the glomeruli. The patient developed progressive renal failure (Andrews et al., 1989). Mesangial IgA glomerulonephritis has been observed in two men 15 and 26 years old, both of whom had hematuria within 2 days of the onset of diarrhea (Carter and Cimolai, 1991; M. B. Skirrow, unpublished data). There is a single report of selflimited tubulointerstitial nephritis diagnosed in a 20year-old man 8 days after the onset of C. jejuni enteritis (Rautelin et al., 1987).

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Prostatitis and urinary tract infection The urinary tract seems an unlikely place to find campylobacters because they do not tolerate acid conditions well, but there are two reports of apparent C. jejuni urinary infection. In the first, the main site of infection was thought to be the prostate (Davies and Penfold, 1979), but in the other, there appeared to be cystitis in a 6-year-old girl (Feder et al., 1986). The authors of that report saw Campylobacter-like organisms in the urine and obtained appropriate cultures for them. As they indicate, infections will be missed unless cultures specific for campylobacters are set up when morphologically suspect bacteria are seen in urine samples. Miscellaneous Extraintestinal Infections Focal infection Focal infection with C. jejuni or C. coli is rare. Most such infections are in subjects with immunodeficiency or other predisposing conditions such as cirrhosis, and presumably they arise from bacteremia. Reports of focal infection, other than infection of the female reproductive tract (see above), are listed in Table 3. Peritonitis There are four reports of spontaneous peritonitis, two due to C. jejuni, one to C. coli, and one in which the differentiation was not made. Three patients had long-standing alcoholic cirrhosis, and the other had cirrhosis due to congestive heart failure (Doming0 et al., 1985; Ho et al., 1987; McNeil et al., 1984; Schmidt et al., 1980). Less rare is Campylobacter peritonitis complicating continuous am-

bulatory peritoneal dialysis, which usually arises during an attack of Campylobacter enteritis. It is unknown whether the bacteria seed the peritoneal cavity from the bloodstream, by transluminal migration from the intestinal, or by contamination of the catheter fittings. A report of eight patients and a review of other case reports are given by Wood et al. (1992), and there has been one other subsequent report (Webster and Farrell, 1993). Myocarditis Myocarditis has been reported within a week of C. jejuni enteritis in two young men. The cardinal feature in both patients was severe chest pain radiating to the right arm. The first patient, aged 27 years, was believed to have perimyocarditis (Ponka et al., 1980). The second patient, aged 23 years, had more convincing evidence of cardiac damage in that his cardiac enzymes were quite high (Florkowski et al., 1984). Electrocardiogram changes were observed in both cases: T-wave inversion, slight increase of ST segment, and lengthening of the PR interval on serial tracings. No signs of lasting damage were found in either patient 2 months after the episode. There is a brief report of transient atrial fibrillation associated with C. jejuni enteritis in three patients, all over the age of 50 years (Kell and Ellis, 1985). Splenic rupture Spontaneous rupture of the spleen was reported in a 71-year-old man 1 week after the onset of explosive diarrhea due to C. jejuni enteritis (Frizellaand Rietveld, 1993). Recovery was uneventful after partial splenectomy. There was no other factor to account for the rupture; the authors indicated that

Table 3. Focal infections due to C. jejuni and C. coli Reference

Focus of infection

Age ( yr) I sex of patient

Predisposing condition

Organism

Muytjens and Hoogenhout (1982) Pedler and Bint (1984)

Chest wall abscess

72/F

Mastectomy scar, postirradiation

C. jejuni

Osteitis of foot

57/M

C. jejuni

Peterson et al. (1993) Pasticci et al. (1992) Unpublished data" Schieven et al. (1991) Norrby et al. (1980) Ritchie et al. (1987) Unpublished data" Unpublished data"

Prosthetic hip sepsis Septic arthritis of knee Septic arthritis of shoulder Acute bursitis Meningitis, nonneonatal Subdural sepsis Empyema Empyema

Site of previously removed histiocytoma AIDS Rheumatoid arthritis Not known Chronic bursitis Long-standing ventricular shunt Previous hemispherectomy Not known Not known

a

Public Health Laboratory Service, London.

60/M 51/F 90lM 81IM 34/M 21/2/F 70/M 52/M

C. jejuni C. jejuni Campylobacter spp. C. jejuni C. jejuni C. jejuni C. jejuni C. coli

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CLINICAL ASPECTS OF C. TETUNI AND C. COLI INFECTIONS

spontaneous splenic rupture has been associated with other enteric pathogens, including Salmonella Dublin. Because Campylobacter infection is so common, it is not certain which of these complications are secondary events and which are unrelated adventitious occurrences. Late-Onset Complications Reactive arthritis and Reiter’s syndrome The reactive arthritis (ReA) that sometimes follows Campylobacter enteritis is no different from that associated with Salmonella or other intestinal bacterial infections. In a review of 29 patients, the mean interval between the onset of bowel symptoms and the appearance of pain and swelling of the joints was 14 days, with a range of 3 days to 6 weeks (Peterson, 1994). Ankles, knees, wrists, and the small joints of the hands and feet are most commonly affected, often in migratory fashion (Peterson, 1994; Schaad, 1982). Little is known about the mechanism by which Campylobacter causes ReA. A recent study examined the lipooligosaccharide biosynthesis gene cluster of infecting C. jejuni strains causing joint disease and uncomplicated gastroenteritis, and the authors identified lipooligosaccharide biosynthesis gene cluster A as a potential bacterial marker for an increased risk of developing postinfectious joint inflammation (Bergman et al., 2007). Campylobacter-associated ReA incidence and prevalence varies widely among reports because of differences in exposure and in case ascertainment, lack of diagnostic criteria for ReA, and perhaps the genetic makeup and the age of exposed individuals. For instance, clinical examination of a subset of patients with self-reported joint symptoms in a recent prospective study revealed joint symptoms from other causes unrelated to the preceding infection in 37% of patients (Schiellerup, 2006). A review of the literature available to date suggests that the incidence of Campylobacter ReA may occur in 1 to 5% of those infected (Pope et al., 2007). The duration of arthritis ranges from several weeks to several months, or occasionally a year. Although the arthritis can be incapacitating, full recovery is the rule. Acute erosions of the hamate bone of the wrist and distal ends of the clavicles were found in a 46-year-old man (Ebright and Ryan, 1984). The full Reiter’s syndrome has been observed in some patients with Cumpylobacter-associated arthritis (Leung et al., 1980; Peterson, 1994; Saari and Kauranen, 1980; Schaad, 1982), and uveitis in the absence of arthritis has been reported in two middle-aged women, one of whom had hypogammaglobulinemia (Lever et al., 1984) and the other of whom had conjunctivitis several weeks before the onset of uveitis (Howard et al., 1987).

109

In a recent prospective case-case comparison study of 1,003 sporadic Campylobacter infections, no significant correlation between HLA B27 and occurrence of ReA was found, whereas in 812 patients with Salmonella, Yersinia, or Shigella infection, there was a significant correlation between ReA and HLA B27 (Schiellerup, 2006). However, possession of the HLA B27 tissue antigen carries a significantly increased risk of a more severe course of ReA, and this also accounts for Campylobacter-associated ReA. Two recent studies also show that a severe acute course of gastrointestinal symptoms is correlated to a higher incidence of ReA (Locht and Krogfelt, 2002; Schiellerup, 2006). The long-term prognosis for postCampylobacter ReA is not defined in the current literature. However, in another study, HLA B27 was associated with a strong predisposition to ReA and patients who possess it had, on average, higher erythrocyte sedimentation rate values (84.6 versus 44.6) than B27-negative patients (Peterson, 1994). At least 60% (and probably closer to 80%) of ReA patients possess the B27 antigen. However, it is difficult to generalize about the frequency of ReA in Campylobucter enteritis patients because much depends on the prevalence of HLA B27 in the population. Estimates ranging from 0 to 1.7% have been given in community outbreaks of the disease (Eastmond et al., 1983; Jones et al., 1981; Melby et al., 1990; Mentzing, 1981; Millson et al., 1991). A calculation of 5% in hospital patients has been reported in Scandinavia, where 14% of the population is HLA B27 positive (Pitkanen et al., 1983). A long-term follow up of 66 patients affected in a food-borne outbreak of C. jejuni enteritis in Sweden suggested that nonspecific rheumatic disorders are a common sequela of infection (Bremell et al., 1991). Four patients experienced chronic or relapsing rheumatic symptoms beginning 3 to 8 months after infection and persisting for 5 years, but in the absence of a control group, the significance of this finding is questionable. Guillain-BarrC syndrome The association of Campylobacter enteritis with Guillain-BarrC syndrome, which emerged during the mid-1980s, greatly improved our understanding of the morbidity of the disease. In view of the importance of Guillain-Barrt syndrome and the complexity of its pathogenesis, it is given special consideration in chapter 13. Postinfectious irritable bowel syndrome Late diarrheal complications are not infrequent after acute episodes of infectious disease (Spiller,

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2007). A major question is whether long-term diarrhea is postinfectious irritable bowel syndrome (PIIBS) or represents closer surveillance of events that are occurring at similar rates in individuals without infectious diarrhea. Among patients with unselected IBS, 6 to 17% believe that their symptoms began after an infection; conversely, prospective studies following patients with acute bacterial gastroenteritis show a 4 to 31% incidence of PI-IBS (Spiller, 2007). An increasing body of evidence Suggests that after Campylobacter enteritis, PI-IBS is occurring at rates greater than expected from controls (Dunlop et al., 2003; Marshall et al., 2006; Thornley et al., 2001). Studies of patients after infection indicate PIIBS rates that are about 5 to 20% after subtracting the rate from unaffected controls (Dunlop et al., 2003; Marshall et al., 2006; Thornley et al., 2001). In a study of individuals 3 months after Campylobacter infection, PI-IBS, chiefly of the diarrheapredominant type, occurred in 103 (13.8%) of the 747 subjects (Thornley et al., 2001). Rectal biopsy samples were examined in patients with new-onset PI-IBS, and they matched with those of asymptomatic patient controls and healthy volunteers. Enterochromaffin cell counts were significantly higher in the PI-IBS patients than in the two control groups. Multivariant analysis showed that both increased enterochromaffin cell counts and clinical depression were important predictors of developing PI-IBS (Thornley et al., 2001). The overlay of physical and psychosomatic syndromes is illustrated by these associations. One other interesting observation is that among C. jejuni isolates from patients, those that were toxigenic to HEp-2 cells were more often isolated from patients who later developed PI-IBS (Thornley et al., 2001). Altogether, a body of evidence is emerging that PI-IBS develops in a proportion of patients after Campylobacter enteritis, in which some level of diarrheal symptoms can persist for months, at least-.

DIAGNOSIS AND CLINICAL INVESTIGATIONS Bacteriology As mentioned previously, a definitive diagnosis of Campylobacter enteritis can only be made bacteriologically. Because the laboratory diagnosis of the infection is described in chapter 12, all that is necessary here is to stress the importance of delivering fecal samples to the laboratory with a minimum of delay. Samples should be refrigerated while awaiting transport. If delays of more than a day are likely, transport medium, such as a standard Cary-Blair type medium, should be used. Rectal swabs as a substitute

for feces should only be used as a last resort, in which case the use of transport medium is essential. Rapid diagnosis may be helpful in patients with suspected appendicitis or with acute colitis masquerading as acute IBD, or for definitive early antibacterial therapy. The microscopic examination of wet feces or fecal smears for campylobacters can provide a rapid diagnosis, but the sensitivity of microscopy is variable (36 to 90%), and it may not be available in all laboratories. Hematology and Biochemistry It is only in patients with complications that hematologic and biochemical analyses are likely to be needed, but it is useful to be aware of the changes to be found in patients with Campylobacter enteritis. These values relate mainly to series of patients sufficiently ill to be treated in the hospital. Leucocyte counts are usually normal or only slightly high (Pitkanen et al., 1983). Mean leucocyte counts (109/liter) in three series of patients were 7.9 (Svedhem and Kaijser, 1980), 8.9 (Pentland, 1979), and 10.0 (DeWitt et al., 1985). A particular feature of Campylobacter enteritis not seen in other forms of acute diarrhea, except shigellosis, was a high percentage of band forms (mean 15.1%), especially in the presence of only a moderate total leucocyte count (DeWitt et al., 1985). The erythrocyte sedimentation rate is usually raised, and high C-reactive proteins were recorded in all acutely ill patients in one series (Pitkanen et al., 1983). Biochemical values in Campylobacter enteritis are usually normal, although metabolic acidosis was detected in 32% of hospital patients in one study (Pitkanen et al., 1983). Mildly increased serum transaminases have been reported in 14 to 25% of patients (Drake et al., 1981; McKendrick et al., 1982; Pitkanen et al., 1983) and slightly increased alkaline phosphatase values in 10% (Drake et al., 1981). Endoscopy and Radiography The role of endoscopy and rectal biopsy in the management of patients has already been described above (see “Colitis”). Only patients with severe or complicated infection are likely to require radiographic investigation. Dilated loops of intestine with fluid levels on plain abdominal radiographs were observed in 5 of 20 patients in one study of hospital patients (Bradshaw et al., 1980), but not in 14 patients in another study (Pentland, 1979). Contrast radiography has shown the presence of pancolitis (Blaser et al., 1980; Lambert et al., 1979), nodular thickening of the terminal ileum (Lambert et al.,

CHAPTER 6

-

CLINICAL ASPECTS OF C. lElUNI AND C. COLI INFECTIONS

1982), right-sided colonic ulceration and dilation (McKinley et al., 1980), and multiple aphthous type ulceration, which in one patient mimicked Crohn’s disease (Bentley et al., 1985). Barium enema examination strongly suggested colonic carcinoma in two patients, so much so that one patient was subjected to hemicolectomy for what was found to be nothing more than inflammatory edema, congestion, and lymphoid hyperplasia (Doberneck, 1983; Noble et al., 1982). The increasing availability and usage of abdominal computed tomographic scanning should facilitate differentiation between appendicitis and mesenteric adenitis.

TREATMENT

No specific treatment is required for most patients with Campylobacter enteritis, other than the oral replacement of fluid and electrolytes lost through diarrhea and vomiting. Antimicrobial therapy plays a limited role because the patient will likely be showing signs of improvement by the time a bacteriological diagnosis has been made. This does not necessarily reflect a slow laboratory service; it has repeatedly been shown that patients seek medical advice only after they have been ill for an average of 4 to 6 days (Anders et al., 1982; Mandal et al., 1984; Pai et al., 1983). This late presentation of patients affected early trials of antibiotic therapy, but anecdotal evidence suggested that antimicrobial agents were effective when given early in the disease. This prompted trials of erythromycin and fluoroquinolones given empirically when patients first sought care for diarrhea. Such studies confirmed that illness was shortened and that campylobacters disappeared from the feces, provided that the infecting strain was susceptible to the antimicrobial used (Dryden et al., 1996; Goodman et al., 1990; Mattila et al., 1993; Pichler et al., 1987; Salazar Lindo et al., 1986). Trials in developing countries showed less effect, but the results were confounded by the simultaneous presence of other pathogens or the high frequency of antibiotic-resistant strains (Taylor et al., 1987). Clear instances of improvement with antimicrobial therapy have been observed in patients with severe or chronic infection, often in cases where other measures have failed (Bentley et al., 1985; Blaser et al., 1980; Noble et al., 1982). Erythromycin Erythromycin was the first antimicrobial agent to be used, and in general, macrolides remain the agents

111

of choice. Table 4 shows data on macrolide resistance among C. jejuni, C. coli, and C. jejuni and C. coli combined, isolated from human sources around the world since 1997 (Engberg et al., 2005). There are notable differences in resistance rates between countries and species. Almost all studies report a higher frequency of erythromycin resistance in C. coli than in C. jejuni, with rates reported in proportions ranging from 0 to 20% in C. jejuni and 0 to 29% in C. coli. In a number of industrialized countries, a higher proportion of C. coli, including macrolide-resistant C. coli, have been reported among travel-related infections than among those domestically acquired. The trend over time for macrolide resistance shows stable, low rates in most countries, which is comforting because erythromycin-or alternatively one of the newer macrolides, such as clarithromycin or azithromycin-is the drug of choice for treating C. jejuni and C. coli enteritis. Development of resistance to macrolides in Cumpylobucter during therapy has not been documented in humans. The origin of resistant strains has been linked to the veterinary use of antibiotics of the macrolide-lincosamide group and is discussed in more detail in chapter 36. Fluoroquinolones and Other Antimicrobials Ciprofloxacin and other fluoroquinolones were hailed with great enthusiasm when they first appeared, but their use has been severely compromised by increasing resistance rates in many countries (Fig. 4). In some countries, the increase in resistance has been remarkably rapid and considerable; in others, resistance rates have increased more steadily. For example, 86% of human C. jejuni isolates were quinolone resistant in Hong Kong (Chu et al., 2004). Thus, in the high-endemic quinolone resistance areas, fluoroquinolones cannot be recommended for community-acquired bacterial diarrhea because the predominant causes are often Campylobacter spp. Although lower frequencies are reported from other regions, recent trends show a clear and worrying tendency of emerging quinolone resistance in many countries. As discussed in chapter 36, the use of fluoroquinolones (mainly enrofloxacin) in veterinary medicine is correlated with an increase in quinolone resistance in food animals; in retail food of animal origin, especially poultry products; and most importantly, in human Cumpylobacter infections. Veterinary use of fluoroquinolones is not the only selection pressure that acts on Campylobacter to

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Table 4. Macrolide resistance among C. jejuni, C. coli, and C. jejuni and C. coli combined, isolated from human sources around the world since 1997" Country

C. jejuni

C. coli

Argentina Australia Austria Belgium Bosnia and Herzegovina Canada Chile Denmark

3b 3

6b

4 20 0-12 6 0-7'1 0-7d

6 25

4-21

Ob

Ob

3 0-4

11 0-29

Egypt Finland France Germany India Indonesia Ireland Italy The Netherlands New Zealand Norway Mexico Spain Sweden Thailand United Kingdom

C. jejuni and C. coli

<1-2 22

0'13'

6

0 2 1 4'13' 3

0-2 '1 <1-3 14 2-5 3 lb-2 1-3

United States Vietnam

24 6"/11'

35 0'15d 17'-26 25

1-5

4-9

0

0

2-1 1

Reference(s) Fernandez (2001) Sharma et al. (2003) Feierl et al. (2001, 2003); Feierl (2004) Vandenberg et al. (2003) Uzunovic-Kamberovic (2003) Gaudreau and Gilbert (2003); Gibreel et al. (2004) Fernandez (2001) Danish Zoonosis Centre (2004a; Engberg et al. (2004); Engberg (2004) Putnam et al. (2003) Rautelin et al. (2003) Mtgraud and Prouzet-Maulion (2004) Luber et al. (2003); Steinbruckner et al. (2001); Wagner et al. (2003) Jain et al. (2005) Tjaniadi et al. (2003) Lucey et al. (2002) Pezzotti et al. (2003) VANTURES (2003b) Goodchild et al. (2001) N O M I N O M - V E T (2004); Afset and Maeland (2001) Tuz-Dzib et al. (1999) Campos et al. (2001); Saenz et al. (2000) Osterlund et al. (2003); Ronner et al. (2004) Bodhidatta et al. (2002); Isenbarger et al. (2002) Campylobacter Sentinel Surveillance Scheme Collaborators (2002); Moore et al. (2001); Rao et al. (2005); Wickins et al. (2001) Centers for Disease Control and Prevention (2004); Gupta et al. (2004); Nachamkin et al. (2002) Isenbarger et al. (2002)

"Reprinted from Engberg et al., 2005. bIsolates exclusively from children. Isolates acquired domestically. dIsolates acquired abroad.

select for quinolone resistance. Resistance occurs naturally, but the selection and dissemination of resistance are inevitable results of antibiotic use. Fluoroquinolone use in humans can in itself lead to the emergence of quinolone-resistant Campylobacter in treated infections. At least five case-control studies have specifically addressed risk factors for quinolone Campylobacter infections in the United States, the United Kingdom, Denmark, and Canada (Campylobacter Sentinel Surveillance Scheme Collaborators, 2002; Engberg et al., 2004; Johnson et al., 2007; Kassenborg et al., 2004; Smith et al., 1999). Four of the five studies evaluated current or recent treatment with antimicrobials. An association between treatment with a fluoroquinolone before stoolspecimen collection and having a quinolone-resistant Campylobacter infection was only observed in the study by Smith and colleagues (1999). They showed that treatment with a fluoroquinolone before stool

culture accounted for a maximum of 15% of resistant isolates in Minnesota during 1996 and 1998. Quinolone use in humans is not the major selective force for quinolone resistance among Campylobacter spp. causing human infection. Foreign travel was identified as a risk factor in all five studies, and this is in agreement with recent surveillance data from a number of countries. The studies also show a significant difference in quinolone resistance rates between travel-related infections and domestically acquired infections, and they document the importance of stratifying susceptibility data by travel status (Engberg, 2006; Engberg et al., 2005). Travel-related infections from destinations with recognized high quinolone resistance in Campylobacter in poultry, and high risk of acquiring Campylobacter infections are unsurprisingly associated with significantly higher prevalence of quinolone resistance compared with infections acquired domesti-

CHAPTER 6

CLINICAL ASPECTS OF C. lETUNI AND C. COLI INFECTIONS

113

100

80 60 40

20

0 r W #

w

Figure 4. Trends in quinolone resistance rates among C. coli and C. jejuni isolates from humans from 11 countries, 1989 to 2006. The bars represent both nalidixic acid and fluoroquinolone resistance and are based on mean values of resistance from numerous reports. Year in parentheses is year of licensure for use in veterinary medicine in each country. Canada and the United States banned veterinary use of fluoroquinolones in 1997 and 2005, respectively. Updated and modified from Engberg et al. (2001). References therein plus the following: Bodhidatta et al. (2002); Boonmar et al. (2005); Centers for Disease Control and Prevention (2003, 2004, 2007); Danish Zoonosis Centre (2004a, 2004b, 2005, 2006, 2007); Engberg et al. (2004); Feierl (2004); Feierl et al. (2001, 2003, 2007); Gallay et al. (2007); Migraud and Prouzet-Maulion (2004); Pezzotti et al. (2003); Rautelin et al. (2003); Sanders et al. (2002); Tribble et al. (2007); VANTURES (2003, 2004, 2005); Wickins et al. (2001); Prouzet-Maulion and Megraud (personal communication); J. Engberg (unpublished data).

cally. The significantly lower prevalence of quinolone resistance among domestically acquired Campylobacter probably reflects less veterinary use of fluoroquinolones in these countries. For example, in Australia, where fluoroquinolones have not been licensed for use in food production animals and only cooked chicken products may be imported, little fluoroquinolone resistance has been found in domestically acquired human infections (Unicomb et al., 2006). In contrast, although foreign travel is associated with quinolone-resistant infections in the United States, the majority of quinolone-resistant infections are domestically acquired (Gupta et al., 2004; Kassenborg et al., 2004). Table 5 summarizes information from casecomparison studies evaluating the duration of illness in patients infected with quinolone-resistant Campylobacter strains versus quinolone-sensitive Campylo-

bacter strains (Campylobacter Sentinel Surveillance Scheme Collaborators, 2002; Engberg et al., 2004; Kassenborg et al., 2004; Smith et al., 1999). The recent study by Nelson et al. (2004) evaluated the duration of illness across a variety of analytical models, including a multivariable analysis of variance model, and identified a consistent correlation between quinolone resistance and prolonged duration of diarrhea. Although the results from these studies are not all statistically significant, the estimates all point in the same direction, and taken together, they suggest that patients infected with quinolone-resistant strains have a longer duration of illness. Whether patients with resistant infections may experience a longer duration of illness because the antibiotic provided to them simply does not work against resistant Campylobacter, whether it may be due to a possible coselection of virulence traits in re-

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Table 5 . Studies evaluating the duration of illness in patients infected with quinolone-resistant or quinolone-susceptible Campylobacter strains a Resistant Reference No. of patients

Smith et al. (1999) Neimann et al. (2001)b The Campylobacter Sentinel Surveillance Scheme Collaborators (2002) Domestically acquired infection Travel-related infection Engberg et al. (2004)” Nelson et al. (2004)d Model A Model B Model C

Sensitive

Duration of diarrhea (days)

No. of patients

Duration of diarrhea (days)

69 5

10 14

115 31

7 9

0.03 0.13

86

12.7 11.8 13.2

381

13.5 11.2 10.3

0.56 0.66 0.001

26 7 9

9 12 8

264 56 76

7 6 6

0.04 0.04 0.2

-

“Reprinted from Engberg et al., 2005. *Stratified by treatment, but not on antimicrobial agent used for treatment. ‘Analysis not stratified by treatment. dModel A, analysis of 290 persons who did not receive antidiarrheal medications; model B, analysis of 63 persons who did not receive antimicrobial agents or antidiarrheal medications; model C, analysis of 85 persons who received only fluoroquinolone antimicrobial agents.

sistant strains, or both remains to be fully determined. Tetracyclines and chloramphenicol are alternative antibiotics when resistance or some other reason excludes the former drugs, but up to 60% of strains may be resistant to tetracyclines. Amoxicillin plus clavulanic acid (but not sulbactam or tazobactam) appears to be effective (Hakanen et al., 2003; Lachance et al., 1993). Serious systemic infection should be treated with an arninoglycoside, such as gentamicin, or imipenem. A macrolide may still be needed to clear infection from the gut.

INDICATIONS FOR ANTIMICROBIAL THERAPY The categories of patients likely to benefit from antimicrobial therapy include those who remain acutely ill and show no signs of improvement at the time a bacteriological diagnosis is made, those who have unusually severe or complicated infection, those who have systemic infection, those who are immunosuppressed or have another predisposition to infection, and those who live in an institution or closed group where the risks of the spread of disease are high. In this last case, treatment is provided to elirninate the organism from the stools of infected patients. Erythromycin was used successfully in this way to control a persistent outbreak of Cumpylobacter infection in a day care nursery in Israel (Ashkenazi et al., 1987).

Acknowledgments. We thank Martin Skirrow for his contributions to this chapter in the second edition and for his many contributions over more than three decades of study to our understanding of the clinical nature of Campylobacter enteritis.

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Antimicrobial Resistance and Antibiotic Usage in Animals in The Netherlands in 2003. http://www.cidc-lelystad.wur.nl/ NR/ rdonlyres/ 7F79ACE6-OFD2-41AB-8lB2-BB17FA89603C/ 11381/MARAN2003webl.pdf. VANTURES, the Veterinary Antibiotic Usage and Resistance Sur"2004. Monitoring of veillance Working Group. 2005. i Antimicrobial Resistance and Antibiotic Usage in Animals in The Netherlands in 2004. http: //www.cidc-lelystad.wur.nl/ NR / rdonlyres / 7F79ACE6-OFD2-41AB-81B2-BB17FA89603C/ 11382/MARAN2004webl.pdf. Vogt, R. L., H. E. Sours, T. Barrett, R. A. Feldman, R. J. Dickinson, and L. Witherell. 1982. Campylobacter enteritis associated with contaminated water. Ann. Intern. Med. 96:292-296. Wagner, J., M. Jabbusch, M. Eisenblatter, H. Hahn, C. Wendt, and R. Ignatius. 2003. Susceptibilities of Campylobacter jejuni isolates from Germany to ciprofloxacin, moxifloxacin, erythromycin, clindamycin, and tetracycline. Antimicrob. Agents Chemother. 47:2358-236 1. Wang, W. L., and M. J. Blaser. 1986. Detection of pathogenic Campylobacter species in blood culture systems. J. Clin. Microbiol. 23:709-714. Webster, P. B., and D. J. Farrell. 1993. Campylobacter peritonitis as a complication of continuous ambulatory peritoneal dialysis. Aust. J. Med. Sci. 14:68-70. Wickins, H. V., R. Thwaites, and J. A. Frost. 2001. Drug resistance in Campylobacter jejuni and Campylobacter coli in England, and Wales 1993-2001. Abstr. 11th Int. Workshop Campylobacter Helicobacter Relat. Organisms, Freiburg, Germany, 1 to 5 September 2001. Wilson, P. G., J. R Davies, T. W. Hoskins, K. P. Lander, H. Lior, D. M. Jones, and A. D. Pearson. 1983. Epidemiology of an outbreak of milk-borne enteritis in a residential school, p. 143. In A. D. Pearson, M. B. Skirrow, B. Rowe, J. R. Davies, and D. M. Jones (ed.), Campylobacter 11. Proceedings of the Second lnternational Workshop on Campylobacter Infections. Public Health Laboratory Service, London, United Kingdom. Wood, C. J., V. Fleming, J. Turnidge, N. Thomson, and R. C. Atkins. 1992. Campylobacter peritonitis in continuous ambulatory peritoneal dialysis: report of eight cases and a review of the literature. Am. J. Kidney Dis. 19:257-263. Youngs, E. R., and C. Roberts. 1985. Campylobacter carriage and pregnancy. Br. J. Obstet. Gynaecol. 9 2 5 4 1 4 4 2 .

Campylobuctcr, 3rd ed. Edited by I. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 7

Clinical Significance of Campylobacter and Related Species Other Than Campylobacter jejuni and Campylobacter coli ALBERT J. LASTOVICA AND BAN MISHU ALLOS

subsp. fetus is now recognized as a major cause of septic abortion in domestic animals (Garcia et al., 1983). Since 1947, C. fetus has also been implicated as a causative agent of a range of human intestinal and extraintestinal illnesses. C. fetus is the type species of the genus Campylobacter (VCron and Chatelain, 1973), and it is separated into two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis. This division stems from the realization that two distinct disease entities could be attributed to two varieties of strains (Florent, 1959), which were subsequently accorded subspecies status on the basis of DNA-DNA hybridization studies (Harvey and Greenwood, 1983). C. fetus subsp. fetus colonizes the intestine and causes sporadic abortion in sheep and cattle, usually late in gestation. C. fetus subsp. venerealis is adapted to the bovine genital tract and causes infertility by destroying the embryo early in gestation. This disease, known as bovine vibriosis, is of major concern to the cattle industry (Garcia et al., 1983). The principal habitat of C. fetus subsp. venerealis is the prepuce of asymptomatic bulls, which form a reservoir of infection for cows. The status of C. fetus subsp. venerealis as a human pathogen is apparently minimal, but because of the difficulty of separating subsp. venerealis from subsp. fetus by phenotypic methods (mainly growth in the presence of 1%glycine), uncertainty remains. Hum et al. (1997) developed a PCR assay for the identification and differentiation of the two subspecies, which, when compared with traditional phenotyping, indicated that strains are often misidentified. Strains confirmed as subsp. venerealis by genotyping methods have been isolated from the vagina of a woman with bacterial vaginosis in Sweden, and from two homosexual men in Australia (Holst et al., 1987;

Campylobacters are the most frequently identified bacteria causing diarrhea in humans, particularly in very young children, in both developed and developing countries (Allos, 2001). Campylobacters have also been associated with other clinical conditions such as bacteremia Guillain-BarrC syndrome, hemolytic-uremic syndrome, pancreatitis, and reactive arthritis. The genus Campylobacter currently comprises 17 species, of which 14 have been isolated from humans. Historically, more than 95% of Campylobacter strains isolated and identified in cases of human disease have been c. jejuni subsp. jejuni or coli. However, the isolation techniques currently used in many diagnostic laboratories may not support the growth of other, potentially pathogenic non-jejuni, non-coli Campylobacter species. These organisms may be fastidious, requiring special atmospheric and temperature conditions as well as prolonged incubation, or they may be unable to tolerate the antibiotics commonly included in selective media plates. The disease potential of these non-jejuni, non-coli Campylobacter species is beginning to be appreciated, particularly in areas where they are being looked for. This chapter will describe the microbiology, epidemiology, and clinical features of infection with Campylobacter species other than C. jejuni subsp. jejuni and C. coli that are associated with human disease.

c.

CAMPYLOBACTER FETUS McFaydean and Stockman (1913) first recognized campylobacters as a causative agent of fetal infection and abortion in sheep. In 1919, these organisms, then called Vibrio fetus, were reported to cause abortion in cattle (Smith, 1919). Campylobacter fetus

Albert J. Lastovica Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, South AfBan Mishu Allos Vanderbilt University School of Medicine, Division of Infectious Diseases, A 2200 MCN, Nashville, TN rica. 37232.

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LASTOVICA AND ALLOS

Salama et al., 1992a). Sexual transmission of C. fetus subsp. venerealis in humans has not been demonstrated. Our concern in this chapter is with C. fetus subsp. fetus, which will hereafter be referred to simply as C. fetus. Microbiology

C. fetus, like other campylobacters, grows in a microaerobic atmosphere (6 to 12% CO,). These organisms do not ferment carbohydrates and are catalase, oxidase, and nitrate reductase positive (Table 1). C. fetus strains grow well at 25 and 37”C, but unlike C. jejuni, will not usually grow at 42°C (Dediste et al., 1998). C. fetus is resistant to nalidixic acid, susceptible to cephalothin, and lacks pyrazimidase activity (Burnens and Nicolet, 1993), which are useful characteristics for distinguishing C. fetus from C. jejuni. Although early investigators (Butzler and Skirrow, 1979; Edmonds et al., 1985) reported that the isolation of C. fetus from stools was rare, its natural habitat is the intestine. Many clinical laboratories incubate samples at 42°C and may use a medium that contains cephalothin, conditions favoring the isolation of C. jejuni but not of C. fetus. C. fetus grows slowly, and isolation from blood culture can take several weeks (Wang and Blaser, 1986). Because other enteric flora grow more rapidly than campylobacteria, antibiotic-containing media traditionally have been used for the isolation of campylobacters from stool specimens. However, isolation of C. fetus and other non-jejuni Campylobacter species from stools may be unsuccessful because of the use of antibioticcontaining media to which these organisms are susceptible. Filtration of stools through 0.65-pm filters onto the surface of antibiotic-free blood agar plates permits the passage of the smaller C. fetus bacteria while retaining larger contaminating microorganisms (Fig. 1) (Lastovica, 2006). PCR assays for C. fetus subsp. fetus and C. fetus subsp. venerealis have been published (Oyarzabal et al., 1997; Schultz et al., 2006). Epidemiology The source of many C. fetus infections in humans is likely zoonotic. The major habitat of C. fetus is the intestine, and it is commonly isolated from healthy sheep and cattle (Table 2). The organism is found in abundance in the genital tracts of aborting sheep and cattle and their products of conception (Garcia et al., 1983). It has also been found in poultry, swine, and reptiles (Smibert, 1984). Feces from infected animals may contaminate soil, freshwater,

and carcasses during abattoir processing (Blaser et al., 1983), and human infection most probably results from consumption of contaminated food or water. The only exposure common to 10 Californian patients with malignancy or other serious disease who developed C. fetus sepsis was “nutritional therapy” that entailed consuming raw calf‘s liver during the week before they became ill (CDC, 1981). Exposure to raw beef, raw beef liver, or improperly cooked pork shortly before illness has been reported in several patients with C. fetus bacteremia (Miki et al., 2005; Rao et al., 1990). Klein et al. (1986) reported an outbreak in Wisconsin of C. jejuni and C. fetus infection caused by drinking raw milk. The presence of C. fetus was detected solely because the C. fetus strains grew (atypically) at 42”C, the standard temperature for isolating C. jejuni. In an outbreak of C. fetus diarrhea that affected 18 of 90 members of a Hutterite colony in Alberta, Canada, persons who worked in the colony’s abattoir were 2.03 times more likely to have diarrhea than others (Rennie et al., 1994). The modes of transmission of C. fetus infection in humans are not well understood. Despite evidence which indicates zoonotic transmission, more than two-thirds of patients with C. fetus bacteremia lived in an urban environment and had no known exposure to farm animals (Bokkenheuser, 1970; Tremblay et al., 2003). Nosocomial spread of C. fetus infection in a neonatal intensive care unit, in which four infants developed meningitis, has been recorded (Morooka et al., 1996). Clinical Features The clinical features of diarrheal disease due to C. fetus infection in healthy individuals is similar to infection by C. jejuni (Rennie et al., 1994). Sequelae are uncommon, and most patients do not require antibiotic treatment. Although C. fetus infection may occur at any age, most infections occur in elderly persons. In an analysis of 111 C. fetus strains isolated in Quebec, Canada, from 1983 to 2000 (Tremblay et al., 2003), 53% occurred in persons older than 70 years. The annual incidence of infection was 0.1 per 100,000 population; however, this is likely an underestimate because many infections may be neither detected nor reported (Tremblay et al., 2003). C. fetus infection usually occurs in compromised patients, more than 75% of whom are men with serious medical conditions (e.g., diabetes mellitus, atherosclerosis, liver cirrhosis, chronic alcoholism, asplenia, and AIDS) and patients being treated with immunosuppressive agents (Devlin and McIntyre, 1983; Guerrant et al., 1976; Monno et al., 2004; Sakran et al., 1999). In a study of almost 100 patients

Table 1. Phenotypic and biochemical characteristics of Campylobacter, Arcobacter, and intestinal Helicobacter species of clinical significance"

Species or subspecies

C. jejuni subsp. jejuni biotype I f C. jejuni subsp. jejuni biotype 2f C. jejuni subsp. doylei C. coli C. fetus subsp. fetus C. upsaliensis C. lari C. hyointestinalis C. sputorum C. concisus C. mucosalis

c. cuwusi

C. rectus' C. showae C. gracilisi.' A. butzleri' A. cryaerophilus' H. cinaedi" H. fennelliae m.n H. pullorum

Catalase

Nitrate reduction

Arylsulfatase

Pyrazinamidase

Hippurate hydrolisis

Nalidixic acidb

Cephalothin'

HIS production Rapid TSIe Lead H,Sd acetate

Growth at:

250c

420c

Indoxyl acetate

+

++ ++

+ +

-

-

+

+ + + + + + + + + + + + + + +

++ +

+

-

+

(+I

+

5+ 5+ 3+ 5+ 5+ 3+ ND ND -

-

(-) -

+ + + ND

+ +

(+)

-

+

+

ND

ND

+, positive; (+), most strains positive; -, negative; (-), most strains negative; ND, not determined; R, resistant; (R), most strains resistant; S, susceptible; ( S ) , most strains susceptible. bNalidixic acid (30-pg disk). Cephalothin (30-pg disk). dMethod of Skirrow and Benjamin (1980). 'Triple sugar iron. "Biotypes 1 and 2 of Skirrow's scheme (1980). 8 Some strains grow better in HI-enhanced rnicroaerophilic conditions. hUPTC variant positive. Biovar paraureolytic positive. iPitting colonies on blood agar. kNonmotile and oxidase negative. 'Aerobic growth at 30°C. Spreading, noncolonial growth. "Hypochlorite odor. a

+ +

-

H~

required

Urease

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LASTOVICA AND ALLOS

Figure 1. A 4-day-old pure growth of C. fetus after filtration of a stool specimen onto an antibiotic-free blood-agar plate.

with C. fetus infection, the male-to-female ratio was 1.1 to 1.0, and 69% of isolates were from blood, 20% from stools, and 11% from other typically sterile sites (Tremblay et al., 2003). Intestinal infections may have serious complications such as toxic megacolon (Kalkay et al., 1983). C. fetus accounts for more bacteremia in adults than any other Campylobacter species except C. jejuni (Lastovica, 1996; Skirrow et al., 1993). Infections due to C. fetus were previously considered primarily to cause bacteremia in elderly men with chronic underlying illness, but AIDS patients may now represent the most typical population (Font et al., 1997; Rao et al., 1990). Infections also may occur in patients who have undergone solid or hematopoietic organ transplantation (Heng et al., 2002; Monno et al., 2004). C. fetus may cause a prolonged, relapsing illness that is characterized by chills, fever, and myalgia, and that lacks an identified focus of infection (Blaser et al., 1983; Guerrant et al., 1976;). Sec-

Table 2. Sources and disease associations of non-jejuni, non-coli Campylobacter and related bacteria" Species or subspecies

Recognized sources

Human disease association

Animal disease association

Cattle Cats, dogs, ducks, monkeys Pigs, cattle, hamsters

Septicemia, enteritis, abortion, meningitis Septicemia (rarely) Enteritis, septicemia Enteritis, septicemia6

Bovine, and ovine spontaneous abortion Bovine infectious infertility Canine, feline gastroenteritis Porcine and bovine enteritis

Pigs, birds (including poultry)

None

?c

Enteritis, septicemia

?

C. concisusd

Cats, dogs, chickens, monkeys, seals, mussels, oysters (river and seawater) Humans

?

C. mucosalis C. sputorum biovar sputorum C. jeiuni subsp. doylei H. cinaedi

Pigs Humans, cattle, pigs, sheep Humans Humans, hamsters

H. fennelliae

Humans

H. pullorurn A. butzleri

A. skirrowii

Poultry Pigs, bulls, horses, cattle, chickens, primates, ostriches, ducks, water, sewage Pigs, bulls, poultry, sheep, sewage, horses Sheep, bulls, pigs, chickens, ducks

Periodontal disease, enteritis, septicemia None Abscesses Enteritis, septicemiab Enteritis, septicemia: proctocolitis Enteritis, septicemia: proctocolitis Enteritis Enteritis, septicemia

H. rappini

Humans, mice, sheep

C. fetus subsp. fetus

Cattle, sheep

C. fetus subsp. venerealis C. upsaliensis C. hyointestinalis subsp. hyointestinalis C. hyointestinalis subsp. lawsonii C. lari

A. cryaerophilus

Enteritis, septicemia None Enteritis, septicemia6

Porcine necrotic enteritis ? ?

Hamster enteritis ?

Avian hepatitis Porcine, bovine, primate gastroenteritis, porcine abortion Bovine, porcine, ovine, equine abortion Porcine, equine abortion; ovine, bovine gastroenteritis Ovine abortion

"Data from Bolton et al. (1987); Flores et al. (1990); Foster et al. (2004); Fox et al. (1989); Garcia et al. (1983); Holst et al. (1987); Lammerding et al. (1996); Lastovica (2006); Patton et al. (1989); Sandstedt and Ursing (1991); Tanner et al. (1984); Vandamme et al. (1992b). 'Children and HIV patients. Includes C. curvus and C. rectus. ?,unknown.

CHAPTER 7

CLINICAL SIGNIFICANCE OF CAMPYLOBACTER AND RELATED SPECIES

ondary seeding to an organ may occur, which can lead to complications and occasionally death (Collins et al., 1964; Viejo et al., 2001). Bacteremia due to C. fetus may be primary, arising from the gastrointestinal tract, or secondary, arising from infection of another site. Infection may occur as a direct result of invasion of the bloodstream via venipuncture or by an intravenous line (Guerrant et al., 1976). Although the organism is rarely isolated from feces, diarrhea precedes or accompanies bacteremia in nearly half the cases (Guerrant et al., 1976; Lastovica, 1996). C. fetus is occasionally isolated from pediatric patients (Table 3). High fevers frequently occur, but usually they are well tolerated, and the mortality is approximately 20% (Dickgiesser et al., 1983; Rao et al., 1990). C. fetus exhibits a distinct affinity for vascular tissue, and infections have been associated with thrombophlebitis, cellulitis, and mycotic aneurysms (Carbone et al., 1985; Cone et al., 2003; Montero et al., 1997). Reviews describe 26 cases of C. fetusassociated endocarditis (Bar et al., 1998; Farrugia et al., 1994); all the patients had some kind of predisposing disorder such as liver or hepatic cirrhosis, rheumatic or ischemic heart disease, or alcoholism. A 52-year-old man developed C. fetus endocarditis when he ate raw meat shortly after having a tooth extracted (Miki et al., 2005). Kanj et al. (2001) described a 14-year-old girl with P-thalassemia and C. fetus pericarditis who improved with pericardiectomy and ampicillin treatment. C. fetus is recognized as a causative agent of septic abortions in animals, and it is now known to cause perinatal sepsis and fetal loss

Table 3. Distribution of Campylobacter and related species isolated from the diarrhetic stools of children at the Red Cross Children’s Hospital, Cape Town, South Africa, from 1 October 1990 to 30 June 2007” Species or subspecies

No.

YO

C. jejuni subsp. jejuni C. concisus C. upsaliensis C. jejuni subsp. doylei H. fennelliae C. coli C. hyointestinalis H. cinaedi CLO / HLO A. butzleri C. fetus subsp. fetus C. curvtrs, C.rectus, and H. rappini C. sputorum biovar sputorum and C. lari Total

1,956 1,503 1,414 43 1 337 181 57 51 31 20 9 9 7 6,006

32.57 25.02 23.54 7.18 5.61 3.01 0.95 0.85 0.52 0.33 0.15 0.15 0.12 100.00

“Data from Lastovica (2006) and unpublished results. bCLOIHLO, Cumpylobucter or Helicobacter organisms that could not be fully characterized.

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in humans. In pregnant animals, ingestion of C. fetus leads to intestinal infection, followed by bacteremia, and because the organism has a high affinity for placental tissues, there is a risk of infection of the placenta and fetus (Miller et al., 1959). Similarly, gastrointestinal colonization, with or without enteritis, followed by a hematogenous spread and then a placental infection, probably occurs in the rare cases of infection in women. C. fetus infection in pregnant women is usually recognized during the third trimester, although abortions at 9.5 and 14 weeks have been reported (Steinkraus and Wright, 1994). The illness is usually mild in the mothers, but outcome is often poor in the infants. Neonates may be infected transplacentally or during delivery. Even with appropriate antimicrobial therapy, the average mortality of fetuses and neonates is about 70% (Sauerwein et al., 1993; Steinkraus and Wright, 1994). Fetal infection and abortion may only become apparent several weeks after maternal infection. Salpingitis and turboovarian abscess due to C. fetus have been described in nonpregnant women (Brown and Sautter, 1977; McGechie et al., 1982). The central nervous system may be infected by C. fetus, with meningoencephalitis being the most common manifestation in adults (Dronda et al., 1998; Gorkiewicz et al., 2002). Subarachnoid hemorrhages, brain abscesses (La Scola et al., 1998), cerebral infarctions, and subdural abscesses (Mendelson et al., 1986) have been reported; although two-thirds of the patients survive, neurological sequelae are frequent. In neonates, the prognosis is usually worse, but not invariably so (La Scolea, 1985). The cerebrospinal fluid typically shows polymorphonuclear pleocytosis. Subdural effusion may also be present. C. fetus may remain latent in an immunocompromised host, after bacteremic seeding in a bony focus, only to be reactivated years later (Neuzil et al., 1994). Eradication of such an infection probably requires surgical excision of the infected area. C. fetus can be an unobtrusive inhabitant of the gut, but with decreased host immunity, the organism may invade the mucosa and cause generalized infection. Postoperative prosthetic hip joint (Chambers et al., 2005) and knee (David et al., 2005) infections due to C. fetus have been reported, as has chronic osteomyelitis of the ankle (Bracikowski et al., 1984) and spine (Yamashita et al., 1999). Other forms of C. fetus infection are septic arthritis (Fick et al., 1979), lung abscess (Lawrence et al., 1971), empyema (Targan et al., 1977), gluteal abscess (Otero et al., 1994), chorioamnionitis (Viejo et al., 2001), cholangitis (Alvarez et al., 2000), and cholecystitis (Takatsu et al., 1997). Infection of the peritoneum by C. fetus has been reported in peritoneal dialysis patients; it was postu-

128

LASTOVICA AND ALLOS

lated that direct contamination of the catheter had occurred (Wens et al., 1985). Spontaneous C. fetus peritonitis has occurred in alcoholic patients with cirrhosis (Targan et al., 1976), possibly as a result of impaired reticuloendothelial clearance of portal bacteremia. The pathogenesis of C. fetus infection is covered in detail in chapter 23. Treatment

C. fetus infections are often prolonged and result in relapse, but most patients will recover with appropriate antibiotic treatment and medical procedures. The prognosis depends on the severity of the underlying illness and the rapidity of application of antibiotic treatment. Infection by c. fetus can be lethal in some debilitated persons and could hasten the death of others. Healthy immunocompetent patients usually have self-limiting enteritis or bacteremia with no sequelae and rarely need antibiotics. However, patients with systemic C. fetus infection often require extended parenteral therapy. Erythromycin may not be sufficient to treat infections, but ampicillin, carbapenems, or third-generation cephalosporins are usually effective against established C. fetus infections (Fujihara et al., 2006; Herve et al., 2004). Although tigecycline has been shown to be effective against C. jejuni and C. coli, its effectiveness in treating infections with C. fetus has not been studied (RodriguezAvial et al., 2006). Patients with hypogammaglobulinemia and persistent C. fetus bacteremia may require lifelong antibiotic therapy. Intravenous immunoglobulins are not effective in treating immunodeficient persons with C. fetus infection because the serum from normal persons usually does not contain opsonizing antibodies to C. fetus (Neuzil et al., 1994).

CAMPYLOBACTER UPSALIENSIS C. upsaliensis is a recognized human pathogen in both healthy and immunocompromised patients. It causes bacteremia and acute or chronic diarrhea (Lastovica, 1996). This organism, originally described as a catalase-negative or weak Campylobacter, has been associated with the hemolytic uremic syndrome (Carter and Cimolai, 1996) and with spontaneous human abortion (Gurgan and Diker, 1994). C. upsaliensis was first isolated from the stools of healthy and diarrhetic dogs in 1983. DNA-DNA hybridization studies indicated that this was a new species (Sandstedt and Ursing, 1991), and the name upsaliensis was adopted in honor of the Swedish town, Uppsala, where the organism was first isolated (International

Union of Microbiological Societies, 199 1). Analysis of 96 C. upsaliensis strains from three continents showed that 84% belonged to one of five serotypes (Lentzsch et al., 2004). These data point to clonal expansion of C. upsaliensis and indicate a high degree of antigen conservation during evolution. A review by Bourke et al. (1998) has summarized available data on C. upsaliensis. C. helveticus is a closely related species found in dogs and cats, but to date, it has not been isolated from humans (Stanley et al., 1992). Microbiology

C. upsaliensis is a thermotolerant Campylobacter species that usually grows well at 42"C, but not at 25°C. Some strains will grow better in an H,enhanced microaerophilic atmosphere (Table 1).This organism is catalase negative or only weakly positive (Sandstedt and Ursing, 1991); it is hippurate and aryl sulfatase negative but nitrate reductase and indoxyl acetate positive. A striking and distinguishing characteristic of C. upsaliensis is its intense susceptibility to both nalidixic acid and cephalothin, with inhibitory zones of up to 80 mm for cephalothin (Lastovica et al., 1989). Because cephalothin is often used in media for the isolation of Campylobacter, cephalothin-susceptible C. upsaliensis cannot be isolated with such media. Detection and the Cape Town Protocol The so-called Cape Town protocol for the isolation of C. upsaliensis and other campylobacters from stool specimens without the use of selective media was developed at the Red Cross Children's hospital in Cape Town. The method involves filtration of stools through a membrane filter onto antibioticfree blood agar plates (Fig. 1) and subsequent incubation in an H,-enhanced microaerobic atmosphere (Lastovica, 2006). With the use of this protocol since 1990, stool cultures positive for Campylobacter or related microorganisms rose to 21.8% from the 7.1% obtained with antibiotic-containing selective plates and conventional microaerobic incubation previously used (Lastovica, 2006). This South African diagnostic microbiology laboratory could only begin to isolate C. upsaliensis from stool specimens once the isolation protocol was changed from antibiotic-containing media to the Cape Town protocol (Lastovica, 2006). The use of membrane filtration in this protocol has the added advantage of permitting the isolation of antibiotic-susceptible campylobacters other than C. upsaliensis. On the basis of the difference in colony

CHAPTER 7

CLINICAL SIGNIFICANCE OF CAMPYLOBACTER AND RELATED SPECIES

morphology on primary isolation and subsequent biochemical and serological confirmation, 16.2% of the stools of South African children with gastroenteritis had multiple isolates of two to five species, or of several different serotypes of C. jejuni (Lastovica, 2006). C. upsaliensis was frequently coisolated with C. jejuni subsp. jejuni, C. jejuni subsp. doylei, and particularly with Helicobacter fennelliae (Lastovica, 2006). If infection by more than one species is suspected, considerable care must be taken to separate the domed colonies of C. upsaliensis from the spreading, noncolonial growth of Helicobacter fennelliae or H. cinaedi before positive identification can be undertaken (Fig. 2). In an Australian study of 676 hospitalized patients with gastroenteritis, 75 Campylobacter strains were isolated on blood-free medium with a selective supplement, but concurrent isolation onto antibioticfree blood agar overlaid with a membrane filter yielded 213 Campylobacter strains. Some strains could only be isolated by the membrane filter technique (Albert et al., 1992). Loades et al. (2005) showed that every commercial Campylobacter selective media tested was inhibitory to some stains in a panel of 59 isolates of a variety of Campylobacter, Helicobacter, and Arcobacter spp. In a direct comparison, the Cape Town protocol proved superior to selective media for the isolation of C. upsaliensis from clinical specimens (Lastovica and Le ROUX,2001). A process of membrane filtration onto antibiotic-free media and incubation in an H,-enhanced microaerophilic atmo-

Figure 2. Culture plate showing the spreading, noncolonial growth of H. fennelliae (top) contrasted with the domed colonies of C. upsaliensis (bottom).

129

sphere is a simple, efficient, and cost-effective alternative to antibiotic-containing selection media, and this method permits the isolation of a whole spectrum of clinically relevant Campylobacter, Helicobacter, and Arcobacter species (Lastovica, 2006). Epidemiology C. upsaliensis is widespread on all continents and is commonly isolated from the intestinal contents of dogs. C. upsaliensis comprised 64% of the 98 Campylobacter strains isolated from dogs in Sweden over a 2-year period (Sandstedt et al., 1983). The organism has been isolated from dogs with chronic diarrhea (Davies et al., 1984; Modolo and Giuffrida, 2004), healthy puppies and kittens (Hald and Madsen, 1997), asymptomatic cats (Fox et al., 1989), and asymptomatic vervet monkeys (Lastovica et al., 1991). Although the exact source of C. upsaliensis infection in humans in unknown, zoonotic transmission is a distinct possibility. Four of seven patients with C. upsaliensis infection in one study reported animal contact (Patton et al., 1989). Stool isolates of C. upsaliensis that appeared to be the same strain were isolated from a 53-year-old man with bloody diarrhea and his healthy 3-year-old dog (Goossens et al., 1991). In another study, identical plasmid profiles were detected in human and dog isolates (Owen et al., 1985). Gurgan and Diker (1994) documented the isolation of C. upsaliensis in blood and fetoplacental specimens of a woman who experienced a spontaneous abortion at 18 weeks’ gestation; a C. upsaliensis strain was isolated from her asymptomatic cat, and protein profile analysis confirmed a strong similarity between the human and feline isolates. Although these observations are suggestive, animal-to-human transmission of C. upsaliensis still remains to be unequivocally proved. Indirect evidence suggests that person-to-person transmission of C. upsaliensis is possible. Goossens et al. (1995) documented C. upsaliensis infection in 34 children in four day care centers in Brussels, Belgium. On the basis of several molecular typing methods, it was demonstrated that the outbreaks of C. upsaliensis infection in three centers were due to the same strain. This C. upsaliensis strain was closely related to the strain isolated from the fourth day care center. The proportion of all campylobacters that are C. upsaliensis is relatively high when fecal samples are prospectively cultured for C. upsaliensis. From 1990 to 2007, at a pediatric hospital in South Africa, 1,414 strains of C. upsaliensis were isolated, which accounted for 23.5% of all the campylobacters and related organisms isolated and identified (Table 3). This

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high isolation rate of C. upsaliensis is attributed to application of the Cape Town protocol (Lastovica, 2006). Only a single C. upsaliensis strain was obtained in a study of 631 stools from Thai children (Taylor et al., 1991). A Canadian study of 915 Campylobacter stool isolates indicated that only 7 (0.1%) were C. upsaliensis (Taylor et al., 1989). In a survey of 3 94 Campylobacter clinical blood culture strains isolated in England and Wales, two (0.8%) were C. upsaliensis (Skirrow et al., 1993). By contrast, a South African study of 221 Campylobacter strains obtained from children's blood cultures indicated that 39 (18%) were C. upsaliensis (Lastovica, 1996). Differences in isolation and culture protocols used in various laboratories could account in part for the wide variation in C. upsaliensis prevalence. Alternatively, data from South Africa compared with other studies may reflect differences in prevalence and exposure to C. upsaliensis, or possibly differences in colonization and the nature of C. upsaliensis infection in different geographical areas. Characterization of Isolates

C. upsaliensis has a plasmid carriage rate of about 90%, much higher than other species of Campylobacter (Da Silva Tatley et al., 1992; Owen and Hernandez, 1990). Digestion of C. upsaliensis chromosomal DNA with HaeIII has indicated that less than 20% of the strains examined were related (Owen and Hernandez, 1990). Although serotyping C. upsaliensis strains has been of limited value (Lastovica et al., 1989), sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) protein profile analysis (Owen et al., 1989) has proved useful for the differentiation of individual isolates of C. upsaliensis. Pulsed-field gel electrophoresis (PFGE)

indicated molecular heterogeneity in a study of 20 C. upsaliensis strains (Bourke et al., 1996). Identification of C. upsaliensis can be done by PCR assays based on the 16s rRNA gene (Linton et al., 1996), the GTPase gene (Van Doorn et al., 1997), or the GlyA gene (Steele et al., 1985b). Miller et al. (2005) have developed a multilocus sequence typing scheme for C. coli, C. lari, C. upsaliensis, and C. helveticus. Realtime PCR may ultimately provide an efficient method for differentiating Campylobacter species (Jensen et al., 2005). Clinical Features The usual symptoms associated with C. upsaliensis infection are gastrointestinal and include watery diarrhea, abdominal cramps, vomiting, and low-grade fever (MCgraud and Bonnet, 1986; Patton et al., 1989; Taylor et al., 1989). Although most patients recover quickly, others may be ill for several weeks (MCgraud and Bonnet, 1986; Patton et al., 1989; Taylor et al., 1989). In a study of 99 patients with C. upsaliensis in their stools, in which the onset of the symptoms was abrupt, 92% had diarrhea, 14% had vomiting, and 6% had fever (Goossens et al., 1990). Symptoms persisted for more than 1 week in 16% of these patients, while 25% had bloody stools and 10% had fecal leukocytes. In a study of 882 C. upsaliensis-infected children (Table 4), the average age was 19.4 months (range, 1 month to 10 years). Loose stools were present in 89% and watery stools in lo%, and they formed in less than 1% of the children. Gastroenteritis symptoms were present in 88%, vomiting in 9%, and fever (temperature >38"C) in 6% of these patients. Less than 4% were coinfected with Salmonella or Shigella. However, 8% of these patients had Ascaris, Tricuris, Cryptosporidium, or

Table 4. Clinical features of infection with non-jejuni, non-coli Cumpylobucter species and related organisms obtained from children in Cape Town, South Africa, 1990 to 2007 % of patients with:

Organism

Coexisting enteric

No. of patients Diarrhea

C. jejuni subsp. jejuni C. concisus C. upsaliensis C. jejuni subsp. doylei H. fennelliae C. hyointestinalis H. cinaedi A. butzleri C. fetus subsp. fetus

1,432 911 882 358 253 51 38 15 6

85 80 88 79 79 74 79 100 100

Vomiting

6 3 9 6 8 12 8 60 17

Fever

5 9 6 4 5 4

8 20 17

pathogen:

Preexisting conditions

Bacterial

Parasitic

6

7 6 6 8 10 12 8 0 0

4

3 4 5 6 8 20 17

9 18 1 10 33 39 26 40 100

CHAPTER 7

CLINICAL SIGNIFICANCE OF CAMPYLOBACTER AND RELATED SPECIES

Giardia detected in their stools. Underlying illnesses such as kwashiorkor, marasmus, convulsions, hepatitis, anemia, or tuberculosis were present in 13% of these children (Lastovica et al., 1989). Two of 8 C. upsaliensis bacteremic patients had recent abdominal surgery (Patton et al., 1989), and 8 of 16 children with C. upsaliensis bacteremia had gastrointestinal symptoms. These observations suggest that the bacteremias may have been the result of intestinal infections (Lastovica et al., 1989). Most patients with C. upsaliensis bacteremia have other serious underlying medical conditions (Lastovica et al., 1989; Patton et al., 1989). C. upsaliensis has been isolated from the breast abscess of a patient who reported no animal contact or gastrointestinal symptoms (Gaudreau and Lamonthe, 1992), and C. upsaliensis has been linked to the hemolytic-uremic syndrome (Carter and Cimolai, 1996). Pathogenesis Because C. upsaliensis may produce both inflammatory and noninflammatory diarrhea in humans, the organism’s virulence may depend in part on hostcell invasion. C. upsaliensis is capable of invading cell lines of gastrointestinal origin more efficiently than nonintestinal cell lines (Mooney et al., 2003), suggesting that the organism has a tropism for the gastrointestinal tract. Specific intestinal cell factors may influence the organism’s ability to be internalized, which likely affects its disease-causing abilities. Sylvester et al. (1996) demonstrated that C. upsaliensis is capable of binding to CHO and HEp-2 cells in tissue culture. Surface proteins in the range 50 to 90 kDa on C. upsaliensis isolates could bind to phosphatidylethanolamine, a putative cell membrane receptor. Biotin-labeled C. upsaliensis strains also bound in a concentration-dependent fashion to human small-intestine mucin, implying that that C. upsaliensis express an adhesin or adhesins capable of recognizing a specific mucin epitope or epitopes (Sylvester et al., 1996). Binding to mucins may possibly influence bacterial access to cell membrane receptors and thus influence host resistance to infection. Mooney et al. (2001) observed that C. upsaliensis releases a cytolethal distending toxin that affected HeLa and T lymphocyte cells. They also described invasion of cultured epithelial and primary human small intestinal cells, and electron microscopy revealed C. upsaliensis within Caco-2 cell cytoplamic vacuoles (Mooney et al., 2003). Fouts et al. (2005) described a novel putative virulence locus, licABCD for C. upsaliensis, similar to genes present in Neisseria (Serino and Virji, 2002). These licABCD genes encode proteins involved in the acquisition of cho-

13 1

line, the synthesis of phosphorylcholine, and the transfer of phosphorylcholine to lipooligosaccharide or teichoic/lipoteichoic acids to facilitate attachment to host cells (Serino and Virji, 2002). Although these observations are suggestive but not conclusive of virulence, proof of C. upsaliensis as a human enteropathogen will require additional studies. Treatment The most active antimicrobial agents available for treatment of C. upsaliensis infection are fluoroquinolones (Preston et al., 1990). Erythromycin was once considered the preferred treatment in Campylobacter infections, but 4 to 18% of C. upsaliensis isolates are resistant to erythromycin (CDC, 1981; Moore et al., 2006; Patton et al., 1989). Infections with C. upsaliensis have been treated successfully with amoxicillin-clavulanate, cefotaxime, and doxycycline (Goossens et al., 1991, 1995; Russell et al., 1992).

CAMPYLOBACTER HYOINTESTINALIS In 1983, C. hyointestinalis was identified and suggested as a possible cause of proliferative enteritis in pigs (Gebhart et al., 1983). This organism has subsequently been isolated from human stools, particularly those from children with watery, nonbloody diarrhea. The name hyointestinalis is derived from the Latin hyo, “hog,” and intestinalis, “pertaining to the intestine.” On the basis of phenotypic and genomic methods, On et al. (1995a) described Campylobacter hyointestinalis subsp. lawsonii, subsp. nov., and Campylobacter hyointestinalis subsp. hyointestinalis subsp. nov. The pathogenic role of C. hyointestinalis subsp. lawsonii in animals and humans is unknown at present (On, 1996). Microbiology and Diagnosis

C. hyointestinalis is closely related to C. fetus, and like C. fetus, it is catalase and nitrate reductase positive, indoxyl acetate negative, susceptible to cephalothin, and resistant to nalidixic acid. C. hyointestinalis differs from C. fetus in its copious production of H,S in triple sugar iron agar in the presence of H,, often entirely blackening lead acetate strips. C. hyointestinalis grows under microaerophilic conditions, but some strains require additional hydrogen (Edmonds et al., 1987; Lastovica, 2006). These isolates can be differentiated from other H,-requiring isolates by the catalase and other tests (Table 1)(Vandamme et al., 1992b). All strains of C. hyointestinalis

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will grow at 37"C, and some strains will also grow at 42°C. Lack of aryl sulfatase activity and intolerance to 3.5% NaCl are useful phenotypic tests for the diagnosis of C. hyointestinalis (Burnens and Nicolet, 1993). Because cephalothin is a constituent of many Campylobacter-selective media, C. hyointestinalis, similar to other cephalothin-susceptible campylobacters, is underdetected. Application of the Cape Town protocol has proved to be efficient for the isolation of C. hyointestinalis (Lastovica, 2006). PFGE and SDS-PAGE have been used to differentiate strains of C. hyointestinalis from each other as well as from other Campylobacter species (Costas et al., 1987; Salama et al., 1992b). DNA probes have been useful for detection of C. hyointestinalis in swine with proliferative enteritis (Gebhart et al., 1990). Oligodeoxynucleotide probes have been constructed for both C. hyointestinalis and C. fetus based on 16s rRNA sequence data (Wesley et al., 1991). A PCR assay based on the 16s rRNA gene has been developed for the detection of C. hyointestinalis (Linton et al., 1996). Epidemiology C. hyointestinalis has been consistently isolated from the intestines of pigs with proliferative enteritis, but not from asymptomatic pigs or from pigs with other enteric diseases (Gebhart et al., 1985). Its role in this disease remains uncertain. C. hyointestinalis has been isolated from hamsters, cattle, reindeer, and nonhuman primates (Table 2) (Hanninen et al., 2002; Russell et al., 1992; Walder et al., 1983). In one study, 17% of fecal samples from 300 steers contained C. hyointestinalis (Inglis et al., 2005). Transmission of C. hyointestinalis from pig to a human with chronic diarrhea and vomiting was verified by whole-cell protein electrophoresis and 16s rRNA gene sequencing data (Gorkiewicz et al., 2002). Ohya and Nakazawa (1992) reported that 21 (77%) of 29 C. hyointestinalis strains from swine with proliferative enteropathy can produce a cytotoxin, although at present, the role of this cytotoxin in human disease is uncertain. Clinical Features and Treatment In 1986, the first reported human illness was obtained with the isolation of a C. hyointestinalis strain from the stool of a homosexual man with proctitis. The patient's symptoms resolved after appropriate antibiotic treatment (Fennel1et al., 1986). Since then, C. hyointestinalis strains have been isolated from stool specimens of four patients, all of whom had

nonbloody, watery diarrhea (Edmonds et al., 1987). An 8-month-old girl, the youngest patient, had drunk unpasteurized milk, and the oldest, a 78-year-old woman, exhibited fever and vomiting. There were no leukocytes present in their stools, and both patients recovered with appropriate antibiotic therapy. The other two patients, both homosexual men, had abdominal cramps, and one was febrile. One patient recovered with trimethoprim-sulfamethoxazoletreatment. No antibiotic treatment was administered to the other patient, who continued to have intermittent diarrhea and cramps for several months (Edmonds et al., 1987). A case of C. hyointestinalis-associated diarrhea was reported in a 52-year-old woman who was immunodeficient because of an evolutive chronic myeloid leukemia (Minet et al., 1988). This patient was febrile and had a nonbloody, watery diarrhea. In another study, five strains of C. hyointestinalis were isolated from five members of the same family who had previously drunk unpasteurized milk (Salama et al., 1992b). In this family outbreak, only in the index case, a 5-month-old girl, was diarrhea present; she was also infected with C. jejuni. These five C. hyointestinalis strains were examined by PFGE. Three of the strains had identical genome patterns; the other two had completely different patterns and appeared to be unrelated (Salama et al., 1992b). In a review of patients infected with C. hyointestinalis, Breynaert et al. (1998) examined the clinical features of nine patients. Seven of the patients (four men and three women with a mean age of 63 years) were adults, and two were young children. Six patients experienced diarrhea, and five had abdominal pain. The youngest asymptomatic patient was a l-year-old girl with constipation, and the oldest was an 89-year-old woman with a myocardial infarction. None of these patients was seriously immunocompromised, but most of the adult patients had a history of neurological or vascular disease (Breynaert et al., 1998). The clinical features of 51 South African pediatric strains of C. hyointestinalis were examined (Table 4). The average age of the C. hyointestinalis patients was 16 months (range, l month to 7 years). Stools from these children were loose in 77%, watery in 21%, and formed in <2% of the cases. Blood was present in 13% of the stools and fecal leukocytes in 18%. Thirty-nine percent of these patients had underlying illness such as anemia, hepatitis, tuberculosis, convulsions, kwashiorkor, and marasmus. Other clinical features are listed in Table 4. Two extraintestinal isolates of C. hyointestinalis were obtained from the blood cultures of a 22-year-old man after bone marrow transplantation and from a 7-monthold girl with chronic diarrhea (Lastovica, 1996).

CHAPTER 7

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C. hyointestinalis is susceptible to a range of antimicrobial agents including ampicillin, amoxicillin, amoxicillin-clavulanic acid, doxycycline, and ciprofloxacin (Inglis et al., 2005; Laatu et al., 2005). Resistance has been observed to metronidazole, lincomycin, tetracycline, and erythromycin (Inglis et al., 2005; Laatu et al., 2005).

CAMPYLOBACTER LARl C. lari is a nalidixic acid-resistant, thermophilic Campylobacter first isolated from seagulls in 1980 by Skirrow and Benjamin (1980). Although the first human isolate was from an asymptomatic 6-year-old boy, C. Zari can produce acute diarrheal illness in normal hosts and can cause bacteremia in immunocompromised patients. A subgroup of C. Zuri has the unusual capability (for a Cumpylobucter) of hydrolyzing urea; strains are usually sensitive to nalidixic acid. Known by the acronym UPTC (urease-positive thermophilic Cumpylobacter), they are plentiful in natural water and shellfish. C. Zari are microaerophilic (some isolates may require H,-enhanced microaerobic growth conditions) campylobacters that grow at 42°C but not usually at 25°C. They are resistant to nalidixic acid, cephalosporins, vancomycin, and trimethoprim. Most are oxidase and nitrate reductase positive and do not hydrolyze hippurate (Table 1) (Tauxe et al., 1985). Epidemiology

C. lari has been isolated from a variety of environmental and animal sources (Table 2). Of 312 riverine samples collected in a United Kingdom study, 134 yielded campylobacters, and 7 (5%) of these were C. Zuri (MCgraud et al., 1988). In another survey of surface waters in Norway, two (2%) of 96 Campylobucter samples cultured were C. Zari (Brennhoud et al., 1992). These isolations may be significant because water is an established vehicle for the transmission of campylobacters to humans (Taylor et al., 1983). C. Zuri isolation rates were 8% from herring gulls, 29% from kittiwakes (Glunder and Petermann, 1989), and 7% from crows (Maruyama et al., 1990). In a study of the prevalence of Campylobacter spp. in oysters and mussels in The Netherlands, Van Doorn et al. (1998) found that of 44 Campylobacter isolates, 38 were C. luri. C. luri has been isolated from pigs in Sweden (Lindblom et al., 1990), poultry in Peru (Tresierra-Ayala et al., 1994) and Tanzania (Kazawala et al., 1993), fresh vegetables in Canada

133

(Park and Sanders, 1992), and occasionally from dogs (Benjamin et al., 1983). Diagnosis Because the spectrum of clinical disease described in association with C. lari is similar to that seen with other campylobacters, the diagnosis of C. luri depends on the isolation and identification of the organisms from cultured specimens. C. luri is distinct from C. jejuni in several ways, most notably in its resistance to nalidixic acid, to which most C. jejuni isolates are sensitive. C. Zari may not be correctly identified because many clinical microbiology laboratories may not routinely test isolates for nalidixic acid resistance. Nalidixic acid-susceptible isolates of C. Zari have been reported (Bolton et al., 1987; MCgraud et al., 1988), as have nalidixic acid-resistant C. jejuni strains (Walder et al., 1983), but C. lari is negative in the hippurate hydrolysis test, whereas C. jejuni is positive (Table l),and C. jejuni strains hydrolyze indoxyl acetate, whereas C. Zari does not (Popovic-Uroic et al., 1990). PCR assays specific for C. Zari based on the 16s rRNA gene (Linton et al., 1996) and the flaA gene (Sekizuka et al., 2005) are available. Van Doorn and associates (Stamp et al., 1993) developed a PCR assay based on a novel GTPase gene that utilizes speciesspecific probes for C. jejuni, C. coli, C. lari, and C. upsaliensis. This PCR hybridization assay offers rapid and specific identification of thermophilic CampyZobacter spp. Of 38 C. lari strains isolated from mussels and oysters, Van Doorn et al. (1998) found that the C. Zuri isolates were a more heterogeneous group of isolates when compared with C. jejuni than previously thought. On the basis of sequence information, a novel PCR reverse hybridization line probe assay was developed by these researchers to permit the specific and rapid detection of different c. lari variants (Van Doorn et al., 1998). A multilocus sequence typing system is available for the identification and differentiation of C. coZi, C. Zari, C. upsuliensis, and C. helventicus in mixed cultures (McGechie et al., 1982). A multiplex of PCR assays have been devised that distinguish C. coli, C. jejuni, C. Zari, and C. upsuliensis (Klena et al., 2004).

Clinical Features

C. luri is an enteric pathogen for both immunocompetent and immunocompromised hosts. Although the first human isolates of C. lari were from asymptomatic persons (Benjamin et al., 1983; Skirrow and Benjamin, 1980), the pathogenicity of C. Zari

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was only realized in 1984 with the description of a fatal case of C. luri bacteremia in a severely immunocompromised patient (Lentzsch et al., 2004). Persistent C. luri bacteremia has been reported in a human immunodeficiency virus (H1V)-infected patient (Vargas et al., 1992) and in a patient with X-linked agammaglobulinemia Uirapongsananuruk et al., 2006). Bacteremia was associated with a purulent pleurisy in an 80-year-old debilitated patient (Bruneau et al., 1998). C. luri accounted for 2 (0.8%) of 394 Cumpylobucter strains isolated from the blood cultures of patients in England and Wales (Skirrow et al., 1993). C. luri is associated with diarrheal disease, as it induced colitis in a 32-year-old HIV-positive woman who required extensive antimicrobial therapy before symptoms improved (Dronda et al., 1998). C. luri was isolated from the diarrhetic stools of five immunocompetent persons, two of whom were hospitalized. Illness lasted from 1 week to 4 months (median, 2 weeks), and all patients recovered completely (Tauxe et al., 1985). None of these patients was febrile, but four had abdominal cramps, four had contact with pets, and four had eaten chicken in the week before symptoms became apparent. A commonsource waterborne outbreak of C. luri infection occurred in Ontario in 1985 (Borczyk et al., 1987b). Gastroenteritis occurred among construction workers who drank water that had become contaminated with surface water from Lake Ontario, which had a large population of seagulls. Of 162 ill persons, 87% had diarrhea, 70% abdominal pain, and 20% fever, and vomiting, nausea, malaise, and headaches were also noted (Borczyk et al., 1987b). Only one patient reported bloody stools, and the mean duration of illness was 4 days (range, 1 to 10 days). Of the 125 stool samples cultured, 7 yielded C. luri, which is probably an underestimate because collection was delayed and specimens were transported in dry containers, reducing the bacterial viability. UPTC variants of C. luri were isolated in France from the stools of two compromised adult patients (an ovarian cancer patient and an alcoholic) with diarrhea, and from the inflamed appendix of an immunocompetent 10-year-old boy (Mtgraud et al., 1988). C. luri was isolated from the blood of a 79year-old woman with leukemia (Schrader et al., 1991). A urinary tract infection due to a UPTC variant was reported in an alcoholic man with cirrhosis (Btzian et al., 1990). C. luri-associated reactive arthritis has been reported (Goudswaard et al., 1995). C. luri strains are capable of producing both cytotoxic and cytotonic factors Uohnson and Lior, 1986), but their role in the disease process is still unknown.

Treatment

C. luri infections involved with uncomplicated diarrhea are usually self-limiting and generally do not require antibiotic therapy. When indicated, erythromycin, aminoglycosides, clindamycin, and chloramphenicol have been successfully used (Evans and Riley, 1992; Nachamkin et al., 1984). C. luri is resistant to third-generation cephalosporins, vancomycin, penicillin, and trimethoprim-sulfamethoxazole (Evans and Riley, 1992;). Fluoroquinolone-resistant strains have been reported in HIV-infected persons (Evans and Riley, 1992;).

CAMPYLOBACTER JEJUNl SUBSP. DOYLE1 On the basis of DNA hybridization studies, C. jejuni has been divided into two subspecies: C. jejuni subsp. jejuni, and C. jejuni subsp. doylei (Smibert, 1984). C. jejuni subsp. doylei was named after L. P. Doyle, who first isolated these organisms (Steele and Owen, 1988). The clinical relevance of C. jejuni subsp. doylei is just beginning to be appreciated. Clinical and other aspects of C. jejuni subsp. jejuni are covered in detail in chapter 6. Microbiology and Diagnosis The inability to reduce nitrate to nitrite (Steele and Owen, 1988) is the determining phenotypic characteristic that distinguishes C. jejuni subsp. doylei from C. jejuni subsp. jejuni and all other campylobacters (Table 1). Although H. fennelliue is also nitrate reductase negative, it has a spreading colony morphology on blood agar plates, has a strong hypochlorite smell (Fennel1et al., 1984), and is resistant to polymyxin B (Burnens and Nicolet, 1993). These characteristics readily differentiate C. jejuni subsp. doylei from H. fennelliue. C. jejuni subsp. doylei grows poorly at 42°C and is usually hippuricase and catalase positive (Table 1).These organisms are sensitive to nalidixic acid, but unlike C. jejuni subsp. jejuni, they are also susceptible to cephalothin (Table 1).Application of the Cape Town protocol is an efficient method for obtaining these microorganisms (Lastovica, 2006). Differences in colony morphology at primary isolation and subsequent characterization by biochemical and serological tests indicate that C. jejuni subsp. doylei can be coisolated with C. jejuni subsp. jejuni, C. upsuliensis, and H. fennelliue (Lastovica, 2006). Care must taken to separate the discrete, domed colonies of jejuni subsp. doylei from the noncolonial spreading growth of H. fennelliue in suspected cases of mixed infection (Fig. 2). Miller et

c.

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135

al. (2007) have developed a nap multiplex PCR assay based on the nitrate reductase locus that can be used to unambiguously subspeciate C. jejuni subsp. jejuni and C. jejuni subsp. doylei. A microarray study by Parker et al. (2007) demonstrated that C. jejuni subsp. doylei lacks various metabolic, transport, and virulence functions that were present in C. jejuni subsp. jejuni.

gest a pathogenic and possibly invasive role for C. jejuni subsp. doylei. Little is known of the virulence mechanisms in C. jejuni subsp. doylei, and in a comparative genomic indexing study (Parker et al., 2007), virulence factors such as cytolethal distending toxin were unable to be detected in the C. jejuni subsp. doylei strains examined.

Epidemiology and Clinical Features

Hz-REQUIRING CAMPYLOBACTERS

Urease-negative, gastric Campylobacter-like organisms, which were originally called GCL02 isolates and subsequently C. jejuni subsp. doylei, were identified in the gastric antral biopsy specimens of six patients (Owen et al., 1985). Similar microorganisms were found in the feces of young Australian children hospitalized with gastritis (Steele et al., 1985a, 1985b). Other studies have shown that this microorganism may be associated with diarrhea in children (FCrnandez et al., 1997; Morey, 1996). Of 631 Thai children with diarrhea, 93 (15%) had campylobacters in their stool, and 1 (1.1%) of these was a C. jejuni subsp. doylei strain (Taylor et al., 1991). In a Belgian study of 15,185 stools, 802 Campylobacter isolates were cultured, and C. jejuni subsp. doylei accounted for 4 (0.5%) of 787 strains. C. jejuni subsp. doylei was detected in 2 (0.7%) of 288 Italian children with enteritis (Musmanno et al., 1998). In a South African study of 6,006 Campylobacter and Helicobacter isolates obtained from the diarrheal stools of children over a 17-year period, 431 (7.2%) of the strains were C. jejuni subsp. doylei (Table 3). These isolates were obtained from gastroenteritis patients whose average age was 20 months (range, 1 month to 11 years). Eighty-two percent of the children had loose stools, 18% had watery stools, 15% had bloody stools, and 24% had fecal leukocytes. Other clinical features are indicated in Table 4. In a separate study of South African pediatric Campylobacter blood cultures, 53 (24%) of 221 isolates were C. jejuni subsp. doylei (Lastovica, 1996). The average age of these children was 12 months (range, 2 to 30 months) Twenty-six of 53 children had diarrhea, often chronic, suggesting that intestinal infection preceded systemic infection. Thirty of the 53 patients had severe protein deficiency diseases, such as marasmus and kwashiorkor. C. jejuni subsp. doylei comprised less than 8% of the campylobacters found in stool (Table 3) but formed 24% of the Campylobacter blood cultures seen at the same pediatric hospital (Lastovica, 1996). Morey (1996) reported that in aboriginal children in central Australia, C. jejuni subsp. doylei accounted for a staggering 85% of Campylobacter bacteremias. These observations sug-

Six Campylobacter species have an essential growth requirement for hydrogen or formate. Five of them are found in the gingival flora of the human mouth, notably in periodontal pockets of diseased gums. C. rectus (Latin rectus, “straight”) and C. curvus (Latin cuwus, “curved”) were originally described as Wolinella species (Tanner et al., 1984, 1987). C. concisus (Latin concisus, “concise”) and C. showae (Latin showae, referring to Showa University, where the organism was first isolated) are biochemically similar organisms (Etoh et al., 1993; Tanner et al., 1981). C. gracilis was formerly regarded as a Bacteroides species but was reclassified in 1995 (Vandamme et al., 1995). A similar organism, also wrongly classified as a Bacteroides sp., “B.” ureolyticus, is now considered to belong to the family Camp y lobacteraceae and is awaiting suitable generic assignation (Vandamme et al., 1995). C. mucosalis is an H,-requiring species that has been associated with proliferative enteritis in pigs (Lawson and Rowland, 1984) (Table 2). Microbiology Apart from the H, or formate requirement of these species, another feature they have in common (except for C. showae) is that they are all catalase negative, Many isolates are susceptible to cephalothin and nalidixic acid (30-pg disks), often with inhibitory zones up to 50 mm in diameter (Lastovica et al., 1993; Vandamme et al., 1991). C. cuwus may be difficult to differentiate from C. concisus because the indoxyl acetate assay is not always infallible (Lauwers et al., 1991), and serological (Tanner et al., 1984) or other methods may be required. Two suspected cases of C. mucosalis enteritis in Italian school children were reported (Figura et al., 1993). However, these isolates were only characterized by variable phenotypic criteria, such as colony color. A subsequent investigation of these presumptive C. mucosalis strains by another researcher who used DNA-DNA hybridization techniques indicated these strains were misidentified C. concisus strains (Lastovica et al., 1993). Occasionally, some isolates of C. hyointestinalis, C.

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jejuni subsp. doylei, C. upsaliensis or C. lari may require an H,-enriched microaerophilic atmosphere for growth. However, these strains can easily be differentiated from the above hydrogen-requiring species by differences in the available phenotypic tests (Lastovica, 2006) (Table 1).C. rectus, C. curvus, C. gracilis and “B.” ureolyticus can produce agar-corroding (pitting) colonies. C. gracilis is unique (for a Campylobacter) because it is nonmotile (aflagellate) and oxidase negative. C. rectus and C. cuwus may appear oxidase negative in traditional tests, but they are positive in the modified test of Tarrand and Groschel (1982). A PFGE study obtained 51 patterns from 53 C. concisus isolates, indicating considerable genetic heterogeneity (Matsheka et al., 2002). A random amplification of polymorphic DNA assay is available for the differentiation of C. concisus strains (Matsheka et al., 2006). Epidemiology and Clinical Features

C. rectus was isolated from 80% of 1,654 adults and children with periodontitis in a 2-year study (Rams et al., 1993) and from periodontitis patients with inflammatory bowel disease (Van Dyke et al., 1986). A recent intriguing study showed C. rectus and C. concisus were isolated from endoscopic biopsy samples of seven patients with Barrett’s esophagitis but not from controls, which is suggestive of a link between these bacteria and the initiation, maintenance, or exacerbation of this disease (Macfarlane et al., 2007). Chronic oral infection with C. rectus also has been postulated as a possible contributor to the cause of the severe vasculopathy known as Buerger’s disease (Iwai et al., 2005). Reports of additional C. rectus isolates associated with gastroenteritis have not been published, possibly because less than optimal conditions were used for the isolation of this fastidious microorganism. C. curzlus has been recovered from periodontal and septicemia patients (Tanner et al., 1987). It has been isolated from diarrheal stools of children in Belgium (Lauwers et al., 1991) and South Africa (Table 3), and from the blood culture of a man with liver abscesses (Wetsch et al., 2006). Oral bacteria are rarely reported to cause extraorbital infection. Han et al. (2005) reported isolating under anaerobic conditions and verified by 16s rRNA assays of C. rectus in a breast abscess and C. cuwus in a liver abscess and a bronchial abscess. All three patients recovered with antibiotic treatment. The association of C. concisus with human periodontal disease is well known (Tanner et al., 1981, 1987), but a direct causal role has not been established. C. concisus may have been responsible for osteomyelitis of the sacrum in a patient with diabetes

and a sacral decubitus ulcer (Engberg et al., 2005; Johnson et al., 1985). Evidence that C. concisus is an enteric pathogen is equivocal. Of 14 C. concisus isolates studied in Belgium, 7 were from patients with diarrhea and 1 was from the blood of a man with carcinoma of the bronchus (Vandamme et al., 1989). One study found no significant difference in C. concisus isolation rates between patients with diarrhea and controls (Totten et al., 1985). Studies in South Africa (Samie et al., 2007) and Belgium (Engberg et al., 2000) of adults and children with diarrhea have shown the prevalence of C. curzlus in patients’ stools was not different from controls. In another South African study of 1,519 Campylobacter isolates, 187 (12.3%) required hydrogen for growth (Lastovica et al., 1993). One isolate was from an adult diarrheal stool, one was from the blood culture of an 18-dayold infant, one was from the duodenal tissue sample of an adult, and 184 were from pediatric diarrheal stools. All 187 H,-requiring Campylobacter isolates were characterized as C. concisus on the basis of phenotypic testing. Ninety-two isolates were tested and confirmed as C. concisus by DNA-DNA hybridization (Lastovica et al., 1993). The mean age of the pediatric patients was 24 months (range, 4 days to 11 years). Twenty-one percent of the stools were watery; the rest were loose. Blood was present in 14% of the stools and fecal leukocytes in 27%. Preexisting clinical problems in 18% of the patients included anemia, metabolic disturbances, tuberculosis, and kwashiorkor. Additional data are provided in Table 4. Istavan et al. (2004) detected and characterized a membrane-bound hemolytic phospholipase in clinical strains of C. concisus isolated from Australian children with gastroenteritis. The presence of this potential virulence factor suggests C. concisus is an opportunistic pathogen. Danish investigators who used molecular techniques have proposed subpopulations of C. concisus (“genomospecies”) that have more pathogenic potential than others (Aabenhus et al., 2002). They found some genomospecies were more likely to be found in stools of patients with diarrhea, whereas others were more likely to be detected only in healthy controls (Aabenhus et al., 2005). Thirty C. concisus isolates from pediatric stools were tested against eight antimicrobial agents, and ciprofloxacin was the most effective agent examined (Greig et al., 1993). All strains were susceptible to tetracycline, ampicillin, and gentamicin. Activity of the cephalosporins was variable. In a study of 457 pediatric C. concisus strains isolated over the period 1998 to 2005, resistance to ciprofloxacin increased from 7 to 18%, and erythromycin resistance increased from 5 to 22% (Moore et al., 2006). Twenty Belgian C. concisus strains were susceptible to tetracycline and am-

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picillin; and resistance to gentamicin, ciprofloxacin, and erythromycin was 5% (Vandenberg et al., 2006). C. gracilis and “B.” ureolyticus are both associated with periodontal disease but have been isolated from other sites. C. gracilis is found in deep-seated abscesses, particularly in the head and neck region, and “B.” ureolyticus from more superficial lesions such as ulcers, soft tissue infections, and urethritis (Akhtar and Eley, 1992; Johnson and Finegold, 1987; Johnson et al., 1985; Vandamme et al., 1995). C. mucosalis can be recovered at high concentrations (up to 10* CFU/g) from the diseased intestinal mucosa of porcine intestinal adenomatosis cases, but experimental infection does not always cause the disease (Lawson and Rowland, 1984). There is no known association of C. mucosalis and human disease.

CAMPYLOBACTER SPUTORUM Although not strictly hydrogen requiring, C. sputorum shares many of the features with the foregoing species, notably in being catalase negative, and it also forms part of the human gingival flora. C. sputorum was considered to have three biovars: biovar sputorum, living in the mouth and intestinal tract; biovar bubulus, living in the genital tract of healthy cattle and sheep; and biovar fecalis, found mainly in the feces of sheep and cattle. The biochemical differences between biovar bubulus and biovar sputorum are unreliable, and biovar bubulus is no longer recognized (On et al., 1998). However, a group of catalasenegative, urease-positive Campylobacter strains isolated from cattle feces and from a patient with enteritis in Canada were found to be C. sputorum and now form a new biovar, paraureolyticus (On et al., 1998). C. sputorum biovar sputorum has been isolated from the human lung and from abscesses of the groin and axillary areas (Borczyk et al., 1987a; On et al., 1992), from gastroenteritis patients (Table 3 ) , and from the blood of a knee abscess patient (Tee et al., 1998b).

ARCOBACTER SPECIES The genus Arcobacter was originally described as aerotolerant campylobacters, but after DNA-rDNA and DNA-DNA hybridization studies, these organisms were moved from the genus Campylobacter to the genus Arcobacter (Vandamme and De Ley, 1991). Arcobacters are morphologically similar to campylobacters, but they differ sharply in that they grow over a wide temperature range (15 to 42°C) (Lehner

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et al., 2005) and under aerobic atmospheric conditions, which Campylobacter and Helicobacter spp. cannot. There are six recognized Arcobacter species: A. butzleri, A. cryaerophilus, A. nitrofigilis, A. skirrowii, A. cibarius, and A. halophilus (Snelling et al., 2006). The type species, A. nitrofigilis, originally isolated from plant roots, has not been isolated from humans or animals. A. skirrowii has been isolated from aborted cattle, sheep and pig fetuses, and humans. A. butzleri was first described by Kiehlbauch et al. (1991a), who recognized two distinct genetic groups among 78 isolates of aerotolerant “campylobacters” referred to the CDC in the United States. Group 1 contained the type strain of C. cyaerophila (now A. cryaerophilus). Group 2 was named C. butzleri (now A. butzleri). Biochemical differences of these two species are outlined in Table 1. A. cyaerophilus and A. butzleri grow poorly or not at all at 42°C on blood agar plates, but they will grow on MacConkey plates and are generally resistant to cephalothin (Table 1).A. cibarius (Houf et al., 2005) was isolated from chicken carcasses, and A. halophilus (Donachie et al., 2005) was isolated from a hypersaline lagoon. Epidemiology Livestock animals and surface and groundwaters represent a significant source of Arcobacter (Lehner et al., 2005). One hundred strains of A. butzleri and 41 strains of A. butzleri-like or Arcobacter spp. were isolated from six drinking water plants in Germany (Jacob et al., 1998). Two strains of A. butzleri were isolated from a contaminated well in Idaho (Moore et al., 2006). Survival studies, conducted at 5”C, a temperature typical of groundwater, indicated that A. butzleri can remain viable for up to 16 days in groundwater (Rice et al., 1999). A. cryaerophilus was first isolated from porcine, equine, and bovine feces and from aborted porcine and bovine fetuses (Boudreau et al., 1991; Ellis et al., 1977), and from urban sewage (Stamp et al., 1993). In a study of 15 broiler chickens obtained from a poultry abattoir and 10 broiler chickens from a supermarket, all 25 carcasses yielded A. butzleri. Three supermarket and 10 abattoir carcasses also carried A. cyaerophilus, and 2 abattoir carcasses carried A. skirrowii (Atabay et al., 1998). A Canadian study indicated that poultry appears to be a major reservoir for A. butzleri: 121 of 125 broiler chicken carcasses examined were positive for A. butzleri after abattoir processing. A. butzleri was recovered from whole chicken and ground chicken and turkey samples from retail stores (Lammerding et al., 1996). Arcobacters also may contaminate the poultry-processing environment (Gude et

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al., 2005). In a study of 130 raw meat samples, a strain of A. butzleri was identified in a pork sample (Zanetti et al., 1996). The organisms may be transmitted to piglets both vertically (i.e., in utero) and horizontally (Ho et al., 2006b). Characterization of Isolates

A multiplex PCR assay has been developed to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters (Harmon and Wesley, 1997). On the basis of 23s rRNA gene polymorphisms, Hurtado and Owen (1997) have proposed a PCR scheme for the rapid identification and speciation of Campylobacter and Arcobacter spp. Rhotyping and restriction fragment-length polymorphism analysis have been reported to be able to distinguish between A. cryaerophilus and A. butzleri (Kiehlbauch et al., 1991b). Clinical Features

Arcobacter butzleri can produce diarrhea and associated gastrointestinal symptoms in humans. A. butzleri was the fourth most common “atypical” campylobacter isolated and was associated with persistent, watery diarrhea in a study of more than 65,000 stool samples (Vandenberg et al., 2004). A. butzleri has been isolated from human diarrhetic stools, blood cultures, and the peritoneal fluid of appendicitis patients (Kiehlbauch et al., 1991). A. butzleri was isolated from the feces of diarrheic children in Thailand (Taylor et al., 1991) and South Africa (Samie et al., 2007) (Table 3). Underlying illness such as anemia, tuberculosis, and pneumonia were present in 40% of these children, whose average age was 19 months. Additional clinical details are provided in Table 4. In a survey of fecal samples in Belgium, Desiste and colleagues (1998) found that A. butzleri made up 7.4% of Carnpylobacter and Arcobacter isolations. The absence of fever (temperature >38”C) was noted in most patients, but watery diarrhea occurred in 13 of the 16 patients, and vomiting and abdominal pain occurred in about half of them. Neither blood nor inflammatory exudates was detected by microscopy of stools. Half the patients were treated with amoxicillin-clavulanate and rapidly recovered; the remainder improved spontaneously with conservative management. An outbreak of A. butzleri infection affected 10 Italian children, aged 2 to 5 years, none of whom had diarrhea or fever, but all had episodes of recurrent abdominal cramps. Each episode lasted up to 2 h and occurred several times a day for up to 10 days

(Vandamme et al., 1992a). These children felt well between attacks, and the illness was self-limiting. All isolates belonged to a single serogroup and had identical protein profiles. Most of the children seroconverted to this strain. The sequential timing of these infections suggested person-to-person spread. A. butzleri was isolated from the blood of a preterm neonate, probably infected in utero (On et al., 1995b). Treatment with penicillin and cefotaxime was successful in resolving the infection. A. butzleri appears to be pathogenic for nonhuman primates. It was found in 14 (6%) of 222 nonhuman primates with diarrhea. A. butzleri was isolated from colonic specimens obtained at necroscopy from 3 (4%) of 76 macaques. All three animals had active colitis (Anderson et al., 1993). The stools of 7 (39%) of 18 infant Macaca nemestrina monkeys (apparently asymptomatic), when cultured weekly from birth to 1year of age, also yielded A. butzleri (Russell et al., 1992). A. cryaerophilus has been isolated from human diarrhetic stools and blood cultures (Dediste et al., 1998; Kiehlbauch et al., 1991). An A. cyaerophilus strain was isolated from the blood of a 72-year-old uremic woman with hematogenous pneumonia that resulted from an infected atrioventricular fistula (Hsueh et al., 1997). She had experienced a period of diarrhea 2 months before this episode, and she was successfully treated with ceftizoxime and tobramycin. A 73-year-old man developed persistent diarrhea attributed to A. skimowii infection (Wybo et al., 2004), an organism rarely isolated from humans. A. butzleri is generally susceptible to fluoroquinolones, aminoglycosides, and tetracyclines; >20% of isolates are resistant to ampicillin and erythromycin (Kabeya et al., 2004; Vandenberg et al., 2006). Pathogenicity Neonatal piglets have been used as models to determine relative pathogenicities based on fecal shedding and tissue colonization of Arcobacter (Wesley et al., 1996). One-day-old cesarean-derived colostrum-deprived piglets were infected by mouth with three field strains and type strains of A. butzleri, A. cryaerophilus, and A. skirrowii. Arcobacter spp. were detected at least once in rectal swab samples of all but one of the experimentally infected piglets. At necroscopy, Arcobacter spp. were cultured from the liver, kidney, ileum, or brain tissue of two infected piglets, but no severe gross pathology was noted. These data suggest that Arcobacter spp., particularly A. butzleri, can colonize neonatal pigs (Wesley et al., 1996). Two recent reviews summarize clinical presentation, pathogenicity, and other aspects of Arco-

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bacter infection (Ho et al., 2006a; Houf and Stephan, 2007).

HELICOBACTER CINAEDI AND HELICOBACTER FENNELLIAE Initially, H. cinaedi and H. fennelliae were called CLOs (Campylobacter-like organisms). Helicobacter cinaedi derives from the Latin word cinaedi, “of a homosexual.” C. fennelliae was named after Cynthia Fennell, the microbiologist who first isolated these organisms from the rectal swabs of homosexual men. In a taxonomic revision, the names of these organisms were subsequently amended to Helicobacter cinaedi and Helicobacter fennelliae (Vandamme et al., 1991). Microbiology

H. cinaedi and H. fennelliae grow poorly if at all under microaerobic conditions and require H,-enhanced microaerobic conditions for optimum growth. These bacteria grow at 37°C but grow poorly or not at all at 42°C (Table 1).H. fennelliae may be differentiated from H. cinaedi by SDS-PAGE profiles (On et al., 1991), serology (Flores et al., 1989), and differences in aryl sulfatase activity (Burnens and Nicolet, 1993) (Table 1).A useful diagnostic test is the smell of a mature growth of H. fennelliae, which has an odor similar to bleach and which is absent in H. cinaedi and campylobacters (Fennell et al., 1984). Although Campylobacter and Arcobacter species form domed colonies on freshly prepared agar plates, both H. cinaedi and H. fennelliae produce a flat, spreading growth, without discrete colonies (Fennell et al., 1984). This growth may be missed on primary isolation plates, particularly if the domed colonies of a Campylobacter species are present as well. H. fennelliae is often coisolated with C. jejuni subsp. jejuni, C. jejuni subsp. doylei, or C. upsaliensis (Albert et al., 1992; Lastovica, 2006). In cases of suspected mixed infection, extreme care must be taken to separate the noncolonial spreading growth of H. fennelliae or H. cinaedi from the domed colonies of Campylobacter (Fig. 2). Strain Characterization Thirty-four human and animal strains of H. cinaedi and four animal and human strains of H. fennelliae were characterized by phenotypic and molecular tests (Kiehlbauch et al., 1995). Results indicated that most isolates of H. cinaedi formed a single group, both phenotypically and by DNA-DNA hy-

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bridization studies. Subgroups were distinguishable by ribotyping. Two human H. fennelliae strains had similar but different ribotyping patterns from those of the type strains and from each other, and had no bands in common with either of the H. fennelliae animal strains. Molecular phylogenic analysis of the 16s rRNA gene of 3 1 South African H. fennelliae clinical isolates indicated that they have a closer affinity to a described Australian strain than to the type strain, NCTC 11612 (Lastovica and Smuts, 2007). Epidemiology and Clinical Features Enteric helicobacters were first recognized as human pathogens when they were isolated from 26 of 158 stools of homosexual men with diarrhea or proctitis examined at a sexually transmitted disease clinic (Lentzsch et al., 2004). These organisms were also isolated from 6 of 75 asymptomatic homosexual men but were not isolated from 150 heterosexual men and women (Lentzsch et al., 2004). In a study of the diarrheal stools of homosexual or bisexual men in Baltimore and Washington, DC, 9 (27%) of 33 patients had Campylobacter or Helicobacter in their stools, 2 patients had H. cinaedi infections, and 1 had a CL03 strain (Laughton et al., 1988). Two CL03 strains were isolated from the stools of homosexual men with proctitis (Flores et al., 1989; Totten et al., 1985). CLOs have been isolated from up to 8% of the stools of homosexual men with diarrhea or proctitis (Laughton et al., 1988). In a California study, CLOs were not identified in the stools or colonic brushings of 27 homosexual men with diarrhea (Wilcox et al., 1990). The clinical features of CLO infections, including diarrhea, abdominal cramps, and hematochezia, were similar to those in C. jejuniinfected patients (Quinn et al., 1984). CLO infections also produced fever, anal discharge, and pain (Quinn et al., 1984). Sigmoidoscopic examinations of infected patients indicated mucosal bleeding and ulcers. Fecal leukocytes were present in most patients, and histological examination revealed crypt abscesses and polymorphonuclear leukocytes scattered through the lamina propria. A subsequent report described HIVinfected patients with H. cinaedi in their stools who experienced chronic but mild symptoms lasting several weeks (Grayson et al., 1989), but without blood or polymorphonuclear leukocyte cells in the stools. Diarrhea consisted of three to four loose stools per day and was not associated with systemic illness. In addition to gastroenteritis, H. cinaedi and H. fennelliae can cause bacteremia, particularly in HIVpositive patients (Burman et al., 1995). Chills, lowgrade fever, lethargy, and malaise are common in patients with H. cinaedi bacteremia, and not all have

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preceding gastrointestinal symptoms. A homosexual man with AIDS developed recurrent H. cinaedi bacteremia during 2 months of intermittent diarrhea, and fecal incontinence. His blood cultures yielded a second organism that was identified as H. fennelliae, and cultures were consistently positive, despite treatment with ciprofloxacin and trimethoprim-sulfamethoxazole. The patient died 4 months later, although the role of H. cinaedi and H. fennelliae was not clearly defined (Ng et al., 1987). Another AIDS patient developed bacteremia due to H. fennelliae (Kemper et al., 1993). In a survey of 394 blood culture Campylobacter isolates from patients in England and Wales, 2 (0.50%) isolates were H. fennelliae and 1 (0.25%) was H. cinaedi (Skirrow et al., 1993). A study of 221 pediatric blood culture isolates in a pediatric hospital in South Africa indicated that 7 (3.2%) strains were H. fennelliae and 5 (2.3%) were H. cinaedi (Lastovica, 1996). At this same hospital, 6,006 Carnpylobacter or Helicobacter isolates were obtained from 26,933 diarrheal stools (Table 3). There were 337 strains (5.61%) of H. fennelliae and 51 (0.95%) of H. cinaedi detected (Table 3). The average age of the infected children was 18 months (range, 2 weeks to 11 years). Seventy-three percent of the stools contained blood, and 8% had fecal leukocytes. More than 2.5% of these patients had underlying illnesses such as anemia, pneumonia, metabolic disturbances, and nutritional problems. Additional clinical details are listed in Table 4. These reports suggest that H. cinaedi and H. fennelliae infections may occur more frequently than previously thought in the immunocompetent and heterosexual populations. Gastroenteritis due to H. cinaedi or H. fennelliae have been reported in heterosexual men, women, and children in various studies (Burnens et al., 1993; Grayson et al., 1989; Lastovica, 2006; Uckay et al., 2006; Wilcox et al., 1990). The source of human infection of these organisms is not known, although apparent transmission from animals has been recorded. H. cinaedi was isolated from the cerebrospinal fluid and blood of a 5day-old neonate whose mother had a mild diarrheal illness during the third trimester of her pregnancy. The mother had cared for pet hamsters during the first two trimesters of her pregnancy (Orlicek et al., 1993). H. cinaedi has been isolated from 72% of commercially available hamsters (Gebhart et al., 1989) and from the stools of diarrheic dogs (Burnens et al., 1992). Pathogenesis Evidence for the pathogenicity of H. cinaedi and H. fennelliae comes from several sources. The asso-

ciation of these organisms with homosexual men who had proctitis and enteritis, but not with asymptomatic men, suggests a causal relationship. Bacteremia in patients who are immunocompromised and the presence of fecal leukocytes indicate that these organisms play a pathogenic role. Flores et al. (1990) studied the effects of experimental H. cinaedi and H. fennelliae infection in infant macaque monkeys. When four monkeys were challenged with H. cinaedi, two animals subsequently developed diarrhea, and H. cinaedi was isolated from the stools and blood cultures of all four. When challenged with H. fennelliae, all four monkeys became bacteremic, and two animals developed diarrhea. H. fennelliae was isolated from their stools, and prolonged rectal colonization was observed in all animals examined (Flores et al., 1990). A putative virulence factor in H. cinaedi has been described by Taylor et al. (2003), who demonstrated the production of cytolethal distending toxin, which causes an irreversible cell-cycle block at the G2/M transition. Treatment Although H. cinaedi and H. fennelliae infections have not been fatal, to date, some patients have displayed a slow clinical response to antimicrobial therapy. Antimicrobial agents that have demonstrated in vitro activity against H. cinaedi and H. fennelliae include ampicillin, tetracycline, rifampin, nalidixic acid, chloramphenicol, and gentamicin (Flores et al., 1985). However, 28% of strains were resistant to erythromycin and clindamycin, and 17% were resistant to sulfamethoxazole in one study (Flores et al., 1985). Like campylobacters, these organisms are resistant to trimethoprim, and most of them are resistant to metronidazole. In South Africa, 13% of H. cinaedi and H. fennelliae pediatric stool and blood culture isolates were resistant to erythromycin (Moore et al., 2006). Fluoroquinolones may be the best treatment for severe or persistent H. cinaedi and H. fennelliae infection. Two patients with persistent H. cinaedi bacteremia were successfully treated with oral ciprofloxacin (Burman et al., 1995).

OTHER HELICOBACTER A N D CAMPYLOBACTER SPECIES

H. canis is a species that resembles H. fennelliae. It was originally found in dogs (with and without diarrhea), but it has also been isolated from a 5%year-old boy with diarrhea (Stanley et al., 1993). H. pullorum was described as a species isolated from poultry liver, duodenum, and cecum, and from hu-

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CLINICAL SIGNIFICANCE OF CAMPYLOBACTER AND RELATED SPECIES

mans with diarrhea (Ceelen et al., 2005; Stanley et al., 1994). H. westmeadii is another newly recognized species isolated from the blood of two AIDS patients with febrile diarrhea in Australia (Trivett-Moore et al., 1997). H. rappini (formerly Flexispira rappini) is a fusiform bacterium with spiral periplasmic fibrils and bipolar tufts of sheathed flagella. These organisms were first isolated from aborted lambs, then subsequently from patients with diarrhea (Romero et al., 1988), from the blood cultures of a 7-year-old girl with pneumonia (Tee et al., 1998a), and from a 65year-old febrile man undergoing hemodialysis (Sorlin et al., 1999). Solnick (2003) has provided a comprehensive review article on Helicobacter species other than H. pylori. In 2000, Logan et al. reported the isolation and characterization of C. lanienae sp. nov. from healthy abattoir workers exposed to cattle or pig carcasses. In 2001, Lawson et al. recovered C. hominis sp. nov. from the feces of healthy humans. In 2004, Foster at al. isolated C. insulaenigrae sp. nov. from three seals and a porpoise. The clinical relevance of these newly recognized Campylobacter species has yet to be determined. PCR assays for the simultaneous detection and differentiation of the genera and individual species of Campylobacter, Helicobacter, and Arcobacter may make this task easier (Hill et al., 2006; Marshall et al., 1999).

CONCLUSION New species of Campylobacter and related genera are being identified on a regular basis. Many of these “atypical” campylobacters may play a greater role in causing human and animal disease than previously recognized. Because methods originally formulated for the isolation of C. jejuni will often fail to support the growth of non-jejuni, non-coli Campylobacter species, these fastidious organisms are most likely underdetected in clinical specimens. Appreciation and application of an efficient protocol is essential for the isolation of non-jejuni, non-coli Campylobacter species for surveillance, epidemiological, and other studies. Reservoirs of newly described non-jejuni, non-coli Campylobacter species have been found in animals such as pigs, cattle, dogs, foxes, and rodents. Nonmammalian species such as birds and shellfish have recently been implicated as reservoirs for these organisms, and surface and groundwater are known to harbor non-jejuni, non-coli Campylobacter species and related organisms. At present, the role that these newly described Campylobacter species play in the disease process is not understood. Additional research and surveillance, such as that provided by FoodNet (Allos et al., 2004), is required to

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better define the prevalence, persistence, and pathogenicity of emerging campylobacters and related genera.

REFERENCES Aabenhus, R., S. L. On, B. L. Siemer, H. Permin, and L. P. Andersen. 2005. Delineation of Campylobacter concisus genomospecies by amplified fragment length polymorphism analysis and correlation of results with clinical data. J. Clin. Microbiol. 43: 5091-5096. Aabenhus, R., H. Permin, S. L. On, and L. P. Andersen. 2002. Prevalence of Campylobacter concisus in diarrhoea of immunocompromised patients. Scand. J. Infect. Dis. 34:248-252. Akhtar, N., and A. Eley. 1992. Restriction endonuclease analysis and ribotyping differentiate genital and nongenital strains of Bacteroides ureolyticus. J. Clin. Microbiol. 30:2408-2414. Albert, M. J., W. Tee, A. Leach, V. Asche, and J. L. Penner. 1992. Comparison of a blood free medium and a filtration technique for the isolation of a Campylobacter spp. from diarrhoea1 stools of hospitalized patients in central Australia. J. Med. Microbiol. 37:176-179. Allos, B. M. 2001. Campylobacter jejuni infections: update on emerging issues and trends. Clin. Infect. Dis. 32:1201-1206. Allos, B. M., R. J. Moore, P. M. Griffin, and R. V. Tauxe. 2004. Surveillance for sporadic foodbourne disease in the 21st century: the FoodNet perspective. Clin. Infect. Dis. 38(Suppl 3):S115s120. Alvarez, 0. A., F. Vanegas, G . L. Maze, G. W. Gross, and M. Lee. 2000. Polymicrobial cholangitis and liver abscess in a patient with the acquired immunodeficiency syndrome. South. Med. J. 93:232-234. Anderson, K. F., J. A. Kielbauch, D. C. Anderson, H. M. McClure, and I. K. Wachmuth. 1993. Arcobacter (Campylobacter) butzleri-associated diarrheal illness in a non-human primate population. Infect. Immun. 61:2220-2223. Atabay, H. I., J. E. Corry, and S. L. On. 1998. Diversity and prevalence of Arcobacter spp. in broiler chickens. J. Appl. Microbiol. 84:1007-1016. Bar, W., G. Mirquez de Bar, H.-M. Nitschke, A. Schiessler, G. Mauff, A. Goldmann, B. Steinbrueckner, G. Harter, and M. Kist. 1998. Endocarditis associated with Campylobacter fetus, p. 162-165. In A. J. Lastovica, D. G. Newell, and E. E. Lastovica (ed.), Campylobacter, Helicobacter and Related Organisms.Institute of Child Health, Cape Town, South Africa. Benjamin, J., S. Leaper, R. J. Owen, and M. B. Skirrow. 1983. Description of Campylobacter laridis, a new species comprising the nalidixic acid resistant thermophilic Campylobacter (NARTC) group. Cur. Microbiol. 8:231-238. BCzian, M . C., G. Ribou, C. Barberis-Giletti, and F. Mtgraud. 1990. Isolation of a urease positive thermophilic variant of Campylobacter lari from a patient with urinary tract infection. Eur. J. Clin. Microbiol. Infect. Dis. 9:895-897. Blaser, M. J., D. N. Taylor, and R. A. Feldman. 1983. Epidemiology of Campylobacter jejuni infections. Epidemiol. Rev. 5: 157-176. Bokkenheuser, V. 1970. Vibrio fetus infection in man. Ten new cases and some epidemiological observations. Am. J. Epidemiol. 91:400-409. Bolton, F. J., D. Coates, D. N. Hutchinson, and A. F. Godfree. 1987. A study of thermophilic Campylobacter in a river system. J. Appl. Bacteriol. 62:167-176. Borczyk, A., H. Lior, A. McKeown, and H. Svendsen. 1987a. Isolations of Campylobacter sputorum associated with human in-

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fetus cholecystitis in a patient with advanced hepatocellular carcinoma. Scand. J. Infect. Dis. 29:197-198. Tanner, A. C. R., S . Badger, C. H. Lai, M. A. Listgarten, R A. Visconti, and S . S . Socransky. 1981. Wolinella gen. nov., Wolinella succinogenes (Vibrio succinogenes Wolin et al.) comb. nov., and description of Bacteroides gracilis sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from humans with periodontal disease. Int. J. Syst. Bacteriol. 31:432-435. Tanner, A. C. R., J. L. Dzink, J. L. Ebersole, and S . S . Socransky. 1987. Wolinella recta, Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens from periodontal lesions, J. Periodont. Res. 22:327-330. Tanner, A. C. R., M. A. Listgarten, and J. L. Ebersole. 1984. Wolinella cuwa sp. nov.: “Vibriosuccinogenes” of human origin. Int. J. Syst. Bacteriol. 34:275-282. Targan, S . R., A. W. Chow, and L. B. Guze. 1976. Spontaneous peritonitis of cirrhosis due to Campylobacter fetus. Gastroenterology 71:3 11-3 13. Targan, S . R., A. W. Chow, and L. B. Guze. 1977. Campylobacter fetus associated with pulmonary abscess and empyema. Chest 71: 105-108. Tarrand, J. J., and D. H. M. Groschel. 1982. Rapid, modified oxidase test for oxidase-variable bacterial isolates. J. Clin. Microbiol. 16:772-774. Tauxe, R V., C. M. Patton, P. Edmonds, T. J. Barrett, D. J. Brenner, and P. A. Blake. 1985. Illness associated with Campylobacter laridis, a newly recognized Campylobacter species. J. Clin. Microbiol. 21:222-225. Taylor, D. E., K. Hiratsuka, and L. Mueller. 1989. Isolation and characterization of catalase-negative and catalase-weak strains of Campylobacter species, including “Campylobacter upsaliensis”, from humans with gastroenteritis. J. Clin. Microbiol. 27:20422045. Taylor, D. N., J. A. Kiehlbauch, W. Tee, C. Pitarangsi, and P. Echeverria. 1991. Isolation of group 2 aerotolerant Campylobacter species from Thai children with diarrhea. J. Infect. Dis. 163: 1062-1067. Taylor, D. N., K. T. McDermott, J. R. Little, J. G. Wells, and M. J. Blaser. 1983. Campylobacter enteritis from untreated water in the Rocky Mountains. Ann. Intern. Med. 99:38-40. Taylor, N. S., G. Zhongming, Z. Shen, F. E. Dewhist, and J. G. Fox. 2003. Cytolethal distending toxin: a potential virulence factor for Helicobacter cinaedi. 1.Infect. Dis. 188:1892-1897. Tee, W., K. Leder, E. Karroum, and M. Dyall-Smith. 1998a. “Flexispira rappini” bacteraemia in a child with pneumonia. J. Clin. Microbiol. 36:1679-1682. Tee, W., M. Luppino, and S . Rambaldo. 1998b. Bacteremia due to Campylobacter sputorum biovar sputorum. Clin. Infect. Dis. 27: 1544-1545. Totten, P. A., C. L. Fennell, F. C. Tenover, J. M. Wezenberg, P. L. Perine, W. E. Stamm, and K. K. Holmes. 1985. Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men. J. Infect. Dis. 151:131-139. Tremblay, C., C. Gaudreau, and M. Lorange. 2003. Epidemiology and antimicrobial susceptibilities of 111 Campylobacter fetus subsp. fetus strains isolated in Quebec, Canada, from 1983 to 2000. J. Clin. Microbiol. 41:463-466. Tresierra-Ayala, A., M. E. Bendayan, A. Bernuy, G. Pereyra, and H. Fernandez. 1994. Chicken as potential contamination source of Campylobacter lari in Iquitos, Peru. Rev. Inst. Med. Trop. Sao Paul0 36:497-499. Trivett-Moore, N. L., W. D. Rawlinson, M. Yuen, and G. L. Gilbert. 1997. Helicobacter westmeadii sp. nov., a new species iso-

lated from blood cultures in two AIDS patients. J. Clin. Microbiol. 35:1144-1150. Uckay, I., J. Garbino, P.-Y. Dietrich, B. Ninet, P. Rohner, and V. Jacomo. 2006. Recurrent bacteremia with Helicobacter cinaedi: case report and review of the literature. BMC Infect. Dis. 6:86. Vandamme, P., M. I. Daneshvar, F. E. Dewhirst, B. J. Paster, K. Kersters, H. Goossens, and C. W. Moss. 1995. Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov. Int. J. Syst. Bacteriol. 45:145-152. Vandamme, P., and J. De Ley. 1991. Proposals for a new family, Campylobacteraceae.Int. J. Syst. Bacteriol. 41:45 1-455. Vandamme, P., E. Falsen, B. Pot, B. Holste, K. Kersters, and J. De Ley. 1989. Identification of EF group 22 campylobacters from gastroenteritis cases as Campylobacter concisus. J. Clin. Microbiol. 27: 1775-1782. Vandamme, P., E. Falsen, R. Rossau, B. Hoste, R. Tygat, and J. De Ley. 1991. Revision of Campylobacter, Helicobacter and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. Int. J. Syst. Bacteriol. 41:88103. Vandamme, P., P. Pugina, G. Benzi, R. Van Etterijck, L. Vlaes, K. Kersters, J.4’. Butzler, H. Lior, and S . Lauwers. 1992a. Outbreak of recurrent abdominal cramps associated with Arcobacter butzleri in an Italian school. J. Clin. Microbiol. 30:2335-2337. Vandamme, P., M. Vancanneyt, B. Pot, L. Mels, B. Holste, D. Dewettinck, L. Vlaes, C. Van den Borre, R. Higgins, J. Hommez, K. Kersters, J.4’. Butzler, and H. Goossens. 1992b. Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. Int. J. Syst. Bacteriol. 42:344-356. Vandenberg, O., A. Dediste, K. Houf, S . Ibekwem, H. Souayah, S . Cadranel, N. Douat, G. Zissis, J. P. Butzler, and P. Vandamme. 2004. Arcobacter species in humans. Emerg. Infect. Dis. 10~1863-1867. Vandenberg, O., K. Houf, N. Douat, L. Vlaes, P. Retore, J. P. Butzler, and A. Dediste. 2006. Antimicrobial susceptibility of clinical isolates of non-jejunilcoli campylobacters and arcobacters from Belgium. J. Antimicrob. Chemother. 57:908-913. Van Doorn, L. J., B. A. Giesendorf, R. Bax, B. A. Van der Zeijst, P. Vandamme, and W. G. Quint. 1997. Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene. Mol. Cell. Probes 11:177-1 85. Van Doorn, L. J., W. G. Verschuuren-Van Haperen, A. Van Belkum, H. P. Endtz, J. S . Vliegenthart, P. Vandamme, and W. G. Quint. 1998. Rapid identification of diverse Campylobacter lari strains isolated from mussels and oysters using a reverse hybridization line probe assay. J. Appl. Microbiol. 84545550. Van Dyke, T. E., V. R. Dowell, S . Offenbacher, S . Synder, and W. Hersh. 1986. Potential role of micro-organisms isolated from periodontal lesions in the pathogenesis of inflammatory bowel disease. Infect. Immun. 53:671-677. Vargas, J., J. E. Corzo, M. J. Perez, F., Lozano, and E. Martin. 1992. Bacteriemia por Campylobacter e infeccion por VIH. Enfern. Infecc. Microbiol. Clin. 10:155-157. VQon, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and VCron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) Sebald and VCron. Int J. Syst. Bacteriol. 23:122-134. Viejo, G., B. Gomez, D. De Miguel, A. Del Valle, L. Otero, and P. De La Iglesia. 2001. Campylobacter fetus subspecies fetus bac-

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CLINICAL SIGNIFICANCE OF CAMPYLOBACTER AND RELATED SPECIES

teremia associated with chorioamnionitis and intact fetal membranes. Scand. J. Infect. Dis. 33:126-127. Walder, M., K. Sandstedt, and J. Ursing. 1983. Phenotypic characteristics of thermotolerant Campylobacter from human and animal sources. Curr. Microbiol. 9:291-296. Wang, W. L., and M. J. Blaser. 1986. Detection of pathogenic Campylobacter species in blood culture systems. 1. Clin. Microbiol. 23:709-714. Wens, R., M. Dratwa, C. Potvliege, C. Hansen, C. Tielemans, and F. Collart. 1985. Campylobacter fetus peritonitis followed by septicaemia in a patient on continuous ambulatory dialysis. J. Infect. 10:249-25 1. Wesley, I. V., A. L. Baetz, and D. J. Larson. 1996. Infection of cesarean-derived colostrum-deprived 1 day-old piglets with Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. Infect. Immun. 64:2295-2299. Wesley, I. V., R. D. Wesley, M. Cardella, F. E., Dewhirst, and B. J. Paster. 1991. Oligodeoxynucleotide probes for Campylo-

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bacter fetus and Campylobacter hyointestinalis based on 16s rRNA sequences. J. Clin. Microbiol. 29:1812-1817. Wetsch, N. M., K. Somani, G. J. Tyrrell, C. Gebhart, R. J. Bailey, and D. E. Taylor. 2006. Campylobacter cuwus-associated hepatic abscesses: a case report. ]. Clin. Microbiol. 44:1909-1911. Wilcox, C. M., B. A. Byford, C. E. Forsmark, W. K. Hadley, J. P. Cello, and M. A. Jacobson. 1990. Campylobacter-like organisms are uncommon pathogens in patients infected with the human immunodeficiency virus. J. Clin. Microbiol. 28:2370-2371. Wybo, I., J. Breynaert, S . Lauwers, F. Lindenburg, and K. Houf. 2004. Isolation of Arcobacter skirrowii from a patient with chronic diarrhea. J. Clin. Microbiol. 42:1851-1852. Yamashita, K., Y. Aoki, and K. Hiroshima. 1999. Pyrogenic vertebral osteomyelitis caused by Campylobacter fetus subspecies fetus. A case report. Spine 24582-584. Zanetti, F., 0. Varoli, S . Stampi, and G. De Luca. 1996. Prevalence of thermophilic Campylobacter and Arcobacter butzleri in food of animal origin. lnt. J. Food Microbiol. 33:315-321.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 8

Burden of Illness of Campylobacteriosis and Sequelae U R E M0LBAK AND

ARIEHAVELAAR

single measure. Burden of disease studies include elements of epidemiology, disease modeling, risk assessment, and burden projections; the latter help policy makers learn where to target preventive strategies and what to expect in terms of future disease burden.

The burden of disease can be defined as the summary of morbidity and mortality associated with different acute and chronic manifestations of Campylobacter infections. Such health effects vary widely in severity and timescale but can be translated to a single summary measure. Burden of disease measures offer a more comprehensive insight into the impact of illness on a population than traditional measures such as the number of fatal cases or the number of life-years lost as a result of premature mortality. Reliable estimates of burden of illness are important to advise risk managers and decision makers on several aspects of public health policy. These include comparison of the public health impact of different diseases, which is an important aspect of priority setting for intervention (Kemmeren et al., 2006; Vijgen et al., 2007). Furthermore, product standards for the microbiological safety of food and water can be defined on a common basis. An example of this application is the World Health Organization (WHO) Guidelines for Drinking-Water Quality (2004), where a health outcome target for all microbial hazards of 1 disability-adjusted life-year (DALY) per 1 million person-years is suggested as a reference level of risk. This level is similar to the generally accepted risk for genotoxic carcinogens of per lifetime. A further application is the balancing of competing risks, as demonstrated by Havelaar et al. (2000), who demonstrated that the positive health effects of inactivation by ozone of Cryptosporidium oocysts in drinking water outweighed the negative health effects of bromate that is produced as a by-product. Disease burden estimates are an important factor in cost-utility analyses to compare various preventive and curative interventions. Burden of disease studies capitalize primarily on existing information and translate these data into a

HOW IS BURDEN OF ILLNESS MEASURED? Most of the information collected by public health laboratories and through other types of disease surveillance and health statistics cannot be directly translated into policy. Health statistics are often fragmented, and in particular for surveillance data, outcomes of disease are rarely reported. Thus, public health laboratories report the annual number of culture-confirmed cases of Campylobacter infections to the public health authority. However, the actual numbers of infections in the community are not known, nor are the different outcomes (duration of illness, admission to the hospital, and long-term complications) of the illness. Moreover, traditional health statistics use measures such as incidence of cultureconfirmed infections per 100,000, which do not permit direct comparisons of the cost-effectiveness of different interventions. Against this background, there was a need to develop a single metric that can be used to compare burden of illness and the cost-effectiveness of different interventions between different populations and between different diseases. To meet this requirement, the DALY measure has become increasingly popular. The DALY methodology has been described by Murray and Lopez (1996) and was adopted by WHO for its reporting on health information in the late 1990s. DALYs express the years of life lost to premature death (YLL) and the years lived with disability (YLD)

-

Klre Mslbak Department of Epidemiology, Statens Serum Institut, Copenhagen, Denmark. Arie Havelaar Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and the Environment, Bilthoven, and Division of Veterinary Public Health Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands.

151

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for varying degrees of severity, making time itself the common metric for death and disability.

DALY = YLL

+ YLD

YLL is the number of years of life lost to mortality, and YLD is the number of years lived with a disability, weighted with a factor between 0 and 1 for the severity of the disability. The YLL due to a specific disease in a specified population is calculated by summation of all fatal cases (d) due to the health outcomes (1) of a specific disease, each case multiplied by the expected individual life span (e) at the age of death: YLL

d, x el

= 1

A high number of YLL can be associated with a high number of fatal cases, with deaths of young children, with a high assumed life expectancy, or a combination of these factors. YLD is calculated as the product of the duration of the illness (t) and the disability weight (w) of a specific disease, accumulated over all cases (n) and all health outcomes (I):

A high number of YLD can be associated with a high incidence of morbidity, a high disability weight, a long duration of illness, or a high prevalence of (chronic) sequelae after primary illness. DALYs can be seen as health gap measures, equating to years of healthy life lost. Disadvantages of the DALY approach include the need for strong value judgments on disability and age, thus placing emphasis on death and morbidity in young adulthood. There are other ways to measure burden of illness than the DALY metric (Gold et al., 2002). Whereas DALY measures health losses, the qualityadjusted life-year (QALY) is a measure of the current health state. DALYs are typically used in public health research, whereas QALYs are used in clinical settings to indicate the potential benefit of treatment. However, under the same assumption, one averted DALY equals one gained QALY. Burden of illness may also be measured in terms of cost of illness. Cost of illness can be calculated as the sum of direct health care costs, direct non-health care costs, and indirect non-health care costs. Direct health care costs include medical services, etc.; direct non-health care costs includes costs of informal care, transport, additional diapers, etc.; and indirect non-

health care costs are the value of production lost to society. The production loss can stem from temporary absence, long-term disability, or premature death. The cost of illness methodology and related monetary approaches such as willingness to pay are based on country- and time-specific cost estimates. They are therefore less consistent than DALY if the aim is to compare burden of illness between different geographical areas and over time.

CHALLENGES IN THE MEASUREMENT OF BURDEN OF ILLNESS A careful assessment of the burden of illness due to Campylobacter should in principle be based on a detailed knowledge of the age-specific frequency of Campylobacter-associated illness in a given community and a detailed understanding of the frequency and the severity of all possible outcomes associated with the infection. The outcomes must include consequences in both the short and long term. In reality, not all these data will be available, and a calculation of burden of illness will be subject to many uncertainties and assumptions. In a pilot study of disease burden of infectious diseases (including campylobacteriosis) in Europe (Van Lier and Havelaar, 2007), major limitations in data availability were as follows: inconsistent information on morbidity, mortality, or both; limited information about patients’ age distribution; no reporting of the incidence of complications and chronic sequelae; and no consistent set of disability weights available. Major limitations with regard to data quality were as follows: no information on underascertainment of morbidity and mortality; no information on possible variation between countries of the duration, severity, and rate of complications and chronic sequelae; and differences between figures obtained from different sources. For a valid estimation of the burden of illness, it is a major challenge to overcome some of these limitations; selected aspects of this problem will be discussed below.

UNDERASCERTAINMENT OF MORBIDITYTHE PYRAMID OF SURVEILLANCE Official numbers of Campylobacter-infections are commonly derived from laboratory-based surveillance, where clinical microbiology laboratories report positive findings to national reference centers. These data are pivotal to measure trends over time but do not reflect the burden of illness. The sensitivity of surveillance differs among countries (chapter

CHAPTER 8

9). The reported incidence is a composite measure of several factors. These factors include the true incidence of Campylobacter infections, health careseeking behavior of patients with gastroenteritis, and the likelihood that the physician will request a stool culture. Furthermore, access to laboratories and the microbiological methods in place vary, and so does the completeness in reporting findings to the public health authorities. Thus, the official numbers therefore represent only the tip of the surveillance pyramid (Fig. 1).Although this top is likely to include the most severe cases, there is a significant amount of illness that is not recorded in the official system. In population-based studies in England and Wales (Wheeler et al., 1999) and in The Netherlands (De Wit et al., 2001), the true incidence of disease was investigated. The studies suggest that for every reported Campylobacter infection, between 7.6 and 19 persons actually fell ill (Table 1). In the United States, an underreporting factor of 38 was assumed on the basis of studies with Salmonella infections (Mead et al., 1999). Part of the difference between the three studies may be the result of different methodology, but it is interesting that there is less variation between estimated incidences in the general population than the reported figures indicate. This suggests that the sensitivity of the surveillance is a critical factor when attempts are made to compare numbers between countries. Flint et al. (2005) reviewed these key national studies and the international efforts that are providing the necessary information and technical re-

BURDEN OF ILLNESS O F CAMPYLOBACTERIOSIS

153

Table 1. Reported and estimated numbers of Cumpylobacterassociated gastroenteritis per 100,000 population No. of cases:

Country England United States The Netherlands

Reported

Estimated

Reference

110 24 36

870 916 685

Wheeler et al. (1999) Mead et al. (1999) Van Pelt et al. (2003)

sources to derive national, regional, and global burden of disease estimates. Integrated food chain surveillance, where community-based, etiology-specific disease estimates are obtained, is subject to several types of bias, is costly, and requires good collaboration with the study population. Seroepidemiology may provide an attractive alternative, in particular if serum samples from a random sample of the general population can be obtained for this purpose. The major limitation of seroepidemiology is that it measures the incidence of Campylobacter-related seroreactions, and some of these may be reactions to previous exposures that took place long ago. Hence, data on the disease-toinfection ratio are needed to translate the seroepidemiology estimates to estimates of morbidity. On the basis of serum samples from Danish blood donors, the yearly incidence of Campylobacter infections was determined to be 138 per 1,000 population (Simonsen et al., 2007). This figure is 249 times

Figure 1. Surveillance pyramid. Only a fraction of the population that is exposed to Campylobucter will become infected, and among those who are infected, only a fraction will experience symptoms. Furthermore, only a fraction of these patients will seek medical care, and only a fraction of those who seek care (whether at the hospital or at their general practitioner’s office) will have the organism culture confirmed and reported, and thus figure in the official statistics. The tip of the pyramid represents the most severe cases.

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higher than the reported incidence. In The Netherlands, the seroconversion rate indicates at least one exposure every 6 to 7 years during childhood (Ang et al., 2007). These high incidences are the same order of magnitude as the results from a risk assessment study, in which the total exposure of the Dutch population to Campylobacter was estimated to average 1 CFU per week. Although this may seem to be a low exposure, application of a dose-response model (Teunis and Havelaar, 2000) results in a predicted estimate of approximately one infection (which may be asymptomatic) per 2 person-years (Evers et al., in press).

THE PROBLEM OF ASCERTAINMENT OF OUTCOMES To assess the burden of disease, the most important or relevant disease outcomes need to be defined. This can be illustrated by an outcome tree, which is a qualitative representation of the progres-

sion of a disease in time. For Campylobacter, main outcomes as defined by Mangen et al. (2005) include gastroenteritis, Guillain-Barrt syndrome, reactive arthritis, inflammatory bowel disease, and death (Fig. 2 and Table 2). It is obvious that constructing such trees requires a simplification of the real world. For example, an outcome such as sepsis (without death) is not included in the tree because preliminary calculations show that this outcome would not substantially add to the burden. On the other hand, the inclusion of inflammatory bowel disease was made on the basis of the observation of a statistical relationship by Helms et al. (2003). In the absence of sufficient mechanistic supportive evidence, such an inclusion might be disputed. New evidence may require additions to the tree, and currently, sufficient evidence for inclusion of irritable bowel syndrome appears to be available (Smith and Bayles, 2007). On the basis of the scope of relevant adverse health outcomes, it is often necessary to collate information from a variety of different sources in order to obtain data on frequency and duration of these

Reactive arthritis

Figure 2 . Example of an outcome tree for Campylobacter-associated gastroenteritis and sequelae. R, recovery. From Van Lier and Havelaar (2007).

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BURDEN OF ILLNESS OF CAMPYLOBACTERIOSIS

155

Table 2. Incidence (cases per year) of Campylobacter gastroenteritis and associated sequelae, The Netherlands, 2004 (population 16 million)“ Incidence estimate (cases per year) Infection Gastroenteritis Without medical care See general practitioner Admitted to hospital Fatal Sequelae Guillain-Barrt syndrome Reactive arthritis Inflammatory bowel disease

Most likely

Low estimate

High estimate

59,000 45,000 14,000 570 25

25,000 19,000 5,000 500 18

140,000 110,000 33,000 650 34

60 1,000 22

40 430 17

85 2,600 29

“From Kemmeren et al. (2006).

outcomes and disability weights. For example, in the project Priority Setting of Foodborne Pathogens in The Netherlands, disability weights were obtained from the Dutch Public Health Status and Forecast studies, other studies from The Netherlands, and the international literature (Kemmeren et al., 2006). In Denmark, a series of registry studies has contributed to a better understanding of the severity of Carnpylobacter infections; these estimates have proved important for improving burden of disease estimates. Data on outcomes were obtained by linking different national registries, including the surveillance registry for gastrointestinal pathogens, the Danish civil registry, and the national hospital discharge registry. Of 16,180 Campylobacter patients, 190 (1.2%) died within 1 year after infection; this was 2.3 times higher than the general Danish population (matched by age and geography). After adjustment for comorbidity, this relative mortality declined to 1.9. The estimate was highest in the acute phase 30 days after diagnosis (relative risk, 5.0) but remained significantly increased up to 1 year after the initial diagnosis. On the basis of a calculation of the attributable risk among exposed patients, it can be estimated that the fatality rate of culture-confirmed Carnpylobacter infection is 0.5%, a figure that is higher than, for example, the FoodNet case fatality rate for Campylobacter, which is 0.10% (Mead et al., 1999). This may suggest that the burden of illness has been somehow underestimated in studies that have ignored mortality as an outcome of campylobacteriosis. Of 17,99 1 patients with Campylobacter, 2,221 (12%) had been admitted to the hospital. Although most (1,937 patients) had a discharge diagnosis of gastroenteritis, other outcomes included irritable bowel syndrome (161 patients), inflammatory bowel disease (72 patients), invasive illness (41 patients), reactive arthritis (22 patients), gastrointestinal compli-

cations (16 patients), and Guillain-Barr6 syndrome (6 patients) (Helms et al., 2006). The burden of illness was measured in terms of numbers of days in the hospital, and the analysis shows that 1,000 Campylobacter patients result in some 700 bed-days (Table 3). In another study, it was shown that antimicrobial drug resistance (fluoroquinolone or macrolide resistance) was associated with an excess mortality compared with infections with drug-sensitive Campylobacter (Helms et al., 2005). This indicates that the burden of illness may increase by increasing levels of drug resistance. A similar observation has been made for duration of gastrointestinal illness among individuals infected with fluoroquinolone-resistant Campylobacter (reviewed by Mlirlbak, 2005). However, excess morbidity and mortality associated with drug resistance in Campylobacter remains a subject of debate (Wassenaar et al., 2007).

THE IMPACT OF IMMUNITY O N THE MEASUREMENT OF BURDEN OF ILLNESS One of the challenges in the measurement of burden of illness is to identify methods to include contribution from immunity. An exposure to Cam-

Table 3. Duration of hospital stay per 1,000 patients with Campylobacter infections Hospital stay

.

Hospital days per 1,000 patients

...

Total.. . . . . . . . . . . . . , , ,., Attributed tv gastroenteritis. . . . Attributed to complications. . . . Attributed to chronic sequelae..

714 days 530 days 30 days 150 days

“Analyses of the national patients registry in Denmark, 1991 to 1999, based on 17,991 patients. From Helms et al. (2006).

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M0LBAK AND HAVELAAR

pylobacter may give rise to an infection that may lead to clinical symptoms or that may be asymptomatic. All things equal, the likelihood that exposure to a certain dose of Campylobacter results in an infection depends on the immunity of the host, and the immunity of the host will also modify the clinical course of infection. In other words, in a population with frequent exposure to Campylobacter, the disease-toinfection ratio will be lower than in an immunologically naive population where a larger proportion of infections will be symptomatic. This concept is corroborated by several lines of evidence. (i) In developing countries, where Campylobacter is ubiquitous, rates of asymptomatic carriers are high, and clinical disease is primarily seen in young children. This may suggest that immunity toward clinical illness is widespread, although strain differences between different areas of the world might contribute (Coker et al., 2002; Oberhelman and Taylor, 2000). (ii) In industrialized countries, chronic exposure to Campylobacter may lead to immunity against clinical disease, as discussed in chapter 9. Indeed, as also reviewed, some case-control studies of sporadic illness suggest that repeated exposure to high-risk food is associated with reduced risk of illness. It has also been found in some studies that chicken eaten outside of the home is a more important risk factor than chicken eaten in the home. These counterintuitive findings corroborate that notion that immunological factors mitigate the risk of clinical disease. (iii) Environmental risk factors for Campylobacter infections may be more important in children than in adults. This may point toward the notion that adults living in a rural community with frequent exposure to Campylobacter may be relatively spared clinical illness, whereas young, disease-naive children become sick (Ethelberg et al., 2005). (iv) Finally, patients with hypo- or agammaglobulinemia, patients who have a defect in humoral immunity, and patients infected with human immunodeficiency virus and who have compromised cell-mediated immunity experience more severe illness than individuals with normal immune response (Perlman et al., 1988; Skirrow and Blaser, 2000). It is beyond the scope of this chapter to discuss these immunological aspects in detail. The point is, however, that the relation between the overall exposure to Campylobacter in a given country and the burden of illness may be nonlinear. In particular, for countries with high rates of diarrheal illnesses or a high risk of frequent exposures to Campylobacter, the demonstration of Campylobacter in the stool of a patient with gastrointestinal symptoms may not be of etiological relevance, but merely a coincidental finding. Hence, for developing countries in particu-

lar, the methodology to determine morbidity from Campylobacter infections needs further development.

DISABILITY WEIGHTS Disability weights reflect the health impact of a condition, and they are based on the preferences of a panel of judges. Disability weights typically range between 0, reflecting the best possible health state, and 1, reflecting the worst possible health state. For example, for Guillain-BarrC syndrome, the severity score may range between 0.01 and 0.94 depending on the functional status of the patient. The assignment of disability weights is a controversial issue. Ideally, disability weights that are based on preferences of the general public are used in burden of disease studies aimed to inform policy making at the national or international level. Disability weights that are based on elicitation panels consisting of laypersons are increasingly becoming available. Previous work has depended on panels of medical professionals. Preferences of patients who actually have the disease are biased because of coping behavior. The international transferability of disability weights is of concern. A study in Western Europe (Schwarzinger et al., 2003) concluded that there was “a reasonably high level of agreement on disability weights in Western European countries with the visual analogue scale (VAS) and time trade-off (TTO) methods, but a lower level of agreement with the person trade-off (PTO) method.” Ustun et al. (1999) concluded that ranking of weights is relatively stable across countries, but differences are large enough to cast doubt on the universality of experts’ judgments of disability weights. Another study concluded that “Meaningful differences exist in directly elicited TTO valuations of EQ-5D health states between the US and UK general populations” (Johnson et al., 2005). Several preference measurement methods are available for panel elicitation, including the standard gamble (SG), TTO, PTO, and VAS. All methods give different results (VAS > TTO > PTO > SG), but they are highly correlated. The SG and VAS are not considered informative because they are only sensitive to severe disease (SG) or very sensitive to mild diseases (VAS), leading to compression at either end of the scale. Additionally, the VAS is not choice based because it does not allow a trade-off. The TTO and PTO methods are generally used. For chronic diseases, most descriptions are based on the impact of a disease in the course of a year. However, many infectious diseases have a rapid course, and consequently the disability weight can be assessed by fo-

CHAPTER 8

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Sahnella

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Iisteria

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Giardia

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I El Undiscounted 0 Discounted (4%)I Figure 3. Disease burden of seven enteric pathogens in The Netherlands, 2004. From Kemmeren et al. (2006) and Vijgen et al. (2007). Bars indicate most likely estimate; error bars, minimum and maximum values. STEC, Shiga-toxin producing Escherichia coli; cong., congenital.

cusing on the phase of acute disease only (period profile) or by focusing on a year in which an episode of acute illness is experienced (annual profile; when using an annual profile, the duration of the illness is 1 year by definition). Both methods have been used in disease burden studies. In practice, large differences may be found between these two methods for diseases that have a high incidence but low severity (e.g., norovirus-associated gastroenteritis). For such diseases, the use of annual profiles may lead to high estimates of disease burden. One proposed solution is to introduce a threshold that is based on the median weight of the respondents in an elicitation study (J. Haagsma et al., unpublished data).

ESTIMATED BURDEN OF CAMPYLOBACTER INFECTIONS AND THE RELATIVE CONTRIBUTION OF ACUTE INFECTIONS VERSUS POSTINFECTIVE COMPLICATIONS Figure 3 shows the estimated burden of seven enteric pathogens in The Netherlands. Campylobacter causes the largest burden of these pathogens, followed by Salmonella and Toxoplasma. It is noteworthy that the burden of enteric viruses is lower, despite their higher incidence. Viral infections such as norovirus or rotavirus are usually associated with a relatively mild clinical course, a low case-fatality ratio, and the absence of complications. Figure 4 shows

Giardia Cryptosporidium Tomplasma (cong.) Listena Rotavirus Norovirus Salmonella STEC 0157

Campylobacter 0

DALYs per year

200

400

600

800

loo0

1200

1400

Figure 4. Breakdown of disease burden of seven enteric pathogens in The Netherlands, 2004. From Kemmeren et al. (2006) and Vijgen et al. (2007). STEC, Shiga toxin-producing Escherichia coli.

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Campylobacter Sabnella

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Figure 5. Cost of illness of seven enteric pathogens in The Netherlands, 2004. From Kemmeren et al. (2006) and Vijgen et al. (2007). Bars indicate most likely estimate; error bars, minimum and maximum values.

that for Campylobacter, the largest contribution to the burden is from complications, in particular Guillain-Barrt syndrome. Mortality also causes a relatively high burden, while the contribution of acute gastroenteritis is relatively low. Data for the cost of illness are shown in Figs. 5 and 6. The enteric viruses have the largest economic impact because their high incidence leads to considerable productivity losses (indirect non-health care costs). The costs of campylobacteriosis are somewhat lower than the viruses but still significant. Direct health care costs (mainly associated with Guillain-

Barrt syndrome) are an important factor in these costs. Table 4 shows that the burden of campylobacteriosis is similar to that of other important bacterial infections such as meningitis, bacterial sexually transmitted infections, and tuberculosis but lower than AIDS, human immunodeficiency virus, and influenza. Collectively, the burden of infectious diseases is lower than that of chronic diseases such as cardiovascular disease and cancer. This does not imply that infectious diseases are unimportant but that in a country such as The Netherlands, infectious disease

Giardia

Rotavirus

Norovirus

Sahnella

0 5 Cost of illness (million €per year)

10

15

20

25

30

Figure 6. Breakdown of cost of illness of seven enteric pathogens in The Netherlands, 2004. From Kemmeren et al. (2006). DHC, direct health care costs; DNHC, direct non-health care costs; INHC, indirect non-health care costs.

CHAPTER 8

BURDEN OF ILLNESS OF CAMPYLOBACTERIOSIS

159

Table 4. Disease burden of Cumpylobacter infections in comparison with selected other diseases in The Netherlands (population 16 million)" Amount of lost DALYs per yr

Infectious diseases

Noninfectious diseases

>100,000

Acute lower respiratory tract infections (e.g., pneumonia and acute bronchi[oli]tis) Influenza HIVIAIDS, upper respiratory tract infections, stomach ulcers Campylobacter infections, bacterial meningitis, bacterial sexually transmitted infections, tuberculosis Shiga toxin-producing Escherichiu coli 0157

30,000 to 100,000 10,000 to 30,000 3,000 to 10,000 1,000 to 3,000

<1,000

Cardiovascular disease, cancer, depression, diabetes, alcohol dependency Traffic accidents, breast cancer, suicide Epilepsy, multiple sclerosis Ulcers of the stomach and duodenum Hip fractures

"From Havelaar et al. (2005).

parties (equity). Risk assessment, epidemiology, and economic analysis are all tools that can be used to aid risk management in such decisions. In The Netherlands, this interdisciplinary approach, which involved different scientific institutes, was applied to the control of Campylobacter on broiler meat. Estimates were made of the potential benefits and the cost-utility ratio of a large number of possible interventions to decrease human exposure to Campylobacter by consumption of chicken meat. For this purpose, a farm-to-fork risk assessment model was combined with economic analysis and epidemiological data (Havelaar et al., 2007; Katsma et al., 2007; Nauta et al., 2007; Mylius et al., 2007). Chicken

control is relatively effective. Maintaining the current level of protection is essential, and cost-effective interventions to further reduce the current burden are available (Figs. 7 and 8).

INTEGRATING BURDEN ESTIMATES WITH RISK ASSESSMENT AND ECONOMICS: THE CARMA PROJECT When evaluating an intervention or policy, decision makers need to determine its ability to positively impact public health (effectiveness)at a reasonable cost (efficiency) in a fair manner for all affected

^ ^

farm hygiene phage therapy red. faecal leakage

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i

crust freezing

i

irradiation : product freezing :

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40%

60%

80%

100%

Figure 7. Risk reduction of campylobacteriosis by interventions in the broiler meat chain. From Havelaar et al. (2007).

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farm tygiene (opbmisbc scenario) phaw tkrapy red. fecal leakage dewnt scald tank decort. rip

decont dip and spray crust freezing irradiation pro&ct freezing prepaed meat hane hezing kitchen tygiene I

-lw

0

100

2w

30

-

400

500

Cost-utility ratio (x 1000 E per DALY averted)

Figure 8. Cost-utility ratio of different interventions to reduce contamination of broiler meat with Cumpylobacter. Data are presented when effects on Dutch consumers only are considered and when effects on all consumers (including exports from The Netherlands) are considered. Only products from flocks that tested positive by PCR are treated. From Havelaar et al. (2005). NL, The Netherlands.

meat consumption accounts for 20 to 40% of all cases of illness in The Netherlands. Reduction of contamination at broiler farms could be efficient in theory. However, it is unclear which hygienic measures need to be taken, and the costs can be high. The experimental treatment of colonized broiler flocks with bacteriophages has proven to be effective in laboratory-scale studies and could also be cost-efficient if confirmed in practice. Because a major decrease of infections at the broiler farm is not expected in the short term, additional measures in the processing plant were also considered. At this moment, guaranteed Campylobucter-free chicken meat at the retail level is not realistic. The most promising interventions in the processing plant include limiting fecal leakage during processing, separating contaminated and noncontaminated flocks (scheduling), and decontaminating a contaminated flock. New, faster, and more sensitive test methods to detect Cumpylobucter colonization in broilers flocks are a prerequisite for successful scheduling scenarios. Other methods to decrease the contamination of meat of colonized flocks, such as freezing and heat treatment, are more expensive and less effective than chemical decontamination. On the basis of the risk models developed for the project, quantitative criteria for Cumpylobacter

on broiler meat are being developed and related to the appropriate level of protection, as defined by Codex Alimentarius Commission (2005).

PERSPECTIVES A N D FUTURE RESEARCH The burden of Cumpylobucter infections and other food-borne disease is not well defined in many countries or regions or on a global level. WHO, in conjunction with other national public health agencies, is coordinating a number of international activities designed to assist countries in the strengthening of disease surveillance and to determine the burden of acute gastroenteritis. This activity is important to enable decision makers to make priorities between different interventions to improve food safety. WHO has recently developed a strategic framework for the estimation of global burden of foodborne diseases from all causes and is in the process of establishing the Foodborne Disease Burden Epidemiology Reference Group. This group will include experts from various disciplines, including microbiology, parasitology, and virology; burden of disease methodologies; microbiological and chemical risk assessment (including toxicological) and source attribution; disease and exposure modeling; statistics and

CHAPTER 8

geographic information systems; clinical medicine and nutrition; and food protection, policy, and regulation. The main aim of the Foodborne Disease Burden Epidemiology Reference Group will be to arrive at DALY estimates for food-borne illness (e.g., bacteria, viruses, parasites, fungi, chemicals). This activity will be closely coordinated with similar WHO initiatives in other domains (such as the Child Health Epidemiology Reference Group) and will also fit in a recent initiative to update the global burden of disease estimates. For developing countries, where deaths from diarrheal illness remain a leading cause of mortality, there is still a lack of data on the relative role of Campylobacter compared with better-described agents such as rotavirus, enterotoxigenic Escherichia coli, and Shigella spp. In addition, postinfective complications have largely been described in studies from industrialized countries. Although these complications also occur in developing countries, they have not been quantified. In the coming years, this project will be an area of intense work. In industrialized countries and countries in the transition phase, recent studies have shed light on the burden of illness. Metrics that allow a comparison of the burden of illness between different countries and over time have contributed to this, but many important areas of research still remain. One particular area of further work is vulnerability and burden of illness in certain population groups at risk. India, as a country in a transitional phase, has a growing population of persons with diabetes. This is one example of a large population group at a high risk of illness and complications from illness. Unless steps are taken to secure safe food, burden of illness may increase as population demographics change. Acknowledgments. We thank Marie-JosCe Mangen, Jeanet Kemmeren, Sylvia Vijgen, and Juanita Haagsma for their contributions to the Dutch disease burden studies; Gerhard Falkenhorst, Morten Helms, and Jacob Simonsen for the Danish studies; and Wilfrid van Pelt and Rob Tauxe for helpful discussions when drafting this chapter. The European Network of Excellence MED-VET-NET supported our work.

REFERENCES Ang, C. W., W. Van Pelt, P. Herbrink, J. Keijser, Y. T. H. P. Van Duynhoven, and C. E. Visser. 2007. Sero-epidemiologyindicates frequent and repeated exposure to Campylobacter during childhood. Zoonoses Public Health 5450. Codex Alimentarins Commission. 2005. Proposed Draft Principles and Guidelines for the Conduct of Microbiological Risk Management (MRM). Codex Committee on Food Hygiene, 37th session, 14 to 19 March 2005. Alinorm 05-28/13, appendix 3, 67-81. Codex Alimentarius Commission, Rome.

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Coker, A. O., R D. Isokpehi, B. N. Thomas, K. 0. Amisu, and C. L. Obi. 2002. Human campylobacteriosis in developing countries. Emerg. Infect. Dis. 8:237-244. De Wit, M. A. S., M. P. G. Koopmans, L. M. Kortbeek, W. J. Wannet, J. Vinjt, F. Van Leusden, A. I. M. Bartelds, and Y. T. H. P. van Duynhoven. 2001. Sensor, a population-based cohort study on gastroenteritis in The Netherlands: incidence and etiology. Am. 1.Epidemiol. 154:666-674. Ethelberg, S., J. Simonsen, P. Gerner-Smidt, K. E. Olsen, and K. Molbak. 2005. Spatial distribution and registry-based casecontrol analysis of Campylobacter infections in Denmark, 19912001. Am. 1.Epidemiol. 162:1008-1015. Evers, E. G., H. J. Van Der Feld-Klerx, M. J. Nauta, J. F. Schijven, and A. H. Havelaar. In press. Campylobacter source attribution by exposure assessment. Int. 1.Risk Assess. Manage. Flint, J. A., Y. T. Van Duynhoven, F. J. Angulo, S. M. DeLong, P. Braun, M. Kirk, E. Scallan, M. Fitzgerald, G. K. Adak, P. Sockett, A. Ellis, G. Hall, N. Gargouri, H. Walke, and P. Braam. 2005. Estimating the burden of acute gastroenteritis, foodborne disease, and pathogens commonly transmitted by food: an international review. Clin. Infect. Dis. 41:698-704. Gold, M. R., D. Stevenson, and D. G. Fryback. 2002. HALYS and QALYS and DALYS, oh my: similarities and differences in summary measures of population health. Annu. Rev. Public Health 23~115-134. Havelaar, A. H., A. E. M. De Hollander, P. F. M. Teunis, E. G. Evers, H. J. Van Kranen, J. F. M. Versteegh, J. E. M. Van Koten, and W. Slob. 2000. Balancing the risks and benefits of drinking water disinfection: disability adjusted life-years on the scale. Environ. Health Perspect. 108:3 15-321. Havelaar, A. H., M. J. Mangen, A. A. de Koeijer, M. J. Bogaardt, E. G. Evers, W. F. Jacobs-Reitsma, W. van Pelt, J. A. Wagenaar, G. A. de Wit, H. van der Zee, and M. J. Nauta. 2007. Effectiveness and efficiency of controlling Campylobacter on broiler chicken meat. Risk Anal. 272331-844. Havelaar, A. H., M. J. Nauta, M.-J. J. Mangen, A. G. De Koeijer, M.-J. Bogaardt, E. G. Evers, W. F. Jacobs-Reitsma, W. Van Pelt, J. A. Wagenaar, G. A. De Wit, and H. Van der Zee. 2005. Costs and Benefits of Controlling Campylobacter in The NetherlandsIntegrating Risk Analysis, Epidemiology and Economics. National Institute for Public Health and the Environment, Bilthoven. Helms, M., J. Simonsen, and K. Melbak. 2006. Foodborne bacterial infection and hospitalization: a registry-based study. Clin. Infect. Dis. 42:498-506. Helms, M., J. Simonsen, K. E. Olsen, and K. Melbak. 2005. Adverse health events associated with antimicrobial drug resistance in Campylobacter species: a registry-based cohort study. 1. Infect. Dis. 191:1050-1055. Helms, M., P. Vastrup, P. Gerner-Smidt, and K. Melbak. 2003. Short and long term mortality associated with foodborne bacterial gastrointestinal infections: registry based study. BM] 326: 357. Johnson, J. A., N. Luo, J. W. Shaw, P. Kind, and S. J. Coons. 2005. Valuations of EQ-5D health states: are the United States and United Kingdom different? Med. Care 43:221-228. Katsma, W. E., A. A. De Koeijer, W. F. Jacobs-Reitsma, M. J. Mangen, and J. A. Wagenaar. 2007. Assessing interventions to reduce the risk of Campylobacter prevalence in broilers. Risk Anal. 27:863-876. Kemmeren, J. M., M.-J. J. Mangen, Y. T. H. P. Van Duynhoven, and A. H. Havelaar. 2006. Priority Setting of Foodborne Pathogens-Disease Burden and Costs of Selected Enteric Pathogens. National Institute for Public Health and the Environment, Bilthoven. Mangen, M.-J. J., A. H. Havelaar, and K. J. Poppe. 2005. Controlling Carnpylobacter in The Chicken Meat Chain-Estimation

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of Intervention Costs. Agricultural Economics Institute, The Hague. Mead, P. S., L. Slutsker, V. Dietz, L. F. Mccaig, J. S. Bresee, C . Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607625. Murray, C . J. L., and A. D. Lopez. 1996. The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries, and Risk Factors in 1990 and Projected to 2020. Harvard School of Public Health, Cambridge, MA, on behalf of the World Health Organization and the World Bank. Mylius, S. D., M. J. Nauta, and A. H. Havelaar. 2007. Crosscontamination during food preparation: a mechanistic model applied to chicken-borne Campylobacter. Risk Anal. 27:803-8 13. Melbak, K. 2005. Human health consequences of antimicrobial drug-resistant Salmonella and other foodborne pathogens. Clin. Infect. Dis. 41:1613-1620. Nauta, M. J., W. F. Jacobs-Reitsma, and A. H. Havelaar. 2007. A risk assessment model for Campylobacter in broiler meat. Risk Anal. 27:845-861. Oberhelman, R. A., and D. N. Taylor. 2000. Campylobacter infections in developing countries, p. 139-154. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Perlman, D. M., N. M. Ampel, R. B. Schifman, D. L. Cohn, C . M. Patton, M. L. Aguirre, W. L. Wang, and M. J. Blaser. 1988. Persistent Campylobacter jejuni infections in patients infected with the human immunodeficiency virus (HIV). Ann. Intern. Med. 108540-546. Schwarzinger, M., M. E. Stouthard, K. Burstrom, and E. Nord. 2003. Cross-national agreement on disability weights: the European Disability Weights Project. Popul. Health Metr. 1:9. Simonsen, J., K. Krogfelt, K. Melbak, and G. Falkenhorst. 2007. Sero-epidemiology as a tool for estimating the incidence of Campylobacter infections, abstr. RR04. 3rd Med-Vet-Net Annu. Sci. Mtng.

Skirrow, M. B., and M. J. Blaser. 2000. Clinical aspects of Campylobacter infection, p. 69-88. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Smith, J. L., and D. Bayles. 2007. Postinfectious irritable bowel syndrome: a long-term consequence of bacterial gastroenteritis. J. Food Prot. 70:1762-1769. Teunis, P. F. M., and A. H. Havelaar. 2000. The beta-Poisson dose-response model is not a single-hit model. Risk Anal. 20: 513-520. Van Lier, E. A., and A. H. Havelaar. 2007. Disease Burden o f Infectious Diseases in Europe: A Pilot Study. National Institute for Public Health and the Environment, Bilthoven. Van Pelt, W., M. A. de Wit, W. J. Wannet, E. J. Ligtvoet, M. A. Widdowson, and Y. T. van Duynhoven. 2003. Laboratory surveillance of bacterial gastroenteric pathogens in The Netherlands, 1991-2001. Epidemiol. Infect. 130:431-441. Vijgen, S. M. C., M. J. M. Mangen, L. M. Kortbeek, Y. T. H. P. Van Duynhoven, and A. H. Havelaar. 2007. Diseuse Burden and Related Costs of Cyptosporidiosis and Giardiasis in The Netherlands. National Institute for Public Health and the Environment, Bilthoven. Wassenaar, T. M., M. Kist, and A. de Jong. 2007. Re-analysis of the risks attributed to ciprofloxacin-resistant Campylobacter jejuni infections. Int. J. Antimicrob. Agents 30:195-201. Wheeler, J. G., D. Sethi, J. M. Cowden, P. G. Wall, L. C. Rodrigues, D. S. Tompkins, M. J. Hudson, and P. J. Roderick. 1999. Study of infectious intestinal disease in England: rates in the community, presenting to general practice, and reported to national surveillance. The Infectious Intestinal Disease Study Executive. BMJ 318:1046-1050. World Health Organization. 2004. Guidelines for Drinking-Water Quality, 3rd ed., vol. 1, Recommendations. World Health Organization, Geneva. Ustun, T. B., J. Rehm, S. Chatterji, S. Saxena, R. Trotter, R. Room, and J. Bickenbach. 1999. Multiple-informant ranking of the disabling effects of different health conditions in 14 countries. WHO/NIH Joint Project CAR Study Group. Lancet 354: 111-1 15.

Campylobacter, 3rd ed. Edited by I. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 9

Epidemiology of Campylobacter jejuni Infections in Industrialized Nations CHRISTINE

K.

OLSON, STEEN

ETHELBERG, WILFRID VAN PELT, AND ROBERTv. TAUXE

Campylobacter organisms have long been recognized as a cause of diarrhea in cattle and of septic abortion in both cattle and sheep. More recently, they have been recognized as an important cause of human illness. Campylobacter organisms may have been what Escherich described as motile Vibrio-like organisms in the stools of infants with diarrhea in Germany in 1881 (Escherich, 1881). However, in 1962, Campylobacter, then still known as “related Vibrio,” was described as a rare and opportunistic human pathogen that was isolated from blood cultures of immunocompromised humans (King, 1962). It was not until 1972 that the most commonly identified species, Campylobacter jejuni, was first isolated from human diarrheal stools by applying a filtration technique that was originally developed for veterinary diagnosis (Dekeyser et al., 1972). The subsequent development of selective Campylobacter stool culture media led to the recognition that C. jejuni was a common cause of human diarrheal illness everywhere, often more common even than Salmonella. Since then, knowledge of the epidemiology of the infections this organism causes has been steadily accumulating. The majority of these infections are sporadic; C. jejuni is far less often recognized as a cause of outbreaks than is Salmonella. As a result, much of what we know has come from the study of sporadic cases with casecontrol methodologies and other epidemiological techniques. Campylobacter is a common cause of diarrheal illness in the developing world, particularly in the first few years of life (Coker et al., 2002). However, the scope of this chapter is restricted to the industrialized world. An excellent chapter in the previous

edition of this book summarized well the available information on Campylobacter in the developing world, and the interested reader is referred to that publication for further information (Oberhelman and Taylor, 2000).

INFECTIOUS DOSE AND IMMUNITY The infectious dose of C. jejuni for humans appears to be low. Robinson (1981) reported his own illness that followed ingestion of 500 organisms. Human volunteer studies at the University of Maryland have shown that half the subjects became infected after ingesting 800 organisms, the lowest dose tested. Approximately half also became ill after exposure to 90,000 organisms, although curiously, illness was less frequent at higher doses (Black et al., 1988). The variation in illness attack rates could reflect prior immunity. Data suggest that in a previously naive population, high rates of illness could well follow low doses (Teunis and Havelaar, 2000; Teunis et al., 2005). Protective immunity to Campylobacter is well documented after chronic exposure to a risky food, such as raw milk (Blaser et al., 1987; Jones et al., 1981). Persons who had already been infected once did not develop illness a second time on rechallenge with the same strain in volunteer trials (Black et al., 1988). In Scotland, older persons tend to be infected with less common serotypes, possibly indicating that repeated exposure may immunize a fraction of the population to the most common serotypes (Miller et al., 2005). Persons with AIDS are at increased risk of acquiring Campylobacter infections (Angulo and

Christine K. Olson Enteric Diseases Epidemiology Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333. Steen Wilfrid van Ethelberg * Departments of Epidemiology and Bacteriology, Statens Serum Institut, Copenhagen, DK-2300, Denmark. Pelt Department of Epidemiology and Surveillance, Rijksinstituut voor Volksgezondheid en Milieu, 3721 MA Bilthoven, The Netherlands. Robert V. Tauxe Division of Foodborne, Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.

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Swerdlow, 1995). The reported incidence of laboratory-confirmed campylobacteriosis in persons with AIDS in Los Angeles County between 1983 and 1987 was 519 per 100,000, much higher than the reported rate in the general population (Sorvillo et al., 1991). Host factors may play a role in the risk of development of C. jejuni bacteremia. In the same Los Angeles study, patients with AIDS were more likely to develop bacteremia from Campylobacter than were similar patients without AIDS who had Campylobacter infection (Sorvillo et al., 1991). However, no association with invasive disease was seen in a study of 3 0 human immunodeficiency virus-infected patients in a study at a London hospital between 1986 and 1991 (Nelson et al., 1992). National Campylobacter Surveillance in the United States Passive surveillance The Centers for Disease Control and Prevention (CDC) began national Campylobacter surveillance in 1982 using weekly written reports from state public health laboratories (Riley and Finch, 1985; Tauxe et al., 1988). In the early 1990s, this was converted to an electronic data transmission that used the Public Health Laboratory Information System (Bean et al., 1992). The data reported from the state public health laboratories include the Campylobacter species isolated, the date of reporting to the CDC, the age and sex of the person from whom the organism was isolated, the person’s county of residence, and the clinical source of the culture. Unlike Salmonella or Shigella, however, public health laboratories do not serotype Campylobacter routinely, and states vary widely in whether they require Campylobacter isolates to be sent to public health laboratories after being isolated in a clinical laboratory. In many states, few isolates are referred from clinical laboratories to state public health laboratories outside of an outbreak setting, so the number of isolates reported through this system greatly underestimates the number of diagnosed cases. For example, in 2005, 7,578 isolates of Campylobacter were reported through this system, one-fifth of the number of Salmonella isolates reported through this system, although the actual incidence of diagnosed infections identified by active surveillance was similar between the two organisms. Requirements for notification of diagnosed cases of Campylobacter infections have also been highly variable, and notification does not happen at all in many states. Until the establishment of the Foodborne Disease Active Surveillance System (FoodNet), accurate estimates of the burden of Campylobacter disease in the United States have not been possible (Scallan, 2007).

Active surveillance In 1996, the CDC, together with the U.S. Department of Agriculture’s Food Safety and Inspection Service, the U.S. Food and Drug Administration, and the state health departments of the Emerging Infections Program sites, developed FoodNet to better determine the frequency and severity of foodborne disease (Angulo et al., 1997). FoodNet is an active population-based surveillance system that began conducting surveillance in 1996 in five sites: Minnesota, Oregon, and selected counties in California, Connecticut, and Georgia. Addition of five more sites (Maryland, New Mexico, Tennessee, and selected counties in Colorado and New York) and expansion of some of the initial sites mean that the current population of the FoodNet surveillance area is 44.9 million persons (15% of the U.S. population) and is relatively closely representative of the U.S. population, except for underrepresentation of the Hispanic population (9% of FoodNet site population versus 14% of the U.S. population) (Scallan, 2007). Public health officials from each site regularly contact microbiology laboratories in their surveillance catchment area to collect information on all culture-confirmed cases of the foodborne pathogens under surveillance and transmit it to CDC. Information is collected on culture-confirmed cases of Campylobacter, Cryptosporidium, Cyclospora, Listeria monocytogenes, Salmonella, Shiga toxin-producing Escherichia coli including 0157:H7, Shigella, Vibrio, and Yersinia infections. The system is active because public health staff reach out to the clinical laboratories to gather reports, rather than waiting passively for reports to arrive. Virtually all clinical laboratories in the FoodNet catchment area routinely culture diarrheal stool specimens for Campylobacter (Angulo et al., 1997). FoodNet thereby provides timely data on the incidence of diagnosed Campylobacter infections. Trends The number of illnesses from Campylobacter reported in the FoodNet population has increased from 1996 through 2005 as more FoodNet sites have been added and the population expanded. However, the crude incidence rate of Campylobacter infection has declined from 23.6 per 100,000 population in 1996 to 12.7 in 2005, with a mean over that 10-year period of 16.3 infections per 100,000 persons (Fig. l). In 2005, Campylobacter had the second highest incidence rate of all pathogens under surveillance in FoodNet after Salmonella infections, which were 14.5 per 100,000 (CDC, 2006). Because the incidence was affected by the variation among sites as

CHAPTER 9

0

C. TETUNI INFECTIONS IN INDUSTRIALIZED NATIONS

___~

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1997

1996

1398

1999

2000

165

2001

2002

2003

2004

2005

Year Figure 1. Reported incidence of Cumpylobucter infections in FoodNet, by month and year, 1996 to 2005.

new sites were added, the overall trend is best estimated by a multivariable model that accounts for those differences. By means of a negative binomial Poisson model, the incidence in FoodNet declined by approximately 30% in 2005 compared with the baseline period of 1996 to 1998 (Fig. 2), with most of

Relative Rate (logscale)

the decline occurring before 2002 (CDC, 2006). This decline corresponds in time with the introduction of new meat and poultry safety regulations in the United States, and to an effort on the part of the industry to reduce contamination of poultry meat at slaughter through increased chlorination and other process

_ _

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1996-1998

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Year Figure 2. Relative incidence rate of Cumpylobucter infections in FoodNet through 2005, compared with baseline period 1996 to 1998, using a negative binomial Poisson regression model to account for effect of new sites added over time (CDC, 2006).

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28.63

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Figure 3. Annual incidence of Cumpylobacter infections, by site, FoodNet, 2005. CA, California; CO, Colorado; CT, Connecticut; GA, Georgia; MD, Maryland; MN, Minnesota; NM, New Mexico; NY, New York; OR, Oregon; TN, Tennessee.

changes, although the impact of these changes on campylobacteriosis is unproven (U.S. Department of Agriculture, 1996).

and thus may reflect real differences in infection rates, although no regional differences in risk factor exposures have been identified.

Regional differences in incidence

Age and sex distribution

Differences in Campylobacter infection rates vary substantially by region. In 2005, rates per 100,000 population demonstrated California, Colorado, and New Mexico to have the highest rates of infection: 28.6, 19.1, and 18.2 cases per 100,000 population, respectively, whereas rates in the Eastern states were substantially lower (Fig. 3 ) . These differences have not been shown to be related to regional differences in rates of culturing or reporting of cases

In 2005, for children younger than 1 year of age, the rate of Campylobacter infection was 27 per 100,000, a rate substantially higher than for other age groups (Fig. 4). The next highest rates in male subjects occurred among those 1 to 4 and 40 to 49 years old and in female subjects among those 1 to 4 and 20 to 29 years old. Rates of Campylobacter infection are in general approximately 20% higher in boys and men than in girls and women, although the

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Figure 4 . Incidence of Campylobacter infections by age group and sex, FoodNet, 2005.

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C. TETUNI INFECTIONS IN INDUSTRIALIZEDNATIONS

prominent peak in young adult men observed in the 1980s has become less evident (Tauxe et al., 1988). The incidence rate of Campylobacter infection was higher for boys and men in all age groups, except for the 20- to 29-year-old age group, in which the female peak rate was 13 per 100,000. Some of this difference may be due to different food consumption and handling practices between men and women, with men tending to engage in riskier practices than women (Yang et al., 1998). Seasonality

Campylobacter infections reported through FoodNet show a consistent seasonal pattern, with rates of infection beginning to rise in March, peaking in June and July, and declining slowly during the fall (Fig. 1).The lowest rates are in December, January, and February. This may be due to higher consumption of contaminated products during the summer months, suboptimal refrigeration or freezing of contaminated foods at that time, and other as yet unidentified factors. Burden of disease The true annual incidence of Campylobacter infection in the United States is much greater than that identified by surveillance based on laboratory-

167

confirmed cases. The so-called surveillance pyramid describes this, with a broad base representing the many infections that actually occur and a narrow peak representing those that are diagnosed and reported (Fig. 5). Only a fraction of those who are ill seek medical care, only a fraction of those who seek care have specimens cultured, and only a fraction of cultures yield the organism, even in the case of true infection. By surveying the population to determine the frequency of diarrheal illness, and by estimating each of the fractions and combining them for bloody and nonbloody diarrhea, it was estimated that Campylobacter caused illness in 34 persons for every reported case in 1998 (Samuel et al., 2004). A similar approach applied to Salmonella developed a multiplier of 38.6 for Salmonella (Voetsch et al., 2004). By assuming that the population in FoodNet sites is representative of the total U.S. population, we can estimate there were 36,624 isolations of Campylobacter in the United States in 2005 (12.7 per 100,000 X 2005 U.S. population), and then, by use of the multiplier of 34, estimate that there were a total of 1,250,000 illnesses that year, an incidence of 432 per 100,000. Such a high rate of infection sustained over many years may mean that a substantial fraction of the population has been infected. During 2005, the vast majority of Campylobacter isolates came from stool specimens, and less than 1% of Campylobacter isolations were from normally

Lab tests for organism

Specimen obtained Person seeks care

Person becomes ill

Illness in the general population Figure 5. Surveillance pyramid used to estimate the burden of food-borne diseases in FoodNet.

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OLSON ET AL.

sterile sites. In 2005, the rate of hospitalization for patients with Campylobacter was 14% in FoodNet sites. The overall case fatality rate from FoodNet data for 1996 to 2005 was 0.11%, or 11 per 10,000 culture-confirmed cases. Applying this case fatality rate to the estimated 36,624 isolations of Campylobacter in the United States, it can be estimated that there were approximately 40 deaths in 2005 due to diagnosed Campylobacter infections. National Campylobacter Surveillance in Other Parts of the Developed World

overall incidence of 51.6 per 100,000 population, including only reporting countries (EFSA, 2006; Gauci and Ammon, 2007). The Czech Republic reported an exceptionally high incidence of 296 cases per 100,000 population. In other European countries that traditionally have well-developed laboratorybased surveillance systems, reported incidence rates range from 50 to 90 cases per 100,000. Australia has a similar level of reported infections, whereas the incidence rate in New Zealand is the highest reported by any country, 396 per 100,000 in 2003 (Anonymous, 2006b; Baker et al., 2007).

Results of national surveillance systems for human campylobacteriosis

Trends

In Europe, the European Centre of Disease Prevention and Control (ECDC) was established in 2005 and has begun to collect and collate national surveillance data. In its first comprehensive epidemiological report covering 2005, campylobacteriosis was the most frequently occurring enteric bacterial infection, second only to Chlamydia infection among all diseases reported (Gauci and Ammon, 2007). There were about 200,000 campylobacteriosis cases reported among 28 countries covered, resulting in an

Most developed countries with long-standing surveillance have experienced an increase in the number of reported cases during the last 25 years (Fig. 6), and several countries have experienced dramatic increases over this period. In some countries, there has been a steady increase during the entire period, whereas in other countries, the increase has primarily occurred in the 1990s. Denmark reported no increase between 1980 and 1990 but had a fourfold increase between 1992 and 2001 (Anonymous, 2006a). New

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Year 1983 1987 1991 1995 1999 2003

Year Figure 6 . Trends in reported incidence of Campylobacter infections for six countries and the European Union, 1979 to 2006. Data sources: Denmark, Statens Serum Institut (http: //www.mave-tarm.dk) and Statistics Denmark; Norway, Folkehelseinstituttet (http://www.msis.no) and Statistics Norway; Sweden, Smittskyddinstitutet (http://www.smittskyddsinstitutet.se/ statistik/), M. Lofdahl (personal communication), and Statistics Sweden; England and Wales, Health Protection Agency (http: //www.hpa.org.uk/infections/topics-az/campy/data-ew.hun); Australia, National Notifiable Diseases Surveillance System (http: // http: //www.health.gov.au/internet/wcms/publishing.nsf/ Content/ health-pubhlth-strateg-communic-index.htm) and J. Musto, NSW Department of Health (personal communication); NZ, M. Baker, University of Otago, Wellington (personal communication) and Statistics New Zealand; ECDC, European Centre of Disease Prevention and Control and A. Amato (personal communication).

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Zealand had a nearly ninefold increase in incidence, from 14 to 120 cases per 100,000 population, between 1981 and 1990, and another threefold increase, from 128 cases to 396 cases from 1991 to 2003 (Baker et al., 2007; Brieseman, 1990). The early phase of this increase in Campylobacter incidence is likely due to increased physician awareness about this pathogen, increased culturing by laboratories, improved laboratory methods for detection, and improved reporting (Brieseman, 1990; Cowden, 1992; Kapperud and Aasen, 1992; Skirrow, 1991). However, the increase observed during the 1990s, when culturing for Campylobacter had become routine in many laboratories, more likely reflects a true increase in infections (World Health Organization WHO], 2001). Since the beginning of the 2000s, the reported number of infections has been stable or has moderately decreased in several countries (Fig. 6), and it is possible that we have now seen the peak of the epidemic of Campylobacter in part of the industrialized world. The mechanisms behind the long-term changes in the number of infections are likely multifactorial and are not understood in detail, although in recent years, consumption and handling of fresh poultry has been generally accepted as the main source of human infections (WHO, 2001). In accordance with this, control measures aimed at reducing the level of contaminated chicken meat have been enacted in some countries, including England and Wales and all five Scandinavian countries. These have included measures such as improved biosecurity barriers at chicken farms, increased hygiene at transport and slaughter, freezing of contaminated poultry, labeling of chicken products, use of leak-proof packaging, and campaigns targeting consumers. These initiatives have generally resulted in a reduction of contaminated poultry sold at the retail level and likely contributed to a recently observed reduction in the number of human infections, although a causal association has been difficult to measure and reductions have also been seen in countries where control programs have not been implemented (Borck et al., 2007; Hofshagen and Kruse, 2005; Stern et al., 2003). In Iceland, after an epidemic increase in Campylobacter infections in the late 1990s, an intensive prevention effort was begun in 2000 that included public education about poultry handling, on-farm biosecurity measures, and freezing carcasses of chickens from flocks known to have Campylobacter. As a result, farmers producing chicken without Campylobacter could sell them at higher prices, as consumers prefer fresh chicken (Stern et al., 2003). The following year, domestically acquired Campylobacter infec-

169

tions declined by 70% as farmers producing chicken without Campylobacter got the premium price for fresh chicken. The reduction in Campylobacter counts after freezing of poultry carcasses has been well documented (Georgsson et al., 2006). In 2003, a similar strategy-freezing all chicken from Campylobacter-positive flocks-was launched in Denmark, along with a multipronged effort to reduce contamination from farm to table. That year, a decrease in Campylobacter infections occurred and has continued, with a decrease of 28% in human cases between 2001 and 2006. This has been accompanied by a decrease from 42 to 30% in the proportion of Danish flocks that yield Campylobacter and from 47 to 12% in the proportion of broiler meat at retail that test positive for Campylobacter (Borck et al., 2007). In New Zealand, where such measures have not been introduced, the annual increase in culture-confirmed Campylobacter cases mirrored the 10-fold increase in the annual consumption of fresh chicken meat from 1981 to 2005, during which time there was little change in the consumption of frozen chicken (Baker et al., 2006). Comparison of disease incidence between countries Although Campylobacter is the most frequently reported cause of acute bacterial gastroenteritis in many developed countries around the world, substantial variation in incidence rates has been observed between countries (Gauci and Ammon, 2007; WHO, 2001). The differences in incidence rates can be difficult to compare because they reflect differences in surveillance that result from varying cultural norms for health care-seeking and diagnostic practices, and in the surveillance systems themselves, as well as reflecting real differences in infection rates (Gauci and Ammon, 2007; van Pelt et al., 2003a). For example, much of the difference in incidence rates between the otherwise comparable countries Denmark and The Netherlands is explained by the fact that all Danish laboratories report Campylobacter diagnoses to the national level, compared with 51% of the Dutch laboratories (Anonymous, 2006a; van Pelt et al., 2003a). In France, reporting is based on the Campylobacter isolates received at the national reference center, which are usually collected from patients who have been diagnosed in hospitals (Mtgraud, 1999). In the Czech Republic, the reported incidence is severalfold higher than in other European countries (Gauci and Ammon, 2007). This may be due at least partly to frequent culturing of patients, to a policy of active case-finding among contacts of index cases, and to efficient reporting. In many industrialized countries, laboratory-based surveillance programs include Cam-

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pylobacter, but they vary in geographic coverage. Some report Campylobacter routinely from all jurisdictions, others are partial or sentinel systems, and for some, the degree of coverage is not reported. A number of European countries do not have routine culturing of diarrheal stool specimens or a national surveillance system for Campylobacter at all. Comparison of the fraction of cases that are captured by surveillance should help international comparisons, although these estimates are just beginning to be systematically developed (Flint et al., 2005). By use of a prospective sentinel clinic-based approach, in England and Wales, laboratory surveillance for Campylobacter has been estimated to capture 13% of all community Campylobacter cases compared with about 5% in The Netherlands (van Pelt et al., 2003a; Wheeler et al., 1999). Some of the difference between the United Kingdom and The Netherlands may be related to partial reporting in the latter country (see above). To be most useful, the multipliers should be estimated in the same way in each country. Differences in the way that partial reporting is accounted for may lead to different multipliers. Even modest variation in the case definition of gastroenteritis may halve or double the number of estimated cases of that condition (Majowicz et al., in press). Countries may also differ in the true incidence of infection and in the degree to which their populations are immune. At one extreme, populations in much of the developing world may be subject to such frequent and recurrent infections as young children that the infections in older children and adults are inapparent (Coker et al., 2002). In the future, serological surveys of populations may enable comparisons to be made. In The Netherlands, the rapid increase in seropositivity between ages 10 and 20 was used to estimate the number of infections that occurred in that age group. It was estimated that 30 to 50 infections may occur for each acute gastroenteritis illness diagnosed as Campylobacter (Nauta et al., 2005; van Pelt et al., 2005). In Denmark, a calculation for Salmonella enterica subsp. enterica serovar Enteritidis used antibody decay profiles after acute infection, combined with cross-sectional seroprevalence data, to estimate the true incidence of that infection (Simonsen et al., in press). In principle, the same can be done for Campylobacter (Strid et al., 2001). Because estimates based on serology capture asymptomatic infections as well as symptomatic ones, the multipliers estimated from seroprevalence data will be greater than the multipliers that are based on symptomatic disease alone. As the efficacy of serological methods for diagnosis of Campylobacter infection are further validated, including appropriate control sera, and as mathematical models advance,

the usefulness of seroepidemiology to study the incidence of Campylobacter infections will become clearer. Age and sex distribution The age distribution of infection in other industrialized nations is similar to that in the United States. Campylobacter affects all age groups, but in many countries, the infection has a bimodal age distribution, with one peak in small children and a second peak at between 15 and 44 years of age. By comparing available data from 14 European countries, the ECDC found the incidence in children under 5 years old to be 3 times higher than in other age groups and found a modest second peak in the 15-to-24 age group (Gauci and Ammon, 2007). A high incidence in small children was also noted for other enteric diseases, including Salmonella, which may be because parents are more likely to seek medical care for sick children and doctors will culture specimens taken from the children, as well as because a genuinely higher incidence in that age group actually exists. It has been suggested that in some countries, the peak in incidence in young adults may be related to their greater likelihood of foreign travel (Kapperud and Aasen, 1992; Stafford et al., 1996). The sex distribution of Campylobacter infection in developed countries in other parts of the world is also similar to the distribution in the United States. The incidence is 1.1to 1.5 times higher in boys and men compared with girls and women. This difference has been more pronounced in persons under 30 years old (Gauci and Ammon, 2007; Kapperud and Aasen, 1992; Skirrow, 1987). Seasonality

Many countries in temperate zones observe a sudden increase in Campylobacter infections in the spring, a well-defined summer peak, and a gradual decrease afterward (Kovats et al., 2005; Miller et al., 2004; Nylen et al., 2002; Skirrow, 1987). In England and Wales, the onset, size, and shape of the summer peak remained remarkably stable within regions over a 10-year period, although the size and shape of the summer peak differed across regions (Louis et al., 2005). When surveillance data from five countries in Europe were compared, seasonal variation became more pronounced with increasing latitude (Nylen et al., 2002). A study conducted in Norway found a similar south-to-north gradient within that country (Kapperud and Aasen, 1992). By comparing 11 European countries, Canada, Australia, and New Zealand, countries with milder winters have peaks of in-

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et al., 2004). In Wales, the seasonal peak in chicken at retail was observed to occur 2 weeks before the peak in human cases (Meldrum et al., 2005). Temperature may play a role in Campylobacter sources in the farm environment that are seasonal, such as insects, rodents, or migratory birds (Jacobs-Reitsma et al., 1994). The consistent sudden increase at the same time each year suggests the presence of a seasonal environmental stimulus. Transmission by flies has been suggested as one such driver of the seasonality of campylobacteriosis in both humans and in poultry, through seasonally dependent fly prevalence (Ekdahl et al., 2005; Hald et al., 2004; Nichols, 2005). Seasonal shifts in vacation travel, and swimming and other exposures may also play a role. In Minnesota, winter spikes in fluoroquinolone-resistant infections were related to a surge in winter holiday travel to Latin America, where fluoroquinolone resistance was more common (Smith et al., 1999). In The Netherlands, a sudden dilution of foodborne Cumpylobacter with those from environmental source may explain why during the sudden increase in Campylobacter infections in the spring, fluoroquinolone resistance drops by 15% and then gradually increases again after the summer peak (Fig. 7) (van Pelt et al., 2007).

fection earlier in the year (Kovats et al., 2005). In New Zealand, the summer peak is pronounced and stable, whereas in Queensland, Australia, the seasonal trend varies from year to year and incidence is only modestly higher during warmer months (Kovats et al., 2005; Stafford et al., 1996). Analysis of infections in Swedish travelers shows a consistent summer peak in travelers returning from temperate zones, and a more variable or absent peak in travelers to the tropics (Ekdahl and Anderson, 2004). Reports from Israel and Hong Kong suggest that those countries experience a higher number of infections in winter and spring (Ho and Wong, 1985; Shmilovitz et al., 1982). Climatic factors such as temperature, sunlight, and humidity may drive the dynamics of Campylobacter transmission by affecting prevalence at the reservoir, and by affecting human behavior that increases exposure to Campylobucter, such as barbecuing meat or swimming in surface waters. In broilers tested at farm, slaughter, and retail, higher carriage rates or levels of contamination are found in the summer (Meldrum et al., 2005; Patrick et al., 2004; van Pelt et al., 2003b; Wilson, 2003). In Denmark and The Netherlands, similar seasonal trends are seen in both the percentage of infected broiler flocks and human infections (Fig. 7) (Patrick et al., 2004; van Pelt et al., 2003b). In Denmark, the seasonality of human infections correlated best with average daytime temperature and sunlight 4 weeks before the date the infection was reported (and thus approximately 1 to 2 weeks before exposure), and for broilers a time lag of 3 weeks before slaughter gave the best fit (Patrick

1-4

5-8

9-12

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171

Burden of illness The burden of gastroenteritis caused by Campylobacter can be estimated by surveying the population to determine the frequency of recent gastro-

25-28

29-32

3336

37-40

41-44

45-48

49-52

4-weekly period (mean for 2002-2006)

Figure 7. Number of human infections with Cumpylobucter, proportion of isolates from humans resistant to ciprofloxacin, and prevalence of cecal carriage of Cumpylobucter in broiler flocks, by 4-week interval, The Netherlands, aggregated over the time period, 2002 to 2006 (van Pelt et al., 2007). Human surveillance coverage is approximately 50%.

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enteritis and constructing a surveillance pyramid, as is done in FoodNet. It can also be measured more directly by monitoring a population prospectively, attempting to identify and culture all episodes of diarrhea, and constructing a surveillance multiplier from those data, as was done in the Infectious Intestinal Disease study in England and the SENSOR study in The Netherlands (de Wit et al., 2001; Wheeler et al., 1999). In these studies, the annual incidence of symptomatic Campylobacter infection in the population was estimated to be 7.6 cases for every one reported to national surveillance in England, and 19 cases for every one reported in The Netherlands (van Pelt et al., 2003a; Wheeler et al., 1999). Estimating the full public health burden due to Carnpylobacter includes hospital admissions, chronic sequelae, and mortality (see chapter 8 for a full description). In Denmark, it has been possible to estimate the frequency of outcomes by linking national registries of gastrointestinal pathogen isolation, hospital admissions and discharges, and deaths (Helms et al., 2003, 2006). Thus, 11% of patients with Campylobacter infection were hospitalized around the time of the acute illness with gastroenteritis or complications, and 0.3% died within a month of the diagnosis (Helms et al., 2006). The mortality in that month was 5 times greater than that observed in a similar population without Campylobacter, and the mortality was still slightly high up to a year after diagnosis (Helms et al., 2003). In Sweden, mortality in the month after domestically acquired Campylobacter infection was 3 times higher than in the population in general but was far lower in travel-associated infections, probably because travelers have fewer underlying health conditions in general (Ternhag et al., 2005). Common-Source Outbreaks Common-source outbreaks caused by Campylobacter are recognized infrequently, particularly in comparison with the frequency of sporadic campylobacteriosis. Unlike Salmonella, Cumpylobacter does not tolerate exposure to atmospheric oxygen or to drying, and does not multiply on foods left out for many hours (Bayliss et al., 2000; Blaser et al., 1980). This could explain the rarity of large outbreaks related to solid foods (Franco, 1988). Unlike Salmonella, serotyping and molecular subtyping methods have not yet been identified that are useful for routine public health surveillance, and outbreaks that are small or dispersed may be missed by current surveillance (Ethelberg et al., 2004; Gillespie et al., 2003). The low infectious dose, ease of cross-contamination,

multiple subtypes contaminating a food product, and relatively long incubation period may also obscure the source of a cluster of related illnesses (Gillespie et al., 2003). Common-source outbreaks in the United States In the United States, the CDC receives reports of foodborne and waterborne disease outbreaks, including those caused by Campylobacter species, as part of the national food- and waterborne disease outbreak surveillance systems (Dziuban et al., 2006; Liang et al., 2006; Lynch et al., 2006). The first reported outbreak was also the largest. It occurred in 1977 in Burlington, Vermont, when a contaminated community water supply affected an estimated 3,000 persons (Vogt et al., 1982). Between 1998 and 2004, a total of 102 outbreaks of Campylobacter infections were reported, or 14 outbreaks per year, affecting a total of 4,587 persons (Table 1).They represented approximately 1% of all foodborne and waterborne outbreaks between 1998 and 2002. Of these, 74 were caused by C. jejuni, one was caused by C. coli, and for 27 the species was not reported. Seven of the 12 waterborne outbreaks included other enteric pathogens in addition to Campylobacter. This was not the case for foodborne outbreaks. The reported sources of outbreaks have changed over time. Between 1978 and 1987, water and unpasteurized milk accounted for 56% of outbreaks, while between 1988 and 1996, other foods ac-

Table 1. Food- and waterborne outbreaks of Campylobacter infections reported in the United States, by vehicle, 1998-2004 Method of transmission Foodborne

Waterborne

Total

Vehicle Beef Dairy Eggs Game Pork Poultry Vegetables Fruit Finfish Shellfish Complex foods No specific food implicated Community water supply Non-community water supply Recreational water

No. of outbreaks

No. of outbreakassociated cases

1 25 0 1 0 11 5 1 0 1 21 24

15 404 0 2 0 71 471 14 0 2 591 405

3

143

7

2,443

2

26

102

4,587

Next Page CHAPTER 9

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C. 7E7UNI INFECTIONS IN INDUSTRIALIZED NATIONS

counted for 83% of all outbreaks. From 1998 to 2004, 49% of all outbreaks were attributed to dairy products and drinking water, while 48% were attributed to other foods (44% of these were related to poultry). In that same time period, the two largest common-source foodborne Campylobacter outbreaks were a lettuce outbreak in 1998 in Minnesota (300 cases), and a tuna salad outbreak in a Washington state prison in 2002 (115 cases). Large waterborne outbreaks have occurred as a result of breaks in system integrity and disruptions in disinfection in municipal water systems, or when a nonchlorinated groundwater supply was contaminated by infiltration from the surface. In 2004, 1,450 people became ill with Campylobacter jejuni and other enteric pathogens after visiting a resort island in Lake Erie (O’Reilly et al., 2007). This outbreak occurred because of fecal contamination of the groundwater aquifer, probably through a combination of limestone sinkholes and poor sewage disposal practices (O’Reilly et al., 2007). Of 10 drinking water source outbreaks reported between 1998 and 2004, only one source (a community water source) was determined to have consistent disinfection. In 2006, the U.S. Environmental Protection Agency published new regulations that require disinfection of both ground and surface water before distributing it in community systems, which may decrease these outbreaks. Among foodborne outbreaks, milk has historically been the most common single food source, usually as raw or incompletely pasteurized milk. Milkor other dairy-associated outbreaks accounted for 38% of the foodborne outbreaks with reported food sources (Table 1).In the 1 9 8 0 ~ a ~common scenario for these outbreaks was a school field trip to a dairy farm where a taste of raw milk was part of the experience (CDC, 1986; Wood et al., 1992). In one outbreak with two different species of Campylobacter linked to raw milk, those with C. jejuni infections were older than those with C. fetus subsp fetus infections, although all consumed milk from the same bucket (Klein et al., 1986). Illness traced to the consumption of raw milk has been a continuing public health challenge, even when sanitary conditions on the farm are exemplary (CDC, 2002; Headrick et al., 1998). Milk can become contaminated by cattle feces or as a result of asymptomatic Campylobacter mastitis in dairy cattle (Hudson et al., 1984; Hutchinson et al., 1985; Sat0 et al., 2004). Raw goat milk has also been a source of an outbreak (Harris et al., 1987). Routine pasteurization of all milk and good dairy plant hygiene can prevent such outbreaks. A broad variety of other foods has been implicated in outbreaks. Campylobacter outbreaks have been associated with produce. Cantaloupe was re-

173

sponsible for an outbreak in Wisconsin in 1985 affecting 16 people and lettuce for an outbreak in Minnesota in 1998 affecting 300 people (Bowen et al., 2006; CDC, unpublished data). In an outbreak in Kansas in 1998, illness was related to eating either canned pineapple or gravy, which were likely to have been contaminated shortly before serving, perhaps by an ill food handler (Olsen et al., 2001). In one outbreak affecting at least 30 patrons of a restaurant, garlic butter used on sandwiches was implicated, perhaps after becoming contaminated in the kitchen (Berg et al., 1996). Campylobacter can survive in refrigerated butter for many days, and can survive the process of melting butter onto garlic bread (Zhao et al., 2000). Milk- and waterborne outbreaks are most likely to occur in the spring and fall but are uncommon in the summer, when sporadic cases are at their height. Foodborne outbreaks due to other vehicles increase in spring and continue steadily through December, without a summer peak. The difference between the seasonality of sporadic cases and that of outbreaks suggests that somewhat different ecological events drive them. In Wisconsin, the incidence of asymptomatic bovine carriage was higher in spring than in fall, possibly explaining the seasonal nature of milkassociated outbreaks (Sato et al., 2004). It is also possible that surface-water sources become contaminated during spring and fall bird migrations, leading to seasonal waterborne outbreaks. Common-source outbreaks in other developed nations Outbreaks reported from other developed nations are also less frequent than those caused by other bacterial foodborne pathogens; they are predominantly caused by contaminated poultry, drinking water, and milk. Starting in 2004, an annual report summarizing foodborne outbreaks in Europe has been produced by EFSA, the European Union Food Safety Agency. In 2005, reporting of foodborne outbreaks by European Union countries became mandatory, and 494 outbreaks of campylobacteriosis involving 2,478 individuals were reported (EFSA, 2006; Frost et al., 2002). The source of infection was reported for 107 outbreaks; 39 were caused by broiler or poultry meat, 5 by dairy products, 2 by drinking water, and the remaining by a variety of other foods. Although reporting from different countries varies in level of detail and 81% of outbreaks were reported by only two countries (Germany and Austria), this reporting system may be an increasingly important source of outbreak information in the future. In England and Wales in the second half of the

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OLSON ET AL.

1990s, 50 Campylobacter outbreaks were reported, representing only 2% of all reported outbreaks of diarrheal disease (Frost et al., 2002). Route of transmission was reported to be foodborne in 70%, waterborne in 8%, and undetermined in 18%. Among 24 foodborne outbreaks where a vehicle of infection was found, poultry was implicated in 14 outbreaks, substantially more often than in foodborne outbreaks due to other pathogens (Frost et al., 2002). In Denmark, three outbreaks due to Campylobacter were reported over a 3-year period, and all were transmitted in workplace kitchens and found to be caused by cross-contamination from raw chicken to ready-to-eat foods in the kitchen (Lewis et al., 2007; Mazick et al., 2006). Cross-contamination in the kitchen may explain a prolonged Australian outbreak traced to cucumber served at a salad bar (Kirk et al., 1997) and an outbreak in England in 1993 associated with a melon-and-prawn appetizer (Pebody et al., 1997). An outbreak in a school in Spain was likely caused by custard contaminated via chicken in the kitchen, and in an Australian restaurant-associated outbreak, chicken was also found to be the source (Black et al., 2006; Jimenez et al., 2005). Raw or inadequately pasteurized milk is often associated with outbreaks. Outbreaks caused by contaminated milk have been seen regularly in Sweden and in the United Kingdom (Pebody et al., 1997; SMI, 1997). Raw milk may frequently contain Campylobacter. In a survey of retail raw milk in southwest England, 5.9% of samples yielded the organism (Humphrey and Hart, 1988). Waterborne outbreaks are often much larger than foodborne outbreaks, affecting several hundreds or thousand of individuals (Anonymous, 2000; Engberg et al., 1998; Kuusi et al., 2005; Maurer and Sturchler, 2000; Melby et al., 1991; Mentzing, 1981). The outbreaks typically result from weaknesses in water supply systems combined with special circumstances, and sometimes involve several pathogens. In Canada, a large outbreak of E. coli 0157:H7 and Campylobacter infections occurred when groundwater supplying the drinking water system of a small town became contaminated after a heavy rainfall (Anonymous, 2000; Clark et al., 2003). In a Danish town, nonchlorinated municipal drinking water supplied from groundwater was contaminated with sewage, leading to a 6-week outbreak with an attack rate of 87.5% (Engberg et al., 1998). A similar outbreak occurred 10 years later in another Danish town (Vestergaard et al., 2007). In a Swiss town, contamination of the groundwater by the sewage system was believed to be the cause of a large outbreak (Maurer and Sturchler, 2000). In a municipality in Finland, water main repair work led to the contamination of drinking water with sewage, sick-

ening an estimated 2,700 persons (Kuusi et al., 2005). Outbreaks have also been traced to drinking water supplied from surface water. In a Norwegian outbreak in 1990, a small unfenced reservoir may have been contaminated by runoff from a nearby sheep pasture after heavy rain (Melby et al., 1990). Campylobacter species are rapidly inactivated by chlorine, and failure to disinfect is a frequent contributing cause. An outbreak in northern Norway took place during 4 weeks in which the chlorination system underwent repairs. Surface water was thought to have been contaminated by sheep cattle or wild animals (Melby et al., 2000). Similar episodes may have contaminated spring water in two outbreaks in Christchurch, New Zealand (Brieseman, 1987; CDC, 1991). During investigation of an outbreak in England in 1993, a dead lamb was found in the water supply of a university dormitory (Pebody et al., 1997). Drinking water supplied from springs, boreholes, or private wells may frequently be contaminated with microorganisms and pose a particular risk of outbreaks. Half of the 25 reported outbreaks associated with private water supplies in England and Wales from 1970 to 2000 were caused by Campylobacter (Said et al., 2003). Two non-drinking-waterrelated outbreaks among participants in an annual bicycle race have been described in Norway. The affected races took place on rainy days, and illness was associated with mud splashes and completing the route at the time of day when it rained most heavily; contamination of the roadway with runoff from adjacent pastures was the presumed mechanism (Kapperud et al., 2000). It appears that the proportion of investigated outbreaks associated with chicken has increased during the last 15 years relative to outbreaks associated with milk or water. The reported chicken outbreaks appear typically to be point-source outbreaks with a setting in a restaurant, cafeteria, or other institution. The data suggest that some Campylobacter outbreaks linked to other foods are the result of cross-contamination from poultry products in kitchens, so that the proportion of outbreaks caused by chicken is underestimated. Campylobacter outbreaks are rarely associated with processed ready-to-eat foods. Sources of Sporadic Infections The vast majority of Campylobacter infections are not linked to outbreaks but occur as sporadic infections. As noted above, the predominant sources of sporadic infections may differ from those of outbreaks. Although it is often difficult to determine the source of an individual infection, epidemiologic investigations that use a case-control methodology can

CHAPTER 9

C. 7E7uNl INFECTIONS IN INDUSTRIALIZED NATIONS

identify exposures that are most likely to be associated with C. jejuni infection, from which sources can be inferred. Ten such published studies have been done in the United States, including two large nationwide studies (Table 2) (Deming et al., 1987; Effler et al., 2001; Engleberg et al., 1984; Friedman et al., 2004; Fullerton et al., 2007; Harris et al., 1986b; Hopkins et al., 1984; Hopkins and Scott, 1983; Potter et al., 2003; Saeed et al., 1993; Schmid et al., 1987). Twenty four similar investigations have been conducted in other developed nations (Table 3) (Adak et al., 1995; Cameron et al., 2004; Doorduyn et al., 2005; Eberhart-Phillips et al., 1997; Ethelberg et al., 2005; Ikram et al., 1994; Kapperud et al., 1992, 2003; Kist, 1982; Lighton et al., 1991; Neal and Slack, 1995, 1997; Neimann et al., 2003; Norkrans and Svedhem, 1982; Oosterom et al., 1984; Rodrigues et al., 2001; Salfield and Pugh, 1987; Schonberg-Norio et al., 2004; Schorr et al., 1994; Southern et al., 1990; Stafford et al., 2007; Studahl and Anderson, 2000; Tenkate and Stafford, 2001; Wingstrand et al., 2006). Although these studies differ in technique and in the array of hypotheses tested, they consistently indicate several dominant routes of transmission: consumption of and contact with poultry, transmission from pets and other animals, and drinking contaminated water and raw milk. Campylobacter is also a major cause of traveler’s diarrhea, probably because similar exposures occur while traveling.

The case-control method as used for sporadic cases Case-control studies of infections collect exposure information from persons with the illness and compare it with similar data from comparable persons without illness. Long established as a technique for outbreak investigations, it has also been applied successfully to sporadic cases. Well-conducted casecontrol studies can offer data to estimate the population attributable risk (PAR), the fraction of cases that can be attributed to a particular exposure (Schlesselman, 1982). However, even in large casecontrol studies, the PARS often only explain a small portion of cases. For example, in New Zealand, where the incidence of campylobacteriosis is the highest in the world and poultry is the dominant source identified, the PAR for poultry just reaches 50% (Eberhart-Phillips et al., 1997). Calculated PARS may be diminished by selective recall, poor memory, unobserved cross-contamination of multiple foods, and prior population immunity. For example, immune persons who are exposed may develop undetected inapparent infection; including them as controls may mask obvious risk factors and lower the

175

estimated PARS (Nauta et al., 2005; Swift and Hunter, 2004). It may be more appropriate to view the associations as general indicators of risk, rather than as precise measures of the magnitude of the risk. Case-control studies in the United States Six early studies were conducted in the 1980s, soon after the importance of Campylobacter infections was recognized (Table 2). In 1981, a large casecontrol study conducted in Seattle, Washington, showed that eating poultry, including chicken, turkey, and Cornish game hen, accounted for over 50% of cases (Harris et al., 1986b). Smaller numbers of cases were attributed to drinking raw milk, including both cow’s and goat’s milk, consuming raw fish or shellfish, having contact with pets, and drinking contaminated surface water, and 9% were related to exposure during overseas travel (Harris et al., 1986b; Saeed et al., 1993). That same year, a study conducted among Hopi and Navajo Native Americans identified ownership of farm animals as a risk factor (Engleberg et al., 1984). Also in 1981, a small study in Colorado identified drinking untreated water or raw milk, having contact with cats, and eating undercooked chicken as risk factors (Hopkins et al., 1984). In a second Colorado study conducted in 1983, handling raw chicken, rather than just eating it, emerged as a risk factor (Hopkins and Scott, 1983). In 1984, in a study conducted among students at a Georgia university, 70% of cases were attributed to eating chicken and 30% to having contact with cats (Deming et al., 1987). In rural parts of the United States, raw milk can be an important source of sporadic cases as well as of outbreaks. In 1983, a study conducted in Iowa identified drinking raw milk as the only risk factor, and this accounted for nearly half the cases (Schmid et al., 1987). In the last decade, four additional case-control studies of sporadic cases have yielded similar results. In a 1998 study conducted in Hawaii, which has the highest incidence of Campylobacter infections in the United States, eating poultry prepared by a commercial establishment was the principal risk factor exposure, and taking antibiotics before onset of illness was also independently associated with illness (Effler et al., 2001). In 1998-1999, FoodNet conducted a large multisite study (Friedman et al., 2004). In this study, 12% of cases were attributed to foreign travel, whereas among the domestically acquired cases, 30% were attributed to chicken or turkey prepared in restaurants and 21% to other meats. Smaller fractions were attributed to drinking surface water (3%) or raw milk (1.5%), eating raw seafood (3%), having contact with a puppy (5%), and having contact with farm

Table 2. Case-control studies of laboratory-confirmed sporadic campylobacteriosis within the United States No. of cases

No. of controls

Yr

Population

Country

% Travel

10 40

10 71

1981 1981

Native Americans Denver and Fort Collins, CO

USA USA

NR“ NR

10

15

1982

Larimer County,

USA

NR

46 218

46 526

1982-1983 1982-1983

Iowa Washington state HMO members

USA USA

NR 9 yo

45

45

1983-1984

USA

0%

211

21 1

1998

University, Georgia Hawaii‘

USA

Exclb

1,316

1,316

1998-1999

7 FoodNet sites

USA

12%

83

122

2000-2001

Michigan rural population

USA

NS

123

928

2002-2004

8 FoodNet sites; O-ll-monthold infants

USA

5Yo

co

“NR, not reported. bExcl, travel-associated cases excluded from study. ‘NS, reported as not significantly associated with illness.

Risk factors identified related to consumption of food or water Untreated water, undercooked chicken, raw milk Chicken preparation Raw milk Undercooked chicken, other meat, raw milk, mushrooms, untreated water, raw seafood Undercooked chicken Chicken from restaurant; antibiotic use; slightly: ham, turkey Poultry prepared at restaurant, other meats prepared at a restaurant, raw milk, raw seafood, untreated water

Untreated well water, contact with meat or poultry in shopping cart, fruit and vegetables at home

Animal,environmental

contact Farm animals Cats

Reference(s) Engleberg et al. (1984) Hopkins et al. (1984)

Hopkins and Scott (1983)

Animals with diarrhea

Schmid et al. (1987) Harris et al. (1986a, 1986b), Saeed et al. (1993)

Cats

Deming et al. (1987)

Puppies, chickens

Effler et al. (2001)

Farm animals, animal stool, puppies

Friedman et al. (2004)

Poultry husbandry; dose-response for contact with poultry Visiting or living on farm, puppies

Potter et al. (2003)

Fullerton et al. (2007)

CHAPTER 9

c. r E r u w INFECTIONS IN INDUSTRIALIZED NATIONS

animals (4%). Nested within this study, the antimicrobial resistance of the isolates was measured, permitting determination of risk factors for fluoroquinolone-resistant infections: eating poultry prepared at a commercial establishment and traveling abroad (Kassenborg et al., 2004). In a 2000-2001 study conducted in rural Michigan, living on a farm and specifically having contact with poultry was associated with illness, and the risk rose with the frequency of poultry contact (Potter et al., 2003). In 2002-2004, FoodNet conducted an eight-site study focusing on infants younger than 1 year (see below) (Fullerton et al., 2007). Case-control studies in other developed nations Among 24 studies conducted in Europe, Australia and New Zealand, consumption of poultry was identified as a risk factor in 16 studies (Table 3). Nine studies identified other meats as a risk factor, most often pork or a variety of barbecued meats. Five studies identified drinking untreated water as a risk factor, and in two, swimming in natural surface waters was a risk. Drinking raw milk was identified in three studies, in Sweden, Denmark, and New Zealand. One study in the United Kingdom identified occupational contact with raw meat as a risk factor for infection, 13 identified contact with pets or farm animals as a risk factor, and 10 identified foreign travel. Pediatric infections Four studies focused on pediatric infections. Three non-U.S. studies all identified contact with dogs as a risk factor; one also identified untreated water, grilled meat, and raw milk; and one curiously identified mayonnaise as the risky exposure (Carrique-Mas et al., 2005; Salfield and Pugh, 1987; Tenkate and Stafford, 2001). The U.S. study found that breast-feeding was highly protective, and that foreign travel accounted for 5% of infant cases (Fullerton et al., 2007). In that study, drinking well water and riding in a shopping cart next to raw meat or poultry were risk factors for infants younger than 7 months, while being on a farm, having a pet with diarrhea, or eating fruits and vegetables prepared in the home were risk factors for infants aged 7 to 11 months. Sporadic infections and poultry The studies above shed some epidemiologic light on how poultry can cause infection. Location and circumstances of preparation often play a role. Some studies find that chicken prepared in the home is a

177

risk factor, but more often it is chicken eaten outside of the home; eating chicken in the home may be protective. Indeed, the large U.S. study found no risk for eating chicken in general, but both strong risk and protective effects were found when that exposure was evaluated by location of consumption (Friedman et al., 2004). It is unclear whether this might reflect differences in the contamination of chicken sold for restaurant use compared with that sold in grocery stores, or differences in variation in preparation practices between homes and restaurants. In Denmark, eating fresh, unfrozen chicken meat was the dominant risk factor, while the risk from other, previously frozen, chicken was only borderline significant (Wingstrand et al., 2006). Eating chicken that is perceived to be undercooked is often a risk factor, although one that accounts for a relatively small number of cases. Because incompletely cooked chicken would typically still be cooked on the surface, this risk factor suggests that there is contamination of deep muscle. Such contamination may be common, although at low counts. One survey found 22% of chicken legs had detectable Campylobacter in deep tissues next to the bone, and a survey of breast meat found internal contamination in 20% (Luber and Bartelt, 2007; Scherer et al., 2006). In both studies, levels of surface contamination were far greater than those of deep tissue contamination, leading the investigators to conclude that cross-contamination from the surface of the meat is the risk. Inapparent bacteremia was recently documented in 5 to 11% of broilers, suggesting a mechanism for deep tissue contamination (Richardson, 2007). In two studies, the risk was associated with handling raw chicken rather than with eating it (Hopkins and Scott, 1983; Norkrans and Svedhem, 1982). Ingestion of a drop of heavily contaminated raw chicken juice could provide a dose of 500 organisms. One study in the United Kingdom measured lo3 to l o 6 Campylobacter organisms on the surface per fresh chicken carcass, and two recent studies from Germany found a median of 250 CFUs per gram of skin on chicken legs and 2,000 CFUs per gram of chicken breast fillet (Hood et al., 1988; Luber and Bartelt, 2007; Scherer et al., 2006). This means that reducing the number of organisms on each carcass might have an important impact on public health even if there is little change in the proportion of carcasses that are contaminated. Other observations underline the importance of the poultry reservoir for human Campylobacter infections. In 1999, an inadvertent experiment occurred in Belgium when locally produced poultry was withdrawn from the market for several months

Table 3. Case-control studies of sporadic campylobacteriosis in industrialized countries outside the United States No. of cases

No. of controls

Yr

Population

Country

% Travel

Risk factors identified related to consumption of food or water

114 55

90 14

1979-1980 1980

Freiburg Gothenburg

West Germany Sweden

NR" u p to 75%

Chicken consumption Chicken preparation

44

54

1982

Rotterdam

The Netherlands

NS

Chicken, pork, food from barbecue

15 52

90 103

1984-1985 1999-2000

1 city, <6 yr 3 counties

England Norway

NR Excl"

32

64

1990

Bridgend

Wales

NR

29

41

1990

Manchester

England

NS

598

598

1990-1991

Nationwide

NR

167

282

1991

7 laboratories

England and Wales Switzerland

100

100

1992-1993

Christchurch

New Zealand

NR

282

318

1993-1994

England

Yes

313

512

1994-1995

Nottingham, >18 yr old Nottingham, >18 yr old

England

Up to 25%

621

621

1994-1995

4 areas

New Zealand

5%

229

229

1993-1996

Nationwide

9%

101

198

1995

1 county

England and Wales Sweden

Up to 46%

Excl

Poultry, sausage, crosscontamination Milk from bird-pecked bottles Milk from bird-pecked bottles Profession working with raw meat; untreated water Poultry liver, foreign citizenship Poultry outside home, undercooked or barbecued poultry Chicken, preparing raw chicken Antisecretory drugs, diabetes mellitus, bird-pecked milk, poultry Chicken pieces, raw or undercooked chicken, chicken eaten outside of home, raw milk, drinking untreated rainwater Chicken in restaurant Raw milk; chicken; pork; barbecue

Animal / environmental contact

Puppies, dogs

Puppies Dogs

Reference(s) Kist (1982) Norkrans and Svedhem (1982) Oosterom et al. (1985) Salfield and Pugh (1987) Kapperud et al. (1992) Southern et al. (1990) Lighton et al. (1991)

Pet with diarrhea

Adak et al. (1995) Schorr et al. (1994) Ikram et al. (1994)

Puppies

Neal and Slack (1995)

Puppies

Neal and Slack (1997)

Puppies, calf feces

Eberhart-Phillips et al. (1997)

Rodrigues et al. (2001) Poultry, live or work on farm

Studahl and Andersson (2000)

282

319

1996-1997

9 counties

Denmark

10%

81

144

1996-1997

Australia

NS

212

422

1999-2000

Brisbane, Queensland; children <3 yr old 3 counties

Norway

Excl

126

270

Children <6 yr old

Sweden

Excl

107

178

Nationwide

Denmark

15%

881

833

8 states 2 5 yr old

Australia

3yo

100

137

2001-2002, 12 mo 2000-2001, 11 mo 2001-2002, 12 mo 2002,3 mo

3 areas

Finland

Excl

1,292 C. jejuni

3,409

2002-2003, 12 mo

Nationwide

The Netherlands

16%

121 C. coli

3,409

2002-2003, 12 mo

Nationwide

The Netherlands

29%

318,958

1991-2001

Nationwide registry

Denmark

Excl

22,066

“NR, not reported. *NS, reported as not significantly associated with illness. Excl, travel-associated cases excluded from study.

Organ meats, undercooked poultry, red meat from barbecue, grapes, raw milk, pork Mayonnaise

Kittens, cows, pigs

Neimann et al. (2003)

Puppies, chickens as pets

Tenkate and Stafford (2001)

Untreated water, barbecues, fresh poultry, undercooked pork Untreated water, grilled meat, raw milk Chicken bought fresh; other poultry borderline Chicken and undercooked chicken, organ meats Undercooked meat or poultry, untreated dugwell water Chicken, undercooked meat or seafood, eating at a restaurant, antacid drugs, exposure to raw meat Game, tripe or organ meats, undercooked meat, food from street stall, antacid drugs Possible drinking water association

Occupational exposure to animals

Kapperud et al. (2003)

Dogs

Carrique-Mas et al. (2005) Wingstrand et al. (2006)

Chickens, puppies

Stafford et al. (2007)

Swimming in natural waters

Schonberg-Norio et al. (2004)

Cats or dogs, ill person outside of household

Doorduyn et al. (2005)

Swimming

Doorduyn et al. (2005)

Rural housing type

Ethelberg et al. (2005)

180

OLSON ET AL.

because of dioxin contamination (Vellinga and Van Loock, 2002). Campylobacter infections decreased by 30%, in parallel with the decrease in sales of local poultry. In 2003, when avian influenza appeared in The Netherlands, poultry production decreased and reported Campylobacter infections also decreased, so that about 14% fewer cases than expected were reported in the next 9 months (van Pelt et al., 2004). Much has been learned about the ecology and transmission of Campylobacter in flocks (Newel1 and Fearnley, 2003). The organisms can rapidly spread horizontally in chickens, presumably through a common water source or fecal contact (Shane, 1991). The observation that source and potability of the drinking water of poultry is associated with flock-to-flock variation in infection in Norway, the United Kingdom, and Iceland suggests that environmental interventions at the flock level may be a useful part of prevention (Guerin et al., 2007; Kapperud et al., 1993; Pearson et al., 1993). Recent trials used insect screens on chicken houses and found that it reduced flock prevalence by up to 70%, suggesting that excluding flies may be another simple and promising control strategy (Hald et al., 2007).

Seattle, FoodNet, Swedish, Norwegian, and New Zealand studies (Carrique-Mas et al., 2005; Eberhart-Phillips et al., 1997; Friedman et al., 2004; Harris et al., 1986a; Kapperud et al., 2003), or in areas where raw milk consumption is common, such as Dubuque, Iowa (Schmid et al., 1987). Other foods have also been implicated, including consumption of sausages at a barbecue, or undercooked pork in Norway (Kapperud et al., 1992, 2003). Consumption of barbecued red meat in Denmark, pork or meat from a barbecue in Sweden, game in The Netherlands, other meats in restaurants in the FoodNet study, or organ meats in Australia may represent direct transmission from those animal reservoirs, as well as possible cross-contamination in the kitchen from poultry (Doorduyn et al., 2005; Friedman et al., 2004; Neimann et al., 2003; Stafford et al., 2007; Studahl and Anderson, 2000). A survey of fresh mushrooms obtained from retail stores found 1.5% of the 200 samples to be contaminated with C. jejuni (Doyle and Schoeni, 1986).

Sporadic infections and cross-contamination

The contribution of drinking water to the burden of sporadic cases may be substantial in many parts of the developed and developing world, and small and unprotected rural water supplies may be a particular problem. Even pristine mountain streams can be a source of infection for unwary backpackers, presumably as a result of contamination by the feces of wild birds (Taylor et al., 1983). In Norway, 53% of cases had exposure to untreated drinking water, either from private wells or drinking river or stream water while hiking (Kapperud et al., 2003). In Denmark, drinking water from a private well that had a bad taste or smell was associated with infection, and in a large registry-based study, the risk varied by the residential address of patients in a way suggestive of, although not proving, an effect of the water supply (Ethelberg et al., 2005; Neimann et al., 2003). In New Zealand, drinking rainwater collected from a roof where birds might roost was associated with infection (Eberhart-Phillips et al., 1997). Routine disinfection of drinking water can prevent these infections at extremely low cost (Mintz et al., 2001). For example, in rural Bolivia, a controlled trial of chlorination of drinking water in the homes reduced overall diarrheal rates in intervention households by 44% and reduced infections with Campylobacter, the most commonly identified pathogen in the control group, by 80% (Quick et al., 1996). Two studies, from Finland and The Netherlands, identified swimming in surface waters as a risk factor, although in Norway, swimming was protective (Doorduyn et al.,

A study in Seattle in 1986 identified an association between infection and not washing the kitchen cutting board with soap, a marker for cross-contamination (Harris et al., 1986b). Quantification studies of cross-contamination in simulated kitchens confirm that it happens easily. In one study, 1 to 3% of the campylobacters present on the surface of poultry parts were transferred to hands or utensils, and from there to other foods with equal or greater efficiency (Luber et al., 2006). In another, 29% of cooking sessions that started with contaminated chicken resulted in documented contamination of other foods or food contact surfaces (Redmond et al., 2004). Campylobacter has been shown to survive on the cut surfaces of inoculated fruit for up to 6 hours at room temperature (Castillo and Escartin, 1994). Contamination may also occur from the outside of a package before it reaches the kitchen, as indicated by the finding that riding in shopping carts with raw meat and poultry is a risk factor for infants (Fullerton et al., 2007). In a survey in the United Kingdom, approximately 3% of retail poultry packages were contaminated on the outside with Campylobacter, as were 24% of packages in New Zealand (Burgess et al., 2005; Wong et al., 2004). Sporadic infections and other foods Raw milk has been implicated in the largest studies, where there is sufficient power, such as the

Sporadic infections and water

CHAPTER 9

C. TETUNI INFECTIONS IN INDUSTRIALIZED NATIONS

2005; Kapperud et al., 2003; Schonberg-Norio et al., 2004). If infection spreads in poultry through drinking water, and if cattle are infected in the spring by drinking contaminated surface water, then the waterborne route may be a common underlying pathway linking waterborne, milkborne and poultry-associated campylobacteriosis in humans to an underlying annual cycle involving water and environmental contamination (Fig. 8).

nation routes to humans have been difficult to document. In the western United Kingdom, three studies have associated illness with drinking pasteurized milk from bottles that were pecked by jackdaws (Lighton et al., 1991; hordan et al., 1993; Southern et al., 1990). The birds learned to pierce the foil tops of bottles delivered to the front door and may have contaminated the milk directly. Secondary transmission

Sporadic infections and contact with animals Several studies identified contact with farm animals, especially poultry but also cattle and pigs, to be a risk factor for Campylobacter infection (Engleberg et al., 1984; Friedman et al., 2004; Neimann et al., 2003; Potter et al., 2003; Schonberg-Norio et al., 2004; Stafford et al., 2007). In rural Michigan, risk increased with the frequency of contact with chickens (Potter et al., 2003). Contact with pets has been demonstrated repeatedly as a risk factor. In Norway, fecal culture yielded Campylobacter jejuni in 3% of cats and 3% of dogs, and the isolation rate was higher in puppies than in adult dogs (Sandberg et al., 2002). The nature of the contact with an infected dog or cat that actually transmits the illness remains to be defined, as does the reason that pets become infected themselves. Anecdotally, contact can be quite casual, the animal need not be sick, and there need be no direct contact with animal feces. In addition to established reservoirs in poultry and other domesticated animals, wild birds often carry Campylobacter, particularly crows, gulls, and shoreline foraging birds (It0 et al., 1988; Kapperud and Rosef, 1983; Waldenstroem et al., 2002). Wild birds may contaminate streams and lakes, from which people may drink, as well as the water collected from rooftops in New Zealand, although direct contami-

Despite the low infectious dose and the abundance of organisms in feces of ill persons, secondary transmission from one human to another is unusual, and sustained transmission among humans has not been documented. In the large U.S. case-control study, preceding diarrheal illness in a household member was not significantly associated with illness (Friedman et al., 2004). In general, outbreaks in settings such as day care centers or mental institutions are rare or unheard of, although person-to-person transmission of Shigella, E. coli 0157, Giardia, hepatitis A virus, and other low-dose pathogens has been well documented in these settings. In Australia, attending a child care center and contact with diapered children were protective (Tenkate and Stafford, 2001). Infection rates in homosexual men have been reported to be high but do not appear to be higher than infection rates in heterosexual men of the same age, and transmission through sexual contact has not been conclusively demonstrated (Quinn et al., 1984). Contamination of food by an infected food handler has apparently occurred but is surprising by its rarity (Olsen et al., 2001). It would appear that the infectiousness of the organism for humans diminishes on passage through the human gut, in contrast to passage through bird or cow.

Poultry meat

(also turkeys)

Chicken Chicken

Human Human

Raw milk

v=Wild birds

d

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3

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Figure 8. Schema of transmission pathways for Campylobacterjejuni, 2007.

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Sporadic infections and foreign travel Many countries identify travel-associated cases as a routine part of surveillance. Antecedent foreign travel has been reported in 10% of cases in Britain, 50% of cases in Norway, and 65% of cases in Sweden (Cowden, 1992; Kapperud and Aasen, 1992; Norkrans and Svedhem, 1982). In Swiss and Danish studies, ill travelers had more frequently been to the developing world and Southern Europe (Neimann et al., 2003; Schorr et al., 1994). In a study of Swedish travelers, the risk of travel-associated campylobacteriosis varied dramatically by travel destination, from 3 of 100,000 travelers to other Nordic destinations to 1.2% of travelers to the Indian subcontinent (Ekdahl and Andersson, 2004). Identifying travel as a risk factor does not identify the actual sources of infection. Travel in other countries is a complex exposure, including consumption of foods that may be unusual and water of unknown quality. A casecontrol study comparing the specific exposures of those who are ill with other travelers to similar destinations who remain well remains to be done. Infections with Campylobacter coli Most studies did not attempt to distinguish Campylobacter coli from C. jejuni and did not identify risk factors specific to the latter. However, in the United Kingdom, a case-case comparison suggested that there may be epidemiologic differences between the two closely related species, and in particular that drinking bottled water and eating piit6 were more common among those with C. coli infection than among those with C. jejuni infection (Gillespie et al., 2002). In The Netherlands, a case-control study of C. coli infections found that 29% were related to travel, nearly twice the frequency as C. jejuni, and linked C. coli infections specifically to eating game, organ meats, tripe, undercooked meat, or food from street stalls (Doorduyn et al., 2005). Little is known about the sources of sporadic infections with other Campylobacter species. Protective factors Many case-control studies have identified exposures associated with reduced risk of Campylobacter infection (Adak et al., 1995; Carrique-Mas et al., 2005; Doorduyn et al., 2005; Eberhart-Phillips et al., 1997; Effler et al., 2001; Friedman et al., 2004; Ikram et al., 1994; Kapperud et al., 2003; Neimann et al., 2003; Rodrigues et al., 2001; Stafford et al., 2007; Tenkate and Stafford, 2001; Wingstrand et al., 2006). Apparently protective factors may have been

identified in other studies as well, but not included in the publication. Factors associated with reduced risk include good kitchen hygiene and breast-feeding that are likely to reflect a true lower risk of infection. Other factors, such as consumption of certain food items (particularly produce, but also poultry, red meat, dairy products, nuts, chocolate, and fish), contact with animals, and swimming are more difficult to interpret. Some recurrent risky exposures may induce protective immunity (Adak et al., 1995; Carrique-Mas et al., 2005; Friedman et al., 2004; Neimann et al., 2003). This might explain why handling raw chicken and being in a profession with frequent animal contact were protective factors in a study in the United Kingdom (Adak et al., 1995). Repeated exposure to one source, such as poultry, might induce enough protective immunity to introduce a misclassification bias, because the exposure of some controls will not be likely to result in illness, which could lower the odds ratio even to below 1 (Swift and Hunter, 2004). Protective immunity does not provide an obvious explanation of why in the same case-control study, chicken appears to be protective if prepared at home and a risk factor if prepared in a restaurant, as demonstrated in several large studies (Adak et al., 1995; Eberhart-Phillips et al., 1997; Effler et al., 2001; Friedman et al., 2004; Ikram et al., 1994). Differential recall bias might play a role, if exposure in one location is more memorable than a similar exposure in another location. It is also possible that there are real protective effects associated with some foods, particularly produce. For example, in a recent large study in Australia, risk decreased as the number of types of uncooked vegetables consumed increased, independent of chicken consumption (Stafford et al., 2007). In New Zealand, the protective effect of salad increased with the frequency of consumption (Eberhart-Phillips et al., 1997). Some have speculated that high levels of plant antioxidants and carotenoids could boost general immunity to infection or inhibit bacterial growth (Kapperud et al., 2003; Neimann et al., 2003). Specific diets may change the intestinal microflora and alter the host susceptibility to infection (Cameron et al., 2004; Rodrigues et al., 2001) or reduce the severity of diarrhea, as has been shown for vitamin A in young children in developing countries (Barreto et al., 1994). Surveillance for Emerging Antimicrobial Resistance Although most infections resolve without specific treatment, antimicrobial treatment can be critical in invasive or severe infections. Fluoroquinolone agents like ciprofloxacin are commonly used for the

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treatment of infections caused by Campylobacter. Antimicrobial resistance has been monitored around the world in several ways, and the frequency of resistance to various agents varies widely by location. In Australia, fluoroquinolone resistance in domestically acquired Campylobacter infections in humans has been largely absent, reported to be 2% of strains in 2002; it has been reported at substantially higher levels from Europe and Asia since the end of the 1980s (Gaunt and Piddock, 1996; Hoge et al., 1998; Piddock, 1995; Unicomb et al., 2003, 2006). In 19891990 in the United States, no C. jejuni or coli strains collected from 19 sentinel counties were resistant to fluoroquinolones (CDC, unpublished data). By 1997, when the National Antimicrobial Resistance Monitoring System for enteric bacteria first began monitoring Campylobacter in humans, 86% of tested strains were resistant to at least one antimicrobial, 24% were resistant to nalidixic acid, and 13% were resistant to ciprofloxacin. The frequency of resistance among C. jejuni strains increased to a peak of 20.7% in 2002 (CDC, 2007) (Fig. 9). In 1997, a review of the fluoroquinolone-resistant cases indicated that foreign travel was an important risk factor, but that most of the fluoroquinolone-resistant infections were acquired in the United States (Friedman et al., 1998). In a case-control study of fluoroquinolone-resistant infections conducted in 1998-1999, nested inside the larger study of all C. jejuni and C. coli infections, 42% of persons with fluoroquinolone-resistant infections reported preceding foreign travel, but only 9% of those with susceptible infections did (Kassenborg et al., 2004). Among the non-travel-associated cases, consumption of poultry in restaurants was the only identified risk factor. The resistance is associated with clinical impact: illness with fluoroquinolone-resistant strains lasted longer than did illnesses with suscepti-

10 Tetracycline

Erythromycin

I

Ciprofloxacin

60

89-90

97

98

99

00 01 02 03 04

Figure 9. Antimicrobial resistance in C. jejuni isolates, United States, 1997 to 2004 (CDC, 2007).

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ble strains in two separate studies, although whether this was due to treatment failure or to a greater virulence of resistant strains or both remains uncertain (Neimann et al., 2001; Nelson et al., 2004). Reports from Europe suggested that the increase in fluoroquinolone-resistant Campylobacter isolations in humans was associated with fluoroquinolone use in poultry that has led to fluoroquinolone-resistant Campylobacter strains in poultry, which have then spread to humans via the food chain (Gaunt and Piddock, 1996; Piddock, 1995). Veterinary use of fluoroquinolones in poultry began elsewhere before 1995, when it was approved in the United States; it has never been permitted in Australia (Unicomb et al., 2003). A 1997 study conducted in Minnesota showed that 20% of 60 C. jejuni isolates obtained from raw chicken bought at grocery stores were ciprofloxacin resistant (Smith et al., 1999). In 2000, the U.S. Food and Drug Administration proposed withdrawing approval for fluoroquinolone use in poultry, and in 2005, this rule was adopted (Nelson et al., 2007). Judicious use of fluoroquinolones and other antimicrobial agents in human and veterinary medicine is essential to preserve the efficacy of these important chemotherapeutic agents.

SUMMARY Campylobacter is the most common bacterial enteric pathogen in many developed countries and is the second most common such pathogen in the United States. Although infection is not usually invasive, it is occasionally fatal. In the last 30 years, Campylobacter rates have risen markedly in many developed countries and have begun to decrease in some. Part of the increase may be due to improvements in detection and reporting, but it also reflects a true increase in infections. The burden of illness of Campylobacter is substantial, and infection may be universal. The vast majority of Campylobacter infections are sporadic individual infections and often affect young adults and young children. Consumption of untreated water, raw milk, or milk products and contact with pets are important sources of infection. However, the most frequently identified source of sporadic disease is consumption of raw or undercooked poultry. Outbreaks of Campylobacter in developed countries are predominantly caused by contaminated water, milk, poultry, and produce. Control of this infection thus depends on a multipronged prevention strategy, including public education to lower the risk of infection and cross-contamination in the kitchen, better slaughter hygiene to reduce the level of carcass contamination, and control measures on

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farms to reduce infection of live animals. Routine treatment of drinking water and pasteurization of milk are core prevention strategies. Veterinary use of fluoroquinolones in poultry explains the recent rise in ciprofloxacin-resistant Campylobacter infections in humans in the United States and may explain much of the geographic variation in resistance around the world. Acknowledgments. We gratefully acknowledge the assistance of Tracy Ayers and Cherie Long. The findings and conclusions in this publication are those of the authors and do not necessarily represent the views of the CDC or other institutions.

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of severe gastroenteritis with multiple aetiologies caused by contaminated drinking water in Denmark, January 2007. Eurosurveillance 12:E070329 1. Voetsch, A., T. Van Gilder, F. Angulo, M. Farley, S. Shallow, R. Marcus, R Cieslak, V. C. Deneen, and R. Tauxe. 2004. FoodNet estimate of the burden of illness caused by nontyphoidal Salmonella infections in the United States. Clin. Infect. Dis. 38:S127-S134. Vogt, R. L., H. E. Sours, T. Barrett, R. A. Feldman, R. J. Dickinson, and L. Witherell. 1982. Campylobacter enteritis associated with contaminated water. Ann. Intern. Med. 96~292-296. Waldenstroem, J., T. Broman, I. Carlsson, D. Hasselquist, R. Achterberg, J. Wagenaar, and B. Olsen. 2002. Prevalence of Campylobacter jejuni, Campylobacter lari, and Campylobacter coli in different ecological guilds and taxa of migrating birds. Appl. Environ. Microbiol. 685911-5917. Wheeler, J., D. Sethi, J. Cowden, P. Wall, L. Rodrigues, D. Tompkins, M. Hudson, and P. Roderick. 1999. Study of infectious intestinal diseases in England: rates in the community, presenting to general practice, and reported to national surveillance. Br. Med. I. 318:1046-1050. World Health Organization. 2001. The Increasing Incidence of Human Campylobacteriosis: Report and Proceedings of a W H O Consultation of Experts. WHO/CDS/CSR/APH 2001.7. World Health Organization, Copenhagen. Wilson, I. 2003. Salmonella and Campylobacter contamination of raw retail chicken from different producers: a six-year survey. Epidemiol. Infect. 130:320-323. Wingstrand, A., J. Neimann, J. Engberg, E. Nielsen, P. GernerSmidt, H. Wegener, and K. Mslbak. 2006. Fresh chicken as main risk factor for campylobacteriosis,Denmark. Emerg. Infect. Dis. 12:280-285. Wong, T., R. Whyte, A. Cornelius, and J. Hudson. 2004. Enumeration of Campylobacter and Salmonella on chicken packs. Br. Food J. 106:651-662. Wood, R., K. MacDonald, and M. Osterholm. 1992. Campylobacter enteritis outbreaks associated with drinking raw milk during youth activities. A 10-year review of outbreaks in the United States. JAMA 268:3228-3230. Yang, S., M. Leff, D. McTague, K. Horvath, T. JacksonThompson, T. Murayi, G. Boeslager, T. Melnik, M. Gildermaster, D. Ridings, s. Altekruse, and F. Angulo. 1998. Multistate surveillance for food handling, preparation and consumption behaviors associated with foodborne diseases: 1995 and 1996 BRFSS food safety questions. Morb. Mortal. Wkly. Rep. CDC Surveill. Summ. 47(4):33-57. Zhao, T., M. Doyle, and D. Berg. 2000. Fate of Campylobacter jejuni in butter. J. Food Prot. 63:120-122.

Campylobacter, 3rd ed. Edited by I. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

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Molecular Epidemiology of Campylobacter Species STEPHEN L. W. ON, NOELMCCARTHY, WILLIAMG. MILLER,AND BRENT J. GILPIN

demonstrated that genetic change in at least C. jejuni and C. coli is driven by recombination events that result in a weakly clonal population structure (Dingle et al., 2001a, 2005). This, alongside other evolutionary drivers including spontaneous mutation and plasmid acquisition and loss (Wassenaar et al., 2000), can make the definitive linkage between any two strains a difficult task because genotyping results of related strains may differ. Simply put, there are many human cases and many possible sources of infection from a genetically diverse organism. It is little wonder that the study of the epidemiology of Campylobacter is complex and challenging. The need for subtyping methods to study Campylobacter epidemiology became evident after the seminal studies of Butzler et al. (1973) and Skirrow (1977) first demonstrated their prevalence and significance. Some of the early phenotypic methods (biotyping, serotyping, phage typing) are still used today. However, although such methods are suited for large-scale screening, their limited discriminatory potential has proved insufficiently informative for resolving any but the most clear-cut issues. Moreover, although serotyping (notably the Penner scheme based predominantly on heat-stable antigens) had for many years been the main tool used to analyze Campylobacter, many laboratories no longer have either the expertise or the antisera to perform serotyping. Other laboratories (based in New Zealand and The Netherlands, for example) only have antisera to determine a limited range of serotypes. Since the 1980s, the increased interest in exploiting molecular methods to determine genetic polymorphisms between bacteria has driven developments in identification and subtyping of Campylobacter spp. with the aim of

There are three major obstacles to the tracing of sources of human campylobacteriosis: the scale of the problem, the possible route or routes of infection, and the population biology of the major pathogenic Campylobacter species. In developed countries with surveillance systems, Campylobacter is known to be the most frequently reported bacterial cause of human gastroenteritis, usually accounting for more cases of disease than Salmonella, Yersinia, and pathogenic Escherichia coli combined. With an incidence of up to 432 cases per 100,000 population (Institute of Environmental Science and Research, Ltd., 2007), subtyping strains to identify sources of infection demands a major effort. Moreover, the potential sources of infection are many. Campylobacter species are widespread in nature and may be found in an extensive range of terrestrial animals where they form part of their normal intestinal microflora. Chickens, turkeys, ducks, geese, ostriches, sheep, cattle, deer, goats, pigs, cats, dogs, rodents, wild birds, and even reptiles harbor Campylobacter species-sometimes different species andl or strains concurrently. Rodents as well as insects such as flies and beetles may also serve as unwitting couriers of the organism (Adhikari et al., 2004; Bates et al., 2004; Hald et al., 2004a), transferring strains to hitherto uncontaminated areas. Consumption of, or contact (direct or indirect, through exposure to aqua- or agricultural environments or contaminated surfaces) with the animal host can result in transmission of the organism to humans and subsequent infection. In addition, the genetic diversity of many campylobacters adds to the dilemma of making an informed decision as to the relatedness of any two isolates. Advances in population biology have

Stephen L. W. On Food Safety Programme, Institute of Environmental Science and Research Ltd., Christchurch, New ZeaNoel McCarthy Peter Medawar Building for Pathogen Research and Department of Zoology, Health Protection Agency land. William G. Miller Produce Safety and Microbiology Research and University of Oxford, Oxford OX1 3SY, United Kingdom. Unit, United States Department of Agriculture, Albany, CA 94710. Brent J. Gilpin Water Management Programme, Institute of Environmental Science and Research Ltd., Christchurch, New Zealand.

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improving our understanding of their epidemiology. Interestingly, although genetic methods almost invariably prove more discriminatory than phenotypic tools, the population genetics of the bacteria (which differ markedly between different Campylobacter species) that drive their evolution often mean that typing results prove as challenging to interpret as the broad picture of the epidemiology of the organisms they aim to elucidate. This chapter contains a summary of the various modern genotypic methods available for subtyping the various Campylobacter species, the use of genetic methods for investigating outbreaks of disease, the application and interpretation of large-scale techniques in broader epidemiological studies, and a perspective of future developments in the field. MOLECULAR TYPING METHODSA BRIEF OVERVIEW Our overview of genotyping methods is divided into two broad sections: the first describes methods that have principally been applied to comparatively small-scale studies, limited to investigations of relatively few strains and/or restricted geographical or chronological origin. Discussions of three methods (pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and amplified fragmentlength polymorphism [AFLP]) used to compare relatively large numbers of strains follow in a section aimed at summarizing our knowledge of Campylobacter molecular epidemiology in a broader perspective.

fla Typing Campylobacter coli and C. jejuni subsp. jejuni (hereafter C. jejuni) have two flagellin genes, designated flaA and flaB. Each gene is approximately 1.7 kb and is separated by an intergenic spacer region of -170 bp. These genes encode repeats of flagellin subunits that form the flagellum found on one or both ends of Campylobacter cells. The 5’ and 3’ regions of each gene are highly conserved, with considerable sequence variation in the region in between. A number of PCR primers have been designed that amplify specific regions of this gene cluster. Variability within each amplicon is identified by either digestion followed by restriction fragment length polymorphism (fa-RFLP) or direct sequencing (fa-amplified short variable regions [fla-SVR]). fla-RFLP Nachamkin et al. (1996) described primers that amplify 1,728 bp of the flagellin A gene @aA) and

flanking sequences. A number of restriction enzymes including AluI, DdeI, EcoRI, HinfI, and PstI have been used either alone or in combination to identify variability in fZa sequences. Digestion of the amplified fragment with DdeI is favored by most, with fragments between 100 and 1,500 bp commonly analyzed. fZaA-RFLP is relatively simple to perform, fairly inexpensive, and well suited to relatively highthroughput analysis. However, standardization of electrophoresis conditions remains an issue, although groups in both Europe and Australia (Djordjevic et al., 2007) have shown that high levels of interlaboratory standardization are possible. Moreover, because polymorphisms within a single genetic locus are being determined, its discriminatory power is inherently limited, and certainly less so than methods determining whole-genome polymorphisms. For example, in a survey of C. jejuni isolates from 100 different poultry samples, 73 different SmaI PFGE types were observed, but only 30 fZaA types (Han et al., 2007). In 179 C. jejuni strains from cattle in Turkey, just 23 different fZaA-RFLPtypes were observed, with 5 fZaAtypes in 30 C. coli strains (Acik and Cetinkaya, 2005). Djordjevic et al. (2007) reported that fZaARFLP was less discriminatory than MLST but could be used to predict MLST clonal complexes in many cases. However, fZaA-RFLP was the method best associated with epidemiology in the Walkerton outbreak in 2000 (Clark et al., 2005), supporting its value in outbreak investigations where rapid results, high throughput, and low cost are important factors for consideration of applicable typing methods. fla-SVR Sequencing Direct sequencing of PCR-amplified short variable regions (SVR) of either the flaA or flaB genes overcomes many of the reproducibility problems which flaA-RFLP can have. An online database has been established (http://hercules.medawar.ox.ac.uk/ flaA/), which as of July 2007 contained 965 fla SVR alleles 321 bp in length. Novel sequences not in the database can have an allele number assigned after submission and validation of DNA trace files. However, the diversity of fZaA-SVR types is less than that observed with fZaA-RFLP types. For example, of 66 Irish human and poultry isolates, 58 different banding profiles with fZaA PCR-RFLP were observed, but just 28 different fZaA-SVR types (Corcoran et al., 2006). Similarly, 20 fZuA-RFLP types were found among 84 human diarrheal strains, but only 15 fZaASVR types (O’Reilly et al., 2006). In contrast, Mellmann et al. (2004) found fZaB-SVR to be slightly less discriminatory than flaA-SVR, but better correlated with outbreak strains.

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Genomic instability of C. jejuni, and in particular the flaA gene, has been described by a number of researchers (Harrington et al., 1997). Although this could potentially affect the reliability of flaA typing, in practice, this appears not to be the case. High levels of genomic instability may be restricted to particular strains or situations. Instability of fla genes does not appear an impediment to typing methods that are based on these genes. fla typing methods can be used as a first screen to identify isolates for further investigation by other typing methods. Even though several researchers have demonstrated by means of fla typing methods that outbreak strains can be distinguished from sporadic strains, this may not hold true if a large number of sporadic strains are examined within the same time frame.

genotypes to flaA typing, with some studies finding ribotyping to be more discriminatory (O’Reilly et al., 2006) and others finding it to be less discriminatory (de Boer et al., 2000). The RiboPrinter system requires a considerable capital outlay, and according to some (O’Reilly et al., 2006), the cost of testing each isolate is twice as expensive as SmaI PFGE and six times more expensive than flaA-RFLP. In combination with other subtyping methods, automated ribotyping will produce valid and reproducible results. However, the low level of diversity and relatively high costs of automated ribotyping do restrict its usefulness for the study of Campylobacter species.

Ribotyping and Riboprinting

Random amplification of polymorphic DNA (RAPD) has been applied to the analysis of campylobacters for at least the last 10 years. DNA is isolated from purified isolates and PCR reactions performed by using a single 10- to 15-bp primer. This primer anneals to multiple sites in the genome, which, when close enough together, will generate amplicons whose size depends on the position of the annealing sites. These amplicons can be visualized by standard electrophoresis on 1.5% (wt/vol) agarose gels (Hernandez et al., 1995). By means of this approach, Acik and Cetinkaya (2006) identified from 348 cattle and sheep C. jejuni and C. coli isolates a total of 87 different RAPD genotypes, each composed of two to eight fragments. Nielsen et al. (2000) performed RAPD analysis with three different fluorescently labeled primers. The resultant PCR products were then mixed and amplicons separated by using the fragment sizing capacity of a DNA sequencer. In an analysis of 80 unrelated strains, 56 genotypes were identified by RAPD analysis-6 more than were identified by SmaI PFGE. RAPD analysis is relatively inexpensive and has the potential to identify a high level of diversity among isolates, but its use has not become widespread because of the justified concern of many researchers that the diversity of patterns may in part arise from variation in the assay rather than the organism. To address this concern, many researchers use some form of standardized, premixed “Ready to Go” RAPD analysis beads to provide a consistent and uniform source of Taq polymerase, deoxynucleoside triphosphates, and buffer conditions. The fact that RAPD analysis requires no prior knowledge of any genome targets makes it particularly applicable to the analysis of the less frequently studied Campylobacter species. There have been few published examples demonstrating the value of RAPD analysis in out-

Ribotyping involves cleaving of whole genomic DNA with a cutting restriction enzyme (e.g., HindIII), subsequent hybridization of a labeled oligonucleotide probe derived from the ribosomal gene to fragments containing the gene, and visualization of the resulting labeled patterns. This technique is technically demanding and difficult to standardize, but a semiautomated system is available that overcomes these difficulties, permitting its wider use. With the Dupont Qualicon RiboPrinter microbial characterization system, bacterial suspensions from isolated bacteria are added to the RiboPrinter, which undertakes in an automated manner lysis of cells, DNA isolation, restriction enzyme digestion, electrophoresis, Southern blot analysis, hybridization with a chemiluminescently labeled 16s to 23s rRNA primer, detection of patterns, and finally comparison with previously analyzed isolates. The automation facilitates a high level of reproducibility with relatively low levels of technical input. Up to eight bacterial isolates can be tested at one time, with results available 8 h from sample input. New batches of isolates can be added every 2 to 3 h. The RiboPrinter has been applied to analysis of Campylobacter strains, most commonly by using the enzyme PstI, which does not cut within the Campylobacter 16s rRNA gene. Most Campylobacter strains have only three copies of the ribosomal genes, which results in the generation of only three to six bands by using PstI. Ribotyping is less discriminatory than PFGE with SmaI. For example, of 120 isolates from meats, 44 SmaI PFGE patterns were found, but only 22 ribotypes (Ge et al., 2006). Of 84 human isolates, O’Reilly et al. (2006) found 53 subtypes by PFGE with SmaI, 33 by MLST, and only 28 by ribotyping. Ribotyping appears to generate a similar number of

Random Amplification of Polymorphic DNA Analysis

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break investigations (Nielsen et al., 2000; Ono et al., 2003). In an outbreak situation, indistinguishable RAPD profiles should be generated, but interpreting the significance of any differences in patterns may be difficult. In common with PFGE and other bandbased methods, inferring phylogenetic relationships on the basis of similarity of RAPD patterns is a potentially misleading exercise, particularly because the risk of obtaining different profiles for the same strain upon reanalysis is comparatively high. RAPD analysis appears to have largely been supplanted by other methodologies by most researchers. Rep-PCR and ERIC-PCR Repetitive extragenic palindromic-PCR (RepPCR) targets conserved repetitive DNA sequences. Rep-PCR has been applied widely to a number of organisms, but there is only a single report of its application to campylobacters (Hiett et al., 2006). In a survey of 48 C. jejuni isolates, Rep-PCR performed using the Uprime DT primer set generated four to eight bands in the 400- to 5,000-bp range. Rep-PCR was less discriminating than flaA-SVR sequencing, with a diversity index of 0.8364 for Rep-PCR compared with 0.9394 for flaA-SVR (Hiett et al., 2006). Rep-PCR does not appear to offer significant advantages over the other, more widely used methods for genotyping campylobacters. Similar to Rep-motifs, enterobacterial repetitive intergenic consensus (ERIC) sequences may also be targeted by PCR assays and used for subtyping. Its application to 107 strains of diverse origin was used to support other genotypic data, suggesting a dichotomy of infectious potential (Moser et al., 2002). In a study of Campylobacter epidemiology in pigs, ERICPCR patterns suggested that transmission could be maternal, although because some patterns resembled those from distinct farms, this hypothesis was not unequivocal (Weijtens et al., 1997). Other researchers have elected to use ERIC-PCR primers with random primers, whereby outbreak strains yield identical or similar patterns that are distinguished from sporadic isolates, which yield unique profiles (Iriarte et al., 1996). Nonetheless, neither Rep- nor ERIC-PCR fingerprinting has proven especially popular with researchers, most likely because of the same pitfalls that are encountered with RAPD fingerprinting. High-Resolution Melt Analysis and Denaturing Gradient Gel Electrophoresis High-resolution melt (HRM) analysis is a postPCR analysis step that uses the fluorescence detectors of real-time PCR machines to characterize PCR prod-

ucts. After PCR amplification, the amplified DNA is denatured by slowly increasing the temperature in small increments (0.01 to 0.05OC). As the DNA denatures, intercalating DNA dyes such as SYBR green are released, causing a change in fluorescence. Precise measurements of the melting point peak and the shape of the curve allow the differentiation of amplicons with different sequences, even if they share the same melting point. HRM analysis could, in principle, be applied to any PCR amplicon with sufficient genetic variation, including fla loci described above. Price et al. (2007) described the use of HRM to analyze PCR amplicons derived from clustered, regularly interspaced short palindromic repeats (CRISPRs). CRISPRs are composed of near-perfect direct repeats interspersed with similarly sized nonrepetitive spacer sequences. New primers were described which amplified CRISPR PCR product from almost all Australian C. jejuni isolates tested, but not from any of the seven C. coli isolates tested (Price et al., 2007). By means of a SYBR green-based HRM real-time PCR assay, Price et al. (2007) were able to identify 36 CRISPR HRM genotypes among 84 strains on the basis of differences in the melt curve generated. Previous analysis of these strains had resolved 53 SmaI PFGE types, 32 MLST types, and 15 flaA SVR types (O’Reilly et al., 2006). HRM analysis of Campylobacter requires evaluation in additional laboratories. Providing that results are reproducible, it may be a useful first screening tool, and in combination with other methods, it provides sufficiently high levels of discrimination. Denaturing gradient gel electrophoresis is an alternative way that sequence polymorphisms in PCR products may be distinguished. Here, differences in the relative stability of DNA sequences are detected by electrophoresis in a denaturing chemical gradient. Separation can be improved by adding a polynucleotide tail, or GC clamp, to the target during the PCR. This approach has been used to type C. jejuni strains by examination of the flaA gene, but with rather poor results: just 13 types from 80 unrelated strains were obtained, compared with 40 types found by fla-RFLP analysis (Nielsen et al., 2000). Single Nucleotide Polymorphism, Binary Typing, and DNA Microarray Methods Genomic analysis of increasing numbers of strains of Campylobacter is identifying an increasing number of gene targets that can be used to develop genotyping assays. Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur in coding and noncoding sequences, and for typing pur-

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poses are best when only two variations are observed (e.g., A or G). SNPs are typically detected by PCRbased assays specific for either the wild-type (0) or the SNP (1)allele. When multiple SNPs are analyzed in parallel, a binary typing system can emerge. Price et al. (2006b) designed seven SNP assays that were based on loci in the C. jejuni MLST database. This research group also developed a binary typing system that was based on the presence or absence of eight specific genes (Price et al., 2006a). Both of these assays utilize real-time PCR-based assays for detection. Among a set of 84 isolates, the binary typing assay identified 27 genotypes, whereas the SNP assay identified 20 genotypes. When combined, 37 genotypes could be resolved-more than obtained by MLST, but less than SmaI PFGE (Price et al., 2006a). Microarray-based technologies offer an alternative detection methodology that is based on hybridization of DNA extracted from isolates to arrays containing specific oligonucleotide arrays. Use of a first-generation whole-genome microarray that is based on the genome library produced during the sequencing of strain NCTC 11168 revealed all distinct isolates to be genetically different, but strains found to be more similar in AFLP profiling (On and Harrington, 2000) were also more similar in genome content in the microarray analysis, giving credibility to the epidemiological validity of this approach (On et al., 2006). A set of 70-mer probes has also been described that not only allows species identification, but through inclusion of probes targeting the lipooligosaccharide biosynthesis locus, also allows a limited genotypic classification (Quinones et al., 2007). With the addition of further probes, microarraybased typing systems may facilitate genotyping goals and permit characterization of virulence and other attributes. The identification of appropriate probes is an area of active research, but to be feasible on a routine basis, it will require technological advances to reduce the cost of the analysis.

PFGE PFGE is widely regarded as among the most discriminatory typing methods generally available for bacteria. It involves the digestion of intact genomic DNA (embedded in agarose to avoid shearing) with rare-cutting restriction enzymes that cut the DNA into relatively few fragments, in contrast with its predecessor, restriction enzyme analysis (e.g., Lind et al., 1996). These fragments are then separated in a specially designed coordinated pulsed electric field, such that the large fragments are gently oriented through the agarose gel matrix. In a typical 18- to 20-h electrophoresis run, the separation of fragments

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in the 20,000- to 1,000,000-bp range is achieved. Widespread, worldwide adoption of the PulseNet protocol developed at the CDC (Ribot et al., 2001) has significantly improved the comparability of PFGE analysis. However, it remains a technically demanding method with a low throughput and is a relatively high consumable and capital item cost. Furthermore, the DNase activity of some strains needs to be deactivated to ensure that DNA samples do not degrade before electrophoresis. A sample preparation protocol has been described that successfully deactivates DNase (Gibson et al., 1994) but involves the pretreatment of bacterial cells with the toxic chemical formaldehyde. On occasion, strains will resist digestion with the endonuclease of choice (On et al., 1999; On and Vandamme, 1997; W. G. Miller, unpublished data). Site-specific DNA methylation and similar processes are likely to be responsible for such phenomena. In the case of C. jejuni, most researchers have used the enzyme SmaI, which typically generates 4 to 10 fragments (Cornelius et al., 2005; Devane et al., 2005). KpnI has been used as a secondary enzyme in a number of cases (Eyles et al., 2006; Gilpin et al., 2006; On et al., 1998), or even as the primary enzyme (Karenlampi et al., 2007a). SalI (Malik-Kale et al., 2007) and BamHI (On et al., 1998) have also been used. For C. jejuni, the number of fragments generated is generally Ken1 > BamHI > SmaI > SalI. Comparisons between large numbers of isolates have been facilitated by the widespread availability of software such as BioNumerics (Applied Maths, Ghent, Belgium), which are able to electronically compare electrophoresis patterns generated. BioNumerics converts band positions into a percentage of migration down the gel. Through use of a standard marker (in PulseNet protocols, Salmonella enterica subsp. enterica serovar Braenderup H9182), gels can be electronically stretched or compressed to adjust the gel so that the bands of the standard migrate in silico to the same common distance. This adjustment for small variations in PFGE patterns that can and do occur between gels and laboratories allows comparison of isolates run on different gels and in different laboratories. This normalization is of course not perfect, so software packages such as BioNumerics allow the setting of a tolerance (variation where two bands are considered the same, typically 1.0%) and optimization values (allowance for movement of whole pattern up and down a gel, typically 1.5%).Setting these values is critical because making these two parameters smaller will split isolates up, and making them bigger will group isolates with patterns that are actually different, especially because identification of common patterns or types is most often attained by use of clus-

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tering algorithms such as UPGMA (unweighted pair group with mathematical average). Different patterns are typically assigned a unique pattern designation by most researchers. There is no common pattern designation internationally for PFGE patterns, nor is there an agreed consensus of what constitutes a different PFGE pattern. Isolates within PulseNet USA are assigned a common pattern number, but other networks and laboratories use their own pattern designations and their own criteria for pattern assignments. In New Zealand, we have decided to take a conservative approach that errs on the side of grouping isolates rather than risking splitting isolates, which may only appear different because of uncorrected gel-to-gel artifacts. However, even strains that give indistinguishable patterns with one restriction enzyme on a single gel may be distinguished with the application of a different enzyme (On et al., 1998). Digestion with one enzyme is sufficient in many cases to show differences between isolates but is insufficient to demonstrate similarity between isolates. The limitation of one enzyme is well illustrated by examination of the most frequent SmaI pattern in the New Zealand databases, Sm0001, which consists of only five bands (Isolates A-I Fig. 1).Some variation is evident within this SmaI pattern, but this variation is insufficient to reproducibly differentiate the isolates by computer-based comparisons as described above. Digestion with KpnI, however, demonstrates the genetic diversity in this group, some of which do form clonal groups (isolates A to I; Fig. l), a wellestablished phenomenon (On et al., 1998). Thus, what is arguably more important than the enhanced discrimination this approach allows is the increased confidence in interpretation that digestion with two enzymes allows. Digestion with a second (or more)

. . 8 : :

enzyme, however, is a strong indicator of a close clonal relationship, and the implied phylogenetic similarity between two enzymes is supported by MLST analysis in a number of situations we have examined (B. J. Gilpin, unpublished data; S. L. W. On, unpublished data). Whenever the intention is to show that two isolates are indistinguishable, PFGE should include the use of two enzymes. Conversely, it must be noted that a clonal relationship may exist between strains with dissimilar PFGE profiles. Several different types of genetic phenomena, including point mutation, transformation, and plasmid acquisition or loss can lead to minor or major changes in profiles (reviewed by Wassenaar et al., 2000). AFLP Profiling First designed for high-resolution genotyping of complex plant genomes, this method was later adapted for discriminating bacteria. Conventional AFLP profiling involves digesting DNA with two enzymes, ligating restriction half-site-specific oligonucleotide adaptors on to the ends of the fragments, and then amplifying the fragments with PCR primers. Typically, one of the primers is labeled with a fluorophore, and hence only a subset of fragments is detected under electrophoresis (normally achieved by use of a sequencer or fragment analyzer). Patterns can be further simplified by using one or both of the PCR primers, which binds to the adaptor with a 1-base overlap (one of A, G, T, or C ) to increase the specificity of the amplification. Moreover, fragments that are too large will amplify poorly or not at all, and those that are too small will not be visualized. There are several variations of fluorescent AFLP analyses. The systems vary in their choice of restriction enzymes, the use of selective bases in the PCR

PFGE-Kpnl

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Figure 1. PFGE patterns of selected New Zealand C. jejuni isolates when digested with SmaI and KpnI. UPGMA (unweighted pair group with mathematical average) dendrograms generated by BioNumerics v4.5, with DICE coefficient, optimization of 1.0%. and tolerance of 1.5%.

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reaction, or both (Duim et al., 1999; Kokotovic et al., 1999; Lindstedt et al., 2000; Siemer et al., 2005). The sensitivity of the methods is well established, as is their potency for identification of related (i.e., outbreak) strains. Comparisons with MLST show a good correlation in identification of phylogenetic relationships (Miller et al., 2005; S. L. W. On, unpublished data). As discussed below, two of the systems described have additional value in identification and typing of the full taxonomic range of Campylobacter species and of the related Arcobacter species also (Aabenhus et al., 2005; On and Harrington, 2000; On et al., 2003; S. L. W. On and B. L. Siemer, unpublished data). Because species-level identification of these taxa is often demanding (On, 2005), this is a useful adjunct. Fluorescent AFLP (fAFLP) systems can be technically demanding and can also require expensive equipment to run. For C. jejuni, the use of a single enzyme combined with one-base PCR selectivity has been used (Champion et al., 2002) to reduce the number of fragments to a number practical for detection in conventional electrophoresis. These authors found that single-enzyme AFLP had a similar discriminatory ability to PFGE with SmaI. At the Institute of Environmental Science and Research, we have further developed this approach to enable the identification and typing of a broad taxonomic range of Campylobacter and Arcobacter species, similar to previous fAFLP methods (Fig. 2). Given the clinical significance of most species (On, 2005), the need for relatively inexpensive methods to identify and genotype a wide taxonomic range remains a priority.

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Multilocus Sequence Typing (MLST) MLST involves the sequencing of genes, typically those with housekeeping functions, whereby each sequence variant represents a distinct allelic type. Combining the analyses of several (typically seven) genes alongside appropriate computational analyses is a powerful tool for the study of population biology, epidemiology, and evolution of microbes that has expanded hugely (Maiden, 2006) since first described in 1998 (Maiden et al., 1998). A general rapid and ongoing decline in the cost of DNA sequencing, and more pertinently the inherent portability of this approach (unlike fragment-based methods, there is no requirement to standardize or normalize data once common gene targets are defined, making sharing and wide-scale data comparisons relatively straightforward) are resulting in an increasing use of MLST globally. This extensive use enhances the MLST potential to address questions regarding geographical, temporal, and host species-related variation in type, all issues central to source attribution of human cases. Moreover, the evaluation of any attempts to control Campylobacter in the food chain will be supported if studies of specific interventions can build on comparable reference data. Further useful features of MLST mainly center on the following: (i) the ability to base analyses on biological models that integrate the growing knowledge of the genetic processes and population biology that create diversity in the nucleotide sequence, (ii) exploitation of advances in analytical approaches that are being made to allow explicit model-based inference from multi-

1

I

C.~

~

I

~I

~

#

i

Figure 2. Novel AFLP method for use in conventional electrophoresis and detection systems aimed at identification and typing Campylobacter and Arcobacter species. Lanes 1 and 27 are 100-bp markers; lanes 2 to 7, A. butzleri strains; lanes 8 to 15, C. coli; lanes 16 to 19, C. jejuni subsp. doylei; lanes 20 to 25, C. jejuni subsp. jejuni. Lanes 9 to 11 and 21 to 22 represent well-characterized outbreak strains and are indistinguishable (E. Podivinsky, K. Thorn, and S. L. W. On, unpublished data).

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ONETAL.

locus sequence data as a result of the enormous amounts of these data being generated in many different fields, and (iii) changing from a typing paradigm that considers whether isolates are identical to (here, assigned to the same sequence type, or ST) or different from one that considers how closely related they are, ancestrally and potentially ecologically. Evaluating the proportion of allelic variants shared between isolates allows their assignation to clonally related groups or clonal complexes from which an objective assessment of strain phylogeny can be made. Information on the seven alleles or 3,309 nucleotides can also be analyzed directly. Either of these approaches has the potential to be useful in source attribution (see below). Limitations of MLST include cost (despite methodological advances, the method remains comparatively expensive) and the degree of resolution that is possible with the quantity of data provided by seven highly conserved gene fragments. Indeed, some studies show the discriminatory index of MLST as somewhat lower than that offered by PFGE (Mellmann et al., 2004; Sails et al., 2003). However, where a group of isolates share a very rare MLST type, some, although not conclusive, evidence for increased likelihood of an epidemiological relationship is given. The role of MLST in understanding the population biology is considered elsewhere in this book (chapter 2).

PEAKS AND TROUGHS: MOLECULAR EPIDEMIOLOGY AND SEASONALITY OF INFECTION The seasonal peak of Campylobacter infection is well recognized and widely studied, but the cause remains uncertain with many theories, none of which have been fully tested (Ekdahl et al., 2005; Hearnden et al., 2003; Hudson et al., 1999; Miller et al., 2004; Nylen et al., 2002; Stanley et al., 1998a, 1998b). Use of genotyping could help answer this question by assessing the extent to which types vary by season and the likely origin of the types involved in the seasonal peak. The ideal data set would be longitudinally sampled isolates from humans and the range of possible sources of infection, which is not currently available. However, some useful data are already available or coming on stream. One study from Finland reported no evidence for different isolates identified from cases of infection observed during the seasonal peak compared with other times in the year (Karenlampi et al., 2007b). An initial report, with a small amount of data, on a United Kingdom study did not report on this outcome directly but displays data indicating that the three largest clonal complexes throughout

the year (ST-21, ST-45, and other/unassigned) all increased during the seasonal peak, in line with the overall increase of cases (Sopwith et al., 2006). Our preliminary analysis of a larger accruing data set also suggests that seasonal variation in humans, at least in the United Kingdom, may be due more to an expansion of the same types that are present during the rest of the year rather than the appearance of novel types (McCarthy et al., 2007). A related finding is that a sample of 725 retail poultry isolates from the United Kingdom showed similar types in spring and early summer compared with the rest of the year, suggesting that the origins of infection in poultry do not vary substantially at this time (Sheppard et al., 2007). The seasonal peak therefore appears most likely to be due to an increase in common types among the sources of Campylobacter infection rather than novel contributions, although this remains to be confirmed, as does the possible contribution of adaptation for environmental survivability to the prevalence of common clonal groups (On et al., 2006).

POINTING THE FINGER OR PASSING THE BUCK? ASSESSING THE CONTRIBUTION OF DIFFERENT SOURCES T O HUMAN CASES OF C. JEJUnrI AND C. COLI INFECTION Source tracking depends, in part, on an accurate estimation of the frequency of different subtypes in each host reservoir. In Salmonella, particular serotypes and phage subtypes are stably found in the same host (Hald et al., 2004b). The biology underlying this success is first that specific clones are well adapted to specific hosts and second that the serophage type is a stable and reliable indicator of a specific clone. For C. jejuni, host-associated markers are extremely rare. Genetic methods of discrimination show enormous diversity, with studies typically reporting about half as many genotypes as there are strains in the study (Dingle et al., 2001a, 2002; French et al., 2005; Hopkins et al., 2004; Manning et al., 2003; Schouls et al., 2003; Siemer et al., 2004). Many common genotypes are broadly distributed (Dingle et al., 2002; Siemer et al., 2004), whereas for rare genotypes, it is not possible to accurately estimate the relative frequency of genotypes in different host reservoirs. These difficulties have meant that although host associations have been identified for particular genotypes, no generally useable approach has been developed. However, several MLST-based studies have shown host association of some clonal complexes such as ST-61 complex (Colles et al., 2003; Dingle et al., 2002; French et al., 2005) and for isolates from these clonal complexes a strong prediction of source

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MOLECULAR EPIDEMIOLOGY O F CAMPYLOBACTER SPECIES

could be made on the basis of clonal complex membership. However, other clonal groups have a wide distribution (Dingle et al., 2002; Miller et al., 2006). When applied as a general approach to attribution of isolates among food animals, clonal complex information alone provides little improvement on random guessing or the use of ST (McCarthy et al., 2007). The poor performance of clonal complexes in supporting attribution of C. jejuni suggests that major clones are not, as a whole, highly adapted to individual host species among food production animals-at least not when considering adaptation at the whole genomic level. Similar observations on the relative specificity of a few genotypes, and the wide host range of others, have been made with other wholegenome typing methods including AFLP, PFGE, and DNA microarray analysis, where major food animal sources such as poultry and cattle are strongly implicated reservoirs of human infection but genotypes from wild bird isolates show little overlap with those from human isolates (On et al., 1998; Petersen et al., 2001; Siemer et al., 2004). Results of the microarraybased study were correlated with strain viability in aerobic conditions, suggesting that major clones are better adapted for environmental survival than those that occur in a more limited host range (On et al., 2006). In contrast, an analysis of 25 Guillain-Bard and Miller Fisher syndrome-related isolates showed that one clonal complex appeared to be overrepresented, but the isolates came from a wide range of clonal groupings (Dingle et al., 2001b); further studies are required to confirm the consistency or otherwise of phenotypic markers of environmental survival

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and pathogenicity among major and minor clonal groups. A somewhat different picture is obtained when considering host adaptation from a phylogenetic perspective. With MLST, analysis at allele level is less prone to the problems of diversity affecting analysis of ST and can use differentiation between C. jejuni gene pools in different host species, even if this differentiation is not based on separation between host species at the clonal complex level. This allows more accurate assignment of isolates to correct host species with 80% accuracy in identifying whether C. jejuni isolates originated from ruminants (cattle and sheep) or chicken (McCarthy et al., 2007). An exploration of the biological processes underlying the greater accuracy from allele-based assignment used the ST-21 complex, a large clonal group that is widespread in many food animals and other sources. There are many STs within the complex, each differing from the central genotype at between one and three of the seven alleles. Prediction used only these discordant alleles (i.e., those that differed from the control genotype) and assigned ST-21 complex isolates to host species on the basis of the frequency of each isolate’s discordant alleles in reference populations composed of non-ST-21 complex isolates from these host species. In Fig. 3 , each box indicates an ST and the shading represents the isolate source with this ST (light for chicken, dark for ruminant and mixed if from both sources). Considering only discordant alleles with strong evidence of differential distribution between chicken and ruminant reference populations, the lines joining STs to ST21 indicate whether it had

Figure 3. Prediction of origin by using only alleles for which substantial reference information is available. Light lines indicate alleles different from ST-21 present mainly in chickens in the reference population (i.e., an allele that would predict chicken origin); dark lines indicate alleles present mainly in bovids (i.e., predicts bovid origin). Light boxes indicate STs found only in chickens, dark boxes indicate STs found only in bovids, and boxes with light and dark shading indicate STs found in bovids and chickens. Figure from McCarthy et al. (2007).

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a discordant allele typical of chicken (light line, e.g., the line going to ST-368) or ruminant (dark line, e.g., the line going to ST-43). Isolates with two such discordant alleles are separated from ST-21 by two lines. The presence of an allele typical of a particular host species reference population predicts the origin of that isolate rather well. All four isolates having two alleles, both of which are suggestive of either chicken or ruminant origin, are indeed from the predicted source. In one case, two potentially informative alleles gave conflicting information, one suggesting bovid origin and the other chicken. Isolates with this ST came from both sources. Of the 24 STs with only one informative allele available, 18 are correctly assigned and only 4 incorrectly assigned with two STs isolated from both chicken and ruminant sources. Formal statistical assessment of the overall predictive power of discordant alleles within the ST-21 complex shows that they far outperform any effect likely by chance alone (P < 0.0001) (McCarthy et al., 2007). This result shows that ST-21 complex isolates coming from chickens are from lineages that have imported genetic material from other, clonally distinct, chicken lineages. This effect identifies host association not on the basis of ancestral (clonal) relationships but on a direct effect of different lineages having shared a host species niche, and by extension the opportunity to share DNA by lateral gene transfer. An extension of this is that isolates giving rise to this signal (as either donor or recipient of recombinant DNA) must, on average, represent lineages that preferentially inhabited chicken for a considerable period of time. This suggests that a clonal model of host association may also be effective, but for more highly resolved clonal groupings than are currently identified. The molecular epidemiology of C. coli is less well studied, but more recent work offers some insights into host association. Miller et al. (2006) applied MLST to the investigation of host association in C. coli in North America by using 484 isolates from cattle, swine, chickens, and turkeys. Substantial diversity was seen, with many STs observed only once. The large majority of STs (and isolates) clustered into a single clonal grouping and the large majority of the rest into a second grouping on the basis of a minimum spanning tree and eBURST clustering. The larger grouping included isolates from all four hosts, and the smaller included mainly turkey and chicken. Large clonal groups are thus not predictive of host in C. coli either. However, individual allele frequencies at all seven loci showed clear differences between hosts that were more striking than in C. jejuni, and there were some sublineages within the main clonal group that appeared to be host associ-

ated. However, a comparison of U.S. with United Kingdom isolates (Miller et al., 2006) revealed that overall host association of alleles was largely consistent, but with some marked exceptions such as tkt allele 35, which was mainly found in swine isolates in the United States but mainly in chicken isolates in the United Kingdom. We have assigned 259 independent United Kingdom C. coli poultry isolates (N. McCarthy, unpublished data) by using the U.S. data set as a reference population and by using previously described assignment methods (McCarthy et al., 2007). Fifty percent of the C. coli isolates were assigned to a chicken origin, 28% to cattle, 13% to swine, and 10% to turkey. Although this result is a lot better than the 25% correct expected by chance-and it shows that host association is geographically robust even between continents-further work is needed before attributing unknown isolates to likely source on the basis of MLST data. An investigation of 174 C. coli strains by AFLP profiling found human C. coli isolates in Denmark to more closely resemble those recovered from chicken, turkey, and duck, despite an overrepresentation of porcine isolates (Siemer et al., 2005). A subsequent MLST-based study of 160 Danish C. coli strains similarly found 38% of human isolates shared the same STs as those from chickens; just 9% of human strains were assigned to STs also found in pigs, and these STs were also detected among poultry isolates (Litrup et al., 2007). Two of seven cattle strains in this study belonged to STs found in humans. Preliminary work that used MLST to compare C. coli strains from human infections and chicken, cattle, and pigs in the United States also suggests that a greater proportion of poultry isolates belong to the same STs found in humans, but that some strains from pigs and especially cattle are also assigned to STs represented in human diarrheal isolates (W. G. Miller, unpublished data). Current results suggest that the attribution of C. jejuni and C. coli isolates from human cases to their source will be best undertaken by means of a phylogenetic approach that uses analysis based on allele frequencies, or that follows robust definition of hostassociated sublineages within the main clonal groups. Larger, more widely sampled reference data, explicit consideration of temporal and geographical aspects, and better analytical models may also be needed. Nonetheless, the consideration of many studies undertaken to date, together with other quantitative risk assessment data (Allen et al., 2007; Luber and Bartelt, 2007; Meldrum et al., 2006; Wingstrand et al., 2006), clearly identify chicken as a major source of human infection in industrialized countries. It is the relative contribution of all other sources to the bur-

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MOLECULAR EPIDEMIOLOGY OF CAMPYLOBACTER SPECIES

den of disease that is in need of most clarity; however, almost all other foods of animal origin (notably other poultry and cattle) are credible sources of infection. MOLECULAR TYPING AND OUTBREAK INVESTIGATIONS An outbreak is defined here as two or more cases of disease that arise from the exposure to a common source of contamination. In contrast with other enteric pathogens such as Salmonella species and pathogenic Escherichia coli, human outbreaks of campylobacteriosis appear relatively uncommon, although the difficulty in linking human cases with, for example, contaminated meat from a single supplier but distributed nationally is a clear obstacle in identifying an outbreak. What is incontrovertible is the fact that the definitive identification of the origin of the outbreak by any typing method requires that the type of an isolate from the source matches those of the outbreak isolates. With Campylobacter outbreaks (irrespective of the species), isolates are rarely recovered from the source of infection. With an incubation period of 2 to 5 days, this is hardly surprising: by the time the patient has manifested diarrhea, the source has most likely been consumed or discarded. Even if samples remain, recovery of Campylobacter from the environment is far from trivial, and because multiple types may be harbored in a single specimen, the isolation of the same type that was recovered from the patient is not guaranteed. Ergo, definitive identification of the source is rarely attained. There are a number of outbreaks documented where the use of molecular typing has definitively identified the source. These are summarized in Table 1. The number is small, but the number of similar outbreaks where phenotypic typing methods have de-

finitively kesolved the outbreak source is little better (On, 2001), proving the challenges posed in epidemiological investigations of Campylobacter outbreaks. There are, however, numerous descriptions of outbreaks whereby the source was not identified but where the typing data did at least alert the authorities to a common cause. Some of these are notable in involving unusual settings (school: Fayos et al., 1993; ostriches: Stephens et al., 1998), strain phenotypes (C. jejuni Penner serotype 55; Harrington et al., 1999), or species (C. fetus, Rennie et al., 1994; C. hyointestinalis subsp. hyointestinalis, Salama et al., 1992). In all but the former study, where plasmid profiling, ribotyping and RAPD were used, PFGE was the method applied. OUTBREAKS AND EVOLUTION: RECONSIDERING THE PARADIGM Given the particularly complex nature of Campylobacter epidemiology from a wider perspective, together with recent advances in, and the increased use of, molecular typing, it may be considered pertinent to review the core question posed in such studies. Rather than ask if cases were caused by isolates from a common source, it may be more prudent to consider whether the isolates from these patients shared a common ancestor within the past 2 to 6 months, if postulating a shared source such as a recent cycle of poultry from a particular producer. In studying C. coli in cattle, U.S. researchers followed up an observation that a single MLST type was identified 47 times over a wide area and in isolates 4 years apart by additionally sequencing three highly variable loci: all 47 isolates were identical at each of the 10 loci (Miller et al., 2006). We have made similar unpublished observations for C. jejuni in chicken where

Table 1. Summary of outbreaks in which the source of infection was definitively identified by use of genotyping" Source

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Year

Method(s)

Reference

Water Cow milk and feces Water

1996 1996 1998

Cattle Infected food handler

2000 2001

Nosocomial infection Milk Water

2003 2003 2004

REA REA PFGE, rihotyping AFLP PFGE PFGE FlaA, SVR, FlaA sequencing PFGE MLST PFGE

Lind et al., 1996 Lind et al., 1996 Engberg et al., 1998 Kokotovic and On, 1999 Lehner et al., 2000 Olsen et al., 2001 Fitzgerald et al., 2001 Llovo et al., 2003 Sails et al., 2003 Kuusi et al., 2004

"An isolate from the implicated source of infection must have been available for comparison with an outbreak strain and a matching genotype must have been obtained.

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isolates with identical sequence at MLST plus additional antigen-endoding gene segments were present in different areas of the United Kingdom several years apart (N. McCarthy and colleagues, unpublished data). Sharing an identical genotype does not therefore of itself indicate a common source, or even a high probability of a common source, and should not be taken to indicate that isolates are epidemiologically related. The main contribution of MLST to outbreak detection to date, including more extensive typing including additional loci over the usual seven, is therefore not in allowing outbreak detection but in making explicit in population genetic terms how very loosely related apparently identical isolates may be. It should be noted that MLST plus two or more sequenced antigen-encoding genes is highly discriminatory, and where high-throughout sequencing facilities and appropriate personnel and financial resources are readily available, this combined approach is thus a highly informative tool in outbreak investigations.

GENOTYPING OF NON-JEjUnrr, NON-COLI CAMPYLOBACTER SPECIES Given the scale of the human problems caused by C. jejuni and C. coli, comparatively little attention has been given to methods for typing other Campylobacter species, although many of these are strongly implicated as human pathogens. The use of improved isolation and detection methods are likely to demonstrate the need for improved typing methods for at least some of these species. The taxonomic diversity of Campylobacter has led to most workers in this area using broad-range whole-genomic methods that may be applied without a detailed knowledge of the genome, although recent advances in genome sequencing have shifted this paradigm somewhat. The major methods are summarized below. PFGE

C. concisus Matsheka et al. (2002) examined 53 C. concisus strains, principally from cases of diarrhea in South African children, by means of NotI-based macrorestriction profiling. Fifty-one distinct patterns were obtained, indicating both the high discriminatory potential of the method and the genetic diversity of the species. Indeed, other studies indicate C. concisus represents a taxonomic complex of several genomospecies (Aabenhus et al., 2005). Formaldehyde pre-

treatment was required to obtain DNA suitable for analysis, suggesting a high level of DNase activity, a trait not detected with standard phenotypic methods. C . fetus

Salama et al. (1992) applied PFGE analysis to strains of C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. venerealis bv. intermedius (i-e., glycine-tolerant variants of subsp. venerealis). The authors distinguished most (but not all) strains assigned to these taxa by differences in their estimated genome sizes (1.1, 1.3, and 1.5 Mb, respectively). Such estimates are likely to have been compromised by the technical limitations of PFGE because the genome sequence of C. fetus subsp. fetus strain 82-40 is 1.77 Mb (NCBI accession no. NC008599). Hum et al. (1997) used visual comparison of C. fetus PFGE profiles to differentiate between the two subspecies; although generally effective, one strain yielded a profile so atypical that it could not be readily assigned to either subspecies. A later study (On and Harrington, 2000) used numerical analysis of PFGE DNA profiles to assign each of 31 strains into one of two major clusters, of which the first contained 19 strains clearly identified as C. fetus subsp. venerealis. The second cluster comprised 12 strains, of which 10 were unambiguously identified as C. fetus subsp. fetus. The remaining two strains were identified as C. fetus subsp. venerealis by either phenotypic or PCR methods, but not both. At higher similarity levels, clusters containing isolates from each of two countries were identified, suggesting that certain clones predominate in certain geographical regions. Results from these studies have indicated that PFGE profiling is an effective means of genotyping C. fetus because most unrelated strains can be readily distinguished. The validity of the method for outbreak investigations of this species has been proven in outbreaks of diarrheal illness in a closed community (Rennie et al., 1994) and nosocomially acquired meningitis in a neonatal intensive care unit (Morooka et al., 1996). In both reports, outbreak strains were readily distinguished from unrelated strains. Ichiyama et al. (1998) used PFGE profiling to show that isolates of C. fetus subsp. fetus from feces, blood, and, in one case, a cellulitis lesion from three patients differed from each other, but that strains found in each of the different sites from a given patient were the same. A detailed discussion about typing of C. fetus can be found in chapter 11.

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MOLECULAR EPIDEMIOLOGY OF CAMPYLOBACTER SPECIES

C. hyointestinalis Twenty-two different SmaI-derived PFGE profiles were obtained in a study of 28 strains representing C. hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, and two distinct but unclassified groups of C. hyointestinalis-like strains (On and Vandamme, 1997). In addition, certain features of the profiles obtained were taxon specific and could thus be used for identification purposes. In this study, restriction enzyme SalI provided discrimination equal to SmaI for PFGE profiling C. hyointestinalis subsp. hyointestinalis but was significantly less effective for typing the other taxa because the DNA of most strains resisted digestion (On and Vandamme, 1997). The converse was observed by Salama et al. (1992) in an epidemiological investigation of a family outbreak of C. hyointestinalis (probably subsp. hyointestinalis) because strain DNA was digested only with SalI, not SmaI. Three of the five outbreak strains yielded the same PFGE profile, and raw milk was considered the most likely vehicle of infection (Salama et al., 1992). It was suggested that multiple strains of C. hyointestinalis may have been present in the milk, accounting for the different patterns obtained. This hypothesis is given credence because a diversity of genotypes has been found among isolates from a single animal herd: 10 SmaI patterns and 11 KpnI patterns were identified among 24 isolates from farmed reindeer (Hanninen et al., 2002). Several genotypes were seen in each herd where more than one isolate was available; the largest herd sample contained nine different genotypes (combined SmaI and KpnI patterns) among 13 isolates.

C. upsaliensis A range of restriction enzymes suitable for PFGE profiling this species were tested by Bourke et al. (1996), who examined 20 strains representing each of the seven C. upsaliensis HL serogroups described thus far. Of the enzymes examined (including SalI, SacII, SmaI, NruI, NarI, RsrII, and BssHII), only XhoI was considered to yield suitable profiles for all strains examined. There is some evidence for clonality as indicated by serotype association of similar macrorestriction profiles (Bourke et al., 1996; Moser et al., 2001), although these studies used different restriction enzymes to reach this conclusion. The more conserved SmaI profiling was used by Hald et al. (2004a) to indicate that domestic pet dogs could harbor the same strain for up to 21 months.

C. sputorum

A study of 18 C. sputorum bv. paraureolyticus isolates obtained from a single dairy cow herd over

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a l-year period indicated that the same SmaI-defined PFGE genotype persisted in some cows for up to 12 months (On et al., 1999). Moreover, differences between banding patterns obtained could be accounted for by up to two mutational events in restriction sites, suggesting the herd infection was clonal in origin. Inclusion of an additional C. sputorum bv. paraureolyticus strain, and five strains of C. sputorum bv. sputorum (all epidemiologically unrelated), and further PFGE profiling that used enzymes SmaI, SalI, KpnI, and BamHI led the authors to propose that each of the C. sputorum biovars studied represented distinct clonal lines of this species. Most notably, KpnI sites were absent (or methylated) in all C. sputorum bv. paraureolyticus strains and SalI PFGE profiles of this biovar comprised just three bands. In contrast, C. sputorum bv. sputorum strains were readily differentiated by these enzymes. The results also illustrated that PFGE profiling was a useful method for genotyping C. sputorum because a total of 11 different profiles were noted among 26 strains examined.

AFLP The use of two restriction enzymes that cleave genomic DNA frequently in AFLP analysis greatly facilitates its use to a wider taxonomic range of species than most other genotyping methods. Three combinations of restriction enzymes have been used in AFLP profiling campylobacters, and each has, to a greater or lesser extent, been found successful in typing a wide range of species. The HindIII-HhaI-based method (Duim et al., 1999) is known to genotype Campylobacter species of animal origin, but to our knowledge, its validity in typing other species that are putative human pathogens such as C. concisus and C. rectus has not been tested (Duim et al., 2001). It has, however, been used to confirm canine-to-human transmission of C. upsaliensis (Wolfs et al., 2001) and applied to examine the taxonomy of C. lari, where four genomospecies were delineated; of these, two groups demonstrated resistance to nalidixic acid, but the two remaining groups lacking this trait were not clearly differentiated in their ability to produce urease (Duim et al., 2004). Conversely, the genetic diversity revealed in C. fetus led the authors to conclude that although it is a useful adjunctive method for discriminating C. fetus subsp. fetus from C. fetus subsp. venerealis, this protocol was likely to be limited in epidemiological studies (Wagenaar et al., 2001). The BglII-CspVI-based protocol (Kokotovic et al., 1999) typed most Campylobacter species satisfactorily (On and Harrington, 2000; Siemer et al., 2004), as well as the related Arcobacter spp. (On et al., 2003), but the limited number of fragments ob-

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tained in profiles for C. helveticus and C. upsaliensis (On and Harrington, 2000) led this group to develop an improved protocol that used BspDI and MfeI (Siemer et al., 2005). Subsequently, the method has been found to successfully genotype Carnpylobacter and Arcobucter species and proved useful in investigating an outbreak of C. fetus abortion in Danish sheep (S. L. W. On, unpublished data). This method was also used to investigate 62 C. concisus strains, 60 of which were of Danish origin, and assign them to one of four major clusters that represented distinct genomospecies with statistically significant differences in host immune status, indicative of differences in pathogenic potential (Aabenhus et al., 2005). Given the correspondence between AFLP and MLST generally (e.g., Miller et al., 2005), the high level of genetic diversity (62 distinct types among 62 strains) suggests a panmictic population structure for C. concisus.

RAPD As described above, the RAPD principle lends itself to genetic analysis of different species, although it has not been widely used for this purpose, arguably because of the difficulties in obtaining stable markers. Matsheka et al. (2006) described the use of a (GTG), oligonucleotide for RAPD analysis of C. concisus isolates, identifying unique profiles for 86% of all the strains typed. A system first applied to C. jejuni (Nielsen et al., 2000) identified unique patterns for all 37 C. concisus isolates examined by Engberg et al. (2005) by means of the same approach.

MLST The availability of whole-genome sequences has played an important role in the ease by which PCR primers can be designed for enabling MLST analysis. This comparative genomics approach was instrumental in the development of an approach that allows MLST of the closely related C. jejuni, C. coli, C. lari, C. helveticus, and C. upsaliensis taxa (Miller et al., 2005). Carnpylobacter insulaenigrae is a recently identified member of this aforementioned clade of species (Foster et al., 2004). Isolated exclusively from marine mammals (Foster et al., 2004; Stoddard, 2005), its closest relative is C. lari. The examination of 71 putative C. insulaenigrae isolates for 12 phenotypic traits identified eight profiles, suggesting that this species is diverse phenotypically and also that some strains of the species could be misidentified as C. lari (Stoddard et al., 2007). By use of MLST, phylogenetic analysis of the 40 C. insulaenigrae STs indicated that all 71 isolates were members of a highly related clade, distinct from related species, such as C.

lari and C. upsaliensis (Stoddard et al., 2007). Interestingly, failure to detect the citrate synthase gene gltA in any of the C. insulaenigrae or C. lari strains, supports further the close association between the two species. In C. lari strain Rh42100, absence ofgltA was confirmed after closure of the genome sequence. Therefore, absence of gltA (and presumably other TCA cycle genes) is likely a genomic feature shared by C. insulaenigrae and C. lari and probably defines a common evolutionary origin for the two species. Differentiation of C. fetus subspecies usually relies on biochemical tests, especially growth in 1% glycine, whereby C. fetus subsp. fetus (which is associated with sporadic abortion in cattle and sheep) grows on 1% glycine medium while C. fetus subsp. venereulis (which causes infectious infertility in said animals) does not. However, the glycine tolerance test may not be robust (On, 1996; On and Harrington, 2001). Additional genotypic tests, such as PCR assays, 16s rDNA sequencing, AFLP, and PFGE profiling, have also proved unable to unequivocally distinguish between the two c. fetus subspecies (On and Harrington, 2001; van Bergen et al., 2005). Therefore, a novel MLST method for C. fetus was developed. Despite the highly clonal nature of C. fetus and minimal sequence diversity between the two subspecies, the C. fetus MLST method defined unambiguously a C. fetus subsp. venerealis-specific ST, ST-4 (van Bergen et al., 2005). Thus, MLST can be used to accurately classify C. fetus strains at the subspecies level and provides an attractive alternative to standard phenotypic testing. As described with C. coli, individual alleles or allelic profiles correlate in some instances with host. However, host association of alleles and STs has not been demonstrated to date in the non-jejuni, non-coli Campylobacter (Miller et al., 2005; Stoddard et al., 2007; van Bergen et al., 2005). For species such as C. lari, lack of host association might be attributed to the small number and low host diversity of strains typed. However, MLST host association in some species can be difficult to identify because the species (or subspecies) in question is itself strongly host associated or host restricted. An example of the former is C. helveticus, which is primarily associated with cats and is rarely isolated from dogs (Moser et al., 2001; Shen et al., 2001; Stanley et al., 1992), and examples of the latter include C. insulaenigrae, isolated exclusively from marine mammals, especially pinnipeds, and C. fetus subsp. venerealis, isolated almost exclusively from the bovine genital tract (Thompson and Blaser, 2000). Van Bergen et al. (2005) demonstrated that Campylobacter fetus ST-4 was restricted to bovine isolates; however, because ST-4 was the C. fetus subsp. venerealis ST, this apparent host restriction of

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ST-4 was attributed instead to restriction of C. fetus subsp. venerealis to cattle. Similarly, little association between ST and the geographical region of isolation has been identified (Miller et al., 2005; van Bergen et al., 2005). Phylogenetic analysis of C. upsaliensis concatenated allele sequences identified two major clades, cluster I and cluster I1 (Fig. 4). Cluster I comprises strains primarily from South Africa and Belgium, while cluster I1 comprises mainly strains from the United States. However, attribution of these clusters to geographic association of C. upsaliensis STs may be premature. It is noteworthy that almost all of the cluster I strains were isolated by the Cape Town protocol filtration method (Lastovica, 2006), which uses passive filtration and antibiotic-free media, while the majority of cluster I1 strains were isolated on agar amended with cefoperazone or cephalothin. Thus, separation of the C. upsaliensis STs into two distinct clusters may re-

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flect isolation methodology rather than the geographical origin of the strains. Additional investigations that used isolation methodologies and strains from diverse geographical origins will be necessary to determine whether C. upsaliensis STs are geographically associated. Separation of C. upsaliensis STs on the basis of isolation methodology might indicate that for some species, MLST STs can associate with phenotype. Indeed, cluster I strains, such as RM3195 (ST-5), are sensitive to cefoperazone (Fouts et al., ZOOS), suggesting that clusters I and I1 represent cefoperazonesensitive and -resistant strains, respectively. Although the Cape Town isolation protocol (based on filtration onto a nonselective blood agar medium in an atmosphere containing 7 to 10% H,) would isolate both cluster I and cluster I1 strains, use of media amended with cefoperazone would isolate necessarily only cluster I1 strains. The existence of an antibiotic-

I

I1

0.002

Figure 4. Dendrogram of Campylobacter upsaliensis STs. Allele sequences from each of the 66 C. upsaliensis STs were concatenated in the order adk-aspA-atpA-glnA-glyA-pgi-tkt and aligned by ClustalX. The dendrogram was constructed by the neighbor-joining algorithm and the Kimura two-parameter distance estimation method. Bootstrap values of >75%, generated from 500 replicates, are shown at the nodes. The scale bar represents substitutions per site. Numeric labels represent STs. Geographic source of the strains was, where indicated: South Africa (solid circles), Belgium (open triangles), United States (crosses), or United Kingdom, France, and Sweden (solid triangles). STs representing Zic+ strains are boxed in gray.

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sensitive subpopulation indicates that subpopulations of some species may be undetected, depending on the isolation method used, especially if the isolation method uses antibiotic resistance. Such undetected subpopulations would have important implications for epidemiology. In addition to the separation of the C. upsaliensis STs by antibiotic resistance, the C. upsaliensis STs segregate also by the presence or absence of another marker, namely the phosphorylcholine locus licABCD (Fouts et al., 2005). In Haemophilus, Neisseria, and Streptococcus spp., the lic locus is involved in phosphorylcholine synthesis and the transfer of phosphorylcholine to the lipooligosaccharide or teichoic and lipoteichoic acids, and is considered to be a putative virulence locus (Fischer, 2000; Tong et al., 2000). Significantly, all cluster I strains contain the lic locus, whereas the lic locus is absent in all but two of the cluster I1 strains. Therefore, the C. upsaliensis STs segregate not only by antibiotic resistance but also by the presence or absence of the putative lic virulence locus. A similar segregation of STs according to surface structure-associated loci was identified in C. fetus (van Bergen et al., 200.5), where serotype A and serotype B C. fetus strains were associated with distinct STs. Lateral gene transfer within Campylobacter has important implications for the epidemiology and indeed phylogeny of the genus. Detection of lateral transfer events between two species could imply that they share, however transiently, the same environmental niche or host. Also, although the MLST genes themselves are housekeeping genes and are not considered to be related to pathogenicity, identification of lateral gene transfer between two species would suggest that other genes, involved putatively in host range, colonization, pathogenicity, antibiotic resistance, etc., might be exchanged between the two species. Lateral gene transfer events cannot be detected by fragment-based typing methods such as PFGE and AFLP. However, the events identified by MLST would represent only a small fraction of the total: the MLST gene set of seven genes is <0.5% of the total number of genes in an average Campylobacter genome. Detection of lateral transfer by MLST is facilitated by the interspecies nucleotide sequence diversity of Campylobacter, typically >10% for any pairwise combination of genes from two different Campylobacter species; thus, sequences from two different species can be identified unequivocally within an alignment. Detection of lateral transfer is facilitated also by the presence of four common loci, i.e., atpA(uncA), glnA, glyA, and tkt, within the MLST typing methods for the seven Campylobacter species (Miller et al., 2005; Stoddard et al., 2007; van Ber-

gen et al., 2005). Two of these loci, atpA(uncA) and glyA, can be extended still further into additional Campylobacter species as a result of a high level of sequence conservation across the genus; for example, atpA(uncA) MLST alleles have been amplified and sequenced from multiple strains of 14 Campylobacter species (data not shown). Given the fact that C. jejuni and C. coli are isolated often from the same host or food source, it is not surprising that lateral gene transfer events between C. jejuni and C. coli have been detected by MLST (Miller et al., 200.5; Schouls et al., 2003). Similarly, it is not surprising that lateral gene transfer between C. upsaliensis and C. helveticus, found together in dogs and occasionally in cats, was identified (Miller et al., 200.5). Other examples of putative lateral gene transfer events exist: C. lari allele p g m l l is identical to C. jejuni allele pgm1 10 and similar to C. jejuni alleles pgm100, pgm108, and pgmZ09 (Miller et al., 2005); the C. showae type strain glyA allele is identical to C. jejuni glyA27 (data not shown); and the C. lari alleles atpA36 and atpA49 are similar to atpA alleles from C. lanienae and C. concisus, respectively. Although it is possible that these lateral transfer events reflect simple strain mistyping, divergent MLST alleles represent generally only one or two alleles out of the seven-gene set. For example, the C. showae type strain atpA allele is only 76.5% identical at the nucleotide level to its nearest C. jejuni homolog but is similar to atpA alleles from the genetically related species C. concisus and C. curuus, with whom a common ecological niche is shared (the human oral cavity). Given recent work that has demonstrated species including C. lari (principally associated with the enteric tract of birds and mammals) in oral rinses from cats and dogs (Petersen et al., 2007), these observations may represent additional evidence of the oral route of infection for wider consideration in Campylobacter epidemiology. The expansion of typing methods into other Campylobacter species and the typing of additional Campylobacter strains by means of existing methodology should provide further insights into the role of lateral gene transfer in Carnpylobacter epidemiology and evolution.

CONCLUSION Molecular epidemiological studies of Campylobacter species have matured considerably since the early applications of plasmid profiling and restriction enzyme analysis in homogeneous electric fields. The more recent advances have facilitated the development of large-scale databases of normalized genotypic

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or sequence data that can be applied at national or international scales to obtain a broad view of epidemiology of the organisms under study. The true value of these standardized methods, and the corresponding databases, will become increasingly evident-and relevant-when their content truly reflects the geographical, temporal, and ecological diversity that is exhibited by Campylobacter species. The role of continuous long-term surveillance at a national level particularly is self-evident here. The intelligent use of the methods currently available will ensure that the next advances in understanding the sources of infection and thereby their relative risk to humans may be made, and consequently will inform the scientific community how best to reduce the burden of disease. There are early signs that some methods are yielding an insight into the underlying biology of major clones widespread in human disease, offering new options for better understanding-and thus controlling-the burden of campylobacteriosis. For acute outbreaks of disease, there remains a need for rapid, discriminatory, but inexpensive methods to help identify the infections source so that appropriate countermeasures can be taken. Combined with the appropriate expertise in epidemiology and public health response, molecular typing can play a vital role in limiting the spread of infection. It is important that each of the stakeholders in the public health system-from nurses to environmental health officers and beyond-understands his or her role in outbreak detection and ensures that the typing tools used can be used to best efficacy. The role of species other than C. jejuni and C. coli as causes of human disease has been contested for some time. Nonetheless, the sheer volume of evidence that has been brought to bear in this area should now convince even the most hardened skeptic that some of the other Carnpylobacter species at least represent important agents of human and animal disease. Advances in genotyping have facilitated the identification and epidemiological typing of these species such that researchers will be able to rapidly address the central epidemiological questions that will arise when the importance of any given taxon becomes evident. With the integration of statistics, epidemiology, biology, and a risk-based paradigm, genotyping campylobacters is about to come of age. All that is certain is that the discoveries will be as thrilling, challenging, and surprising as the developments leading up to them have been in the 25 or so years that genetic typing methods have been applied. REFERENCES Aabenhus, R., S. L. W. On, B. L. Siemer, H. Permin, and L. P. Andersen. 2005. Delineation of Campylobacter concisus geno-

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methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle. J. Clin. Microbiol. 38:3800-3810. Nylen, G., F. Dunstan, S. R. Palmer, Y. Andersson, F. Bager, J. Cowden, G. Feierl, Y. Galloway, G. Kapperud, F. Megraud, K. Molbak, L. R. Petersen, and P. Ruutu. 2002. The seasonal distribution of Campylobacter infection in nine European countries and New Zealand. Epidemiol. Infect. 128:383-390. Olsen, S. J., G. R. Hansen, L. Bartlett, C. Fitzgerald, A. Sonder, R. Manjrekar, T. Riggs, J. Kim, R. Flahart, G. Pezzino, and D. L. Swerdlow. 2001. An outbreak of Campylobacter jejuni infections associated with food handler contamination: the use of pulsed-field gel electrophoresis. J. Infect. Dis. 183:164-167. On, S. L. W. 1996. Identification methods for campylobacters, helicobacters, and related organisms. Clin Microbiol. Rev. 9:405422. On, S. L. W. 2005. Taxonomy, phylogeny, and methods for the identification of Campylobacter species, p. 13-42. In J. M. Ketley and M. E. Konkel (ed.), Campylobacter: Molecular and Cellular Biology. Horizon Press, Norfolk, United Kingdom. On, S. L. W. 2001. What have we learned about Campylobacter coliljejuni from bacteriological typing studies? p. 53-57 In World Health Organization Department of Communicable Disease Surveillance and Response, The Increasing Incidence of Human Campylobacteriosis. Report and Proceedings of a WHO Consultation of Experts, 21 to 25 November 2000, Copenhagen, Denmark. World Health Organization, Geneva. On, S. L. W., H. I. Atabay, and J. E. Corry. 1999. Clonality of Campylobacter sputorum bv. paraureolyticus determined by macrorestriction profiling and biotyping, and evidence for longterm persistent infection in cattle. Epidemiol. Infect. 122:175182. On, S. L. W., N. Dorrell, L. Petersen, D. D. Bang, S. Morris, S. J. Forsythe, and B. W. Wren. 2006. Numerical analysis of DNA microarray data of Campylobacter jejuni strains correlated with survival, cytolethal distending toxin and haemolysin analyses. lnt. J. Med. Microbiol. 296:353-363. On, S. L. W., and C. S. Harrington. 2000. Identification of taxonomic and epidemiological relationships among Campylobacter species by numerical analysis of AFLP profiles. FEMS Microbiol. Lett. 193:161-169. On, S. L. W., and C. S. Harrington. 2001. Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16s rDNA sequencing methods. J. Appl. Microbiol. 90:285-293. On, S. L. W., C. S. Harrington, and H. I. Atabay. 2003. Differentiation of Arcobacter species by numerical analysis of AFLP profiles and description of a novel Arcobacter from pig abortions and turkey faeces. J. Appl. Microbiol. 95:1096-1105. On, S. L. W., E. M. Nielsen, J. Engberg, and M. Madsen. 1998. Validity of Sma-defined genotypes of Campylobacter jejuni examined by Sull, Kpnl,and BamHl polymorphisms: evidence of identical clones infecting humans, poultry, and cattle. Epidemiol. Infect. 120:231-237. On, S. L. W., and P. A. Vandamme. 1997. Identification and epidemiological typing of Campylobacter hyointestinalis subspeciies by phenotypic and genotypic methods and description of novel subgroups. Syst. Appl. Microbiol. 20:238-247. Ono, K., T. Kurazono, H. Niwa, and K. Itoh. 2003. Comparison of three methods for epidemiological typing of Campylobacter jejuni and C. coli. Curr. Microbiol 47:364-371. O’Reilly, L. C., T. J. Inglis, and L. Unicomb. 2006. Australian multicentre comparison of subtyping methods for the investigation of Campylobacter infection. Epidemiol. Infect. 134:768779. Petersen, L., E. M. Nielsen, J. Engberg, S. L. On, and H. H. Dietz. 2001. Comparison of genotypes and serotypes of Campylobacter

jejuni isolated from Danish wild mammals and birds and from broiler flocks and humans. Appl. Environ. Microbiol. 67:31153121. Petersen, R. F., C. S. Harrington, H. E. Kortegaard, and S. L. W. On. 2007. A PCR-DGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic pets. J. Appl. Microbiol. 103:2601-2615. Price, E. P., F. Huygens, and P. M. Giffard. 2006a. Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies. Appl. Environ. Microbiol. 72: 7793-78 03. Price, E. P., H. Smith, F. Huygens, and P. M. Giffard. 2007. Highresolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni. Appl. Environ. Microbiol. 73:343 1-3436. Price, E. P., V. Thiruvenkataswamy, L. Mickan, L. Unicomb, R. E. Rios, F. Huygens, and P. M. Giffard. 2006b. Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing. J. Med. Microbiol. 55:1061-1070. Quinones, B., C. T. Parker, J. M. Janda, Jr., W. G. Miller, and R. E. Mandrell. 2007. Detection and genotyping of Arcobacter and Campylobacter isolates from retail chicken samples by use of DNA oligonucleotide arrays. Appl. Environ. Microbiol. 73: 3645-3655. Rennie, R. P., D. Strong, D. E. Taylor, S. M. Salama, C. Davidson, and H. Tabor. 1994. Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism. 1. Clin. Microbiol. 32:721-724. Ribot, E. M., C. Fitzgerald, K. Kubota, B. Swaminathan, and T. J. Barrett. 2001. Rapid pulsed-field gel electrophoresis protocol for subtyping of Campylobacter jejuni. J. Clin. Microbiol. 39: 1889-1894. Sails, A. D., B. Swaminathan, and P. I. Fields. 2003. Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni. J. Clin. Microbiol. 41:4733-4739. Salama, S. M., M. M. Garcia, and D. E. Taylor. 1992. Differentiation of the subspecies of Campylobacter fetus by genomic sizing. Int. J. Syst. Bacteriol. 42:446-450. Schouls, L. M., S. Reulen, B. Duim, J. A. Wagenaar, R. J. Willems, K. E. Dingle, F. M. Colles, and J. D. Van Embden. 2003. Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J. Clin.Microbiol. 41:15-26. Shen, Z., Y. Feng, F. E. Dewhirst, and J. G . Fox. 2001. Coinfection of enteric Helicobacter spp. and Campylobacter spp. in cats. J. Clin. Microbiol39:2 166-2 172. Sheppard, S., N. D. McCarthy, M. C. Maiden, C. Little, R. Elson, R. J. Owen, and R. J. Meldrum. 2007. Multilocus sequence typing and analysis of Campylobacter isolates from the current retail poultry surveys (Project code B15011). Study report to the Foods Standards Agency Siemer, B. L., C. S. Harrington, E. M. Nielsen, B. Borck, N. L. Nielsen, J. Engberg, and S. L. On. 2004. Genetic relatedness among Campylobacter jejuni serotyped isolates of diverse origin as determined by numerical analysis of amplified fragment length polymorphism (AFLP) profiles. J. Appl. Microbiol. 96:795-802. Siemer, B. L., E. M. Nielsen, and S. L. On. 2005. Identification and molecular epidemiology of Campylobacter coli isolates from human gastroenteritis, food, and animal sources by amplified fragment length polymorphism analysis and Penner serotyping. Appl. Environ. Microbiol. 71:1953-1958.

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Skirrow, M. B. 1977. Campylobacter enteritis: a “new” disease. Br. Med.]. 2:9-11. Sopwith, W., A. Birtles, M. Matthews, A. Fox, S. Gee, M. Painter, M. Regan, Q. Syed, and E. Bolton. 2006. Campylobacter jejuni multilocus sequence types in humans, northwest England, 20032004. Emerg. Infect. Dis. 12:1500-1507. Stanley, J., A. P. Burnens, D. Linton, S. L. On, M. Costas, and R. J. Owen. 1992. Campylobacter helveticus sp. nov., a new thermophilic species from domestic animals: characterization, and cloning of a species-specific DNA probe. J Gen Microbiol 138(Pt. 11):2293-2303. Stanley, K. N., J. S. Wallace, J. E. Currie, P. J. Diggle, and K. Jones. 1998a. The seasonal variation of thermophilic campylobacters in beef cattle, dairy cattle and calves. J. Appl. Microbiol. 85:472-480. Stanley, K. N., J. S. Wallace, J. E. Currie, P. J. Diggle, and K. Jones. 1998b. Seasonal variation of thermophilic campylobacters in lambs at slaughter. J. Appl. Microbiol. 84:1111-1116. Stephens, C. P., S. L. On, and J. A. Gibson. 1998. An outbreak of infectious hepatitis in commercially reared ostriches associated with Campylobacter coli and Campylobacter jejuni. Vet. Microbiol. 61:183-1 90. Stoddard, R. A. 2005. Salmonella and Campylobacter spp. in Northern Elephant Seals, California. Emerg. Infect. Dis. 11: 1967-1969. Stoddard, R. A., W. G. Miller, J. E. Foley, J. Lawrence, F. M. Gulland, P. A. Conrad, and B. A. Byrne. 2007. Campylobacter insulaenigrae Isolates from Northern Elephant Seals (Mirounga angustirostris) in California. Appl Environ Microbiol 73 :17291735. Thompson, S. A., and M. J. Blaser. 2000. Pathogenesis of Campylobacter fetus infections, p. 321-347. In I. Nachamkin and

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M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Tong, H. H., L. E. Blue, M. A. James, Y. P. Chen, and T. F. DeMaria. 2000. Evaluation of phase variation of nontypeable Haemophilus influenzae lipooligosaccharide during nasopharyngeal colonization and development of otitis media in the chinchilla model. Infect. Immun. 68:4593-4597. van Bergen, M. A., K. E. Dingle, M. C. Maiden, D. G. Newell, L. van der Graaf-Van Bloois, J. P. van Putten, and J. A. Wagenaar. 2005. Clonal nature of Campylobacter fetus as defined by multilocus sequence typing.]. Clin. Microbiol 435888-5898. Wagenaar, J. A., M. A. van Bergen, D. G. Newell, R. GrogonoThomas, and B. Duim. 2001. Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals. J. Clin. Microbiol. 39:2283-2286. Wassenaar, T. M., S. L. W. On, and R Meinersmann. 2000. Genotyping and the consequences of genetic instability, p. 369380 In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Weijtens, M. J., J. van der Plas, P. G. Bijker, H. A. Urlings, D. Koster, J. G. van Logtestijn, and J. H. Huis in’t Veld. 1997. The transmission of Campylobacter in piggeries; an epidemiological study.]. Appl. Microbiol. 83:693-698. Wingstrand, A., J. Neimann, J. Engberg, E. M. Nielsen, P. GernerSmidt, H. C. Wegener, and K. Molbak. 2006. Fresh chicken as main risk factor for campylobacteriosis, Denmark. Emerg. Infect. Dis. 12~280-285. Wolfs, T. F., B. Duim, S. P. Geelen, A. Rigter, F. Thomson-Carter, A. Fleer, and J. A. Wagenaar. 2001. Neonatal sepsis by Campylobacter jejuni: genetically proven transmission from a household puppy. Clin. Infect. Dis. 32:E97-E99.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 11

Isolation, Identification, Subspecies Differentiation, and Typing of Campylobacter fetus MARCEL

A. P.

VAN

BERGEN,Jos P. M.

VAN PUTTEN, KATEE. AND JAAPA. WAGENAAR

DINGLE,MARTIN J. BLASER,

as well as humans (Dennis, 1967; Harvey and Greenwood, 1985; Meinershagen et al., 1965; Tu et al., 2004; Watson et al., 1967). Carrier states of other animals probably play a role in transmission. C. fetus subsp. fetus is generally orally transmitted by the ingestion of food or water contaminated by feces, aborted fetuses, membranes, or discharges (Garcia et al., 1983).

Campylobacter fetus is recognized as an important veterinary and human pathogen (Vandamme, 2000). It is associated with genital infections in livestock, resulting in different clinical presentations including abortion and infertility. The species is divided into two subspecies: C. fetus subsp. venerealis and C. fetus subsp. fetus. C. fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, also known as bovine venereal campylobacteriosis, bovine vibriosis, infectious infertility, and enzootic sterility (Dekeyser, 1984). It is mainly restricted to the bovine genital tract (Table 1);however, in rare cases, C. fetus subsp. venerealis has been isolated from other hosts, including humans (Eaglesome and Garcia, 1992; Salama et al., 1992). C. fetus subsp. venerealis is mainly transmitted venereally both naturally or by artificial insemination with contaminated semen (Stegenga and Terpstra, 1949). The main differences between C. fetus subsp. fetus and C. fetus subsp. venerealis are the clinical manifestation and the host and niche where the bacteria reside. C. fetus subsp. fetus causes abortion in cattle and sheep, and sporadic infections in humans. Sheep may serve as the primary reservoir of C. fetus subsp. fetus (Garcia et al., 1983), whereas other animals probably serve as secondary (less important) reservoirs (Dennis, 1967; Meinershagen et al., 1965; Watson et al., 1967) (Table 1). C. fetus subsp. fetus can be isolated from different animal species, including cattle, sheep, goats, pigs, horses, fowl, and reptiles,

HISTORIC CLASSIFICATION AND NOMENCLATURE OF C. FETUS The bacterial species now known as Campylobacter fetus was first identified in 1913 and named Vibrio fetus. It was known as a causative agent of abortions in cattle and sheep (McFadyean and Stockman, 1913). Florent (1959) observed that venereally transmitted enzootic infertility in cattle was caused by a V. fetus variant that he designated V. fetus venerealis, whereas sporadic abortions in cattle were caused by another variant of intestinal origin named V. fetus intestinalis. He described several biochemical tests to differentiate the two variants. V. fetus venerealis did not produce H,S and would not grow in a semisolid medium containing 1%glycine, whereas V. fetus intestinalis produced H,S and was resistant to glycine, as earlier described by Lecce (1958; Florent, 1959). On the basis of these clinical and biochemical observations, the species was split into two subspecies.

Marcel A. P. van Bergen and Jaap A. Wagenaar Division of Infectious Diseases, Animal Sciences Group, OIE Reference Laboratory Jos for Campylobacteriosis and WHO Collaboration Centre for Campylobacter, P.O. Box 65, 8200 AB Lelystad, The Netherlands. P. M. van Putten and Jaap A. Wagenaar Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, OIE Reference Laboratory for Campylobacteriosis and WHO Collaboration Centre for Campylobacter, P.O. Box 80.165,3508 Kate E. Dingle Department of Microbiology, John Radcliffe Hospital, Nuffield Department of TD Utrecht, The Netherlands. Clinical Sciences, Oxford University, OX3 9DU Oxford, United Kingdom. Martin J. Blaser Departments of Medicine and Microbiology, New York University School of Medicine, New York NY 10016.

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Table 1. Overview of the subspecies and their host preference and their clinical importance“ _ _ _ _ _ _ ~ ~

Species

Disease and/or commensal status

Principal host@)

C. fetus subsp. venerealis

Cattle

C. fetus subsp. fetus

Sheep Cattle Humans Cattle/sheep

Bovine genital campylobacteriosis: infertility, early embryonic death, and occasional abortion Ovine genital campylobacteriosis: outbreaks of abortion Sporadic abortions Sporadic infections mainly in immunocompromised people Commensal in the intestinal tract

“From Quinn et al. (1994).

In 1973, the results of a taxonomic study of a newly defined genus, “Campylobacter,y’ were published by Vtron and Chatelain (1973). They proposed that Vibrio fetus intestinalis should be renamed Campylobacter fetus subspecies fetus, and Vibrio fetus venerealis should be renamed Campylobacter fetus subspecies venerealis. An H,S-positive intermediate of C. fetus subsp. venerealis was designated Campylobacter fetus subspecies venerealis biovar intermedius (Vtron and Chatelain, 1973). An overview of the historic classifications and nomenclature of C. fetus is shown in Table 2. The current recognized nomenclature for Campylobacteraceae was officially accepted in 1980, published in the approved list of bacterial names (Skerman et al., 1980), and in accordance with that used in Bergey’s Manual of Systematic Bacteriology (Smibert, 1984). Both the nomenclature and diagnostic methods for C. fetus have changed over time, complicating the interpretation of previous studies and their comparison with more recent work (Penner, 1988). C. fetus is the earliest Campylobacter identified, and a considerable number of studies, especially those concerning pathogenesis, were performed in the 1950s to 1970s. At that time, identification, subspecies differentiation, and typing results were based on phenotypic methods that are limited in both reliability and interpretation.

rooka et al., 1996; Rennie et al., 1994). Reliable confirmation of the diagnosis of a C. fetus infection requires the detection of the bacterium by direct or indirect methods. This can be done by culturing, serological testing, immunofluorescence testing, or using a DNA-based assay. Recently, a DNA-based detection assay (TaqMan PCR) was described for the detection of C. fetus subsp. venerealis in crude clinical extracts (McMillen et al., 2006). However, this PCR uses a target sequence, which may give false results as discussed in the subspecies differentiation section. Serological and immunofluorescence testing detection methods also have been described (Ardrey et al., 1972; Bokkenheuser, 1972; Brooks et al., 2004; Camper0 et al., 2005; Hum et al., 1991; OIE, 2004), but culture has significant advantages because isolates can then be used in further tests and characterized more fully in terms of their antimicrobial susceptibility, subspecies differentiation, and epidemiological typing. Therefore, the first step is usually isolation, followed by identification and possibly subspecies differentiation, typing, or both. However, if different methods are used, they may in some cases give contradictory results (van Bergen et al., 2005a, 2005b). Recently, several additional molecular methods have been developed and assessed in comparison with the generally accepted methods. Practical considerations on the use of the best available methods for C. fetus diagnosis and epidemiology are described below.

C. FETUS DIAGNOSTICS Isolation, identification, subspecies differentiation, and to a lesser extent typing are important in C. fetus diagnostics. Screening programs aiming to control bovine genital campylobacteriosis and limit the economic impact are performed by veterinary health services. Veterinary clinical laboratories use these methods for confirmation of the isolated causative agent from clinical samples (e.g., aborted fetuses). Among medical clinical microbiology laboratories, reliable diagnosis of C. fetus infections is important to ensure correct antimicrobial treatment and to trace outbreaks and sources of infection (Mo-

ISOLATION OF C. FETUS

C. fetus subsp. venerealis detection is performed on specimens submitted to the laboratory for veterinary health screening programs or on samples from suspected clinical cases. For bovine samples, sampling methods are described in the chapter entitled “Bovine Genital Campylobacteriosis” in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE, 2004). Because C. fetus subsp. fetus is not a microorganism for which notification is required, no pre-

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scribed methods for its isolation are described. However, in practice, the same methods as for C. fetus subsp. venerealis are used to culture C. fetus subsp. fetus from the genital tract and from aborted fetuses. The latter are the most common type of veterinary clinical sample, whereas human specimens usually include, among others, blood or fecal samples. Collection of veterinary or clinical samples, treatments applied (such as the use of filters), and the choice of transport and isolation media all affect C. fetus isolation rates (Atabay and Corry, 1998; Garcia et al., 1984; Hum et al., 1994; Lander, 1990; Monke et al., 2002). These factors are critically important because successful isolation of this fastidious bacterium requires not only its survival and propagation, but also inhibition of contaminating biota. When confirmation of the diagnosis of C. fetus infection by bacteriological culture is sought, their failure to survive suboptimal conditions should be borne in mind. If the sample is unlikely to be contaminated with other microorganisms, nonselective blood agar plates can be used; however, selective plates (without cephalosporins) must be used for fecal samples. Unlike thermophilic campylobacters, C. fetus can grow at 25 and 37"C, and although thermotolerant strains have been described, they do not generally survive at 42°C (Barrett et al., 1988; Firehammer and Berg, 1965; Harvey and Greenwood, 1983; Leaper and Owen, 1981; Roop et al., 1984). Recent evaluation of a global diverse C. fetus collection showed that the majority of C. fetus subsp. fetus isolates (from recent decades) do grow at 42°C (C. Fitzgerald and M. van Bergen, unpublished data), whereas C. fetus subsp. venerealis isolates generally do not grow at 42°C. Visible growth is usually obtained in 48 to 72 h, longer than most of the other campylobacters (Smibert, 1984).

s

s

s IDENTIFICATION AND CONFIRMATION OF C. FETUS Phenotypic Identification

d

ci

The accepted phenotypic identification scheme for C. fetus is described in Bergey's Manual of Systematic Bacteriology (Smibert, 1984). Traditional biochemical assays (Holt et al., 1994) have the advantages of simplicity and discriminatory power. Conventional assays are also relatively cheap because they do not require sophisticated hardware. However, reliance on this approach can lead to misidentifications (Hum et al., 1997; On, 1996). A 3-day incubation period under standardized conditions of constant atmosphere and temperature is also required

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for a reliable outcome. Two immunological approaches have been described that can be used. Immunofluorescence testing (OIE, 2004) can be a valuable tool, but it requires antiserum that is difficult to obtain commercially and that may cross-react with other Campylobacter spp. Antigen capture immunological assays that use C. fetus-specific monoclonal or polyclonal antibodies are useful for identification (Ardrey et al., 1972; Brooks et al., 2004; Camper0 et al., 2005; OIE, 2004). Molecular Identification The C. fetus genome has a mean G+C content of 33 to 36% (Charlier et al., 1974; Owen, 1983; Viron and Chatelain, 1973), varying from 1.1 to 1.6 Mb in size (Salama et al., 1992), although a larger genome of 2.0 Mb has also been described (Fujita and Amako, 1994). A variety of methods for the molecular identification of C. fetus have been developed that monitor either cellular protein or fatty acid composition, or that detect C. fetus by DNA amplification, sometimes together with DNA sequencing. Catalase-positive C. fetus strains (Ferguson and Lambe, 1984) can be identified by whole-cell protein analysis by polyacrylamide gel electrophoresis. This provides reproducible banding patterns of soluble protein extracts. Numerical analysis of the protein profiles then allows a C. fetus cluster, separate from other Campylobacter species to be identified (Vandamme et al., 1990). Most Campylobacter species, including C. fetus and C. jejuni, can be distinguished by analysis of the cellular fatty acid composition (Blaser et al., 1980; Vandamme, 2000). This parameter is stable, provided the analysis is performed on isolates cultured under standardized conditions. DNA-based methods include 16s and 23s rDNA sequencing (Moureau et al., 1989), amplified fragmentlength polymorphism (AFLP) (Duim et al., 2001; Wagenaar et al., 2001), and multilocus sequence typing (MLST) (van Bergen et al., 2005a). The latter allowed clear differentiation of C. fetus from other species and showed that it is more homogenous (Fig. 1). The majority of the above methods are sensitive and can be used to identify C. fetus. However, several are laborious, time-consuming, and relatively expensive and therefore are not well suited to routine diagnostic laboratories. PCR is another DNA-based method that is simple, robust, and quick. A number of PCR-based assays with various target genes including the 16s ribosomal gene-specific region (Linton et al., 1996; Oyarzabal et al., 1997) and others (Casadtmont et al., 1998; Hum et al., 1997) have been developed. PCR probe-based methods are also avail-

able to detect C. fetus (Blom et al., 1995; Logan et al., 2001; Wesley et al., 1991). The combination of PCR and nonradioactive microplate hybridization is convenient for rapid C. fetus identification (Casadtmont et al., 2000), and also a PCR-enzyme linked immunosorbent assay has been described (Metherell et al., 1999). PCR followed by restriction enzyme analysis has differentiated C. fetus from other organisms, but some cross-reaction with other campylobacters was observed (Eaglesome et al., 1995). Other restriction enzyme analysis methods revealed many similarities in the patterns obtained for all strains of C. fetus (Collins and ROSS,1984). The PCR described by Hum et al. (1997) currently appears the most evaluated and is therefore the most useful method for C. fetus identification (Muller et al., 2003; Newel1 et al., 2000a; On and Harrington, 2001; Schulze et al., 2006; van Bergen et al., 2005a, 2 0 0 5 ~Wagenaar ; et al., 2001; Willoughby et al., 2005). SUBSPECIES DIFFERENTIATION OF C . FETUS Identification of C. fetus is relatively straightforward, but reliable subspecies differentiation is much more difficult. The two subspecies are closely related genetically and hence are indistinguishable by DNADNA hybridization (Harvey and Greenwood, 1983; Roop et al., 1984; Thompson et al., 1988). Even so, several phenotypic, animal, and genotypic assays have been evaluated for their ability to differentiate C. fetus subsp. venerealis and C. fetus subsp. fetus. Phenotypic Subspecies Differentiation The currently recommended World Organization for Animal Health assay to discriminate the two subspecies is the 1% glycine tolerance test (C. fetus subsp. venerealis negative; C. fetus subsp. fetus positive) (Lecce, 1958). However, doubts exist as to the stability of this marker because tolerance may be phage mediated or may occur by point mutation (Chang and Ogg, 1970, 1971). The biochemical H,S test on sensitive medium is also described as able to differentiate C. fetus subsp. fetus (positive) from C. fetus subsp. venerealis (negative). C. fetus subsp. venerealis biovar intermedius strains were so called because of their positive reaction in this assay (Vtron and Chatelain, 1973). Other methods have been described but did not provide definitive discrimination. Parameters assayed include growth rate, size, and type of the spiral, aerotolerance, and growth at 42°C (Dekeyser, 1984; George et al., 1978; Karmali et al., 1981). A list of seven apparent key phenotypic tests that derived from probability matrices (including selenite reduc-

C. FETUS ISOLATION. IDENTIFICATION.AND TYPING

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217

C-76 C-116 ~-118--

. lati

h-7 h-6 h-2 v h-8

C.jejuni

I I

0.02

U-6

C.upsaliensis

h

f-3 f-2 f- 1

C.fetus Figure 1. Lack of genetic diversity in the C. fetus glnA locus compared with other Curnpylobucter species. Radial neighborjoining tree comparing 477 nt sequences at the glnA locus of C. fetus with those of five other Cumpylobucterspecies. Alleles are numbered as in the MLST databases at http: //pubmlst.org/and prefixed by a letter to indicate the species. Bootstrap values are shown. Reprinted from the Journal of Clinical Microbiology (van Bergen et al., 2005a).

tion, growth at 42"C, 1% glycine tolerance, susceptibility for metronidazole and cefoperazone, basic fuchsin, and KMnO,) proved indicative for subspecies differentiation but revealed different classifications per test (On and Harrington, 2001). Susceptibility to cephalosporins-cefoxitin and cefamandole (Varga et al., 1990), cefaperazone, and cephalothin (Harvey and Greenwood, 1983) and penicillin (Dekeyser, 1984)-has been described as discriminatory, but again, contradictory results have been noted (Tremblay and Gaudreau, 1998; Tremblay et al.,

2003), suggesting that antibiotic resistance is not a useful marker for subspecies. Subspecies Differentiation Using Animal Models Animal models for C. fetus infections are used primarily in pathogenesis and vaccine studies (Bryner et al., 1978; Grogono-Thomas et al., 2003; Schurig et al., 1973). However, one study has investigated the use of animal models for subspecies differentiation, on the premise that C. fetus subsp. venerealis and C.

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fetus subsp. fetus have differences in their preference for host and niche (Bryner et al., 1971). Isolates of C. fetus subsp. fetus should be able to persist in the gallbladder and intestines, whereas C. fetus subsp. venerealis, because of its niche restriction, should not be able to colonize these sites. The method was evaluated for cattle, sheep, rabbits, guinea pigs, and mice, and the latter were concluded to be most suitable. The use of animals for subspecies differentiation purposes is not practical and has never been publicly evaluated. One study found the assay not to be reproducible (van Bergen, 2005). Molecular Subspecies Differentiation Only a few molecular tools to discriminate C. fetus subsp. fetus and C. fetus subsp. venerealis have been developed to date. Analysis of a specific AFLP region provided the first reliable molecular method to differentiate C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. venerealis biovar intermedius (Fig. 2) (van Bergen et al., 2005a; Wagenaar et al., 2001). This was confirmed by MLST data, which demonstrate an evolutionary relationship among strains consistent with the divergence of the two subspecies (van Bergen et al., 2005a). However, MLST does not separate C. fetus subsp. venerealis biovar intermedius from C. fetus subsp. venerealis (van Bergen et al., 2005a) because isolates of both groups are ST-4 (or, exceptionally, ST-7 or ST-12) (Fig. 3). The use of pulsed-field gel electrophoresis (PFGE) for subspecies differentiation has been described; the resulting banding patterns enable genome sizing and provide data for numerical analysis. However, neither is optimal for routine application. Although numerical analysis of PFGE DNA profiles is effective, it requires sophisticated software and a database (On and Harrington, 2001). Genome sizing is less useful because inconclusive results are often obtained (On and Harrington, 2001; Salama et al., 1992; Vargas et al., 2003). Routine diagnostic laboratories would be best served by a simple PCR for differentiation of the subspecies. Several PCRs have been developed and claimed to be subspecies specific (Hum et al., 1997; Tu et al., 2005; van Bergen et al., 2 0 0 5 ~ ;Wang et al., 2002). The multiplex PCR described by Hum et al. (1997) is currently the most cited. It amplifies a C. fetus-specific DNA fragment approximately 200 bp smaller than the 960 bp described in the original publication (Muller et al., 2003; Wagenaar et al., 2001; Willoughby et al., 2005), as well as a 142-bp fragment thought to be specific to C. fetus subsp. venerealis. C. fetus subsp. venerealis biovar intermedius strains were not evaluated in the study of Hum and

colleagues, but results presented by van Bergen et al. (2005a) show that C. fetus subsp. venerealis biovar intermedius isolates (identified by AFLP) are either C. fetus subsp. fetus or C. fetus subsp. venerealis in the PCR of Hum and colleagues (van Bergen et al., 2005a). Comparison of AFLP and MLST data confirm that this PCR can give false-positive and falsenegative reactions (van Bergen et al., 2005a), as previously shown in comparisons with other assays (Hum et al., 1997; Willoughby et al., 2005). The PCR described by Wang et al. (2002) was thought to amplify only a C. fetus subsp. fetusspecific product, but these results were obtained for a small number of strains. More recent evaluation that used a larger number of strains yielded both false-positive and false-negative reactions (van Bergen et al., 2 0 0 5 ~Willoughby ; et al., 2005). The PCR that gives data most consistent (140 of 140) with C. fetus subsp. venerealis subspecies as defined by AFLP was described by van Bergen et al. (2005~).However, C. fetus subsp. venerealis biovar intermedius, as defined by AFLP, is not identified by this PCR. The random amplification of polymorphic DNA (RAPD) PCRs described by Tu et al. (2005) were evaluated with a small number of C. fetus subsp. venerealis strains, so further evaluation with a larger group of strains is required. In conclusion, AFLP and MLST are currently the best methods for definitive subspecies differentiation. AFLP and a combination of MLST with the PCR as described by van Bergen et al. allow differentiation of C. fetus subsp. venerealis and C. fetus subsp. venerealis biovar intermedius. For laboratories without molecular facilities, unfortunately, there are no alternatives to the unreliable 1% glycine tolerance test. Subspeciation Justified? A subspecies is by definition a group of organisms that are genetically closely related but that diverge in phenotype (Staley and Krieg, 1984; Wayne et al., 1987). The current classification of C. fetus into the two subspecies-C. fetus subsp. venerealis and C. fetus subsp. fetus-meets this definition and was therefore officially accepted in 1980 (Skerman et al., 1980). Ever since, doubts have been cast whether subspeciation is justified (Harvey and Greenwood, 1983; Penner, 1988), especially because the outcome of the 1% glycine tolerance assay appears unreliable as well as sensitive to transduction and point mutation (Chang and Ogg, 1970, 1971). Therefore, research has focused on identification of additional differences between the subspecies. Berg et al. (1971), who combined serotyping and biochemical results, reported strain characteristics that crossed the classical subspecies barriers. Currently, the clearest di-

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C. FETUS ISOLATION. IDENTIFICATION. AND TYPING

Figure 2. Dendrogram showing the AFLP banding patterns of 69 C. fetus strains. Cluster analysis was based on the similarity levels among bands in region 841 to 879 of the banding patterns (arrow). The different clusters of C. fetus subsp. fetus and C. fetus subsp. venerealis are indicated. The percentage of genetic similarity among banding patterns is shown. Reprinted from the Journal of Clinical Microbiology (Wagenaar et al., 2001).

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Figure 3. Congruence of sequence type (ST), sup type, and subspecies. Analysis of concatenated MLST sequences by split decomposition. The three STs identified in association with C. fetus subsp. venereulis are indicated by the dotted line, the remainder being C. fetus subsp. fetus. The correlation among ST and sup type is marked; solid line, supA; dashed line, supB. A radial neighbor-joining tree constructed by using the same data (inset) is shown to indicate the corresponding treelike phylogenies obtained by both methods. This supports the idea that C. fetus subsp. fetus and C. fetus subsp. venereulis evolved recently, with little (if any) evidence of recombination, and that genetic changes have accumulated by the vertical transmission of point mutations, yielding a clonal structure to the population. Reprinted from the ]ournu1 of Clinical Microbiology (van Bergen et al., 2005a).

chotomy in C. fetus is the division into the LPS/sap type A or B (Dworkin et al., 1995; Moran et al., 1994; Perez-Perez, 1986; Tu et al., 2001). C. fetus subsp. fetus can be either type A or By whereas C. fetus subsp. venerealis is always type A, confirming the crossing of the classical subspecies barrier. The fact that the serotype A C. fetus subsp. fetus and C. fetus subsp. venerealis strains are on the same branch suggests that their differentiation occurred after the type A-type B split (Tu et al., 2001) (Fig. 4). Because different results have been obtained with MLST and AFLP compared with the currently applied classical phenotypic and PCR analysis, it seems appropriate to reconsider the basis of C. fetus subspecies classification.

C. fetus subsp. venerealis Biovar Intermedius: to Which Subspecies Do These Strains Belong? A group of strains that reacted positively in the “sensitive” H,S test, and whose characteristics resembled those of C. fetus subsp. venerealis, was designated as C. fetus subsp. venerealis biovar intermedius (VCron and Chatelain, 1973). Taxa below the rank of subspecies are not covered by the rules of the Bacteriological Code (1990 revision, http: //www. bacterio.cict.fr/) but were introduced to determine infrasubspecific subdivisions. Biovar intermedius is therefore not recorded in the approved list of bacterial names (Skerman et al., 1980), but it is mentioned in Bergey’s Munuul (Smibert, 1984). It is not

CHAPTER 11

Ancient

C. FETUS ISOLATION. IDENTIFICATION. AND TYPING

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I I

I I

i Ancestral Mammal type A C. fetus (Mammal 16s rRNA sequence)

- - I I.. - I - -

1-----------I

Modern

presented as a separate biovar in most of the C. fetus studies described to date, probably because of the lack of obvious reference strains, its questionable clinical relevance, and its incomplete original description. Yet it has been reported that C. fetus subsp. venerealis intermedius strains cover a substantial number of a diverse collection of C. fetus strains (van Bergen et al., 2005a). The existence of biovar intermedius was initially proposed on the basis of its identical properties to C. fetus subsp. venerealis, except for a single biochemical trait (positive in the H,S assay) (Vtron and Chatelain, 1973), and it was distinguished from C. fetus subsp. fetus only on the basis of a different result in the glycine tolerance test. As a result of the discrepancies described for the biological niche of this biovar (genital and/or intestinal) (Bryner et al., 1964; Elazhary, 1968; Garcia et al., 1983; VCron and Chatelain, 1973) and the ambiguous interpretation of the subspecies differentiation assays, it has been difficult to decide whether these strains are more closely related to C. fetus subsp. venerealis or C. fetus subsp. fetus. Vkron and Chatelain (1973) initially described this group as a different subspecies (subsp. intermedius Elazhary), but they

h

TypeB

eventually opposed the biovar because of the few available differentiating tests and the ecological niche shared with C. fetus subsp. venerealis. Recent AFLP data (van Bergen et al., 2005a) support the definition of the biovar intermedius as a separate subspecies, according to the definition of a subspecies (genetically homolog, with a phenotypic difference). However, MLST data showed that these strains were the same sequence type as C. fetus subsp. venerealis isolates and therefore not allow to differentiate these strains from C. fetus subsp. venerealis (van Bergen et al., 2005a). Better evidence that biovar intermedius is or is not a separate group of strains should be obtained by detailed genetic analyses. Differentiation of C. fetus Isolates of Classical and Reptile Origin

A group of C. fetus isolates of reptile origin has been identified containing isolates that are genetically distinct from the classical C. fetus isolates. A reptile strain isolated from a turtle was suggested to be the cause of an acute diarrheal illness in a child (Harvey and Greenwood, 1985); however, no causative agent

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was isolated from the child. The first confirmed human infection with a C. fetus strain with markers of reptile origin was described by Tu et al. in 2004. To date, several reptile and human isolates have been identified (C. Fitzgerald, personal communication). To further investigate the genetic diversity among C. fetus strains of different origins, Tu et al. (2005) performed multiple genetic analyses, including RAPD, DNA-DNA hybridization, and sequence analysis of 16s rRNA. All RAPD primers used distinguished C. fetus of classical and reptile origin. DNA-DNA hybridization demonstrated substantial genomic homology differences between strains of mammal and reptile origin. Sequence analysis of 16s rRNA also showed that the reptile strains form a distinct phylotype between mammalian C. fetus and Cumpylobucter hyointestinalis (Tu et al., 2001). Isolates from mammals and reptiles formed two distinct tight phylogenetic clusters that were well separated (Tu et al., 2001). In total, these data suggest that C. fetus subsp. fetus strains of reptile and mammal origin have genetic divergence more extensive than between the two subspecies and than between the type A and type B strains (Fig. 4) (Tu et al., 2005). Currently, a C. fetus MLST scheme is being applied to the previously described reptile isolates and human isolates with reptile markers, as well as to newly identified organisms.

TYPING OF C. FETUS Although identification and subspecies differentiation of C. fetus is performed routinely, typing is generally used only for epidemiological purposes, outbreak identification being the most important. Typing by Phenotypic Methods Phenotyping of C. fetus has been confined to serotyping. Several serotyping schemes have been described on the basis of different isolated antigens (e.g., heat-labile antigen, heat-stable antigen, 0 antigen, and whole-cell antigen). However, the discriminatory power of serotyping is low because of the small number of serotypes. The currently accepted serotyping scheme is based on the heat-stable 0 antigens and consists of only two serotypes, A and B (Berg et al., 1971; Morgan, 1959). The different serotypes correlate with the surface array protein (sap) types. The sup typing scheme is confined to two types, A and B (Yang et al., 1992), and is thus considered to not be very informative. Phage typing has been proposed as a practical supplemental typing

method for C. fetus (Bryner et al., 1973) but has never been developed. The same is true for flagellin typing. Although C. fetus has polar flagella at one or both ends, no flagellin-based typing (flu typing) system has been developed. Typing by Molecular Methods The introduction of molecular methods has allowed new approaches for typing of C. fetus because they allow further discrimination within the subspecies. MLST results showed that C. fetus has a more clonal structure compared with most other species within the genus Campylobucter. This clonality limits differentiation within the C. fetus species. Nevertheless, a PCR-based method focusing on the sap genes was developed, which classified strains into type A or B (Tu et al., 2001). This classification correlated with MLST types (Fig. 3) (van Bergen et al., 2005a). One study that used restriction enzyme analysis showed patterns that could be divided into a limited number of different groups (Collins and ROSS,1984). Typing of strains on the basis of the diversity of the molecular weight of the Sap protein also has been reported, but because frequent shifts in the molecular weight of Sap occur during in vitro and in vivo passage, this typing scheme is not reliable (Fujimoto et al., 1991; Wang et al., 1993). Southern blot hybridization with a sup probe appeared useful for typing and involved the use of a supA probe in combination with PFGE (Fujita et al., 1995b), the whole supA gene as a probe (Denes et al., 1997), and the use of a supB2 probe (Casademont et al., 1998). PFGE was found to be suitable in several epidemiological studies and is currently the best available method for veterinary and human applications (Fujita et al., 1995a; Mannering et al., 2004; Morooka et al., 1996; Newel1 et al., 2000b; Rennie et al., 1994; Vargas et al., 2003). Other molecular methods such as AFLP (Wagenaar et al., 2001) and MLST (van Bergen et al., 2005a) appeared less useful as a result of limited discriminatory power compared with PFGE. For future epidemiological studies, c. fetus microarrays may prove to be a good alternative to detect differences between strains. However, this awaits completion of C. fetus whole-genome sequences. Currently, the genome of one human C. fetus subsp. fetus isolate (82-40) has been deposited in GenBank.

CONCLUSIONS For species identification, the specific PCR described by Hum et al. (1997) is the best for routine use. For subspecies differentiation, AFLP is the best

CHAPTER 11

C. FETUS ISOLATION. IDENTIFICATION. AND TYPING

because C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. venerealis intermedius strains can be distinguished (van Bergen et al., 2005a). Alternatively, MLST in combination with a C. fetus subsp. venerealis-specific PCR (van Bergen et al., 2005c) may be applied. Currently, the best method for typing purposes is PFGE. REFERENCES Ardrey, W. B., P. Armstrong, W. A. Meinershagen, and F. W. Frank. 1972. Diagnosis of ovine vibriosis and enzootic abortion of ewes by immunofluorescence technique. Am. 1.Vet. Res. 33: 2535-2538. Atabay, H. I., and J. E. L. Corry. 1998. The isolation and prevalence of campylobacters from dairy cattle using a variety of methods. J. Appl. Microbiol. 84:733-740. Barrett, T. J., C. Patton, and G. K. Morris. 1988. Differentiation of Campylobacter species using phenotypic characterization. Microbiology 19:96-102. Berg, R. L., J. W. Jutila, and B. D. Firehammer. 1971. A revised classification of Vibrio fetus. Am. J. Vet. Res. 32:ll-22. Blaser, M. J., C. W. Moss, and R. E. Weaver. 1980. Cellular fatty acid composition of Campylobacter fetus. 1.Clin. Microbiol. 11: 448-45 1. Blom, K., C. M. Patton, M. A. Nicholson, and B. Swaminathan. 1995. Identification of Campylobacter fetus by PCR DNA probe method. J. Clin. Microbiol. 33:1360-1362. Bokkenheuser, V. 1972. Vibrio fetus infection in man: a serological test. Infect. Immun. 5:222-226. Brooks, B. W., J. Devenish, C. L. Lutze-Wallace, D. Milnes, R H. Robertson, and G. Berlie-Surujballi. 2004. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. Vet. Microbiol. 103:77-84. Bryner, J. H., P. C. Estes, J. W. Foley, and P. A. O’Berry. 1971. Infectivity of three Vibrio fetus biotypes for gallbladder and intestines of cattle, sheep, rabbits, guinea pigs, and mice. Am. ]. Vet. Res. 32:465-470. Bryner, J. H., J. W. Foley, W. T. Hubbert, and P. J. Matthews. 1978. Pregnant guinea pig model for testing efficacy of Campylobacter fetus vaccines. Am. ]. Vet. Res. 39:119-121. Bryner, J. H., A. H. Frank, and P. A. O’Berry. 1962. Dissociation studies of Vibrios from the bovine genital tract. Am. /. Vet. Res. 23 :32-4 1. Bryner, J. H., P. A. O’Berry, and A. H. Frank. 1964. Vibrio infection of the digestive organs of cattle. Am. ]. Vet. Res. 25: 1048-1050. Bryner, J. H., A. E. Ritchie, G. D. Booth, and J. W. Foley. 1973. Lytic activity of vibrio phages on strains of Vibrio fetus isolated from man and animals. Appl. Microbiol. 26:404-409. Campero, C. M., M. L. Anderson, R. L. Walker, P. C. Blanchard, L. Barbano, P. Chiu, A. Martinez, G. Combessies, J. C. Bardon, and J. Cordeviola. 2005. Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions. J. Vet. Med. B Infect. Dis. Vet. Public Health 52:138-141. Casadkmont, I., C. Bizet, D. Chevrier, and J.-L. Guesdon. 2000. Rapid detection of Campylobacter fetus by polymerase chain reaction combined with non-radioactive hybridization using a oligonucleotide covalently bound to microwells. Mol. Cell. Probes 14:233-240. Casadtmont, I., D. Chevrier, and J. Guesdon. 1998. Cloning of a sapB homologue (sapB2) encoding a putative 112-kDa Campylobacter fetus S-layer protein and its use for identification and

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molecular genotyping. FEMS Immunol. Med. Microbiol. 21: 269-281. Chang, W., and J. E. Ogg. 1971. Transduction and mutation to glycine tolerance in Vibrio fetus. Am. J. Vet. Res. 32:649-653. Chang, W., and J. E. Ogg. 1970. Transduction in Vibrio fetus. Am. 1.Vet. Res. 31:919-924. Charlier, G., P. Dekeyser, A. Florent, R. Strobbe, and J. De Ley. 1974. DNA base composition and biochemical characters of Campylobacter strains. Antonie Van Leeuwwenhoek 40: 145151. Collins, D. M., and D. E. Ross. 1984. Restriction endonuclease analysis of Campylobacter strains with particular reference to Campylobacter fetus ss. fetus. 1.Med. Microbiol. 18:117-124. Dekeyser, J. 1984. Bovine genital campylobacteriosis, p. 181-191. In J. P. Butzler (ed.), Campylobacter Infection in Man and Animals. CRC Press, Boca Raton, FL. Denes, A. S., C. L. Lutze-Wallace, M. L. Cormier, and M. M. Garcia. 1997. DNA fingerprinting of Campylobacter fetus using cloned constructs of ribosomal RNA and surface array protein genes. Vet. Microbiol. 54:185-193. Dennis, S . M. 1967. The possible role of the raven in the transmission of ovine vibriosis. Aust. Vet. /. 43:45-48. Duim, B., P. A. Vandamme, A. Rigter, S . Laevens, J. R. Dijkstra, and J. A. Wagenaar. 2001. Differentiation of Carnpylobacter species by AFLP fingerprinting. Microbiology 147:2729-2737. Dworkin, J., M. K. Tummuru, and M. J. Blaser. 1995. Segmental conservation of sapA sequences in type B Campylobacter fetus cells. J. Biol. Chem. 270:15093-15101. Eaglesome, M. D., and M. M. Garcia. 1992. Microbial agents associated with bovine genital tract infections and semen. I. Brucella abortus, Leptospira, Campylobacter fetus, and Tritrichomonas foetus. Vet. Bull. 62:743-775. Eaglesome, M. D., M. I. Sampath, and M. M. Garcia. 1995. A detection assay for Campylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA. Vet. Res. Commun. 19: 253-263. Elazhary, M. 1968. An assay of isolation and identification of some animal vibrios and of elucidation of their pathological significance. Med. Veeartschool Rijksuniv Gent 12: 1-80. Ferguson, D. A., and D. W. Lambe. 1984. Differentiation of Campylobacter species by protein banding patterns in polyacrylamide slab gels. 1.Clin. Microbiol. 20:453-460. Firehammer, B. D., and R. L. Berg. 1965. The use of temperature tolerance in the identification of Vibrio fetus. Am. J. Vet. Res. 26~995-997. Florent, A. 1959. Les deux vibriosis ginitales: la vibriose due Q V. fetus venerealis et la vibriose d’origine intestinale due Q V. fetus intestinalis. Med. Veeartschool Rijksuniv. Gent 3: 1-60. Fujimoto, S., A. Takade, K. Amako, and M. J. Blaser. 1991. Correlation between molecular size of the surface array protein and morphology and antigenicity of the Campylobacter fetus S layer. Infect. Immun. 59:2017-2022. Fujita, M., and K. Amako. 1994. Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA. Arch. Microbiol. 162:375-80. Fujita, M., S . Fujimoto, T. Morooka, and K. Amako. 1995a. Analysis of strains of Campylobacter fetus by pulsed-field gel electrophoresis.]. Clin. Microbiol. 33:1676-1678. Fujita, M., T. Morooka, S. Fujimoto, T. Moriya, and K. Amako. 1995b. Southern blotting analyses of strains of Campylobacter fetus using the conserved region of sapA. Arch. Microbiol. 164: 444-447. Garcia, M. M., M. D. Eaglesome, and C. Rigby. 1983. Campylobacters important in veterinary medicine. Vet. Bull. 53:793818.

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Garcia, M. M., R. B. Stewart, and G. M Ruckerbauer. 1984. Quantitative evaluation of a transport-enrichment medium for Campylobacter fetus. Vet. Rec. 115:434-436. George, H. A., P. S. Hoffman, R. M. Smibert, and N. R Krieg. 1978. Improved media for growth and aerotolerance of Campylobacter fetus. J. Clin. Microbiol. 8:36-41. Grogono-Thomas, M. J. B., M. Ahmadi, and D. G. Newell. 2003. Role of S-layer protein antigenic diversity in the immune responses of sheep experimentally challenged with Campylobacter fetus subsp. fetus. Infect. Immun. 71:147-154. Harvey, S., and J. R. Greenwood. 1985. Isolation of Campylobacter fetus from a pet turtle. J. Clin. Microbiol. 21:260-261. Harvey, S., and J. R. Greenwood. 1983. Relationships among catalase-positive Campylobacters determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization. lnt. J. Syst. Bacteriol. 33:275-284. Holt, J. G., N. R Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams (ed.). 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed., vol. 1. Lippincott, Williams and Wilkins, Baltimore, MD. Hum, S., J. Brunner, A. McInnes, G. Mendoza, and J. Stephens. 1994. Evaluation of cultural methods and selective media for the isolation of Campylobacter fetus subsp venerealis from cattle. Aust. Vet. J. 71:184-186. Hum, S., K. Quinn, J. Brunner, and S. L. W. On. 1997. Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies. Aust. Vet. J. 759327-83 1. Hum, S., L. R. Stephens, and C. Quinn. 1991. Diagnosis by ELISA of bovine abortion due to Campylobacter fetus. Aust. Vet. J. 68: 272-5. Karmali, M. A., A. K. Allen, and P. C. Fleming. 1981. Differentiation of catalase-positive campylobacters with special reference to morphology. Int. J. Syst. Bacteriol. 31:64-71. Lander, K. P. 1990. The application of a transport and enrichment medium to the diagnosis of Campylobacter fetus infections in bulls. Br. Vet. J. 146:334-340. Leaper, S., and R. J. Owen. 1981. Identification of catalaseproducing Campylobacter species based on biochemical characteristics and on cellular fatty acid composition. Curr. Microbiol. 6:31. Lecce, J. G. 1958. Some biochemical characteristics of Vibrio fetus and other related Vibrios isolated from animals. J. Bacteriol. 76: 312-316. Linton, D., R. J. Owen, and J. Stanley. 1996. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Res. Microbiol. 147:707-718. Logan, J. M. J., K. J. Edwards, N. A. Saunders, and J. Stanley. 2001. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR. J. Clin. Microbiol. 39:2227-2232. Mannering, S. A., D. M. West, S. G. Fenwick, R. M. Marchant, N. R. Perkins, and K. O’Connell. 2004. Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand. N. Z. Vet. J. 52:358-363. McFadyean, J., and S. Stockman. 1913. Appendix to part 111, Abortion in sheep. In Report of the Departmental Committee Appointed by the Board of Agriculture and Fisheries to Inquire into Epizootic Abortion. Her Majesty’s Stationery Office, London. McMillen, L., G. Fordyce, V. J. Doogan, and A. E. Lew. 2006. Comparison of culture and a novel 5’ Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle. 1. Clin. Microbiol. 44:93 8-945. Meinershagen, W. A., D. G. Waldhalm, F. W. Fank, and L. H. Scrivner. 1965. Magpies as a reservoir of infection for ovine vibriosis. J. Am. Vet. Med. Assn. 147:843-845.

Metherell, L. A., J. M. J. Logan, and J. Stanley. 1999. PCRenzyme-linked immunosorbent assay for detection and identification of Campylobacter species: application to isolates and stool samples. J. Clin. Microbiol. 37:433-435. Mitscherlich, E., and B. Liess. 1958. Die serologische differzierung von Vibrio fetus-Stammen. Dtsch. Tierartzl. Wochenschr. 65:25, 36-39. Mohanty, S. B., G. J. Plumer, and J. E. Faber. 1962. Biochemical and colonial characteristics of some bovine vibrios. Am. J. Vet. Res. 23554-557. Monke, H. J., B. C. Love, T. E. Wittum, D. R. Monke, and B. A. Byrum. 2002. Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.J. Vet. Diagn. Invest. 14:35-39. Moran, A. P., D. T. O’Malley, T. U. Kosunen, and I. M. Helander. 1994. Biochemical characterization of Campylobacter fetus lipopolysaccharides. Infect. Immun. 62:3922-3929. Morgan, W. J. 1959. Studies on the antigenic structure of Vibrio fetus. J. Comp. Pathol. 69:125-140. Morooka, T., A. Umeda, M. Fujita, H. Matano, S. Fujimoto, K. Yukitake, K. Amako, and T. Oda. 1996. Epidemiologic application of pulsed-field gel electrophoresis to an outbreak of Campylobacter fetus meningitis in a neonatal intensive care unit. Sand. J. Infect. Dis. 28:269-270. Moureau, P., I. Derclaye, D. Gregoire, M. Janssen, and G . R. Cornelis. 1989. Campylobacter species identification based on polymorphism of DNA encoding rRNA. J. Clin. Microbiol. 27: 1514-1517. Muller, W., H. Hotzel, and F. Schulze. 2003. Identification and differentiation of Campylobacter fetus subspecies by PCR. Dtsch. Tierarztl. Wochenschr. 11055-59. Newell, D. G., B. Duim, M. A. P. van Bergen, R. GrogonoThomas, and J. A. Wagenaar. 2000a. Speciation, subspeciation and subtyping of Campylobacter spp. associated with bovine infertility and abortion. Cattle Pract. 8:421-425. Newell, D. G., J. A. Frost, B. Duim, J. A. Wagenaar, R. H. Madden, J. van der Plas, and s. L. W. On. 2000b. New developments in the subtyping of Campylobacter species, p. 27-44. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. OIE. 2004. Chapter 2.3.2. Bovine Genital Campylobacteriosis, p. 439-450. In OIE (ed.), Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th ed. World Organisation for Animal Health, Paris. On, S . L. W. 1996. Identification methods for Campylobacters, Helicobacters and related organisms. Clin. Microbiol. Rev. 9: 405-422. On, S . L. W., and C. S. Harrington. 2001. Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16s rDNA sequencing methods.]. Appl. Microbiol. 90:285-293. Owen, R. J. 1983. Nucleic acids in the classification of Campylobacters. Eur J. Clin. Microbiol. 2:367-377. Oyarzabal, 0. A., I. V. Wesley, K. M. Harmon, L. SchroederTucker, J. M. Barbaree, L. H. Lauerman, S. Backert, and D. E. Conner. 1997. Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16s rDNA. Vet. Microbiol. 58:6 1-71. Penner, J. L. 1988. The genus Campylobacter: a decade of progress. Clin. Microbiol. Rev. 1:157-172. Perez-Perez, G. I., M. J. Blaser, and J. H. Bryner. 1986. Lipopolysaccharide structures of Campylobacter fetus are related to heat-stable serogroups. Infect. Immun. 51:209-212. Plastridge, W. N., L. F. Williams, and D. G. Trowbridge. 1964. Antibiotic sensitivity of physiological groups of microaerophilic vibrios. Am. 1. Vet. Res. 25:1295-1299. Quinn, P. J., M. E. Carter, M. E. Markey, and G. R. Carter. 1994. Campylobacter species, p. 268-272. In P. J. Quinn, M. E. Car-

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C. FETUS ISOLATION. IDENTIFICATION. AND TYPING

ter, M. E. Markey, and G. R. Carter (ed.), Clinical Veterinary Microbiology. Mosby, London. Rennie, R. P., D. Strong, D. E. Taylor, S. M. Salama, C. Davidson, and H. Tabor. 1994. Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism. J. Clin. Microbiol. 32:721-724. Roop, R. M., 11, R. M. Smibert, J. L. Johnson, and N. R. Krieg. 1984. Differential characteristics of catalase-positive Campylobacters correlated with DNA homology groups. Can. J. Microbiol. 30:938-951. Salama, S. M., M. M. Garcia, and D. E. Taylor. 1992. Differentiation of the subspecies of Campylobacter fetus by genomic sizing. Int. J. Syst. Bacteriol. 42:446-450. Schulze, F., A. Bagon, W. Muller, and H. Hotzel. 2006. Identification of Campylobacter fetus subspecies by phenotypic differentiation and PCR. J. Clin. Microbiol. 44:2019-2024. Schurig, G. D., C. E. Hall, K. Burda, L. B. Corbeil, J. R. Duncan, and A. J. Winter. 1973. Persistent genital tract infection with Vibrio fetus intestinalis associated with serotypic alteration of the infecting strain. Am. J. Vet. Res. 34:1399-1403. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath. 1980. Approved list of bacterial names. Int. J. Syst. Bact. 30:225-420. Smibert, R. M. 1974. Genus I1 Campylobacter Sebald and Veron 1963, 907, p. 207-212. In R. E. Buchana and N. E. Gibbons (ed.), Bergey’s Manual of Determinative Bacteriology, 8th ed. Williams and Wilkins, Baltimore. Smibert, R M. 1984. Genus I1 Campylobacter Sebald and VCron 1963, p. 111-118. In N. R. Krieg and H. G. Holt (ed.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, MD. Staley, J. T., and N. R. Krieg. 1984. Classification of procaryotic organisms: an overview, p. 1-4. In N. R. Krieg and J. G. Holt (ed.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkins, Baltimore, MD. Stegenga, T., and J. I. Terpstra. 1949. Over Vibrio fetus infecties bij het rund en “enzootische” steriliteit. Tijdschr. Diergeneesk. 74~293-296. Thompson, L. M., 111, R. M. Smibert, J. L. Johnson and N. R. Krieg. 1988. Phylogenetic study of the genus Campylobacter. lnt. J. Syst. Bacteriol. 38:190-200. Thompson, S. A., and M. J. Blaser. 2000. Pathogenesis of Campylobacter fetus infections, p. 321-347. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd. ed. American Society for Microbiology, Washington, D. C. Tremblay, C., and C. Gaudreau. 1998. Antimicrobial susceptibility testing of 59 strains of Campylobacter fetus susbp. fetus. Antimicrob. Agents Chemother. 42:1847-1849. Tremblay, C., C. Gaudreau, and M. Lorange. 2003. Epidemiology and antimicrobial susceptibilities of 111 Campylobacter fetus subsp. fetus strains isolated in Quebec, Canada, from 1983 to 2000. J. Clin. Microbiol. 41:463-466. Tu, 2.-C., F. E. Dewhirst, and M. J. Blaser. 2001. Evidence that the Campylobacter fetus sap locus is an ancient genomic constituent with origins before mammals and reptiles diverged. Infect. Immun. 69:2237-2244. Tu, 2.-C., G. Zeitlin, J-P. Gagner, T. Keo, B. A. Hanna, and M. J. Blaser. 2004. Campylobacter fetus of reptile origin as a human pathogen. J. Clin. Microbiol. 42:4405-4407. Tu, Z. C., W. Eisner, B. N. Kreiswirth, and M. J. Blaser. 2005. Genetic divergence of Campylobacter fetus strains of mammal and reptile origins. J. Clin. Microbiol. 43:3334-3340. van Bergen, M. A. P. 2005. Subspecies differentiation and typing of Campylobacter fetus. Ph.D. thesis. Utrecht University, Utrecht, The Netherlands. van Bergen, M. A. P., K. E. Dingle, M. C. Maiden, D. G. Newell, L. van der Graaf-Van Bloois, J. P. M. van Putten, and J. A.

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Wagenaar. 2005a. Clonal nature of Campylobacter fetus as defined by multilocus sequence typing. J. Clin. Microbiol. 43: 5 888-9 8. van Bergen, M. A. P., S. Linnane, J. P. M. van Putten, and J. A. Wagenaar. 2005b. Global detection and identification of Campylobacter fetus subsp. uenerealis. Rev. Sci. Tech. 24:1017-26. van Bergen, M. A. P., G. Simons, L. van der Graaf-van Bloois, J. P. M. van Putten, J. Rombout, I. Wesley, and J. A. Wagenaar. 2005c. Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies. J. Med. Microbiol. 54: 1 217-24. Vandamme, P. 2000. Taxonomy of the family Campylobacteraceae, p. 3-26. In I. Nachamkin and M. J. Blaser (ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC. Vandamme, P., B. Pot, E. Falsen, K. Kersters, and J. De Ley. 1990. Intra- and interspecific relationships of veterinary Campylobacters revealed by numerical analysis of electrophoretic protein profiles and DNA:DNA hybridizations. Syst. Appl. Microbiol. 13:295-3 03. Varga, J., B. Mezes, L. Fodor, and I. Hajtos. 1990. Serogroups of Campylobacter fetus and Campylobacter jejuni isolated in cases of ovine abortion. Zbl. Vet. Med. B 37:148-152. Vargas, A. C., M. M. Costa, M. H. Vainstein, L. C. Kreutz, and J. P. Neves. 2003. Phenotypic and molecular characterization of bovine Campylobacter fetus strains isolated in Brazil. Vet. Microbiol. 93:121-132. VCron, M., and R. Chatelain. 1973. Taxonomic study of the genus Campylobacter Sebald and VCron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) Sebald and VCron. Int. J. Syst. Bacteriol. 23:122-134. Wagenaar, J. A., M. A. P. van Bergen, D. G. Newell, R. GrogonoThomas, and B. Duim. 2001. Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals. J. Clin. Microbiol. 39:2283-2286. Wang, E., M. M. Garcia, M. S. Blake, 2. H. Pei, and M. J. Blaser. 1993. Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus. J. Bacteriol. 175:49794984. Wang, G., C. G. Clark, T. M. Taylor, C. Pucknell, C. Barton, L. Price, D. L. Woodward, and F. G. Rodgers. 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis and C. fetus subsp. fetus. J. Clin. Microbiol. 40:4744-4747. Watson, W. A., D. Hunter, and R. Bellhouse. 1967. Studies on vibrionic infection of sheep and carrion crows. Vet. Rec. 81:220226. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, P. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Triiper. 1987. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37: 463-464. Wesley, I. V., R. D. Wesley, M. Cardella, F. E. Dewhirst, and B. J. Paster. 1991. Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16s rRNA sequences. J. Clin. Microbiol. 29:1812-1817. Willoughby, K., P. F. Nettleton, M. Quirie, M. A. Maley, G. Foster, M. Toszeghy, and D. G. Newell. 2005. A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies uenerealis: use on UK isolates of Campylobacter fetus and other Campylobacter spp. J. Appl. Microbiol. 99:758-766. Yang, L. Y., Z. H. Pei, S. Fujimoto, and M. J. Blaser. 1992. Reattachment of surface array proteins to Campylobacter fetus cells. J. Bacteriol. 174: 1258-1267.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

Chapter 12

Diagnosis and Antimicrobial Susceptibility of Campylobacter Speciest COLLETTEFITZGERALD, JEANWHICHARD, AND IRVINGNACHAMKIN

Campylobacter jejuni subsp. jejuni (referred to as C. jejuni throughout this chapter) and C. coli have been recognized since the late 1970s as important agents of gastrointestinal infections. Within the genus Campylobacter, C. jejuni and C. coli are the most common species associated with diarrheal illness in humans and cause clinically similar illnesses (Gupta et al., 2004) Although most laboratories do not routinely distinguish these organisms, 80 to 90% of Campylobacter infections in industrialized countries are thought to be due to C. jejuni and 5 to 10% due to C. coli when the diagnosis is performed solely on selective media. The distribution of species may be different in other parts of the world and when a nonselective isolation technique, such as the filter technique, is applied in conjunction with a selective medium (Allos et al., 1995; Le Roux and Lastovica, 1998; Taylor, 1992). C. jejuni and C. coli cause a spectrum of illnesses in humans. Fever, abdominal cramping, and diarrhea (with or without blood or fecal white cells) are prominent features of uncomplicated illness and may last for as little as a few days to more than 1 week. Campylobacter infections are generally self-limited with a relapse rate of 5 to 10% in untreated patients (Blaser, 1995). Patients with Campylobacter infection may manifest signs and symptoms of acute appendicitis and result in unnecessary surgery. Bacteremia, endocarditis, meningitis, urinary tract infection, and other extraintestinal diseases may result from Campylobacter infection (Blaser, 1995). Bacteremia occurs at an estimated rate of 1.5 per 1,000 intestinal infections, with the elderly having the highest rate (Sltirrow et al., 1993). Patients with immunodeficien-

cies, such as in patients with human immunodeficiency virus, may develop persistent diarrheal illness and bacteremia (Perlman et al., 1988). C. jejuni is now the most recognized antecedent cause of Guillain-BarrC syndrome, an acute paralytic disease of the peripheral nervous system, which is reviewed in chapter 13. Death after C. jejuni infection is rare but does occur (Font et al., 1997). CLASSIFICATION

Campylobacter, Arcobacter, and Sulfurospirillum are members of the family Campylobacteraceae (Vandamme et al., 1991, 1992; Vandamme and De Ley, 1991). An extensive review on the taxonomy of Campylobacter is in chapter 1. Specimen Considerations Fecal samples should routinely be submitted to the laboratory for isolation of Campylobacter species from patients with diarrheal illness. Although not ideal, rectal swabs are also acceptable for culture. Like the other common enteric pathogens (e.g., Salmonella and Shigella), Campylobacter infections are usually community acquired, and therefore, cultures for Campylobacter should not be routinely performed on hospitalized patients with diarrhea according to the 3-day rule (Hines and Nachamkin, 1996). Although culture of a single fecal specimen during acute illness has high sensitivity, two fecal samples may be required to rule out infection (Valenstein et al., 1996).

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Collette Fitzgerald and Jean Whichard Enteric Diseases Laboratory Branch (Proposed), Centers for Disease Control and Prevention, Irving Nachamkin Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Atlanta, GA 30333. Medicine, Philadelphia, PA 19104-4283. +This chapter contains information presented in chapter 3 by Irving Nachamkin, Jorgen Engberg, and Frank Moller Aarestrup in the 2nd edition of this book.

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Numerous formulations of transport media have been used for Campylobacter, such as alkaline peptone water with thioglycolate and cystine (Wang et al., 1983), modified Stuart medium (Ah0 et al., 1988), and Cary-Blair medium (Cary and Blair, 1964). Buffered glycerol saline and Stuart medium do not have good performance characteristics (Wang et al., 1983). As a single transport medium, modified Cary-Blair medium containing reduced agar (1.6 g/liter) is quite useful and is suitable for Campylobacter and other enteric pathogens (Wang et al., 1983). If cultures are not performed immediately, specimens received in Cary-Blair medium should be stored at 4°C. Supplementation of Cary-Blair medium with laked sheep’s blood aids in prolonged storage of stool samples and recovery of C. jejuni (Wasfy et al., 1995).

Blood Samples Campylobacter species are occasionally isolated from blood cultures, primarily C. fetus, C. jejuni, and C. upsaliensis, but the optimal conditions for isolating Campylobacter from blood culture systems have not been evaluated to any great degree. Commercial blood culture systems such as the BACTEC system (BD, Sparks, MD) (aerobic bottles) and Septi-Chek system (BD, Sparks, MD) support the growth of the common Campylobacter species (Kasten et al., 1991; Lastovica et al., 1989; Wang and Blaser, 1986). Anaerobic broth or lysis centrifugation may not be as sensitive for the isolation of Campylobacter species from blood samples (Kasten et al., 1991). Direct Detection in Stool Samples A rapid and sensitive method for presumptive diagnosis of Campylobacter enteritis may be accomplished by performing Gram stain analysis of stool samples. The most commonly used Gram-stain procedure uses safranin counterstain, which does not visualize Campylobacter well; however, carbol fuchsin or 0.1% aqueous basic fuchsin counterstain works well for visualizing organisms in smears of stools or pure cultures (Park et al., 1983; Sazie and Titus, 1982; Wang and Murdoch, 2004). The sensitivity and specificity of stool Gram stain for detecting Campylobacter during the acute stage of illness is high, with sensitivity ranging from 66 to 94% and >95% specificity (Park et al., 1983; Sazie and Titus, 1982; Wang and Murdoch, 2004). Other methods have been used to directly detect organisms in fresh stool, such as phase contrast and dark-field microscopy; however, these methods are more technically de-

manding (Karmali and Fleming, 1979; Paisley et al., 1982). Some clinicians look for the presence of fecal white cells as an indicator of bacterial infection and may be present during Campylobacter infection, with positive results in 25 to 80% of culture-proven cases (Hines and Nachamkin, 1996). The presence of fecal white cells (or other white cell markers) may indicate a higher likelihood of bacterial infection; however, the lack of white cells (or white cell markers) does not discount the diagnosis (Valenstein et al., 1996). Further, there is no clinical evidence that management of bacterial infections with Campylobacter should be different on the basis of the presence or absence of fecal white cells. Thus, we do not recommend routine examination of stool samples for fecal leukocytes for predicting bacterial infection. Further, fecal white cell analysis should not be used for selective culturing for Campylobacter or other stool pathogens (Guerrant et al., 2001). Stool antigen tests for Campylobacter have been developed and are commercially available. The ProSpecT Carnpylobacter immunoassay (AlexonTrend, Inc., distributed through Remel) varies in sensitivity from 80 to 96% compared with culture and has a specificity of >97% (Dediste et al., 2003; Hindiyeh et al., 2000; Tolcin et al., 2000). There are some reported cross-reactions with C. upsaliensis, and antigen may be detected after cold storage for several days (Dediste et al., 2003; Endtz et al., 2000). Another commercial assay, Ridascreen Campylobacter (R-Biopharm, Darmstadt, Germany), was recently shown to have 69% sensitivity and 87% specificity in an analysis of 1,050 stool samples submitted for culture (Tissari and Rautelin, 2007). There is a need for molecular-based assays to detect Campylobacter in stool samples (Persson and Olsen, 2005). Commercial molecular systems are not yet available for detecting Campylobacter or for other enteric pathogens directly in fecal specimens. Laboratories continue to have difficulty isolating and identifying Campylobacter spp. in stool cultures, and molecular approaches may improve the ability of laboratories to diagnose these infections. PCR-based detection, identification to the species level, and typing of campylobacters directly from stool samples has been reported (Amar et al., 2007; Best et al., 2007; Linton et al., 1997b). Advantages of molecular approaches over culture include same-day detection, additional data regarding mixed infections, and uncommon Campylobacter species that are often missed when routine culture and procedures that are amenable to automation and high-throughput capabilities are used (Kulkarni et al., 2002; Lawson et al., 1999; Maher et al., 2003). However, PCR does not provide

CHAPTER 12

an isolate for further characterization such as susceptibility testing or subtyping. In addition, diagnostic laboratories are required to comply with clinical laboratory improvement amendments regulations in the United States, so it is important to ensure that quality standards are first established for use of molecular diagnostic assays to maintain the accuracy, reliability, and timeliness of patient test results.

CULTURE AND ISOLATION Enrichment A number of enrichment broths have been formulated to improve the recovery of Campylobacter from stool samples, including Preston broth (Bolton and Robertson, 1982), Campy-thio (Rubin and Woodard, 1983), and Campylobacter enrichment broth (Martin et al., 1983). It is unclear whether enrichment cultures are useful for primary isolation of Campylobacter during acute illness because of the high concentration of organisms present in the stool. However, enrichment cultures may enhance the yield of culture where low numbers of organisms are expected because of delays in transport of the sample to the laboratory. After the acute stage of disease when the concentration of organisms decrease, or in the investigation of Guillain-BarrC syndrome after acute Campylobacter infection, enrichment cultures may be necessary to recover Campylobacter (Nachamkin, 1997). Filtration In addition to direct plating of samples, filtration techniques were designed to isolate Campylobacter species including C. jejuni and C. coli as well as other Campylobacter species (Engberg et al., 2000; Goossens et al., 1986; Vandenberg et al., 2004) and Arcobacter spp. (Kiehlbauch et al., 1991; Vandenberg et al., 2004) that are susceptible to antibiotics present in most selective media (Vandenberg et al., 2004). Filtration methods should be used as an adjunct method in addition to direct plating for the assessment of clinical specimens. Because of the small size of Campylobacter, organisms pass through membrane filters with pore sizes of 0.45 to 0.65 pm. Wells et al. (1989) recommend cellulose acetate membrane filters (0.65-pm pore size) for routine use, and they are available commercially. Mixed cellulose ester membrane filters (0.60-pm pore size) were found to be optimal in the filtration method described by Lastovica (2006; see chapter 7). The method is easily performed by placing a sterile 0.65-pm cellulose acetate filter onto the

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surface of an antibiotic-free agar medium, placing 10 to 15 drops of fecal suspension on the filter, and incubating the plate at 37°C for 60 min. The filter is removed and the plate incubated at 37°C under microaerobic conditions. Because many of the species not isolated on routine selective media require increased hydrogen environments, the filtration method is best used if an atmosphere containing increased hydrogen can be used. Filtration is a relatively insensitive method and requires that the stool samples contain -lo5 CFU/ml of Campylobacter in order to detect the organism (Goossens et al., 1990b).

ISOLATION PROCEDURES A microaerobic atmosphere containing approximately 5% 0,, 10% CO,, and 85% N, is required for optimal recovery of most Campylobacter species. Gas generator packs that generate suitable environments are available from commercial sources. Specialized incubators such as the tri-gas incubator and evacuation replacement procedures may also be used (Morris and Patton, 1985; Thompson et al., 1990). Candles jars should not be used for isolation and maintenance of Campylobacter species because of the suboptimal environment produced with this method (Luechtefeld et al., 1982). An increased hydrogen concentration in the incubation gas mixture is required to isolate several Campylobacter species, including C. sputorum, C. concisus, C. mucosalis, C. curvus, C. rectus, and C. hyointestinalis (Vandenberg et al., 2004). Unfortunately, commercial gas pack systems do not generate sufficient hydrogen for effective isolation of hydrogen-requiring Campylobacter species. An environment containing 10% CO,, 6%H2, and the balance N, should be sufficient for isolating hydrogen-requiring species (Bolton, 2001). Campylobacter jejuni and C. coli are readily isolated on a variety of selective media. Charcoal cefoperazone deoxycholate agar (Hutchinson and Bolton, 1984) and charcoal-based selective medium (Karmali et al., 1986) are blood-free media, and Campy-CVA medium (Reller et al., 1983) and Skirrow medium (Skirrow, 1977) are blood-containing media used for routine isolation procedures. A selective medium for isolation of C. upsaliensis (CAT medium) has also been described (Aspinall et al., 1996), but its effectiveness has not been regularly demonstrated (Engberg et al., 2000; Hindiyeh et al., 2000). C. upsaliensis may be isolated by other media or filtration methods. A hydrogen-enriched atmosphere may enhance growth of some strains (Goossens et al., 1990b; Lastovica and Skirrow, 2000).

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Several studies have shown that the use of a combination of media, including either charcoal cefoperazone deoxycholate agar or charcoal-based selective medium, achieves a higher yield of Campylobacter from stool samples (Endtz et al., 1991; Gun-Monro et al., 1987) than the use of only a single medium. As a single medium, charcoal cefoperazone deoxycholate agar medium was found to be the most sensitive for detecting C. jejuni and C. coli compared with Skirrow’s medium, CAT agar, and filtration technique (Engberg et al., 2000). Most selective media for isolation of Campylobacter contain antibiotics to inhibit the growth of normal enteric bacteria. Some antibiotics including cephalothin, colistin, and polymyxin B that are present in some formulations inhibit some strains of C. jejuni and C. coli and C. fetus subsp. fetus (Goossens et al., 1986; Ng et al., 1988). Cephalothin will inhibit C. jejuni subsp. doylei, C. upsaliensis, and A. butzleri, and thus, we do not recommend cephalothincontaining culture media such as Campy-BAP for isolation of Cumpylobacter and Arcobacter from fecal samples.

IDENTIFICATION

Cumpylobacter species are difficult to identify phenotypically because of relative biochemical inactivity, special growth requirements, and complex taxonomy. Numerous schemes are thus available for phenotypic and genotypic characterization of these bacterial species (On, 1996). Campylobacter colonies may have different appearances and depend on the type of medium used for primary isolation. Cumpylobucter spp. usually produce gray, flat, irregular, and spreading colonies on most isolation media and nonhemolytic colonies on blood-containing media. When utilizing freshly prepared media, spreading along the streak line is commonly seen. With decreased moisture content, reduced spreading is observed, and colonies may form a round, convex, and glistening appearance. Thus, for optimal isolation and identification of Campylobacter species, proper storage of media to control moisture content is essential. Arcobacter colonies are similar in appearance to Campylobucter (Vandamme, 2000; Vandamme et al., 1992). At primary isolation, colonies should be examined by Gram stain, and an oxidase test should be performed. Campylobucter species give a positive reaction with oxidase reagent. Colonies that exhibit positive oxidase reactions along with a typical Gram stain appearance can be presumptively reported as Cumpylobacter species subject to further testing with

the hippurate hydrolysis test (MacFaddin, 2000a). C. jejuni is distinguished from other species on the basis of the hippurate hydrolysis test. Oxidase-positive, curved (or S-shaped), gram-negative rods that grow at 42°C and are hippurate positive can be reported as C. jejuni subsp. jejuni, and further characterization is not routinely necessary. Not all strains of C. jejuni are hippurate positive by conventional biochemical assays and may require alternative assays, such as gas-liquid chromatography for detecting benzoic acid (liberated from hydrolysis of sodium hippurate) (Steinbruckner et al., 1999). Molecular detection of the hip0 (hippuricase) gene (Hani and Chan, 1995) or other C. jejuni-specific DNA markers (Table 1) by PCR may also be useful for identifying phenotypically hippurate-negative isolates (Totten et al., 1987), weakly positive isolates (Burnett et al., 2002a) and to clarify false-positive results for non-C. jejuni species (Denis et al., 1999). However, evaluation of C. jejuni-specific PCR tests has showed no single test to be entirely specific or sensitive; therefore, the use of more than one target for molecular identification of C. jejuni is recommended, and assay validation is important (Burnett et al., 2002b; On and Jordan, 2003). In addition, purified DNA rather than crude cell lysates may be needed for some PCR assays (Mohran et al., 1998; On and Jordan, 2003). C. coli is similar to C. jejuni biochemically except for hippuricase activity, for which C. coli is lacking. Molecular methods are recommended to accurately identify C. coli (Siemer et al., 2005), and most have proved both accurate and sensitive (On and Jordan, 2003). Disk antimicrobial susceptibility tests (nalidixic acid, cephalothin) have traditionally been used to aid in the identification of Cumpylobacter jejuni; however, disk tests are no longer appropriate for species identification because of increasing resistance to fluoroquinolones worldwide. The identification of species other than C. jejuni is difficult by using phenotypic characterization, and an algorithm for identification of the thermophilic Campylobucter spp. was recently published (Fitzgerald and Nachamkin, 2007). Phenotypic tests that may be useful for initial identification include temperature growth studies, catalase production, hippurate hydrolysis, indoxyl acetate hydrolysis (MacFaddin, 2000b), and production of H,S (Barrett et al., 1988). Some species of thermophilic Cumpylobucter may be differentiated by the indoxyl acetate hydrolysis test. Commercial systems for identifying Campylobacter species are available. In the United States, there are two immunologic reagents for culture identification: INDX Campy-JCL (Hardy Diagnostics and

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Table 1. Commonly used gene targets and PCR assays for genus and species differentiation of Campylobacter spp. Gene target

hipO" mapA" ceuE" , asp' nap locus (multiplex) Unknown (multiplex)" 1 6 s rRNA

Species detected

Reference(s)

C. jejuni C. jejuni C. coli C. coli C. jejuni subspecies C. jejuni and C. coli Genus Campylobacter C. upsaliensis, C. helveticus, C. fetus, C. hyointestinalis, and C. lari C. hominis C. lanienae

Linton et al. (1997a) Stucki et al. (1995) Gonzalez et al. (1997) Linton et al. (1997a) Miller et al. (2007) Vandamrne et al. (1997) Linton et al. (1996); Vanniasinkarn et al. (1999) Linton et al. (1996)'

C. gracilis C. rectus

Siqueira and Rocas (2003) Ashimoto et al. (1996); Siqueira and Rocas (2003) A1 Rashid et al. (2000) Maher et al. (2003)

Lawson et al. (1998) Inglis and Kalischuk (2003); Logan et al. (2000)

dYAb 16123s rRNAb GTPase' lpxA (multiplex) Unknown Unknown 23s rRNA GyrB/ hypothetical protein

C. jejuni, C. coli, C. lari, and C. upsaliensis Genus Campylobacter, C. jejuni, C. coli, C. upsaliensis, and C. lari C. jejuni, C. coli, C. lari, and C. upsaliensis C. jejuni, C. coli, C. lari, C. upsaliensis C. fetus C. fetus subsp. venerealis C. concisus C. concisus

van Doom et al. (1999) Klena et al. (2004) Hum et al. (1997) van Bergen et al. (2005) Bastyns et al. (1995) Matsheka et al. (2001)

PCR sensitivity and sensitivity evaluated by On and Jordan (2003). PCR probe-based methods. "PCR assays for C. upsuliensis and C. helveticus or C. fetus and C. hyointestinulis are duplex reactions that use a common forward primer and species-specific reverse primers.

Fisher Scientific) and Dry Spot Campylobacter Test Kit (Remel). INDX Campy-JCL does not differentiate between C. jejuni and C. coli (Nachamkin and Barbagallo, 1990). The Dry Spot Campylobacter latex test identifies, but does not differentiate, C. jejuni, C. coli, C. lari, or C. upsaliensis with variable results for C. fetus subsp. fetus (Oxoid USA, http:// www.oxoid.com/). Several studies have evaluated nucleic acid probes (Accuprobe, Gen-Probe Inc., San Diego, CA) for genus-level identification of Campylobacter and detection of C. jejuni subsp. jejuni, C. jejuni subsp. doylei, C. coli, and C. lari (PopovicUroic et al., 1991; Tenover et al., 1990); it may also hybridize with C. hyointestinalis strains (PopovicUroic et al., 1991). If phenotypic tests are inconclusive, immunologic and molecular probe identification may be useful for confirming Campylobacter to the genus level. The problems associated with the standard phenotypic methods for species identification of Cumpylobacter spp. have led to the development of a growing number of PCR assays for species-specific identification of Campylobacter. The 16s rRNA gene and 23s rRNA gene are two widely used targets for the design of species-specific tests; PCR assays based on these targets have been described for 12 different

Cumpylobacter species (On, 2005). Other gene targets used for species-specific PCR assays include gyrA (Menard et al., 2005), glyA (A1 Rashid et al., 2000), ceuE gene (Gonzalez et al., 1997), asp (Linton et al., 1997a), lpxA (Klena et al., 2004), and a GTPase gene (van Doorn et al., 1999). Subspecies identification by PCR within C. fetus (Hum et al., 1997; van Bergen et al., 2005) and C. jejuni (Miller et al., 2007) has also been described (see also chapter 11).Other assays involve restriction fragment analysis of PCRamplified regions of the 16s or 23s rRNA genes for differentiation of the thermotolerant Campylobacter spp. (Fermer and Engvall, 1999; Hurtado and Owen, 1997). These assays require further validation to fully determine their specificity and sensitivity (On, 2005). More recently, species-specific microarrays have been described for identification of several Campylobacter species including C. jejuni, C. coli, C. lari, and C. upsaliensis (Keramas et al., 2003; Quinones et al., 2007; Volokhov et al., 2003). Although these methods are promising tools for both identification and further genetic characterization of Campylobacter spp., the cost and limited availability of the technology in the clinical laboratory makes this approach not currently practical for routine application in the clinical setting.

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EPIDEMIOLOGIC TYPING SYSTEMS

Serology

Numerous typing systems have been developed to study the epidemiology of Campylobacter infection. They vary in complexity and ability to discriminate between strains. Some of the more common phenotypic methods used in studies include biotyping, phage typing, and serotyping, but the latter is the most commonly used phenotypic system (Newell et al., 2000; Patton and Wachsmuth, 1992). Originally described by Lior et al. (1982), the heat-labile system detects over 100 serotypes of C. jejuni, C. coli, and C. lari. The seroderminants of the heat-labile system include uncharacterized bacterial surface antigens and in some serotypes, flagellar antigens (Alm et al., 1991). The serotyping system described by Penner and Hennessy (1980), the heat-stable serotyping scheme, detects 60 types of C. jejuni and C. coli (Patton and Wachsmuth, 1992). The major serodeterminant of the heat-stable system is a Carnpylobacter capsular polysaccharide (Karlyshev et al., 2000). Because of the complexity of performing serotype analysis, only a few reference laboratories worldwide perform serotyping studies. A commercially produced serotyping kit is also available (Denka Seiken USA Inc., Campbell, CA). In recent years, numerous molecular subtyping systems have been developed, including restriction endonuclease analysis, ribotyping, PCR-based techniques, pulsed-field gel electrophoresis of macrorestricted chromosomal DNA, and amplified fragmentlength polymorphism (Newell et al., 2000; Patton and Wachsmuth, 1992). Rapid 1-day standardized PFGE protocols (Ribot et al., 2001) have facilitated the use of PFGE in the U.S. National Surveillance network, PulseNet, for food-borne pathogens (http:// www.cdc.gov/pulsenet/) (Olsen et al., 2001), but interpretation of PFGE profiles can be challenging (Fitzgerald et al., 2005). DNA sequence-based technology has emerged in recent years, and multilocus sequence typing has been useful for studies of the population structure and molecular epidemiology of C. jejuni (Dingle et al., 2001), although recognition of outbreaks caused by strains with common sequence types may be problematic (Clark et al., 2005; Sails et al., 2003). Genomotyping based on microarray technology has also been described for Campylobacter (Klena and Konkel, 2005). Unfortunately, a single method for determining strain relatedness and investigating outbreaks is not adequate, and multiple approaches may be required (Newell et al., 2000). A detailed discussion about typing systems and approaches to studying the epidemiology of Campylobacter can be found in chapter 10.

During the first few weeks of infection, fecal and serum immunoglobulin (Ig) A antibodies are apparent and then decrease precipitously, and as expected, serum IgG, I@, and IgA levels increase in response to infection (Black et al., 1992; Taylor et al., 1993). The performance characteristics of serum antibody assays are variable, and sensitivity, specificity, and predictive value will be population dependent. Patients with Campylobacter infection may have false-positive legionella antibody tests (Boswell and Kudesia, 1992). Serologic testing is not recommended for routine diagnosis of Campylobacter infection but appears to be useful for epidemiologic investigations (Taylor et al., 2004) (see chapter 6). Susceptibility Testing of Campylobacter The Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) has published an agar dilution method for testing Campylobacter spp. and includes quality control (QC) ranges for several antimicrobial agents (CLSI, 2005; McDermott et al., 2005a). CLSI has also recently approved a disk diffusion and broth microdilution method. QC ranges for eight antimicrobial agents are given for C. jejuni ATCC 33560 (McDermott et al., 2005b) (CLSI, 2006). This publication also includes interpretive criteria for erythromycin, ciprofloxacin, tetracycline, and doxycycline. Tests can be incubated at 36°C for 48 h or 42°C for 24 h. In our experience, 36°C for 48 h results in optimal growth for test interpretation. CLSI recommends performing primary testing of clinical isolates with erythromycin and ciprofloxacin. The Etest (PDM Epsilometer, AB Biodisk, Solna, Sweden) method, which uses Mueller-Hinton agar with 5% sheep’s blood, has been shown to compare favorably with the agar dilution method (Baker, 1992).

In Vitro Susceptibility Profiles C. jejuni and C. coli are almost universally resistant to penicillins, cephalosporins (except a few third-generation cephalosporins), trimethoprim, sulfamethoxazole, rifampicin, and vancomycin. Originally described as highly susceptible to erythromycin, fluoroquinolones, tetracyclines, aminoglycosides, and clindamycin and moderately susceptible to chloramphenicol, cefotaxime, ceftazidime, and cefpirome, increasing resistance to a variety of antimicrobial agents is occurring with increased frequency (McNulty, 1987; Skirrow and Blaser, 1995).

CHAPTER 12

-

Macrolides and Clindamycin Since the recognition of Cumpylobucter enteritis in the 1970s, erythromycin has commonly been used for treating patients with uncomplicated enteritis (Elharrif et al., 1985; Karmali et al., 1981; Nolan et al., 1983; Vanhoof et al., 1978). Erythromycin resistance rates in C. jejuni range from 0 to 12% and are generally higher in C . coli, ranging from 0 to 50% (Gibreel and Taylor, 2006) (chapter 6). Trends over time for erythromycin resistance show stable and low rates in countries like Japan, Canada, Finland, and Denmark, whereas recent data from Thailand and Sweden document development of resistance (Harnett et al., 1995; Sjogren et al., 1997). It is unclear whether erythromycin resistance develops during therapy, although posttreatment resistant isolates have been described (Funke et al., 1994; Pitkanen et al., 1982; Snijders et al., 1997). The activity of clindamycin against C. jejuni is equivalent to that of erythromycin, and the drug may be a useful alternative for the treatment of serious infections in children. However, reported resistance rates are variable (CDC, 1998; Guevremont et al., 2006; Li et al., 1998; Papavasileiou et al., 2007; Reina et al., 1994; Taylor et al., 1987). Quinolones The fluoroquinolones have activity against most major pathogens causing bacterial enteritis. Early clinical trials of both community-acquired acute diarrhea and traveler’s diarrhea demonstrated good clinical response (Piddock, 1995; Wistrom and Norrby, 1995). Fluoroquinolones had good in vitro activity for all Cumpylobacter species as well as for members of the family of Enterobucteriuceue. Unfortunately , it soon became apparent that resistance in Campylobacter spp. can arise in vivo, sometimes even after just one or two administrations of fluoroquinolones (Adler et al., 1991). Additionally, an increasing number of reports claimed that fluoroquinoloneresistant strains were isolated from patients who had not received any medical treatment for their illness, suggesting that strains were already fluoroquinolone resistant before causing the infection (Chatzipanagiotou et al., 1993; Gaunt and Piddock, 1996; Li et al., 1998; Piddock, 1995; Reina et al., 1994; Sanchez et al., 1994). The 1990s were notable for a striking emergence of resistance among campylobacters and other enteric pathogens to nalidixic acid and fluoroquinolones. Before 1990, quinolone resistance among campylobacters was rarely reported. With the introduction of enrofloxacin and sarafloxacin (derivatives of cipro-

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floxacin) in veterinary medicine, and less importantly, fluoroquinolones in human medicine in mainland Europe, a rapid emergence of quinolone resistance in Cumpylobucter isolates from patients was registered (Adler et al., 1991; Chatzipanagiotou et al., 1993; Rautelin et al., 1991; Reina et al., 1992). Surveillance data on resistance rates in human isolates from Asia soon indicated an equal and worrisome increase (Kuschner et al., 1995; Tee et al., 1995). Studies in the late 1990s from Taiwan, Thailand, and Spain showed rates of fluoroquinolone resistance in C. jejuni or C. jejuni and C. coli of 57, 84, and 88%, respectively (Gallardo et al., 1998; Hoge et al., 1998; Li et al., 1998). With the approval of quinolones for veterinary use in the United Kingdom and in the United States in late 1993 and 1995, respectively, reports from these areas showed increasing quinolone resistance profiles (CDC, 1998; Frost and Thwaites, 1998; Nachamkin, 1994). Antimicrobial resistance in animals and relevance to human infections are discussed in more detail in chapters 6 and 36. More recent studies indicate variable rates of ciprofloxacin resistance, in some cases higher among travel-associated infections. Ciprofloxacin resistance in France in 2004 was 25.3% (Gallay et al., 2007). During the same year, isolates collected in the United States exhibited 19% ciprofloxacin resistance, higher in C. coli (30.8%) than C. jejuni (18.1%) (CDC, 2007). The U.S. Food and Drug Administration proposed withdrawal of fluoroquinolone approval for use in poultry in 2000. A federal administrative law process upheld the proposed withdrawal, which took effect in September 2005 (Nelson et al., 2007). Australia continued to show low prevalence of ciprofloxacin resistance in 2001 to 2002-only 2% for domestically acquired cases (Unicomb et al., 2006). By contrast, 82% of isolates collected in Thailand in 2002 to 2003 were ciprofloxacin resistant (Boonmar et al., 2005). In Denmark, 26.7% of domestically acquired infections in 2005 were ciprofloxacin resistant; 7 of the 10 infections acquired abroad were resistant. The Netherlands documented 33% resistance among domestically acquired infections from 2002 to 2004, but 54% among travel-associated infections for the same time period (van Hees et al., 2007). Although ciprofloxacin resistance correlates well with nalidixic acid resistance (Boonmar et al., 2007; Kinana et al., 2006), some ciprofloxacin susceptibility among nalidixic acid-resistant isolates is commonly reported (Englen et al., 2007; Gallay et al., 2007; Jesse et al., 2006; McGill et al., 2006; National Antimicrobial Resistance Monitoring System, 2006; Varela et al., 2007) Occasionally ciprofloxacinresistant isolates are reported to be nalidixic acid susceptible (Bachoual et al., 2001; Reina et al., 1996).

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Induction of fluoroquinolone resistance during treatment is well recognized (Adler et al., 1991; Ellis et al., 1995; Segreti et al., 1992; Tee and Mijch, 1998). It has been predicted that in 10 to 20% of patients treated with a fluoroquinolone for Cumpylobacter enteritis, the Campylobacter strains will develop quinolone resistance (Piddock, 1997; Wistrom and Norrby, 1995). However, in a study by EllisPegler and colleagues (1995), between 18 and 28% of the patients in their prospective trial developed fluoroquinolone resistance. Development of resistance has been registered in short-term treatments, but prolonged therapy, i.e., in immunosuppressed patients, is a risk factor and has been associated with both clinical and bacteriological failures (Adler et al., 1991; Molina et al., 1995; Segreti et al., 1992; Tee and Mijch, 1998; Tee et al., 1995). In conclusion, the available information on fluoroquinolone resistance, frequent and rapid in vivo development of high-grade resistance and lack of clear clinical efficacy on community-acquired diarrhea suggest that fluoroquinolones may now be of limited use in the treatment of Campylobacter infections in many regions (Harnett et al., 1995; Hoge et al., 1998; Kuschner et al., 1995; Li et al., 1998; Mandal et al., 1984; Murphy et al., 1996; Pigrau et al., 1997; Rautelin et al., 1991; Ruiz et al., 1998; Sjogren et al., 1997). /.?-Lactam Agents With the exception of imipenem, the majority of C. jejuni and C. coli strains are resistant to p-lactam agents, principally penicillins and cephalosporins. However, they are moderately susceptible to cefotaxime, ceftazidime, and cefpirome (Van der Auwera and Scorneaux, 1985). p-Lactamase-producing strains of C. jejuni and C. coli are frequently identified (reviewed by Li et al., 2007). The chromosomal molecular class D p-lactamase of C. jejuni and C. cob seems to play a role only in resistance to amoxicillin, ampicillin, and ticarcillin, and a novel enzyme, OXA61, has also been described in a clinical C. jejuni isolate collected in 2001 (Alfredson and Korolik, 2005). With penicillin G, piperacillin, and cephalosporins, the mechanism of resistance in C. jejuni and C. coli is primarily considered to depend on their limited ability to bind to penicillin-binding proteins and permeability (Lachance et al., 1991, 1993; Tajada et al., 1996). Ampicillin is not recommended for use in the treatment of C. jejuni infections (Gaudreau and Gilbert, 1998). The reported rate of resistance to amoxicillin and clavulanic acid has been low (Gaudreau and Gilbert, 2003; Rodriguez-Avial et al., 2006), but data on the efficacy for treatment of Campylobacter

bacterernia are sparse (Pigrau et al., 1997). Imipenem may be useful for serious systemic infections in patients with renal damage, where the use of aminoglycosides may be problematic (Gaudreau and Gilbert, 2003; Skirrow and Blaser, 1995). Tetracycline Although tetracyclines have been suggested as an alternative choice in the treatment of C. jejuni and C. coli enteritis, large geographical differences on their susceptibility have been reported. Rates of resistance reported in the 1990s in Denmark were 0 to 11% (Aarestrup et al., 1997; Danish Zoonosis Centre, 1998), in Spain 25% (Gomez et al., 1995), in the United States 48% (CDC, 1998; Nachamkin, 1994), in Israel 70% (Schwartz et al., 1993), in Singapore 79% (Lim and Tay, 1992), and in Taiwan 85 to 95% (Li et al., 1998). In a Canadian study, Gaudreau and Gilbert (1998) noticed a significant increase in the resistance of C. jejuni to tetracycline from 19.1% in 1985 to 1986 to 55.7% in 1995 to 1997. In 2005, prevalence in Denmark was 16% for domestically acquired infections and 50% among those acquired abroad (Danish Zoonosis Centre, 2006). Forty-six percent of isolates in the United States were resistant in 2004, and 70% of clinical isolates collected in the Madrid area of Spain in 2003 were resistant (Rodriguez-Avial et al., 2006). Tetracyclines are ecologically disadvantageous drugs with a broad antibacterial spectrum, and their use is contraindicated in children. General use of tetracycline is not recommended but can be used in areas of low resistance to the agent, or better, after susceptibility testing of the clinical isolate in situations when other agents are contraindicated because of strain resistance or idiosyncratic responses in patients. Multidrug Resistance Multidrug resistance in Campylobacter appears to be occurring more frequently and poses the risk that an effective antimicrobial regimen to treat infection may be lacking. Hoge and colleagues (1998) from Thailand found 100% coresistance between azithromycin and ciprofloxacin in the last 2 years of a 15-year investigation (1981 to 1995). In addition, the level of tetracycline and ampicillin resistance in this area is now so high that these agents have no role in the treatment of Campylobacter or noncholera diarrhea in general. Chung-Chen and colleagues from Taiwan (Li et al., 1998) reported that concomitant resistance rates among their nalidixic acidresistant C. jejuni isolates from patients (exclusively children) were as follows: gentamicin 2%, erythro-

CHAPTER 12

mycin 12%, clindamycin 12%, tetracycline 97%, and ciprofloxacin 66%. A total of 100% of their human erythromycin-resistant C. jejuni isolates and 90% of their C. coli isolates were concomitantly resistant to clindamycin. Erythromycin and ciprofloxacin coresistance was found among C. jejuni collected in Quebec from 1998 to 2001, some of which were also resistant to tetracycline (Gaudreau and Michaud, 2003). Among ciprofloxacin-resistant C. jejuni collected in Finland from 1995 to 2000, Hakanen et al. (2003) found 3% coresistance to erythromycin or clindamycin, 68% coresistance to tetracycline, and 25% coresistance to ampicillin. Six of the eight erythromycin-resistant strains were ciprofloxacin resistant.

Campylobacter lari Until the 1990s, C. lari could easily be distinguished from other thermophilic species on the basis of intrinsic resistance to nalidixic acid (Nachamkin et al., 1984). However, since then, resistance to nalidixic acid and fluoroquinolones has emerged among C. jejuni and C. coli isolates, and nalidixic acidsensitive biovars of C. lari have been described (Megraud et al., 1988). Thus, more reliable tests are required to identify this species. The indication for treatment with antimicrobial agents is similar to that of C. jejuni and C. coli, and with the same classes of drugs, e.g., macrolides, aminoglycosides, imipenem, clindamycin, tetracycline, and fluoroquinolones according to susceptibility testing results (Bruneau et al., 1998; Chiu et al., 1995; Evans and Riley, 1992; Simor and Wilcox, 1987). As for the other thermophilic Campylobacter spp., fluoroquinolone resistance has also been described for this species (Evans and Riley, 1992). Thwaites and Frost (1999) reported no erythromycin resistance among C. lari isolated in England, but 100% resistance to nalidixic acid and ciprofloxacin.

Campylobacter upsaliensis No controlled trials of antibiotic treatment for C. upsaliensis enteritis have been performed, and experience with antimicrobial treatment is limited. The organism is typically sensitive to aminoglycosides, cephalosporins, tetracycline, and nalidixic acid and resistant to vancomycin, methicillin, piperacillin, and chloramphenicol (Bourke et al., 1998; Preston et al., 1990). C. upsaliensis is usually sensitive to erythromycin, but up to 10% resistance has been observed (Goossens et al., 1990a). Jenkin and Tee (1998) reported resistance to fluoroquinolones in three isolates from two human immunodeficiency virus-

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seropositive patients, suggesting that routine susceptibility testing for this species, as for any other species in this genus, is advised. Vandenberg et al. (2006) found 12% nalidixic acid resistance, 6% ciprofloxacin resistance, and 13% erythromycin resistance among 85 C. upsaliensis isolated from humans in Belgium from 1995 to 2005.

Campylobacter fetus subsp. fetus Reported C. fetus cases most often involve bloodstream and extraintestinal infections that are often prolonged and relapsing. The prognosis depends largely on the severity of underlying illnesses. Immunocompetent patients with uncomplicated enteritis may not require antibiotics. Systemic infections require parenteral therapy, but erythromycin is not always effective and is no longer a recommended treatment for systemic C. fetus infections (Allos et al., 1995; Francioli et al., 1985; Kwon et al., 1994; Tremblay and Gaudreau, 1998). Appropriate antimicrobial therapy for serious infections due to fetus is not fully assessed for two reasons. First, C. fetus infection is rare, and second, many patients die despite intensive therapy as a result of associated predisposing factors (Goossens et al., 1989). However, patients with endovascular infections due to C. fetus require at least 4 weeks of treatment, and gentamicin with ampicillin seems to be the combination of choice (Blaser et al., 1978; Goossens et al., 1989).Imipenem may be a useful alternative (Dronda et al., 1998; Tremblay and Gaudreau, 1998). An abdominal aortic aneurism infected by C. fetus was successfully treated with surgery and ciprofloxacin (la Scola et al., 1998). A 2003 study of C. fetus subsp. fetus isolated from humans in Quebec found resistance to ciprofloxacin (5%), erythromycin (71%), and tetracycline (34%) (Tremblay et al., 2003). Most studies have used agar dilution to assess the susceptibility of C. fetus. However, Tremblay and Gaudreau (1998) compared minimal inhibitory concentration (MIC), disk diffusion, and Etest on 59 C. fetus subsp. fetus isolates. They found relative good agreement between the three methods for most antimicrobial agents. However, results for cefotaxime and erythromycin varied from 0 to 90% and 39 to 100% susceptible, respectively. In general, C. fetus were found to be susceptible to most antimicrobials, except tetracycline, with 27% resistant isolates.

c.

Arcobacter butzleri and A. ctyaerophilus Kiehlbauch and colleagues (1992) studied 78 human and animal isolates of Arcobacter butzleri and A. cyaerophilus for susceptibility to 22 antimicrobial

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agents. Most isolates were resistant to macrolides including erythromycin, cephalosporins except cefotaxime, ampicillin, ampicillin-sulbactam, clindamycin, chloramphenicol, and trimethoprim-sulfamethoxazole, whereas the fluoroquinolones, aminoglycosides, and one tetracycline (minocycline) demonstrated most activity. Fluoroquinolone resistance has also been reported (Lerner et al., 1994). Two studies have addressed MIC distributions and resistance prevalence among Arcobacter isolated in Belgium (Houf et al., 2004; Vandenberg et al., 2006). Houf et al. (2004) found that the nalidixic acid MICs for human isolates collected from 1995 to 2001 were 1 to 64 pg/ml, whereas all ciprofloxacin MICs were 50.25. Erythromycin MICs ranged from 2 to 32 pg/ml. The MIC distributions for poultry isolates were found to be similar, except that the ciprofloxacin MICs for 3 of the 68 A. butzleri isolates from poultry products were 16 pg/ml. Vandenberg et al. (2006) found no antimicrobial resistance among the 10 human clinical A. cryaerophilus isolates tested, whereas resistance was found among A. butzleri: nalidixic acid 18%, ciprofloxacin 3%, and erythromycin 21%. No tetracycline resistance was found among A. butzleri. C . concisus

Little information is available on the antimicrobial susceptibility of C. concisus. Greig and colleagues from South Africa have tested the MIC values of eight isolates and found that ciprofloxacin was the most active agent. All strains were also sensitive to tetracycline, ampicillin, and gentamicin. In contrast, all but one of the strains were resistant to erythromycin. Activity of cephalosporins was variable. Vandenberg et al. (2006) found 16 of 20 C. consisus isolates tested were resistant to nalidixic acid, but only one of these was resistant to ciprofloxacin. Acknowledgments. Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.

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Vandenberg, O., A. Dediste, K. Houf, S. Ibekwem, H. Souayah, S . Cadranel, N. Douat, G. Zissis, J. P. Butzler, and P. Vandamme. 2004. Arcobucter species in humans. Emerg. Infect. Dis. 10~1863-1867. Vandenberg, O., K. Houf, N. Douat, L. Vlaes, P. Retore, J. P. Butzler, and A. Dediste. 2006. Antimicrobial susceptibility of clinical isolates of non-jejuni/coli campylobacters and arcobacters from Belgium. J. Antimicrob. Chernother. 57:908-913. Vanhoof, R., M. P. Vanderlinden, R. Dierickx, S . Lauwers, E. Yourassowsky, and J. P. Butzler. 1978. Susceptibility of Campylobacter fetus subsp. jejuni to twenty-nine antimicrobial agents. Antimicrob. Agents Chemother. 14553-556. Vanniasinkam, T., J. A. Lamer, and M. D. Barton. 1999. PCR for the detection of Cumpylobucter spp. in clinical specimens. Lett. Appl. Microbiol. 2852-56. Varela, N. P., R. Friendship, and C. Dewey. 2007. Prevalence of resistance to 11 antimicrobials among C. coli isolated from pigs on 80 grower-finisher farms in Ontario. Can. J. Vet. Res. 71: 189-194. Volokhov, D., V. Chizhikov, K. Chumakov, and A. Rasooly. 2003. Microarray-based identification of thermophilic Campylobacter

DIAGNOSIS AND ANTIMICROBIAL SUSCEPTIBILITY

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jejuni, C. coli, C. lari, and C. upsaliensis.1.Clin. Microbiol. 41: 4071-4080. Wang, H., and D. R. Murdoch. 2004. Detection of Cumpylobacter species in faecal samples by direct gram stain microscopy. Pathology 36:343-344. Wang, W.-L. L., and M. J. Blaser. 1986. Detection of pathogenic Cumpylobucter species in blood culture systems. I; Clin. Microbiol. 23:709-714. Wang, W.-L. L., L. B. Reller, B. Smallwood, N. W. Luechtefeld, and M. J. Blaser. 1983. Evaluation of transport media for Campylobacter jejuni in human fecal specimens. ]. Clin. Microbiol. 18~803-807. Wasfy, M., B. Oyofo, A. Elgindy, and A. Churilla. 1995. Comparison of preservation media for storage of stool samples. J. Clin. Microbiol. 3 3 :2 176-21 78. Wells, J. G . N. D. Puhr, C. M. Patton, M. A. Nicholson, M. A. Lambert, and R. Jerris. 1989. Comparison of selective media and filtration for the isolation of Campylobucter from feces, abstr. C231. Abstr. Ann. Meet. Am. Soc. Microbiol. Wistrom, J., and S . R. Norrby. 1995. Fluoroquinolones and bacterial enteritis, when and for whom? J. Antimicrob. Chemother. 36~23-39.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser 0 2008 ASM Press, Washington, DC

ChaDter 13

Guillain-Barre Syndrome and Campylobacter Infection BART c. JACOBS,

ALEX VAN

BELKUM,AND HUBERT P. ENDTZ

GBS is presently a descriptive disease entity defined by a set of clinical, laboratory, and electrodiagnostic criteria (Table 1). GBS is a polyradiculoneuropathy characterized by an acute progressive and symmetrical motor weakness of the extremities with loss of tendon reflexes (Asbury and Cornblath, 1990). A specific subform of GBS, the Miller Fisher syndrome (MFS), is characterized by areflexia, ataxia, and ophthalmoplegia (Miller Fisher, 1956). Surveillance studies from widely scattered geographical areas showed that the annual incidence rate of GBS per 100,000 subjects ranges from 0.6 to 1.9 (Schonberger et al., 1981). In some areas of the world, the incidence of GBS may be higher, as has been suggested for endemic forms in rural areas in northern China (Ho et al., 1995; Liu and Jou, 1993), or may fluctuate in time, as was demonstrated in CuraSao (van Koningsveld et al., 2001). On the basis of the abovementioned incidence figures, 40,000 to 120,000 new cases are being diagnosed per year worldwide. GBS may affect persons of all ages but has an increased incidence in the elderly (Dowling et al., 1977; Schonberger et al., 1981; van Koningsveld et al., 2000). GBS occurs slightly more often in male subjects than in female subjects (Hurwitz et al., 1983; van Koningsveld et al., 2000). Multiple cases of GBS within one family have been described, although this appears to be an infrequent event (Geleijns et al., 2004). Two-thirds of patients with GBS report symptoms of a preceding gastrointestinal or respiratory infectious illness from which they usually recover spontaneously (van Koningsveld et al., 2000; Winer et al., 1988a, 1988b). One to 3 weeks later, the patient may notice numbness or tingling in the arms or legs with symmetrical loss of strength in hands or feet. The

Guillain-Barrt syndrome (GBS) is the most common form of acute neuromuscular paralysis in countries where poliomyelitis has been eradicated. The major clinical features of the syndrome were first united by J.-B. 0. Landry in 1859. The syndrome was named after G. Guillain and J. A. Barrt, two French army neurologists, who in 1916 described, together with A. Strohl, the typical findings in the cerebrospinal fluid (Guillain et al., 1916). This dissociation albumino-cytologique, the combination of high protein level and normal cell count in cerebrospinal fluid, was in those days an important diagnostic feature that distinguished the syndrome from other neurological diseases, including poliomyelitis. GBS was early recognized as a typical postinfectious disease, although it took several decades before the types of antecedent infection were identified. In 1982, shortly after the development of adequate culture systems, Rhodes and Tattersfield (1982) reported the first isolation of Campylobacter jejuni from the stools of a patient with GBS. This discovery set the stage for research on the pathogenesis of C. jejuni-related GBS. At present, there is convincing evidence that C. jejuni is the predominant preceding infection in GBS and that this infection triggers the production of crossreactive antibodies to gangliosides that are damaging peripheral nerve tissue. GBS is currently considered to be a true case of molecular mimicry mediated disease, at least in those patients with a preceding jejuni infection. All these discoveries were made in the last 25 years. One of the challenges of the next decennium will be to translate the newly acquired knowledge into interventions in order to prevent disease, or to improve recovery and lower morbidity and mortality in GBS patients.

c.

Bart C. Jacobs Departments of Neurology and Immunology, Erasmus University Medical Center, Rotterdam, 3015 CE The Netherlands. Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Alex van Belkum Rotterdam, 3015 CE The Netherlands. Hubert P. Endtz International Centre for Diarrhoea1 Disease Research, Bangladesh, Dhaka 1212, Bangladesh, and Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, 3015 CE The Netherlands.

-

245

246

IACOBS ET AL.

Table 1. Diagnostic criteria for Guillain-Barrt syndrome" Features required for diagnosis Progressive motor weakness of more than one limb Low or absent reflexes * No other identifiable cause Features strongly supporting the diagnosis Clinical Progression of less than 4 weeks Relative symmetry Mild sensory symptoms and signs Cranial nerve involvement Recovery beginning 2 weeks to months after progression ceases Autonomic dysfunction Absence of fever at onset Cerebrospinal fluid (CSF) High concentration of CSF protein after the first week Less than 50 mononuclear cells per pl CSF Electrodiagnostic Conduction slowing or block Features casting doubt on the diagnosis Marked, persistent asymmetry of weakness Persistent bladder or bowel dysfunction Bladder or bowel dysfunction at onset More than 50 mononuclear leukocytes per pl CSF Presence of polymorphonuclear leukocytes in CSF Sharp sensory level Onset with respiratory failure disproportionate to limb weakness "Adapted from the criteria supported by the National Institute of Neurological and Communication Disorders and Stroke (Asbury et al. 1990).

weakness spreads during the next few hours to weeks and may finally lead to a paralysis of limb, trunk, extraocular, facial, pharyngeal, and tongue musculature. Because of the involvement of respiratory muscles, up to one-third of the patients need to be ventilated (Winer et al., 1988a, 1988b). Patients may also have severe pain, diminished sensibility, or autonomic dysfunction. Clinical examination of the patients shows poor or absent tendon reflexes. The diagnosis is further supported by a high protein level in combination with normal cell count in cerebrospinal fluid and by features of demyelination and axonal degeneration in electrodiagnostic studies. The progression of weakness ceases by definition within 4 weeks. Recovery usually begins after a plateau phase of 2 to 4 weeks but may be delayed for months (Fig. 1). General medical support is of the utmost importance in treating GBS patients, especially to prevent secondary complications such as infections and decubitus. Additionally, patients will recover more rapidly from their paresis and have less residual deficits after treatment with plasma exchange (French Cooperative Group on Plasma Exchange in GBS, 1987;

GBS Study Group, 1985) or intravenous immunoglobulins (Plasma ExchangelSandoglobulin GBS Trial Group, 1997; van der MechC et al., 1992; van Koningsveld et al., 2004). The mortality is usually less than 5 to 10% and is mostly the result of respiratory or autonomic complications (Ropper et al., 1991). Recovery may be complete, but most patients have some residual handicap. Approximately 20% of patients have a severe functional deficit and remain unable to walk or bedridden, and they even may need continuous artificial ventilation (Ropper et al., 1991; van Koningsveld et al., 2007). A prognostic model was developed that accurately predicts which patients will have a poor outcome (van Koningsveld et al., 2007). GBS usually has a monophasic course, but treatment-related fluctuations occur in 10 to 15% of patients (Kleyweg and van der Mech6, 1991), and about 1 to 5% may experience relapse (Ropper et al., 1991). HETEROGENEITY AND SUBGROUPS GBS is an accepted disease entity, but there is marked patient-to-patient variation regarding disease severity, distribution of neurological deficits, electrophysiological findings, type of antecedent infection, and specificity of autoantibodies (Table 2). The extent of the motor deficits ranges from minimal weakness of the legs to a total paralysis of all limb, trunk, eye, facial, and bulbar muscles, a grave situation in which the patient has full awareness but is unable to communicate. A vivid description of such a situation was given by Joseph Heller (Heller and Vogel, 1986). The distribution of motor deficits varies between patients, as illustrated by the selective weakness of proximal limb muscles in some cases and of distal muscles in others. Sensory involvement may be completely absent, as in the case of a pure motor GBS, or may be severe and long-lasting and may include all sensory modalities. Some patients may rapidly recover without residual deficits, even from severe GBS, whereas others need ventilation for months or remain bed-bound for years. This clinical heterogeneity is further illustrated by the presence of distinct variants of GBS such as the MFS, in which the paresis is restricted to the muscles involved in eye movements (ophthalmoplegia), frequently in combination with bulbar or facial weakness (Fisher, 1956). Other subforms are the pharyngeal-cervical-brachial and the lower bulbar variants of GBS (Ropper et al., 1991). The extent of demyelization and axonal degeneration of peripheral nerves may also differ considerably between individual patients. The acute inflammatory demyelinating polyneuropathy (AIDP) variant, characterized by segmental demyelination

CHAPTJZR 13

SYNDROME AND CAMPYLOBACTER

GUILLAIN-BA&

247

................... i Admission i

1

,

,

~

,

,

,

,

,

,

,

lInfeaionl I

,

,

,

,

,

,

,

,

,

,

,

,

Weeks Serum antibodies to clanclliosides

1-

I

Plateau phase

with no or minor secondary axonal degeneration, is the predominant type of GBS in Western countries (Hadden et al., 2001; van Koningsveld et al., 2004). The acute motor axonal neuropathy (AMAN) and acute motor sensory axonal neuropathy variants, characterized by predominant axonal involvement,

I Recovery phase

Disability

are more frequent in Asia and developing countries (Griffin et al., 1995; Islam et al., 2007; McKhann et al., 1993). The involvement of axonal degeneration is indicated by electrophysiological findings but can only be proven in histological studies (Cros and Triggs, 1994). It is as yet unclear whether the AIDP

Table 2. Heterogeneity in Guillain-BarrC syndrome Variation Neurological deficits

Subforms

......... Motor-sensory

form Pure motor variant Miller Fisher syndrome (MFS) Lower bulbar variant Pure sensory variant Clinical severity .............Mild variant Total paralysis with respiratory insufficiency and autonomic dysfunction Electrophysiology/ histology .. .Acute inflammatory demyelinating polyneuropathy (AIDP) Acute motor axonal neuropathy (AMAN) Acute motor sensory axonal neuropathy (AMSAN) Antecedent infections ........Campylobacter jejuni Cytomegalovirus Epstein-Barr virus Mycoplasma pneumoniae Haemophilus influenzae Anti-ganglioside antibodies. ...GM1, GMlb, (GalNAc-)GDla (pure motor variant, AMAN) GQlb, GTla, GD3, GD2 (MFS, lower bulbar variant) GM2, GM3, LM1 Clinical outcome ............Spontaneous and full recovery Severely disabled Treatment-related fluctuations Relapse

248

TACOBS ET AL.

and axonal forms are absolute distinct entities resulting from exclusive pathogenic mechanisms, or whether they represent extreme forms in a continuous spectrum of a syndromic disease entity.

IMMUNOPATHOLOGY There is convincing evidence from extensive histology, serology, and animal model studies that GBS is caused by an autoimmune response. This parallels the failure of natural immune tolerance in other disorders such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, which are generally classified as autoimmune diseases. However, most of these classic autoimmune diseases have a specific age at onset, are more frequent in female subjects, and are associated with family, human leukocyte antigen (HLA) haplotype, and other autoimmune diseases, and usually show a relapsingremitting or chronic course that frequently improves after treatment with corticosteroids. None of these characteristics, however, is evident in GBS (Table 3). Instead, GBS is strongly associated with preceding acute infections, which are usually much less evident in these classic autoimmune diseases. GBS is therefore frequently classified as a typical postinfectious d’isease. In about half of GBS patients, serum antibodies to various gangliosides can be demonstrated in the acute stage of disease (Willison, 2005; Willison and Yuki, 2002; Yuki, 2007). Gangliosides are glycolipids that are highly enriched in neural membranes (Ledeen, 1985). They consist of a ceramide tail inserted in the lipid bilayer and a highly variable oligosaccharide moiety containing sialic acid that protrudes extracellulary (Ledeen and Yu, 1982). Gangliosides are organized in specialized functional microdomains called lipid rafts, where they play a role in mainte-

nance of the cell membrane structure and are implicated in cell growth, differentiation, and cell-cell recognition. A variety of gangliosides have been identified in the nerve myelin sheets and axons of human peripheral nerves, each with a specific tissue distribution (Ledeen, 1985). Antibodies to more than 20 different gangliosides have been identified in various frequencies in serum from GBS patients. The most frequent antibodies in GBS are directed to the gangliosides LM1, GM1, GMlb, GM2, GDla, GalNAc-GDla, GDlb, GD2, GD3, GTla, and GQlb (Willison and Yuki, 2002). These antibodies specifically recognize parts of the oligosaccharide moieties and may bind to various gangliosides with the similar carbohydrate structures. Other antibodies bind to mixtures or complexes of different gangliosides instead of individual gangliosides (Kaida et al., 2006, 2007; Kuijf et al., 2007). The highest titers of serum antiganglioside antibodies are usually found in the acute stage of disease (Fig. 1).These antibodies usually disappear within weeks, although they may persist for more than 6 months, and antibodies may also occasionally persist in patients who have completely recovered. The antiganglioside antibodies are predominantly immunoglobulin (Ig) G antibodies and usually of the IgGl and IgG3 subclass, but IgM and IgA antibodies can also be demonstrated. Typically they are oligoclonal or even monoclonal, polyreactive, and have a moderate affinity (Boffey et al., 2005; Townson et al., 2007). Antibodies to other glycolipids and even antibodies and T cells to peripheral nerve proteins have also been demonstrated in serum from GBS patients. Some antiganglioside antibodies are highly toxic for peripheral nerves. Antibodies to GQlb and other disialylated gangliosides have an a-latrotoxin-like effect in mice characterized by a dramatic release of acetylcholine, resulting in a depletion of this neurotransmitter at the nerve terminals, final blockade of

Table 3. General comparison between classic autoimmune diseases and Guillain-Barri syndrome“ Classic AID

Characteristic Demographic associations Predominant age at onset Predominant sex Segregation within families Immunologic associations Preceding acute infections Association with HLA types Association with other AID Clinical course Progression Response to corticosteroids

GBS

20-40 yr Females Yes

None; frequently at older age Males Usually not

Weak Strong Yes

Strong None or weak None

Relapsing-remitting, chronic Frequently

Monop hasic None

“AID, autoimmune diseases (classic AID such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis); GBS, Guillain-BarrC syndrome; HLA, human leucocyte antigen.

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GUILLAIN-BARF& SYNDROME AND CAMPYLOBACTER

249

PRECEDING INFECTIONS ASSOCIATED WITH GBS

nerve transmission, and paralysis of the nerve-muscle preparation (Jacobs et al., 2002; Plomp et al., 1999). There is additional destruction of the architecture of these nerve endings (O’Hanlon et al., 2001). Antibodies to GM1 affect the sodium channels in the nodes of Ranvier of rabbit peripheral nerves (Susuki et al., 2007). All these effects are complement dependent. After binding of the antibodies to their targets on peripheral nerves, there are local depositions of complement factors leading to the formation of the membrane attack complex (Halstead et al., 2004). Obduction studies also demonstrated that local complement activation occurs at the site of nerve damage, which is the axolemma in patients with Ah4AN and the Schwann cell membrane in patients with AIDP (Hafer-Macko et al., 1996a, 199613). The neurotoxic effects of serum anti-GQlb antibodies were inhibited by immunoglobulins, which also have a therapeutic effect in patients with GBS (Jacobs et al., 2003). These studies show that at least some of the serum antiganglioside antibodies are highly neurotoxic in ex vivo models, and probably also in GBS patients. After these antibodies have disappeared, have lowered in titer, or are neutralized by the treatment, the peripheral nervous system has the capacity to slowly recover, provided that some of the nerve architecture has remained intact.

After the first isolation of C. jejuni from stool specimens from a GBS patient in 1982, studies in controlled series of patients confirmed the association between Cumpylobacter infections and GBS (Table 4). These studies indicate that a C. jejuni infection precedes GBS in 20 to 50% in Europe, North and South America, Japan, and Australia. In other areas, the frequency of C. jejuni infections preceding GBS may be even higher, such as in northern China and Bangladesh (Ho et al., 1995; Islam et al., 2007). C. jejuni can been cultured from stool specimens from GBS patients, although this appears to necessitate laborious isolation protocols including enrichment media. Still, stool cultures are often negative at the onset of the neurological symptoms, considering the mean period of bacterial excretion after onset of diarrhea is only 16 days (Svedhem and Kaijser, 1980). GBS typically starts 1 to 3 weeks after a Campylobacter infection. Studies based on stool culture alone will therefore underestimate the frequency of C. jejuni infections in GBS. Recent studies, however, have shown that Campylobacter serology is highly sensitive and specific in GBS patients (Ang et al., 2007). The C. jejuni infections preceding GBS are usually

Table 4. Frequency studies on Campylobacterjejuni infections in patients with Guillain-Barrt syndrome“ Reference

Frequencyb

Axonal‘

Kaldor and Speed (1984) Speed et al. (1987) Winer et al. (1988a) Walsh et al. (1991) Gruenewald et al. (1991) Boucquey et al. (1991) Nobile-Orazio et al. (1992) Gregson et al. (1993) Kuroki et al. (1993) Enders et al. (1993) Vriesendorp et al. (1993) Mishu and Blaser (1993) Von Wulffen et al. (1994) Rees et al. (1995b)

21/56 (38%)” 22/45 (49%)” 14/99 (14%)’ 14/94 (15%)’ 3/17 (18%)’.f 6/42 (14%)’ 3/16 (19%)’ 15/42 (36%)’ 14/46 (30%)f 15/38 (39%)e 10158 (17%)e 431118 (36%)’ 11142 (26%)c 25/96 (26%)e,f 217 (29%)‘ 25/38 (66%)’ 461154 (32%)’ 531229 (23%)’ 11311049 (ll%)f 19/80 (26%)’ 32/78 (41%)”

NT NT NT

Ho et al. (1995) Jacobs et al. (1996) Hadden et al. (2001) Takahashi et al. (2005) Sinha et al. (2006) Nachamkin et al. (2007)

+

NT NT NT NT NT -

+

NT NT

+ + NT + NT + NT

-

Clinical association

Poor outcomee

Severe weakness NT NT NT NT NT NT NT Severe weakness NT NT Pure motor MFS

NT

-

-

Severe, pure motor Pure motor NT NT NT

+

NT NT NT NT NT -

+

NT NT

+

NT

+ after PE; - after IVIg + NT NT NT

a NT, not tested; +, present; -, absent; MFS, Miller Fisher syndrome; PE, plasma exchange; IVIg, intravenous immunoglobulins. “Number of patients in case reports, or number of C. jejuni-infected patients per number of tested patients in frequency studies. ‘Association with severe axonal degeneration. dDetermined at least 3 months after diagnosis of GBS. eDetermined by serology. fDetermined by stool culture.

250

IACOBS ET AL.

self-limiting, and treatment with erythromycin does not prevent the development of weakness (Constant et al., 1983; Kohler and Goldblatt, 1987; Pryor et al., 1984; Rhodes et al., 1982; Ropper, 1988; Sovilla et al., 1988). A remarkable diversity of antecedent infections other than those caused by C. jejuni has been reported in relation with GBS. Most of these microorganisms were reported in case reports only and may represent coincidental findings. In a large comparative and prospective study that tested for 16 different infectious causes, C. jejuni infection was found to be the predominant antecedent event (Jacobs et al., 1996). Other infections associated with GBS in these case-control studies were cytomegalovirus, EpsteinBarr virus, and Mycoplasma pneumoniae. Infections with Huemophilus influenzae (Houliston et al., 2007; Ju et al., 2004; Koga et al., 2001; Mori et al., 2000), human immunodeficiency virus, and vaccinations have also been reported in association with GBS. Other types of infection may also be related to GBS, but their frequency is probably too low to produce a significant association in case-control studies. This wide spectrum of preceding infections in GBS may partly explain the patient-to-patient variation in clinical and electrophysiological phenotype. Patients with a C. jejuni infection may represent a clinical and pathological subgroup distinct from other patients with GBS (Table 4). Some studies in larger series found an association between C. jejuni infection and a more severe form of GBS (Jacobs et al., 1996; Kaldor and Speed, 1984; Visser et al., 1995), with less sensory deficits (Hadden et al., 2001; Jacobs et al., 1996; Rees and Hughes, 1994; Visser et al., 1995), more frequent or extensive axonal degeneration (Hadden et al., 2001; Ho et al., 1995; Rees et al., 1995a; Sinha et al., 2006; Vriesendorp et al., 1993; Walsh et al., 1991), or poorer outcome (Hadden et al., 2001; Jacobs et al., 1996; Rees and Hughes, 1994; Rees et al., 1995; Vriensendorp et al., 1993). In a more recent study including two large independent cohorts of patients, preceding diarrhea or positive C. jejuni serology was identified as a strong prognostic factor, independent of other prognostic factors, which can be used to predict the chance of recovery toward independent walking after 6 months (van Koningsveld et al., 2007). In Northern China, C. jejuni infections are more frequent in patients with AMAN than AIDP (Ho et al., 1995). Also in Japan, C. jejuni infections are strongly associated with the axonal variants of GBS, and some studies concluded that this type of infection is exclusively found in patients with axonal forms of GBS (Kuwabara et al., 2004). Geographical differences in the incidence of C. jejuni infections may therefore also

influence the predominant clinical and electrophysiological phenotype. Whether this exclusive association between C. jejuni and axonal variants is also present in other geographical areas remains to be determined. Preceding infections with C. jejuni are also the predominant type of preceding infection in patients with the MFS (Koga et al., 2005a). Preceding infections with cytomegalovirus, on the other hand, are related to a different phenotype. These patients have severe sensory symptoms and cranial nerve deficits (Jacobs et al., 1997b; Yuki and Tagawa, 1998).

WHOLE-GENOME POLYMORPHISMS IN GBS-RELATED C. JEJUNI C. jejuni is an organism with a relatively wellconserved genome size and composition (Parkhill et al., 2000). Despite this apparent conservatism, many variable target regions suited for bacterialepidemiologic and functional typing have been identified. Such tools are of importance in defining the determinant of GBS-inducing capacities in GBSassociated bacterial isolates. Targets harboring variable DNA sequences range from distinct regions such as the flagellin gene locus or the clustered, regularly interspaced short palindromic repeat domain to variable restriction sites more suited for pan-genomic restriction fragment length polymorphism assessment or pulsed-field gel electrophoresis analyses. The current trend toward direct DNA sequence-based typing has also penetrated the Campylobacter research field. A variety of more or less conserved genes has been identified and used in large-scale genetic typing studies of clinical, veterinary, or specific diseaseassociated collections of Carnpylobacter strains. We will here briefly discuss the international literature on epidemiological and high-throughput functional typing of C. jejuni strains associated with postinfectious neurological syndromes including GBS and MFS (Allos, 1998; Tam et al., 2006). Initial studies pointed in the direction of putative clonality of the possibly neuropathogenic GBS- and MFS-associated strains. Studies from different geographical locations revealed that most of the clinical isolates belonged to a restricted set of serotypes, e.g., HS:19 and HS:44 (Allos et al., 1998; Lastovica et al., 1997; Nishimura et al., 1997; Saida et al., 1997; Yuki et al., 1992). These data strongly proposed clonal dissemination of neuropathogenic strains across restricted areas. In turn, this suggested the existence of specific traits, not widely disseminated among all Campylobacter strains, which were causally related to the postinfectious neurological complications associated with Campylobacter enteritis. Unfortunately, these data were in a sense misleading. Subsequent re-

CHAPTER 13

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search revealed that C. jejuni strains collected within restricted time frames and geographic locales could also display extensive diversity or polyclonality (Endtz et al., 2000; Engberg et al., 2001). This latter observation suggested that the capacity of strains to cause neuropathological syndromes was more widely disseminated than previously anticipated on the basis of the clonality hypothesis. Because GBS- and MFSassociated strains were now considered to derive from a variety of different genetic backgrounds, focused experimental searches for non-serotypedependent factors important in the induction of such diseases were facilitated.

GENE-SPECIFIC VARIATION IN GBS-RELATED C . JEJUNI Obviously, hunting for genetic traits by epidemiological comparisons was initially focused toward the genes encoding the enzymes responsible for the biosynthesis of the lipooligosaccharides (LOS), the core element in the molecular mimicry hypothesis (Ang et al., 2002a; Aspinall et al., 1994b; Yuki et al., 2004). Further screening for the presence of LOSassociated genes that were identified through genome sequencing (Parker et al., 2005) revealed that several genes were selectively present or absent, depending on the composition of the LOS gene cluster. A diversity of gene clusters has now been identified (ranging from type A to type R), all of which have a characteristic set of core LOS biosynthesis genes present. These genes may be polymorphic with respect to their primary structure, but absence or presence appeared to be strongly correlated with the propensity to cause GBS or MFS. The Campylobacter sialic acid transferase (cstll) gene turned out to be a particularly interesting one. It was discovered, again by comparative genomics technologies, that the presence of the gene and the presence of G Q l b epitopes on the Campylobacter outer LOS surface were intimately linked to the development of MFS, thereby for the first time linking bacterial gene variation to the capacity to cause neurological damage through autoimmune antibody induction (van Belkum et al., 2001). This for the first time supported the molecular mimicry hypothesis from the bacterial genomics perspective. After these initial explorations, several additional large-scale comparative genetic screenings have been performed for C. jejuni, but none of these yielded other molecular markers for GBS and MFS besides the LOS biosynthesis genes. Multilocus sequence typing and flagellin gene sequencing confirmed the polyclonality previously assessed among GBS- and MFS-related Campylobacter strains (Dingle

25 1

et al., 2001). Amplified fragment-length polymorphism assessment also did not contribute substantially. Although its appropriateness for identifying clonal expansion among clinical, veterinary, and environmental isolates was convincingly demonstrated, no specific markers could be identified on the basis of the DNA fingerprints developed for the neuropathogenic isolates (Duim et al., 2000, 2003; Endtz et al., 2003). Even the application of high-throughput amplified fragment-length polymorphism rendering thousands of markers for individual strains failed to add novel markers to the existing ones (Godschalk et al., 2006a). The use of whole-genome DNA arraysthe typing tool used after whole-genome sequencing-did not reveal any GBS-specific markers when comparing gene content for strains derived from noncomplicated enteritis cases versus GBS (Leonard et al., 2004). More recently, Taboada et al. (2007) determined the gene conservation profile of 1,712 virulence and other genes in 56 GBS-associated strains and 102 control strains. They confirmed that genes involved in the sialylation of LOS are significantly associated with neuropathogenic strains. However, some enteritis control strains still bear these genes and share remarkable levels of genomic similarity with their neuropathogenic counterparts. In addition, they described two genes involved in the capsular biosynthesis (Cj1421c and Cj1428c) that also were significantly associated with GBS. Any potential involvement of these genes in the pathogenesis of GBS is yet to be determined.

BACTERIAL GENE CHARACTERISTICS, CLINICAL SYMPTOMS, AND GBS VARIANTS There appears to be an association between the specific clinical symptoms, GBS variants, and particular characteristics of C. jejuni strains that primarily resides in the LOS biosynthesis genes. This has been demonstrated by gene-specific knockout mutagenesis in the locus itself (Godschalk et al., 2005; Perera et al., 2007; Xiang et al., 2006). Typing studies also revealed that different types of C. jejuni are capable of inducing different disease presentations (Kuwabara et al., 2004; Takahashi et al., 2005). The presence of certain genes could be associated with a specific epitope at the bacterial cell surface, even corroborated with sophisticated mass spectrometry techniques, which could in turn be coupled to various classes of autoimmune antibody reactions. In this respect, it is important to note that C. jejuni can acquire large portions and even entire LOS gene clusters from external sources through horizontal transfer (Gilbert et al.,

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2004; Phongsisay et al., 2006). Interestingly, specific point mutations within specific genes could also be associated with LOS variation and hence different clinical presentations of the diseases induced (Kimoto et al., 2006). The LOS locus classification and some highly specific amino acid changes in, for instance, the Cstll gene ultimately determine the LOS biosynthetic capacity. The Thr51 as opposed to the Am51 variant of cstll is responsible for the capacity to synthesize GM1 and GDla ganglioside mimics, respectively, and as such constitutes an important GBS risk factor (Hye and Nachamkin, 2007; Koga et al., 2 0 0 . 5 ~2006). ~ It has to be noted, however, that both these studies were biased because specific, clonally related C. jejuni strains were overrepresented in the stain collections.

EVIDENCE FOR A CAUSAL RELATIONSHIP BETWEEN C. JEJUNI AND GBS Classically, the fulfillment of Koch's postulates confirms infectious causation, but this has become increasingly difficult during the last century (Fredricks and Relman, 1996). Sequence-based identification of microbial pathogens has led to a reconsideration of Koch's postulates. Reliance on Koch's postulates as critical evidence for causation has thus diminished. In fact, the postulates have not been fulfilled for most diseases for which infectious causation has been accepted, including cervical cancer, liver cancer, T-cell leukemia, and AIDS. Therefore, because of the limitations of these postulates, the emphasis has been on alternative ways to identify causative organisms (Cochan et al., 2000). The discovery of C. jejuni as the predominant infection preceding GBS stimulated the research on the pathogenesis of the disease and its role in causing the postinfectious neuropathy. Early reports speculated that the C. jejuni infection provides the antigenic trigger for a transient immune response to peripheral nerves. The antibody response to C. jejuni generally peaks at 1 to 2 weeks after infection (Blaser and Duncan, 1984), which coincides with the delay between infection and the first neurological symptoms in GBS. After eradication of the C. jejuni, the antigenic drive of this immune response would be lost, possibly explaining the monophasic course of GBS. There is now convincing evidence from previous studies that antibodies to gangliosides in patients with a C. jejuni-related GBS are induced by molecular mimicry (Table 5 ) . In short, these studies described the following: (i) among GBS patients, recent C. jejuni infection are associated with the presence of serum antibodies to distinct gangliosides; (ii) C.

Table 5 . Evidence for a causal role of Cumpylobucter jejuni infections in the pathogenesis of Guillain-Barre syndrome" Association between C. jejuni infections and GBS Molecular mimicry between C. jejuni LOS and peripheral nerve ganglisoides Cross-reactive antibodies to C. jejuni LOS and gangliosides in serum from GBS patients Induction of similar cross-reactive antibodies in animals after immunization with C. jejuni LOS Pathogenicity of these cross-reactive antibodies in animal models Prevention of disease in animal models after blocking pathogenic effects of these antibodies a

GBS, Guillain-Barre syndrome; LOS, lipooligosaccharide.

jejuni isolates from GBS patients frequently express a LOS that mimics gangliosides; (iii) among GBS patients, serum antibodies to C. jejuni LOS usually cross-react with gangliosides; (iv) animals immunized with C. jejuni LOS produce similar cross-reactive antiganglioside antibodies as found in humans; and (v) these immunizations induce neurotoxic antibodies or an axonal neuropathy with limb paresis in certain infection models. Association between C. jejuni Infection and Serum Antiganglioside Antibodies Initial studies reported an association between various types of antecedent infection and the presence of specific serum antiganglioside antibodies (Jacobs et al., 1998; Willison and Yuki, 2002). C. jejuni infections are frequently associated with antibodies to GM1, GMlb, GDla, and GalNAc-GDla in patients with the pure motor or axonal variants of GBS (Ang et al., 1999; Ho et al., 1999; Jacobs et al., 1996; Ogawara et al., 2000, 2003; Yuki et al., 1990). C. jejuni infections are also associated with antibodies to GQlb and GTla and oculomotor weakness in patients with GBS and MFS. In contrast, patients with a recent cytomegalovirus infection more frequently have antibodies to GM2 (Jacobs et al., 1997b; Yuki and Tagawa, 1998), and patients with a recent M. gneumoniue infection frequently have antibodies to galactocerebroside (Ang et al., 2002b; Kusunoki et al., 2001), although this was not confirmed by others (Susuki et al., 2004). Molecular Mimicry between C. jejuni LOS and Peripheral Nerve Gangliosides Mass spectrometry studies of the C. jejuni isolates from several patients with GBS and MFS showed a similar carbohydrate moiety in LOS and peripheral nerve gangliosides (Aspinall et al., 1994a, 1994b; Yuki et al., 1993) (Fig. 2). In a recent study,

GUILLAIN-BARF& SYNDROME AND CAMPYLOBACTER

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C. jejuni isolate

GB2

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LOS outer core

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GMI

GDla

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Severe, pure motor

N-Acetyl-galactosamide

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G D3

1 MFS

KAcetylneurarninic acid,

0 Heptose,

Glucose, Cer Ceramide

Figure 2. C. jejuni genotype and LOS phenotype in relation to serum antiganglioside antibodies and clinical features in GBS and MFS. Two illustrative examples of C. jejuni strains isolated from a patient with a severe, pure motor form of GBS (strain GB2) and from a patient with MFS (strain MF6) (Godschalk et al., 2007). Strain GB2 with a class A genotype produces a GM1- and GDla-like LOS, which induced cross-reactive antibodies to the peripheral nerve ganglioside GM1 and GDla. Strain MF6 with a class B genotype produces a GMlb- and GDlc-like LOS, which induced cross-reactive antibodies to the peripheral nerve ganglioside GD3 and GQlb.

21 of 26 GBS- and MFS-related C. jejuni strains expressed a ganglioside-like LOS, with up to five identical glycan structures (Godschalk et al., 2007). The majority of these strains expressed a heterogeneous LOS, in which the combination of GM1- and GDlalike structures was most frequently found. This combination was associated with the presence of serum antibodies to GM1 and GDla and with the occurrence of severe pure motor GBS. Other strains showed mimicry with disialylated gangliosides and were associated with antibodies to GQlb, GTla, and GD3 and with the occurrence of oculomotor weakness and the MFS. The LOS of some strains contained no sialic acids and showed no mimicry with gangliosides. Previous studies on C. jejuni isolates from enteritis controls without GBS already found a large heterogeneity in the carbohydrate structure of LOS. LOS from serostrain PEN HS:3 is not sialylated, and this Penner serotype is not associated with GBS (Aspinall et al., 1995). These biochemical studies indicate that C. jejuni strains isolated from GBS and MFS patients more frequently have a ganglioside-like

LOS than strains isolated from enteritis controls. The association, however, is not absolute: GBS-related strains may not have ganglioside mimicry (Godschalk et al., 2006a), whereas some strains from enteritis controls do (Godschalk et al., 2006a). In addition, on infection with multiple strains of C. jejuni, the isolation of only one strain may easily lead to a wrong conclusion with respect to absence or presence of molecular mimicry involved in the pathogenesis of GBS (Godschalk et al., 2006b). Cross-Reactive Antibodies to C . jejuni LOS and Gangliosides Serological studies have clearly demonstrated that antiganglioside antibodies in serum from patients with GBS and MFS cross-react with the LOS from autologous C. jejuni isolates (Jacobs et al., 1995, 1997a; Yuki et al., 1994). The serum antibodies to LOS in these patients can also be depleted by gangliosides. The variation in antibody specificity observed in GBS patients is related to the variation in gangli-

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oside mimicry found in LOS from C. jejuni isolates. Serum antibodies to GM1 and GDla are specifically adsorbed by C. jejuni strains expressing LOS with GM1 and GDla mimics. On the other hand, antibodies to G Q l b are adsorbed by strains expressing a GD3 or GTla mimic (a GQlb-like structure in C. jejuni LOS has not been found). Even antibodies to ganglioside complexes cross-react with C. jejuni LOS (Kuijf et al., 2007). These studies strongly indicate that in C. jejuni-related cases of GBS, the carbohydrate moiety of the LOS determines the specificity of the antiganglioside antibodies and the related neurological deficits (Fig. 2). Induction of Cross-Reactive Antibodies in Animals Immunized with C. jejuni LOS Two conditions need to be fulfilled to induce a cross-reactive immune response against autologous antigens by molecular mimicry. First, the microbial and autologous antigens need to be sufficiently similar to induce antibodies that can cross-react. C. jejuni LOS shows a high similarity with gangliosides, and rabbits immunized with LOS produce antiganglioside antibodies with a similar specificity and crossreactivity as found in the sera of patients with GBS and MFS (Ang et al., 2000a, 2001; Bowes et al., 2002; Goodyear et al., 1999). Second, the microbial antigen needs to be sufficiently different from the autoantigen to break the tolerance of the immune system and induce a specific immune response. This second condition explains why the production of antiganglioside antibodies in wild-type mice may be low or even absent, and why GalNAc transferase knockout mice, which lack complex gangliosides and only express high levels of GM3 and GD3, produce much higher titers (Bowes et al., 2002). Also, the antiganglioside antibodies induced in those experiments cross-react with C. jejuni LOS. Induction of Neurotoxic Antibodies in Animals Immunized with C. jejuni LOS Monoclonal antibodies to GQlb and other disialylated gangliosides raised in mice after immunization with C. jejuni LOS are highly toxic for mouse perisynaptic Schwann cells, peripheral nerve endings, and neuromuscular junctions (Goodyear et al., 1999; Halstead et al., 2004; O’Hanlon et al., 2001). Antibodies to GDla have similar neurotoxic effects on motor nerve terminals in GD3 synthetase knockout mice that overexpress GDla (Goodfellow et al., 2005). These toxic effects are all fully complement dependent and require the presence of a human com-

plement source to exert their neurotoxic effects. In a rabbit model of GBS, immunization with a GM1-like LOS from a C. jejuni isolated from a GBS patient with anti-GM1 antibodies induced anti-GM1 IgG antibodies, an axonal neuropathy, and limb weakness (Yuki et al., 2004). Studies of biopsy samples showed that at the nodes of Ranvier in peripheral nerves of these affected rabbits (Susuki et al., 2007). Complement activation by anti-GM1 antibodies may have precipitated these effects because depositions of IgG and complement products were found along the nodes of Ranvier. Passive transfer studies with antiganglioside antibodies are needed to confirm this hypothesis.

UNRESOLVED ISSUES IN THE PATHOGENESIS OF C. JEJUNI-RELATED GBS Why Do Infections with Ganglioside-Mimicking C. jejuni So Rarely Induce GBS? If C. jejuni is directly involved in the pathogenesis of GBS, how can such a common infection, with an estimated incidence of 1 per 100 individuals per year, be followed by such a rare disease as GBS, with an incidence of 1 to 2 per 100,000 per year? Assuming that one-third of GBS patients have an antecedent C. jejuni infection, one may calculate that approximately only 1 in 1,000 to 5,000 subjects with C. jejuni infection will subsequently develop GBS (Mishu and Blaser, 1993). This discrepancy is further illustrated by a report of a single case of GBS in a family outbreak of C. jejuni enteritis (Ang et al., 2001). Thus, GBS is a rare complication of a Cumpylobucter enteritis. This infrequent event may be partly explained by the observation that ganglioside-like structures are expressed only by a proportion of C. jejuni strains. Other virulence factors might also be involved in triggering GBS because C. jejuni isolates from patients with uncomplicated enteritis without GBS also express ganglioside-like LOS. Presumably, the immunogenicity of these structures may differ, or alternatively, the expression may depend on other bacterial factors-for instance, the LOS itself or other, yet unknown structures. In addition, host factors may also play an important role in the production of antiganglioside antibodies and the development of GBS. This would also explain the absence of epidemics or outbreaks of C. jejuni-associated GBS. Single nucleotide polymorphisms (SNPs) in immune response genes determine the variation in immune responses to infections in general and may also influence the susceptibility to develop GBS after an infection with C. jejuni. Two studies reported an association between C. jejuni infections and specific

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class I or I1 HLA types in, respectively, Japanese and European patients with GBS (Koga et al., 1998; Rees at al., 1 9 9 5 ~ )A. study on GBS patients from northern China reported an association between distinct HLADQ and HLA-DR types and the occurrence of AIDP (Magira et al., 2003). In a Dutch study, no association between these HLA class I1 alleles and AIDP or preceding infection with C. jejuni was found (Geleijns et al., 2005). Another Japanese study also found no association between HLA and GBS (subgroups) (Ma et al., 1998). In conclusion, at present there is no consistent evidence that HLA alleles are a general susceptibility factor for GBS. Other SNPs may be related to the development of GBS, such as those in interleukin 10 (Myhr et al., 2003). There is more evidence, however, that SNP may play a role as diseasemodifying factors. An association has been demonstrated between disease severity or outcome and SNP in genes coding for mannose binding lectin, Fc gamma receptor 111, matrix metalloproteinase 9, and tumor necrosis factor alpha (Geleijns et al., 2006, 2007; van Sorge et al., 2005). SNP analysis may provide insight in the role of host factors in the pathogenesis of GBS, although these studies require confirmation in large and unselected groups of patients and the demonstration of a functional effect of these genetic associations. What Cellular Mechanism Drives the Production of Cross-Reactive Antibodies to Gangliosides after Infection with a Ganglioside-Mimicking C. jejuni? There is convincing evidence that an immune response to C. jejuni LOS by molecular mimicry leads to the production of cross-reactive and neurotoxic antibodies to peripheral nerve gangliosides. The immune competent cells and mechanisms involved in antigen presentation and B-cell activation, however, are currently unknown. There is increasing evidence that C. jejuni antigens, including LOS, can induce maturation and cytokine production in human dendritic cells (Hu et al., 2006). Whether C. jejuni antigens are presented to T cells and whether T cells are required to provide help to B cell to produce cross-reactive anti-ganglioside antibodies has not been elucidated. T cells reactive to gangliosides have been identified that are activated by bacterial infections (de Libero et al., 2005). Antigen-presenting cells mediate this activation by increasing the endogenous synthesis of glycosphingolipids, including GM1, which are presented by CD1 and activate antiglycosphingolipid T cells. In a patient with a c. jejuni-related GBS, T cells recognizing a C. jejuni “nonprotein antigen” were identified (Cooper et al., 2002). Oligoclonal expansions of T cells in periph-

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eral blood are frequent in patients with GBS, although the role of this T-cell activation and proliferation in the pathogenesis remains to be determined (Koga et al., 2003b). The immune response responsible for the production of antiganglioside antibodies in GBS is unusual given the subclass distribution, the polyreactivity, the moderate affinity, the oligoclonality, and the temporary presence of these antibodies. In humans, IgG2 is the predominant subclass of IgG antibodies to carbohydrates, but in GBS, the antibodies to C. jejuni are usually IgGl and/or IgG3. The IgG subclass of these antibodies may also indicate the involvement of T-cell help, although the immune response in GBS does not lead to sustained high titers. If T cells indeed are activated and provide help to B cells, the recognized C. jejuni T-cell epitope is unknown. It has been hypothesized that a yet unknown Campylobacter protein, possibly a toxin, may function as a carrier protein to provide adequate T-cell help (Willison and Kennedy, 1993). However, there is as yet no conclusive evidence in support of this hypothesis. Do C. jejuni Infections Exclusively Trigger the Onset of Axonal Forms of GBS?

Many studies have reported an association between preceding C. jejuni infections and axonal forms of GBS. A study from Japan suggested that C. jejuni infections there are exclusively found in patients with axonal variants, although the first electrophysiological studies may show features compatible with demyelination (Kuwabara et al., 2004). In Western countries, however, axonal variants are rare, although preceding C. jejuni infections are found in 20 to 50% of patients with GBS (Hadden et al., 1998). This finding may suggest that C. jejuni may also trigger AIDP, at least in Western countries. In addition, it remains unclear whether all electrophysiological findings in patients with GBS can be attributed to axonal degeneration or demyelination. Is There a “Safe” C. jejuni Strain that Cannot Trigger GBS? C. jejuni isolates from GBS patients more frequently have a distinct LOS genotype and a related ganglioside-like LOS as compared with isolates from patients with uncomplicated enteritis (Ang et al., 2002a; Godschalk et al., 2006a, 2007; van Belkum et al., 2001). These associations, although statistically significant, are far from absolute, and other bacterial factors may also trigger GBS. Moreover, occasionally, C. jejuni strains that do not express ganglioside mimicry are isolated from the stools of GBS patients

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(Godschalk et al., 2006b, 2007). Extensive genotyping studies have shown that these strains are almost indistinguishable from enteritis control strains (Taboada et al., 2007). Identification of additional and as yet unknown bacterial factors involved in the pathogenesis is highly relevant for future vaccine development and the designation of safe challenge strains in chicken but also in human experiments. Further understanding of the microbe-host interaction, including the involvement of the cellular immune response, remains a challenging objective. Can a Single Animal Model Be Developed T o Demonstrate That Antiganglioside Antibodies Triggered by a C . jejuni Infection Induce a Neuropathy Similar to That of Patients with GBS? Studies in current animal models have come a long way in providing evidence for the molecular mimicry hypothesis for the pathogenesis of C. jejunirelated GBS. All these models, however, have some limitations. In the mouse model, the C. jejuni LOSinduced cross-reactive antibodies to gangliosides have an evident and transferable toxic effect on the neuromuscular junction and perisynaptic Schwann cells (Halstead et al., 2004; Jacobs et al., 2002; O’Hanlon et al., 2001; Plomp et al., 1999). To induce these effects, however, the presence of a human complement source is required, and these mice do not develop a similar polyneuropathy as observed in GBS patients. In the rabbit model, immunization with C. jejuni LOS leads to the production of cross-reactive antibodies to gangliosides that induce a complementmediated disruption of sodium channel clusters in peripheral motor nerve fibers and a neuropathy that shows some similarities with AMAN (Susuki et al., 2007; Yuki et al., 2004). Passive transfer studies, however, have not been reported, and because of that, the role of antibodies in the pathogenesis of this axonal polyneuropathy model remains elusive. How Can These Discoveries Be Used to Improve Outcome in Patients with GBS? The progress made in the understanding of the pathogenesis of C. jejuni-related GBS until now has not influenced the treatment and outcome of these patients. C. jejuni-related GBS patients in particular need better treatment because prognosis in these patients in general is poor. About one-third of these patients will be unable to walk independently 6 months after onset of disease. Considering the crucial role of antiganglioside antibodies in the pathogenesis of these forms of GBS, patients may profit from se-

lective depletion of antibodies. Artificially produced ganglioside carbohydrates were found to be effective in depleting antiganglioside antibodies from the serum of GBS patients (Townson et al., 2007; Willison et al., 2004). Alternatively, treatment with recently developed complement inhibitors may block the pathogenic actions of these antibodies (Halstead et al., 2005). New treatment regimens will be further developed and tested in future clinical trials. The above-mentioned two future strategies appear within our abilities and may have a significant impact on the outcome of autoimmune neuropathies, and GBS in particular.

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Barrt syndrome associated with high titers of anti-GM1 antibodies. J. Neurol. Sci. 109:200-206. Ogawara, K., S. Kuwabara, M. Koga, M. Mori, N. Yuki, and T. Hattori. 2003. Anti -GMlb IgG antibody is associated with acute motor axonal neuropathy and Campylobacter jejuni infection. J. Neurol. Sci. 210:41-45. Ogawara, K., S. Kuwabara, M. Mori, T. Hattori, M. Koga, and N. Yuki. 2000. Axonal Guillain-Barrt syndrome: relation to anti-ganglioside antibodies and Campylobacter jejuni infection in Japan. Ann. Neurol. 48:624-631. O’Hanlon, G. M., J. J. Plomp, M. Chakrabarti, I. Morrison, E. R. Wagner, C. S. Goodyear, X. Yin, B. D. Trapp, J. Conner, P. C. Molenaar, S. Stewart, E. G. Rowan, and H. J. Willison. 2001. Anti-GQlb ganglioside antibodies mediate complement-dependent destruction of the motor nerve terminal. Brain 124:893906. Parker, C. T., S. T. Horn, M. Gilbert, W. G. Miller, D. L. Woodward, and R. E. Mandrell. 2005. Comparison of Campylobacter jejuni lipooligosaccharide biosynthesis loci from a variety of sources. J. Clin. Microbiol. 43:2771-2281. Parkhill, J., B. W. Wren, K. Mungall, J. M. Ketley, C. Chucher, D. Basham, T. Chillingworth, R. M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A. V. Karlyshev, S. Moule, M. J. Pallen, C. W. Penn, M. A. Quall, M. A. Rajandream, K. M. Rntherford, A. H. M. van Vliet, S. Whitehead, and B. G. Barrtll. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665-668. Perera, V. N., I. Nachamkin, H. Ung, J. H. Patterson, M. J. McConville, P. J. Coloe, and B. N. Fry. 2007. Molecular mimicry in Campylobacter jejuni: role of the lipooligosaccharidecore oligosaccharide in inducing anti-ganglioside antibodies. FEMS Immunol. Med. Microbiol. 50:27-36. Phongsisay, V., V. N. Perera, and B. N. Fry. 2006. Exchange of lipooligosaccharide synthesis genes creates potential Guillan Barrt syndrome-inducible strains of Campylobacter jejuni. Infect Immun. 74:1368-1372. Plasma Exchange/Sandoglobulin Guillain-Barrt Syndrome Trial Group. 1997. Randomised trial of plasma exchange, intravenous immunoglobulin, and combined treatments in Guillain-Barrt syndrome. Lancet 349:225-230. Plomp, J. J., P. C. Molenaar, G. M. O’Hanlon, B. C. Jacobs, J. Veitch, M. R. Daha, P. A. van Doorn, F. G. A. van der Mech&, A. Vincent, B. P. Morgan, and H. J. Willison. 1999. Miller Fisher anti-GQlb antibodies: alpha-latrotoxin-like effects on motor endplates. Ann. Neurol. 45:189-199. Pryor, W. M., J. S. Freiman, M. A. Gillies, and R. R. Tuck. 1984. Guillain-Barrt syndrome associated with Cumpylobucter infection. Aust. N.Z. J. Med. 14:687-688. Rees, J. H., and R. A. C. Hughes. 1994. Campylobacter jejuni and Guillan-Barrt syndrome. Ann. Neurol. 35:248-249. Rees, J. H., N. A. Gregson, and R A. C. Hughes. 1995a. Antiganglioside GM 1 antibodies in Guillain-Barrt syndrome and their relationship to Campylobacter jejuni infection. Ann. Neu701. 38:809-816. Rees, J. H., S. E. Soudain, N. A. Gregson, and R. A. C. Hughes. 1995b. Campylobacter jejuni infection and Guillain-Barrt syndrome. N. Engl. J. Med. 333:1374-1379. Rees, J. H., R. W. Vaughan, E. Kondeatis, and R. A. Hughes. 1995c. HLA-class I1 alleles in Guillain-Barrt syndrome and Miller Fisher syndrome and their association with preceding Campylobacter jejuni infection. J. Neuroimmunol. 62:53-57. Rhodes, K. M., and A. E. Tattersfield. 1982. Guillain-Barrt syndrome associated with Campylobacter infection. Br. Med. J. 285: 173-1 74. Ropper, A. H., E. F. M. Wijdicks, and B. T. Truax. 1991. Guillain-Bad Syndrome. F. A. Davis Company, Philadelphia.

Ropper, A. H. 1988. Campylobacter diarrhea and Guillain-Barrt syndrome. Arch. Neurol. 45:655-656. Saida, T., S. Kuroki, Q. Hao, M. Nishimura, M. Nukina, and H. Obayashi. 1997. Campylobacter jejuni isolates from Japanese patients with Guillain-Barrt syndrome. J. Infect. Dis. 1765129S134. Schonberger, L. B., E. S. Hurwitz, P. Katona, R. C. Holman, and D. J. Bregman. 1981. Guillain-Barrt syndrome: its epidemiology and associations with influenza vaccination. Ann. Neurol. 9:3 138. Sinha, S., K. N. Prasad, D. Jain, C. M. Pandey, S. Jha, and S. Pradhan. 2006. Preceding infections and anti-ganglioside antibodies in patients with Guillain-Barrt syndrome: a single centre prospective case-control study. Clin. Microbiol Infect. 13:3 16346. Sovilla, J. Y., R. Regli, and P. B. Francioli. 1988. Guillain-Barrt syndrome following Campylobacter jejuni enteritis. Report of three cases and review of the literature. Arch. Intern. Med.148: 739-74 1. Speed, B. R., J. Kaldor, J. Watson, H. Newton-John, W. Tee, D. Noonan, and B. W. Dwyer. 1987. Campylobacter jejunilcampylobacter coli-associated Guillain-Barre syndrome. Immunoblot confirmation of the serological response. Med. 1.Aust. 147: 13-16. Susuki, K., M. Odaka, M. Mori, K. Hirata, and N. Yuki. 2004. Acute motor axonal neuropathy after Mycoplasma infection. Evidence of molecular mimicry. Neurology 62:949-956. Susuki, K., M. N. Rasband, K. Tohyama, K. Koibuchi, S. Okamoto, K. Funakoshi, K. Hirata, H. Baba, and N. Yuki. 2007. Anti-GM1 antibodies cause complement-mediated disruption of sodium channel clusters in peripheral motor nerve fibers.J. Neurosci. 27:3956-3976. Svedhem, A., and B. Kaijser. 1980. Campylobacter fetus subspecies jejuni: a common sause of diarrhea in Sweden. J. Infect. Dis. 142: 353-3 59. Taboada, E. N., A. F. van Belkum, N. Yuki, R. R. Acedillo, P. C. Godschalk, M. Koga, H. P. Endtz, M. Gilbert, and J. H. Nash. 2007. Comparative genomic analysis of Campylobacter jejuni associated with Guillain-Barrt and Miller Fisher syndromes: neuropathogenic and enteritis-associated isolates can share high levels of genomic similarity. BMC Genomics 8:359. Takahashi, M., M. Koga, K. Yokoyama, and N. Yuki. 2005. Epidemiology of Campylobacter jejuni isolated from patients with Guillain-Barrt and Fisher syndromes in Japan. J. Clin. Microbiol. 43:335-339. Tam, C. C., L. C. Rodrigues, I. Petersen, A. Islam, A. Hayward, and S. J. O’Brien. 2006. Incidence of Guillain Barrt syndrome among patients with Campylobacter infection: a general practice research database study. J. Infect. Dis. 194:95-97. Townson. K., J. Boffey, D. Nicholl, J. Veitch, D. Bundle, P. Zhang, E. Samai, T. Antoine, A. Bernardi, D. Arosio, S. Sonnino, N. Isaacs, and H. Willison. 2007. Solid phase immunoadsorption for therapeutic and analytical studies on neuropathyassociated anti-GM1 antibodies. Glycobiology 17:294-303. Turck, D. C. 1984. The pathogenesis of Haemophilw infection. J. Med. Microbiol. 18:l-16. Winer, J. B., R. A. C. Hughes, M. J. Anderson, D. M. Jones, H. Kangro, and R P. Watkins. 1988a. A prospective study of acute idiopathic neuropathy. 11. Antecedent events. J. Neurol. Neurosurg. Psychiatry 5 1:6 13-6 18. Winer, J. B., R. A. C. Hughes, and C. Osmond. 1988b. A prospective study of acute idiopathic neuropathy. I. Clinical features and their prognostic value. J. Neurol. Neurosurg. Psychiatry 51: 605-612. van Belkum, A., N. van den Braak, P. Godschalk, W. Ang, B. Jacobs, M. Gilbert, W. Wakarchuk, H. Verbrugh, and E. Endtz.

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2001. A Campylobacter jejuni gene associated with immunemediated neuropathy. Nut. Med. 7:752-753. van der Mecht, F. G. A., P. I. M. Schmitz, and the Dutch GuillainBarrt Study Group. 1992. A randomized trial comparing intravenous immune globulin and plasma exchange in Guillain-Barrt syndrome. N. Engl.]. Med. 326:1123-1129. van Koningsveld, R., R. Rico, I. Gerstenbluth, P. I. Schmitz, C. W. ang, I. S. Merkies, B. C. Jacobs, Y. Halabi, H. P. Endtz, F. G. van der Mecht, and P. A. van Doorn. 2001. Gastroenteritisassociated Guillain-Barrt syndrome on the Caribbean island Curafao. Neurology 56:1467-1472. van Koningsveld, R., P. I. M. Schmitz, F. G. A. van der MechC, L. H. Visser, J. Meulstee, and P. A. van Doorn, for the Dutch GBS Study Group. 2004. Effect of methylprednisolone when added to standard treatment with intravenous immunoglobulin for Guillain-Barrt syndrome: randomised trial. Lancet 363:192196. van Koningsveld, R., E. W. Steyerberg, R. A. C. Hughes, A. V. Swan, P. A. van Doorn, and B. C. Jacobs. 2007. A clinical prognostic scoring system for Guillain-Bard syndrome. Lancet Neurol. 6:5 89-594. van Koningsveld, R., P. van Doorn, P. I. Schmitz, C. W. Ang, and F. G. S. van der Mecht. 2000. Mild forms of Guillain-Barrt syndrome in an epidemioloc survey in The Netherlands. Neurology 54:620-625. van Sorge, N. M., W. L. van der Pol, M. D. Jansen, K. P. Geleijns, S. Kalmijn, R. A. Hughes, J. H. Rees, J. Pritchard, C. A. Vedeler, K. M. Myhr, C. Shaw, I. N. van Schaik, J. H. Wokke, P. A. van Doorn, B. C. Jacobs, J. G. van de Winkel, and L. H. van den Berg. 2005. Severity of Guillain-Barrt syndrome with Fc gamma Receptor 111 polymorphisms. J. Neuroimmunol. 162:157-164. Visser, L. H., F. G. A. van der Mecht, P. A. van Doorn, J. Meulstee, B. C. Jacobs, P. G. Oomes, and R. P. Kleyweg for the Dutch Guillain-Barrt Study Group. 1995. Guillain-Barrt syndrome without sensory loss (acute motor neuropathy). Brain 118: 84 1-847. Von Wulffen, H., C. Hartard, and E. Scharein. 1994. Seroreactivity to Campylobacter jejuni and gangliosides in patients with Guillain-Barrt syndrome. 1.Infect. Dis.170:828-833. Vriesendorp, F. J., B. Mishu, M. J. Blaser, and C. L. Koski. 1993. Serum antibodies to GM1, GDlb, peripheral nerve myelin, and Campylobacter jejuni in patients with Guillain-BarrC syndrome and controls: correlation and prognosis. Ann. Neurol. 34:130135.

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Walsh, F. S., M. Cronin, S. Koblar, P. Doherty, J. Winer, A, Leon, and R. A. C. Hughes. 1991. Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barrt syndrome. J. Neuroimmunol. 34:43-5 1. Willison, H. J., and P. G. Kennedy. 1993. Gangliosides and bacterial toxins in Guillain-Barrt syndrome. J. Neuroimmunol. 46: 105-112. Willison, H. J., K. Towson, J. Veitch, J. Boffey, N. Isaacs, S. M. Andersen, P. Zhang, C. Ling, and D. R. Brundle. 2004. Synthetic disialylgalactose immunoadsorbents deplete anit-GQlb antibodies from autoimmune neuropathy sera. Bruin 127:680691. Willison, H. J., and N. Yuki. 2002. Peripheral neuropathies and anti-glycolipid antibodies. Brain 125 :2591-2625. Willison, H. J. 2005. The immunobiology of Guillain-Barrt syndromes. J. Peripher. New. Syst. 10:94-112. Xiang, S. L., M. Zhong, F. C. Cai, B. Deng, and X. P. Zhang. 2006. The sialic acid residue is a crucial component of Campylobucter jejuni ganglioside mimicry in the induction of the Guillain Barrt syndrome.]. Neuroimmunol. 174:126-132. Yuki, N. 2007. Ganglioside mimicry and peripheral nerve disease. Muscle Nerve 35:691-711. Yuki, N., S. Sato, S. Fujimoto, S. Yamada, Y. Tsujino, A. Kinoshita, and T. Itoh. 1992. Serotype of Carnpylobacter jejuni, HLA, and the Guillain-BarrC syndrome. Muscle Nerve 15:968969. Yuki, N., K. Susuki, M. Koga, Y. Nishimoto, M. Odaka, K. Hirata, K. Taguchi, T. Miyatake, K. Furukawa, T. Kobata, and M. Yamada. 2004. Carbohydrate mimicry between human ganglioside GMI and Campylobacter jejuni lipooligosaccharide causes Guillain-Barrt syndrome. Proc. Natl. Acad. Sci. USA 101:1140411409. Yuki, N., and Y. Tagawa. 1998. Acute cytomegalovirus infection and IgM anti-GM2 antibody. J. Neurol. Sci. 154:14-17. Yuki, N., T. Taki, F. Inagaki, T. Kasama, M. Takahashi, K. Saito, S. Handa, and T. Miyatake. 1993. A bacterium lipopolysaccharide that elicits Guillain-Barrt syndrome has a GMI ganglioside-like structure. 1.Exp. Med. 178:1771-1775. Yuki, N., T. Taki, M. Takahashi, K. Saito, H. Yoshino, T. Tai, S. Handa, and T. Miyatake. 1994. Molecular mimicry between G Q l b ganglioside and lipopolysaccharides of Campylobacter jejuni isolated from patients with Fisher’s syndrome. Ann. Neurol. 36:79 1-793. Yuki, N., H. Yoshino, S. Sato, and T. Miyatake. 1990. Acute axonal polyneuropathy associated with anti-GM1 antibodies following Campylobacter enteritis. Neurology 40: 1900-1902.

Campylobacter, 3rd ed. Edited by 1. Nachamkin, C. M. Szymanski, and M. J. Blaser Q 2008 ASM Press, Washington, DC

Chapter 14

Mechanisms of Antibiotic Resistance in Campylobacter QIJINGZHANG

AND PAUL J. PLUMMER

organisms (Appelbaum and Hunter, 2000; Hooper, 1998). They are among the most valuable antimicrobials available to treat bacterial infections because of their broad spectrum of activity, pharmacodynamics, safety, and ease of administration (Appelbaum and Hunter, 2000; Chu, 1999b). Once inside bacterial cells, FQ antimicrobials interact with two target enzymes, DNA gyrase and topoisomerase IV (Drlica and Zhao, 1997; Hooper, 2001), forming stable complexes with these enzymes and trapping the enzymes on DNA, which results in double-stranded breaks in DNA and bacterial death (Drlica and Malik, 2003; Shea and Hiasa, 1999; Willmott et al., 1994). In gram-negative bacteria, DNA gyrase is the primary target of FQ antibiotics, while topoisomerase IV is the main target of FQs in gram-positive bacteria (Hooper, 2001). FQ antimicrobials are important in the treatment of enteric infections including campylobacteriosis (Adachi et al., 2000). The mechanisms involved in FQ resistance include modification of DNA gyrase and/ or topoisomerase IV, active efflux, altered membrane permeability, and reduced target enzyme expression (Hooper, 2001; Levy, 1992; Poole, 2000). All of these resistance mechanisms are encoded by chromosomal elements. However, plasmid-mediated quinolone resistance has been recently reported in Klebsiella pneumoniae and Escherichia coli, in which the plasmid-carried quinolone-resistance gene encodes a protein (Qnr) that protects DNA gyrase from inhibition by the drug (Tran et al., 2005; Wang et al., 2003, 2004). Alterations in target enzymes (DNA gyrase and topoisomerase IV) have been studied extensively in bacterial pathogens and are mainly due to spontaneous mutations in the quinoloneresistance-determining region of the genes encoding GyrA or ParC (Khodursky et al., 1995; Yoshida et

Campylobacter spp., especially Campylobacter jejuni and Campylobacter coli, are increasingly resistant to antimicrobials, which has become a significant concern for public health. To counteract the selection pressure from antimicrobial usage, the Campylobacter pathogens have evolved multiple mechanisms for antimicrobial resistance, including (i) synthesis of antibiotic-inactivating/modifying enzymes (e.g., plactamase), (ii) alteration or protection of antibiotic targets (e.g., mutations in gyrA or 23s rRNA genes), (iii) active extrusion of drugs out of bacterial cells through drug efflux transporters (e.g., CmeABC), and (iv) reduced permeability to antibiotics due to unique membrane structures. Some of the resistanceassociated traits are endogenous to Campylobacter, whereas others are acquired by mutation or genetic transfer. In this chapter, we will discuss the mechanisms involved in Campylobacter resistance to various antibiotics, with a particular emphasis on new information obtained during the last few years. It should be pointed out that our understanding of the resistance mechanisms is limited to C. jejuni and C. coli, and little information is available on antimicrobial resistance in other Campylobacter spp. In addition, the epidemiology of antibiotic resistance and the transfer of antibiotic-resistant Campylobacter from animals to humans through the food chain are discussed elsewhere (chapter 36) and will not be covered in this chapter.

FLUOROQUINOLONE (FQ) RESISTANCE FQ antimicrobials, such as ciprofloxacin, enrofloxacin, and levofloxacin, are a class of structurally related synthetic compounds with potent bactericidal activity against both gram-positive and gram-negative

Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 1116 Veterinary Medicine, Qijing Zhang Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 2180 Ames, IA 50011. Paul J. Plummer Veterinary Medicine, Ames, LA 50011.

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al., 1990). Mutations in the quinolone-resistancedetermining region drastically reduce the affinity of gyrases to quinolone antimicrobials, resulting in resistance to the bactericidal action. Resistance-associated mutations can also occur in GyrB and ParE (Hooper, 2001). Campylobacter has become increasingly resistant to FQ antimicrobials (Aarestrup and Wegener, 1999; Bachoual et al., 2001; Ge et al., 2003; Gupta et al., 2004; Nachamkin et al., 2002; Piddock, 1995; Rautelin et al., 1991; Ruiz et al., 1998; Saenz et al., 2000; Sanchez et al., 1994; Smith et al., 2000; Van Looveren et al., 2001). The resistance is mediated by point mutations in the gyrA gene in conjunction with the function of the multidrug efflux pump CmeABC (Ge et al., 2005; Luo et al., 2003; Zhang et al., 2003). Specific mutations at positions Thr-86, Asp90 and Ala-70 in GyrA have been linked to FQ resistance in C. jejuni (Engberg et al., 2001; Luo et al., 2003; Wang et al., 1993). Specifically, the Thr-86-Ile change (mediated by the C257T mutation) is the most commonly observed mutation in FQ-resistant Campylobacter isolates and confers high-level (ciprofloxacin MIC 2 1 6 pg/ml) resistance to FQs, while the Asp-90-Asn and Thr-86-Lys mutations are less common and are associated with intermediate-level FQ resistance (Gootz and Martin, 1991; Luo et al., 2003; Ruiz et al., 1998; Wang et al., 1993). No mutations in gyrB have been associated with FQ resistance in Campylobacter (Bachoual et al., 2001; Payot et al., 2002; Piddock et al., 2003). Although a single report documented the involvement of ParC mutations in the resistance to FQs (Gibreel et al., 1998), subsequent work revealed that Campylobacter lack the parC and parE genes (Bachoual et al., 2001; Cooper et al., 2002; Luo et al., 2003; Parkhill et al., 2000; Payot et al., 2002; Piddock et al., 2003). Thus, it can be concluded that parC and parE mutations are not involved in Campylobacter resistance to FQ antimicrobials. The contribution of an efflux mechanism to FQ resistance in Campylobacter was reported in an early study (Charvalos et al., 1995), but the identity of the efflux pump was not determined. It has been found that the CmeABC efflux pump contributes significantly to both intrinsic and acquired resistance of C. jejuni to FQ antimicrobials (Ge et al., 2005; Lin et al., 2002; Luo et al., 2003; Pumbwe and Piddock, 2002). This efflux pump reduces the accumulation of FQs in Campylobacter cells and functions synergistically with the gyrA mutations in conferring and maintaining high-level FQ resistance in clinical isolates (Ge et al., 2005; Luo et al., 2003). Overexpression of CmeABC reduces the accumulation of ciprofloxacin in C. jejuni and increases the resistance

conferred by various types of GyrA mutations (Yan et al., 2006). This efflux pump also plays an important role in the emergence of FQ-resistant Campylobacter under selection pressure because many of the spontaneous gyrA mutants cannot survive the selection by ciprofloxacin in the absence of CmeABC (Yan et al., 2006). On the other hand, overexpression of CmeABC increases the frequency of emergence of FQ resistant mutants that are highly resistant to ciprofloxacin (Yan et al., 2006). One unique feature of FQ resistance in Campylobacter is that acquisition of high-level resistance does not require stepwise accumulation of point mutations in gyrA. Instead, a single point mutation in gyrA can lead to clinically relevant levels of resistance to FQ antimicrobials (Ge et al., 2005; Gootz and Martin, 1991; Luo et al., 2003; Ruiz et al., 1998; Wang et al., 1993; Zhang et al., 2003). In vitro, the frequencies of emergence of FQ-resistant GyrA mutants range from approximately to lo-', depending on the concentrations of ciprofloxacin used on the enumerating plates (Yan et al., 2006). The higher the selection pressure, the lower the frequencies of emergence. The reason for the variable frequencies is that multiple types of spontaneous point mutations occur in the gyrA genes, but different point mutations confer varied levels of FQ resistance and thus survive different levels of selection pressure (Yan et al., 2006). When 4 pg/ml of ciprofloxacin was used in the plating media, the frequency of mutant emergence was consistently measured in the order of lo-', and the mutants predominantly harbor the C257T mutation in gyrA (Gootz and Martin, 1991; Yan et al., 2006). In vivo, FQ-resistant Campylobacter mutants occur rapidly in chickens treated with enrofloxacin and reached to as high as lo7 CFU/g feces within 24 to 48 h after the initiation of treatment (Farnell et al., 2005; Griggs et al., 2005; Luo et al., 2003; McDermott et al., 2002; van Boven et al., 2003). FQ-resistant C. jejuni could also rapidly develop in Campylobacter-infected patients treated with ciprofloxacin (Ellis-Pegler et al., 1995; Segreti et al., 1992; Wretlind et al., 1992). These findings suggest that Campylobacter is highly mutable to FQ treatment and that FQ antimicrobials may not be the ideal antibiotics for clinical therapy of campylobacteriosis. The GyrA mutations not only confer FQ resistance, but also affect the fitness of C. jejuni in the chicken host. Luo et al. (2005) examined the fitness of FQ-resistant Carnpylobacter in the absence of selection pressure by using clonally related and genetically isogenic mutants. When monoinoculated into the chickens, FQ-resistant Campylobacter was able to colonize and persist in chickens as efficiently as the

CHAPTER 14

FQ-resistant strains in the absence of FQ antimicrobials. The prolonged colonization in vivo did not result in the reversion or loss of the specific resistance-conferring mutation (C257T) in gyrA. When coinoculated into chickens as pairwise competition, the FQ-resistant Campylobacter outcompeted the FQ susceptible in the majority of pairs. The fitness change was directly linked to the C257T mutation in gyrA. These findings suggest that the resistanceassociated gyrA mutation does not incur a fitness cost and even enhances the fitness of Campylobacter in the absence of selection pressure. This notion is supported by two surveys conducted in the United States (Price et al., 2005, 2007), which revealed that FQresistant Campylobacter was still prevalent in poultry from farms that had not used FQ antimicrobials for several years. The physiological and molecular basis for the enhanced fitness in FQ-resistant Campylobacter is unknown and remains to be determined in future work.

MACROLIDE RESISTANCE Macrolide antibiotics, such as erythromycin, clarithromycin, azithromycin, and telithromycin, are important drugs for the treatment of respiratory tract infections and gastric diseases caused by Helicobacter pylori and Campylobacter in humans (Chu, 1999a; Kasbekar and Acharya, 2005). This class of antibiotics including erythromycin, tylosin, spiramycin, tilmicosin, and roxithromycin are also approved for growth promotion and therapeutic purposes in animal agriculture (McEwen and Fedorka-Cray , 2002; Prescott, 2000). Macrolide antibiotics target the 50s subunit of bacterial ribosome and produce antibacterial effects by inhibiting protein synthesis (Poehlsgaard and Douthwaite, 2005). Bacterial resistance to macrolides is mediated by three mechanisms, including antibiotic modifications, target site modification or alteration, and drug efflux (Leclercq, 2002). Some bacteria produce enzymes that inactivate macrolide antibiotics by phosphorylation or glycosylation (Kuo et al., 1989; Noguchi et al., 1995). Target modification/alteration is via either rRNA methylases encoded by the erm genes or mutations in 23s rRNA or ribosomal proteins L4 and L22 (Jacobs and Johnson, 2003; Maravic, 2004). These changes interfere with the binding of macrolides to the 50s ribosomal subunit and confer macrolide resistance. Effluxmediated macrolide resistance is through various efflux pumps that reduce accumulation of the drugs in bacterial cells Uacobs and Johnson, 2003). Macrolides such as erythromycin are considered the drug of choice for treating human campylobac-

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teriosis, and there is a trend of rising resistance in Campylobacter to erythromycin and related macrolides (Engberg et al., 2001; Gibreel and Taylor, 2006). Two mechanisms, target modification and efflux, are involved in macrolide resistance in Campylobacter, but there is no evidence that Campylobacter produces macrolide-modifying enzymes (Payot et al., 2006). Target modification involves chromosomal mutations in the 23s rRNA gene or the genes encoding ribosomal proteins L4 and L22. Several point mutations in 23s rRNA have been associated with Campylobacter resistance to macrolides (Engberg et al., 2001; Gibreel et al., 2005; Harrow et al., 2004; Jensen and Aarestrup, 2001; Lin et al., 2007; Mamelli et al., 2005), which occur at the base position 2074 (A2074C, A2074G, or A2074T) or 2075 (A2075G, A2075T, or A2075C) or both. These positions correspond to the base positions 2058 and 2059, respectively, in E. coli. Both the A2074G and A2075G changes can confer high-level resistance to macrolide antibiotics, but the A2075G mutation is more frequently observed than the A2074G mutation (Gibreel and Taylor, 2006). Although a previous study indicated that the A2074G mutation was unstable (Gibreel et al., 2005), recent work on the development of macrolide-resistant Campylobacter revealed the predominance of the A2074G mutation in chickens fed with tylosin, and this mutation was stably maintained in antibiotic-free media (Lin et al., 2007). There are three copies of the 23s rRNA gene in the chromosome of C. jejuni and C. coli (Fouts et al., 2005; Parkhill et al., 2000). In most cases, all three copies of the 23s rRNA gene carry the resistanceassociated mutations, but both the wild-type and mutated alleles can coexist in a single macrolide-resistant mutant (Gibreel et al., 2005; Jensen and Aarestrup, 2001; Lin et al., 2007). These point mutations in the 23s rRNA likely block the interaction of macrolide antibiotics with the 50s ribosome and thus confer resistance to this class of antibiotics. In addition to the 23s rRNA mutations, mutations in the ribosomal protein L4 (G74D) and L22 (insertions at position 86 or 98) were also shown to confer macrolide resistance in Campylobacter (Cagliero et al., 2006). The contribution of efflux systems to macrolide resistance in Campylobacter has been described in several recent studies (Cagliero et al., 2005; Gibreel et al., 2007; Lin et al., 2007; Mamelli et al., 2005; Payot et al., 2004). Inhibition by efflux pump inhibitors decreased the MICs of macrolides in various Campylobacter isolates and also significantly decreased the frequency of emergence of erythromycinresistant C. jejuni mutants (Cagliero et al., 2005; Martinez and Lin, 2006; Payot et al., 2004), indicating the involvement of efflux pumps in macrolide re-

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sistance. By use of insertional mutagenesis, CmeABC was found to be a contributor to the intrinsic resistance to macrolides in Campylobacter (Lin et al., 2002; Pumbwe and Piddock, 2002). Subsequently, it was found that CmeABC also contributes significantly to the acquired resistance to macrolide antibiotics (Cagliero et al., 2005, 2006; Lin et al., 2007). In the macrolide-resistant mutants that lack target mutations and show low to intermediate resistance to macrolide, inactivation of CmeABC completely reversed the susceptibility (Cagliero et al., 2005; Lin et al., 2007), while in those mutants that harbor resistanceassociated mutation in L4, L22, or 23s rRNA, CmeABC functions synergistically with the target mutations in conferring the acquired resistance to macrolides (Cagliero et al., 2005, 2006; Lin et al., 2007). These findings clearly indicate that CmeABC is a significant player in conferring the acquired resistance to macrolides. However, there is also some evidence that a CmeABC-independent efflux system also contributes to macrolide resistance in Campylobacter (Mamelli et al., 2005), but the identity of this efflux pump has not been defined. Similar to the situation in other bacteria, the target mutations and efflux pumps in Campylobacter confer cross-resistance to different members of the macrolide family (e.g., erythromycin, clarithromycin, azithromycin, and tylosin), including ketolides (e.g., telithromycin) of this class (Cagliero et al., 2005; Mamelli et al., 2005). It is also reported that crossresistance between macrolides and lincosamides occurs in Campylobacter (Luangtongkum et al., 2006; Taylor and Courvalin, 1988), suggesting that use of lincosamides may potentially affect the development of macrolide resistance in Campylobacter. This crossresistance is probably due to the fact that both classes of antibiotics interact with the same site on the bacteria ribosomal subunit (Poehlsgaard and Douthwaite, 2003). Different from FQ resistance conferred by gyrA mutations, the spontaneous mutation rates for macrolide resistance appear to be low in C. jejuni and C. coli when measured by both in vitro and in vivo studies. As shown by experimental study, treatment of Campylobacter-infected chickens with a therapeutic dose of tylosin (a macrolide) did not select for erythromycin-resistant Campylobacter even after three courses of treatment (Lin et al., 2007). This is in clear contrast to the development of FQ-resistant Campylobacter in chickens treated with enrofloxacin, in which FQ-resistant Campylobacter developed rapidly-in some cases within 24 h (Luo et al., 2003). However, when Campylobacter-infected chickens were fed tylosin at a growth-promoting dose, macrolide-resistant Campylobacter occurred in the

birds after several weeks of exposure, suggesting that emergence of macrolide-resistant Campylobacter requires a prolonged selection process (Lin et al., 2007). The low spontaneous mutation rate and the slow process of resistance development may explain why macrolide-resistant Campylobacter isolates are generally less prevalent than FQ-resistant Cumpylobacter.

TETRACYCLINE RESISTANCE Tetracyclines are an important class of antibiotics widely used in both human and animal medicine. The lipophilic nature of tetracyclines allows them to freely diffuse through the cell membrane of grampositive bacteria and accumulate in the cytoplasm (Chopra and Roberts, 2001). In contrast, transfer across the outer membrane of gram-negative bacteria appears to involve binding of the drug to Mg2+cations and passage through outer membrane porins into the periplasmic space, where the drug dissociates from the magnesium and moves passively through the inner membrane, as is the case in gram-positive bacteria (Chopra and Roberts, 2001). Once in the cytoplasm, the drug binds reversibly with the ribosome, inhibiting peptide elongation by preventing the incoming aminoacyl-tRNA from binding to the A site of the ribosome (Connell et al., 2003a, 2003b). A number of tetracycline resistance genes have been identified in various bacteria to date (Chopra and Roberts, 2001). General mechanisms of tetracycline resistance can be classified into four major categories: efflux pumps, ribosomal protection proteins, chemical modification of the drug, and mutations of the ribosome that alter the ability of the antibiotic to bind. The ribosomal protection protein class of molecules represents a common mechanism of resistance and often confers a broader range of resistance to the tetracycline class of antibiotics (Chopra and Roberts, 2001). To date, two mechanisms have been described for Campylobacter resistance to tetracyclines. The first mechanism involves a ribosomal protection protein termed Tet(0) (Manavathu et al., 1988; Sougakoff et al., 1987; Taylor et al., 1987). The molecular mechanisms underlying the resistance conferred by the Tet(0) protein has been documented recently (Connell et al., 2003a, 2003b). The current model suggests that Tet(0) recognizes the presence of an unoccupied A site on tetracycline blocked ribosomal complexes. Upon binding of the Tet(0) protein, a conformational change occurs in the region of the decoding site and results in the sequential release of the tetracycline molecule and the GTPase-mediated

CHAPTER 14

release of the Tet(0) protein. Interestingly, the conformational state that is induced by the binding of Tet(0) is able to persist after the release of the protein and thus allows the A site to function in protein elongation. The second mechanism of tetracycline resistance in Campylobacter involves the efflux system. The multidrug efflux pump CmeABC has been shown to contribute to both intrinsic and acquired resistance to tetracycline (Gibreel et al., 2007; Lin et al., 2002; Pumbwe and Piddock, 2002). Different from the tetracycline-specific efflux pumps, such as Tet(A) and Tet(B), CmeABC is not tetracycline specific and has a broad substrate spectrum. CmeABC functions synergistically with Tet(0) in conferring high-level resistance to tetracyclines (Gibreel et al., 2007; Lin et al., 2002). The t e t ( 0 ) gene is mainly encoded on transferable plasmids of various sizes, but the gene can be chromosomally encoded in some strains (Akhtar, 1988; Dasti et al., 2007; Manavathu et al., 1988; Ng et al. 1987; Pratt and Korolik, 2005; Sougakoff et al., 1987; Taylor et al., 1987). The pTet plasmid carrying t e t ( 0 ) was fully sequenced, revealing the colocalization of t e t ( 0 )with conjugation-associated genes on the same plasmid (Batchelor et al., 2004). te t (0 )carrying plasmids can be transferred from donor strains to recipient strains via conjugation in laboratory systems (Batchelor et al., 2004; Gibreel et al., 2004b; Nirdnoy et al., 2005; Pratt and Korolik, 2005). In addition, in vivo transfer studies have demonstrated rapid transmission of the t e t ( 0 ) gene between Campylobacter strains via horizontal gene transfer in the intestinal environment of chickens, a natural host for this organism (Avrain et al., 2004). Evolutionarily, the t e t ( 0 )gene likely originated from gram-positive bacteria (Sougakoff et al., 1987; Zilhao et al., 1988). In support of this theory is the fact that the nucleotide sequence of the Campylobacter t e t ( 0 ) gene shares 99.4% homology (98.9% amino acid identity) with that of the t e t ( 0 ) gene in Streptococcus mutans DL5; the G+C content (40%) of the t e t ( 0 ) gene is substantially higher than the remainder sequences of Campylobacter plasmids or genomes (approximately 30%); the codon usage of t e t ( 0 ) is more similar to that of Streptococcus spp. than to that of Campylobacter spp.; and the ribosomal binding site of t e t ( 0 ) matches complementarily to the 3' end of the 16s rRNA of Bacillus subtilis (Batchelor et al., 2004; Khachatryan et al., 2006; Sougakoff et al., 1987; Taylor et al., 1983). In addition, Southern blot experiments have demonstrated the hybridization of the Campylobacter t e t ( 0 )gene to genomic DNA obtained from isolates of tetracycline-resistant Streptoco