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METHODS IN

MICROBIOLOGY

This Page Intentionally Left Blank

METHODS IN

MICROBIOLOGY Volume 14

Edited by

T. BERGAN Department of Microbiology, Institute of Pharmacy and Department of Microbiology, Aker Hospital, University of Oslo, Oslo, Norway

1984

ACADEMIC PRESS (Harcourt Brace Javanovich, Publishers) London Orlando San Diego San Francisco New York Toronto Montreal Sydney Tokyo Siio Paulo

ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road London WI 7DX

US.Edition published by ACADEMIC PRESS INC. (Harcourt Brace Jovanovich, Inc.) Orlando, Florida 32887

. Copyright 0 1984 by ACADEMIC PRESS INC. (LONDON) LTD.

All Rights Reserved

No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

ISBN &12-32151&2 LCCCN: 68-57745 ISSN 058&9517

Filmset in Monophoto Times New Roman by Latimer Trend & Company Ltd, Plymouth Printed in Great Britain by St Edmundsbury Press, Bury St Edmunds, Suffolk

CONTRIBUTORS

T. Bergan Department of Microbiology, Institute of Pharmacy, P.O. BOX1108. University of Oslo, Blindern, Oslo 3. and Department of Microbiology, Aker University Hospital, Oslo, Norway W. H. Ewing 2364 Wineleas Road, Decatur, Georgia 30033, USA

P. Larsson Departments ofClinical Bacteriology and Clinical Immunology, Institute of Medical Microbiology, University of Goteborg. Guldhedsgaten 10, S-41346 Goteborg, Sweden A. A. Lindberg Department of Bacteriology, National Bacteriological Laboratory, and Karolinska Institute. Department of Clinical Bacteriology, Huddinge University Hospital. Huddinge. Sweden. F. Orskov Collaborative Centre for Reference and Research on Escherichia and Klebsiella, (WHO), Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen, Denmark

I. Orskov Collaborative Centre for Reference and Research on Escherichia and Klebsiella, (WHO), Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen, S Denmark

R. Sakazaki Enterobacteriology Laboratories, National Institute of Health, 10-35 Kamiosaki, 2-Chome, Shinagawa, Tokyo 141, Japan

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PREFACE

This volume of “Methods in Microbiology” continues the presentation of epidemiological typing methods of bacteria. In Volume 11 0rskov and 0rskov presented an excellent overview of the serology of the enterobacteria. This has stimulated feedback to the effect that this important group of bacteria should be further described. We have, therefore, included in this and the next volume serological methods for the enterobacteria which have known serology. Chapter 1 is devoted to the general taxonomy of the enterobacteria. Chapter 2 describes the serology of Escherichia coli. The genus Shigella, which is one of the best examples of the diagnostic use of serology, is described in Chapter 3. Chapters 4 and 5 deal with the species which have to be differentiated from these bacteria: Klebsiella, Enterobacter and Hafnia. The protea have been the subject of surprisingly few studies, but Proteus mirabilis and P . vulgaris are presented in detail in Chapter 6. Taxonomic problems and a lack of studies have excluded P . morganii, P . rettgeri and P . inconstans from this volume. In the course of this series it has become apparent that workers dealing with different groups of bacteria use conflicting terminology. One example is the use of the words phase and form. The term phase is used by all for the two Hantigen states of Salmonella cells which have serological changes without altered colony morphology. The S-R variation which has distinct changes in colony morphology, for instance colony form, is accompanied by antigenic changes. This is, for example, the case for Shigella and Proteus, but this antigenic change is called both form variation and phase variation. Since colony form variation is the primary event and takes precedence regarding the publication date the word form will be used for the antigen state of S-R variation. The terminology used to describe different antigen classes is also conflicting. One general comment is needed on the terminology of bacterial antigens. The different classes of antigen can be depicted as for example 0:1,2,3;K:1,2,3;H:a,b,c;F: 1,2,3 or 01,2,3:K1,2,3:Ha,b,c:F1,2,3. The latter is used in the USA and laboratories elsewhere. The former retains the philologically more correct use of punctuation marks and takes precedence

...

Vlll

PREFACE

because of its extensive use from the beginning of bacterial serology. In many groups this has been retained as, for example, 0:1,2,3. In this volume the former alternative will therefore be used. Since specific antigens have been attached tofimbriae we have also touched on the problem of whether to use this or the term pili. Fimbriae has been criticized as not being as linguistically correct as pili, which means hairs (Latin) (C. C. Brinton, 1965, Trans. N . Y . Acad. Sci. Ser. 2 27, 1003). Although fimbriae is derived from Latin and denotes threads, fibres and fringes in the Oxford English Dictionary there is no doubt that fimbriae was used before pili (1966, J. Pathol. Bacteriol. 92, 137-138). The term fimbriae was introduced into the literature by J. P. Duguid and collaborators in 1955 (J. Parhol. Bacteriol. 70,335) and therefore takes precedence over the term pili which was introduced into the literature by C. C. Brinton in 1959 (Nature 183, 782). In adherence to the general principles of precedence described in the International Code of Nomenclature and Taxonomy of Bacteria we use the term jimbriae. Oslo November 1983

T . Bergan

CONTENTS

Contributors Preface

Classification of Enterobacteriacea T. Bergan Serotyping of Escherichia coli F. Orskov and I. 0rskov

V

vii 1

43

Serology of Shigella W. H. Ewing and A. A, Lindberg

113

Serotyping of Klebsiella I. Orskov and F. Orskov

143

Serology of Enterobacter and Hafnia R. Sakazaki

165

Serology of Proteus rnirabilis and Proteus vulgaris P. Larsson

187

Index

215

Contents of published volumes

223

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1 Classification of Enterobacteriaceae T. BERGAN Department of Microbiology, University of Oslo, Norway I. Introduction. . 11. Taxonomy . A. Escherichia and Shigella . . . B. Klebsiella and Enterobacter C. Hafnia . D. Citrobacter . E. Salmonella . F. Serratia . G. Morganella, Proteus and Providencia . H. Yersinia . I . Edwardsiella, Erwinia and Pectobacterium . J. Kluyvera . K. Rahnella L. Cedecea . References .

. .

1 3 1 7 15 19 23 25 28 29 32 32 35 31 31

I. Introduction Without unwarranted exaggeration merely to stress this topic, it is true to say that there is more interest in the classification of Enterobacteriaceae than in any other group of bacteria. This is understandable since these organisms grow readily, are among the most common isolates studied in medical bacteriology and cause a range of serious infections (e.g. urinary tract infections, wound infections and septicaemia) including epidemic diseases such as salmonelloses or dysenteria (requiring constant public monitoring, which is co-ordinated by the World Health Organization). Escherichia coli and Salmonella species are important tools for the geneticist, molecular biologist, cloner and biochemist. The importance of these organisms motivates this discussion of typing methods for the Enterobacteriaceae. However, although the relationships between species within enterobacteria seem closer than is the case for most bacterial genera, a sharp increase in the number of proposals for new taxa within the Enterobacteriaceae has appeared recently. This situation follows the application of new methods such as DNA METHODS IN MICROBIOLOGY VOLUME 14

Copyright 0 1984 by Academic Press, London All rights of reproduction in any form reserved.

2

T. BERGAN

hybridization, the availability of objective numerical grouping procedures, a rising interest in habitats outside traditional human medicine and the fact that more research groups are involved in the taxonomy of these microbes. The changes in nomenclature of these organisms appear in Table I. One consequence of the rising interest is the presentation of diverse taxonomic schemes and names. Edwards and Ewing (1972), Ewing (1963, 1972) and Ewing and collaborators (Edwards et al., l972,1978a,b; Ewing and Fife, 1966, 1972; Ewing et al., 1971) as well as various other groups at the Pasteur Institute in Paris (e.g. Grimont and collaborators, Le Minor and collaborators and Veron and collaborators) have contributed greatly to our improved understanding of the relationships between the enterobacterial taxa. Computer taxonomy and identification combined with DNA relationships (Brenner, 1981a,b, 1983) have completely changed the taxonomy as presented, for instance, in the eighth edition of “Bergey’s Manual of Determinative Bacteriology” (Cowan, 1974a,b). In this chapter, consequently, an overview of the present status of the taxonomy and classification of Enterobacteriaceae is presented. The major characteristics definable by traditional bacteriological determinative methods are presented. The purely phenetic relationships of enterobacteria have been studied by numerical taxonomy only to a limited degree (Baer and Washington, 1972; Bascomb et al., 1971;Colwell and Mandel, 1964; Colwell et al., 1974;Johnson et al., 1975; McKelfand Jones, 1976). We also demonstrate in this volume the degree of phenetic relationship between the major taxa as assessed by a collection of clinical isolates. Finite agreement is missing on the criteria for the definition of species or genera and the extent of distinction required between the respective taxonomic ranks. The high phenetic similarity between the species of enterobacteria is related to their wide occurrence and because they exhibit relatively diverse metabolic activity. Research into enterobacteria has led to the working hypothesis that a DNA-DNA homology above 70% can serve as a practical and enforceable break point between species (Brenner, 1981a). This allows both further subdivision of recognized species and recognition of previously unclassified groups of bacteria, and would allow lumping together of some recognized species, both as members of the same species and as belonging to the same genus. For instance, Klebsiella oxytoca, which has been considered a subspecies of K. pneumoniae, has less than 70% DNA homology with the type strain of the latter justifies recognition as an independent species. On the other hand, the four species of Shigella and E. coli have high DNA homology which would be consistent with placing them in the same genus although it is unacceptable to the clinical bacteriologist who requires a practical tool for the differentiation between pathovars. DNA-associated

1 . CLASSIFICATION OF ENTEROBACTERIACEAE

3

approaches can resolve the classification or position of biochemically aberrant strains or questionable catch-it-alls as in the original concept of the species Enterobacter agglomerans. In many published studies one or rather few reference DNAs have been taken to represent one species. Heterogenicity may require more labelled DNAs in taxonomic studies employing DNA hybridization. This would improve on the “representativeness” of the labelled DNAs. For instance, one single reference DNA was used for the delineation of Yersinia frederikseni (Bercovier et al., 1978, 1980a,b), which in other studies has been questioned because of phenetic overlapping to Y . enterocolitica and was originally considered distinct (Kapperud et al., 1981). Perhaps four or five labelled reference DNAs should be formalized as a minimum before the subdivision of a single species can be accepted. A numerical grouping method such as principal components analysis employed to sort out the large body of quantitative data efficiently and, not the least, objectively, would serve as a useful tool for such identification schemes. Further phenetic and genetic analysis of this type is being carried out on strains from the typical clinical laboratory and from less traditional sources (Kapperud et al., 1981; Kapperud and Bergan, 1984). The achievements demonstrated by the thorough study of Grimont and Popoff (1980) applying principal components analysis to the interpretation of DNA relatedness are pioneering in this regard. Besides the formal and academic professionalism required of orderly taxonomy, it is important that agreement on genus classification is universal, particularly for epidemic and pathogenic species, such as Salmonella typhi, S. arizonae, Shigella sonnei or S . dysenteriae. Therefore the pragmatic aspects of species differentiation should be considered by appropriate selection of the bacterial characteristics. 11. Taxonomy

The taxonomic structure of the enterobacteria of the 8th edition of “Bergey’s Manual of Determinative Bacteriology” (Cowan, 1974a) is reflected by the criteria listed in Table 11. Species have been designated by subjective, and thus necessarily arbitrary, assessment of the differences separating them from existing taxa. Therefore, conflicting data have repeatedly arisen from objective criteria, such as phenetic affinity assessed by numerical taxonomy or genetic relationships reflected by DNA-DNA or RNA-DNA hybridization. DNA relatedness has caused the family to be extensively revised and suggested new species (Brenner, 1981a; Brenner et al., 1976, 1977a). Species such as E. coli and Y. enterocolitica are rather heterogeneous phenetically. Unusual biotypes may be shown by the nearest outlier principle. Brenner (1981a) indicates that further revision could be made for E. coli, Salmonella

TABLE I Changes in the nomenclature of species within the Enterobacteriaceae Genus

Species in “Bergey’s Manual of Determinative Bacteriology” (1974)

Species modified since 1974

Buttiauxella Cedecea

B. agrestris C . davisae, C. lapagei, C. neteri, Cedecea species 4, Cedecea species 5

Citrobacter

C. freundii

Edwardsiella Enterobacter

E. tarda (synonym : E. anguillimortijera) E. aerogenes (synonym : K. mobilis) E. agglomerans (synonyms: Envinia E. cloacae herbicola, Erw. stuartii, Erw. uredovora, Escherichia adecarboxylata), E. sakazakii (synonym: yellow-pigmented E. cloacae) E. amylovora, E. earotovora (synonym: Pectobacterium carotovorum), E. chrysanthemi (synonym : P. chrysantherni), E. cvpripedii (synonym: P. cypripedii), E. herbicola (synonym : Enterobacter agglomerans), E. nigripuens, E. quercina, E. rhapontici, E. rubrifaciens, E. salicis, E. stewartii (synonym : Enterob. agglomerans), E. tracheiphila, E. uredovora (synonym : Enterob. agglomerans)

Erwinia

Not listed in “Bergey’s Manual of Determinative Bacteriology” (1974)

C. amalonaticus (listed as C. intermedius biotype a; synonym: L. amalonatica) C . diversus (listed as C. intermedius biotype b; synonym: L. malonatica)

E. hoshinae, E. ictaluri, E. amnigenus, E. gergoviae, E. intermedium, Enterobacter species 1

E. carnegieana (synonym : Pectobacterium carnegieana), E. mallotivora

Escherichia

E. coli

Hafnia Klebsiella

H. alvei K. ozaenae, K. oxytoca (synonym : K. pneumoniae, indole positive), K. pneumoniae, K. rhinoscleromatis

E. adecarboxylata (synonym : Enterob. agglomerans), E. blattae, E. hermannii, E. ewing, E. fergusonii, E. vulneris, Escherichia species 1, Hafnia species 1 , Hafnia species 2, K. planticola, K. terrigena, Klebsiella species 1

KluyVera Morganella M . morganii (synonym: Prof. morganii) Obesumbacterium Proteus Prot. mirabilis, Prot. vulgaris, Prot. rettgeri (synonym : Prvd. rettgeri) Providencia Prvd. alcalifaciens (synonym : Prot. inconstans biotype A), Prvd. stuartii (synonym : Prot. inconstans biotype B) Rahnella See (Bergan, 1984) Salmonella Serratia S. liquejaciens, S. marcescens Shigella Tatumella Xenorhabdus Yersinia

S.boydii, S.dysenteriae, S.Jexneri, S. sonnei

Y. pestis, Y . pseudotuberculosis

From Brenner (1981a. 1983).

Y. enterocolitica

K. ascorbata, K. cryocrescens, Kluyvera species 1 0.proteus Prot. myxofaciens, P. penneri Providencia species 1

R. aquatilis

S.ficaria, S.fonticola, S.odorifera, S. proteamaculans, S.plymuthica, S. rubidaea (synonym : Serr. marinorubra) T. ptyseos X . luminescens,.'A nematophilus, Xenorhabdus species 1 Y . frederiksenii, Y . intermedia, Y . kristensenii, Y. ruckeri, Yersinia species I

TABLE II Differentiation of genera within Enterobacteriaceae and Pasteurella multocida _

Characteristic Indole UICaV Mannitol H,S (agar slant) Motility 35-37°C 22-28°C Acetoin production (Voges-Roskauer) 35-37°C 25-28°C CitEe KCN PhenyIaIanine deaminase Mucate

Escherichia

Shigella

Cirrobacrer

+/-(70-80)

Klebsiella Enterobacter Hafnia Edwardsiella Serratia Salmonella

+ -

+

Protnrs

Erwinia

-If+) -I(+)

-

+/K:-

+

+/+/-

-

+'

+/+/-

-/+

+/-

+-

+ +

+/-

+ +'

_

-/+ -I+ -I+

~

Yersinio

Posreurella multocida

+I(-) +/-

-

-/+

-

-

+ (Y. pestis-)

-

-

-

+-

+ -

+/-

-

-( Y. pestis?)

Symbols: +, most strains have positive reactions; -, most strains have negative reactions; -/+, variable results for different isolates, dominant reaction indicated inverse of previous sign. first; +/( -), most species positive reaction but species with mostly negative reaction included; -I(+), 'S. dysenreriae:mannitol-(0). other species 83-W/, mannitol positive. Shigellae species strains mucate negative except S. sonnei, 16%of which is mucate positive. ' C. fieundii 96% KCN positive, C. diversus KCN negative. Proreus spccies mannitol negative except P. rerrgeri,which is 99%positive; includes P. morganii and previous Providencia. Proteus species indole positive except P. mirabilis, which is 2% positive. P. mirabilis reportedly 16%positive. * Negative Simon's citrate occurs in P. mirabilis and P. vulgaris. Negative strains on Christensen's urea found in P. inconstans (5-lVA+),P. mirabilis (88%+) and P. vulgaris (95%+). H,S reaction may be negative in S. ryphi (94%+) and S. choleraesuis (60"/.+). Among other Salmonella 98% are positive. Citrate (Simmons) positive in 99.3% of Salmonello strains except S. ryphi and S. choleraesuis, where the reaction is always negative after overnight incubation. ' Mucate positive in 88% of Salmonella strains except S. ryphi and S. choleraesuis, where the reaction is always negative.

'

'

' '

I . CLASSIFICATION OF ENTEROBACTERIACEAE

7

and Shigella, but since the current classification is established clinically any changes may cause considerable confusion. In the following tables species are characterized with respect to the percentage of strains which demonstrate the properties. These characterizations follow particularly the extensive studies of Edwards and Ewing( 1972), Ewing (1963,1972) and collaborators and Ewing and collaborators (Edwards et al., 1972, 1978a,b; Ewing and Fife, 1966, 1972; Ewing er al., 1971, 1972, 1978). These percentages are necessarily influenced by the randomness of the selecting strains studied in the respective laboratories. Some species have not been formally designated but their existence was published by Brenner (198 1a, 1983). A. Escherichia and Shigella

E. coli overlaps genetically (DNA relatedness) with the four Shigella species. But since these five entities have such a long standing, and define distinct pathovars, their continued recognition as five separate species is maintained (Table 111) (IZlrskov, 1981). Strains related to both E. coli and Shigella are commonly recognized as Alkalescens-Dispar. They can be classified as nonaerogenic E. coli and are both non-motile and lactose negative (but ONPG positive). The relationship between the genera Escherichia and Shigellu is further reflected by pathogenicity. The shigellae are typically enteroinvasive, as are also certain enteropathogenic E. coli. The strains belonging to the tribe Escherichiae (which includes both E. coli and Shigella) (Cowan, 1974a,b) do not form acetoin, are methyl red negative, lack both phenylalanine deaminase and urease, show variable growth in the presence of KCN and have 50-53% (G+C), DNA. The genus Escherichia has recently been supplemented by further species, such as E. adecarboxylata, E. blattae, E. ewing, E. fergusonii, E. hermanni and E. vulneris (Brenner, 1981a,b, 1983; Burgess et al., 1973). Only the characteristics of E. blattae and E. adecarboxylata have been published so far. E. ewing has a DNA relatedness of 41-47%, E. fergusonii of about 56% and E. blattae of 42%. Sakazaki el al. (1976) considered that H,S-positive strains of E. coli should continue to belong to this species, and not to Citrobacter. This has been verified by Gavini et al. (1981) who found that this property was vested in a plasmid. This is akin to the fact that atypical strains of all enterobacterial species do occur, for instance, plasmid-mediated lactose fermentation even in isolates of notoriously B-galactosidase-negative species like Morganella morganii, S. typhi and H . alvei (Le Minor and Coynault, 1976).

B. Klebsiella and Enterobacter The characteristics presently recognized for Klebsiella appear in Table IV.

TABLE 111

Characteristic H,S (TSI)

Urease Indole Methyl red (37°C) Aatoin (VogesPtoskauer. 37°C) Citrate (Simmon’s, 37°C) (Christensen’s) KCN Motility (37°C) Gelatin (22°C) Lysine decarboxylase Arginine dihydrolase Ornithine dccarboxylase Phenylalanine deaminase Glucose,acid Glucose, gas Lactose b-Galactosidase (ONPG.37 C) sucrose Mannitol Dulcitol Salicin Adonitol Inositol Sorbitol Arabinose

Biochemicalkultural characteristics of Escherichia and Shigella E. colib

E. blattae

E. adecarboxylara

Shigella sp.

S . dysenteriae

S.flexneri

S . boydii

S . sonnei

Raffinose Rhamnose Malonate Mucate Xylose Trehalose Cellobiose Maltose - CH,-glucose Erythritol Esculin Glycerol Tartrate (Jordan’s) Acetate Alginate Lipase (corn oil) NO, reduction to NO, Polypectate Pigment (G+C) mole % DNA Type strain

d

+

ATCC 13313

ATCC 29930

ATCC 8700

From Edwards and Ewing (1972). Ewing er al. (1971). Brenner (1981). Nonnore (1973) and Burgess er a/. (1973). positive within 48 h; (+). positive reaction within three or more Reactions of biochemicalxultural determinative tests carried out at 36 or 37 C (unless noted). +. W o days; -,no reaction in 90% or more: or -.majority of strains positive, some isolates negative; - or +.majority of isolates negative. some positive; ( + ) o r +,majority of reactions delayed. some occur within two days; - or ( ). negative after two days. positive reactions in majority later.Figures in parentheses indicate percentage of positive reactions. a See text. *E.ewing differs from E. roli in being yellow pigmented, growing in the presence of KCN and being cellobiose positive. E.fergusonii differs from E. roli in the reactions for adonitol (+), cellobiose (+), lactose (-). mucate (-)and sorbitol (-). E. blottoe is nonmotile differs from E. coli in malonate (+). mannitol (-). tartrate (-)and j-galactosidase ( -). ‘d signifies different results.

+

+

TABLE I V Biochemical-cultural characteristics of Klebsiella species Characteristic

H 8 (‘W Urease Indole Methyl red (37°C) (28°C) Acctoin (VogesProskauer. 37°C) (28OC)

Citrate (Simmon’s. 37°C) (28°C) (Christensen’s) KCN Motility (37°C) (28°C) Gelatin (22°C) Lysine decarboxylase A r ~ n i n edihydrolase Ornithine decarboxylase Phenylalanine deaminase Glucose, acid Glucose, gas Lactose B-Galactosidase (ONPG.37 C) Sucrose Mannitol Dulcitol Salicin Adonitol tnositol Sorbitol

K. pneumoniae K . pneumoniae (smsu lato) ( m s u m i c r o )

K.awogenes

K. oxytoca

K. ozaenae

K . rhinoscleromaris K. ferrigeno K . planricolu

hbinose RahOse

Rhamnose Malonate Mucate Starch Xylose Trebalose Celiobiose MaItose -CH,-glucose Erythritol

Esculin Melibiose Glycerol Tartrate (Jordan’s) Acetate Alginate Lipast (wrn oil) NO, reduction to NO, Polypatate (G+ C)mole % DNA Type strain a

See text.

+

-t

The K. pneumonioe strains with positive gelatin reaction presumably belong to what is now recognized as K. oxyroca. From Edwards and Ewing (1972). Fife el ai. (1965). Brenner (1981). Bascomber al. (1971). Izard er a/. (1981). Gardaer and Kado (1972). Stenzel er a / . (1972) and Bagley et 4 i . (1981).

12

T. BERGAN

DNA relatedness has had major consequences for the klebsiellae. The klebsiellae (to which the genera Klebsiella and Enterobacter are assigned) produce 2,3-butanediol, have variable acetoin and methyl red reactions, lack phenylalanine deaminase, may produce urease, grow in the presence of KCN and have 52-59x (G+C) in their DNA. The two general Klebsiella and Enterobacter are separated by more than the lack of motility traditionally considered a major criterion of the former. DNA relatedness has set the two genera distinctly apart (Brenner et al., 1972). Nonmotile strains of traditional Enterobacter species have now by DNA-DNA hybridization been duly demonstrated as distinct from their potential Klebsiella counterpart. Motile strains with a high DNA relatedness, above 80%,with K . pneumoniae, K. rhinoscleromatis and K . ozaenae (but not with K . oxytoca) have now been isolated. These observations indicate that a lack of motility cannot any more be considered as a mandatory trait of Kfebsiella and that this genus needs further taxonomic assessments (Ferragut and Leclerc, 1978). Two new taxa given the provisional names group D and group J are compared with K . pneumoniae in Table V. Within Klebsiella the disagreement on recognition of a narrow K . pneumoniae (sensu stricto) as used by Cowan et al. (1960) and Cowan (1974b) or a broader K . pneumoniae (sensu lato) has largely been resolved in favour of the latter, which originated from the studies of Ewing (1963). DNA TABLE V Differentiation between Group D and Group J klebsiellae from Klebsiella pneumoniae

Characteristic

“Group D ’

Motility KCN Lysine Arginine Ornithine Gelatin Urease Indole Methyl red Acetoin Citrate (Simmon’s) D-Tartrate Tartrate (Jordan) Mucate Malonate From Ferragut and Leclerc (1978).

“Group J”

K . pneumoniae

I . CLASSIFICATION OF ENTEROBACTERIACEAE

13

relatedness of K . aerogenes, K . rhinoscleromatis and K . ozaenue with K . pneumoniae (sensu lato) has motivated the proposed lumping together of these species into one species: K . pneumoniae. Many bacteriologists prefer to continue recognizing K . aerogenes as a separate entity (Barr, 1977). Although international use of one and the same system is to be recommended rather than using two systems, to minimize confusion use of the partially subjective synonyms K . aerogenes and K . pneumoniae is in standing with the code of nomenclature, and this practice is consistent with the recommendations that S. shigella and E . coli should continue as distinct species although their DNA relatedness would indicate otherwise. K . edwardsii and K . atlantae would also be classifiable as K . pneumoniae (sensu lato), since they were considered close to K . aerogenes (Bascomb et al., 1971). The DNA homology study on which this lumping together is based showed 80-90% homology of K . ozaenae, K . rhinoscleromatis and K . atlantae to K . pneumoniae (sensu lato), but no reference strains of K . pneumoniae (sensu stricto) in the sense of Cowan (1974b) or Cowan et al. (1960) were included in this particular study. Since the phenetic distinction of K . pneumoniae (sensu stricto) from K . aerogenes, and thus K . pneumoniae (sensu lato), has been supported by extensive numerical taxonomic studies (Bascomb et al., 1971), further elucidation of the DNA relatedness between K . pneumoniae (sensu stricto) on the one hand and K . aerogenes, K . rhinoscleromatis and K . ozaenae on the other would be useful. On the other hand, DNA relatedness has shown that the K . pneumoniae (sensu lato) as defined in the eighth edition of “Bergey’s Manual of Derminative Bacteriology” (I. IZlrskov, 1974) was too broad (sensu lato) and should not continue to include K . oxytoca, which has been recognized as a separate species (Jain et al., 1974). Indeed it has been proposed that it could form a distinct genus (Oxytocum) (Jain et al., 1974). Seidler et al. (1975) in reviewing the properties of K . pneumoniae isolated from both clinical and environmental sources concluded that the species was rather heterogeneous and included strains with a DNA relationship which would justify subdivision if one employed the suggested (Brenner, 1981a) niveau of 70%. Brenner has suggested that further Klebsiella species are emerging from his DNA hybridization data, both indole-positive and indole-negative species. Table VI shows the criteria that differentiate K. aerogenes from K . pneumoniae (sensu stricto). The recent addition to the Klebsiella species, K . planticola, is difficult to distinguish from K . pneumoniae by traditional biochemicalkultural tests and is encapsulated, although the two species are distinct in DNA relatedness (Bagley et al., 1981). The presently recognizable species of Klebsiella are generally considered to be K . pneumoniae, K . oxytoca, K . rhinoscleromatis, K . ozaenae and K . terrigena. My laboratories continue to recognize K. aerogenes, since strains of K . pneumoniae (sensu stricto) serotype 3 do not appear to have been used as

TABLE VI Differentiation between Klebsiella species, Enterobacter aerogenes and E. cloacae K.pneumoniae K . pneumoniae Characteristic Motility KCN Lysine Arginine Ornithine Gelatin Urease ONPG Indole Methyl red Acetoin Citrate (Simmon’s) Tartrate (Jordan) Mucate Malonate Adonitol Dulcitol Inositol

( s e w lato) (sensu stricto), K . rhino- K . terriK . aerogenes serotype 3 K. oxytoca K . ozaenae scleromatis gena Group D

-

-

+ +-

+ + -

- (+I

-

+-

-

+ +

-

+ + + ++ +

+ +d

+ + + + + + d .+

d d

+ + +

+ + +

+ + + + + d + + ++

Group J E. aerogenes E. cloacae

d

+

d d d

+

+ d

+ + + + +

d d

+

+ + + +

+ ++ +

+-

+-

+ + d + d ++

+ +

- (+) -

-

- (+) -

-

d d d d d d

1 . CLASSIFICATION OF ENTEROBACTERIACEAE

15

labelled DNA in studies on DNA relatedness and strains recognizable as K . pneumoniae have been heterogeneous even in terms of DNA-DNA hybridization. In Enterobacter species the classical E. aerogenes and E. cloacae are maintained and new species have been suggested (Table VII). The heterogeneous E. agglomerans that was proposed by Ewing and Fife (1972) is in the process of becoming redefined with two subspecies (Table VII) and otherwise Erw. adecarboxylata, Erw. herbicola, Erw. stewartii and Erw. uredova which emanate from the original complex. The continued recognition of the epithet agglomerans could create considerable confusion since the name was originally used for such a heterogeneous entity. The transfer of E. aerogenes to Klebsiella as K . mobilis suggested by numerical taxonomy (Bascomb et al., 1971) has been opposed by DNA relatedness data, which since have verified its recognition as an Enterobacter species (Brenner, 1981a,b). Differentiation between K . pneumoniae (sensu lato), E. aerogenes and E. cloacae is particularly required in the medical laboratory (key reactions in Table VII). Besides motility, gelatin liquefaction, ornithine dehydrolase and urease are useful criteria for the separation of the two genera. E. aerogenes and E. cloacae are distinguished by acid formation from adonitol, by inositol in the former, and the different breakdown of arginine and lysine.

C. Hafnia The genus Hafnia was suggested by Msller (1954) and remains recognized as such although Enterobacter has been suggested as suitable (Sakazaki, 1961). Strains of Hafnia are peritrichously flagellated, produce H2S, have variable acetoin and methyl red reactions, have no phenylalanine deaminase or urease and grow in the presence of KCN like the klebsiellae. The (G + C) content of their DNA has been reported differently as 52-57% (Sakazaki, 1974) and 4849% (Ritter and Gerloff, 1966), probably due to differences in methods and the selected strains examined. The characteristics of Hafnia species appear in Tables VIII, IXA and IXB. The species H. alvei contains two groups of phenotypically indistinguishable bacteria, which belong to two distinct DNA relatedness groups (Brenner, 1981a). A third H. alvei related group awaits designation (Brenner, 1981a). H. protea (Priest et al., 1973), which corresponds to the previously described Obesumbacterium proteus (Priest et al., 1973), is divisible into two biotypes. The subdivision was reflected by DNA relatedness, but the difference was in the view of Priest et al. (1973) consistent with recognition as one species, whereas Brenner (1981a,b) considered that biotype 1 corresponds to H. alvei on the basis of DNA relatedness while its biotype 2 is distinct. The DNA relatedness of the two laboratories differs, probably because of different

TABLE VII Biochemicalkultural characteristics of Enterohacrer Characteristic

H,S (TSI) Urease Indole Methvl red (37°C) Acetiin (Voges-Proskauer. 37°C) (30°C) Citrate (Simmon's, 37'C) KCN Motility (37°C) (30°C) Gelatin (22 C) Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase Phenylalanine deaminase Glucose, acid Glucose, gas Lactose /I-Galactosidase (ONPG. 37 C) Sucrose Mannitol Mannose Dulcitol Sa1icin Adonitol

E. cloacae

E. aerogenes

E. agglomerans E. agglomerans (aerogenic) (anaerogenic)

E . sakazakii

E . gergoviae

E. i d w r i s

E. amnigenus

TABLE VIII Differentiation between Enterobacter species and Hafnia K . pneumoniae Characteristic Lysine Arginine Ornithine Urease Acetoin Mucate Adonitol Dulcitol Inositol Sorbitol Deoxyribonuclease Yellow pigment Motility Gelatin a

E. aerogenes E. cloacae E. sakazaki E. agglomerans E. gergoviae E. vulneris E. amnigenus H . alvei

++

-

+ + +

+ +'

Slow, requiring two to four days.

-

+

d d d d d d d

-

d

d d d d

-

-

+ + +-

+ +'

-

+ +.

-

a

+ + d + +

-

+ + +d

+

d

-

+

(sensu lato)

+-

I . CLASSIFICATION OF ENTEROBACTERIACEAE

19

methods and reference strains employed for H. alvei. H. protea biotype 1 was “indistinguishable” (Brenner, 1981a,b) from H. alvei by a DNA relatedness of 72% with H. alvei biotype 1, whereas the relationship to H. alvei biotypes 2 and 3 was only 55% and 30%. The question requires further studies with labelled DNA from more strains of the respective taxa to resolve the question. The biochemical reactions of H. protea biotypes 1 and 2 of H. protea differ from the three biotypes described for H. alvei (Priest e f al., 1973; Brenner, 1981a,b; Sakazaki, 1981).

D. Citrobacter The genus Citrobacter is far from its final state. The eighth edition of “Bergey’s Manual of Determinative Bacteriology”, listed C. freundii and C . intermedius biotypes a and b (Sedlak, 1974). In the same order the names C. freundii, C. diversus and C . amalonaticus apply (Brenner, 198la,b; Farmer, 1981). C.freundii consists of three biotypes with distinct DNA relatedness, but at a rather high level, which is consistent with continued recognition as only one species (Popoff and Stoleru, 1980). The reactions most efficiently separating the species within this genus are H2S, indole, malonate, KCN and adonitol (Tables IXA and IXB). The genus Levinea has been suggested (Young et al., 1971) for strains related to Citrobacter. The species described as Levinea malonatica and L . amalonatica (Young et al., 1971) correspond to C. diversus and C . amalonaticus respectively (Crosa et al., 1974; Farmer, 1981). Sakazaki et al. (1976) considered C . intermedius synonymous with C ..freundii. The nomentaxa C . diversus and C . koseri are synonymous and both are in use (Brenner, 1981a;Cowan, 1974b), although C . diversus had priority before the Approved List of Bacterial Names (Burkey, 1928; Werkman and Gillen, 1932; Frederiksen, 1970), if the same biovar is meant. Based on an extensive phenetic analysis of Levinea, Citrobacter and Enterobacter, Gavini et al. (1976b) could separate the three entities. However, a revision of Levinea and Citrobacter has followed determinations of % (G+C) in their DNA and DNA-DNA hybridization. These methods have shown similar % (G+C) DNA ranges and DNA affinity for Citrobacter and Levinea (Brenner, 1981; Gavini et al., 1976a; Leclerc and Buttiaux, 1965). This situation makes it difficult to separate the two suggested nomentaxa as was originally done on the basis of % (G+C) DNA (Farmer, 1981), and other distinctive criteria are wanted.

TABLE IXA Biochemicalkultural characteristics of Citrohacter, Hafnia and Salmonella Characteristic

C.freundii

H,S (Kliegler'r)

d (82) d (87)

Urease lndol Methyl red (37'C) Aatoin ( W C ) (22C) Citrate (Simmon's. 37'C) (22T) (Christensen's)

-(7)

+(loo) - (0) NI

+(W) NI

+(96) +(96)

i%ty (37'c) Gelatin (22C) - (0) Lysine decarboxylasc -(O) Argininc dihydrolase d (43)(44) Ornithine decarboxylase d (17) -(O) Phenylalanine deaminase Glucose, acid +(loo) Glucose, gas +(91) Lactose dor(+) (39) (51) P-Galactosidase(ONPG.

C . diver.sus/ koseri

C. mtiuloiiurrrus

-

-

-(O)

d

d

-(3) - (0) d (54) d (65)

+ + -

+

-

-

NI

NI

NI d01+(49) (M)

NI NI

+

-

+

+

d

+

-

+(loo)

+-

-

+(loo)

+ +

+ -

d (IS)(%

+(W

d (60)

-(O)

-(9)

- (0)

d

d

d

d (12)(61)

d

+

-

+(W -(I)

-(O) NI - (0) NI NI - (0)

NI

-(0.3) +(95) - (0) +(95) dor(+)(59) +(93)

+(I00 -or (d) (0)(23)

+

-(O)

NI NI NI

NI NI

NI (

+ MO) (90) NI

NI

NI

+(loo) -

-(I)

-(O) -(O)

NI

+ +

d

+(94)

-(O)

NI d (80)

+

S. pararyphi A

d (60)

-(O)

+(W

S. !?phi

+(92) - (0)

+(loo)

-or(d)(O)(%) (4(3) ( 7 9 )

-

+

Sulmonellu rp. S. rholerae-ruis

+(%I

+(96) +(93)

37'C)

sucrose Mannitol Dulcitol

H. alvei

-(O)

+(loo)

NI

NI NI NI (34) +(loo) NI

+(loo)

+(92) -(0.8)

+(loo)

-(0.5)

-

+(W d (87)

NI

+(W

N1 d (5)(15)

- (0) - (2) + ( 100) - (0)

NI NI

NI NI

NI

NI

NI

+(W

NI NI

NI

NI NI

NI

NI NI -(9)

- (0)

NI

NI NI NI

++(92) ('O0) +(loo)

NI

N1

NI

N1

NI

NI

NI NI NI NI NI

NI NI NI

dor(+)(13)(85)

NI

NI NI NI NI

+(loo)

NI

NI

d (61)(17)

NI NI

-0)

NI

NI

NI NI

+(loo)

NI

N1

NI

40)

NI

N1

NI

NI

-

NI

-(O)

NI

NI

- (0) -(O)

+(loo)

+(W

NI

+(loo)

d (6)(31)

NI

NI

NI

- (0) - (0)

-(O)

S. hourenae

NI - (0)

(+)@)@I)

NI

S. ari:onue

-(I31

+(loo)

+(loo) - (0)

S. .salaitiue

N1

+(loo) +(W

+(93)

-

N1

Salmn Adonitol lnositol Sorbitol Arabinose Rafinose Rhamnose Malonate Muate Xylose Trehalose Cellobiose Maltose - CH,-glucoside Erythritol Esculin Melibiose Glycerol Tartrate (Jordan’s) Acetate Alginate Lipase (cron oil) NO, reduction to NO, Yellow pigment ( G + C ) mole O 0 DNA Type strain

-

+ + + + +

+ +

+ + +

+

N1

NI

-

+

-

NI

d

+ +

Id I

NI

NI

NI

+

None 52-53 ATCC 8090

-

+ +

NI

+

N1 NI None None 54 5657 ATCC 271561 ATCC 25405 ATCC 27028

.

NI N1 NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI NI -(O) -(O) - io)

None 47-51 ATCC 13337

From Farmer (1981). Sakazaki (1974). Le Minor and Rohde (1974) and lzard PI ol. (1978). NI. no information.

+Iloo) NI None

22

T. BERGAN

TABLE IXB Further biochemical-cultural characteristic of Salmonella and Hufhia Characteristic

S. enteritidis"

H,S (TSI) +(98) Urease lndole Methyl red (37
H . protea Group 1

H. protea Group 2

-

++ +

+ ++ +

None

None

23

1. CLASSIFICATION OF ENTEROBACTERIACEAE

TABLE MB- continued H . protea

Characteristic

S. enteritidis“

Group 1

(G +C) mole % DNA Type strain

50-53 ATCC 13076

48 NCIB 8771

H . protea Group 2

48

From Priest et al. (1973), Edwards and Ewing (1972), Popoff and Stoleru (1980). Le Minor and Rohde (1974). In the sense of Ewing, for instance subgenus I excluding S. cholerae-suis and S. typhi, but uniting further serotypes such as S. paratyphi A as S. enteritidis serotype A, S. schottmuelleri as S. enteritidis serotype B, S. hirschfeldii as S. enteritidis serotype paratyphi C , S. typhimurium as S . enteritidis serotype typhimurium, S.enteritidis as S. enteritidis serotype enteritidis and S. oranienburg as S. enteritidis serotype oranienburg. (I

E. Salmonella The major species of Salmonella as commonly recognized appear in Table IX. There are presently at least six taxonomic systems of the salmonellae under scrutiny. The taxonomy of Salmonella has been a constant source of controversy. The most widely used system blatantly violates the Bacterial Code of Nomenclature, and assigns Latinized species names to each serotype within subgenus I. Two other major alternatives recognize four subgenera according to Kauffman (1963), in essence as presented in the eighth edition of “Bergey’s Manual of Determinative Bacteriology” and as proposed by Edwards and Ewing (1972). A fourth system has been proposed by Le Minor et al. (1979) basing the recognition of four Salmonella species upon the subgenera of Kauffmann: S. kauffmannii (subgenus I), S . salamae (subgenus II), S. arizonae (subgenus 111) and S. houtenae (subgenus IV). This is essentially as adopted in the latest edition of “Bergey’s Manual of Determinative Bacteriology” (Le Minor and Rohde, 1974), except that each serotype in subgenus I was still assigned binomials because of the widespread use and acceptance of the Kauffman-White schema and the decision of the Subcommittee on the Taxonomy of Enterobacteriaceae at that time that the nomenclatural practice of giving binomials to each serotype of subgenus I ( S . kauflmannii) should continue in spite of the rules specified by the Code of Nomenclature. The policy may change in the future. P-Glucuronidase, gammaglutamyltransferase (GGT) and the ability to ferment galacturonate may be used as simple tools to differentiate between the Salmonella subgenera (Giammanco et al., 1980; Le Minor et al., 1979).

24

T. BERGAN

P-Glucuronidase Salmonella subgenus I Salmonella subgenus I1 Salmonella subgenus tIII, diphasic Salmonella subgenus 111, monophasic Salmonella subgenus IV, except S. houtenae S. houtenae Cirrobacter H . alvei

+ or - depending upon serotype or - depending upon serotype

GGT

Galacturonate

+ (93%)

-

+

+ + +(loo%) +(loo%)

+ +

+ +/-

+ +

In the USA S. arizonae is often referred to a separate genus as Arizona himshawii. Since this was proposed in 1966 (Ewing and Fife, 1966) it becomes a later synonym to the name S. arizonae published in 1964 (Kauffman, 1964) in the genus Salmonella. DNA relatedness above 70% opposes Arizona as a separate genus. S. cholerae-suis and S . typhi are recognized in all alternate systems, but otherwise S. enteritidis is an overriding species name for all other serotypes in the USA. This system is consistent with DNA relatedness. Strains of salmonella studied so far have exhibited more than 70% DNA affinity (Brenner, 1981a; Crosa et al., 1973; Le Minor, 1981; Stoleru et al., 1976). A sixth system would emerge by referring all salmonella isolates to one and the same Salmonella species. The A.pproved List of Bacterial Names indicates S. cholerae-suis as the type species of the genus Salmonella (Skerman et al., 1980). S . typhi is, since Opinion 18 of the Judicial Commission, the oldest recognizable epithet for a species within this genus (Bacillus typhi Schroeter 1886) (Le Minor and Rohde, 1974). In this system the serotypes would be shown by adding e.g. “serotype typhi” for the typhoid-inducing species or “serotype typhimurium” for the murine-derived species and so on. Adding the antigenic formula as a suffixwould seem advisable. Presently in excess of 2000 serotypes have been found as presented in Bergan (1984). For the serotypes without designated names, the antigenic formula should be added. DNA relationships has led to subgenera I, I1 and IV and has differentiated two entities among subgenus 111. The S. arizona is characterized by or production of P-galactoside (positive o-nitrophenyl-/I-D-galactopyranoside ONPG reaction), is subdivided into one group which has monophasic H-

1. CLASSIFICATION OF ENTEROBACTERIACEAE

25

antigens and ferments lactose slowly, and into one group with has diphasic Hantigens and are more rapid at acid production from lactose. It is important that taxonomic changes within Salmonella be made with due respect to maintaining their identification reasonably accomplishable under pragmatic conditions and compatible with both pathogenetic and epidemiological properties of the species. F. Serratia

The genus Serratia has been considerably modified during the last decade, particularly as a consequence of the continued research of the Grimonts and collaborators (Grimont and Grimont, 1981). The subdivision follows phenotypic studies, DNA hybridization and analysis of the fatty acid composition. The characteristics of species within this genus appear in Table X. S. fonticola was described as Citrobacter-like and has been isolated exclusively from water (Gavani et a / . , 1979). There is disagreement regarding the proper name for the species which Brenner (1981a) calls S. rubidae after Ewing et al. (1972), and Grimont and Grimont (1981) call S. marinorubra in accordance with the rational explained previously (Grimont et a / . , 1977). The Approved List of Bacterial Names has employed one and the same type strain for both species and has by implication given S. rubidae preference instead of S. marinorubra. One entity which has been the subject of considerable controversy is S. liquefaciens. It has recently been recognized as an Enterobacter (Ewing, 1963; Cowan, 1974a,b), but DNA relatedness has now supported its recognition as S. liquefaciens. Valid publication follows the numerical taxonomical study of Bascomb et a/. (1971). Fatty acid composition of the type strain and related isolates (Bergan et a / . , 1983) as well as DNA relatedness indicate that part of the entity could be recognized as a separate species named S . proteamaculans in recognition of the first description of the species under the name Pseudomonas proteamaculans in 1919 (Paine and Stansfield, 1919)and later as Erwinia proteamaculans as explained elsewhere (Grimont et a / . , 1978b). A disagreement between research groups is suggested by continued use of the name S. liquefaciens in spite of the years which have elapsed since the welldocumented exposure in favour of S. proteamaculans. The situation may now be resolved. Among a large collection of strains bearing the name S. liquefaciens were differentiated three DNA relatedness groups and the names S. proteamaculans, S. liquefaciens and S . grimesii applied (Grimont et a / . , 1982a,b). Consequently, the situation should now be considered resolved, but the practising microbiologist must hence forth beware that S. liquefaciens may for some time still be used in the sensu stricto

TABLE X Biochemical-cultural characteristics of Serratia and Rahnella ~~

Characteristic

~

~~~~

S. marcescens

~

~~

~

S. ruhidw

S. prorearnaculans subsp. suhsp. S. liquefaciens proteamaculans quimvora

S . grimcsii

FNCIOX

sucroae

Mannitol Mannose Dukitol Sslicin

S. odorifera

S. fonricola

d

d d

+ +

d

d

+

+

+

+

R. aquarilis

+-

-(0)

-

LacIosc

fl-Galactosidase (ONPG. 37°C) Galactose

S. plymurhica

+

n,s (TSI)

Urease lndolc Methyl red (37°C) Aatoin (VogcsRoskauer, 37°C) (22°C) Citrate (Simmon's, 3TC) (Christensen's) KCN Motility (37°C) Gelatin (22'12) Lysinc dccarboxylasc Arginine dihydrolase Ornithine dcearboxylase Phenylalanine dearninasc Glucose, acid Glucose,gas

S./icoriu

+ +

i

+

+

-(0)

+ +

+

+

+ +

+ +

+ +

+

+

+

d or+

+

+

+ +

+ +

+

+ +

+

- (0)

- (0)

+

d

+

+

-

+ + +++ +-

+(+)

-

+-

+ + + + +

Adonitol Inositol Sorbitol Arabinose Raffinosc Rhamnosc Ribose Malonate Mucate Xylosc Trehalose Ccllobiose Maltose - CH,-glucooide Erythritol Esculin Melibiose GIyarol Xylitol D-Melezitose Arabitol Adonitol Tartrate (Jordan's) Acetate Alginatc Lipax (Tween 80) Dcoxyribonucleasc NO, reduction l o NO, Polypcaate Red Dipment (G+'Cimole 3 . DNA Type strain From lzard

*see text.

YI

+

+ +

+ +

+ + + + + +

d

+

+

+

+ -

+

+

+

+ +

+

+

+ +(loo)

56-60

53-58

ATCC 13880

ATCC 27593

53-54 ATCC 27592

51-54

ATCC 19323

01. (1978. 1979). Grimont and Grimont (1981) and Grimont Y I ol. (1979. 1982).

NI. no information.

-

++

5ATCC 33105

5s57

ATCC 183

54-55 ATCC 33077

51-55

CIP78-25

28

T. BERGAN

sense of Grimont et al. (1982a,b) or in the sensu lato sense used until now by the Centres for Disease Control (Brenner, 1981a). The differentiation between these taxa is distinct in terms of DNA relatedness but routine biochemical-cultural tests distinguish less efficiently between the entities (Table X). Grimont et al. (1982b) found that assimilation tests were more useful than traditional determinative methods for the distinction between the species in S. liquefaciens (sensu lato). In that situation, a way out in clinical microbiology is to diagnose isolates with the corresponding biotypes as S. liquefaciens-like. The three species in this complex are readily distinguished by their fatty acid composition (Bergan et al., 1983). The currently recognizable species of Serratia are S. marcescens (type species), S . Jicaria, S. fonticola, S. grimesii, S. liquefaciens, S. odorifera, S. plymuthica, S. proteamaculans subsp. proteamaculans, S. proteamaculans subsp. quinovora and S. rubidea. The species S. liquefaciens (sensu stricto), S. grimesii and S. proteamaculans have emanated from what was formerly known as S. liquefaciens. These conclusions are the results of developments over the last few years (Grimont et al., 1978a,b, 1979a,b, 1981, 1982a,b; Steigerwalt et al., 1976). S. proteamaculans was earlier recognized as Erwinia proteamaculans (Dye, 1966). The entities S. fonticola and S. proteamaculans require further assessment. Accordingly, the genus Serratia can now be defined more closely as strains conforming in general to the definition of the family Enterobacteriaceae, which produce acetoin from pyruvate and ferment maltose, mannitol and trehalose. These compounds and caprylate and L-fucose are utilized as the sole sources of carbon. DNAase, lipidases (corn oil, tributyrin), gelatinase, caseinase and the hydrolysis of o-nitro-B-D-galactoside (ONPG), but not starch, are further characteristics.

G . Morganella , Proteus and Providencia The proteae are foremost characterized as the phenylalanine- and tyrosinehydrolysing Enterobacteriaceae. They grow in the presence of KCN, most species (except biotypes of Providencia) produce urease, do not utilize malonate or acetoin and are methyl red negative. Lysine decarboxylase and arginine dehydrolase are not present, whereas ornithine decarboxylase is variable. Acid is produced f r o p glucose, but not from lactose, dulcitol, sorbitol or melibiose. The characteristics of currently recognized proteae appear in Table XI. The proteae have been recognized with fairly stable species descriptions and are easy to differentiate. However, throughout the years changing subjective synonyms have been proposed. For instance, the eighth edition of “Bergey’s

1. CLASSIFICATION OF ENTEROBACTERIACEAE

29

Manual of Determinative Bacteriology” recognized the species Proteus mirabilis, Prot. vulgaris, Prot. rettgeri and Prot. morganii within the same genus. Providencia stuartii and Prvd. alcalifaciens were designated Prvd. inconstans biotypes A and B. Prvd. stuartii is a urease-positive biogroup of P . inconstans (Hickman et al., 1980; Lautrop, 1974). The species Prot. morganii has been identified as somewhat more distinct in terms of DNA relatedness than the other species. Brenner et al. (1978) considered that this was substantiable phylogenetic evidence necessitating reassigning the species to a separate genus and, consequently, followed the suggestion of Fulton (1943) that it be recognized as Morganella morganii. M . morganii has 50% (G+C) DNA such as most of the other most common enterobacteria and in contrast to the markedly lower 39% found for Prot. mirabilis (Falkow et al., 1962). This change of name is opposite to the current and widespread practice which has been the result of extensive biochemical and serological studies (Johnson et al., 1975; Lessel, 1971; Raus, 1962; Rustigan and Stuart, 1941, 1943, 1945). Since the definition of the species is not in focus, no change in the strains assigned to each species as such is involved. Thus for the purpose of routine clinical microbiology it may be questioned whether it is necessary or advisable merely to change the name since any change will create confusion. Although the basic arguments in favour of Morganella are convincing and the name may in time become commonly used, the individual microbiologist may choose between subjective synonyms. The theoretical borderline of 70% DNA relatedness is not followed consistently even within the enterobacteria ( E . coli versus Shigella, see Section 1I.A). Consequently, the rules of nomenclature allow the continued use of the name Prot. morganii for those who wish to do so, for example for historical reasons. Accordingly, the proteae can be considered to contain either the three genera Proteus, Providencia and Morganella or only the first two mentioned ones. Unusual biotypes of M . morganii (s. Prot. morganii) have been described (Hickman et al., 1980). A recent addition to the proteae is Prot. myxofaciens (Cosenza and Podgwaite, 1966), which is isolated from larvae of the gypsy moth. It swarms on the surface of moist agar. It has been recognized as identical to Erwinia herbicola (or the heterogeneous taxon recognized as Enterobacter agglomerans) (Lautrop, 1974). DNA relatedness supports the association with Proteus (Brenner et al., 1978).

H. Yersinia The genus Yersinia can be defined as organisms with mixed acid fermentation, a positive methyl red reaction without acetoin production, lack of phenyla-

TABLE XI Biochemical-cultural characteristics of Proteus, Providencia and Morganella Characteristic

H,S (TSI) Urease Indole Methyl red, 37°C Acetoin (Voges-Proskauer, 37°C) (28°C) Citrate (Sirnmon’s, 37°C)

KCN

Motility (37°C) Gelatin (22°C) Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase Phenylalanine dearninase Glucose, acid Glucose, gas Lactose /?-Galactosidase(ONPG,37 C)

Sucrose

Mannitol Mannose Dulcitol Salicin

P . mirabilis

P . vulgaris

P . myxofaciens

-

+ + + +

Prov. alcalifaciens

Prov. stuartii

Prov. rettgeri

M . morganii

Adonitol Inositol Sorbitol Arabinose Raffinose Rhamnose Malonate Mucate Xylose Trehalose Cellobiose Maltose - CH,-glucoside Erythritol Esculin Melibiose Glycerol Tartrate (Jordan’s) Acetate Lipase (corn oil) Deoxyribonuclease (25’C) NO, reduction to NO, Polypectate Pigment (G+C) mole % DNA Type strain

-

+

ATCC 19692

ATCC 299 14

From Edwards and Ewing (1972). Brenner rt ul. (1977, 1978),Lautrop (1974) and Gardner and Kado (1972). ‘See text.

32

T. BERGAN

lanine deaminase but urease in occasional strains, lack of growth in the presence of KCN and 4547% (G + C) DNA. This genus has recently been the subject ofconsiderable interest (Brenner et al., 1980a,b; Kapperud et al., 1981; Ursing et al., 1980a,b). The subdivision of this genus is apparent from Table XII. The taxonomy of this genus appears in Volume 15 of this series (Kapperud and Bergan, 1984).

I. Edwardsiella, Erwinia and Pectobacterium The genera Edwardsiella, Erwinia and Pectobacterium all mostly contain phytopathogenic species, but vertebrate pathogens belonging to these genera have been described (Starr, 1981). The taxonomic status of many nomenspecies requires further elucidation. Pectobacteria and edwardsiellae appear to have shuttled between the genera. Brenner et al. (1977b) upon studying DNA relatedness found that P. carotovorum should be referred to as P. chrysanthemi and that the genus affiliation of this species should not be Erwinia. Edw. anguillimortiferum corresponds to Edw. tarda, but it was first described as Paracolobactrum anguillimortiferum (Hoshina, 1962), however without designation of type strain, deposition in a culture collection or maintenance of reference strains. Since the description of Hoshina (1962) is close to the subsequently described Edw. tarda (Ewing et al., 1965), Sakazaki and Tamura (1975) suggest that the epithet anguillimortiferum has obvious priority over the epithet tarda (Farmer et al., 1976; Sakazaki and Tamura, 1978). The recognition of the two as synonymous has been acknowledged by Grimont et al. (1980). Since the recognition of the two names as objective synonyms was stressed (Grimont et al., 1980), the species Edw. anguillimortiferum would have the priority of publication. The Approved List of Bacterial Names has resolved the problem, in favour of Edw. tarda. Grimont et al. (1980) described as new Edwardsiella species, Edw. hoshinae and two DNA relatedness groups among Edw. tarda (Edw. anguillimortiferum) (Table X I I I ) . Erw. rubrifaciens is distinguished by DNA relatedness from Erw. amylovora and Erw. quercina (Azad and Kado, 1980). The classification of Erwinia and Pectobacteriurn particularly, but also of Edwardsiella, are at a stage where further data are required before a good subdivision can be achieved. The reader is referred to the contributions by Azad and Kado (1980), Brenner (1981a, 1983), Dye (1968a,b,c,d), Farmer et al. (1976, 1980), Grimont et al. (1980) and Starr (1981). J. Kluyvera The genus Kluyvera was proposed by Fanning et al. (1979) and contains two

TABLE XI1 Biochemicalkultural characteristics of Yersinia species Y . enrerocolitica

Characteristic Acetoin (Voges-Proskauer) (37T) (28°C) Arginine dihydrolase Catalase Citrate (Simmon's, 37°C) (28'C) (Christensen's) Deoxyribonuclease P-Galactosidase (ONPG. 37 C ) Gelatin (film) H,S (Kliegler's) Indole KCN Lipase (Tween 80) Lysine decarboxylase Malonate Methyl red (37'C) (28°C) Motility (37°C) (28'C) Mucate NO, reduction to NO, Ornithine decarboxylase Phenylalanine Polypectate Urease (G+C) mole % DNA Type strain From Ewing rr

ti/.

(1978)

Y . f r rderiksenii

Y . intermedia

Y . kristensenii

Y . pseudotuberculosis

Y . pestis

Y . ruckeri

T. BERGAN

34

TABLE XI11 Biochemicalkultural characteristics of Edwardsiella

Characteristic HIS CrSU urease

Indole Methyl red (37°C) Acetoin (Voges-Proskauer, 37°C) Citrate (Simmon’s, 37°C) KCN Gelatin (22°C) Lysine decarboxylate Arginine dihydrolase Ornithine decarboxylase Phenylalanine deaminase Glucose, acid Glucose, gas Lactose B-Galactosidase (ONPG, 37°C) Sucrose Mannose Dulcitol Adonitol Inositol Sorbitol Raffinose Rhamnose Malonate Galactose Xylose Trehalose Cellobiose Esculin - CH,-glucoside Mucate Melibiose Glycerol Deoxyribonuclease (25°C) NO, reduction to NO, Type strain From Grimont er ol. (1980).

E . tarda (subsp. anguillimort$era) (typical) (atypical)

E . hoshinae

1 . CLASSIFICATION OF ENTEROBACTERIACEAE

35

species. Biochemically they are related to Citrobacter and Enterobacter. Sputum from man is the most common source of these organisms.

K. Rahnella A new member of the Enterobacteriaceae is Rahnella aquatilis (Izard et al., 1979). The relationship of this species to Enterobacteriaceae has been verified by DNA-DNA hybridization and numerical taxonomy of phenetic data (Table XIV). R . aquatilis resembles E. cloacae phenetically, but DNA relatedness to this species was only 11-37% to the centrostrain (most typical according to similarity of a collection of R . aquatilis strains) of R . aquatilis.

TABLE XIV DNA relationship between Rahnella aquatilis and other enterobacteria assessed by DNA-DNA hybridization

Species E. coli E. cloacae H . alvei E. agglomerans S. liquefaciens E . aerogenes Shigella E. tarda S. arizona S . typhimurium C .freundii C . diversus C . amalonaticus K . pneumoniae (sensu lato) K . oxytoca K . ozaenae E. adecarboxylata S . marcescens S. marinorubra S. plymuthica E . amylovora P . rettgeri P . mirabilis Y . pseudotuberculosis From hard er a / . (1979).

Percentage hybridization 19 11-37 9-18 6

40

17-32 10-28 6 17 8 1&14

3&36 13-25 1 5-2 1 33-44 12 12 22-38 8-27 39 20 25 20 8

TABLE XV Biochemical-cultural characteristics of Cedeceu Characteristic Urease Indole Methyl red (37°C) Acetoin (Voges-Proskauer, 37°C) Citrate KCN Motility (37°C) Gelatin (22°C) Lysine decarboxylase Arginine dehydrolase Ornithine decarboxylase Phenylalanine deaminase Glucose, acid Glucose, gas Lactose /3-Galactosidase (ONPG, 37' C) Sucrose Mannitol Mannose Dulcitol Salicin Adonitol Inositol Sorbitol Arabinose Raffinose Rhamnose Malonate Mucate Xylose Trehalose Cellobiose Maltose - CH,-glucoside Erythritol Esculin Melibiose Glycerol Tartrate (Jordan's) Acetate Lipase (corn oil) Deoxyribonuclease NO, reduction to NO, Arabitol Pigment (G+C) mole % DNA Type strain From Grimont e t a / . (1981).

C. davisae

(+) -

+-

49-50 ATCC 33431

C . lapagei

I . CLASSIFICATION OF ENTEROBACTERIACEAE

37

L. Cedecea A group of unidentified enterobacteria formed the basis for proposal of the genus Cedecea (Grimont et al., 1981) (Table XV). This genus contains strains that are lipase positive, lack deoxiribonuclease, gelatin liquefaction and utilization of L-arabinose and L-rhamnose. The strains of this genus have shown DNA relatedness ranging from 32 to 100% within the genus and less than 23% to members of other genera of the Enterobacteriaceae. Two species have been named: Cedeceae davisae and C. lapagei. The DNA relatedness within these species is above 80%. References Azad, H. R. and Kado, C. I. (1980). J. Gen. Microbiol. 120, 177-129. Bagley, S. T., Seidler, R. J. and Brenner, D. J. (1981). Curr. Microbiol. 6, 105-109. Baer, H. and Washington, L. (1972). Appl. Microbiol. 23, 108-112. Bascomb, S., Lapage, S. P., Curtis, M. A. and Willcox, W. R. (1971). J . Gen. Microbiol. 66, 279-295. Bercovier, H., Brault, J., Barre, N., Treignier, M., Alonso, J. M. and Mollaret, H. H. (1978). Curr. Microbiol. 4, 201-206. Bercovier, H., Brenner, D. J., Ursing, J., Steigerwalt, A. G., Fanning, G. R., Alonso, J. M., Carter, G. P. and Mollaret, H. H. (1980a). Curr. Microbiol. 4, 201-206. Bercovier, H., Mollaret, H. H., Alonso, J. M., Brault, J., Fanning, G. R., Steigerwalt, A. G. and Brenner, D. J. (1980b). Curr. Microbiol. 4, 225-229. Bergan, T. (Ed.) (1984). “Methods in Microbiology”, Vol. 15. Academic Press, London and New York. Bergan, T., Grimont, P. A. D. and Grimont, F. (1983). Curr. Microbiol. 8, 7-11. Brenner, D. J. (1981a). I n “The Procaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria” (M. P. Starr, H. Stolp, H. G. Truper, A. Balows and H. G. Schlegel, Eds), pp. 1105-1 127. Springer-Verlag, Berlin and New York. Brenner, D. J. (1981b). In “The Procaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria” (M. P. Starr, H. Stolp, H. G. Truper, A. Balows and H. G. Schlegel, Eds), pp. 1173-1 180. Springer-Verlag, Berlin and New York. Brenner, D. J. (1983). ASM News 49, 58-63. Brenner, D. J., Steigerwalt, A. G. and Fanning, G. R. (1972). In?. J . Syst. Bacteriol. 22, 193-200. Brenner, D. J., Steigerwalt, A. G., Falcao, D. P., Weaver, R. E. and Fanning, G. R. (1976). In?. J. Syst. Bacteriol. 26, 18&194. Brenner, D. J., Farmer, J. J., Hickman, F. W., Ashbury, M. A. and Steigerwalt, A. G. (1977a). “Taxonomic and Nomenclature Changes in Enterobacteriaceae”. Center for Disease Control, Atlanta, Georgia. Brenner, D. J., Fanning, G. R. and Steigerwalt, A. G. (1977b). Int. J . Syst. Bacteriol. 27,211-221. Brenner, D. J., Farmer, J. J., Fanning, G. R., Steigerwalt, A. G., Klykken, P., Wathen, H. G., Hickman, F. W. and Ewing, W. H. (1978). Int. J. Syst. Bacteriol. 28, 269-282. Brenner, D. J., Ursing, J., Bercovier, H., Steigerwalt, A. G., Fanning, G. F., Alonso, J. M. and Mollaret, H. H. (1980a). Curr. Microbiol. 4, 195-200. Brenner, D. J., Bercovier, H., Ursing, J., Alonso, J. M., Steigerwalt, A. G., Fanning, G. R., Carter, G. P. and Mollaret, H. H. (1980b). Curr. Microbiol. 4, 207-212.

38

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Burgess, N. R. H., McDermott, S. N. and Whiting, J. (1973). J. Hyg. 71, 1-7. Burkey, L. A. (1928). Iowa State Coll. J . Sci. 23, 57-100. Colwell, R. R. and Mandel, M. (1964). J . Bacteriol. 87, 1412-1422. Colwell, R. R., Johnson, R., Wan, L., Lovelace, T. E. and Brenner, D. J. (1974). Int. J . Syst. Bacteriol. 24, 422-433. Cosenza, B. J. and Podgwaite, J. D. (1966). Antonie van Leeuwenhoek J . Microbiol. Serol. 32, 187-191. Cowan, S. T. (1974a). In “Bergey’s Manual of Determinative Bacteriology” (R. E. Buchanan and N. E. Gibbons, Eds), 8th edn, pp. 29&293. Williams & Wilkins, Baltimore, Maryland. Cowan, S. T. (1974b). “Cowan and Steels Manual for the Identification of Medical Bacteria”, 2nd edn, Cambridge University Press, Cambridge. Cowan, S. T., Steel, K. J., Shaw, C. and Duguid, J. P. (1960). J . Gen. Microbiol. 23, 601-6 12. Crosa, J. H., Brenner, D. J., Ewing, W. H. and Falkow, S. (1973). J. Bacteriol. 115, 307-3 15. Crosa, J. H., Steigerwalt, A. G., Fanning, G. R. and Brenner, D. J. (1974). J . Gen. Microbiol. 83, 271-282. Dye, D. W. (1966). N . Z . J. Sci. 9, 843-854. Dye, D. W. (1968a). N . Z . J . Sci. 11, 59&607. Dye, D. W. (1968b). N . Z . J. Sci. 12, 81-97. Dye, D. W. (1968~).N . Z . J. Sci. 12,223-236. Dye, D. W. (1968d). N . Z . J. Sci. 12, 833-839. Edwards, P. R. and Ewing, W. H. (1972). “Identification of Enterobacteriaceae”, 3rd edn. Burgess, Minneapolis, Minnesota. Edwards, P. R. and Fife. M . A. (1952). J. Infect. Dis. 91, 92. Ewing, W. H. (1963). Int. Bull. Bacteriol. Nomencl. Taxon. 13, 95-110. Ewing, W. H. (1972). “Isolation and identification of Salmonella and Shigella”. Center for Disease Control, Atlanta, Georgia. Ewing, W. H. and Fife, M. A. (1966). In?. J. Syst. Bacteriol. 16, 427435. Ewing, W. H. and Fife, M. A. (1972). In?. J. Syst. Bacteriol. 22, 4-11. Ewing, W. H. et al. (1971). “Biochemical Reactions of Shigella”. Center for Disease Control, Atlanta, Georgia. Ewing, W. H., Davis, B. R. and Fife, M. A. (1972). “Biochemical Characterization of Serratia liquefaciens and Serratia rubidaea”. Center for Disease Control, Atlanta, Georgia. Ewing, W. H., Ross, A. J., Brenner, D. J. and Fanning, G. R. (1978). Int. J. Syst. Bacteriol. 28, 3 7 4 4 . Falkow, S.,Ryman, 1. R. and Washington, 0. (1962). J . Bacteriol. 83, 1318-1321. Fanning, G . R., Farmer, J. J., Parker, J. N., Huntley-Carter, G. P. and Brenner, D. J. (1979), Abst. Annu. Meet. Am. SOC.Microbiol. 1979 100. Farmer, J. J. (1981). I n “The Procaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria” (M. P. Starr, H.Stolp, H. G. Triiper, A. Balows and H. G. Schlegel, Eds), pp. 1135-1 147. Springer-Verlag, Berlin and New York. Farmer, J. J., Brenner, D. J. and Clark, W. A. (1976). Int. J. Syst. Bacteriol. 26, 293-294. Farmer, J. J., Asbury, M. A., Hickman, F. W., Brenner, D. J. and the Enterobacteriaceae study group. (1980). Int. J. Syst. Bacteriol. 30, 569-584. Ferragut, C. and Leclerc, H. (1978). Antonie van Leeuwenhoek J . Microbiol. Serol. 44, 407424.

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Fife, M. A,, Ewing, W. H. and Davis, R. B. (1965). “The Biochemical Reactions ofthe Tribe Klebsielleae”. CIC Publications. Frederiksen, W. (1970). Publ. Fac. Sci. Univ. J . E. Purkyne, Brno 47, 89-94. Fulton, M. (1943). J. Bacteriol. 46, 79-82. Gardner, J. M. and Kado, C. I. (1972). Int. J. Syst. Bacteriol. 22, 201-209. Gavini, F., Ferragut, C., Izard, D., Trinel, P. A., Leclerc, H., Lefebvre, B. and Mossel, D. A. A. (1979). Int. H. Syst. Balt. 29, 92-101. Gavini, F., Ferragut, C. and Leclerc, H. (1976a). Ann. Microbiol. 127B, 317-335. Gavini, F., Lefebvre, B. and Leclerc, H. (1976b). Ann. Microbiol. 127A, 275-295. Gavini, F., Izard, D., Trinel, P. A., Lefebvre, B. and Leclerc, H. (1981). Can. J. Microbiol. 27, 98-106. Giammanco, G., Buissiere, J., Toucas, M., Brault, G. and Le Minor, L. (1980). Ann. Microbiol. 131A, 181-187. Grimont, P. A. D. and Grimont, F. (1981). In “The Procaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria” (M. P. Starr, H. Stolp, H. G. Triiper, A. Balows and H. G. Schlegel, Eds), pp. 1187-1203. Springer-Verlag, Berlin and New York. Grimont, P. A. D. and Popoff, M. Y. (1980). Curr. Microbiol. 4, 337-342. Grimont, P. A. D., Grimont, F. and Dulong de Rosnay, H. L. C. (1977). J. Gen. Microbiol. 98, 39-66. Grimont, P. A. D., Grimont, F., Richard, C., Davis, B. R., Steigerwalt, A. G. and Brenner, D. J. (1978a). Int. J. Syst. Bacteriol. 28, 453-463. Grimont, P. A. D., Grimont, F. and Starr, M. P. (1978b). Int. J. Syst. Bacreriol. 28, 503-510. Grimont, P. A. D., Grimont, F. and Starr, M. P. (1979a). Curr. Microbiol. 2,277-282. Grimont, P. A. D., Grimont, F., Le Minor, S., Davis, B. and Pigache, F. (1979b). J. Clin. Microbiol. 10, 425432. Grimont, P. A. D., Grimont, P., Richard, C. and Sakazaki, R. (1980). Curr. Microbiol. 4, 347-35 1. Grimont, P. A. D., Grimont, F., Farmer, J. J. and Asbury, M. A. (1981). Int. J . Syst. Bacteriol. 31, 317-326. Grimont, P. A. D., Irino, K. and Grimont, F. (1982a). Curr. Microbiol. 7, 63-68. Grimont, P. A. D., Grimont, F. and Irino, K. (1982b). Curr. Microbiol. 7, 69-74. Hickman, F. W., Farmer, J. J., Steigerwalt, A. G. and Brenner, D. J. (1980). In?. J. Syst. Bacteriol. 12, 88-94. Hoshina, T. (1962). Bull. Jpn, SOC.Sci. Fish. 28, 162-164. Izard, D., Feragut, C., Gavini, F. and Leclerc, H. (1978). Int. J. Syst. Bacteriol. 28, 449452. Izard, D., Gavini, F., Trinel, P. A. and Leclerc, H. (1979). Ann. Microbiol. 130A. 163- 177. Izard, D., Gavini, B., Trinel, P. A. and Leclerc, H. (198 la). Int. J. Syst. Bacteriol. 31, 3542. Izard, D., Ferragut, C., Gavini, F., Kerstens, K., De Ley, J. and Leclerc, H. (198 I b). Int. J . Syst. Bacteriol. 31, 11C127. Izard, D., Gavini, F., Trinel, P. A. and Leclerc, H. (1981~).Int. J. Syst. Bacteriol. 31, 3542. Jain, K., Radsak, K. and Mannheim, W. (1974). Int. J. Syst. Bacteriol. 24,402407. Johnson, R., Colwell, R. R., Sakazaki, R. and Tamura, K. (1975). Int. J . Syst. Bacteriol. 25, 12-37. Kapperud, G., and Bergan, T. (1984). Meth. Microbiol. 15, 295-343.

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Sedkak, J. (1974). In “Bergey’s Manual of Determinative Bacteriology” (R. E. Buchanan and N. E. Gibbons, Eds), 8th edn, pp. 296-299. Williams & Wilkins, Baltimore, Maryland. Seidler, R. J., Knittel, M. D. and Brown C. (1975). Appl. Microbiol. 29, 819-825. Skerman, V. B. D., McGowan, V. and Sneath, P. H. A. (1980). (Approved list of bacterial names) Int. J. Syst. Bacteriol. 30,225420. Starr, M. P. (1981). In “The Procaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria” (M. P. Starr, H. Stolp, H. G. Truper, A. Balows and H. G. Schlegel, Eds), pp. 1260-1271. Springer-Verlag, Berlin and New York. Steigerwalt, A. G., Fanning, G. R., Fife-Ashbury, M. A. and Brenner, D. J. (1976). Can. J. Microbiol. 22, 121-137. Stenzel, W., Burger, H. and Mannheim, W. (1972). Zentralbla Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A , 219, 193-203. Stoleru, G. H., Le Minor, L. and Lheritier, A. M. (1976). Ann. Microbiol. 127A, 477486. Ursing, J., Brenner, D. J., Bercovier, H., Fanning, G. R., Steigerwalt, A. G., Brault, J. and Mollaret, H. H. (1980a). Curr. Microbiol. 4, 213-217. Ursing, J., Steigerwalt, A. G. and Brenner, D. J. (1980b). Curr. Microbiol. 4,231-233. Werkman, C . H. and Gillen, G. F. (1932). J . Bacteriol. 23, 167-182. Young, V. M., Kenton, D. M., Hobbs, B. J. and Moody, M. R. (1971). In?. J. Syst. Bacteriol. 21, 58-63.

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2 Serotyping of Escherichia coli" F. 0RSKOV and I. 0RSKOV Collaborative Centre for Reference and Research on Escherichia and Klebsiella, ( W H O ) , Statens Seruminstitut, Copenhagen, Denmark ~~

I. Introduction. . 4 4 A. Position of Escherichia coli in the family Enterobacteriaceae . . 4 4 B. General principles for subdivision . . 45 11. Surface structures . . 45 A. Morphology . . . 45 B. Chemistry . . 46 C. Genetics . . 55 111. Serotype definition . . 55 IV. Antigenic scheme . . . 55 . . 56 V. History of typing procedures . A. Serotyping . . 56 B. Phage typing . . 65 C. Colicin typing . . 66 D. Biotyping . . 66 E. Typing by outer membrane protein (OMP) patterns . 68 F. Typing by antibiotic resistance patterns . . 68 G. Typing by direct haemagglutination (HA) . . . 68 H. Type stability. . . 69 VI. Methods . . I0 A. Bacterial agglutination . . I0 B. Indirect haemagglutination (HA) . . 14 C. Co-agglutination . . 74 D. Immunofluorescence . . 14 E. Gel precipitation . . 15 VII. The antigens . . 19 A. 0-antigens . . 19 B. K-antigens . . 81 C. F-antigens . . 82 D. H-antigens . . 82 VIII. Variational phenomena of importance for serotyping . . 83 A. Lipopolysaccharide 0-antigens . . . 84 B. K-antigens . . 85 C. F-antigens . . 86 The terminology used to describe the different classes of bacterial antigens is explained in the Preface. However, the authors would like to mention that a different convention is used in some laboratories, for example O:I, K:l, H:7 is equivalent to 01:KI:H7. METHODS IN MICROBIOLOGY VOLUME 14

Copyright Q 1984 by Academic Press, London All rights of reproduction in any form reserved.

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IX. X.

XI.

XII. XIII.

D. H-antigens . E. M-antigens . Antisera . A. Production of antisera . B. Absorbed antisera . Cross-reactions . A. 0-antigens . B. K-antigens . C. H-antigens . D. F-antigens . Association of serogroups and serotypes of E. coli with pathological conditions . A, E. coli from human sources . B. E. coli from cattle, pig and poultry . . The bacterial clone concept in epidemiology and taxonomy Concluding Remarks . References .

87 88 88 88 91 92 92 94 96 96 97 97 101 103 104 105

I. Introduction

Since the first days of bacteriology attempts to differentiate Escherichia coli strains by serological methods have been carried out. However, only after the pioneering work of Kauffmann and co-workers in the beginning of the 1940s has serotyping become soundly established (Kauffmann, 1943, 1944; Knipschildt, 1945; Vahlne, 1945). Highly recommendable for anyone who wishes to work in this field is “Identification of Enterobacteriaceae” by Edwards and Ewing (1972). Kauffmann’s (1966) own description in “The Bacteriology of Enterobacteriaceae” also contains much important information and can be recommended particularly for readers who want a first-hand account of the historical development within this field. Orskov and Orskov (1978), in Volume 11 of this series, described more broadly the serotyping of Enterobacteriaceae, but with special emphasis on K-antigen determination. A. Position of Escherichia coli in the family Enterobacteriaceae

The genus Escherichia is one of the genera of tribus Escherichieaeas defined in the eighth edition of “Bergey’s Manual of Determinative Bacteriology” (1974). It contains one species Escherichia coli, which consists of Gramnegative, peritrichously flagellated rods that conform to the family Enterobacteriaceae. E. coli can be differentiated from other genera in the tribus Escherichieae by indole production, fermentation of lactose, negative reactions in KCN, gelatine and malonate tests. E. coli is methyl red positive, Voges-Proskauer negative and urease negative. The (G +C) content of DNA is 50-51%.

2. ESCHERICHIA COLI

45

B. General principles for subdivision Escherichia as a genus is defined by a series of physiological and morphological traits. However, it has been recognized for a long time that this genus is quite heterogeneous and that additional characters have to be introduced for an accurate description of single strains. It was soon realized that an attempted subdivision into biotypes based on fermentation or nonfermentation of carbohydrates would not help epidemiological investigations except within narrow topographical limits such as outbreaks in a special hospital at a certain time (Crichton and Old, 1979). It was at the same early date understood that the immunogenicity of the bacterial surface structures would constitute a solid base for a refined subdivision into serotypes. Later it was realized that even this level of refinement in many epidemiological situations did not provide the guidance needed. Thus some 0:K:H-serotypes have been further subdivided into biotypes, phage types, colicin types, antibiotic sensitivity types and, recently, types according to outer membrane protein patterns and plasmid DNA (see below). In the following we discuss the principles and procedures for serotyping. In order to make the serotyping principles intelligible short descriptions of the morphology and immunochemistry of those surface structures that are important in serotyping are given.

11. Surface structures

A. Morphology

Several review papers have been published on the structure of the bacterial cell surface (Freer and Salton, 1971; Shands, 1971). Electron microscopy of enterobacteria shows a rigid double layered cell wall outside the cytoplasmic membrane. The rigidity is determined by the mucopeptide layer. Lipopolysaccharide (LPS) is found, probably fixed by non-covalent bonds, within this layer and at the same time covering it. The polysaccharide part of LPS contains the serological determinants for the 0-antigens, the basis of E. coli serotyping (Luderitz et al., 1968; Jann and Westphal, 1975; Jann and Jann, 1977). The mucopeptide layer and other deeper antigenic substances do not play any role in ordinary serotyping. Lipoproteins are also found in this region but they are not involved in the routine serotyping process except as possible causes of 0-inagglutinability. While these are basic structures found in all typical Enterobacteriaceae, additional structures important for setotyping exist, which are not necessary for the life and multiplication of the organisms and which are not found in all

46

F. ORSKOV AND 1. ORSKOV

organisms. The structures in question are (a) a polysaccharide, most often acidic, found as a capsule or micro-capsule which probably to some extent covers the LPS (Luderitz et al., 1968; Jann and Westphal, 1975;Jann and Jann, 1977). The capsular substance can be more or less tightly bound to the surface and, if loosely bound and produced in great amounts, some may be characterized as slime; (b) motile organisms with flagella. Because of the rich variety in chemical fine structures and because of the fundamental genetic stability (i.e. determination by chromosomal genes), the three structures, the LPS (0-antigen), the polysaccharide capsule (K-antigen) and the protein flagella (H-antigen) make up the three fundamental serotyping antigens. In addition to the above-mentioned structures many enterobacteria carry fimbriae (or pili), which are thread-like protein structures protruding from the surface as seen in electron micrographs and probably arising in or close to the cell membrane. We label these fimbriae the F-antigen (0rskov et a / . , 1980b). B. Chemistry

1 . Lipopolysaccharide ( L P S ) Several review papers on this theme exist (Luderitz et al., 1966, 1968, 1971; Nikaido, 1973; Jann and Westphal, 1975; 0rskov et al., 1977). ( a ) General. The enterobacterial LPS is composed of three structural regions (Luderitz et al., 1966, 1971). Region Ill (lipid A) is the part that is buried in the outer membrane lipoprotein layer of the bacterial cell wall. It consists of glucosamine phosphate and fatty acids, the most prominent of which is /?-hydroxymyristic acid (Luderitz et al., 1973). Lipid A is responsible for the biological (endotoxic) properties of LPS. Region I1 (core) is linked to lipid A via a carbohydrate component which is typical for the LPS and some K-antigens of Gram-negative bacteria: 2-keto-3-deoxy-mannulosoctonic acid (KDO). While there is only one structure of lipid A, found in all enterobacterial LPS, five different core oligosaccharides have been described so far.,The core maylexpress R-antigenic specificity which, however, is hidden in wild type S-forms because of the substitution of the core with region I. Region I is the 0-specific polysaccharide of the S-forms. It consists of oligosaccharide repeating units. This structure is found in most bacterial polysaccharides. Composition and structure of region I are the chemical bases of the 0-antigenic specificity of Gram-negative bacteria. Unsubstituted core LPS can be found in S-LPS, indicating the presence of R-LPS (lacking 0specific polysaccharide), and also in the S-forms of E. coli (Jann et a / . , 1975). Furthermore, S-forms often produce LPS molecules with different chain lengths (Morrison and Leive, 1975).

2 . ESCHERICHIA COLI

47

Since the discovery that serological specificity correlates with small structural units in the side chains, an understanding of the background for the overwhelming diversity among the O-antigens of Enterobacteriaceae has now been achieved. At the same time the many cross-reactions within the genera, between genera and between enterobacteria and other bacteria can be explained simply. ( b ) Neutral LPS. It is noteworthy that unusual amino sugars and also rhamnose occur frequently (Jann and Jann, 1977). Up to six sugar constituents may be present in an O-specific polysaccharide. Only two homopolysaccharides have been found so far in E. coli, i.e. the 0:8-and 0:9specific mannans. Table I shows the structure of some neutral E. coli O-antigens. ( c ) Acidic LPS. Several O-specific polysaccharides have been found to contain acidic components. Table I1 shows the structure of some acidic 0antigens. For serological analysis using immunoelectrophoresis (IE) the existence of these negatively charged LPS molecules is important because they may be misinterpreted as acidic polysaccharide K-molecules (see later). (d) R-LPS. All Salmonella strains have an identical or very similar basal core but in Escherichia other basal core structures in addition to that found in Salmonella can be found. The R-antigens (see later), which generally are not used for serotyping purposes, have their chemical basis in the core polysaccharide. However, when many independent R-mutants are examined it will be found that they can be grouped chemically (and serologically) into five chemotypes: Ra, Rb, Rc, Rd and Re, each one corresponding to a mutational step which leads to a simpler core chain. Thus the LPS of the Rforms contains in its side chain only the innermost part of the polysaccharide chain found in the O-form. When hidden, as it normally is in the O-form, the R-side chain is not, or is only weakly, immunogenic and does not react with possible R-antibody. (e) Chemotypes. Most O-specific polysaccharides isolated from Enterobacteriaceae strains contain five or more sugars. Based on sugar patterns it is possible to establish chemotypes of which more than 50 different types are known from examination of Salmonella and Escherichia strains. For a discussion of the current concepts of chemotypes see Jann and Westphal (1975). It is characteristic that strains from one O-serogroup contain the same sugars; however, many examples can be found where O-antigens belong to the same chemotype but do not cross-react serologically. In such cases the single sugars must be linked differently and undetected constituents or confor-

TABLE I Structures of some neutral 0-antigens of Escherichia coli” 0-antigen

Repeat unit 3

12

12 a

Reference

1

Reske and Jann (1972)

8

+Man

-an

LMan

9

+Man L M a n --+Man-Man

3

13

1,2

a

a

a

4 12 . 1 --Galp L R i b f

75

--GlcNac -Gal

a

3

1,3 a

B 86

--t

Gal

-

a

1,2 a

-B

20

-----)

a

1 a

4

Prehm et a / . (1976) Vasilieu and Zahkarova (1976)

-

1,4

1

+L-Rha a

Erbing et a / . (1975)

194

Man GalNac -GalNac

t

L-FUC 111

-+

Springer (1970)

t

Glc d

Col

1,2

-+Man

Edstrom and Heath (1965)

Col

Col (colitose), 3,6-dideoxy-~-galactose. If not otherwise indicated, the symbols for sugars used in this and the following tables are those suggested by IUPAC-IUB (1966).

TABLE I1 Structures of some acidic 0-antigens of Escherichia cob 0-antigen

Repeat unit

Reference Dmitriev et a/. (1977)

58

RhaLA 100

3

14

Ac

1,2

1,4

Jann et a/. (1970)

-+

t

P-Glyc 124

1

+Gal L G L c N A c A R h a -Rha

3

13

16

B

B

U

4 a l N A c pL G a l p A G a l f

1

Dmitriev et a/. (1976)

B

11,4

GlcLA

Glc GlcUA

141

1 41,3M a n 1,3 GlcNAc 1 t Rha

2 12 13 13 +GlcUA L M a n A M a n L G l c N A c -Man 4 1-6

t

Rha

---c

--c

Jann et a/. (1966)

RhaLA (rhamnolactylic acid), 2-04 l’tarboxyethy1)-L-rhamnose;GlcLA (glucolactylic acid), 4-04 1’-carboxyethy1)-D-glucose.

50

F. ORSKOV AND I. ORSKOV

mational differences must exist. Complete understanding of the background for serological specificity is therefore only possible after chemical structural analysis. More and more such analyses have been carried out. A few examples have been given above. With modern chemical techniques it is possible to explain any 0-antigen factor. Liideritz et al. (1966) summarized their review wifh the following statements: (1) the serological specificitiesdemonstrable on the surface of the bacterial cell by agglutination, i.e. the 0-factors of the Kauffmann-White scheme, are carried by specific polysaccharides; (2) the different 0-factors of a given serotype are not part of different molecules but they are carried by the same polysaccharide molecule. 2. Capsular polysaccharides (K-antigens) Capsular and micro-capsular polysaccharides are found in many genera of Enterobacteriaceae (Liideritz et al., 1968;Jann and Westphal, 1975;Jann and Jann, 1977). The term capsule is most often used for an external substance which covers the complete surface of the bacterium and is bound so strongly to other surface structures that it is not solubilized completely in fluid medium. This last qualification keeps slime substances out of the definition. Many textbooks will also include in the definition that the capsule can be made visible under the microscope. These two requirements are difficult to maintain as immunochemically identical polysaccharides can be found in otherwise identical serotypes, such as (1) capsule, (2) slime, (3) mixtures of capsule and slime or (4) micro-capsules, which can hardly be detected under the microscope but only can be detected by serological or chemical means. In fact, all capsular substances are solubilized to some extent from the living bacteria, and probably both the magnitude of the natural solubilization of this substance and the quantity produced are genetically determined. We would therefore use the term capsule polysaccharide when a surface polysaccharide is detected which is independent of the LPS. We would not include the proteinaceous fimbria or fimbria-like antigens (K:88 or the like) among capsule antigens, although they may constitute a veritable fur around the bacterium and so in this respect they concur with the definition of K-antigens. One important characteristic of polysaccharide capsules, already mentioned above, is the quantitative variations found within the same capsular type. In some instances from one and the same E. coli strain, capsule type variants (mutants) may be isolated concomitantly. In one variant the capsule is easily detected in the light microscope, whereas micro-capsules (less developed capsular Kantigens) in the other variants are only detectable by serological or chemical techniques, in some instances by serology alone. Hitherto described K-antigens have been acidic polysaccharides, except the

2. ESCHERICHIA COLl

51

proteinaceous K:88 (F:4) and K:99 (F:5) (Stirm et al., 1966, 1967b; Isaacson, 1977) which should be moved to the new group of antigens, the F-antigens (Section VI1.C). However, it cannot be excluded that neutral capsular antigens exist also in E. coli strains. Some strains were previously thought to possess K-antigens, e.g. E. coli 0:1 11 but no surface antigen other than the LPS-0-antigen was found. Polysaccharide K-antigens can be obtained by fractionated precipitation with cetyltrimethylammonium bromide (Cetavlon) (Hungerer et al., 1967; Westphal and Jann, 1965). Tables I11 and IV illustrate the combinations of polysaccharide antigens in E. coli. (a) The most important group contains those found in immunoelectrophoretic groups 1A and 2a (see later). They are acidic polysaccharides of low molecular weight. They contain sugar constituents which are not frequently found in bacterial polysaccharides, such as N-acetylneuraminic acid in K: 1 (Barry and Goebel, 1957), N-acetylmannosaminuronic acid in K:7 (Mayer, 1969) or KDO in K:13 (Vann and Jann, 1979). The amino acid threonine is found in K:54 (Table IV). The K:l-antigen, also known as colominic acid (Barry and Goebel, 1957) is a polyneuraminic acid. A type of form variation has been described in this K-antigen as 0-acetylated, and non-0-acetylated variants of the same polysaccharide are found in the same culture (0rskov et al., 1979) (Section VIII.B.2). K:2ab is similarly an 0-acetylated version of the K2a-antigen (Jann et al., 1980; Larsen et al., 1980)and K:13 is an 0-acetylated form of the K:23-antigen (Vann et al., 1983). No form variation, however, has been found in these antigens. (b) Another large group of capsular polysaccharides, generally of high molecular weight, are those found in IE group IBa and always together with 0-antigens 0:8, 0:9, 0:lOl and 0:20. They can be further subdivided chemically into two groups, one with amino sugars and one without. Those without amino sugars are physically heterogeneous and resemble Klebsiella K-antigens (Jann and Westphal, 1975; Jann and Jann, 1977). The K-antigens containing amino sugars were found to consist of either long chains of acidic polysaccharides or just one repeating unit attached to core lipid A. Thus strains with such K-antigens carry two serologically different LPS molecules in their cell wall: one determining 0-specificity (0:8,0:9,0:20 or 0:lOl) and the other acting as a K-antigen (Jann and Jann, 1977). (c) The M-antigen is a common acid polysaccharide surface antigen found in many coli strains, and other Enterobacteriaceae, and often present as a thick slime layer around the cells giving rise to slimy growth on culture plates. The regulation of its synthesis (Markovitz, 1977) and its chemistry (Garegg et al., 1971) have been extensively studied. This antigen is also described as colanic acid (Goebel, 1963).

TABLE 111 Polysaccharide patterns (combinations of polysaccharide antigens) in Escherichia coli based on chemical and immunoelectrophoretic characterizations ~~~

~

Chemical characterization

In 0-groups In many 0In 0-groups 0 : 8 , 0:9, groups 0:8, 0:9, 0:101, (0:20) 0:101, (0:20) Neutral Lps Neutral LPS Neutral LPS Neutral LPS Acidic LPS Acidic capsular Acidic capsular polysaccharide pol ysaccharide

Immunoelectro phoretic characterization

0-line : cathodic slow K-line : anodic fast

0-line : cathodic slow

mmunoelectrophoretic groups"

1A

1Ba

In many 0groups

In many 0groups

In many 0groups

Acidic LPS

Acidic LPS Acidic capsular polysaccharidc

0-line : cathodic 0-line : anodic slow slow

K-line : anodic slow 1Bb

2b

0-line : anodic slow K-line : anodic fast 2a

TABLE IV Composition of some Escherichia coli K-antigens Neutral sugars K-antigen

Acidic sugar

1

5 7 13 54

GlcUA Mannosami neuronic acid KDO* GlcUA

' N-acetylneuraminic acid.

Amino sugar

Ribose

Gal

Glc

Man

Fuc

NA"

-

-

-

-

-

-

-

GlcNac

-

-

-

-

-

-

-

-

-- +- -

-

-

-

GlcNac

' 2-keto-3deoxy-mannulosoctonicacid.

+ -

-

-

-

-

Rha Threonine

+

+

Reference Barry and Goebel(l957) McGuire and Binkley (1964) Vann et al. (1981) Mayer (1969) Vann and Jann (1979) B. Jann (unpublished data)

54

F. ORSKOV AND 1. ORSKOV

3. Flagellar proteins (H-antigens)

The extensive studies on structure and function of flagellar proteins will not be reviewed here and the reader is referred to reviews by Iino (1969, 1977) and Silverman and Simon (1977). 4 . Fimbrial proteins (F-antigens)

Fimbrial antigens are proteins and much information about the detailed structure of these adhesive organelles has been gathered recently. The complete amino acid sequence of the K:88ab-antigen was presented by Klemm (1981). This plasmid-determined protein is composed of about one hundred identical subunits. It exists in several antigenic variants, four of which, namely K:88ab, K:88ac, K:88ad and K:88ad(e), have been partly characterized (Mooi and de Graaf, 1979).The immunological differences have been shown to be related to differences in the primary structure (Gaastra et al., 1979). All four antigenic variants of K:88 (F:4) possess the same C-terminal structure, implying that this part of the protein is essential for the function of the fimbriae. The N-terminal amino acid sequence and the C-terminal part of the K:99 protein subunit were investigated by de Graff et al. (1981) and Gaastra and de Graaf (1982). Comparison of N-terminal and C-terminal sequences showed that there is probably no homology between the primary structures of the fimbrial proteins characterized so far. For details about the chemical nature of the K:99 protein the reader is also referred to Isaacson (1977) and Morris et al. (1977, 1980). Evans et al. (1979) and Klemm (1979) presented data on the CFAl-antigen found in human enterotoxigenic strains. The N-terminal amino acid sequence of E. coli type 1 fimbriae was presented by Hermodson et al. (1978) who also compared it to fimbriae from Neisseria and Moraxella.

5 . Outer membrane proteins It is a well known fact that many additional proteins exist in the outer membrane (Dirienzo et al., 1978), and it is now also recognized that they are immunogenic not only when injected into animals (Nurminen et al., 1979; Maarten et al., 1979; 0rskov er al., 1981) but also in the normal animal host. Probably they play an important role in the host-parasite relationship and they may be important constituents of future vaccines (Lindberg and Svenson, 1982). Hitherto they have not been used for typing purposes. One reason for this is that many of them will behave as common antigens found in many different serotypes. Recently it has been elegantly shown that different E. coli strains can be characterized by their outer membrane protein (OMP) pattern, as seen in polyacrylamide gel electrophoresis (PAGE). Such patterns

2. ESCHERICHIA COLI

55

give important epidemiological information about the geographical spreading of subclones of specific well characterized 0:K:H-serotypes (Achtman er a /. , 1981 and Stenderup and 0rskov 1983) (Section XII). C. Genetics

The genetics of E. coli antigens will not be covered here and the reader is referred to recent reviews on 0-and K-antigens (0rskov et al., 1977) and Hantigens (Iino, 1977).

111. Serotype definition

Before turning to a detailed description of the single antigens a few general remarks about the term serotype may be appropriate. In E. coli serology a serotype is defined by 0-,K- and H-antigens, i.e. 0:K:H and an additional F if fimbrial virulence factors are present: 0:K:H:F. Examples: 0:6;K:2;H: 1;F:7 (0rskov et al., 1980a),a typical strain from pyelonephritis; 0:111;H:2; no K-antigen; F!', from infantile diarrhoea; 0:I8ac;K: 1;H:7, from neonatal meningitis. The term 0:H-serotype, e.g. 0:75;H:5 is often used and may cover an 0:K:H type where the K-antigen has not been examined. However, it is misleading to refer to 0-group determination as serotyping.

IV. Antigenic scheme As explained above a common K-determination has only been carried out to a limited extent and a complete antigenic scheme comparable to that in Salmonella cannot be presented today. Because of the many single antigens now described in the Escherichia group and because most of these can be found in different combinations leading to several different 0:K:H combinations an antigenic scheme such as that of Kauffmann-White would be unwieldy. Detailed serological analysis of Escherichia strains has only been carried out on a limited number of strains in comparison with the large number of Salmonella strains examined over the years. However, even this low number of E. coli strains shows a rich variety of antigenic combinations. As an illustration it can be mentioned that 0-group 111 was found in combination with 0:14 and 0:8 together with 29 different H-antigens; 0:8 is found in many different O:K combinations, and the number of permutations accordingly is

56

F. ORSKOV AND 1. ORSKOV

large. If we compare the situation in Salmonella and Escherichia it should also be remembered that each E. coli 0-group covers several non-identical 0antigens with a common main factor and some partial factors, i.e. E. coli 0groups 1,2,3 could be compared to Salmonella 0-groups A, B, C and so on. What is presented in Table V is a combined, restricted antigenic scheme for all E. coli antigenic test strains.

V. History of typing procedures A. Serotyping

1 . 0-antigen The first successful attempt to classify E. coli by serological methods was carried out by Kauffmann (1943, 1944). By methods which he had used in Salmonella investigations he was able to subdivide a limited number of strains into 20 0-groups. He used a boiled culture for 0-antiserum production and agglutination tests. The first antigenic scheme was thus established by Kauffmann (1944) and was soon extended by Knipschildt (1945) to contain 25 0-groups. Since then many 0-antigens have been added so that the present antigenic scheme comprises 171 0-antigens (Orskov et al., 1977, in press). 2. K-antigens (polysaccharides)

During his investigations Kauffmann observed that many freshly isolated strains were not agglutinated when examined in the non-heated state in 0antiserum. A similar phenomenon was well known, e.g. from Salmonella typhi, which is also inagglutinable in 0-antiserum (0-inagglutinability) when equipped with the Vi surface antigen. This inhibition of agglutinability in 0serum could be overcome by heat treatment. Based on this observation Kauffmann was able to define a number of additional surface antigens in E . coli with somewhat different physical and serological characters, all causing inagglutinability in the homologous 0-antiserum that could be overcome by heat treatment. In 1945 Kauffmann and Vahlne introduced the term Kantigen (from the German word for capsule, Kapsel) as a symbol for envelope or capsular antigens. Their definition was exclusively based on tests using bacterial agglutination and the above described inagglutinability was considered to be the most important criterion for the presence of a K-antigen (Kauffmann, 1943,1944,1966). Table VI gives a schematic presentation of the agglutination results on which the previously used definitions of K-antigens

2. ESCHERICHIA COLI

57

were based. Three different K-antigens were described: A, B and L. Already Kauffmann and Vahlne (1945) pointed out the difficulties that could be encountered in distinguishing between L- and B-antigens. Many investigators have later run into the same type of problems. It was found that an E. coli strain, which according to the original definitions should only have one (L) antigen, contained two K-antigens, e.g. B (K:87) and L (K:88) (0rskov et al., 1961). Sometimes repeated examinations of one and the same strain would show the L-antigen on one occasion and the B-antigen at another. One reason for this was that in the classical “L or B experiment” (Table VI) in which an OK-serum was absorbed by homologous boiled bacteria the outcome was influenced by variable factors, e.g. the amount of boiled bacteria used for absorption. Most often all antibodies against the polysaccharide K-antigen were removed by the standard absorption and, if not, repeated absorptions would remove the rest. The antibodies found in socalled pure L-sera were therefore either a mixture of residual antibodies against the polysaccharide K-antigen and different thermolabile surface antigens or simply antibodies against such thermolabile structures (fimbriae, fimbria-like antigens, H-antigens and other less well-defined outer membrane proteins). If no antibodies were left against the live culture the K-antigen of the strain was labelled B. The chemical nature of the surface antigens was not recognized when the A, B and L definitions were established, and it was not understood that the polysaccharide K-antigens in principle are thermostable and just eluted from the bacterial surface during boiling, making Kagglutination of boiled bacteria impossible. Although an agglutination experiment can be helpful during a preliminary examination, it cannot be relied on as the sole test for K-antigen analysis. Methods which can differentiate between the many single antigen-antibody reactions are required. At present different agar precipitation techniques are preferred. By means of such techniques three representative E. coli K-antigens of the L-variety, K:12, K:51 and K:52, were re-examined by 0rskov and 0rskov (1968,1970). In all three strains the presence of heat-stable extractable K-antigens was demonstrated by passive haemagglutination (HA), double diffusion in gel and IE. In IE they showed a high electrophoretic mobility towards the anode. These three K-antigens were acidic polysaccharides. Similarly, the K:2ac-antigen, now K:2a (Larsen et al., 1980), originally described as an L-antigen, was studied by Holmgren et al. (1969) and found to be a polysaccharide of high electrophoretic mobility retaining its precipitating and agglutinin fixing capacity after boiling. Later IE studies in agar of all E. coli K-antigenic test strains have shown anodic precipitation arcs in extracts heated to 100°C which were due to Kantigens in all the former A- and L-containing strains, except O: 137;K:79(L) (0rskov er al., 1971). From 33 strains in which B-antigens had been described,

TABLE V

E. coli antigenic scheme comprising all 0-,K-and H-antigenic test strains (arranged according to 0-antigen numbers) 0

K

H

I I

I 51 I

-

2 2 2 2 2 3

3 3 4

4 4 4 5 6

7 (56) I 7

ne 2cih ne ne 3 6

12 52 4

2a

6 6 6 6 6

I3 I5 53

7

7

7 US-41

4

7 6

I 8

2 31

44 5 5 -

4 I I 16

54

10

13

49

I

-

-

8

7 8 25 27 (A) 40 41 42 (A) 43 44 (A) 45 46 47 48 49 50 84 (A)

8

87

19

8 8 8 8 8 8 8 8 8 8 8 8 8 8

8

8 8

860 9

9a 9ab 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10

I1 II II 12 I3 14'

102 (A) ne nc

ne 9 26 (A) 28 (A) 29 (A) 30 (A) 31 (A) 32 (A)

33 (A) 34 (A) 35 (A) 36 37 (A) 38 (A) 39 (A) 55 57 nc 5 10

ne nc 5 I1 7

Culture number

4 4 9

9 II -

II

9 30 2 9 21

-

-

20 21 51 12 -

A1838 U9-41 H17b A2oa su1242 Ap32oc u14-41 KIS(=HW33) 781-55 U4-41 Bi7457-41 Su65-42 A103 UI-41 Bi7458-41 su4344-41 F8316-41 PA236 Al2b 2147-59 Bi7509-41 Pus3432-41 G3404-41 Bi7575-41 E56b ASld A433a A295b A195a AI68a A169a A236a A282a A290a A180a PA80c H308V D227 (=G:7. K:88.) 6CBIO-I H3Mb Ulla-44 C218-70 Bi316-42 Bi449-42

- K14a

- 81161-42

I2 E69 - su3973-41

19 H36 - Ap289 - E75 - AI& 19 A!98a - A% - A2628 9 Al2la - N24c 32 HWM 19 A I M 4 81833741 10 Bi623-42 33 K181(=HW35) 52 (2187-69 - 81626-42 I 1 su4321-41 - su4411-41

O I5 IS 15

I5 16

16 17 18uh I Sac l9iih

20 20 20 20 21

22 23 23

23 24 25

25 26

26 26 27 28

28 29 30 32 33 34 35 36 37 38

38 39 40 41 42 43 44 45

45 46 48 49 50 51 51 52

52 53 54 55

55 56 57 S8 59

60 61 62 63 64 65

K

H

Culture number

4 F7902-41 17 PI2b 25 N234 ( - HW26) 27 K50 ( = HW28) ~

F11119-41

48 P4 18 Kl2a I 4 F10018-41

7 D-M32 19-54 7 F8188-41 - P7a 26 CDC134-51 26 CDC2292-55 1413 E19a I E14a I5 E39a IS H38 IS H67 - E41a I2 E47a I H54 H311b - F41 46 5306-56 - F9884-41 - Kla - Katlwijk 10 Su4338-41 - P2a 19 P6a - E40 10 H304 10 E77a 9 H502a 10 H510c 26 F11621-41 3ff N157(=HW32) - H7 4 H316 40 H710c 37 P I l a 2 Bi7455-41 18 H702c 10 H61 23 K42(=.HW23) 16 PIC - U8-41 I2 u12-41 4 U18-41 24 U19-41 24 K72(=HW25) 10 u20-41 45 4106-54 3 817327-41 2 su3972-41 - su3912-41 6 AbcrdeenIW - su3684-41 - F8198-41 27 F8962-41 19 F9095-41 33 F10167a-41 19 F10167b-41 30 F10524-41 - F10598-41 - K6b - Klla ~

~

~

TABLE V O 66 68

69 70 71 73

73 74 75

75 76 77 78 79 80 XI 82 83 83 84 85 86

86 86 86 86 86 87 88 89 90

91 92 95 96 97 98 99 loo I01 101 101

102 103 104

I05 106 107 108 109 110 111

112ab I l2ac 113 I I4 115 116 117 II 8 119

K

-continued H

Culture numher

25 Pla 4 P7d P9b P9c 12 Plod 31 Pl2a 34 6181-66 39 E3a 5 E3b 5 F147 8 E5d El0 E38 40 E49 26 E71 HS HI4 31 H17d 31" H45 21 HI9 I H23 25 H35 - E990 2 F1961 36 5017-53 34 BPI2665 47 1755-58 I2 H40 25 H53 16 H68 - H77 - H307b 33 H308a 33 H311a 19 H319 - H320a 8 HSOld 33 H W c 2 H5Wa 33 H5lOa 38 42

~

~

- 841 - 8CE275-6

40 HSII 8 H515b I2 H519 8 H52Ob 33 H52la 27 H705 10 H708b 19 H709c 39 H711c - Stoke W 18 1411-50 - Guanabara ( 1 6 8 5 ) 21 6182-50 32 26w(=KIO= HW34) 18 21w 10 28w 4 3ow - 31w 27 34w

' Former test strain of 0:93 (Orskov PI~ 1 . 1977). .

O

H

K

120

6 35w

121 123 124 l25uh 125uc. 126 127u

127ab I28 129 1311 I31 I32 133 I34 135 I 36 137 I38 139

Culture numher

10 39w

16 30 19 6 2 -

4 2 II 9 26 28 29 35 41

I39

I 56

140 141

43 4

43w Ew227 Canioni Ew21.29-54 E611 4932-5.i 2160-53 Cigleris Sceliger 178-54 Ew4866-53 s239 ( = ~ ~ 2 7 ) N87(=HW30) N282(=HW31) 4370-53 COll Pecs I I 11-55 RVC1787 CDC62-57 CDC63-57 SN3NII CDC149-51 RVC2907

141 142 I43 144 145 146

147

4 E68 6 C771 4608-58 1624-56 E1385(3) 21 CDC2950-54 19 D357(=Gl253. K 88.) ~

~

147 148

148a

19 GI253 28 E5l9-66 53 E480-68

149 10 Abbotstown A l I50 I51

152 I53 I54 I55 156 157 I58 159 160 I6 I

162 163 I64 165 166 167 168 169 I 70

6 1935 10 880-67 ~

7 4 9 47 19 23 20 34 54 10

19 -

4 5

16 X

I

1184-68 14097 E1541-68 E1529-68 EIS85-68 A2 E1020-72 E2476-72 E110-69 E223-69 lOBI/l SN3B/I 145146 E78634 3866-54 El0702 El0710 1792-54' 745-54

* 0:14 does not contain S-LPSbur R-LPS. 'Strain H :38 has lost its K :21-antigen. K :21 has therefore been deleted. Test strain for K :24 ("H :45"). formerly assigned to 0-group 22. Numbers in italics are test (reference) antigens. Numbers in ilalia in parentheses are former reference antigens. now deleted. These numbers will not be used in the future (Section V.A.). The following polysaccharide K-antigens have been found lo be closcly related or identical (Scmjtn er al., 1977): K:2abK :Zac-K :62. K :7=K 5 6 . K 92-K 182. K 9 3 - K23. K18- K:22, K :16- K 3 7 - K:97. K 5 3 - K :93. K54-K:%. This scheme only contains the xrotype formulae of the reference strains used at the WHO Collaborative Centre for Referencc and Ruearch on Esrherirhia. I t therefore contains all hitherto officially established Escherichia antigens. It is evident that nothing about prevalence of single antigens can be deduced from the data given in this scheme. The strains have been collected over more than 30years and many different considerations have determined the selection.

'

60

F. ORSKOV AND 1. ORSKOV

TABLE VI Schematic presentation of the agglutination results on which hitherto used definitions of K-antigens (A, B and L) were based

0, K-serum

Preparation of antigen

K-type

Absorbed by culture heated to 100°C 0-serum for 1 h Unabsorbed -

-

-I+

-

-

+

Live

A

Boiledlautoclaved

+ +

Live

B Boiled

Live

L Boiled (100°C for 1 h)

‘6

+

7.

-

+ +

Negative or significantly lower than that of the boiled culture. - ,N o agglutination. +, Agglutination.

‘I-’’,

the presence of K-precipitation arcs could only be demonstrated in a few cases. These were the three first established B-antigens, K:25, K:56, K:57, and in addition K:82, K:83, K:84 and K:87. When Knipschildt described the three first B-antigens he was able to produce pure B-antisera by absorption with other strains of different K-types. However, when the next B-antigens were numbered in strains from infantile diarrhoea, 0:111;K:58 (B) and 0:55;K:59 (B:5), this was done with great reluctance because there was uncertainty as to the existence of a separate K-antigen, since it was not possible to produce a pure B-antiserum by absorption (Kauffmann and Dupont, 1950). The presence of B-antigens in these cases and with a few exceptions in all later cases (B:6 to B:22) was thus based solely on the inagglutinability of the live culture in an 0-antiserum. Several substances and structures can cause inagglutinability in the homologous 0-serum and often it is not a simple matter to point out which factor(s) is(are) responsible in a certain case. The antigen which most often interferes is the acidic polysaccharide K-antigen. However, many strains have been found to agglutinate in 0-serum even though they are equipped with such an antigen, as shown by IE (Orskov and Orskov, 1972) or by agar electrophoresis combined with Cetavlon precipitation in the second dimension (Orskov, 1976). H-antigens (flagella), fimbriae (Aleksic et al., 1978) and

2. ESCHERICHIA COLI

61

perhaps other surface structures can cause 0-serum inagglutinability. Furthermore, it is a common experience that such an inagglutinability can be overcome by changes in the growth medium or growth temperature. Another common experience is that strains which are originally inagglutinable in 0serum become agglutinable after passage in the usual media. This applies to several of the test strains for former B-antigens, found in enteropathogenic (EPEC) strains. In such cases, however, it is not possible to detect any serological differences between older 0-agglutinable laboratory strains and freshly isolated 0-inagglutinable. strains of the same serotype. We do not know which factor(s) cause(s) this inagglutinability of the freshly isolated strains, but we believe that the variable agglutinability in 0-serum alone does not warrant the description of a special K-antigen. The existence of two types of LPS, one with long side chains and one with only one repeating unit, and the almost total elution of the first-mentioned type at 100°C may be the explanation of the different behaviour of live and boiled culture in anti-0-sera of strains with no K-antigens, such as 0:111 strains (Morrison and Leive, 1975; Goldman and Leive, 1980; Goldman e f al., 1982). We therefore find it misleading to continue the labelling of K-antigens according to the classical A, B and L criteria and propose to restrict the use of the term K to the (acidic) polysaccharide K-antigens. These K-antigens can be subdivided into two groups, one found in combination with 0:8, 0:9, 0:20 and 0:lOl and another found probably in all other combinations. Several capsulated strains belonging to 0:8, 0:9, 0:20 and 0:lOl are equipped with capsules that make them inagglutinable in 0-serum even after boiling, but heating at 120°C for 2 h will make them agglutinable. For practical reasons the K-antigens of such strains should still be denoted K(A). Such K(A)containing strains may loose the special heat resistance by mutation, and it is not uncommon in the same specimen to find variants of the same 0:K:Hserotype with and without the special heat resistance of the K-antigen. 3. F-antigens (proteins)

For some years adhesive fimbria (pilus) antigens which are found in E. coli strains of special pathogenic serotypes have been of great interest not only for epidemiological studies but also for studies of the pathophysiological process and as possible candidates for constituents in vaccines. The first antigen of this type was found in some enterotoxigenic strains belonging to a limited number of E. coli strains from piglet diarrhoea (0rskov et al., 1961; Stirm et al., 1966, 1967a,b). At the time when this fimbrial antigen was detected its chemical nature and genetic background were not known and it was therefore confined to the K-antigens of the L-variety (Table VII) based on bacterial agglutination experiments and was labelled K:88(L). A similar fimbrial antigen found in

62

F. ORSKOV AND 1. BRSKOV

strains from diarrhoea1 disease in calves was labelled K:99 (Orskov et a / . , 1975a). The fimbrial factor antigens (also called colonization factors), CFA: 1 and CFA:2, were demonstrated in human enterotoxigenic strains (Evans et a / ., 1975,1977; Evans and Evans, 1978) and type E8775 (Thomas et al., 1982). Since then other adhesive fimbrial antigens have been found with adhesiveness for urinary epithelial cells and with serotypes typical of urinary infections (Orskov et a / . , 1980b, 1982a,b; Orskov and Orskov, 1983). TABLE VII

F-antigens of Escherichia coli Proposed antigen number F:l

F:2 F:3 F:4 F:5

F:6

Erythrocytes Present designation Type 1 (CFA:l) (CFA:2) (K:88) (K:99) (987)

F :7 F :8

-

F :9 F:10 F:ll F:12

-

-

-

used for

Test strain

agglutination"

E. coli BAM H 10407

Guinea-pig Human

PB-176 E 68 B 41

Guinea-pig Horse, sheep

987 c 1212 C 1254-79

3669 C 19679 C 1976-79 C 1979-79

ox

-

Human Human Human Human Human Human

Reference Brinton (1965) Evans et a / . (1975) D. G. Evans et a / . (1979) Orskov et al. (1961) Orskov et a / . (1975a) Isaacson et a / . (1977) Orskov et a / . (1980b) Orskov et a / . (1982a) Korhonen et a/. (1982) Orskov et a / . (1982b) Orskov et al. (1982b) Orskov et a / . (1982b)

Only the most characteristic type of animal erythrocytes used is listed.

All these fimbrial antigens are proteinaceous and many of them may be found in addition to a polysaccharide K-antigen. They have genetic determinants which are different from those of the capsule antigens. Some are plasmid determined and others directed by chromosomal genes completely different from those of the K-antigens. Based on these differences in morphology, chemistry, genetics and function it has been proposed (0rskov et al., 1982a; Orskov and Orskov, 1983) to establish a new group of surface antigens called F-antigens and so at the same time to confine the term K-antigen to the capsular or micro-capsular polysaccharide antigens. The hitherto described F-antigens are listed in Table VII. Antigen 987 (F:6) was first found in strains from piglet diarrhoea which did not carry the antigen K:88 (F:4) (Isaacson, 1977). Antigens CFA:1 (F:2) and CFA:2 (F:3) which were described as colonization factors were associated with enterotoxigenic strains from human diarrhoea (Evans et al., 1975, 1977; Evans and Evans,

2. ESCHERICHIA COLI

63

1978). F:7 was the first adhesive antigen found in strains from urinary tract infections (0rskov et a/., 1980b) but has now been followed by F:8, F:9, F:10, F : l l and F:12 (0rskov et a/., 1982a,b). The authors realize the controversial aspects of Table VII, but nevertheless feel that the time is ripe for a proposal. These antigens might be called F, P, CFA or something else, but probably a one-letter symbol is preferable and we have chosen F for filaments and fimbriae. The question is whether the mannose-sensitive (MS) common type 1 fimbriae should be listed together with the many mannose-resistant (MR) adhesive (virulence factor) fimbriae. By giving them the number 1 we feel that they have got the special position they deserve. Another problem concerns the other already named antigens, K:88, K:99,987, CFA:1 and CFA:2. One possibility would be to stick to the present numbers where possible, i.e. F:88, F:99, F:987, and to change CFA:l and CFA:2 to F:2 and F:3. However, we would propose to follow the procedure in Table VII and give these antigens new numbers and use the old numbers in parentheses for a transitional period. It is a characteristic of antigens F:2 to F:12 that they enable the respective bacterial strains to agglutinate animal erythrocytes and that this capacity cannot be inhibited by mannose, i.e. they show mannose-resistant haemagglutination (MRHA) (Duguid and Gillies, 1957; Evans et al., 1977; 0rskov and 0rskov, 1977). It is also typical that the MR fimbrial antigens are not, or only poorly, developed at a low temperature (18°C). The receptor for the urinary tract infection (UTI) fimbrial antigen has recently been described as a glycosphingolipid (Leffler and Eden, 1980) and, similarly, it was found by Kallenius et a / . (1980) that the Pk glycosphingolipid is a receptor for a number of MRHA E. coli from urinary tract infections. So far, only few of the F-antigens listed have been used on a greater scale for typing procedures, i.e. the former K:88 now F:4, K:99 now F:5 and 987 now F:6, which have been used extensively by veterinary bacteriologists for epidemiological studies. Future investigations will show whether the other Fantigens will be of similar significance in the serotyping of human strains. In this connection it should be emphasized that several reports seem to show that disease may be associated with enterotoxigenic strains that d o not carry adhesive fimbriae. In a special position among the fimbrial antigens are the so called type 1 fimbriae (Brinton, 1965; Duguid et al., 1955), which were the first described fimbriae. This type, which is now very well examined morphologically, is similar to the other fimbriae and agglutinates erythrocytes in an MS manner (MSHA) (Duguid and Gillies, 1957). Usually type 1 fimbriae (F:l) are well developed also at lower growth temperatures, and they are surprisingly resistant to heating (Gillies and Duguid, 1958), a character which they have in common with some of the chromosomically determined MR fimbriae.

64

F. ORSKOV AND I. ORSKOV

Genetically they are chromosomically determined and are more or less ubiquitously found among Enterobacteriaceae. Not all type 1 fimbriae are serologically identical, but heavy cross-reactions between them make them rather unuseful for serotyping purposes. While several MR fimbriae can be considered as virulence factors, type 1 fimbriae have not yet been found to play any role of virulence in a pathological process. However, recently it has been suggested by 0rskov et al. (1980a) that the specific binding of F:l fimbriae to urinary mucus, i.e. the Tamm-Horsfall protein, and to mucus in the respiratory tract and on other epithelial surfaces plays an important role in the relationship between enterobacteria and their animal hosts. 4 . H-antigens (proteins)

H-antigens are the serological determinants of the protein flagellar organs found in motile organisms. Since Kauffmann and co-workers published the first E. coli antigenic scheme comprising 21 H-antigens, the number has increased to 53, as shown in the present scheme. H-antigens are usually completely destroyed by heating to 100°C. Phase variation between H:3 and H:16 and H:4 and H:17 has recently been described (Rather, 1982), thus demonstrating that E. coli H-antigens are not always monophasic as hitherto believed.

5 . Common antigens Although some of these antigens have been mentioned previously, they are listed again under this heading because they belong to a group of antigens which serologically, due to their ubiquity, have one important character in common, namely they interfere with the normal serotyping process. ( a ) Polysaccharide antigens. Kunin antigen. This antigen, often called the common antigen or CA, was detected by Kunin et al. (1962) using the indirect haemagglutination technique, and is only one of several common antigens. In fact, it rarely interferes with ordinary typing procedures since it is not immunogenic in strains having an 0-specific polysaccharide. The E. coli strain generally used for production of CA antiserum, i.e. the test strain for E. coli antigen 0:14, is, when examined today, devoid of an 0-specific polysaccharide chain in its LPS. The Kunin antigen, which is found in most, if not all Enterobacteriaceae strains, is normally hidden by the 0-polysaccharide chain (Edwards and Ewing, 1972; Mayer and Schmidt, 1979). M-antigen. This polysaccharide antigen has been described under many different names: M-antigen (Kauffmann, 1935, 1936), capsular antigen (Markovitz, 1964), colanic acid antigen (Goebel, 1963), slime wall antigen, mucous antigen and several others. This group of closely related antigens has

2. ESCHERICHIA COLI

65

been examined serologically by Henriksen (1949, 1950) and Kauffmann (1966). The genetics has been examined in great detail by Markovitz (1964, 1977). Variational phenomena, especially the influence of media and growth temperature, were investigated by Anderson and Rogers (1963) (Section VIII.B.3). For information on chemical composition see Markovitz (1977). The M-antigen is found in many genera of Enterobacteriaceae. ( b ) Thermolabile antigens (proteins). From our own experience we know that non-immunized rabbits may often contain gel precipitating antibodies to one or more of at least four different thermolabile antigenic determinants found in most E. coli strains examined. After immunization with an unheated Escherichia strain the precipitation lines will generally be stronger from sera which were positive before immunization and they will also be found in previously negative rabbits. It is likely that some of these common thermolabile precipitating antigens are similar to some of those mentioned below (Orskov et al., 1981). u-Antigen (Stamp and Stone, 1944). u-Agglutinins can be found in unimmunized rabbits but mostly after immunization. The agglutinating and agglutinin binding capacity is destroyed by heating at 100°C for 15 min. aTitres are often high (1:20 OOO or more). For details see Edwards and Ewing (1972).

/?-Antigen (Mushin, 1949,1955). This antigen is similar in some respects to the a-antigen of Stamp and Stone. The agglutinating and agglutinin binding properties are destroyed by heating at 100°C for 1 h. Both a- and /3antigens are widespread among Enterobacteriaceae (Edwards and Ewing, 1972).

Fimbrial antigens (Section VII.C). Several fimbrial antigens within E. coli have been described. The type 1 (F:l) antigen could be considered as a common antigen. Fimbrial antigens may cause 0-inagglutinability. ATA antigen (anodic thermolabile antigen). Seltmann (1971), Seltmann and Reissbrodt (1975) and Larsson et al. (1973) have described a thermolabile antigen which moves towards the anode during IE. It is probably a glycoprotein and has been detected in all Enterobacteriaceae strains examined. A similar antigen was examined by Kaijser (1975) and Sompolinsky et al. (1980).

B. Phage typing Phage typing of E. coli has been used to a limited extent for typing purposes and there are no generally accepted phage schemes. The most important use of

66

F. ORSKOV AND 1. ORSKOV

phage typing is probably as a means for subdivision of serotypes or groups. In recent years phages specific for some polysaccharide K-antigens of the A type have been isolated (Stirm, 1968), but they have not been used for typing purposes. Gross et al. (1977) have described K:l-specific phages which help to detect E. coli with that antigen. This method is particularly important because it is difficult to obtain a good K:l antiserum. Similarly, Gupta e l al. (1982) have found a K:Sspecific phage which can be used to isolate K5containing strains that have escaped detection hitherto because the K:5-antigen, like K: 1, is only weakly immunogenic in rabbits. Since K:l and K:5 undoubtedly are the most prevalent K-antigens involved in extraintestinal diseases, these two new capsular antigen-specific phages will be of great importance. A very extensive review of the present state of phage typing of E. coli was published a few years ago in Volume 11 of this series and the interested reader is referred to that review (Milch, 1978). C. Colicin typing

This method based on either the sensitivity to a series of known colicins or to the varying capacity of strains to produce colicins has hitherto received even less general acceptance than phage typing. In the review by Milch (1978) on phage typing colicin typing is also covered.

D. Biotyping Attempts to differentiate between epidemiologically different E. coli clones by comparison of the outcome of fermentation tests and other biochemical traits were undoubtedly of some value until serotyping was developed. However, it is generally accepted that biotyping as the only method is not very useful for E. coli differentiation, except under certain special conditions, e.g. at a certain hospital at a certain time in order to follow the spread of a certain clone or to verify that successive urinary tract infections are re-infections or recurrences (Crichton and Old, 1979; Old et al., 1980). It should, however, be strongly emphasized that biotyping is extremely useful in the subdivision of serotypes. Kauffmann and Orskov (1956) described the close correlation between characteristic biotypes and some 0:H-serotypes of strains isolated from cases of infantile diarrhoea, now labelled EPEC strains. In order to illustrate this, a number of such fermentation types is given in Table VIII. Today, more than 20 years after this publication, it is still true that for example 0:111;H:2 or 0:lll;H:12 or 0:26;H:ll are usually found to have biotypes as shown in Table VIII. Recent comparisons of serotypes and biotypes of enterotoxigenic strains have confirmed the frequent correlation of such phenotypic characters

TABLE VIII Biotypes of some selected EPEC serotypes 0 : l l l ; H:2

Adonitol Dulcitol Sorbitol Sorbose Xylose Rhamnose Maltose Salicin Sucrose

+2-6 + 1-9

+ 1-3

+

0 : 5 5 ; H:6

0 : 5 5 ; H:7

-

-

V

+ 2- 1 0

+ 1- 7

+ 1- 2

-

+ 1-2

0 : l l l ; H:12"

+ +

+ 1-6

+*5 + 1- 5

+ 1-6

+ 1-2

+ 1-4

+ + +

V

+ +

V

V

-

+e5

+ 1-2

+I-2

0.26; H : l l

+ + + + x +

+ + + + + + x +

0 . 8 6 ; H:34

0:127; H-

+ 1-4

+ 1-3

+ + + + X

+ +

+ + +

From Kauffmann and Orskov (1956). +', Positive after one day; positive after two to six days; negative after130 days; V, different reactions ( + . - or x); x . late and irregularly positive or negative. "Except for 0 1 11;H:12, all these serotypes are frequently associated with serious cases and outbreaks of infantile diarrhoea. Several other serobiotypes from EPEC diarrhoea have been described. The table is therefore restricted and only demonstrates the usefulness of biotyping.

+'-",

-.

68

F. ORSKOV AND I. ORSKOV

(0rskov and Orskov, 1980). For a more detailed discussion see Section XI1 describing the clone concept. For many years we have quite naturally relied on the most discriminating reactions, i.e. those which ideally would divide randomly isolated E. coli into two groups which were not too different in size (Crichton and Old, 1979; Old et a / . , 1980). The bioreaction, which by this criterion has been most useful in our laboratory, is the fermentation of dulcitol, maltose, rhamnose, xylose, sucrose, sorbose and further prototrophy. Fermentation of adonitol and lactose is generally less useful, although the adonitol-positive and lactosenegative reactions are very important in screening certain strains possessing these rare traits.

E. Typing by outer membrane protein (OMP) patterns Recently Achtman et a/. (1981) and Stenderup and 0rskov (1983) have used the different migration patterns of outer membrane proteins in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to subdivide E. coli strains. In this way it was possible to define precise epidemiologically related subtypes of several E. coli 0:K:H-serotypes associated with neonatal meningitis and infantile diarrhoea. Correlations between biotype and OMP type was found (Section XII). Similar investigations using plasmid DNA patterns have shown that also this approach may be useful for subdivision of serotypes (Silver et a / . , 1980). However, when this method was applied on the collection of strains examined for OMP patterns as mentioned above, the outcome was more complex, particularly when strains from different geographical areas were compared (Achtman, et a / . , 1981).

F. Typing by antibiotic resistance patterns Different bacterial clones can be characterized by their antibiotic resistance pattern. At a certain place, in a certain hospital or in a series of specimens from the same patient a specific pattern may be helpful in the search for an association between strains. It will be important, of course, to remember that resistance characters are transmissible and by nature not stable. G . Typing by direct haemagglutination (HA)

Bacterial fimbriae have been described above by their antigenicity as Fantigens. The adhesive capacity of most fimbriae also enables them to

2. ESCHERICHIA COLI

69

agglutinate erythrocytes. Two main types of direct HA have been described (Duguid, 1964; Brinton, 1965): Mannose-sensitive (MSHA) and mannoseresistant (MRHA). The MSHA is used for characterization of type 1 (F:l) fimbriae by means of guinea-pig erythrocytes. Because of the widespread occurrence of F:l fimbriae they are not fitted for typing purposes. It is important to remember that these fimbriae are found in most E. coli strains and often at the same time as other F-antigens both in the same culture and in the same bacterium (0rskov er a/., 1982a). The HA abilities of MR fimriae can primarily be used to screen for certain pathogenic clones. Many virulent E. coli serotypes associated with extraintestinal diseases will carry MR fimbriae which agglutinate human erythrocytes (0rskov er a / . , 1982a). By means of panels of erythrocytes from different animals typing schemes have been established and used for subdivision of pathogenic E. coli (Evans et a/., 1979,1980,1981). Enterotoxigenic E. colt' from piglet diarrhoea equipped with K:88 (F:4) antigen will show MR-agglutination of guinea-pig cells (Stirm et al., 1966) and K:99 (F:5) will show MR agglutination of horse erythrocytes (Tixier and Gonet, 1975). Since several clones with different genetic and phenotypic traits may carry the same MRHA pattern, this method cannot be used for epidemiological purposes except under special circumstances.

H. Type stability A few words should be said about the stability of types within the different type systems. 0-,K- and H-antigens are primarily determined by chromosomal genes and are therefore highly stable. We use today the same cultures as those established as antigen test cultures 30-40 years ago. Until recently they have been kept together as agar stab cultures or as Dorset egg cultures at room temperature and have only been recultured with several years' intervals. It is, however, always possible to detect mutants, e.g. R forms or Kforms from any culture which is more than a few weeks old. The important thing is therefore to avoid colonies that represent such loss mutations when further subcultures are made. Similarly, most of the biotype characters are chromosomally determined and highly stable, as mentioned above. It is well known that raffinose fermentation may be plasmid determined and this character may therefore be lost in such cases. As raffinose and sucrose fermentation are both usually either positive or negative (0rskov and Orskov, 1973) a raffinose fermentation occurring alone may suggest that this character in the strain in question is plasmid determined. For a discussion of the stability of phage types the reader is referred to Volume 11 of this series (Milch, 1978).

70

F. ORSKOV AND 1. ORSKOV

VI. Methods A. Bacterial agglutination

I . General considerations

Bacterial agglutination is defined as the clumping of bacteria caused by a specific antiserum. The method is simple and has been used since the early days of bacteriology for identification within many bacterial families. It is still the fundamental method for refined antigenic subdivision of Enterobacteriaceae. The Kauffmann-White scheme covering the serological types of Salmonella is the most brilliant example of how successfully detailed agglutination and the agglutination absorption technique can be applied. The agglutination test is carried out in tubes, in plastic trays or on glass slides and consists of mixing bacteria suspended in saline (0.15 M sodium chloride) with appropriate dilutions of antiserum. At neutral pH most Enterobacteriaceae carry a negative surface charge which, together with a number of other physical factors, is responsible for their ability to remain evenly suspended for long periods. Higher salt concentrations may modify this negative charge, thus causing agglutination without the presence of antibody. Contrarily, lower salt concentrations or distilled water can be used to counteract spontaneous agglutination as seen with rough mutants (no 0polysaccharide side chains in LPS), which at the same time lack acidic polysaccharide capsules. If the salt concentration in the mixture is too low the agglutinability of smooth cultures in their specific antisera may also be impaired. It should be emphasized that bacterial agglutination can be used quantitatively only with difficulty and without great precision. It is a qualitative or, at the most, semi-quantitative procedure, where the primary antigen-antibody reaction occurs on the surface of the comparatively large bacterial body and the reaction is the secondary clumping of these corpuscles. It should also be stressed that agglutination is only as simple as it sounds in cases where one is certain that not more than one antigen-antibody system is involved. One such important case is the routine diagnosis of E. coli 0antigens. The sera used for this purpose are 0-sera, i.e. rabbit sera generally produced by immunization with the culture heated to 100°C for 2.5 h. From a practical point of view such antisera contain only 0-antibodies. Agglutination reactions found in these sera could be considered as 0-antigen reactions as long as no other thermostable highly immunogenic antigen is left on the bacterial surface after heating to 100°C for 2.5 h. This is true for many E. coli strains, but not for the heavily capsulated strains with K-antigens of the A variety. 0-sera produced with such strains may contain detectable amounts of

2. ESCHERICHIA COLI

71

antibody against the polysaccharide K-antigen and antibody against the 0antigen at the same time. Likewise the boiled bacteria used as antigens may contain remnants of capsular polysaccharide on their surface. It should also be mentioned here that F:l- and other F-antigens are quite heat stable and so may interfere during the standard 0-determination. Usually the antigenic determinants of the core part of the LPS are hidden in normal smooth strains, but it is not uncommon to find that 0-antisera produced against such smooth strains contain R-antibodies. Since the number of different R-antigens is much more restricted than that of 0-antigens, the existence of R-antibodies in 0-antisera may confuse the 0-determination when unknown smooth strains with some R-LPS determinants exposed are examined. The situation is more complex with so-called OK-sera, i.e. sera produced with a live or formalin-treated culture. Such sera, e.g. typical E. coli OK-sera, contain antibodies against most of the known and unknown structures found on the surface. If examined with homologous live culture, the agglutination observed is the combined consequence of several independent antigen-antibody systems. If the same antiserum is used for the examination of an unknown culture, and if agglutination is observed, it is not simple to demonstrate whether one or more possible surface antigens of the unknown culture are involved. With the aid of appropriately absorbed sera (Section 1X.B) and/or by using several OK-sera produced with different known combinations of the antigens involved it will, however, in many cases be possible to reach a reliable typing result. It is possible by certain cultivation procedures to inhibit the development of a special surface structure (see below) and thereby eliminate its interference with agglutination, e.g. to inhibit the development of the fimbrial F:4 (K:88) antigen by culturing at 18°C (0rskov et al., 1961). 2. Slide agglutination Slide agglutination is an extremely simple, sensitive and for many purposes useful method. Most simply a small amount of culture from an agar plate is transferred and suspended by a straight platinum wire into a droplet of diluted serum on a microscopic slide. The slide is rocked by hand and after a short time (10 or 20 s) the agglutination is read by the naked eye with the aid of a hand lens. Preferably it should be read against a black background using some sort of indirect light. Instead of direct suspension from an agar plate culture, heavy suspensions of bacteria can also be used, transferred either by a loop or pipette. Wooden tooth picks can, with great saving of time, replace the straight wire, also eliminating the Bunsen burner. Tooth picks can be autoclaved and used many times without previous cleaning.

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F. ORSKOV AND 1. ORSKOV

Slide agglutination is primarily used to screen many colonies in a limited number of sera, e.g. when primary cultures are examined for EPEC serotypes or special K-antigens. Usually the slide agglutination test needs confirming by an agglutination titration under more standardized conditions in tubes or trays. Slide agglutination can be much influenced by the ratio between number of bacteria and antibody content and, particularly in cases where only low titre sera are available, negative reactions may be replaced by clear-cut positive reactions by a reduction in the amount of bacteria used; this is very likely the result of a prozone phenomenon. Another pitfall hidden in this technique, which, however, could also be looked upon as a special asset, is caused by the variability in the reaction between single colonies: variation within a culture which cannot be detected by examination of a fluid culture or of confluent growth from an agar plate can be revealed by slide agglutination of several single colonies. A negative slide agglutination test based on the examination of one single colony or of the mass culture may also be misleading because of this variation phenomenon, e.g. when cultures are examined for the K:l antigen (see below). 3. Tube agglutination

Tube agglutination or agglutination in plastic trays can be used both as a primary screening method, e.g. when heated cultures of E. coli strains are examined in pooled 0-antisera (see below), and for titration. Most frequently, similar amounts (e.g. 0.2 ml) of bacterial suspensions and serum dilutions are mixed and then incubated at the recommended temperature and time. ( a ) Titration. The agglutination technique is widely used as a titration assay, generally in tubes, but nowadays quite often in plastic trays. To a series of tubes containing 0.2 ml antiserum diluted in two-fold dilution steps the same amount of a bacterial suspension of a certain density is added; most often 10' to lo9 \organisms ml- is suitable. It is advisable to use a standard density of bacteria. Depending on the antigens examined, the mixture is incubated at 37°C or 50°C for a defined period of time. The titre which indicates the relative strength of the serum is expressed as the reciprocal of the highest dilution causing agglutination which can be seen with the naked eye. Due to slight variation in dilutions agglutination titres are not accurate ( &one titre step). A comparison of the titres of an unknown and the homologous strain in a test serum with known potency is a rough guide to the relationship between the two antigens. However, it should be borne in mind that this is only true in well-defined systems such as the 0-system. In agglutination systems involving unheated bacteria and OK-sera it is extremely difficult to draw any conclusions from the titres about the closeness of a possible

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antigenic relationship of two strains. It is for example a well known fact already demonstrated by Felix (1938) for the Vi-antigen and by Vahlne (1945) for E. coli that well-capsulated bacteria will give a lower titre than mutants of the same strain with less capsular substance in one and the same homologous OK-serum. Likewise, an actively motile culture may give a higher titre than a less motile variant of the same strain when examined in one and the same Hserum.

( b ) Prozone phenomenon. From a theoretical point of view, no agglutination would be expected to occur when antibody molecules are in excess relative to antigenic determinants on the bacterial surface because the simultaneous binding of the two binding sites of antibody molecules to different bacteria would be unlikely. In this situation a tube titration may show no agglutination in the tubes with low serum dilutions but increased agglutination in tubes with higher dilutions. This phenomenon, called the prozone effect, is not a problem in routine 0-antigen serotyping, but, it is not at all uncommon when working with live (or formalin-treated) bacteria and OK-sera, as in K-antigen typing of E. coli. ( c ) Macroscopic appearance of agglutinates. A few words should be said about the macroscopic appearance of agglutinates. They may look very different from one another and textbooks often state that 0-agglutinates are “granular”, H-agglutinates are “floccular” and capsular antigen agglutinates are “disc-like”. These descriptions are only true with strong qualifications. 0agglutinates are granular and difficult to disperse by shaking when boiled suspensions are examined in 0-sera. H-agglutinates are more fluffy, like very loose cotton balls when highly motile cultures are examined in specific sera that contain no antibodies against other surface structures of the agglutinated organism. But, as mentioned above, if live or formalin-treated cultures, and in fact also boiled cultures, are assayed in OKH-sera it is difficult to tell anything about the antigens and antibodies involved from the appearance of the agglutinates. Fimbria-like antigens, e.g. F:4 (K:88), give distinct floccular agglutinates which, however, can easily be disrupted to find granules. Special agglutinoscopes have been developed. We have no personal experience with such agglutinoscopes which presumably are of special value when using a micro-titre system in plastic trays.

4 . Automated typing methods

The typing procedure, particularly of 0-and H-antigens, can be automated, thus saving labour. The interested reader is referred to papers by Bettelheim et al. (1975) and Guinee et al. (1972) who have both developed such systems.

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F. BRSKOV AND 1. BRSKOV

B. Indirect haemagglutination (HA) Indirect or passive HA has not been much used for serotyping in E. coli, but it has been widely used for detection of antibodies against such bacteria and also in immunochemistry as a quantitative measure for antigens. The principle is that certain antigenic macro-molecules will bind to the surface of mammalian erythrocytes, most frequently human blood group O-cells or sheep erythrocytes. Either extracts of bacteria treated in different ways or purified substances are used. In the classical technique described by Neter et al. (1952) for E. coli, boiled extracts of bacteria are employed. After heat treatment of the culture or after treatment of the purified LPS with alkali (Neter et al., 1956) it will bind to the erythrocyte surface. When the strain, and the extract, also contains a capsular polysaccharide this too will bind to the surface. In experiments with OK-sera, the agglutination reaction observed will be a mixed 0-and capsular K-antigen reaction. If a non-boiled extract is used, the capsular K-antigen will usually fix to the erythrocyte surface. Ordinarily, proteinaceous antigens in unheated extracts will not coat the red cell surface. However, a tannic acid treatment of erythrocytes developed by Boyden (1951) has made it possible to coat red cells with protein antigens. Passive HA is most frequently carried out in plastic trays and the settling pattern of the red cells at the bottom of the cups is used as an indication of the degree of agglutination obtained. Latex particles have been used in the same way as red cells (Kabat and Mayer, 1961). C. Co-agglutination Co-agglutination, described by Kronvall(1972), is based on the principle that a certain staphylococcal strain, Cowan I, on its surface carries protein A which unspecifically binds the Fc parts of IgG. Hovanec and Gorzynski (1980) have used this method successfully to detect E. coli belonging to common urinary tract infection O-groups. The method could be modified to cohaemagglutination by coating the erythrocytes with bacterial extracts. Danielson et al. (1979) used the same principle for K-antigen typing. D. Immunofluorescence

This technique has been little used for general serotyping of E. coli. However, it can be useful as a screening method for a limited number of serotypes, such as the special EPEC O-groups (e.g. 0:111,0:55) which are associated with infantile diarrhoea in nurseries and hospitals (Le Minor et al., 1962). The basic principle is that antibodies coupled to fluorescent dyes such as fluorescein or rhodamine will keep their specificity and so after binding to the

2. ESCHERICHIA C O L I

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bacterial cell can be seen under the fluorescence microscope. For serotyping purposes the test can be carried out in two ways. 1. Direct test

Antibacterial rabbit antibody is directly conjugated with the fluorochrome and applied directly to for example a smear from faeces. 2. Indirect test

Antibacterial rabbit serum is applied to the smear and the antibody specifically bound to bacteria is demonstrated by the application of fluorochrome conjugated antirabbit immunoglobulin. The reader is referred to (Le Minor et al. (1962)) giving details of these techniques. E. Gel precipitation

Until recently, precipitation techniques have not been used for serotyping of Enterobacteriaceae. With the development of gel precipitation techniques and the increased interest in distinguishing between different antigens on the bacterial surface such techniques have acquired increasing importance. For serotyping bacterial extracts are more useful than purified antigens. Simple extracts, produced by suspension of overnight 37°C growth from broth agar plates in saline (10'o-lO1l bacteria/ml), can be recommended. The suspension is heated to 60°C for 20min in a water bath followed by centrifugation at 30 OOO g for 20 min. Half of the supernatant is used as the 60°C extract, whereas the other half is treated to 100°C for 1 h and labelled the 60/100"C extract. The 60°C extract will contain LPS, a possible acidic polysaccharide K-antigen and different protein antigens, whereas the 60/100"C extract from a serotyping point of view will only contain the LPS and acidic polysaccharide antigens. Other similar extracts have been described by Holmgren et al. (1969) and 0rskov et al. (1980b). 1. Gel precipitation in two dimensions

Gel precipitation in two dimensions can be carried out according to the original description of Ouchterlony (1958), but for many purposes a simple method with filter paper discs (0rskov and Orskov, 1970) can be used. Filter paper discs, 6 mm in diameter, are dipped into antigen or serum solution until soaked with fluid. The soaked discs are placed in any chosen pattern drawn on a piece of paper placed underneath a 14-cm diameter plastic Petri dish

76

F. ORSKOV AND I. ORSKOV

containing a 2-mm layer of agar. A high quality agar, or agarose recommended for gel precipitation, should be used and 0.065%sodium azide added. The Petri dishes are placed in a plastic? bag and incubated at 37°C overnight followed by incubation at room temperature for three to four days. The plates will not dry out for several weeks. The precipitation results are recorded by drawing after 24 h and repeatedly during the following days. The plates are not easy to stain, but since the method is primarily a qualitative one, we feel that drawings give as much, or perhaps more information than classically stained preparations. A still simpler, and at the same time more gentle, way of preparing an antigenic extract is to place the unused filter paper discs directly onto confluent growth on an agar plate. With forceps the discs are moved to another unused location of the confluent growth. This procedure may be repeated two or three times. Finally, the bacteria-soaked filter paper discs are transferred to the gel precipitation plate. In order to increase the amount of antigens released from the bacteria the broth agar plate with bacterial growth and filter paper discs can be placed in an oven, e.g. for 1 h at 60°C. For the interpretation of precipitation patterns when used to describe the relationships between cross-reacting antigens and sera see Ouchterlony (1958). 2. Immunoelectrophoresis (IE) Immunoelectrophoresis, as described by Grabar and Williams (1953), is a most useful technique when the problem is to distinguish between antigens which move differently in an electrical field. It has been extremely useful for differentiation between LPS and other polysaccharide antigens in Enterobacteriaceae. In our laboratory the micro-method developed by Scheidegger (1955) is used. In this chapter we discuss on several occasions the problems involved in the classical agglutination technique when applied to non-heated bacteria agglutinated by OK(H)-sera. Using IE and the 60°C and 60/100"C extracts described above a clear-cut separation and thus an easily interpretable result (Fig. 1) is often obtained. For a definite diagnosis of capsular acidic polysaccharide antigens in for example E. coli we believe at present that IE is indispensable. GuinCe and Jansen (1979) used IE to demonstrate the K88 (F:4) antigens.

Fig. 1. Immunoelectrophoretic patterns of Escherichia coli 0- and K-antigenic test strains. In the trough is homologous 0-or OK-antiserum. In the well above the trough is 60"C/20-minextract. In the well below the trough is the 60/1OO0Cextract. The right column contains the OK-serotypes of all E. coli 0- and K-antigenic test strains. Underlined antigens are test antigens. In the test strains of 0-antigens 0:24 and 0 5 6 anodic lines close to the basin are seen; K-antigens have been demonstratedchemically in these strains but, since we do not know whether the lines in immunoelectrophoresis represent 0-or K-lines, the strains have been omitted from the scheme. The K-antigens

L 0

/

L

-

u

belonging to immunoelectrophoretic groups 1Bb and 2b' most likely represent 0specificities (LPS) and so can be omitted from the serotype formula (Section IV and Table V). (Note that 0:155,K- antigen should be moved from group 1Bb to 2b.) Recent additions: seven new 0-antigens, 0:164-0:170, have been described since the completion of this figure. None has polysaccharide K-antigens and all, except 0:166, are acidic. Thus test strains 0:164, 0:165, 0:167, 0:168, 0:169 and 0:170 belong to group 2b, whereas 0:166 belongs to group 1Bb.

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F. ORSKOV AND 1. ORSKOV

3. Crossed immunoelectrophoresis (CIE)

A further development of immunoelectrophoresis was conceived by Laurel1 (1966) who added a second electrophoretic step to the first separation of differently charged molecules, run at right angles to the first step on an antibody-containing gel. This technique, which makes quantitative studies possible, has many variations allowing direct examination or absorption directly on the electrophoresis plate. For very precise and easy-to-follow accounts of this technique the reader is referred to Axelsen (1973) and Weeke (1973). A manual covering all the latest developments in this rapidly expanding field is edited by Axelsen (1 978). CIE undoubtedly is the most useful technique for examining charged E. coli surface antigens of both polysaccharide and protein nature. Polysaccharide K-antigens, such as K:2, which has been described earlier in two varieties, K:2ab and K:2ac based on agglutination experiments, was redefined since it was found that not three determinants, 2a, b and c, existed but only two, 2a and 2b, which were found as 2ab and 2a (Larsen et al., 1980; Jann et al., 1980). A series of fimbrial antigens (F-antigens), mostly type MR, was successfully analysed using CIE (Orskov et al., 1980b, 1982a,b). Bacterial surface proteins were also examined by CIE (Orskov et al., 1981; Hofstra et al., 1980). 4 . Counter-current immunoelectrophoresis (CCIE)

Another technique developed for examining capsular antigens such as those of Neisseria meningitidis and Haemophilus injiuenzae has also been adapted for the determination of acidic polysaccharide K-antigens of Enterobacteriaceae. The principle is that in a gel subject to an electrical potential difference the negatively charged acidic polysaccharide will move towards the anode, whereas the precipitating antibodies will move towards the cathode. The antigen and the antibody are therefore loaded in two wells close to each other in the direction of the current, in such a position that they will meet in high concentrations in a narrow area between the wells during the electrophoretic run. This method has only been adapted to E. coli polysaccharide K-antigens and offers a fast and reliable way of typing this group of antigens. The first step is to produce suitable extracts. For E. coli polysaccharide K-antigens the 60/100°C extracts described above are suitable; however, a suspension heated to 100°C can be used directly without centrifugation (Semjen et al., 1977). Using such boiled extracts or suspensions may be difficult in the cases where the polysaccharide K-antigens are partly destroyed or denatured by heating, e.g. the K: 1-antigen. To simplify the procedure the unabsorbed OK(H)-sera are pooled with four sera in each pool, each serum being diluted 1:4. The extract is first used at a dilution of 1:200 or higher. If this test is negative lower dilutions of 1:100 or 1:50 can be tried. If the dilution is 1 5 0 or lower possible

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precipitations will often occur close to or in the serum well. The results may be read within 1 h. High quality agarose, e.g. Litex agarose (Litex, Glostrup), is used instead of agar for both CIE and CCIE.

VII. The antigens A. O-antigens

The LPS O-antigens are heat stable so that boiled bacteria are suitable for immunization, agglutination and agglutinin absorption. For serological purposes smooth colonies are selected that are not spontaneously agglutinable in saline. Cultures are heated to 100°C for 2.5 h for O-antiserum production and similar cultures treated with formalin or heated to 100°C are used for agglutination. The result is read after incubation at 50°C overnight. For the actual O-antigen determination the many O-sera are mixed in pools of cross-reacting O-antisera. We carry out the primary steps of the O-antigen determination in Perspex plates, whereas the final titration is made in glass tubes (Orskov and Orskov, 1975). Many cross-reactions exist between the single O-groups. A record of cross-reactions between 0:1 to 0:148 is given by Edwards and Ewing (1972). In Table IX we show significant O-cross-reactions detected in our laboratory. A number of cross-absorbed O-sera are therefore required when new strains are to be grouped within the O-system. At present we employ more than 300 such O-sera in conjunction with the 170 standard-0sera, for details see Orskov and Orskov (1975) and Edwards and Ewing (1972). Even such a determination does not decide if the O-antigens of the unknown strain and that of the O-group test strain in question are identical. For a definite determination O-antiserum production with the unknown strain and mutual O-absorptions are necessary. For examination of O-antigens of heavily encapsulated cultures, such as E. coli A forms, even prolonged boiling will not destroy the O-inhibitory capacity of the capsule. In such cases it is necessary either (1) to isolate K- mutants or (2) to heat the culture to 120°C for 2 h. The simplest and often most efficient way to isolate K- mutants from mucoid cultures is to let the agar plate, which has been incubated overnight at 37"C, stand for one or more days at room temperature (i.e. 21°C). At this time non-mucoid sectors will often appear at the edge of colonies still growing at this low temperature; such K-sectors will be directly agglutinable in O-serum and give typical O-agglutinations after heating to 100°C for 1 h. Occasionally, non-mucoid sectors will consist of mutants which produce less capsule substance and are only agglutinable in 0serum after heating at 100°C for 1 h. The agglutination obtained with a mucoid culture heated to 120°C for 2 h will often be weak but generally sufficiently strong for O-grouping purposes.

TABLE M

Cross-reactionsbetween Escherichia coli 0-antigenic test strains" 0-serum

0-antigens

0-serum

0-antigens

1

2 3

4 5 6 7 8 9

2, 10. 14.50.53,107, 115. 117, 148, 149,150, 154 I. 50.53.74. 117 13.23.53. I I5 12. 13, 16, IS, 19, 102 7,65.70.71, 114 57 5, 19,25,36,71,116. 141 32.46.60

10 1 1 I25 12 4. 15. 16,123 13 3, 16, 18, 19, 50, 62, 69, 129, 133, 147 14 24 IS I2,40,45,143 16 4.46. 129, 135 17 18.44.62.73, 77, 106 18 4, 13, 16, 19,23, 133, 138, 147 19 13.33, 39,133, 147 20 21 22.32.83 22 76.83 23 3, 13, 18,38,68 24 14.56 25 4.7. 13, 18. 19.26.36.68. 102, 133,138,147,158 26 4. 13.25.32,100. 102 27 28 29 30 I69 32 8.21.26.83 33 34 4.85, 140 35 36 25,43.109 37 48 38 23 39 7.91 40 I5 41 42 43 36.118 44 68,73,77,106 45 15,54,66 46 8, 16, 134 48 19.54. 59 49 50 1, 2, 13, 19, 44, 53, 107, 117, 133. 135 51 52 53 I, 2,3,50. 149 54 45.48.59

55 56 24 57 6 58

59 60 61 62 63 64 65 66 68 69 70 71 73 74 75 76 77 78 79 81 82 83 84 85 86 87 88 89 90 91 92 95 96 97 98 99

48.54 8 I08 13, 16,17.40.68.73, 106

154 5.70.71 45 7. 13, 18,25,36,44,62, 102 13.51, 150 5.65.70.74. 116 5.7.65.70 13, 17,44,56,62.68,77,106 2.40 1,163 22 17,44,66.73 92,116, 137 41 37.51 21.22. 32.46 34, 140,167 19.48.90, 127 41,48,76,96,116, 170 141 115 19,86,127 39 19,78,91 87.170 13.63

100 26

101 117,162 102 4.25,26,36 103 104 105 106 17.44.62.73 107 50, 102, 117. 123

108 61 109

110 166 111

112 113 114 115

144,149 112, 117,131 5,48 1,3, 152

0-serum

116 117 118 119 120 121 123 I24 125 126 127 128 129 130 131 132 133 134

0-antigens

7, 123 50,76,101,122 121,123 3.48 53, 102,105, 115, 117 101,116, 123 12, 116, I21 11.73 86.90, 128 13, 16,133,135

113, 125. 143

13, 129, 135, 147 46 135 13, 16, 17.50, 129, 133 I36 I37 78 138 7, 18.25,148,150 I39 102 I 4 0 34 141 7.88 142 143 3, IS, 131 144 112,149 145 13 146 147 19,102,133 I48 1, 138 I49 I, 50,53, 112,144 150 1.69. 138 151 I52 3,115 153 I54 1.64 155 156 157 7 158 25 I59 168 160

161 162 163 164 165 166 167 168 169 170

101 75

85 96

a These data have been collected mainly from examinations over many years in the laboratory in Copenhagen. However, some of the data of Edwards and Ewing (1972)are included, especially those reactions that have been detected in both laboratories,even if titres may have been low. Several strong reactions found in their laboratory but not in Copenhagen are also listed. Titres are not recorded, as it is our experience that titres of cross-reacting antigens can vary greatly among sera produced at different times. The reactions recorded are generally those that have been found in serum dilutions not less than five two-fold titre steps below the homologous titre. The papers by Kampelmacher (1959)and Glantz (1968), who have made similar studies, should also be consulted by those who are interested in cross-reacting E. coli 0-antigens.

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B. K-antigens Earlier we gave a definition of K-antigens that restricts the term K-antigen to polysaccharide capsular or micro-capsular antigens (Section V). Originally these antigens were determined by slide or tube agglutination of live or formalin-treated cultures in OK-sera. Tube agglutinations are read after incubation for 2 h at 37°C and then incubated at room temperature overnight. In well-controlled systems, e.g. when a few well-examined strains and sera are involved, slide agglutination is a useful and simple procedure. Furthermore, slide and tube agglutination can be used occasionally for more general K-typing, but the result should be confirmed by a gel precipitation technique such as immunoelectrophoresis (IE) or counter-current immunoelectrophoresis (CCIE) (Section V1.E). CCIE is presently the method of choice for general K-antigen determination (see above). In order to determine if a strain has an acidic polysaccharidecapsule, one can examine the heated extract, as used for CCIE, in two-dimensional gel electrophoresis with Cetavlon in the second dimensional trough (IZlrskov, 1976), but it should be remembered that only acidic polysaccharides precipitate with Cetavlon. However, until now no neutral capsular polysaccharides have been found in E. coli. It is also important to remember that acidic LPS will move towards the anode and will precipitate with Cetavlon. Usually LPS will not move as far towards the anode as the more low molecular weight K-antigen polysaccharides. It is helpful to consult the IE patterns in Fig. 1 for the charge on the 0-antigen when strains with known 0-antigens, but unknown K-antigens, are tested. A few words should be said about antigenic characterization of what is commonly referred to as enteropathogenic serotypes, such as 0:111;H:2 and 0:55;H:6. Traditionally, these strains are detected by slide agglutination in antisera produced by immunization with unheated organisms (OK-sera). In our experience, such strains do not contain special polysaccharide K-antigens (earlier called 0:111;B:4,0:111;K:58(B:4) or 0:111;K:58) (Section V.A.2). In spite of this fact, 0-antisera are most often of no use in the primary slide agglutination test because of 0-inagglutinability. What is determined by slide agglutination in OK-sera is the 0-antigen found in the living unheated cells. A positive slide agglutination in OK-serum should always be confirmed by agglutination titration of the culture boiled to 100°C for 1 h. This step is introduced in order to prevent the many possible false positive results caused by anti-flagellar, anti-fimbrial and other antibodies found in OK-serum. The K-antigens at present established run from 1 to 103. However, since several of the K(B)-antigens have been deleted and some K-antigens have been removed for other reasons the actual number is 72.

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F. ORSKOV AND 1. ORSKOV

C. F-antigens Fimbrial antigens may be tested by slide agglutination when wellcharacterized F-antigens, usually found in special serotype combinations and from special disease conditions such as F:4 (K:88) and F:5 (K:99), are examined. They should be examined in OK-sera, preferably produced with immotile strains using agar plate cultures grown at 37°C. When unabsorbed OK-or OK(H)-sera are used, it is recommended that two different antisera produced with two strains, both containing K:88 (or K:99) but differing in OK-polysaccharides and H-antigens are used. As these antigens are not developed at 18°C (Orskov et a/., 1961) cultures grown at this temperature can be used as controls. For the determination of F:5 (K:99) special media are recommended (Guinee et a / . , 1976, 1977), Smyth (1982) and Craviots et al. (1982) have analysed the CFAI and CFAII antigens. Even'though slide agglutination may be of some help for serotyping of the less well-established F-antigens of E. cofi from extraintestinal disease, we have based most of our examinations on CIE (Orskov et a/., 1980b, 1982; Orskov and Orskov, 1983). The absorption in situ method (Axelsen, 1973) has been particularly suited to these investigations. A few words should be said about direct haemagglutination of erythrocytes from different animals, as this test is widely used for characterization of E. coli strains with fimbrial antigens. A majority of E. coli have the capacity to produce MS type 1 (F:l) fimbriae. Many strains associated with toxigenic diarrhoea1 disease or extraintestinal disease produce in addition MR fimbriae (Section V.A.3). When erythrocytes from different animals are tested with such strains, these agglutinate different erythrocytes, and characteristic HA patterns therefore can be described for the different MR fimbriae (Orskov and Orskov, 1977; D. G. Evans et al., 1979, 1980). Use of a panel of animal erythrocytes is often helpful when characterizing an unknown MR fimbriated E. coli strain. As mentioned above, hitherto found MR fimbrial antigens are not developed when bacteria are grown at 18°C (Orskov et a/., 1961). D. H-antigens

The H-antigens are the serological determinants of the protein flagellar organs found in motile organisms. Usually they are completely destroyed by heating at 100°C for 1 h. Slide or tube agglutination is used for their determination (Section VI.A, 32-35). It is highly important for a successful determination that all the organisms are actively motile. Only some E. coli strains will be actively motile on ordinary agar plates. However, it is usually possible to get

2. ESCHERICHIA COLI

83

well-flagellated bacteria by passing the organisms through semi-solid agar media. We do not use U-tubes (Kauffmann, 1966) but the simpler procedure (Orskov and Orskov, 1975): with a straight wire a 3- to 4-cm long stab is made in an ordinary tube containing 10 cm of semi-solid agar. After incubation at 37°C or at 30°C (Chandler and Bettelheim, 1974; Aleksic et al., 1973) for 20 h, most motile cultures will have spread to the bottom ofthe tube. With a Pasteur pipette a 2- to 3-cm long agar cylinder is withdrawn from an area close to the front edge of the advancing culture. The pipette is withdrawn and one or two drops of its contents delivered to a tube containing broth (5-7 ml), care being taken not to dip the pipette into the broth, as its outside is covered by culture from the surface growth in the semi-solid agar tube. The broth culture is incubated at 37°C (or 30°C) for 5 4 h, at which time motility can be checked under the microscope. If all organisms are actively motile the culture is ready after the addition of 0.5% formaldehyde. Generally, one passage is sufficient. When this is not the case, two or more passages in semi-solid agar may be helpful. Other methods for obtaining motile cultures can be found in Edwards and Ewing (1972). H-agglutination occurs rapidly and is therefore read after incubation in a water bath at 50°C for 2 h, thereby avoiding interference by the late developing 0-agglutination. The typical agglutinates are loose and fluffy but, like agglutinates of other surface antigens, can be strongly influenced in shape and structure by the other antigens present. When strains are consistently sluggishly motile, one may determine the Hantigen by looking for immobilization in H-serum. One drop of motile culture is mixed with diluted H-serum (e.g. 150) and examined microscopically for the typical immobilization picture. Fifty-six H-antigens have been described, but two, H:13 and H:22, have been removed as being C. freundii, and H:50 has been withdrawn (0rskov et al., 1975b). The H-sera are combined in pools for primary H-antigen determination. A recent paper describes the pools and further procedures as used in our laboratory (0rskov and Orskov, 1975). Cross-reactions between E. coli antigens H:l to H:51 can be found in Edwards and Ewing (1972).

VIII. Variational phenomena of importance for serotyping It is not possible to write about serotyping without stressing the great influence that different variational phenomena can have on the outcome of such examinations. A thorough description is given by Edwards and Ewing (1972) and Kauffmann (1966) and only an annotated list of the most important phenomena is given here.

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F. ORSKOV AND 1. ORSKOV

A. Lipopolysaccharide 0-antigens 1. Smooth ( S ) to rough ( R ) variation

Smooth (S) to rough (R) variation might be called 0 ' to 0-variation. This type of variation is caused by mutational events which block different steps in the synthesis of the 0-specific polysaccharide chain or the basal core (Section I1 .B). The typical rough strain is spontaneously agglutinable in physiological concentrations of saline but may be stable in lower salt concentrations. It is devoid of the typical 0-antigen. However, many strains exist that are only partly rough, either because they are a mixture of smooth and rough variants or because the enzymic block is incomplete. Therefore, it is of the utmost importance for 0-antigen determination, and indeed for all serotyping procedures, to select smooth-looking colonies whenever subcultures are prepared. It should be kept in mind that mutants which have lost their 0specificity, but have retained their capsular antigen, frequently look like smooth colonies and are stable in saline suspension when the cells are not boiled, but are auto-agglutinable after boiling. 2. Form variation Form variation, first described by Kauffmann (1940) in Salmonella, is a nonmutational variation involving certain 0-antigenic factors manifested by different degrees of agglutinability (from + t o + +) of single colonies from the same strain in homologous 0-antiserum; the changes are reversible. For details see the description by Kauffmann (1966) and Edwards and Ewing (1972). A probably related phenomenon has been described in E. coli 0:141 by Orskov et al. (1961) (at the time when this form variation phenomenon was described it was considered to be a variation in the K:85-antigen, an antigen which today is regarded as part of the 0-antigen). 3. Phage-induced changes in 0-antigens, antigenic (lysogenic) conversion

This type of variation, which has been thoroughly examined in certain Salmonella serotypes, has not yet been described in E. coli strains (Edwards and Ewing, 1972; Stocker and Makela, 1978). 4 . Influence of composition of growth media

Schlecht and Westphal(l966) showed that an increase in glucose in the growth medium will be followed to a certain extent (0.344%) by a relative increase in the amount of LPS produced by Salmonella. It is our experiencce that the 0-

2. ESCHERICHIA COLI

85

inagglutinability of some E. coli will be decreased, i.e. living bacteria will be more agglutinable in 0-serum when grown on media with such high glucose content (I. Orskov and F. Orskov, unpublished data). 5 . Influence of growth temperature As far as we know, no systematic studies on the influence of growth

temperature on the development of the 0-antigen characters have been carried out. Orskov et al. (1961) demonstrated that two types of 0-antibodies were elicited in rabbits with 100°C treated cultures of E. coli strain G7 (0:8;K:87;H:19) after growth at 18"C, but only one type after conventional growth at 37°C. For a discussion of this special phenomenon, see Orskov et al. (1977). B. K-antigens I. K

+

to K - variation

K to K - variation, earlier sometimes called KO- to 0-variation, is determined by mutational blocks in the synthesis of the polysaccharide Kantigen. It is characteristic that intermediate forms containing decreasing amounts of K-antigen can often be isolated. It is also characteristic that reversion of K- to K+ is a rare event, normally not observed. The K-antigen is one of the factors that can make a strain inagglutinable in 0-serum and therefore, when the K-antigen is lost (K+ to K- mutation), the non-heated strain will often show stronger agglutination in 0-serum. It should be remembered that several other surface components such as flagella and fimbriae may also be responsible for 0-inagglutinability. K + -organisms can often be selected by their inagglutinability in 0-serum, but one can also take advantage of the colony morphology since the K+ colonies are generally more opaque and dome-shaped than the corresponding K- colonies. When it is important to select K- forms it is advantageous to let agar plates with wellseparated colonies stand for several days at 21°C (room temperature). The colonies will continue to grow and often produce acapsular mutant sectors which are easy to isolate. +

2. Form variation

Orskov et al. (1971, 1979) described a variational phenomenon in the K:lantigen. In the same K:l- antigen culture two types of colonies with different serological reactivity, K:l+ and K:l-, were found. Either colony form will,

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F. ORSKOV AND 1. ORSKOV

upon subcultivation to single colonies, give rise to both K:l+ and K:lmutants. Chemical analysis has shown that the K:l+ form is an 0-acetylated form of the same polyneuraminic acid polysaccharide which is non-0acetylated in the K:l- colonies. 3. Influence of composition of growth media

No systematic studies have been carried out, but it is a common experience that capsular development may vary on different media and also that the capsule is best developed on rich media with a high sugar (glucose) content. For the special polysaccharide K-antigen, the M-antigen (Section V,A,5), Anderson and Rogers (1963) have shown that solid media with concentrations of solutes giving a high osmotic pressure (either single or different mixed simple salts or sucrose will do) will cause many, normally non-mucoid, Enterobacteriaceae to produce abundant amounts of mucous substance. 4 . Influence of growth temperature

Orskov et al. (in preparation) investigated the development of polysaccharide K-antigens of E. coli and found that many of these are less well, or not at all, developed at 18"C, whereas others are produced at both 18 and 37°C. The polysaccharide M-antigen found in many E. coli can be developed at 37"C, but is better at room temperature.

C.F-antigens I . Plasmid-determined mannose-resistant ( M R ) F-antigens ( a ) F to F - variation. The plasmid-determined F-antigens, e.g. E. coli F:4(K:88) antigen, can be lost when the plasmids are lost and can only be acquired again after reinfection with the plasmid. +

( b ) Influence of growth media. The E. coli F:5- (K:99) antigen, often associated with strains from calf and lamb diseases, can be difficult to detect because of the richly developed polysaccharide capsules often present in such strains. Experience has 'shown that some media are better than others for demonstration of this antigen (Orskov et al., 1975a;GuinCe et a!., 1976,1977). ( c ) Influence ofgrowth temperature. The E. coli F:4 (K:88), F:5 (K:99) and other MR F-antigens are not developed at 18"C, a characteristic which can be very useful for serotyping (Orskov et al., 1975a). This fact is also important when agar plates have been kept at room temperature for some time following

2. ESCHERICHIA COLl

87

incubation; under such circumstances separated colonies will continue to grow and the temperature-sensitive F-antigens will not be formed. However, it will frequently be possible on the same plate to detect these antigens in the areas of confluent growth, where little subsequent growth has occurred. Glucose and some other carbon sources will repress the development of F:5 fimbriae (Isaacson, 1980).

2. F:l-antigen mannose-sensitive ( M S ) type I j m b r i a e Development of F: 1-antigen depends on growth conditions (Duguid and Campbell, 1969). It is, however, our experience that it is not easy to obtain cultures totally devoid of F: 1, even when following the directions given in the literature. For further details and references see Edwards and Ewing (1972).

D. H-antigens 1.

H+

to H - variation

H+ to H- variation is a mutational event which changes a motile strain into a non-motile one, most often caused by the loss of the capacity to form flagella. Unfortunately, no simple routine technique exists for the selection of Hmutants.

2. Phase variation Phase variation has recently been described between H:3 and H:16 and H:4 and H:17, Ratiner (1982), thus demonstrating that E . coli H-antigens are not always monophasic as hitherto believed. 3. Influence of growth media Serotyping of flagellated strains involves two particular problems. One is the growth of sufficiently motile strains so that abundant flagellar antigens are present for dependable H-antigen determination and good production of antiH-antibodies. This may be solved by procedures such as passage through semi-solid agar (Section VI1.D; Edwards and Ewing, 1972). The other problem involves the prevention of developing flagella which will disturb the serological examination of other surface antigens when live or formalintreated cultures and OK(H)-antisera are employed. Different chemicals have been used as additives to agar media to stop swarming and the development of flagella, e.g. 0.1% phenol or short ethanol treatment of the agar surface. We have been satisfied with the addition to the agar medium of 0.1% of the anion-

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F. ORSKOV AND I. ORSKOV

active detergent Pril or 0.01%of a similar substance (Maranil). Motile E. coli cultures grown on such plates generally show no H-agglutination.

E. M-antigens The polysaccharide M-antigen (colanic acid) found in many E. coli strains is often not developed when grown at 37°C. However, when left at room temperature (20"C), many of these strains will develop mucoid mutants that' will grow as mucoid cultures at this lower temperature. Some strains will also grow in mucoid cultures with well-developed M-antigen at 37°C.

IX. Antisera A. Production of antisera 1 . General procedures In general, the production of E. coli antisera in rabbits is simple. The methods used are similar to those used for other Enterobacteriaceae, although they may vary somewhat from author to author (Kauffmann, 1966; Edwards and Ewing, 1972).On the whole, the sera obtained against 0-and polysaccharide K-antigens are equally useful for bacterial agglutination and gel precipitation. We give here the main guidelines for production of 0-,K- and H-antisera with whole-cell vaccine. An important first step in antiserum production is to select suitable strains, culture media and incubation conditions in which the relevant antigen is well developed. Two or more rabbits are injected in their marginal ear vein with the antigen preparation, usually five or six injections with intervals of three to four days. The rabbits are given increasing doses (e.g. 0.25,0.5, 1.0;l.S and 2.0 ml) of the antigen preparation containing 5 x 10' to 5 x 10" bacteria/ml-' and exsanguinated a week after the last injection. If the antibody titre is not satisfactory after the last injection no further injection is recommended. To preserve the antisera we add both merthiolate and chloroform at a final concentration of 0.01% and 1% respectively. Some research workers use equal volumes of glycerol -a preservation method which cannot be recommended if the antiserum is to be used in gel precipitation tests. If it is important to avoid additives the sera should be kept at -20°C or lower. 2. 0-antisera

To produce 0-antisera it is important that the antigen culture is smooth (S)

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89

and not rough (R) or on the verge of becoming R because presence of Ragglutinins in the antisera can lead to confusing cross-reactions. Care must be taken to inspect and examine single colonies for smooth appearance and for stability in suspension after heating to 100°C. A rough culture may look smooth on an agar surface if it has retained a K-antigen and this fact may only be disclosed by autoagglutinability of the culture after boiling. 0-antigens are generally immunogenic after heating to 100"C,in contrast to H- and K-antigens, except K(A)-antigens. Therefore, 0-antisera are prepared against cultures heated to 100°C for 2.5 h, either as a suspension from an agar surface or as a broth culture incubated at 37°C for 18-20 h. Higher doses than usual can be given if the LPS (endotoxin) solubilized by heating is removed by centrifugation. Cultures with K-antigens of heat-stable immunogenicity, K(A)-antigens, should be autoclaved at 121°C for 2 h to prevent the production of K-antibodies. When possible, however, K- forms should be selected for 0-antiserum production. The boiled 0-antigen preparation may be preserved with formalin and then it can be used for the whole series of vaccinations. 0-antisera may also be produced according to the method of Roschka (1950). In this method the sediment of a heated bacterial suspension is treated with alcohol and acetone. For details see Edwards and Ewing (1972). Schlecht and Westphal(l967,1968a,b) have made a systematic study of the influence of various parameters on the production of Salmonella 0-antibodies (agglutinating and precipitating) which indicates that maximum 0-antibody titres are already developed after the first three immunizations. Recent observations by Orskov et a / . (1981) have confirmed this finding when three different E. coli strains were examined. 3. OK-antisera

For OK-antiserum production rabbits are immunized with unheated cultures. Generally, a fresh suspension from an agar plate culture is prepared for each inoculation. Prior to immunization the culture should be examined for the presence of K-antigen, if its identity is known. If only the 0-antigen is known, colonies with the least possible agglutination in 0-antiserum should be selected. Although such 0-inagglutinability is no sure proof of K-antigen presence, it is a useful aid in selecting K+ colonies. Another way of detecting clones with high polysaccharide K-antigen is to examine extracts of selected single-colony cultures for their ability to precipitate with Cetavlon in IE, as described in Section VI1.B (Orskov, 1976). If available, non-motile cultures are preferable. Many authors recommend that formalin-killed antigen preparations be injected at the beginning of the immunization course and live suspensions

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F. ORSKOV AND I. ORSKOV

thereafter. The antisera thus produced may have higher titres than those obtained using formalin-treated suspensions alone. However, it should be emphasized that live vaccine may be difficult to administer in a completely aseptic manner and, unless special precautions are taken, live bacteria may spread in the animal house. Since OK-antisera are produced with non-heated cultures, they contain antibodies additional to those required. They always contain O-antibodies and, in case of motile strains, varying amounts of H-antibodies, even if Prilsupplemented plates are employed for cultivation (Section VII .D.3); therefore the designation OK(H) is frequently used. H-antibodies may be disturbing, but no certain method is known which can prevent some formation of H-specific immunoglobulins. However, the antibodies causing most trouble are those elicited by inadequately defined heat-labile bacterial constituents, e.g. different types of fimbriae (Section V.A.2), and other proteinaceous surface structures (Orskov et a / . , 198 1). 4 . F-antisera

For the determination of F-antigens by gel precipitation we have found that a larger number of immunizations in two series some weeks apart can increase the amount of precipitating antibodies to such antigens. 5 . H-antisera

For production of H-antisera preparations of actively motile cells inactivated by formalin should be used as antigen. Insufficient motility may be improved by passage through semi-solid agar. The preparation can be a young broth culture or a suspension from an agar plate which must be freshly prepared and moist before use. Aleksic et a / .(1973) recommend suspensions prepared from semi-solid agar plate cultures grown for not more than 16 h at 30°C. By this change from the usual 20 h at 37°C they obtained H-sera with four times higher titres. 6 . Pooled and polyvalent antisera

When it is desirable to use several antisera simultaneously a pool consisting of a mixture of single antisera can be used. As the titre of each serum is decreased by this pooling, it may be preferable to produce polyvalent antisera. The antigens of the mixture are prepared as for monovalent antisera, but the injection volume is increased, at least after the first few injections, and the course of the immunization is prolonged to obtain higher amounts of antibodies. If a trial bleeding shows a low titre for a particular component the

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amount of that component is increased. If no improvement is obtained a monovalent antiserum of this specificity should be added to the polyvalent antiserum.

B. Absorbed antisera Absorption of antisera is carried out: (1) if unwanted antibody specificitiesare present, for instance 0-antibodies may be removed from OK- or H-antisera (2) if a factor serum is required, e.g. sera with specificity against a particular determinant or (3) if a closer identification is required. The antiserum to be absorbed will generally be used diluted 1 :5 or more, or undiluted if it is to be used in gel precipitation. Most workers employ suspensions of agar plate cultures for all kinds of absorptions. 0-antibodies are usually removed by heated cultures (boiled for 1-2 h). Unheated cultures can be employed but other antigens in such cultures may prevent the binding of 0-antibodies. For absorption of K- or H-antibodies unheated live or formalin-treated cultures are used. Antibodies against polysaccharide K-antigens, however, are most often also removed by a boiled absorbing culture, whereas this is not the case with H-antibodies. For 0-antibody absorption we use growth from evenly inoculated plates which are suspended in saline, heated to 100°C for 2 h and centrifuged. Antiserum is mixed thoroughly with the cells. For antiserum dilutions phenoltreated (0.25%) physiological concentrations of saline are used. The mixture is kept at 37°C for 1-2 h and usually stored in the refrigerator overnight. Then the bacteria are removed by centrifugation. Absorptions of K- and Hantibodies are performed in the same way, except that the culture suspensions are not boiled but killed by addition of formalin (0.5%) or the bacteria are suspended directly in the antiserum. Plates for the H-absorbing culture must be inoculated with an actively motileculture and, the plates not too well dried. When unheated cultures are used for absorption the absorbed antisera should be preserved, e.g. by adding a small amount of chloroform, particularly if they are not diluted in phenol-treated saline. For all absorptions a two-step procedure is recommended, as this is more effective than a single absorption. The same amount of a cell suspension as for a single absorption is divided into two tubes before centrifugation. Antiserum is added to the sediment of one tube, centrifuged the next day and mixed with the sediment of the other tube which has been kept in the cold after the first centrifugation. The amount of antigen used for each absorption must be sufficient to remove all homologous antibodies. This amount varies with different antisera and different cultures. In our laboratory we use the growth from ten large (14-

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cm diameter) thickly poured broth agar plates as a standard amount to absorb the content of O-agglutinins in 10ml of a 1:lO dilution of antiserum. This process generally removes all homologous O-agglutinins, but in many cases less antigen would suffice. For H-antibody absorption the same amount of growth is added to lOml of H-antiserum, but the antiserum is diluted 1:lOO. For absorption of K-antibodies no rule is given regarding the required amount of absorbing culture, as this varies considerably with different Kantigens. However, smaller amounts than the growth from ten plates (14-cm diameter) - often from one or two plates -will generally suffice to remove the antibodies against polysaccharide K-antigen in 1 ml of serum.

X. Cross-reactions

A. O-antigens 1. Cross-reactions within the E. coli group

Table IX shows the cross-reactions between O-antigens in E. coli test strains. As mentioned in Section VI1.A a high number of cross-absorbed O-antisera are necessary for an accurate O-antigen diagnosis. However, it is important to remember that even using these absorbed antisera it is not possible to tell whether an unknown strain assigned to a certain O-group is really identical with the test strain of that O-group. Only serum production with the strain in question, followed by mutual cross-absorptions, can give the definite answer. The results shown in Table IX are based primarily on results from our laboratory, but they also include data from investigations of Edwards and Ewing (1972). 2. Cross-reactions between O-antigens of E. coli and Shigella, Salmonella, Klebsiella and other Enterobacteriaceae

Many O-antigenic cross-reactions have been found between E. coli and Shigella O-antigens (Table X ) . Ewing et al. (1952) have gathered data which show that only two Shigella serotypes are not related to one or more of the E. coli serotypes 0:l to 0:148 and, furthermore, that a number of subjudice Shigella serotypes are related to E. coli O-groups. The O-antigen relationships between E. coli 0 :149 to 0 :163 and Shigella have been reported by Rowe et al. (1976) and Toledo et al. (1980). These findings stress the well known close relationship between the two genera. A number of enteroinvasive E. coli (EIEC), which can give rise to dysentery-like disease, have O-antigens closely related to Shigella O-antigens (Table X ) . Kauffmann (1954) and Frantzen

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93

(1950) have described 0-antigenic cross-reactions between E. coli and Salmonella, and Refai and Rohde (1975) have carried out similar investigations with 142 E. coli 0-antigenic test strains in available Salmonella and Arizona sera. Table XI shows the results of Refai and Rohde together with TABLE X Cross-reactions between 0-antigens of Escherichia coli from dysentery-likedisease and Shigella"

E . coli

Shigella

O:28ac (Kattwijk) 0:112ac (Guanabara) 0:124 0:136 0:143 0:144 0:152

S. boydii 13 S. dysenteriae 2, identical S. dysenteriae 3, identical S. boydii 8, identical S. dysenteriae 10 Not examined

From Edwards and Ewing (1972). Other strong 0-antigen cross-reactions between E . coli and Shigella are 0:32/S. boydii 14,0531s. boydii 4,0581s. dysenteriae 5, 0:79/S. boydii 5, 0:87ab/S. boydii 2, 0:lOSabS. boydii 1 1 , 0:112ab/S.boydii 15, 0:129/S.Jexneri 5, O:SS/S.Jexneri 4b and 0:1671s. boydii 3. TABLE XI Cross-reactions between 0-antigens of Escherichia coli and Salmonella

E. coli

Salmonella

0:l

0 :42, 0 :55

0 :2 0 :6 0:15 0:21 0 :23 0 :44,:62,0 0 :68 0 :70,0 : 7 3 , 0 :99 0:106 and 0:129 055 0 :75 0 :85 0 : 8 6 , 0 :90 0:111 0:132 0:134

0:40,*40,

I

,

0 :59 0 :38 051 0 : 6 , 14 0:50,.50,+50, 0:11 0:17 0 :43 0 :35 0:17 0 :36

From Kauffmann (1954). Frantzen (1950) and Refai and Rohde (1975).

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F. ORSKOV AND I. ORSKOV

earlier results in a condensed form. Cross-reactions between 0-antigens of E. coli and Klebsiella have been described by Kauffmann (1949) and 0rskov (1954) (Table XII). Most of these cross-reactions were reciprocal but identity was not found in all cases. TABLE XI1 Cross-reactions between Escherichia coli and Klebsiella pneumoniae 0-antigens E. coli

K . pneumoniae

0:19ab 0 :9 0 :20 0 :8

0:1, identical to0:19b 0 :3, identical 0 :4, strong relationship 05,identical

3. Cross-reactions between E. coli 0-antigens and 0-antigens outside the Enterobacteriaceae group Winkle et al. ( 1972) described 0-antigenic relationships between Vibrio cholerae and E. coli, Salmonella and Citrobacter strains (see also Kilian, 1978). Springer (1967, 1970, 1971) has analysed the cross-reactions between E. coli 0:86-antigen and human blood group B-antigen. Reports also exist which describe cross-reactions between E. coli antigens and surface antigens of mammalian cells (Drach et al., 1971; Hirata et a/., 1973; Holmgren et al., 1971), and Hanson et al. (1979) have described cross-reactions between Tamm-Horsfall protein and E. coli LPS.

B. K-antigens 1 . Cross-reactions between E. coli K-antigens

Many E. coli strains will agglutinate on slides in a great number of E. coli OKantisera and many kinds of surface antigens may participate in these reactions. Fewer cross-reactions are found by gel precipitation, such as double diffusion in gel or CCIE. Cross-reactions between polysaccharide K-antigens have been found among' the following K-antigens: K: 18-K:22-K:1W, K:13-K:20-K:23, K:53-K:93, K:54-K:96, K:16-K:97, K:37-K:97, K:12-K:82, K:2-K:62 and K:7-K:56. K:2 is so closely related to K:62, K:7 to K:56 and K:12 to K:82 that K:62, K:56 and K:82 should be deleted in favour of K:2, K:7 and K:12 respectively. The relationships between K:13-K:20-K:23 and K:2-K:62 have been reported in detail (Vann et al., 1983; Larsen et al., 1980).

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2. Cross-reactions between E. coli 0-antigens and K-antigens

The increased significance of K-typing has increased the importance of these cross-reactions. Identity has been described between an unnumbered 0antigen in strain “145” and the polysaccharide K:87 found in strain G:7 (0:8;K:87, K:88;H:19) (0rskov et al., 1961). Furthermore, an antiserum raised with a boiled culture grown at 37°C of strain G:7 contained both agglutinins and precipitins against 0:8 but not against the antigen with K:87 specificity. However, an 0-antiserum prepared with a boiled culture of G:7 grown at 18°Cdid contain agglutinins against the K:87 antigen. The test strain of K:87 was formerly strain “145”, but it is now an F:4-(K:88) negative mutant of G:7 called D:227 (0:8;K:87;H:19). Examples of the relationship between 0- and K-antigens are crossreactions between the following: K:31 and 0:120, K:9 and 0:104, K:44 and 0 5 3 and K:45 and 0:74. All the cross-reacting 0-antigens mentioned are acidic LPS and their presence may confuse the interpretation of K-antigen determination by CCIE because they move towards the anode. Strains such as the test strain for 0:120 may have a K-antigen in addition and will thus give two precipitation lines in CCIE. It is important, therefore, to consult the list of acidic 0-antigens (Fig. 1) before labelling an unknown strain. Probably several not yet described relationships between acidic 0-antigens and Kantigens -- usually K(A) will be found. ~

3. Cross-reactions between E. coli K-antigens and polysaccharide Kantigens outside the E. coli group Morch and Knipschildt (1944) described cross-reactions between some E. coli strains having K(A)-antigens and capsule antigens 9 and 23 of Streptococcus pneumoniae. Unfortunately, these strains have not been kept. Heidelberger et a / . (1968) described relationships between E. coli K:30-polysaccharide and pneumococcal polysaccharide SnII and SV and E. coli K:42 and SnXXV, as well as K:85 and SII and SV. Niemann et a / . (1978) found cross-reactions between E. coli K:42 and Klebsiellu K:63. Grados and Ewing (1970) and Kasper et a / . (1973) demonstrated cross-reactions between E. coli K:l and meningococcal group B-polysaccharide. Bradshaw et ul. (197 1) used agar plates containing anti-Huemophilus influenme type b serum to detect bacterial colonies producing cross-reacting antigens. Schneerson et a / .( 1972) described an E. coli strain (F:147=0:75;K:lOO;H:5) with a polysaccharide K-antigen closely related to H. injluenzue type b capsular antigen. Cross-reactions between the capsular antigen of Neisseriu meningitidis group C and the Kantigen in strain Bos 12 (0:16;K:92;H-) was reported by Robbins et ul. (1972), who also described cross-reactions between S. pneumoniue type 3 capsule and E. coli K:7 (Robbins et a / . , 1975).

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Since some of such cross-reactions most likely play an important role in the non-specific protection from virulent capsulated invasive bacteria, these host-parasite relationships have been studied intensively, reviewed by Robbins et al. (1975). C. H-antigens

Cross-reactions between E. coli H-antigens are listed in Table XIII. Crossabsorbed sera are used for definite H-typing. Cross-reactions to flagellar antigens of other Enterobacteriaceae have not been recorded. TABLE XIII Cross-reactions between Escherichia coli, H-antigens H-antigen

H-serum

1 2 4 5 6 7 8 11 12 21 24 27 29 30 32 33 40 40 43 51

12 51 17 29 11 8 40 21 1 11 27 24 38 32 30 43 8 33 33 2

Significant H-cross-reactions are based on examinations in our laboratory ; see also Edwards and Ewing '(1972).

D. F-antigens This field is new and expanding, and only a limited number of strains have been examined as yet. The F:l fimbriae could be considered as a common

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antigen (Section V.A.3) consisting of a series of non-identical but related antigens with a common function. The exact antigenic relationship between E. coli F:l antigen and between type 1 antigens outside the E. coli group has not been worked out (Duguid and Campbell, 1969). Serological analysis of the plasmid-determined F:4 (K:88) antigen has shown that this is a heterogeneous group of related antigens (0rskov et al., 1964; Guinee et al., 1972). Detailed serological analyses of F:3 (CFA:2) have been published by Cravioto et al. (1982) and Smyth et al. (1982). 0rskov et al. (1980b, 1982a,b; 0rskov and Orskov, 1983) have described a series of new F-antigens in E. coli from urinary tract infections..These studies are necessarily based on a limited number of strains from a limited geographical area.

XI. Association of serogroups and serotypes of E. coli with pathological conditions

Only little is known about the distribution of serotypes. Most knowledge is collected on strains from man and domestic animals in temperate climates, whereas we know little about serogroups and serotypes in warmer regions. It is not possible to state whether there are significant differences in 0-group prevalence between different animal groups. It has been expressed that any difference in this respect is not likely between man and cattle (Hartley et af., 1975). Many 0-groups are the same, but we would hesitate to draw this conclusion until further examinations have been carried out. It is surprising for example that 0:4- and 0:6-antigens, which are so common in man, hardly occur among frequent cattle strains. When the prevalent 0-groups from diseased animals and man are compared it is evident that most of the strains associated with diarrhoea in piglets and human infants are different, even though the two diseases have much in common. A. E. coli from human sources Table XIV summarizes the findings from extra-intestinal infections including only the most common 0-antigen groups. The percentages for the single 0groups differ, but it is probably true that with the use of those 0-sera that correspond to the 0-groups listed, it should be possible to determine the 0antigen of at least half of all coli strains from urinary and other extra-intestinal infections. It should be noted that these common 0-antigen groups are the

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TABLE XIV Escherichia roli 0-groups from extra-intestinal infections in man

0-groups” (1) Urinary tract infectionb

(2) Septicaemia (3) Other (4) Neonatal meningitis‘

(5) Faeces (healthy adults and children)

1,2,4,6,7,8,9,11,18,22,25,62,75 1,2,4,6, 7, 8,9, 11, 18, 22,25, 75 1,2,4,6,8,9,11,21,62 1 , 6, 7, 16, 18, 83 1, 2,4,6,7, 8, 18,25,45, 75,81

References: ( I ) Ewing and Davis (1961). Turck and Petersdorf (1962). Gruneberg rr ul. (1968), Ganguli (1970). Mabeck et a/. (1971). Nimmich et a/. (1975). Lidin-Janson et a/. (1977a), Sietzen (1979). (2) Ewing and Davis (1961), Golubeva et a / . (1975), Orskov and Orskov (1975). (3) Vahlne (1945), Ujvary (1958), Ewing and Davis (1961). (4) Sarff et a / . (1975). ( 5 ) Kauffmann (1944), Vahlne (1945), Sears (1950), Ujvary (1958), Rantz (1962), Wiedemann and Knothe (1969). Lidin-Janson et a/. (1977b). Since the prevalence rates have been compiled from many different investigations the 0-groups are listed numerically. A limited number of polysaccharide K-antigens are found to dominate among strains from urinary tract infections: K:l,K:2,K:5,K:12and K:13.Thesame K-antigensarealso frequently found among stool isolates from healthy persons (Vahlne, 1945; Mabeck et a/., 1971; Kaijser et a / . , 1977). The prevalent 0-groups listed here are characterized by having the same K-antigen, K :1, when found in this disease.

same as those prevalent in the normal intestine but the percentages for faeces are lower. Similarly, only a few polysaccharide K-antigens (K: 1, K:2, K:3, K:5, K:12 and K: 13) are found with high frequency in urinary tract infections and other extra-intestinal infections. It is likely that invasiveness is parallelled by a further selection of strains which are already present in the intestine (Vahlne, 1945; Mabeck el al., 1971; Kaijser et al., 1977). Recently, it has been shown that strains from severe urinary tract infections often have adhesive fimbriae (F-antigens). Some 0:K:H-serotypes, e.g. 0:6;K:2;H:1 with F:7 fimbriae (0rskov rt a/., 1980b) and 0:18;K:5;H:I with F8 antigen, can be characterized as pyelonephritic strains (0rskov et a/., 1982a,b) (Section V.A.3). Today most authors agree that generally the same 0-antigen groups are present in the normal healthy intestines and in extraintestinal infections. However, in the discussion about the involvement of E. coli in urinary tract infections, two seemingly different views have been expressed. One is named the “special pathogenicity theory”, stating that some serotypes are more frequent in the urinary tract than in the intestine due to the special

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pathogenicity of such strains. The other, called the “prevalence theory”, says that the serotypes found in urinary infections represent the most prevalent types in the gut (Griineberg et al., 1968). Unfortunately, comparable data that could make possible a definite choice between the two theories are still missing. Some authors have indicated that differences in 0-antigen group distribution exist, e.g. between Europe and the United States and even within different areas in London (Griineberg and Bettelheim, 1969). Others have suggested that prevalence rates of certain 0-antigen groups could vary with time in the same area (Mabeck et al., 1971). Even though such differences occur they can probably be explained by the use of different techniques and sera. The overall picture of a similar E. coli group prevalence in Western developed countries in temperate climates is not disturbed by these discrepancies. The results of serotyping carried out on different types of diarrhoea1 diseases in man are summarized in Table XV. The left column lists the most common enteropathogenic serotypes associated with infantile diarrhoea (EPEC). They were all originally isolated from severe outbreaks of infantile diarrhoea in instjtutions and are also today most often found in such places. Only the 0-antigen groups have been listed, i.e. 0:26 and 0 5 5 , since complete serotyping of such strains unfortunately has been carried out to a very limited extent. However, it was found (Ewing et al., 1963; Orskov, 1956) that some OH-antigen combinations, e.g. 0:55;H:6, 0:111;H:2 and 0:86;H:34, are much more frequently associated with clear-cut outbreaks than others. The LPS of these strains are neutral, and they have no acidic polysaccharide Kantigens (Orskov and Orskov, 1972), which seems to be in accordance with their non-invasive character. Cravioto et al. (1 979) have recently described adhesive factors in traditional EPEC serotypes. Such strains were adhesive to HEp-2 tissue culture cells in a higher percentage than control strains. The factor(s) responsible is (are) not yet known. Some evidence has also come forward which shows that EPEC strains may produce a special enterotoxin (Levine et al., 1978; Klipstein rt al., 1978; Scotland et al., 1980). A few years ago there was a tendency to reduce the importance of these special EPEC serotypes because only a few of them produced the LT or ST enterotoxins found in enterotoxigenic E. coli. Presumably this trend will stop in the light of the new development described above. It should be remembered that E. coli serotyping has been of great importance for the elucidation of many serious outbreaks and epidemics of infantile diarrhoea (Sack, 1976; Rowe, 1977; Wachsmuth, 1980). On the other hand, it is of course true that plain serotyping of the EPEC serotypes, particularly when limited to 0typing, is probably of little value for the examination of sporadic cases of infantile diarrhoea.

TABLE XV 0-groups, OH- and OKH-types from Escherichia coli enteropathies'

Diarrhoea in adults and children Infantile diarrhoea EPEC 0:20,0:26, 0:44,0:55, 0:86,0:111,0:114,0:119, 0:125,0:126,0:127, 0:128,0:142, 0:158

ETEC 0:6;K15;H16, 0:8;K:40;H:9, 0:8;K:47;H-, 0:8;K:25;H:9, 0:1 1;H:27, 0:15;H:ll, 0:20H-, 0:25;K:7; H:42, 0:25;K:98;HP, 0:27;H:7, 0:27;H:20, 0:63;H: 12, 0:73;H:45, 0:85;H:7, 0:78;H: 11, 078;H:12. 0:114:H:21. 0:I15:IH.Sl). 0:13.8.A.7. 0.17R.w.17 0 :128 ;H : 2 1 , 0 :139;H 28, 0 :148 ;H :28,0 :149;H : 4 , 0 :159 ;H:4, 0:159;H:20,0:159;H:34, 0:166;H:27,0:167; H:5,0:168; H:16,0:169;H-

EIEC O:28ac, 0:112,0:124, 0:124,0:136,0:143, 0:144,0:152,0:164 0:167

EPEC, enteropathogenic E. coli. The 0-groups are listed; however, only a limited number ot0;H-types have been shown to have an association to infantile diarrhoea(Section V.D).ETEC, enterotoxigenic E. coli. The data presented are primarily from Orskov and Orskov (1980). See also Gross er a / . (1978) and Merson er a/. (1979). Earlier designations: 0:166=OX8 and 0:169=OX2. EIEC, enteroinvasive E. coli. Square brackets means that non-motile variants exist.

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Table XV also lists OKH-serotypes frequently found among enterotoxigenic E. coli (ETEC) isolated from adults and children mostly in warm climates. Some such serotypes have been isolated from cases of travellers’ diarrhoea (Orskov and Orskov, 1980). ETEC strains may also be the cause of food and water-borne diseases (Sack er ul., 1977). 1 A third group of E. coli serotypes is associated with dysentery-like disease, i.e. the enteroinvasive E. coli (EIEC) strains (Table XV). In geographical regions where S. dysenteriue is a common cause of diarrhoea these E. coli serotypes are also found to be associated with dysentery. By usual laboratory procedures they will be labelled E. coli, but most of them have close 0antigenic relationships to different Shigellu serogroups (Table X). They have the same invasiveness as Shigellu strains, are positive in the Sereny test (Sereny, 1967) and cause primarily a disease of epithelial layers of the colon. The serotypes from infantile diarrhoea and the Shigellu-like EIEC serotypes are rare in healthy intestines. The 0-antigen groups in strains from enterotoxin-determined diarrhoea are not rare, but some of them represent well-defined combinations of special 0-,H- and K-antigens and have special fermentation patterns that are not common in strains isolated in developed countries (Orskov er a / . , 1976; Orskov and Orskov, 1977; Scotland et a/., 1977). Strains associated with diarrhoea in adults and children which do not fit into the three groups of Table XV have also been described, e.g. 0:150;H:10 (Golubeva et ul., 1976; Steinruck et ul., 1980).

B. E. coli from cattle, pig and poultry When Jensen in 1893 described the aetiology of diarrhoea and septicaemia in calves it was the first attempt to associate E. coli bacteria with a special disease syndrome. Table XVI summarizes the knowledge about the association between E. coli 0-antigen groups and certain diseases in cattle. As in the previous similar tables in this paper, results from different investigations have been combined because a general prevalence of some frequent 0-antigen groups is apparent. The differences in prevalence in various reports may be real but can, most simply, be explained by differences in the selection of strains and in the typing techniques. It is apparent from Table XVI that few examinations of the E. coli 0-antigen groups in healthy calves have been carried out. If the 0-antigen groups from sick and healthy calves are compared only few significant differences in 0-group prevalence can be found, the only important one being

102

F. ORSKOV AND

1. ORSKOV

TABLE XVI

Some Escherichia coli 0-groups and serotypes from bovine disease (1) Diarrhoea and

septicaemia (2) Enterotoxigenic diarrhoea (ETEC) (3) Mastitis

0 :1, 0:2, 0:8, 0 :9, 0:15, 0 :20, 0 :26, 0 :35, 0 5 5 , 0:73, 0:78,0:86,0:87,0 :lOl, 0 :114,0:115,0 :117,0:119,0:137 0 : 8 ; K:25; H - , 0 : 8 ; K:28; [H:19], 0 : 8 ; K.85; H - , 0 : 9 ; K:30; H-, 0 : 9 ; K:35; H-, 0 : 9 ; K:103; H - , 0 : l O l ; K.28; H - , 0 : l O l ; K:30; H (usually K :99+ and “STonly”) 0:2,0:8,0:21,0:81,0:86

References: (1) Wramby (1948), Bokhari and Orskov (1952), Fey (1957a, b), Dam (1960), Gay (1965), Sojka (1965), Soderlind (1965). Vallee e t a / . (1970). (2) Myers and Guinee (1976), I. Orskov and F. Orskov (unpublished data). (3) Fey (1955), Linton et a/.(1979). Square brackets indicate that nonmotile variants exist.

that Wramby (1948) never found 0:78-antigens in healthy animals. Several strains from neonatal calf diarrhoea produce enterotoxin, usually ST, and carry adhesive fimbriae, e.g. F:5 (K:99) and F:6 (987). Table XVII shows the E. coli 0-antigen groups that are frequently found in young pigs. For an extensive review see Sojka (1965). The strains from diarrhoea, particularly in newborn piglets, very often carry the fimbria-like antigen F:4 (K:88). Most of the 0-antigen groups listed TABLE XVII Escherichia coli serotypes from pig disease

Serotype Enterotoxic enteropathy

Enterotoxaemic enteropathy (oedema disease)

0 : 8 ; K:87; H:19 0 1 9 ; - 135; H 0 : 9 ; K:103; H 0:20; K:101; H 0:45; H 0:lOl; K:30; H 0:138; H:14 0:141; H:4 0:147; H:19 0:149; H:10 0:157; H:19

Fimbrial antigen“ F :4 (K :88) F:5 (K:99) F :6 (987) F :6 (987) F :4 (K :88) F :5 (K :99) F :4 (K :88) F :4 (K :88) F :4 (K :88) F :4 (K :88) F :4 (K :88)

0 :138;H 0:139; K:82; H:l 0:141; H:4 ~

~~

From Kelen et a / . (1959), Kramer (1960), Sojka et a/. (1960), Orskov et a/. (1961), Sojka (1965). Sweeney (1970). Soderlind (1971). Dam and Knox (1974), Moon (1974). Isaacson et a/. (1977). a Present numbers in parentheses, F-numbers are those proposed in this paper (Table VII).

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in Table XVII are usually found as well-defined serofermentative types, i.e. 0:138;H:14 (earlier 0:138;K:81;H:14), with a typical fermentative pattern independent of their geographical place of isolation. Among E. coli from poultry 0:2;K:1 in most reports is the dominant OKtype from septicaemia, and next in frequency are 0:78 and 0:l;K:l (Table XVIII). For an extensive review see Sojka (1965).

TABLE XVIII Some Escherichia coli 0-groups from poultry (1) Coli septicaemia (e.g. septic 0 : 1 , 0 : 2 , 0 : 8 , 0 : 7 1 , 0 : 7 3 , 0 : 7 8 pericarditis, air sacculitis) 0:8,0:9,0:16 (2) Hjarre’s disease, coli granuloma disease, mucoid strains 0 : 3 , 0 : 8 , 0: 9 , 0 :18,O :44,:0 7 7 , 0:83,0 :96, (3) Faeces 0:103,0:114 References: (1) Harry (1964). Heller and Perek (1968), Takahashi and Miura (1968), Heller and Smith (1973). (2) Wramby (1945). (3) Howe er a / . (1976).

XII. The bacterial clone concept in epidemiology and taxonomy Perhaps the best known enterobacterial serotype is Salmonella typhi. For a long time it has been known that many other phenotypic traits, also quite unusual ones such as tryptophan dependence, were closely associated with this special combination of surface antigens. Probably most bacteriologists also agree that epidemiological connections between single cases and outbreaks could be traced across borders and from continent to continent. Likewise, nobody would doubt that the movement of a cholera pandemic across the continents was caused by the progeny of the same bacterium which was disseminated through human cholera cases. It is also true that when an E. coli serotype, with a characteristic biotype like 0:11 1;H:2, was found in a certain nursery at a certain time associated with diarrhoea, nobody doubted that such isolates were derived from the same “mother” strain. However, little thought has been given to the implications that may arise if this concept of a global distribution of bacterial clones is also extended to opportunistic pathogens such as E. coli and not only the few well-established virulent bacteria. Our investigations on the EPEC serotypes from infantile diarrhoea (Section X1.A) and examinations of characteristic serotype strains from piglet diarrhoea (e.g. 0:141;H:4, 0:8;K:87;H:19, 0:149;H:10) already many years ago have told us that there was a very close association of OKH- or OHserotypes and special biotype patterns. An 0:111;H:2 or an 0:55;H:6 strain

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isolated in different parts of the world usually had very similar biotypes characteristic for each serotype. This observation was very strongly supported when studies on E. cofi from enterotoxic diarrhoea in different parts of the world were compared. We therefore put forward the hypothesis that such enterotoxigenic serotypes (ETEC) were the progeny of the same clones spread over the world (0rskov and IZIrskov, 1977, 1980). Recent work by Achtman et a f . (1981), which added outer membrane proteins to the phenotype pattern, showed that this concept is also highly plausible when applied to E. cofi serotype strains such as 0:18ac;K:l;H:7, a common serotype from cases of neonatal meningitis all over the world (Sarff et af., 1975). Some non-published evidence from investigations of special E. cofi serotypes from other extra-intestinal diseases also seems to support the idea of a clonal connection between such strains. Future studies will show whether this concept is also true for E. cofi serotypes which have no pathogenic properties. The fact that difference can be found in less stable phenotypic traits, such as phage sensitivity patterns (Milch, 1978, this series), resistance patterns and plasmid DNA patterns (Achtman, 1981), when strains from different geographical areas are compared does not invalidate the general idea of the global connection between clones identified by their bio-serotype. As stressed above the concept is not new and probably raises more questions than answers. When did this spreading occur? Why is it that some highly characteristic bio-serotypes are selected as carriers of virulence determinants such as the plasmids determining enterotoxin production and adhesive fimbriae. Are there for example in these strains special combinations of phenotypic traits which are important for their position as ETEC serotypes? For future taxonomic considerations it may be important to bear in mind that the widespread occurrence of a characteristic bio-serotype (phenotype) may be caused by the spreading of a single clone. XIII. Concluding Remarks

From the above descriptions it is apparent that the E. cofi group is heterogeneous and contains a very high number of stable subtypes. Therefore, it will be a difficult task to command all available methods in a single laboratory, if a complete typing of strains is needed. On the other hand, it is also obvious that a limited number of serotypes (0:K:H) or sero-biotypes are much more common than others and, most important, that only a limited number of such highly defined types are closely associated with disease processes. It will often be possible, therefore, for the local laboratory to screen

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for strains which are of special interest in that area, whereas it will be the task of a central reference laboratory to carry out the complete typing. It is important to remember that it will often be of high value for the work done at the local laboratory to carry out the typing far enough to make it useful as an epidemiological tool, e.g. 0-grouping alone will often be of little value, but combined with biotyping a more accurate description of a strain can be obtained. It should not be necessary to add that typing procedures should never be carried out further than needed to solve the relevant problems, e.g. if the question is whether two strains are different an ordinary examination of the fermentation patterns will usually give the answer.

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3 Serology of Shigella W. H. EWING and A. A. LINDBERG Decatur, Georgia, USA and Department of Bacteriology, National Bacteriological Laboratory and Karolinska Institute, Department of Clinical Bacteriology, Huddinge University Hospital, Huddinge, Sweden

I. Introduction. 11. Taxonomy and identification 111. Antigenic analysis of cultures

. . . . . . . .

.

.

General comments . S . dysenteriae. , S.fiexneri . S . boydii . E. S . sonnei . F. Cross-reactions . IV. Chemical structure of Shigella 0-antigens . A. Approaches towards chemical analysis polysaccharides . B. S. dysenteriae. . C. S.fiexneri . D. S. boydii . E. S. sonnei . V. Production of antisera for typing . References . A. B. C. D.

. .

of

0-

. .

. . .

. .

113 114 115 115 118 418 119 119 124 126 126 126 121 131 132 132 140

I. Introduction The Shigella are members of the family Enterobacteriaceae and the tribe Escherichieae. This tribe is composed of two genera, Escherichia and Shigella. If one forgets, for a moment, lactose fermentation, gas production and motility, and compares the aggregate biochemical reactions of members of these two genera, it is seen that the shigellae and escherichiae are quite similar; the shigellae are somewhat less active physiologically, of course. Furthermore, the 0-antigens [heat-stable somatic lipopolysaccharide (LPS) cell wall antigens] of known serotypes of Shigella are closely related to those of one or METHODS IN MICROBIOLOGY VOLUME 14

Copyright Q 1984 by Academic Press. London All rights of reproduction in any form reserved.

114

W. H. EWING AND A. A. LINDBERG

another of the numerous 0-antigen groups of Escherichia coli (Ewing, 1953; Edwards and Ewing, 1972; Rowe et al., 1976, 1977). The validity of the classification of shigellae in the tribe Escherichieae is confirmed by work published during the last ten years on the relatedness of the deoxyribonucleic acids (DNAs) of various kinds of Enterobacteriaceae. The work of Brenner (1978) indicates that the DNAs of E. coli and members of the four species of Shigella are so intimately related that conceivably there could be just one species that would include all of them, both shigellae and E. coli. This would be impractical, of course, since certain serotypes of E. coli must be separated from the rest of E. coli and the shigellae must be differentiated for clinical and epidemiological purposes.

11. Taxonomy and identification

Because of the close relationships between Shigella and Escherichia it is essential that research workers determine whether a particular unknown culture is a Shigella or an Escherichia before attempting to determine its serotype. Otherwise, mistakes may be made as regards identification, and time and antisera are wasted. To assist in accomplishing this differentiation certain definitions and tabular data are included herein. The tribe Escherichieae is defined as follows (Ewing, 1972a; Edwards and Ewing, 1972): Escherichieae are motile or non-motile bacteria that conform to the definition of the family Enterobacteriaceae. The methyl red reaction is positive and the Voges-Proskauer test is negative. Urease, phenylalanine deaminase and hydrogen sulphide are not produced; sodium malonate is not utilized, gelatin is not liquefied and growth does not occur on Simmons’ agar or in medium containing potassium cyanide. The following definition of the genus Shigella is a slight modification of one given earlier (Ewing, 1967, 1972a). The genus Shigella is composed of nonmotile bacteria that conform to the definitions of the family Enterobacteriaceae and the tribe Escherichieae. With the exception of a few aerogenic biotypes of Shigella flexneri serotype 6 visible gas is not formed from fermentable carbohydrates by shigellae. Shigella sp. are less active in utilizing carbohydrates than E. coli. Salicin, adonitol and inositol are not fermented. Cultures of S. sonnei ferment lactose upon extended incubation, but other species do not utilize this substrate in conventional medium. Lysine is not decarboxylated, the majority of strains do not possess a demonstrable arginine dihydrolase system and ornithine is decarboxylated only by S. sonnei and Shigella boydii serotype 13. The type species is Shigella dysenteriae (Shiga), Castellani and Chalmers 0:1. Evidence of hydrogen sulphide production in triple sugar, iron or peptone

3. SHIGELLA

115

iron agar media, or of urease formation aid in the elimination of many microorganisms from the genus Shigella. Moreover, any culture that decarboxylates lysine, produces phenylalanine or tryptophan deaminase, grows on Simmons’ citrate agar or in Koser’s citrate medium, or is motile, does not belong to the genus Shigella. Members of the genus Providencia sometimes are mistaken for shigellae, presumably because they usually fail to ferment mannitol and may be anaerogenic. Providencia can be excluded from the genus Shigella on the basis of motility, growth on Simmons’ citrate medium, adonitol or inositol fermentation and by deamination of phenylalanine and tryptophan. Means by which differentiation within the tribe Escherichieae may be accomplished are presented in Table I (Ewing, 1972a,b, 1973; Edwards and Ewing, 1972). Additional tests and substrates that are of value for the differentiation of the four Shigella species are given in Table 11. All tests and substrates in which shigellae yield uniformly positive or negative results are omitted from Table 11. Otherwise, the data presented in these two tables are self-explanatory. Fuller definitions for each of the four species of Shigella are also available (Ewing, 1972a). Soviet investigators regard S.Jlexneri 0 : 6 as a subspecies of S.Jlexneri or as a separate species, Shigella newcastle (Petrovskaya and Bondarenko; 1977). Although designation of S. Jlexneri 0:6 as a subspecies on the basis of differences between it and S. Jiexneri 0 : l to 0 : 5 could be done, such differences do not make the biochemical differentiation of S.flexneri 0 : 6 and S . boydii easier. This separation is accomplished with ease by means of serology (see below). Perhaps it might have been better had the late Sir John Boyd classified his serotype 88 with his serotypes 170, P288 etc. (later S. boydii serotypes 0:1,2 etc.; Ewing, 1949). DNA-DNA hybridization studies (Brenner, 1978) indeed indicate that all shigellae are related at the species level (see above). For practical purposes we recommend preservation of the generally accepted classification. Biotypes of S.Jlexneri are characterized in Table I11 (Edwards and Ewing, 1972).

111. Antigenic analysis of cultures

A. General comments

Serotyping of shigellae depends upon determination of 0-antigens and 0-antigen factors. Since shigellae are non-flagellar, H-(flagellar)- antigens are not involved. Fewer antisera are therefore required for complete serotyping of Shigella than for Salmonella. Three species of Shigella are divided into a number of serotypes, as

116

W. H. EWlNC AND A. A. LINDBERC

TABLE I Differentiation within the tribe Escherichieae Escherichia

Shigella

Substrate or test Gas from glucose Lactose Sucrose Salicin Motility Indol Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase Esculin Sodium acetate Christensen's citrate Mucate

+ .

+

,

d d +or-

+

d d d d +or( +) d

+

90.7 90.8 48.9

40 69.1 99.2 87.9 17.2 63.4 30.9 83.9 24.4 96.3

-

-or+

d

-

-

-

-

2.1 0.3 0.9 9 0 39.8 0 9.5 20' 0

(11.4)c (31.1)c

(17.3)

O*

0

OC

From Ewing (1972a, 1972b, 1973) and Edwards and Ewing (1972). Figures in parentheses indicate percentages of delayed reactions (three days or more). * Certain biotypes of S.Jexneri serotype 6 form gas. Strains of S. sonnei usually ferment lactose and sucrose slowly and cultures of this species decarboxylate ornithine. Some strains of S.Jexneri serotype 4a, particularly the mannitol-negativebiotype, grow on sodium acetate medium. 'Percentage of strains with corresponding property. Obviously there is no difficultyin differentiatingtypical E. coli cultures and shigellae. However, the anaerogenic nonmotile varieties of E. coli may require closer examination before they can be definitelyclassified as E. coli. In attempting to classify a particular strain as E. coli or as a member of the genus Shigella the biochemical reactivities of the culture should be considered as a whole. Shigellaeare much less reactive than E. coli and a culture that produces acid promptly (i.e. within 24 h) from all, or most, of a wide variety of carbohydrates, such as maltose, rhamnose, xylose, sorbitol and dulcitol, undoubtedly is not a member of the genus Shigella. Symbols: +, 90% or more positive within one or two days' incubation; (+), positive reaction after three or more days (decarboxylase tests : three or four days); - , no reaction (90% or more); +or - ,majority of strains positive, some cultures negative; -or+, majority of cultures negative, some strains positive; +or(+), majority of reactions delayed, some occur within one or two days; d, different reactions: + ,(+), - ; w, weakly positive reaction.

'

indicated in Table IV. There is only one serotype of S.sonnei,although it exists in at least two forms* I, smooth and I1 which is an intermediate colony form between smooth and rough (R).The species designations and serogroups correspond. Accordingly serogroup A strains belong to S. dysenreriae, serogroup B to S.Jlexneri, serogroup C to S. boydii and serogroup D to S. sonnei. * In shigellaecolony forms I and I1 have also been referred to as phases I and 11 but the former is used here.

TABLE I1 Differentation of the species of Shigeh ~~

~~~~

S . pexneri serotypes 1-5

S . dysenreriae Test or substrate

lndol Arginine dihydrolase Ornithine decarboxylase Mucate Jordan's tartrate Gas from glucose Lactose sucrose

Sign

%+

(%+)'

-or+ d

43.7 1.5

(11.3)

-

0

-

+or-

-

Mannitol Dulcitol Sorbitol Arabinose Raffinose Rhamnose Maltose Xylose Trehalose

d d d +or(+)

Celloboise

-

Glycerol &Galactosidase (ONPG)

d d

-

d -or+

0 78 0 0 0

0 4.5 29.2 43.6 0 32.4 12 3.9 89.8 0

12.3 49.9b

Sign

%+

+or-

61.5

-

(0.5) (29.5) (7.2)

(%+)

Ob

-

0

-

0

-

0 0 (<0.1) 1.5 (41.9) 93.7 0 30.6 (1.5) 65 (8.7) 52.8 (28.4) 6 (6.2) 28.4 (45.3) 1.8 (0.4)' 77.8 (12.2) 0 0 0.8

(1.6)b (4.2)

S.pexneri serotype 6

d

+ d d d d d

(5.5) (77) (7.6) (7.5) +or(+) (72.5) -

-

0

Sign

%+

(%+)

Sign

%+

-

0 48.9

(10.3)

-or+ d

28.8 18.1 2.5' 0 13 0 1 0 97.6 6.7 41.8 94.1 0 0.2 16.6 11.2 85.2

d -or+

+ord (+)or+ +or(+)

-

-

(+)or+ d (+)or+

+or(+)

-

-

0 0 0

-or+ -

18.1 0

0 82.5 9.4 30.2 54.6 0

1.6 16 0.5 7.4 0

60 0

-

(72.2) (59.8) (39.3)

S.sonnei

S.boydii

+ d d

+ -

(3.7) (74.4) d (18.2) d (92.6) +or(+) -

(31.1) +or(+) -or+

0

55.5 11.1

(%+)

(31.9)

Sign

%+

-

0 0.5 99.4 16.4 100 0 1.8 0.1 98.9 0

+ -or+

+

d d

(10.4) (36.3)

+ -

+ d

1

94.2 2.5 77.1 86.4

(1.6) +or(+) (66) +or(+) (57.2) 1 (11.2) + 100 d 10.6 (34.8) d 13 95

+

From Ewing (1972,1973)and Edwards and Ewing (1972). Figures in parentheses indicate percentages of delayed reactions (three or more days). Some strains of S. dysenrerioe serotype 1 ferment lactose slowly; all are ONPG positive. ' A few doubtful reactions occurred but these were regarded as negative. 'Xylose was fermented by some cultures of the mannitol-negative bio-serotype of S.pexneri serotype 4 but not by other strains of serotypes 1-5. Only cultures of S . boydii 13 are positive. See Table I for description of symbols.

(%+)

(5)

(88.1) (85.4) (1) (1)

(2.9) (81.5) (21) (6.8) (1.8) (32.7)

118

W. H. EWING AND A. A. LINDBERG

In practice, polyvalent antisera are employed for preliminary serological examination of cultures thought to be shigellae. These are produced by injection of polyvalent vaccines and subsequently absorbed to eliminate certain unwanted cross-reactions. The S. sonnei antiserum used at this juncture generally is a mixture of antisera for forms I and I1 of the microorganism since the transitional form (11) occurs frequently in carriers. TABLE 111 Biotypes of Shigellajexneri serotype 6 Biotypes

Glucose

Mannitol

Dulcitol

From Edwards and Ewing (1972). Symbols: A, acid production within 24 h ; Ag, acid production and gas formation within 24 h; (A), (Ag), reactions delayed three or more days; - , no reaction (30 days).

B. S. dysenteriae With a few exceptions, antisera for the serotypes of S. dysenteriae may be used in the unabsorbed state if cross-agglutination reactions determined during evaluation of the unabsorbed antisera are kept in mind. Minor relationships, indicated by titres of 1:160 or less, usually do not interfere with slide agglutination when the antisera are diluted 1:lO or more. It is advisable to prepare absorbed antisera for the serotypes of S. dysenteriae listed in Table V. It should go without saying that all newly produced antisera must be ecraluated for all known relationships, minor or not. Such antisera then must be absorbed as required. C. S.flexneri

Since there are extensive intra-specific antigenic relationships among serotypes of S.Jlexneri using absorbed antisera is imperative (Table 64, Edwards and Ewing, 1972). Each serotype of S.Jlexneri possesses a single type-specific antigen that occurs in only one serotype. These antigens are qualitatively different in each serotype. Roman numerals are used for expression of these

3. SHICELLA

119

type-specific antigens in the antigenic formulae of the serotypes (Tables VI and VII). In addition, each serotype of S..flexneri possess a number of group antigens, labelled with Arabic numerals, which are common to more than one serotype (Tables VII and VIII). These factors are responsible for the intraspecific serological relationships among the serotypes of S. Jlexneri. Use is made of the group factors in the delineation of subserotypes ( la , 1b, 2a, 2b etc.). For this delineation it is necessary to employ antisera that are absorbed in such a way as to remove agglutinins for the type-specific antigens as well as for those group factor antigens that interfere with determination of the subserotype of cultures (Table VII and VIII). It is notable that quantitative differences occur in the group factor antigens in different cultures of the same subserotype. In practice, a culture thought to be S.Jlexneri is first tested in the six absorbed type-specific antisera and then in the three absorbed factor antisera (Table VII).

D. S.boydii The species S. boydiicurrently is composed of 15 serotypes (Table IV), the first six were isolated in India and were described by J. S . K. Boyd during the late 1920s and the 1930s (Edwards and Ewing, 1972; Ewing, 1972a). Antisera for S. boydii serotypes 2, 3, 7 , 8 and 14 generally may be used in the unabsorbed state, but antisera for the remaining ten must be absorbed in the manner given in Table IX. The reciprocal antigenic relationship between S. boydii 1 and 4 and E. coli 0-group. 1 is of particular importance in diagnostic work because these serotypes are among the most commonly occurring of the S. boydii serotypes and E. coli 0:1 is common. Hence, their antisera must be absorbed as indicated in Table IX. Antisera produced with S. boydii 0:6 (a rare serotype) generally contain rough agglutinins that agglutinate cultures of S . sonnei that are in the transitional forms (I1 or SR). Therefore, it usually is necessary to absorb antiserum for S. boydii 0 : 6 with a culture of S. sonnei 11. There is no antigenic relationship between the 0-(smooth) antigens of S. boydii 0 : 6 and S . sonnei I ( S ) .

E. S.sonnei A mixture of antisera produced with cultures of S . sonnei form I ( S ) and I1 (SR)is all that is needed in practice. However, antisera that are specific for each of the two forms can be produced and absorbed for special use. In acute infections of S. sonnei, the form I ( S ) colonies predominate, whereas the form I1 (SR)*colonies or transitional forms usually predominate in carriers. *Although labelled here as SR the form I1 bacteria in chemical analyses have all the characteristics of a R strain, i.e. makes a core LPS without 0-antigenic polysaccharide chains.

TABLE IV Nomenclature and taxonomy of Shigella Species and serotypes S . dysenteriae

1

2. 3 4 5 6 7 8 9 10

S.jexneri

la ab 2a 2b 3a 3b 3c 4a 4b 5 6

Ewing (1949) (with additions)

Boyd (1940, 1948)

Boyd (1938)

English (older)

I I1 111

Bacterium shigae S . ambigua, S . schmitzii Q771, type 8524, S . arabinotarda A Q1167, S . arabinotarda B Q 1030 Q454 4902 Serotype 599-52 Serotype 58 Serotype 2050-50

rv

V VI VII

Tyw I I I1 I1 I11 I11 I11 IV IV V VI -

Older designations

Abbreviated formula 1:4.. ? I:6.. . 11:3,4 . . . 11:7,8 . . . I11 :6 :7.8 . . . III:3.4:6 . . . III:6. . . IV:3,4.. . IV:6.. . V:7 . . . v1:-. . . - :7,8. . . -3.4. ..

I

V

I1

W

vz W wx

111

Z

Z

IV IV V VI

103 1032 PI19 88 X Y

Flexner Strong, Hiss-Russell

Lentz Y2 S . newcastle

Y Y

Hiss-Russell

S. boydii

S. sonnei

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

170 P288 D1 P274 P143 D19

I I1 I11 IV V VI VII

S. sonnei

Type T, Lavington, S. etousa Serotype 112 Serotype 129617 Serotype 430 Serotype 34 Serotype 123 Serotype 425 Serotype 2770-51 Serotype 703 Sonne-Duval, Sonne 111, Kruse E. S. ceylonensis A

From Edwards and Ewing (1972). a The dots in I :4 . . .,etc., refer to minor antigens which have not been given an official labelling.There is a strain to strain variation in the appearance of these minor antigens.

TABLE V Absorption of antisera for Sbigella dysenteriae Antiserum for

Absorbing cultures

S. dysenteriae 1 S. dysenteriae 2

E. coli 01 S . boydii 1 plus S . boydii 15 S. dysenteriae 8a, 8c S . dysenteriae 8a, 8b plus S. boydii 15 S . dysenteriae 2 plus S . boydiifplus E. coli 01

S . dysenteriae 8a, 8b S . dysenteriae 8a, 8c S . dysenteriae 10

From Edwards and Ewing (1972).

TABLE VI Preparation of specific absorbed typing antisera for Sbigella Jlexneri Antiserum for S.Jlexneri serotype la lb

2a 2b 3 4a

4b 5

6

Absorbing cultures (S.Jlexneri) 2a or Y ( - :3,4) plus 5 or X ( - :7,8) plus 6 2a or Y (- :3,4) plus 3 plus 5 or X ( - :7, 8) plus 6 la or l b plus Y ( - :3,4) la or l b plus X ( - :7,8) 1b 6 plus 2a or Y ( - :3,4) plus 2b or X (- :7,8) l a or l b or X ( - :7,8)plus 2a or Y (- :3,4) 1b plus 2a or Y ( - :3,4) plus 3 la or 15 plus 2b or X (- :7,8) plus 3 la or l b

From Edwards and Ewing (1972).

Specific factor

I I

I1 I1 I11 IV

IV V

VI

TABLE VII Shigulla Jluxrtrri: Agglutination in typing antisera (slide tests) Absorbed antisera Type specific S.Jfexrieri serotype

Test antigens (antigenic foimutae)

la Ib 2a 2b 3a 3a 3b 4a 4b 5 6 Xvariant Y variant

I : 1.2.4.5.9 . . . I : 1.2.4.5.6.9 . . . 11: 1. 3,4 . . . 11: 1.7.8.9 . . . 111: 1.6.7.8.9 . . . 111: 1,3,4.6,7.8.9 . . . 111: I. 3.4.6 . . . IV: I , 3,4 . . . IV: 1.6 . . . v : 1,5,7,9 . . . VI : I , 2,4 . . . - : 7.8 . . . - : 3.4 . . .

I

I1

++++ ++++

-

-

-

-

-

Ill

IV

Group factors

V

IV

++++ ++++

3.4

6

++++ ++(-) ++++

++++ -

-

-

-

++

++

++++(-) -(++)

-(++I

++(-)

-

++++

-

7. 8 -(+)

-

++++ ++++ + + + + + + + + + + -+ + ++++ -(+ +) ++++ + + + +(-) ++++ -

From Ewing (1972a) and Edwards and Ewing (1972). The complete antigenic formulae are given. The dots in I : 1.2.4.5.9 . . . indicate that minor non-labelled antigen determinants can be present, or absent. in any given strain. no agglutination. Symbols in parentheses indicate Degrees of agglutination: + + + +. complete reaction; + to ( + +). relatively weaker reactions; occasional reactions.

-.

124

W. H. EWlNG AND A. A . LINDBERG

TABLE VIII Antisera for group factors of Shigellaflexneri Antiserum for type 2a I1 :1 , 3 , 4 . . . lbI:l,2,4,5,6,9.. 2bII:1,7,8,9 . . .

Absorbed by

Factor(s) remaining

2bII:1,7,8,9 . . . la1:1,2,4,5,9.. . la or l b plus 2a

.

3,4 6 7,8

From Ewing (1972b) and Edwards and Ewing (1972).

TABLE IX Preparation of absorbed antisera for identification of serotypes of Shigella boydii

S.boydii serotype 1 4 5

6 9 10 11 12 13 15 Provisional serotype 3615-53

Absorbing cultures S . dysenteriae 2 plus" S . boydii 4 plus E. coli 01 S . boydii 1 plus E . coli 01 S.boydii 9 plus E. coli 025 S . sonnei I1 S . boydii 5 plus S . boydii 13 plus E. coli 025 S . boydii 11 S. boydii 10 plus E. coli 01 E. coli 07 S . boydii 9 plus E. coli 01 S . dysenteriae 2 plus S . dysenteriae 8a, 8c S . dysenteriae 3

From Ewing (1972a) and Edwards and Ewing (1972). Additional absorptions may be necessary.

F. Cross-reactions There are numerous antigenic relationships between the genera Shigella and Escherichiu. These are shown in Table X. It must be stressed that most likely only a minority of the cross-reactions observed have been reported. This underlines the importance of establishing the Shigella diagnosis on the basis of biochemical reactions (Table I).

TABLE X Relationship of the 0-antigens of Shigella and Escherichio coli Shigella species S . dysenteriae

S.Jlexneri

Serotype

Related to E. coli 0-group@)

1 2 3 4 5 6 7 8 9 10 la lb 2a 2b

3 (111 : 6,7, 8)

S . boydii

3 (111: 3,4,6,7,8) 4a 4b 4 5 6 X variant Y variant 1 2 3 4 5 6 7 8 9 10 11 12 (and M) 13 14 15

S . sonnei

1, reciprocal, a, b-a, c? 120, unilateral 112a, 112c, identical 124, identical 88, unilateral; coliform 3588-51, identical 58, identical 130, reciprocal, a, b-a, c 121, reciprocal, a-a, b 38, reciprocal, a, b, c-a, b; 23, a, d No relationship, 1-148 144, reciprocal, a, b-a, c 1, 19a, 62,69,73 1, 16, 19a, 62,69,73 13, reciprocal, a, b-a, c 13, reciprocal, a, b-a, 3; 73; 147a. 147b, identical 13, 16 (identical culture 5444-80) 13,16 1, 13 135, identical, 13 1, 17, 19a, 73 129, identical 19a; coliform 3438-51 reciprocal, a, b-a, c 1,2, 13, 19 1,2, 13, 19 2 (SO), reciprocal, a, b-a, c 87a, 87b, identical; 87a, 87b, reciprocal, a, b-a, c; 96, reciprocal, a, b-a, c 85, reciprocal, a, b-a, c 53, identical 79, identical 76, reciprocal, a, b-a, c; 14 4838-69, identical 143, identical 114 minor 102, reciprocal, a, b-a, c 105a, 105c, reciprocal, a, b-a, c 105a, 105b, identical 7, reciprocal, a, b, c-a, d (M=a, b) 28a, 28c, reciprocal, a, b-a, c; 98 reciprocal a, b-a, c 32, identical; 83, reciprocal, a, b-a, c 112a, 112b, identical No relationship 1-148 Identical, type C274 (Aeromonas shigelloides) (Plesiomonas (Aeromonas) shigelloides)

From Edwards and Ewing (1972) and Rowe et al. (1976). These arbitrary formulae are used merely as an aid to make the relationships clearer and they should not be interpreted as permanent designations for the antigens involved. a

W. H. EWING A N D

126

A. A.

LINDBERG

IV. Chemical structure of Shigella 0-antigens A. Approaches towards chemical analysis of 0-polysaccharides

Analysis of lipopolysaccharides from Shigella have in recent years started to unravel the structure of the 0-antigenic polysaccharide chain in the LPS of the bacterial outer membrane. Such information is valuable since it gives the structural background to the complex antigenic relationships both within the genus Shigella and between Shigella and other enteric organisms. Such knowledge makes it possible to identify specific antigenic determinants, and to isolate or synthesize them and use each of the saccharide determinants also as haptens in immunogenic conjugates for production of monospecific diagnostic antisera (Ekborg et af., 1977).

B. S. dysenreriae The structures of known S. dysenteriae 0-antigens are given in Table XI. The structure of the chemical repeating unit has been completely elucidated for eight of the ten different serotypes. The oligosaccharide repeating unit contains from four to six sugar residues, some of which are very uncommon. For instance, the acidic 4-0-[( R)- 1-carboxyethyll- glucose and 4-0-[(R)- 1carboxyethyll-~-rhamnoseresidues in the serotype 3 and 5 polysaccharide chains are each composed of a monosaccharide and a lactic acid moiety. The muramic acid, which together with N-acetylglucosamine forms the rigid shape-determining peptidoglycan layer of microbial cell walls, is similarly constituted. In the serotype 9 polysaccharide chain pyruvic acid is linked by ketal-type bonds to 0:4 and 0 : 6 of a b-D-galactopyranosyl residue. A detailed comparison of the structures given in Table X reveals that no structural element is identical in the various serotypes. This is in agreement with common experience that, with few exceptions, antisera for S. dysenteriae can be used in the unabsorbed state (see above). Cross-reactions with members of other genera or species do occur, however. The homologous cross-reactivities seen between S. dysenteriae serotype 3 and E. coli 0:124 (Table X),and between S. dysenteriae serotype 5 and E. coli 0 : 5 8 were readily explained when structural studies revealed that the 0antigens, indeed, are identical (Dmitriev et al., 1976b, 1977~).Thus identical 0-antigenic polysaccharide chains can occur in different genera. Most likely complete identities or partial relationships will be revealed when structural studies are undertaken in instances of observed cross-reactions between S. dysenteriae and other enteric organisms (Edwards and Ewing, 1972; Ewing, 1953; Rowe et al., 1976).

3. SHIGELLA

127

C. S.flexneri The extensive intra-specific 0-antigenic relationship among serotypes of S. ,flexneri was understood when the structure of the polysaccharide chains of these serotypes was established (Kenne et al., 1978). Structural studies have indicated that all the polysaccharides in strains of sero- and subserotypes 1-5 are polymers of the basic tetrasaccharide repeating unit: +3)-P-~-GlcpNAc-(1 -,2)-a-~-RhapI(1 -2)-a-~-RhapII( 1 -,3)-a-~-RhapI I I ( 1 -, The individual specificity of the sero- and subserotype is a consequence of attachment of a-D-glucopyranosyl and 0-acetyl groups to different positions on the basic repeating unit. The structural and immunochemical data suggest that for some type antigens, labelled with Roman numerals, the immunodominant region can be given (Table XII) (Kenne et u/., 1978; Carlin and A. A. Lindberg, unpublished data). Definitive proof of this hypothesis will not be obtained, however, until specific antisera elicited by immunization with the disaccharide structures as haptens linked to immunogenic carriers have been tested. So far the determinant for type antigen I11 has not been discovered. In addition to the type-specific determinants each serotype possesses a number of group antigens, labelled with Arabic numerals (Tables VII and XII). For group antigens 0:6 and 0:7,8 the structures given in Table XI1 have been deduced (Kenne et af., 1978). For 0:3,4 no determinant has been identified so far. Examination of the table of S.JIexneri serotypes (Table VII) and of the inferred immunodeterminant structures (Table XII) shows that all serotypes from 1 to 5 have one, and only one, of the type-specific antigens I-V, and that for four of these antigens the determinant structure is inferred to be a glucosyl or 0-acetyl substituent linked to the basic tetrasaccharide repeating unit. As the sites of substitution are different for each of factors I, 11, IV and V it would theoretically be possible for more than one kind of substitution to occur in the repeating unit of a single strain, which would presumably result in presence of two different type-specific antigens in a single strain. No such strains have been found. Observations on phages causing lysogenic conversion in respect of type-specific factors (described below) give a hint as to a possible reason for non-existence of strains with more than one type-specific antigen. One may also note that in terms of antigenic determinants the two group-specific factors whose chemical basis is known i.e. antigens 6 and 7,8 are essentially similar to type-specific determinants in being the result of substitutions (0-acetyl and a-glucosyl respectively) of the basic repeating unit. The distinction of factors I-V as defining types, from factor 6 and 7 3 , considered to define subtypes, is in terms of chemical basis an arbitrary one.

TABLE XI Structure of the serotype determining 0-antigenic polysaccharide chain in Shigella dysenteriae S. dysenteriae serotype

Structure"

Reference

1

+3)-a-L-Rhap (1 +3)-a-~-Rhap (1 +2)-a-D-Galp ( 1 +3)-a-~-GlcpNAc ( 1 +

Dmitriev et a / . (l976a)

2

+3)-a-~-GalpNAc ( 1 +3)-a-~-GalpNAc ( I + 4 ) - a - ~ - G k p( I +4)-p-~-Galp ( 1 +

Dmitriev et a/. (1977b)

1:

AcO 3/4-a-~-GlcpNAc 3

+3)-p-~-GalpNAc ( I +3)-a-~-Galp ( 1 +6)-p-~-Ga!j'( I +

I:

Dmitriev et a/. ( 1 977e)

B-D-GIcPLcA ( 1-6)-a-~-Galp 4

+3)-a-~-GlcpNAc( 1 +3)-a-~-GlcpNAc ( 1 +4)-a-~-GlcpA ( 1 +3)-a-~-Fucp ( 1 +

1,f

Dmitriev et a/. ( I 9770

AcO-a-L-Fucp 5

+3)-p-~-GlcpNAc ( 1 +4)-a-D-Manp ( 1 +4)-a-~-Manp( 1 +

a-L-R hapLcA

I

OAc

Dmitriev et a / . (l977a)

Drnitriev et a / . (l975b)

XI I 8

+4)-l)-~-GlcpA ( 1 -3)-l)-~-GalpNAc ( I + 3 ) - f l - ~ - G a l p N A c( I +

1:

Dmitriev et a / . (1978b)

S - D - G I L ~ N A (C1 +4)-fl-D-Gkp Drnitriev et a / . (l978a)

OAC

4CH,

HOzC

Dmitriev et a(. (l977d) D - GD-glucose; ~ D-G~cA.D-glucosuronicacid; D-GIcLcA, Symbols: AcO, 0-acetyl group; D-Gal, D-galactose; ~ - G a l N a cN-acetyl-~-galactosamine; , 4-0-[(R)-1tarboxyethyll-~-glucose;D-GlcNac. N-acetyl-D-glucosamine; D-Man, D-mannose; D-ManNAc. N-acetyl-D-mannosamine; L-FUC.L-fucose; p and findicates if the sugars are in the pyranose or furanose forms, respectively; L-Rha, L-rhamnose; L-RhaLcA. 3-0-((R)-l-carboxyethyl]-~-rhamnose; X,unidentified component.

W . F. EWlNG AND A. A. LINDBERG

130

TABLE XI1 Suggested structures of Shige/ln,flexneri type and group-specific antigens Serotype antigen I II Ill IV V Serogroup antigen 3,4 6 7,8

Structure" x-D-GIc~( 1 +4)-/h-GlcpNAc z-D-GIc~( 1 +4)-a-~-Rhap111 Not identified z-D-GIc~( 1 +6)-/&~-GlcpNAc z-D-GIc~( I +3)-cc-~-RhapI I Not identified AcO-2-a-~-RhapI I I E-D-GIc~( 1 +3)-a-~-RhapI

For abbreviations see Table XI.

Pragmatically it is justified by the absence of strains with more than one of determinants I-V compared with the presence of strains with both factor 6 and factor 7,8 (type 3a). It is evident that the bacteria also possess other antigenic determinants in addition to the type and group antigens mentioned above (see complete formulae in Table VII). It is likely that at least some of'these antigenic specificities are a consequence of the presence of 0-acetyl groups which have been found in varying proportions in the polysaccharide chains (Kenne et al., 1978). Since so few S..fle.meriLPS have been analysed, usually only one strain for each serotype, it is not possible to correlate a chemical structure to any of these minor antigenic determinants. The 1 determinant present in all S. flexneri serotypes, including type 6 which has a repeating unit which differs from that seen in types 1-5 (see below), could possibly be ascribed to the only known common structural element L-Rhap 1 ,-2 L-Rhap (however, below see discussion on agglutination of serotype 6 bacteria in group 3,4 serum). Quantitative as well as qualitative differences in the expression of the type and group antigens of S.Jle.uneri frequently causes problems in the diagnostic laboratory. The fact that quantitative differences do occur in the expression of the group factor antigens in different cultures of the same subserotype is well known (Edwards and Ewing, 1972). This can possibly be explained on the basis of quantitative differences in expression of a type-specific determinant, e.g. differences in efficiency of a glucosylating enzyme structural gene. However, there are also differences between colonies within a single strain. This phenomenon is similar to form variation seen in Salmonella and probably is caused by gene rearrangements which regulates the gene expression (Green, 1977). A qualitative 0-antigenic modification can also occur as a result of lysogenic conversion following bacteriophage infection.

131

3. SHIGELLA

This event usually means a glycosylation, acetylation or other structural modification of the 0-polysaccharide chain of the host, which leads to expression of a new 0-antigen specificity and sometimes also to loss ofexisting 0-antigen specificity (Lindberg, 1977). Shigellu phage Sf6, which comes from a type 3a strain, converts Y strains which retain their group 3,4 antigenic specificity at the same time as group 6 antigen specificity appears. This new specificity is probably a result of phage-directed 0-acetylation of the L-Rhap I11 residue (Gemski et d . ,1975). Other S.,flexneri phages have been reported to cause lysogenic conversion of subserotypes la-+4a, lb+4b, 2a-+4a and 3b-+4b (Matsui, 1958; Iseki and Hamano. 1959). Although the 0-antigenic polysaccharides of these and other phage-converted strains (Gemski et d . , 1975)have not been subjected tochemical studies, the serological data suggest that 0-acetylations and glucosylations are responsible for the changes in antigenic specificity. The structure of the S.,flexneri serotype 6 (previously called S . newcastle among other names) tetrasaccharide repeating unit differs from that of serotypes 1-5 (Dmitriev er d . , 1979): +4)-P-~-GalpA( 1 -+3)-P-~-GalpNAc( 1 +2)-a-~-R!ap 3

(1 +2)-a-~-Rhap(1

-+

OAc The weak serological cross-reactivity between serotype 6 bacteria and a group factor 3,4-specific antiserum (Table VII) is most likely attributed either to the structural element a-L-Rhap ( 1 -+2)-a-~-Rhap( 1 -+) common to the basic repeating unit of types 1-5 and to serotype 6, or to 0-acetyl groups attached to the rhamnosyl residues (Kenne et ul., 1978). D. S. boydii

S. boydii is divided into 15 recognized and one provisional serotypes (Tables IV,IX and X). So far only the structure of the serotype 6 0-antigen has been studied (Dmitriev et al., 1975b): -*3)-b-~-GalpNAc(1 -+3)-a-~-Galp(1 -+6)-a-~-Manp(1 +2)-a-~-Manp(1 +

T:

a-~-GlcpA Several complex antigenic relationships exist within S. boydii, between S.

132

W. H. EWlNG AND A.

A.

LINDBERG

boydii and other Shigella species and with other enterobacteria, primarily E. cofi (Table IX; Edwards and Ewing, 1972). It is conceivable that several interesting structures will be revealed when chemical studies are undertaken. E. S. sonnei S. sonnei is found both as colony form 1 (S) and colony form I1 (R). The rapid and irreversible switch from form I to form I1 was recently shown to be caused by loss of a plasmid (Kopeck0 et al., 1980). The plasmid thus controls synthesis of the 0-antigenic colony form I polysaccharide chain. The disaccharide repeating unit of the polysaccharide contains the unusual sugars 2-acetamido-2-deoxy-~-altruronic acid and 2-acetamido-4-amino-2,4,6trideoxy-D-galactose which are a- 1,4 linked, whereas the disaccharides are joined by 8-1,3 linkages (Kenne et af., 1980). There is only one known antigenic relationship between S. sonnei form I antiserum and other bacteria and that is with one 0-antigen group of Pfesiomonas shigelloides (Table X). It is most likely that the 0-polysaccharide chain of the Plesiomonas strains is identical to that of S. sonnei. The form I1 structure, i.e. the complete basal core, is also known (Jansson et af., 1977). This structure is found as core in S. boydii serotype 6 and in several E. coli, notably E. cofi 0:14 and 0:78. The use of a mixture of forms I and I1 sera may thus occasionally lead to erroneous conclusions because of the broad specificity of the form I1 specific antibodies.

V. Production of antisera for typing Within the Shigefla species, confirmed by their biochemical reactions, a subdivision into serotypes is based on the reactivity of isolated bacteria with a set of antibody preparations. Only the antigenic specificity found in the polysaccharide 0-antigenic part of the lipopolysaccharide (LPS) of the cell envelope is used for typing purposes. The methods used for the protection of antisera for Shigeffaare similar to those outlined for the production of 0antisera for Salmonella (Lindberg and Le Minor, 1984) or E. coli (0rskov and 0rskov, 1978). 1. Bacterial strains

There are no standard strains that can be used for immunization of rabbits. Authentic cultures can be obtained from the American Type Culture Collection, Rockville, Maryland, USA or the National Collection of Type Cultures, London. Strains may also be obtained from the International

3. SHIGELLA

133

Shigella Centers at the Center for Disease Control, Atlanta, Georgia, USA and the Central Public Health Laboratory, Colindale.

2. Immunization procedures Cultures to be used for immunization are selected for specificity, smoothness and agglutinability. Strains that have been selected for antiserum production should be stored lyophilized or in liquid nitrogen. Strains to be used for immunization are subcultured on nutrient agar (or infusion agar or similar medium). Smooth colonies are selected and transferred either to a new nutrient agar plate or nutrient broth. After incubation for 16 to 18 h the growth is tested in a 1:lO or 1:20 dilution of homologous antiserum for agglutinability. The importance of selection and control of the use of smooth cultures cannot be overemphasized because the Occurrence of rough agglutinins in produced antisera leads to confusing cross-reactions. This is so because both within Shigella and between Shigella and E. coli the same core structures, i.e. the rough character, appear (Jansson et al., 1981). The acriflavine test is useful for detecting incipient roughness before this is demonstrable in physiological concentrations of saline. This is particularly important when selecting colonies for preparation of immunogens for antiserum production, or reference agglutinating suspensions. Only rarely will freshly isolated Shigella strains give a positive test. Acriflavine solution O.% Acriflavine lf.3 Physiological concentrations of saline 500 ml Store the solution in the dark and discard if any deposit occurs.

Using a loop emulsify a little growth from a fresh nutrient agar slope or plate in a drop of acriflavine solution on a slide. The suspension remains homogeneous if the organism is smooth. Granularity or clumping indicates incipient or complete roughness. The test must not be done with a mercuric iodide suspension since chemical precipitation will occur. The selectedculture is used to inoculate a liquid culture of nutrient broth (or meat infusion broth). The culture is incubated for 6-8 h, or until it reaches the late logarithmic growth phase, and then heated to 100°C for 2 h and centrifuged at 3000g at +4"C for 20min. After centrifugation the supernatant fluid is discarded and the bacteria are resuspended in phosphatebuffered saline. The density of the suspension should approximate 2-5 x lo9 bacteria ml- l . The broth cultures may instead be sterilized by suspension in phosphatebuffered saline (PBS, ph 7.2) containing 0.5% formalin and used as vaccines in the unheated state. Since heat-labile surface antigens, particularly K-antigens

134

W . H . EWlNG AND A. A. LINDBERG

and fimbrial, are not destroyed by this inactivation procedure the resulting antisera may contain cross-reacting K- and fimbrial antibodies. It also seems that fewer rabbits are lost during the course of immunization when heated immunogens are used. Before immunizations are started rabbits are test bled and agglutinin titres are determined so that unnecessary absorbtions can be avoided after completion of the immunization scheme. The set-up of antigens to be used varies depending on the immunogens, i.e. the type of antiserum being produced. The immunogens are given by intravenous inoculation. Immunization schemes vary widely between different laboratories. One scheme is to give a 1 .O-ml injection twice weekly (interval one of three to four days) for three to four weeks. The rabbits are test bled five to seven days after the last injection. If titres are satisfactory the animals are exsanguinated. When titres are deemed too low immunizations can be continued for another week or two before the rabbits are test bled again. It must be emphasized, however, that not infrequently one observes that rabbits that fail to respond initially also are poor responders even when the immunization period is extended.

3. Polvvulent grouping untiseru These are produced for an initial screening of the antigenic characteristics of the strain under investigation. Most laboratories use the following pools. S. dysenteriae S . dysenteriae S.JIexneri S . boydii S . boydii S . sonnei

A,: types 1-5 A,: types 6-10 B : all types C,: types 1-7 C,: types 8-15 D : S and R

S. dysenteriae subgroup A, antiserum is produced by injection of a mixed vaccine containing equal amounts of bacteria of the first five serotypes of the species S. dysenteriae. Similarly the subgroup A, antiserum is produced by injection of a mixture of bacteria of S. dysenteriae serotypes 6-10. Polyvalent antiserum for S.fEexneri is produced by injection of a mixture of the various serotypes of that species. For S. boydii two pools are used: C, contains types 1-7 and C, contains types 8-15. A mixed antiserum is recommended for S. sonnei, e.g. the vaccine should be composed of bacteria expressing both the 0 chain (form I) and rough core (form 11). After production polyvalent antisera should be tested with suspensions of each of the serotypes that were used in the vaccine. Slide tests may be used for this purpose and each suspension should be tested in antiserum dilutions of 1:5, l:lO, 1:15 and 1:20. For use, a dilution should be selected which causes rapid and complete agglutination of each component serotype. Usually this is

3. SHIGELLA

135

a 1 5 or a 1:10 dilution. If the antibody titre for one of the serotypes is too low the grouping antiserum can be supplemented by addition of a volume of undiluted high titred antiserum against the particular serotype. The grouping antisera may be used either in the unabsorbed or the absorbed state. If used unabsorbed certain cross-reactions have to be borne in mind. Results that may be expected are listed in Table XIII. If deemed necessary the grouping antisera may be absorbed in such a manner as to free them of cross-reactions. The manner in which these absorptions may be made is given in Table XIV and the results obtained with some such absorbed antisera are given in Table XIII. Each grouping antiserum must be tested for specificity before being put into use, and the absorptions directed accordingly. 4. Antisera for type determination The methods used for serotyping cultures of Shigella are based upon the fact that each serotype contains a specific or major 0-antigen which is characteristic of the serotype. Certain shigellae also contain group or minor antigens, which in many cases are shared by other serotypes. For example, serotypes of S. Jlexneri contain common group antigens and, consequently, each type of this species reacts to some extent in antisera prepared against other serotypes of the species. The extent of reactivity varies because of the degree of substitution of the common basic repeating unit of the S. Jlexneri 0polysaccharide chain (see above) and depending on if the form variable determinant is expressed or not. The 0-antigenic relationships within S. Jlexneri make the use of absorbed and well-characterized factor sera a necessity. The cross-agglutination reactions of shigellae are listed in Tables V-X. From these tables it may be deduced which antisera must be absorbed before they can be used for identification.

5 . Technique for absorption of antisera Absorptions of antisera are carried out in order to remove unwanted antibody specificitiespresent. For instance, K-antibodies may have to be removed from a S. Jlexneri antiserum if an unheated vaccine preparation was used. Absorptions must also be done for preparation of a factor serum, e.g. a serum with specificity against a particular determinant. The antiserum to be absorbed will generally be used at a dilution of 15, or more. Most microbiologists use suspensions of agar plate cultures for the absorptions. However, broth cultures can be used as well. For absorption of K-or fimbrial antibodies unheated live or formalin-treated cultures are used. For absorption of 0-antibodies heated cultures are used. Smooth cultures of the strain to be used for absorption are tested for agglutinability and then inoculated into tubes of nutrient or infusion broth.

136

W. H. EWING AND A. A. LINDBERG

TABLE XI11 Reactions obtained with polyvalent Shigella antisera (slide tests) Unabsorbed Living cultures S . dysenteriae

1 2 3 A1 4

5, 6 7

S.Jlexneri

8 A2 9 10 la lb 2a 2b 3 4a 4b

A1 2 4 4s 4 4 4

1s Is

5 6

X Y

1 2 3 5 6 7

S . sonnei

C1

C2

A1

A2

B

C1

d d

-

-

3

-

-

4

-

-

-

-

-

3

-

-

-

2

4 4 4 4 4 4 4 4 4 4 4 4

1s 1s 2s 1s ds

4 ,c1

8 9 10 11 12 pc2 13 14 15 I

B

4

4

S . boydii

A2

Absorbed

-

-

-

-

-

lvs Ivs

-

-

3

-

-

4 4 4 4 -

-

-

-

-

3

-

-

-

-

-

-

-

-

-

2vs 1vs 1vs

-

1s 4 4 4 4 4 4 4

1s 1s

From Edwards and Ewing (1972). I , 2, 3 and 4 indicate degrees of agglutination; d, doubtful; s, slow; vs, very slow.

4 4 4 4

-

C2

3. SHIGELLA

137

TABLE XIV Absorption of Shigella polyvalent antisera

Antisera

Absorbing cultures

Polyvalent A1 (S.dysenteriae 1-5)

S . dysenteriae lo" plus S . boydii 1" plus S . boydii 15 plus A-D 01* S. dysenteriae 2 plus S . boydii 1" plus S . boydii 15"

Polyvalent A2 (S.dysenteriae 6-10) Polyvalent B (S.flexneri 1-6) Polyvalent C1 (S. boydii 1-7) Polyvalent C2 (S. boydii 8-15)

A-D 01 PIUS A-D 03 plus A-D 04 A-D 01 PIUS A-D 03 PIUS A-D 04 PIUS S. sonnei, R (11) S . boydii 1 plus S . dysenteriae 2 plus S . boydii 4 plus S . dysenteriae 8" plus A-D 01 plus A-D 02 PIUS A-D 07

From Edwards and Ewing (1972). Absorption with cultures of serotypes may not be required. Antisera should be tested and absorbed as required. A-D= E. coli sub sp. Alkalescens-Dispar.

After incubation for 6-8 h at 37°C the broth cultures are used to seed nutrient or infusion agar plates. Standard 90-mm Petri dishes containing about 40 ml of the agar medium are employed. Each plate is seeded with 0.3-0.4ml of broth culture and the inoculum is spread over the entire agar surface. The plates are incubated for 18-20h at 37°C. If the cultures to be used for absorption are 0-agglutinable, i.e. react well in antiserum in the unheated state, the growth from the plates may be harvested in formalin-treated (0.5%) phosphate-buffered saline. However, if the cultures contain antigens that inhibit 0-agglutination the growth from the plates should be harvested in phosphate-buffered saline and the tubes containing the harvested growth heated at 100°C for 2 h. The heat treatment inactivates (destroys) labile Kand fimbrial antigens and the procedure usually results in a more effective absorption of 0-agglutinins. Since shigellae are infectious to laboratory

138

W. H. EWING AND A. A. LINDBERG

workers some laboratories always harvest the growth in a mercuric iodide solution to kill the bacteria. Mercuric iodide solution Stock (1:lOO)

Mercuric iodide Potassium iodide Distilled water

Ig 4g 100 ml

Working solution

Mercuric iodide (stock 1:lOO as above) Physiological concentrations of saline Formalin (40%)

10 ml 90 ml 0.5 ml

After flooding the agar plates with the mercuric iodide solution the slants are allowed to stand at room temperature for at least 0.5 h to allow the mercuric iodide to kill the organisms completely. The suspension can be used either heated or unheated for the absorptions. The bacterial suspension is then centrifuged to pellet the bacteria and the supernatant is discarded. The diluted antiserum (usually 1 5 to 1: 10) is added to the packed bacteria, and the mixture shaken thoroughly to resuspend the sedimented bacteria. The absorptive dose is calculated to be in excess of that required. Better results are obtained if the bacteria to be used for absorption are divided into two portions. Antiserum is added to the sediment of one tube, kept at 37°C for 1-2 h and then at +4"C overnight. Then the bacteria are removed by centrifugation. The supernatant is mixed with the sediment of the other tube (which has been kept in the cold after the first centrifugation), incubated at 37°C for 1-2 h and then at +4"C overnight. The supernatant obtained after a subsequent centrifugation to pellet the bacteria is collected and titrated by slide or tube agglutination. The amount of antigen used for each absorption must be sufficient to remove all homologous antibodies. This amount varies with different antisera, e.g. different amounts of antibodies present, and different bacterial strains, e.g. amount and accessability of the antigen. In most cases the growth from one 90-mm Petri dish effectively removes the 0-agglutinins from 0.1 ml of an undiluted 0-antiserum. Absorbed antisera are tested by tube agglutination in serial two-fold dilution steps (1:40-1:10,240) with dense suspensions prepared from several strains of the homologous'and absorbing bacteria. Absorbed antisera should be free of 0-agglutinins for the absorbing culture, or cultures, at a dilution of 1:40 or less. If not the absorption procedure has to be repeated. The absorbed sera should then be tested by slide agglutination in dilutions of 1 5 , l:lO, 1:lZ 1:20 etc. A dilution of antiserum that causes rapid and complete agglutination of the homologous serotype should be selected for use. For polyvalent

3. SHIGELLA

139

grouping antisera the dilutions usually range from 1:5 to 1:25. For factor sera for specific determinants the dilutions range from 1:5 to 1:lOO. 6 . Slide agglutination for type determination

It is recommended that a suspension is used rather than straight sweeps from the living culture. There are several advantages of using a suspension (i) a convenient volume of the strain is available for multiple tests, (ii) sooner or later a heated suspension will be required for agglutination and (iii) since shigellae are infectious to laboratory workers a suspension inactivated by the mercuric iodide solution (see above) provides safety in the laboratory work. The strain suspended in the mercuric iodide solution is diluted to approximately 5 x lo9 bacteria ml-'. This rather heavy suspension can easily be assessed by eye after short experience. The Shigella suspension is also used at a concentration of approximately 2 x lo8 bacteria ml- in tube agglutination tests. First examine on the slide a drop of the agglutinating suspension for autoagglutinability. If this is positive no further testing can be done with that suspension. Try rapid serial subculturing on nutrient agar which may produce a smoother suspension, or try growth at 2 0 T , or wash off the growth with an 0.2% solution of formalin in distilled water instead of mercuric iodide and check again for auto-agglutinability. Equal sized drops of the appropriate sera and the suspension are put on to the glass slide and mixed with a wire. The slide is then gently rocked and observed for agglutination for not more than 2 min. The drops of serum and suspension should not be too small or the results may be difficult to interpret. Similarly, the suspension should not be too dense or positive reactions may be masked. Never use a dirty serum. The sera may reveal a precipitate after storage. This precipitate may give rise to the report of false-positive agglutinations if not detected. The precipitate may originate because of (i) residues after the absorptions which were not completely removed by centrifugation, (ii) formation of lipoprotein aggregates in the rabbit serum or (iii) growth of a contaminating micro-organism. If growth has occurred discard the serum at once. The other types of precipitates may be removed by centrifugation. Test the titre of the antiserum after centrifugation. The bacterial suspension is first tested using polyvalent grouping antisera. I f agglutination is found with a particular grouping antiserum the suspension should be tested with the factor or type sera represented within the polyvalent antiserum. If the testing is done with an unheated suspension which fails to agglutinate in any of the polyvalent antisera it should be heated in a beaker of boiling water for about 15 min, cooled and retested in the same antisera.

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W. H. EWING AND A. A. LINDBERG

Many shigellae possess envelope or capsular* substances that inhibit agglutination of living (unheated) bacteria in 0-antisera, i.e. in antisera produced with heated ( 100°C, 1 h) vaccines. These substances are inactivated by heat. A suspension that fails to agglutinate in polyvalent antisera for Shigella should be tested in polyvalent and grouping antisera for samonellae. In particular, such a suspension should be tested in somatic group D (9,12) and Vi-antisera, since the culture may be a strain of Salmonella typhi that fails to produce hydrogen sulphide. In the authors’ experience, about 5% of cultures of S. typhi fail to form discernable hydrogen sulphide in triple sugar agar or similar media. Cultures that give the appearance of shigellae but fail to agglutinate in any of the antisera mentioned above should be tested in antisera for serotypes of Shigella not represented in the polyvalent sera and should be subjected to additional biochemical tests. Such strains may be shigellae, agglutinins for which are not contained in the polyvalent antisera, or they may be members of another genus. Regardless of the outcome of serological tests cultures suspected of being shigellae must be subjected to biochemical tests, since the 0-antigens of the serotypes of Shigella are identical with, or closely related to, those of E. coli.

Acknowledgements Lindberg’s work was supported by the Swedish Medical Research Council (Grant No. 16X-656) and the Swedish Board for Technical Development (Grant No. 78-4039).

Referencest Brenner, D. J . (1978). Prog. Clin. Pathol. 7 , 71-117. Boyd, J. S. K. (1938). J . Hygiene 38, 4 7 7 4 9 9 . Boyd, J. S. K. (1940). Trans. R. SOC.Trop. Med. Hyg. 33, 553-571. Boyd, J. S. K . (1948). J. Trop. Med. Hyg. 51, 169-170. Dmitriev, B. A., Bakinovsku, L. V., Lvov, V. L., Knirel, Y. A., Kochetkov, N. K. and Khomenko, N. A. (1975a). Carbohydr. Res. 41, 329-333. Dmitriev, B. A., Knirel, Y. A., Kochetkov, N. K. and Hofman, I. L. (1975b). Carbohydr. Res. 44, 77-85. Dmitriev, B. A., Knirel, Y. A., Kochetkov, N. K. and Hofman, I. L. (1976a). Eur. J . Biochem. 66, 559-566. Dmitriev, B. A., Lvov, V. L., Kochetkov, N. K., Jann, B. and Jann, K. (1976b). Eur. J . Biochem. 64, 491498. Dmitriev, B. A., Bakinovsku, L. V., Knirel, Y. A., Kochetkov, N. K . and Hofman, I. L. (1977a). Eur. J . Biochem. 78, 381-387. *There are occasional strains with visible capsules. The chemical nature of these surmized capsules has not been investigated. t The literature search for this chapter was concluded in March 1981.

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Dmitriev, B. A., Knirel, Y. A., Kochetkov, N. K., Hofman, I. L. and Capek, K. (1977b). Eur. J. Biochem. 76, 433-440. Dmitriev, B. A., Knirel, Y. A., Kochetkov, N. K., Jann, B. and Jann, K. (1977~).Eur. J. Biochem. 79, 11 1-1 15. Dmitriev, B. A., Knirel, Y. A., Sheremet, 0.K., Kochetkov, N. K. and Hofman, I. -. (1977d). Bioorg. Chem. 3, 1219-1225. Dmitriev, B. A., Lvov, V. L. and Kochetkov, N. K. (1977e). Carbohydr. Res. 56, 207-209. Dmitriev, B. A., Lvov, V. L., Kochetkov, N. K. and Hofman, I. L. (19770. Bioorg. Chem. 3, 12261233. Dmitriev, B. A., Knirel, Y. A., Vinogradov, E. V., Kochetkov, N. K. and Hofman, 1. L. (1978a). Bioorg. Chem. 4, 4 0 4 6 . Dmitriev, B. A., Lvov, V. L., Ramos, E. L., Kochetkov, N. K. and Hofman, 1. L. (1978b). Bioorg. Chem. 4, 760-766. Dmitriev, B. A., Knirel, Y. A., Shermet, 0.K., Shashkov, A. A., Kochetkov, N. K. and Hofman, I. L. (1979). Eur. J. Biochem. 98, 309-316. Edwards, P. R. and Ewing, W. H. (1972). “Identification of Enterobacteriaceae”, 3rd edn. Burgess, Minneapolis, Minnesota. Ekborg, G., Eklind, K., Garegg, P. J., Gotthammar, B., Carlsson, H. E., Lindberg, A. A. and Svenungsson, B. (1977). Immunochemistry 14, 153-157. Ewing, W. H. (1949). J. Bacteriol. 57, 633-638. Ewing, W. H. (1953). J . Bacteriol. 66, 333-340. Ewing, W. H. (1954). J . Immunol. 72, 404410. Ewing, W. H. (1967). “Revised Definitions for the Family Enterobacteriaceae, its Tribes and Genera”. CDC Publication. Ewing, W. H. (1972a). Public Health Laboratory (Journal of the Conference of Public Health Laboratory Directors) 30, 146-160. Ewing, W. H. (1972b). “Isolation and Identification of Salmonella and Shigella”. CDC Publication. Ewing, W. H. (1973). “Differentiation of Enterobacteriaceae by Biochemical Reactions”. CDC Publication. Ewing, W. H., Tatum, H. W. and Davis, B. R. (1956). “Studies on the Serology of Escherichia coli”. Gemski, P. Jr, Koeltzow, D. E. and Formal, S . B. (1975). Infect. Immun. 11,685491. Green, M. (1977). I n “DNA Insertion Elements, Plasmids and Episomes” (A. I. Bukhari, J. A. Shapiro and S. L. Adhya, Eds), pp. 437445. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Iseki, S. and Hamano, S . (1959). Proc. Jpn. Acad. 35, 407412. Jansson, P.-E., Lindberg, B., Bruse, G., Lindberg, A. A. and Wollin, R. (1977). Carbohydr. Res. 54, 261-268. Jansson, P.-E., Lindberg, A. A., Lindberg, B. and Wollin, R. (1981). Eur. J. Biochem. 115, 571-577. Kenne, L., Lindberg, B., Petersson, K., Katzenellenbogen, E. and Romanowska, E. (1978). Eur. J. Biochem. 91, 279-284. Kenne, L., Lindberg, B., Petersson, K., Katzenellenbogen, E. and Romanowska, E. (1980). Carbohydr. Res. 78, 119-126. Kopecko, D. J., Washington, 0.and Formal, S. B. (1980). Infect. Immun. 29,207-214. . . Lindberg, A. A. (1977). In “Surface Carbohydrates of the Procaryotic Cell” (I. W. Sutherland, Ed.). Academic Press, New York and London. Lindberg, A. A. and Le Minor, L. (1984). I n “Methods in Microbiology” (T. Bergan, Ed.), Vol. 15, 1-142. Academic Press, London and New York.

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Matsui, S. (1958). Antigenic changes in Shigellafle.meri group by bacteriophage. Jpn. J. Microbiol. 2, 153-158.

Orskov, F. and Orskov, I. (1978). In “Methods in Microbiology” (T. Bergan and J. R. Norris, Eds), Vol. 1 1 , pp. 1-77. Academic Press, London and New York. Petrovskaya, V. G. and Bondarenko, W. M. (1977). Int. J. Sysf. Bacreriol. 27, 171-175. Rowe, B., Gross, R. J. and Guiney, M. (1976). h i . J . Syst. Bacferiol. 26, 76-78. Rowe, B., Gross, R . J. and Woodroof, D. P. (1977). Inf.J. Sysr. Bacreriol. 27, 15-18.

4 Serotyping of Klebsiella I. ORSKOV AND F. ORSKOV Collaborative Centre for Reference and Research on Escherichia and Klebsiella, ( W H O ) , Statens Seruminstitut, Copenhagen, Denmark

. I. Taxonomy . Surface structures . . . . A. Morphology . B. Chemistry of K-antigens . . C. Chemistry of 0-antigens . . . 111. Antigenic scheme . . . IV. Cross-reactions between Klebsiella antigens A. Cross-reactions between K-antigens . . . B. Cross-reactions between 0-antigens . . . V. History of typing procedures . . . A. Serological methods . . B. Non-serological methods . . . VI. Antiserum production . . A. K-antiserum production . . . B. 0-antiserum production . . . VII. Serological K-antigen determination . . A. Capsular swelling reaction . . B. Agglutination. . . C. Agglutination and capsular swelling reaction . . D. Indirect immunofluorescence . . E. Counter-current immunoelectrophoresis . . VIII. Serological 0-antigen determination . . A. Agglutination. . . IX. K-antigen type stability . . X. Evaluation . . . XI. Correlation with pathogenicity - special K-antigen types . XII. Cross-reactions between Klebsiella K-antigens and antigens of other bacteria . . XIII. Cross-reactions between Klebsiella and eucaryotic cells . . . References . 11.

143 145 145 146 146 147 150 150 151

151 151 152

153 153 154 155 155 155 155

156 156 151 151 151 157 158

159

160 161

I. Taxonomy Klebsiellu bacteria are Gram-negative, capsulated non-motile rods which constitute the genus Klebsiella in the family Enterobacteriaceae (Buchanan METHODS IN MICROBIOLOGY VOLUME 14

Copyright L 1984 by Academic Press. London All rights of reproduction in any form reserved.

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I. ORSKOV AND F. ORSKOV

and Gibbons, 1974). The following characters are useful for distinction from other members of the family: acid produced from inositol, urea hydrolysed, citrate utilized, hydrogen sulphide and ornithine decarboxylase not produced. In general, the methyl red (MR) test is negative and the Voges-Proskauer (VP) test is positive. However, too much importance should not be attached to these two tests, as some Klebsiella strains are MR positive and VP negative and both tests may be positive or negative. Buchanan and Gibbons (1974) defines three species: K. pneumoniae, K. ozaenae and K. rhinoscleromatis. However, division into several species has been proposed; Cowan et al. (1960) recognized five species: K. aerogenes, K. edwardsii, K. pneumoniae (sensu stricto), K. ozaenae and K. rhinoscleromatis. Later Bascomb et al. (1971) defined six taxa in the genus Klebsiella based on numerical taxonomy. One of these taxa was K. pneumoniae (sensu stricto), another taxon comprised K. aerogenes, K. edwardsii and indole-forming Klebsiella strains ( K . oxytoca). K. oxytoca was excluded from the Klebsiella group by Cowan et al. (1960). Using DNA hybridization experiments K. pneumoniae, K. ozaenae, K. rhinoscleromatis and K. edwardsii were shown to be highly related (Brenner et al., 1972). In a large study on numerical taxonomy of 180 clinical and non-clinical Klebsiella isolates, on which 99 tests were used, Naemura et al. (1979) concluded that K. pneumoniae (sensu stricto) should include K. aerogenes, K. edwardsii and K. pneumoniae (sensu stricto), as described by Cowan et al. (1960) and the groups K. pneumoniae (sensu stricto) and K. aerogenesloxytocaledwardsii (except oxytoca), as described by Bascomb (197 1). Most strains in the K. pneumoniae (sensu stricto) group of Naemura et al. (1979) were of clinical origin. Strains which were selected from this group exhibited 73-100% relative association in DNA hybridization studies to the K. pneumoniae reference strain (Woodward et al., 1979). Indole-positive and gelatine-liquefying strains have been a problem for several years. Some authors have considered them as biotypes of K. pneumoniae (Edwards and Ewing, 1972; Buchanan and Gibbons, 1974), others as a separate group (Lautrop, 1956; Stenzel et al., 1972) and others again have excluded such strains from their Klebsiella studies (Cowan et al., 1960). Based on DNA hybridization experiments it has been proposed removing these strains from the genus Klebsiella (Jain et al., 1974) or considering them as a separate Klebsiella species ( K . oxytoca) (Brenner et al., 1977). In the study by Woodward et al. (1979) the K. oxytoca group exhibited 84-100% relative reassociation to a strain previously referred to as K. oxytoca. Strains of the K. oxytoca group were of both clinical and environmental origin and were different biochemically from those of the K. pneumoniae (sensu stricto) group (Naemura et al., 1979) in that they produced indole, degraded

4. KLEBSIELLA

145

pectate (Von Riesen, 1976),grew at 10°C (Naemura and Seidler, 1978)and did not ferment lactose with gas production at 44°C (faecal coliform test). A third and a fourth proposed species of Klebsiella mainly comprising strains of non-clinical origin are K . terrigena (Izard et al., 1981) and K. planticola (Bagley et al., 1981). According to numerical taxonomy, (Gavini et al., 1977; Naemura et al., 1979) and DNA-DNA hybridization studies (Woodward et al., 1979; Izard et al., 1981; D. J. Brenner, personal communication) they form species distinct from each other and from K. pneumoniae and K . oxytoca. At present it seems most reasonable to distinguish between the following groups in the genus Klebsiella: 1. K. penumoniae (including K . ozaenae and K . rhinoscleromatis),mainly of clinical origin; 2. K. oxytoca, of both clinical and environmental origin; 3. K . terrigena (new proposed species) primarily from soil and aquatic environments; 4. K . planticola (new suggested species) primarily from soil and botanical environments.

11. Surface structures

A. Morphology

-

The cell wall of Klebsiella is structured as that of other Enterobacteriaceae (Jann and Jann, 1977). It consists of three layers: the cytoplasmic membrane; the peptidoglycan layer (murein sacculus), which maintains the rigidity and shape of the cell and the outer membrane consisting of a complex of lipopolysaccharide (LPS), phospholipid and protein. The LPS forms the 0(or R) antigen. Twelve Klebsiella 0-groups have been numbered (see later). Furthermore, Klebsiella are enveloped by a polysaccharide capsule of considerable thickness and because of this Klebsiella form glistening mucoid colonies of viscid consistency. Antigenic capsular material is termed the capsular or K-antigen. Eighty-two K-antigens have been reported (see later). The capsular polysaccharide also diffuses freely into the surrounding liquid medium as extracellular capsular material. In addition, many Klebsiella strains possess fimbriae which protrude on the surface of the cells. All fimbriate Klebsiella strains show one or the other or both of two kinds of adhesive property: one attributed to an “MSadhesin”, susceptible to inhibition by D-mannose and associated with a thick variety of

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1. ORSKOV

AND F. ORSKOV

fimbriae termed type I , the other due to an “MR adhesin”, resistant to mannose and associated with thinner fimbriae, termed type 3. Bacteria with these fimbriae do not agglutinate fresh erythrocytes unless these are first treated with tannic acid (Duguid, 1959; Duguid and Old, 1980). Fimbriae are composed of protein (Brinton, 1965).

B. Chemistry of K-antigens The capsular polysaccharides of about 80 different K-antigen types have been qualitatively analysed (Nimmich, 1968, 1971). Today the structure of a majority of these types has been determined. The types proposed before 1976 (25 in all) were summarized by Heidelberg and Nimmich (1976). References and structures of an additional 15 K-antigen types can be found in the paper by Rieger-Hug and Stirm (1981). Reports on structures of a further five Klebsiella K-antigens have appeared recently (Dutton and Paulin, 1980a,b; Dutton and Fabio, 1980; Dutton and Savage, 1980; Okutani and Dutton, 1980). The Klebsiella capsular polysaccharides, as reviewed by Jann and Jann ‘(1977), are acidic and composed of repeating units. In spite of the many serologically different K-antigens only few sugar constituents are found. The majority of K-antigens contain only a charged monosaccharide constituent most often glucuronic acid -and hexose(s); 6-deoxyhexoses may also be present. Furthermore, non-carbohydrate constituents, such as formyl or acetyl groups and ketal-linked pyruvate are found. These may function as antigenic determinants and may be the cause of cross-reactions of some Klebsiella polysaccharides. Very little is known, however, about the immunochemical specificity of Klebsiella polysaccharides. The repeating unit of three Klebsiella capsular polysaccharides are shown in Table I (K: 1, Erbing et al., 1976; K:2, Gahan el al., 1967; Sutherland, 1972; K:5, Dutton and Yang, 1973). C. Chemistry of 0-antigens

The LPS forming the 0-antigens is composed of three regions, I, I1 and Ill (Liideritz er al., 1966). The outermost part or region I consists of oligosaccharide repeating units which are the chemical basis of the 0-antigen; this region is called the 0-specific polysaccharide. The middle part or region 11 is an oligosaccharide termed the core oligosaccharide, which expresses the R (rough) antigen specificity. The innermost part or region Ill is the lipid moiety of &hemolecule. I t is termed lipid A and this is the part of the LPS which by hydrophobic interaction is anchored to the lipoprotein of the outer cell membrane of the bacterial cell (Braun and Hantke, 1974).

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4. KLEBSIELLA

TABLE 1

Structures of K-antigen polysaccharides from Klebsielln K-antigen

Repeat unit

3 1

- - 1,4

+ Glc

1,4

GlcA

P

II

P

1

FUC a

2 , 3 Pyr

4 2

+

1,3

Glc-

1,4

B

a

1

Man-

Glc

a11,3

GlcA

4 5

-+

1,4

GlcA

1,3

P

Ac

1

Man

Glc

B

+

II

4,6Pyr

Symbols for sugars as suggested by IUPAC-IUB (1966).

The oligosaccharide repeating units of the 0-antigens consist of only a few sugar components in Klebsiella, in contrast to Salmonella, and only a small number of different 0-antigens exist in Klebsiella. The structure of nine Klebsiella 0-antigens is shown in Table I1 (0:10,0:1,0:6and 0:4, Bjorndal et al., 1970, 1971, 1972; 0:3 and 023,Curvall et al., 1973a,b; 0 : 9 and 0:5, Lindberg et al., 1972a,b; 0:7, Simmons et al., 1965). It is seen that three of the are homopolysaccharides nine 0-antigens, 0:1 (identical to 0:6), 0:3 and 05, being either galactans or mannans. Two 0-specific homopolysaccharides are found in E. coli, 0:sand 0:9, and they are both mannans (Reske and Jann, 1972; Prehm et al., 1976). Klebsiella 0 : 3 and E. coli 0 : 9 are identical both serologically (Kauffmann, 1949) and in their chemical structures. Klebsiella 0 : 5 was found to be serologically identical with E. coli 0:s(IZlrskov, 1954a) and their structures are very similar but not identical. Klebsiella 0 : 4 is serologically closely related to E. coli 0:20 (IZlrskov, 1954a); its chemical structure seems, however, identical with that described for E. coli 0:20 (Vasilieu and Zakharova, 1976). Klebsiella 0:3 and 0:5 are very closely related chemically, as seen in Table 11, but serologically they are quite distinct.

111. Antigenic scheme

Although Klebsiella strains possess both 0-and K-antigens serological typing is based on examination of the K-antigen for two reasons: (1) the number of

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1. ORSKOV AND F. ORSKOV

TABLE I1 Structures of 0-antigen polysaccharidesfrom Klebsiella 0-antigen

Repeating unit 3

1,6

+

1 Gal + U

3

5

- - - -

3 1,2 1,3 1,2 1,2 1 Man Man Man Man Man--,

+

U

2 4

+

1,4

Ribf

U

U

- - 1,2

+ L-Rhap

U

U

1 Galp +

B

2 7

U

1,3

Ribf

U

2/6OAc

B

1,3

L-Rhap

1 L-Rhap 4 a

U

2OAc 2160.4~ I

3 8

+

f

3 9

+

10

+

I

-

; 1,3

1,3

Galp

i1,3 f 1 GalfGalp + fl. 3 Galp

- - - 1,3

L-Rhap

1

Galp +

214 OAc 214 OAc 214 OAc

Galf-

3

1

1,3

Galf-

214 OAc i

i

1,3

Galp-

1,4

Ribf

U

B

1,3

L-Rha

194

Ribf

U

B

1 L-Rha + U

different K-antigens is higher than that of different 0-antigens (82 K-antigens have been described but only 12 0-antigens); (2) 0-antigen determination is difficult because it is hampered by the heat stable K-antigens. Table 111 gives an antigenic scheme which lists strain number, K-antigen and 0-antigen. Several authors have contributed to the establishment of

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4. KLEBSIELLA

TABLE I11 0 : K-serotypes of Klebsiella K-antigen reference strains 0-antigen 1 1 2 2 2 2 1 1

R"

1 3 1

R" R"

4 1 R" R" 1 1 1 1 1 1 3 1 2 2 1 1 3 1 3 1 2 R" 1

R" 1 R" 1

K-antigen

Strain number

0-antigen

K-antigen

Strain number

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=Br lSb 27=Br 16 28=Br 17 29=Br 18 30=Br 19 31=Br21 32=Br 22 33=Br 23 34 = Br 24 35=Br 25 36 =Br 26 37=Br 27 38=Br 28 39=Br 29 40=Br 31 41=Br 32

A 5054 B 5055 C 5046 D 5050 E 5051 F 5052 Aerogenes 4140 Klebsiella 1015 Klebsiella 56 Klebsiella 919 Klebsiella 390 Klebsiella 313 Klebsiella 1470 Klebsiella 1193 Mich. 61 2069149 2005149 1754149 293150 889150 1702149 1996149 2812150 1680149 2002149 5884 6613 5758 5725 y 7824 6258 6837 6168 7522 7444 8306 8238 8414 7749 8588 6177

4 2 1 1 1 1 3 3 3 3 R" 3 3 3

42=Br 33 43 =Br 34 44=Br 35 45=Br 36 46 =Br 38 47=Br 39 48=Br 40 49=Br 41

1702 2482 7730 8464 5281 9682 1196 6115 1303/50 47 15/50 5759150 1756151 Stanley 3985151 3534151 4425151 636152 22 12/52 4463152 5710152 5711/52 5845152 NCTC 8172 sw4 438 (3a) 264 (1) 265 (1) 889 167 4349 1205 337 371

R" 5 3 2

R" 5 1 1 6 o r 1' 1 1 7 1 8 or 2' 1 5 9 or 2' 10 3 2,4 1 1 11 1 12

R" R"

50

51 52 53 54 55 56 57 58 59

60 61 62 63

64

65 66 67 68 69 70 71 72 73' 74 75' 76' 77' 78' 79 80 81 82

-

378 325 708 370 3454-70

Rough IR) or not determined. Br 15 was a provisional name, K 2 6 is the present number (Kauffmann, 1954). See text.

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I. ORSKOV AND F. ORSKOV

Kfebsieffu K-antigens: K: 1-K: 14, Kauffmann (1949); K: 15, Worfel and Ferguson (1951); K: 16-K:25, Edwards and Fife (1952); K:26-K:49, Brooke (1951); K:50-K:57, Edwards and Fife (1952); K:58-K:63, Edwards and Fife (1955); K:64-K:69, Edmunds (1954); K:70-K:72, Orskov (1955); K:73-K:76, Maresz-Babczyszyn (I 962); K:77-K:80, Durlakowa et al. ( I 963); K:8 1, Nimmich and Miinter (1975) and K:82, Orskov and Fife-Asbury (1977). The 12 0-antigens were established as follows: 0:1-0:3, Kauffmann (1949); 0 : 4 0 : 5 , Brskov (1954a); 0:6-0:9 and 0:ll-0:12, Durlakowa et ul. (1960, 1963) and 0:10, Maresz-Babzyszyn (1962). 0-antigen determination of the Klebsiella K-antigen test strains was carried out by the following authors: K: 1-K:14, Kauffmann (1949); K: 15-K:62, Orskov (1954a); K:63-K:72, Durlakowa et al. (1960); K:73-K:76, Maresz-Babczyszyn (1962) and K:77-K:80, Durlakowa e f al. (1963). Antigens K:73 and K:75-K:78 could not be confirmed by Orskov and FifeAsbury (1977) for the following reasons: K:73 was motile and so did not belong to the Klebsielfa group; K:75, K:77 and K:78 were found to be serologically identical with K:68, K:39 and K: I5 respectively, and K:76 was only slightly different from K:43. By examination of cross-reactions between pneumococci and Kfebsiellu Heidelberger et ul. (1978) found that K:76 and K:43 had definite chemical differences in their capsular polysaccharide. Based on serological (Kduzewski, 1965) and chemical (Nimmich and Korten, 1970; Bjorndal et al., 1971) similarity 0 : l and 0 : 6 have been combined and it was proposed by Kafuzewski (1965) to regard 0:8 and 0 : 9 as 0:2.

IV. Cross-reactions between Klebsiella antigens A. Cross-reactions between K-antigens

Cross-reactions among established Klebsielfa antigens are numerous (Kauffmann, 1949; Henriksen, 1954a; Edwards and Fife, 1952, 1955; 0rskov, 1955). The majority of the K-antigen types will react with one or more heterologous antisera, and in several cases it is necessary to absorb the antisera for diagnostic use. It is a well known fact that different animals have different capacities to form antibodies; such differences were found to be most pronounced in overlapping reactions (Orskov, 1955). Henriksen (1954d,e) pointed out the failing similarities between the cross-reactions found with antisera produced in different laboratories and suggested that antigenic variations might be the most important factor for this phenomenon. Since the number of cross-reactions varies it is impossible to give a table of such reactions. However, some strong reactions which have been reported more than once are the following: K: 1-K:6, K:2-K:69, K:2-K: 13-K:30, K:3-K:68, K:7-K:10, K:ll-K:21, K:12-K:29-K:42, K:l&K:64, K:18-K:44,

4. KLEBSIELLA

151

K:22-K:37, K:24-K:43, K:27-K:46 and K:70-K:72 (Brooke, 1951; Henriksen, 1954a; Riser et al., 1976b; Palfreyman, 1978; Orskov and Orskov, 1978; Edmondson and Cooke, 1979b).

B. Cross-reactions between 0-antigens The relationships between the 12 established Klebsiella 0-antigens are not fully clarified serologically. The 0-antigens of 0-group 2 are heterogeneous. Kauffmann (1949) demonstrated 0-antigen factor 2a, 2b and 2c. Later an even more complex structure of 0-group 2 was found when additional strains were examined (Orskov, 1954a). Regarding 0 : 6 , 0 : 8 and 0 : 9 see the antigenic scheme in Section 111.

V. History of typing procedures A. Serological methods

Klebsiella strains can be divided into types by serotyping their K- and 0antigens, phage and bacteriocin typing and biochemical typing. The first serological differentiation of Klebsiella strains was carried out by Julianelle (1926) based on a determination of the K-antigen. He divided Klebsiella strains (Friedlander’s bacillus) isolated from patients with pneumonia into types A, B, C and a heterogeneous group X. By means of agglutination, precipitation, absorption and mouse protection tests these capsular antigens were shown to be highly specific. Later, Goslings (1933) and Goslings and Snijders (1936) established types D, E and F, all originating from cases of ozaena. In 1949 Kauffmann extended the number of K-antigens to 14. K:l-K:6 were those previously designated A-F. Kauffmann used both the bacterial agglutination and the capsular swelling technique, but preferred the latter because a specific K-antigen reaction was obtained with no involvement of 0-antigen. However, the capsular swelling method has disadvantages, such as difficulties in distinguishing between the many cross-reacting types and in detecting weak reactions; and so decisions about the labelling of unknown strains to a certain K-type can be subjective. Therefore, other methods have been considered. An indirect fluorescent-antibody technique (IFA) for Kantigen typing of Klebsiella was described by Riser et al. (1976a,b) who reported that this method gave close correlation with the swelling technique and had certain advantages. It allowed more economical use of expensive antisera because higher dilutions could be employed, thereby excluding many cross-reactions, and the results were easier to read. Klebsiella serotyping by the gel precipitation technique, called counter-

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current immunoelectrophoresis (CCI), was reported by Palfreyman (1978) to be more specific, more economical and less time-consuming than the swelling method. As mentioned above, Klebsiella also possess 0-antigens. Kauffmann was the first to demonstrate that in addition to K-antigens Klebsiella contain 0antigens. He described the first three 0-antigen groups (see above).

B. Non-serological methods 1 . Phage typing

Differentiation by phages selected from stools has been attempted and a typing scheme using 14 selected phages has been proposed (Slopek et al., 1967; Slopek, 1978). Only in few cases did the phage typing agree with serological Kantigen typing. However, Rieger-Hug and Stirm (1981) examined 55 Klebsiella K-antigen specific phages, which means that these phages induce formation of enzymes that depolymerize the capsular polysaccharides. Thirty-three of these phages were active against only one and 18 against only two K-antigen types. 2. Bacteriocin typing Bacteriocin typing of Klebsiella has been proposed by some authors (Slopek and Maresz-Babczyszyn, 1967; Hall, 1971; Buffenmeyer et al., 1976; Slopek, 1978; Edmondson and Cooke, 1979a). Receptor sites for bacteriocins are not capsular specific, but Edmondson and Cooke (1979a) reported that there was a generally close agreement between the results from K-serotyping and the bacteriocin (klebecin) typing methods. In a later report on occurrence of Klebsiella in hospitals and patients primarily the capsular swelling but also the klebecin typing method were used, and it was stressed that the use of more than one typing method allows better discrimination between strains (Cooke ef al., 1980). 3. Biochemical typing

Subdivision of Klebsiella can also be carried out by biochemical typing. According to Kauffmann (1949) the fermentation of dulcitol and organic acids, particularly D-tartrate, sodium citrate and mucate, the decomposition of urea, as well as the Voges-Proskauer test, are especially suitable for differentiation. Orskov (1957) found 26 biotypes among 125 indole- and gelatin-negative strains belonging to 68 K-antigen types, based on the dulcitol, adonitol, sorbose, urease, D-tartrate and sodium citrate tests. Fifty

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indole- and/or gelatin-positive strains of 29 capsule types belonged to ten different biotypes; the highest number of K-antigen types within a single biotype was 11. When the two typing methods were combined 149 K-antigen biotypes could be differentiated. The total number of strains was 220, including the 72 K-antigen reference strains. Another report described 27 distinct biotypes among 270 Klebsiella strains belonging to 40 K-antigen types. This result was based on the indole, Voges-Proskauer, citrate, lactose, sucrose, malonate, gluconate, dulcitol, lysine and urea tests. The two typing methods varied independently, and when combined more than 100 types were distinguished (Rennie and Duncan, 1974).

VI. Antiserum production A. K-antiserum production For serotyping of Klebsiella K-antigens a K-antiserum is prepared against non-heated cultures. In our laboratory two rabbits are injected intravenously five times at three- to four-day intervals with 0.2, 0.5, 1.O, 1.5 and 2 ml of a freshly made bacterial suspension. In general, we use bromothymol blue lactose agar as solid medium for growth because capsules are well developed on this carbohydrate-containing medium and a good distinction is obtained between colonies producing various amounts of capsular substance. A suitable colonly, i.e. a colony not too mucoid, is selected and used for inoculation of a fresh plate which is incubated at 37°C overnight. A suspension in saline with 0.5% formalin is used as vaccine. The bacterial density for the two injections. is about 5 x 108/mI and 1 x 109/ml for the following injections. The rabbits are exsanguinated one week after the fifth injection, as further injections with these polysaccharide-containingvaccines seldom increase the titres. A higher yield of antiserum can be obtained if 40 ml of blood is taken from the ear vein a day or two before the final bleeding. Our antisera are preserved by 0.01% merthiolate and 1% chloroform. Some workers use sodium azide (0.08%) for preservation (Palfreyman, 1978). Edmondson and Cooke (1979b) recently compared three methods of Kantiserum production involving vaccination with formalin-treated broth cultures. They concluded that the poorest antibody result was obtained when the same culture was used for each inoculation and that a freshly prepared broth culture gave better results after growth for 16 h than after growth for 4-6 h. An evaluation of the antisera produced is carried out by a titration. For this purpose the capsular swelling reaction (or another K-antigen-antibody

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reaction) is examined in two-fold dilutions of the antiserum and the titre is expressed as the reciprocal value of the highest dilution still causing a positive reaction. Depending on this result the antisera are pooled or kept separately and used at a certain dilution. The titres of Klebsiellu K-antisera rarely exceed 128 and are usually about 32. In some cases it is difficult to obtain titres higher than 4. Cultures producing moderately sized capsules are better immunogens than those producing large capsules (Perch, 1950). Huge capsules or abundant extracellular capsular substance may cause immunological tolerance, previously called immunological paralysis, and is shown with capsule type 5 (Orskov, 1956). In that case a better antibody response was obtained if most of the solubilized capsular material was removed by centrifugation before formalin was added or, even better, if the culture was heated to 100°C before centrifugation in order to eliminate as much of the capsular substance as possible. Klebsiellu K-antisera can be obtained commercially from Difco. Personally, we have no experience with such antisera (Casewell, 1972, 1975).

B. 0-antiserum production For determination of Klebsiella 0-antigens it is strongly recommended to use 0-antisera although K-antisera in reality are OK-antisera since they also contain 0-antibodies. The antigenicity and probably also the immunogenicity of Klebsiellu capsules are preserved after heating to 100°C. Therefore, Klebsiella cultures should be heated to 121°C for 2 h for production of 0antisera in order to prevent formation of K-antibodies. When possible, however, K- mutants should be selected for 0-antiserum production as the immunogenicity of the 0-antigen is somewhat impaired by treatment at 121 C. The best method for isolation of K-mutants is t o leave a plate culture at room temperature after primary incubation at 37°C. Translucent sectors may then eventually be formed at the edge of the mucoid growth. Such sectors usually, but not necessarily, contain K - mutants, i.e. 0- (smooth) or R(rough) forms. They may still form a small, but sufficient, amount of capsular material for production of K-antibodies. After purification of the supposed 0-form, antisera are prepared against culture heated to 100°C for 2.5 h either as a suspension from an agar surface or as a broth culture incubated at 37°C for 18-20 h. The boiled 0-antigen preparation is preserved with formalin (0.5”/,),allowing it to be used for all injections. The vaccination schedule is the same as mentioned above for K-antiserum production.

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VII. Serological K-antigen determination A. Capsular swelling reaction

K-antigen determination of Klebsiella strains has traditionally been carried out by the capsular swelling technique (Kauffmann, 1949; Edwards and Ewing, 1972). This test is performed by mixing equal volumes, i.e. standard loopfuls, of the bacterial suspension (1 x 108/ml) and the antiserum on a glass slide. After application of a coverslip the preparation is examined under the ordinary light microscope or the phase-contrast microscope using an oil emersion objective ( x 90). A preparation with saline substituted for the antiserum is examined simultaneously for the control. By addition of the specific K-antiserum a precipitin reaction occurs at the bacterial surface. This makes the capsule highly refractile and easily visible microscopically as a thin black line delineating the edge of the capsule. If an Indian ink preparation is compared with the antiserum preparation it will be seen that addition of antiserum to very large capsules seems to cause shrinkage, whereas small capsules may look larger. An Indian ink preparation is made by mixing a small loopful of Indian ink with a large loopful of the bacterial suspension. Care should be taken not to use too much Indian ink in order not to cover the marginal periphery of the capsule with black particles. Some Klebsiella strains are very stringy and produce an abundant intercellular capsular-specific substance which, after addition of specific antiserum, is seen as finely granulated precipitates or as bands of a thread-like material which has entangled swelled or unswelled bacteria.

B. Agglutination Serological K-antigen determination may also be carried out using the bacterial agglutination technique either on the slide or in tubes. However, as both 0-and fimbrial antigen-antibody reactions will take place at the same time it cannot replace a K-specific method.

C. Agglutination and capsular swelling reaction In practice, the K-serotyping can be carried out as follows. The culture is grown on a medium considered to be useful for capsule development, such as bromothymol blue lactose plates (0rskov et al., 1976) Worfel-Ferguson agar plates (Difco) or other plates with a carbohydrate-rich medium.

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If the culture appears poorly capsulated after incubation at 37°C for 16-20 h it may be advantageous to leave it at room temperature for an additional 24-48 h. A dense suspension (10’’ bacteria/ml) in 10% formalintreated phosphate-buffered saline is made for a slide agglutination test. This suspension can be kept for several days in the cold. For the capsular swelling reaction the suspension is diluted to approximately lo8 bacteria/ml. The serotyping procedure is commenced by a slide agglutination test, which is easy and fast to carry out. In a positive agglutination reaction coarse granulates appear rapidly. If serum pools are used single sera of the positive pools are tested. Since, however, a positive agglutination reaction is not necessarily caused by the K-antigen it is always followed by a capsular swelling test with the antisera which brought about the agglutination. In case the culture reacts with two or more antisera of known relationship, absorbed antisera should be tested when such are available. Otherwise the degrees of swelling in the “positive” antisera are compared or the culture is examined in serial dilutions of the antisera, in order to see in which serum it reacts to the homologous titre. If the culture reacts with two or more antisera which are not known to be related, it is assigned to both or all the capsular types corresponding to the antisera with which it reacted. It should be emphasized that assigning a strain to a certain K-antigen type does not mean that the capsular antigen is identical to that of the reference strain; true identity can only be demonstrated by crossabsorption. Several Klebsiellu cultures agglutinate with most antisera, probably because of a fimbria-anti-fimbria reaction; in such cases only the capsular swelling method should be used. D. Indirect immunofluorescence

This technique for Klebsiellu K-antigen determination, as described by Riser er ul. (1976a), is briefly mentioned here. A slide containing the bacterial smear is incubated with antiserum, rinsed, air-dried and then incubated with a sheep anti-rabbit fluorescent conjugate, rinsed and air-dried again. The slides are read using a fluorescence microscope. When it is stated (Riser er ul., 1976b) that it is possible to distinguish more easily between cross-reacting types with the fluorescent antibody than with the capsular swelling method, it is probably because it is felt easier by some people to grade the intensity of a staining reaction than a swelling reaction. E. Countercurrent immunoelectrophoresis

This gel precipitation technique has been used by Palfreyman ( 1978) for Klebsiellu K-antigen serotyping. The method described is as follows: glass

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plates, 100 mm square, are coated with a 1-mm thick layer of 1% agarose in Tris-EDTA-NaC1 buffer, pH 8.6, and five pairs of rows with 15 wells in each row are cut. The wells are filled with whole cell suspensions and antisera, and the electrophoresis is run at 20 mA per plate for 90 min.

VIII. Serological 0-antigen determination A. Agglutination The best way to determine the Klebsiella 0-antigen is to isolate nonencapsulated mutants as described above and to test boiled cultures for agglutination in 0-antisera produced with non-encapsulated mutants. If no such antiserum is available an OK-antiserum against the capsulated form may be used since 0-antibodies are present in addition to K-antibodies. The 0antigen determination can probably be carried out, as this is made with E. coli strains possessing heat-stable type A K-antigens, which means that heating to 120°C for 2 h should render the strain agglutinable in 0-antiserum. However, it should be pointed out that using OK-antisera for 0-antigen typing is dangerous, unless one is sure that the culture is non-encapsulated or the antigenicity of the capsular material has been completely destroyed.

IX. K-antigen type stability As mentioned above, cross-reactions among K-antigens are very frequent in the Klebsiella group and the fact that these-especially the weaker ones-vary from time to time, from laboratory to laboratory etc., caused Henriksen (1954d,e) to question the stability of these antigens. He suggested the possibility that the antigens undergo variations which may lead even to change of one type into another, and until the question of stability was studied, a serological classification scheme should be postponed. No such investigation has been carried out. However, when the varying cross-reactions are taken into consideration the K-serotyping of Klebsiella strains is more than feasible. X. Evaluation For the study of epidemiology and for prevention and control of Klebsiella infections serotyping of the K-antigens is valuable because of the great diversity of these antigens. In contrast, 0-antigen determination is of much

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less importance since only few different 0-antigens exist in the Klebsiella group and their determination is hampered by the heat-stable K-antigens. Whether the capsular swelling, the indirect immunofluorescent, the countercurrent precipitation or another serological method will be the choice in the future is difficult to know at present. If phages that are monospecific for all Kantigen types could be isolated the phage typing method might prove to be very useful because it is easy and not costly. However, it should be stressed that K-antigen identity between strains can only be shown by antiserum production and cross-absorption. A biochemical and perhaps a bacteriocin or a non-K-specific phage typing method carried out in conjunction with the Kantigen serotyping would increase the possibility for a greater discrimination between the strains, well knowing that extrachromosomal factors which come and go, such as plasmids and lysogenic phages, may obscure the uniformity of two chromosomally identical strains. Neither biochemical typing nor bacteriocin or bacteriophage typing alone is sufficiently sensitive for epidemiological purposes, except under certain conditions, e.g. in a certain hospital at a certain time.

XI. Correlation with pathogenicity-special K-antigen types Klebsiella are opportunistic pathogens that can give rise to bacteriaemia, pneumonia, urinary tract infections and several other infections. In recent years there has been an increase in Klebsiella infections, particularly in hospitals, and many of the strains isolated have been resistant to multiple antibiotics, mainly because of the presence of R-factors (Montgomerie, 1979). The most severe infections have been in newborns and in patients in intensive care units. Several epidemics of Klebsiella infections in urological wards have been described, one of the earlier ones from Denmark (IZlrskov, 1952, 1954b). In some cases, many different capsular serotypes have been involved and in other cases only one or two types have been isolated from clusters of infections in hospitals. Some capsular types may be incriminated more than others as nosocomial types. Some types which have been reported to be epidemic within hospitals in different countries are types 2, 8, 9, 21 and 24 (IZlrskov, 1952; Steinhauer et al., 1966; Richard, 1973; Comninos, 1977; Riser et al., 1978; Casewell and Talsania, 1979). Strains of these types may have certain advantages in the hospital environment, not because of the particular capsular type, but because of their whole genetic make up. They may spread easily because they have the ability to colonize the bowel, they may be more virulent or they may have other capacities not common to all Klebsiella strains. Klebsiella strains of capsular types 1-6 are on the whole most frequently

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associated with various infections of the upper respiratory tract. Steinhauer et al. (1966) and Eickhoff et al. (1966) did not consider these respiratory Klebsiella types to be acquired in hospitals. Strains of capsular types 1-6 are rarely isolated from faecal or urinary specimens. E. coli strains with type 1 fimbriae are trapped by tracheal slime (0rskov et a / . , 1982), and Duguid (1959), who examined 154 Klebsiella strains, found that 29 permanently nonfimbriated strains belonged to K-serotypes 1-6. Once having entered the respiratory tract strains of these types may have difficulties in being removed from that area because they do not attach to the slime which is constantly being moved upwards by the cilia1 action. It is generally accepted, but has never been proven, that Klebsiella rhinoscleromatis belonging to capsule type 3 or occasionally type 4 is the causative agent of rhinoscleroma, which is a chronic and contagious disease of the mucosa of the upper respiratory tract most commonly confined to the nasal cavity. Sporadic cases are seen all over the world, but the disease is endemic in certain areas in the eastern and central part of Europe, in the northern part of Africa and in South and Central America (Levine et al., 1947; Altmann et al., 1977). Ozaena, which is an atrophic rhinitis with an unpleasant smell, has been considered to be associated with Klebsiella ozaenae of capsular types 4 , 5 and 6. It was reported by Goldstein et al. (1978) that the spectrum of disease caused by K . ozaenae is more extensive (e.g. including bacteriaemia and urinary tract infections) than had been appreciated previously. However, the identification as K . ozaenae was based on biochemical criteria and no serotyping was carried out. Klebsiella strains have also been isolated from infections in animals. Some strains of capsular types 1,2 and 5 have been found particularly in association with metritis in mares (Dimock and Edwards, 1927; Edwards, 1928; Platt et al., 1976).

XII. Cross-reactions between Klebsiella K-antigens and antigens of other bacteria One of the earliest cross-reactions noted between Klebsiella K-antigens and polysaccharide antigens of other bacteria was that between Klebsiella K:2 and Streptococcus pneumoniae type 2 (Avery et al., 1925). Extensive crossreactions between polysaccharides of Klebsiella and pneumococci have later been demonstrated (Heidelberger et al., 1975, 1978; Heidelberger and Nimmich, 1976). Kauffmann (1949) showed that four Klebsiella K-antigens were serologically related to four different E. coli K-antigens: Klebsiella K:7, K:8, K:10 and

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K:l 1 cross-reacted with E: coli K55, K:34, K:39 and K:37 respectively. Henriksen (1954b,c) found that several E. coli strains gave capsular swelling with Klebsiella antisera, and serological identity between Klebsiella K:63 and E. coli K:42 was demonstrated by Niemann et al. (1978). As far as we know, no systematic examination of these cross-reactions has been undertaken, but results from our laboratory confirm the presence of several such cases. Serological cross-reactions between Klebsiella K: 13 and the slime wall antigen or M-antigen of S. paratyphi B was reported by Perch (1950). Regarding cross-reactions between Klebsiella and E. coli 0-antigens see chemistry of 0-antigens in Section 1I.C.

XIII. Cross-reactions between Klebsiella and eucaryotic cells About 97% of patients with ankylosing spondylitis have the histocompatibility antigen B:27 in their tissue type (Brewerton et al., 1973) and this disease is extremely uncommon in B:27-negative individuals. A serological crossreactivity between HLA-B:27-positive lymphocytes and Klebsiella has been reported by Ebringer et al. (1976). Sera from rabbits immunized with HLAB:27-positive lymphocytes showed activity against antigens from K. pneumoniae, Enterobacter aerogenes, Shigella sonnei and Yersinia enterocolitica (Welsh er a/., 1980). Since Klebsiella organisms had been found frequently in the faeces of patients with active ankylosing spondylitis (Ebringer et al., 1978) human monospecific typing sera were examined against Klebsiella. These sera were found to react to a larger extent than antisera produced against non-B:27 HLA antigens. It was suggested that these cross-reactions might be relevant in the pathogenesis of ankylosing spondylitis (Avakian et a / . , 1980). However, this view was modified by Geczy et al. (1980b) who found that the relationship was more complex than simple cross-reactions. These authors have shown that sera against certain Klebsiella isolates lysed the lymphocytes of HLA-B:27-positive patients with ankylosing spondylitis, but not of B:27positive or B:27-negative healthy controls (Seager et al., 1979). Three out of 36 randomly selected Klebsiella isolates were able by adsorption to remove the lymphocytotoxic activity of the antiserum produced against one of the three strains (Geczy and Yap, 1979). Furthermore, it was found that culture filtrates of two Klebsiella strains “modified” lymphocytes from B:27-positive healthy persons in such a way that they were now lysed by antisera to the “modifying” strain, i.e. they had become serologically similar to the cells of B:27-positive patients with ankylosing spondylitis. Culture filtrates of two other Klebsiella strains did not have the same effect (Geczy et al., 1980a). Additional experiments suggested that a cell wall component of 40-52 kilodalton was the

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modifying factor in the Klebsiella isolate assigned to K:43 (Druery et al., 1980). The capsular antigen as such has probably nothing to do with a relationship between Klebsiella and ankylosing spondylitis. References Altmann, G., Ostfeld, E., Zohar, S. and Theodor, E. (1977). Isr. J. Med. Sci. 13,62-64. Avakian, H., Welsh, J., Ebringer, A. and Entwistle, C. C. (1980). Br. J. Exp. Pathol. 61, 92-96. Avery, 0.T., Heidelberger, M. and Goebel, W. F. (1925). J. Exp. Med. 42, 709-725. Bagley, S. T., Seidler, R. J. and Brenner, D. J. (1981). Curr. Microbiol. (in press). Bascomb, S., Lapage, S. P., Wilcox, W. R. and Curtis, M. A. (1971). J. Gen. Microbiol. 66,279-295. Bjorndal, H., Lindberg, B. and Nimmich, M. (1970). Acta Chem. Scand. 24, 3414-34 15. Bjorndal, H., Lindberg, B. and Nimmich, W. (1971). Acta Chem. Scand. 25, 750. Bjorndal, H., Lindberg, B., Lonngren, J., Nilsson, K.and Nimmich, W. (1972). Acra Chem. Scand. 26, 1269-1303. Braun, V. and Hantke, K.(1974). Am. Rev. Biochem. 43, 89-121. Brenner, D. J., Steigerwalt, A. G. and Fanning, G. R. (1972). In?.J. Syst. Bacteriol. 22, 193-200. Brenner, D. J., Farmer, J. J. 111, Hickman, F. W., Asbury, M. A. and Steigerwalt, A. G. (1977). Hew Publication No. (CDC) 78-8356, Atlanta, Georgia. Brewerton, D. A., Caffrey, M., Hart, F. D., James, D. C. O., Nichools, A. and Sturrock, R. D. (1973). Lancet 1, 904-907. Brinton, C. C. (1965). Trans. N . Y. Acad. Sci. 27, 1003-1054. Brooke, M. S. (1951). Acta Pathol. Microbiol. Scand. 28, 313-327. Buchanan, R. E. and Gibbons, N. E. (Eds) (1974). “Bergey’s Manual of Determinative Bacteriology”, 8th edn, pp. 290-340. Williams & Wilkins, Baltimore, Maryland. Buffenmeyer, C. L., Rycheck, R. R. and Yee, R. B. (1976). J. Clin. Microbiol. 4, 239-244. Casewell, M. W. (1972). J . Clin. Pathol. 25, 734737. Casewell, M. W. (1975). J. Clin. Pathol. 28, 33-36. Casewell, M. and Talsania, H. G. (1979). J. Infect. 1, 77-79. Comninos, G. (1977). Thesis, Juris, Zurich. Cooke, E. M., Sazegar, T., Edmondson, A. S., Brayson, J. C. and Hall, D. (1980). J. Hyg. 84, 97-101. Cowan, S. T., Steel, K.J., Shaw, C. and Duguid, J. P. (1960). J. Gen. Microbiol. 23, 601-6 12. Curvall, M., Lindberg, B., Lonngren, J. and Nimmich, W. (1973a). Acta Chem. Scand. 27, 2645-2649. Curvall, M., Lindberg, B., Lonngren, J. and Nimmich, W. (1973b). Acta Chem. Scand. 27, 40 19-402 1. Dimock, W. W. and Edwards, P. R. (1927). J. Am. Vet. Med. Assoc. 70, 469480. Druery, C., Bashir, H., Geczy, A. F., Alexander, K.and Edmonds, J. (1980). Hum. Immunol. 1, 151-160. Duguid, J. P. (1959). J. Gen. Microbiol. 21, 271-280.

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5 Serology of Enterobacter and Hafnia R. SAKAZAKI Enterobacteriology Laboratories, National Institute of Health, Tokyo, Japan I. Introduction. 11. Cultural and biochemical characteristics . 111. Serology and antigenic schema . A. General principles . B. Preparation of diagnostic antiserum . C. Determination of antigens . IV. Epidemiology of E. cloacae and H. alvei and occurrence o f their serovars . References . Appendix1 . . . Appendix I1 . Appendix 111 .

165 167 167 167 170 171

180 181 183 183 185

I. Introduction

The history of the taxonomy of the bacteria classified as Enterobacter and Hafnia is complicated. There was a trend before 1960 that most motile enterobacteria giving a positive reaction in the acetoin production and gelatin liquefaction tests had been classified .as “Aerobacter” cloacae or “Cloaca” cloacae, which is now called Enterobacter cloacae. Although only two species, E. cloacae and E. aerogenes (Hormaeche and Edwards, 1960a,b), were recognized in the genus Enterobacter in the eighth edition of “Bergey’s Manual of Determinative Bacteriology”. The genus Enterobacter has been enlarged recently to include four more species: E. sakazakii (Farmer et a / . , 1980), E. gergoviae (Brenner et al., 1980), E. agglomerans (Ewing and Fife, 1972) and E. intermedium (Izard et a/., 1980). E. sakazakii was formerly included in E. cloacae as a yellow-pigmented variety of the latter species. E. gergoviae was separated from E. aerogenes. The bacteria included in E. intermedium was probably classified into E. cloacae or E. aerogenes as atypical strains of these two species. Therefore, information in older literature concerned with the bacteriology of the genus Enterobacter is generally useless at present because different bacteria belonging not only to E. cloacae or E. aerogenes but also to members of other species or genera might be classified as “Aerobacter” cloacae. E. agglomerans was proposed by Ewing and Fife (1972) METHODS IN MICROBIOLOGY VOLUME 14

Copyright 1984 by Academic Press. London All rights of reproduction in any form reserved. t(’

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for organisms of the Lathyli-Herbicola group which had been classified in the genus Erwinia. Thus this species may be in a different position to other Enterobacter species. In the light of this, previous serological studies on Enterobacter cultures before 1960 should be assessed with care. Edwards and Fife (1955) described that 50% of “Aerobacter” cloacae were encapsulated, 30% of them typed with Klebsiella capsular sera. Sakazaki and Namioka (1957) pointed out, however, that organisms of the Cloaca group were generally non-capsulated and were distinctly different from motile Kfebsieffa-like organisms, which are now classified in E. aerogenes by the amino acid decarboxylases and sugar fermentation tests. Stuart et al. (1943) studied organisms of “paracolon Aerobacter” (their terminology) biochemically and serologically without differentiating the 0-and H-antigens. Deacon (1952) reported 12 0-antigens and six H-antigens in the cultures of slow lactose-fermenting “Aerobacter” cloacae including cultures of biotype 32011 (terminology of Stuart et af., 1943). Eveland and Faber (1953) also studied cultures of biotype 32011 serologically and reported 21 0-antigens and 22 H-antigens among their cultures. It was confirmed by Sakazaki and Namoika (1957), however, that the cultures of biotype 3201 1 studied by Eveland and Faber (1953) included not only E. cloacae, but also Hafnia alvei and even Citrobacter freundii. Thus the serological studies performed before 1960cannot provide information for systematic serology of Enterobacter species. In 1960 Sakazaki and Namoika carried out serological studies on E. cloacae using I70 cultures which possessed typical characteristics based on modern taxonomic criteria of the species. It does not appear that systematic studies on the serology of Enterobacter species have been carried out on species other than E. cloacae. In this chapter, therefore, the serology of Enterobacter is described only for E. cloacae. Since Msller (1954) proposed the name H. afvei, organisms of this taxon have been described under various other names, Enterobacter alvei (Sakazaki, 1960), E. aerogenes subsp. hafniae (Ewing, 1963) and E. hafniae (Ewing and Fife, 1968). However, H. alvei Msller, 1954 is the only correct name. Mraller (1954) described a new group of Enterobacteriaceae in which a presumably authentic strain of Bacillusparatyphi-alvei of Bahr (1919) was included. Ewing and Fife (1968) pointed out that the Bahr’s strain was not the authentic strain of B. paratyphi-alvei and therefore considered that the specific epithet alvei was not legitimate. However, the name H. afvei must be legitimate since there is no doubt that the Bahr’s strain studied by Mraller (1954) was a new bacterium at that time. The numerical taxonomy study by Johnson et al. ( 1 975) and Gavini et al. (1976) and DNA reassociation studies by Steigerwalt et a f .(1976) appear to justify the status of Hafnia as a separate genus from Enterobacter.

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167

11. Cultural and biochemical characteristics

The bacteria belonging to the genera Enterobacter and Hafnia grow profusely in/on ordinary nutrient media. They grow on MacConkey agar. Although highly selective media such as Salmonella-Shigella agar, bismuth sulphite agar and brilliant green agar may be more or less inhibitory for Enterobacter cultures most strains of H . alvei can grow on Salmonella-Shigella agar. Members of E. cloacae are as a rule motile. They give positive reactions in the Voges-Proskauer, ornithine decarboxylase and arginine dihydrolase tests but negative results for the lysine decarboxylase, indole, DNAase and lipase tests. H. alvei may give positive lysine and ornithine decarboxylase reactions, but a negative result for the arginine dihydrolase test is observed. The failure of H. alvei to ferment raffinose, sorbitol, adonitol and inositol are particularly valuable characters when differentiating H. alvei from members of Enterobacter and Serratia. The incubation temperature has a remarkable effect on the results of the Voges-Proskauer, methyl red, and citrate utilization tests. The appropriate incubation temperature is 22-25°C for these tests. The biochemical characteristics of E. cloacae and H . alvei studied by the author are given in Table I, where they are compared with those of E. aerogenes, E. sakazakii and E. gergoviae. According to Guinee and Valkenburg (1968) all strains of H. alvei are lysed by a single phage that does not lyse other members of the Enterobacteriaceae. Routine application of the phage could provide a reliable tool for the identification of H. alvei strains.

111. Serology and antigenic schema

A. General principles 1. Enterobacter cloacae

Sakazaki and Namioka (1960) reported three antigenic components, 0-,Hand K-antigens, in the bacteria of E. cloacae. In most strains cultures boiled to 100°C for 1 h are strongly agglutinated with the homologous 0-antisera, whereas living cultures are agglutinated weakly or not at all, suggesting the presence of a K-like antigen. Strains which are inagglutinable in 0-antisera as living cultures become groupable after heating to 100°C for 1 h, or treatment as suspensions in either 50% ethanol or 1 N HCl at 37°C for 18 h, but not by heating to 60°C for 1 h. The ability of the masking antigens to bind

TABLE I Biochemical characteristics of Enterobacter cloacae, Hafnia alvei and related organisms ~~

H . alvei

E . cloacae

Characteristic

Sign %+ %(+) E. aerogenes E. sakazakii E.gergoviae Sign

%+ %(+)

Indole Voges-Proskauer (22°C) Voges-Proskauer (35°C) Citrate (Simmons’) (22°C) Citrate (Simmons’) (35°C) H,S VSI) Urease (Christensen’s) Gelatinase Phenylalanine deaminase Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase Malonate

1.3 99.3 97.5 98.0 2.0 98.0 2.0 0 d 22.5 (+) 0.5 90.5 0 0 + 98.1 98.5 d 82.0

0 98.0 59.0 9.6 0 0 2.0 0 0

-a

+ + + +

+

+ + + +

+ + + + +

d

++ +

+ + d

+ + + +

-

+ d d d -

-

+ + d

100.0 0 100.0

83.4

65.8 62.5

D-Iartrate Mucate Lipase (Tween 80) DNAase Esculin ONPGb Fermentation Arabinose Lactose Raffinose Rhamnose Sucrose Adonitol Dulcitol Sorbitol Inositol Salicin

d d

-

-

+ + (+) + + + + +

+-

15.0 81.1 0.5 0 9.8 99.6

97.0 5.8 90.5 97.5 95.8 4.0 93.8 2.5 95.5 0.8 5.3 0.3 100.0 d 33.0 0.5 (+) 22.3 75.8

(+I

+

+

+ + + + + ++ + +

-

d

+

-

+ + + + + + + -

d

+

-

0 0

-

0

-

d

+ -

-

+ d d

0

4.8 73.2

1.2

99.0 0 8.0 0 82.0 17.2 8.4 68.0 0 0 0 0 15.8 4.2

symbols: +, 90% or more positive; -, YO”/, or more negative; d, dinerent reactions ( 1 149% positive); (+), delayed positive reactions. Ortho-nitrophenol-8-D-galactopyranoside (p-galatosidase test).

170

R. S A K A Z A K I

to homologous agglutinins is destroyed by heating to 100°C for 2 h and HCl treatment. Antisera prepared with boiled suspensions of the organisms, accordingly, do not contain K-agglutinins. These findings suggest that the masking antigen of most E. cloacae may be the slime or M-antigen. Therefore, the serological typing of E. cloacae involves 0-and H-antigens. Fifty-three 0antrgen groups and 56 H-antigens have been reported by Sakazaki and Namoika (1960). 2. Hafnia alvei

Sakazaki (1961) reported 29 0-antigen groups and 23 H-antigens in cultures of H. alvei in which 0- and H-antigens of the “32011 group of paracolon bacteria” described by Eveland and Faber (1953) were included. Matsumoto (1963, 1964) expanded the serological characterization of H. alvei, and reported further 0- and H-antigens increasing the total number to 68 0antigens and 34 H-antigens. Although the majority of H. alvei strains are agglutinated by homologous 0-antisera, not only after boiling for 1 h, but also as living cultures, some cultures are 0-inagglutinable. This suggests the presence of K-antigens. Deacon (1952) suggested the possibility that Kantigens of his cultures might be A-type rather than true O-antigens. However, Sakazaki (1961) could not confirm the presence of such an A-type K-antigen and suggested that the K-antigen within Hafnia seemed to be the M-antigen. H. alveimay possess the alpha-antigen of Stamp and Stone (1944) (Sakazaki, 1961; Emslie-Smith, 1961).

B. Preparation of diagnostic antiserum 1. 0-antiserum

The antigens used to prepare 0-antisera are cultured in 18-h infusion broth or suspensions of agar heated to 100°C for 2.5h. Rabbits received four intravenous injections of 0.5m1, 1.0m1, 2.0ml and 4.0ml of the heated antigens. Four injections usually result in the production of an antiserum of satisfactory titre (1: 1000 or more). If the titre is low at test bleeding one or two additional injections may be given. The rabbits are bled on the sixth day after the last injection. 2. H-antiserum

Suitable antigens for H-antiserum production are cultured in infusion broth containing 0.2% glucose incubated at 30°C for 6 h followed by addition of 0.3% formalin. The development of H-antigens is so poor in the majority of

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171

the cultures that several passages of the organism through a semi-solid medium is necessary to ensure maximum motility. Motility test medium in a Craigie-tube may be used for the passage. The passage should be repeated until the organisms inoculated into the small inner tube reach the surface of the medium of the outer tube after overnight incubation. For some cultures seven to ten or more successive passages may be necessary to obtain suitable H-antigens. The immunization schedule described above for 0-antisera is also suitable for the production of H-antisera. A titre of 10 000 or more against the homologous H-antigen is satisfactory.

3. Preservation of antiserum 0-and H-antisera are preserved by adding an equal volume of glycerol and stored at 4 C. 4 . Absorption of antiserum

Absorption is necessary in some 0-or H-antisera to remove cross-reactions and to obtain specific sera. For the absorption of 0-agglutinins 5ml of antiserum diluted to 1 5 are mixed with packed cells from saline suspensions in which a 24-h culture of five to ten agar plates is harvested and heated to 100°C for 1 h. After 2 h incubation in a water bath at 50°C the mixture is allowed to stand overnight at 4°C. The mixture is centrifuged and the supernatant serum preserved with thimerosal. The absorbing H-antigen cultures are obtained from overnight growth of active motile organisms on a soft infusion agar plate containing 1% agar and 0.2% glucose. Packed cells harvested from saline suspensions of the flagellar cultures obtained from 10-15 agar plates are added to 10ml of H-antiserum diluted to 1:lO. In some instances, double absorption of H-antiserum may be necessary to remove a cross-reaction completely.

C. Determination of antigens 1. Enterobacter cloacae

( a ) 0-antigen. Slide agglutination is the method of choice for determining 0-antigen of E. cloacae in routine procedures. Dense saline suspensions of an overnight agar culture heated to 100°C for 1 h and washed by centrifugation and the working dilution of 0-antisera are used for agglutination. Although antisera with homologous titres of 500-1000 diluted to 1:lO are customarily used for the slide test, it is more desirable to use higher dilutions still giving a

R. SAKAZAKI

172

strong homologous reaction within a few seconds, but with less crossreactions. Lateral and unilateral reactions may be observed between different 0antigens. The relationships observed among the 53 0-antigen groups established by Sakazaki and Namoika (1960) are shown in Table 11. Although

TABLE I1 Enterobacter cloacae 0-antigen relationships 0-antiserum

Homologous titre

1

5000

2

lo00

3 4

5000 2000 so00

5

Titre with other 0-antigens” 5 :50 23:100

1 :500 31 :SO 16:100 3:100 1 :loo

20:1000

6 7

5000 2000

11 12 13

8

2000 2000 1000 lo00

14 16 18 19

2000 1000 2000 1000

20 21 22

1000 1000 2000

23 29 20 32 34 36 37 39 42 45 51

2000 1000 1000 1000 2000 2000 2000 2000 2000 2000 1000

4:100 1 :200 22 :200 3:100 49:100 30 :100 2 :200 23 :50 1 50 3 :200 8:1000 2:200 1850 I :I00 23:50 1 :loo 23 :200 1 :I00 5 :200 12500 7:SO 7:lOOO 3 :200 29:100 2:200 19:SO 50 :2000 4 500

Reciprocal relationships are italicized.

6:100 29 :50 13:100 32:SO 18:500 51 :200 13:100 9:200 2:100 34 :500 36 :200 3:100

32 :50 2:200

18:500 36:1o00 5 :SO 23 :50 9:200 34:so 5 :200 34:50 19:200

13:lOO 37:50 14 :200

20:100 47:200 30:200

29 :200

4s:50

22:100

23 :SO

3:2000 40:100

13:200

1950

14 :50 40:50 13:100

41:50

13:100 4050

14:50 41:SO

47:200 7:2OOO 40:200

13:SOO

29:100 13 50 4050 8 500

47:200 14:50

38:100

39 :100

23 :100

36:100

18:200

40:100

5 . ENTEROBACTER AND HAFNIA

173

most 0-antigen groups can be determined by appropriately diluted unabsorbed antisera, the use of group-specific antisera obtained by absorption is often necessary for specific 0-antigen determination. Some 0-antigen groups can be subdivided. Sakazaki and Namoika (1960) reported on the presence of subgroups in 0-groups 1-6, 9-11 and 13-15 although they were not at the time shown in their antigenic schema of E. cloacae. They did not then consider that 0-group subdivision would be useful epidemiological tools, but their subgroups were: 0-group 1: la,lb; la,lc and la,ld 0-group 2: 2a,2b; 2a,2b,2d and 2a,2c

0-group 3: 3a,3b; 3a,3c and 3a,3d 0-group 4: 4a,4b; 4a,4c and 4 a , k 0-group 5: 5a,5b and 5a,5c 0-group 6: 6a,6b; 6a,6c and 6a,6d 0-group 9: 9a,9b and 9a,% 0-group 10: 10a and 10a,10b 0-group 1 1 : l l a and l l a , l l b 0-group 13: 13a,13b and 13a,13c 0-group 14: 14a,14b and 14a,14c 0-group 15: 15a and 15a,15b

( b ) H-antigen. To determine H-antigens, tube agglutination is the preferred routine procedure. An overnight broth culture of the actively motile organisms is used. Trypticase soy broth or infusion broth containing 0.2% glucose is satisfactory culture media. After incubation an equal volume of 0.6% formal-treated saline is added to the broth culture. Unabsorbed antisera of homologous titre of 1-20000 are usually used at dilutions of 1:1000, although the dilution may be different with particular H-antisera. Then 0.1 ml of a 1: 100 dilution of H-antiserum is placed into a small tube and 1.O ml of the formal-treated broth culture is added. The test-tubes are incubated in a 50°C water bath for 1 or 2 h before reading. Sakazaki and Namoika (1960) designated 56 H-antigens of E. cloacae. Table 111 shows reciprocal and unilateral relationships among the 56 Hantigens. Although the relationships are minor in most cases extensive crossreactions may be seen between some H-antisera to which absorption is necessary to prepare specific H-antisera. In addition to this, the agglutinability of individual H-antigens in the same H-type may be fairly weak even though the maximum motility of the cultures is taken into consideration. Form variation of H-antigens was reported by Sakazaki and Namoika (1960) between H-antigens 12 and 13 in a strain. Moreover, the segregation phenomenon in H-antigens which was first reported by Edwards (1946) in organisms belonging to the Ballerup group (Cirrobacter freundii) was also demonstrated by partial factors of some H-antigens. ( c ) Antigenic schema for E. cloacae. An antigenic schema for E. cloacae

174

R. SAKAZAKI

TABLE 111 Enterobacter cloacae H-antigen relationships H-antiserum

Homologous titre

1

50 OOO

2 3 4

20 OOO 10OOO 5OOO 10OOO 5OOO 20 OOO

5

6 7 8

9 10 12 16 17 18 20 22 23 25 26 27 28 29 30 31 32 35 37 38 39

40

43 44 46 48 50

51 53 54

Titre with other H-antigens"

5:100 42:1OOO 42:500 48:1OOO 5 :200 48500

19:500

30500

33500

6500

35:2000

5O:lOO

5:200

5O:lOO

39:500

8:lOOo

9500

35:2000 20:2000

265000

20 000 20 OOO 10OOO

37:5000 7:5000 7:1000 31 :5000

17:100

20500

37:200

37:1000

20:2000

20 OOO 20 OOO 10OOO 10OOO 5OOO

35:2OOO

44:2OOO

7500

27:200 8500

48:200 26500

30:500

39:1000

54:100

34:200

47:500

48:2OO

445000

47:100 35:1000

50 000

20 000 5OOO 50 OOO 10OOO 50 OOO 20OOO 5000

2000 50 OOO 20 OOO

5OOO 20OOO 20 000 20 OOO 10OOO 10OOO 5OOO 10OOO 20 OOO 10OOO 20OOO 10OOO

4 :2000

19:lOOO 27:100 24:1000 5 :200

35:500 21 :500 42500 24:5OOO

39:2000

48:lOOO 27:200 17:200

31 :2000

Reciprocal relationships are italicized.

33:200

47:200

47:500 5 :200

33 :200 27:500 23 :SO00 19:500 41:200 7:200

54:2000

37:100

9:lOOO 7:200 47:100 24:5OOO 24:lo00 285000 53:1OOO 2:200 1 :lo00 10:20oo 35:200 27:200 65000 4 5000 8:IOOO 7:1000 48:1OOO 53:2OOO

30 500

27:5OO

35:5000

35:500 33:lOOO 47:100 20:lOOO 48:200 9 :200

39:2OOO 44:200 47:1000 30:lOOO

20:1000

5. E N T E R O B A C T E R

A N D HAFNIA

175

consisting of 53 0-antigen groups, 56 H-antigens and 79 serovars was established by Sakazaki and Namoika (1960) as shown in Table IV. Sedlak (1968) reported 19 0-antigen groups but unfortunately no comparison of the 0-grouping system has been carried out. ( d ) Intergeneric and extrageneric relationships of E. cloacae antigens. Although five species are now recognized in the genus Enterobacter, E. cloacae is the only species in which antigenic studies have been performed. Therefore, the antigenic relationships are presently unknown between E. cloacae and other Enterobacter species. Among the five Enterobacter species, E. sakazakii formerly belonged to E. cloacae. It is possible that strains of E. sakazakii were involved in the studies of Sakazaki and Namoika (1 960) and Sedlak (1968). Further study is necessary to clarify this question. Edwards and Fife (1955) reported that most “Aerobacter” cloacae were TABLE IV

Antigenic schema for Enterobacter cloacae 0-group 1 2 3 4 5 6

1 8 9 10 11 12 13 14 15 16 11

18 19 20 21 22 23 24 25 26

H-antigen 1 4 1

12 14 8 18 20 21 2 2 26 2 28 30 31 23 21 32 33 10 34 35 52 39 25

2 5 8 12-13” 36 15 22 23 24

0-group 3 6 9

40

55 22 44

56

11

26

54

25

21 29 39

51 10, I l b

Form variation occurred between these H-antigens. Segregation of the two H-antigens occurred. N M ,non-motile.

21 28 29 30 31 32 34 35 36 31 38 39

40 41 42 43 44 45 46 41 48 49 50 51 52 53

H-antigen 53 20 16 26 31 38 19 2 8 2 41

NM 42 43

44 46 41 48 49

NM 50 51 7 1 55 45

176

R. SAKAZAKI

typed with Klebsiella capsular sera. However, Sakazaki and Namoika (1960) described that E. cloacae cultures produced slime rather than a real capsule. No significant 0- and K-antigen relationships were recognized between Klebsiella and E. cloacae. It is assumed that Edwards and Fife (1955) dealt with E. aerogenes of which many strains are encapsulated. Kauffmann (1954) separated not only “Aerobacter” cloacae but also motile cultures of “Aerobacter” aerogenes, later named E. aerogens by Hormaeche and Edwards (1960a,b), from Klebsiella and placed them in the Cloaca group. The 0-subgroups 2a,2b,2d and 43 as designated by Sakazaki and Namoika (1960) were identical with 0-subgroups 8a,8b and 19a of the “3201 1 group of paracolon bacteria” as studied by Eveland and Faber (1953). Close relationships are recognized between E. cloacae and H. alvei 0-antigens (Section III.C.2). 2. Hafnia alvei ( a ) 0-antigen. 0-antigen determination of H. alvei follows the general manner described for E. cloacae above. Antigenic relationships among 0antigens of H. alvei are shown in Table V. Most of the 0-antigens could be determined by appropriately diluted unabsorbed antisera although absorption of agglutinins against minor antigens is sometimes necessary for specific 0-antisera. The following 0-subgroups have been recognized in H. alvei: 0-group 1: la,lb and 0-group 2: 2a,2b and 0-group 3: 3a,3b and 0-group 4: 4a,4b and 0-group 5 : 5a,5b and 0-group 6: 6a,6b and 0-group I: 7a,7b and

la,lc 2a,2c 3a,3c 4a& 5a,5c 6a,6c 7a,lc

These 0-subgroups were not shown in the antigenic schema originally presented for H. alvei by Sakazaki (1961). ( b ) H-antigen. H-antigen determination of H. alveican be performed in the same manner as described for E. cloacae above. Although many H-antigens react specifically, it is sometimes necessary to use absorbed H-antisera. The relationships among H-antigens of H. alvei observed by Sakazaki (1961) and by Matsumoto (1963, 1964) are summarized in Table VI. Deacon (1952) described form variation in H-antigens from four of the 17 cultures employed in his study. When descriptions of H-antigens from the six cultures commonly employed in studies by Deacon (1952), Eveland and Faber (1953) and Sakazaki (1961) are compared, the results obtained in Sakazaki’s

177

5 . E N T E R O B A C T E R AND HAFNIA

TABLE V

Hafnia alvei 0-antigen relationships ~~

Homologous 0-antiserum titre 1 4 5

6 7 10 17 23 26 29 34 43 45 46 47 48 49 51 53 54

5000

2000 1000 1000 2000 1000 1000 2000 2000 2000 5000 5000 5000 10 000

55

2000 10 000 2000 5000 5000 5000 5000

56 59

5000 5000

Titre with other 0-antigens 2:1000 7:50 12:500 18:1000 2:500 11 :500 9 :200 6:200 2:100 15 :2000 56 :500 30 :200 38 :100 47 :500 30:100 32 :100 47 :100 30 :200 54:100 50:1000 30 :200 34 :200 33 :1000

4:100

19:500

54:1000 54 :200

45 :200

53 :1000

study agree with those of Eveland and Faber (1953) but disagree with Deacon’s results. It is suggested that all H. alvei strains possess monophasic H-antigens. ( c ) Antigenic schema for H. alvei. Sakazaki (1961) established an antigenic schema for H. alvei which consisted of 29 0-antigen groups, 23 H-antigens and 5 1 serovars. The antigenic schema was expanded by Matsumoto (1963, 1964) to a total number of 68 0- and 34 H-antigens. Table VII shows a composite antigenic schema of 197 serovars described by both investigators. Independent of Sakazaki and Matsumoto, Baturo and Raginskaya (1978) have recently published an antigenic schema of H. alvei consisting of 39 0antigens and 35 H-antigens. However, no comparison of the two antigenic schemata has been performed.

178

R. SAKAZAKI

TABLE VI Hafnia alvei H-antigen relationships

H-antiserum

Homologous titre 10 OOO 10 OOO 10 OOO 10 OOO 2000 10 Ooo

7 8 9 10 11 12 13 14 15 16 17 20 21 23 26

20 OOO 10 OOO 10 OOO 2000 5OOO 5Ooo 20 OOO 10 OOO 10 OOO 10 OOO 10 Ooo 20 OOO 10 OOO 5OOO

10 OOO

Titre with other H-antigens" 4:200 4:lOOO 6:2000 23 :200 7:100 3:1000 19:SOOO 3:lOOO 3:lOOO 65000 3:100 1 :So00

14:2000

5:lOOO

12:lOOO

15:lOOO

4:500 4:500

8 :200 6:200

7:2000

4 :200

5 500

5 :200

11:100

4:100 6:200 4:100

23 :100 7:500

15:1000

7:lOOO 2:lOOO

19 :200

12:200

1 :200 3 :200 3 :200 3:500 18:SOOO 9:2OOO

Reciprocal relationships are italicized.

( d ) Extrageneric relationships of H. alvei antigens. A number of 0antigenic cross-reactions have been reported between H. alvei and Salmonella (Eveland and Faber, 1953; van Oye, 1968). Most of these are only one-side or reciprocal relationships of a minor nature (Sakazaki, 1961). Significant reciprocal 0-antigenic relationships have been demonstrated between H. alvei and Escherichia coli (Sakazaki, 1961; Matsumoto, 1963, 9 0 5 2 related to E. 1964). H. a l v e i 0 : 5 , 0 : 2 1 , 0 : 2 3 , 0 : 3 2 , 0 : 3 3 , 0 : 3 8 , 0 : 3and coli 0:26,0: 103,0:7,0: 102,0:37,0:30,0: 1 13 and 0:139 respectively, in the a,b-a,c or the a,b-a variation of relationships. H. alvei 0:6 related to both E. coli 0:89 and 0:91. These reciprocal relationships were also recognized between H. alvei 0:28 and both E. coli 0 : 5 and 0:65, between H. alvei 0:48 and both E. coliO:7 and 0:36 and between H. alvei 0 5 5 and both E. coli 0:22 and 0:76.O-antigens of H. alvei 0:27 were identical with that of E. coli 0:105. It is obvious that close 0-antigenic relationships exist between H. alvei and E. cloacae from the results of studies by Deacon (1952) and Eveland and

TABLE VII Antigenic schema for Hufniu alvei 0-group 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 45 46 47

H-antigen 1 2 1 4 10 1 1 1 2 3 2 5 2 17 18 1 3 7 2 1 1 18 22 3 3 1 3 23 NM 1 2 3 6 3 1 29 1 21 3 3 21 1 1 12 4 1 1

3 3 2 8 11 2 2 3 12 NM 9 16

11 5 3

13 7 7

30 9 9

NM 11 11

14 12

20 32

NM

15 3 3

27 7 4

29 8 7

NM 9 8

21 9

26 20

31 24

29 26

NM

7 1 1 20 27

30

NM

28 6

12

19 11 11 21

20 17

16 3 7

4 9

3 3 5 12 11 11

9 6 11 17 17 18

21 11

23 12

24 20

21

25

30 20 20

24 24

26

32

34

3 29

22

24

24

26

NM

4 NM 3 4 NM 11 26 11

6 1 7 33

NM 12

17

NM

NM

180

R. SAKAZAKI

TABLE VII -continued O-group 48 49 50

51 52 53 54 55

56 57 58 59 60 61 62 63

64

65 66 67 68

H-antigen NM 1 6 7 1 32 26 7 3 3 NM 21 27 12 23 24 NM 11 7 33 9

12 3

29

17 21 24

34 24

NM,non-motile.

Faber (1953). In the study of Sakazaki (1961), E. cloacae 051 was related to H . alvei 0 : l and 0 : 2 in the a,b-a,c variation of relationships. An antigenic relationship of the a,b-a,c variation was recognized between H. alvei 0 : 2 and E. cloacae 0:34, and H . alvei 0:20 and E. cloacae 0:21. H. alvei 0:25 was almost identical to E. cloacae 0:19. Some antigenic cross-reactions between H. alvei and Citrobacter freundii and ShigellaJlexneri were reported (van Oye and Ghysels, 1961; Sedlak and Slajsova, 1966).

IV. Epidemiology of E. cloacae and H. alvei and occurrence of their seravars Enterobacter and Hafnia are found not only in soil and water but also in human and animal intestines. In recent years, these organisms, especially E. cloacae, have more often than formerly been reported as opportunistic agents of hospital patients, most often causing urinary tract infections, septicaemia, meningitis, bronchitis and cholecystitis. E. cloacae is less common than

5. ENTEROBACTER A N D HAFNIA

181

Escherichia and Klebsiella. Washington et al. (1971) reviewed the epidemiology of nosocomial H. alvei infections and concluded that most isolates were found in the respiratory tract and were considered commensals, whereas a few were secondary invaders. Previous administration of ampicillin or cephalosporins may precede nosocomial infections with H. alvei. There have been controversies regarding enteropathogenicity of Enterobacter and Hafnia. Many investigators have emphasized the role of H. alvei in enteric disorder (Stuart et al., 1943; Stuart and Rustigan, 1943; Deacon, 1952; Eveland and Faber, 1953; Emslie-Smith, 1961; Sedlak and Slajsova, 1966; Kolta and Weiner, 1967; Kalashinikova et al., 1974; Serebrykov and Blochov, 1972). On the other hand, Matsumoto (1963), who investigated the occurrence of H. alvei in stool specimens from healthy persons and used Salmonella-Shigella agar, reported that the organisms were isolated from 13% of 1913 stool specimens. He concluded that probably the majority of H. alvei would not be enteropathogenic. R. Sakazaki (unpublished data) found H. alvei in 42% of normal stool specimens. Klipstein et al. (1973) and Klipstein and Engert (1976) reported the presence of a heat-stable enterotoxin in E. cloacae, similar to that produced by E. coli. Thus, no conclusive evidence on enteropathogenicity of Enterobacter and Hafnia has been obtained from any of the previous studies. Although many incidences of nosocomial infections with Enterobacter and Hafnia have been reported, there has been no attempt to apply any serotyping system as an epidemiological tool.

References Bahr, L. (1919). Scand. Vet. Tidschrifr 9,25-40 and 4 5 4 0 . Baturo, A. P. and Raginskaya, V. P. (1978). Int. J . Syst. Bacteriol. 28, 126127. Brenner, D. C., Richard, C., Steigerwalt, A. G., Asbury, M. A. and Mandel, M. (1980). Int. J. Syst. Bacteriol. 30, 1-6. Deacon, W. E. (1952). Proc. SOC.Exp. Biol. Med. 81, 165-170. Edwards, P. R. (1946). J. Bucteriol. 51, 523-529. Edwards, P. R. and Fife, M. A. (1955). J. Bacteriol. 70, 382-390. Emslie-Smith, A. H. (1961). J. Pathol. Bacteriol. 81,534-536. Eveland, W.C. and Faber, E. (1953). J. Infect. Dis. 93,226-235. Ewing, W.H. (1963). Int. Bull. Bacteriol. Nomencl. Taxon. 13, 110. Ewing, W.H. and Fife, M. A. (1968). Int. J. Syst. Bacteriol. 18,263-271. Ewing, W.H. and Fife, M. A. (1972). Int. J. Syst. Bacteriol. 22,4-11. Farmer, J. J., 111, Asbury, M. A., Hickman, F. W., Brenner, D. J. and the Enterobacteriaceae group (1980). Int. J. Syst. Bacteriol. 30,569-584. Gavini, F., Ferragut, C., Lefebre, B. and Leclerc, H. (1976). Ann. Microbiol. 127B, 3 17-335.

Guinee, P. A. M. and Valkenburg, J. J. (1968). J . Bacteriol. 96, 564.

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Hormaeche, E. and Edwards, P. R. (1960a). Int. Bull. Bacteriol. Nomencl. Taxon. 10, 71-74. Hormaeche, E. and Edwards, P. R. (1960b). f n t . Bull. Bacteriol. Nomencl. Taxon. 10, 75-76. Izard, D., Gavini, F. and Leclerc, H. (1980). Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Abt. I Orig. C1, 51-60. Johnson, R., Colwell, R. R., Sakazaki, R. and Tamura, K. (1975). Int. J. Syst. Bacteriol. 25, 12-37. Kalashinikova, G. K., Lokosova, A. K., Sorokina, R. S.,Brodova, A. I., Grivzova, A. S., Baturo, A. P. and Baginskaya, V. P. (1974). J. Microbiol. (Moscow) 6, 78-81. Kauffmann, F. (1954). “Enterobacteriaceae”, 2nd edn. Munksgaard, Copenhagen. Klipstein, F. A. and Engert, R. F. (1976). Infect. Immun. 13, 1307-1314. Klipstein, F. A., Holdeman, L. V., Corcino, J. J. and Moore, W. E. C. (1973). Ann. Intern. Med. 69, 632-641. Kolta, F. and Weiner, G. (1967). Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I ReJ 207, 413. Matsumoto, H. (1963). Jpn. J. Microbiol. 7 , 105-1 14. Matsumoto, H.(1964). Jpn. J. Microbiol. 8, 139-141. Msller, V. (1954). Acta Pathol. Microbiol. Scand. 35, 259-277. Oye, E. van (1968). In “Enterobacteriaceae Infektionen” (J. Sedlak and H. Rische, Eds), pp. 603406. Thieme, Stuttgart. Oye, E. van and Ghysels, G. (1961). J. Bacteriol. 82, 313. Sakazaki, R. (1961). Jpn. J. Med. Sci. Biol. 14, 223-241. Sakazaki, R. and Namoika, S. (1957). Jpn. J. Exp. Med. 27, 273-282. Sakazaki, R. and Namoika, S. (1960). Jpn. J. Med. Sci.Biol. 13, 1-12. Sedlak, J. (1968). In “Enterobacteriaceae Infektionen” (J. Sedlak and H. Rische, Eds), pp. 595-601. Thieme, Stuttgart. Sedlak, J. and Slajsova, M. (1966). J. Gen. Microbiol. 43, 151-158. Serebrykov, L. W. and Blochov, W. P. (1972). Woennij Med. J. 7 , 60-63. Stamp, L. and Stone, D. M.(1944). J. Hyg. 43, 266-272. Stuart, C. A. and Rustigian, R. (1943). Am. J. Public Health 33, 1323-1325. Stuart, C. A., Wheeler, K. M., Rustigian, R. and Zimmerman, A. (1943). J. Bacteriol. 45, 101-109. Washington, J. A., 11, Bink, R. J. and Ritts, R. E., Jr (1971). J. Infect. Dis. 124, 379-386.

5 . E N T E R O B A C T E R AND HAFNIA

Appendix I. Type strains Enterobacter cloacae ATCC 13047 Hafnia alvei ATCC 13337

Appendix 11. Enterobacter cloacae 0-and H-antigenic reference strains

Antigenic formula 0 H 1

1

2

2 2

3

3

4

4 4 4 5 5

6

6 6 7

8

9

10

I1 12 13 14

14 15 16 17 18 19 20 20 21

I 3 4 5 6 9 22 12 13 40 56 14 36 15 17 54 18 20 21 23 24 26 27 28 29 30 31

23 27

32 33 51 10

Strain

713-53 1287-53 868-53 153-52 712-53 543-53 1640-53 186-52 564-53 2-51 117-53 375-53 398-53 524-53 621-52 535-53 1356-53 546-53 553-53 557-53 332-52 567-53 589-53 2015-53 603-53 605-53 1349-53 715-53 716-53 717-53 718-53 1346-53 1036-53

Arranged by 0-antigen numbers. Numbers italicized indicate reference antigens. Continued

183

R. SAKAZAKI

184

Appendix I1 -continued

Antigenic formula 0 H

Strain

~

21

I1

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

34 35 52 39

25 53 20

16 26

37 38 39 19 2 8 2 41

-

42 43 44 46 47 48 49

-

50 51

7 1

55 45

867-53 1015-53 871-53 121-52 568-53 14-52 41 1-52 903-53 1038-53 1511-53 1611-53 1615-53 88-55 1608-53 181-52 483-52 300-52 650-52 1876-53 113-52 1325-53 1337-53 1897-53 2096-53 291-52 1363-53 1921-53 1330-53 1888-53 1325-53 1609-53 1640-53 29-52

5 . E N T E R O B A C T E R AND HAFNIA

Appendix 111. Hafnia alvei 0-and H-antigenic reference strains

Antigenic formula 0 H I 1 2 2 3 4 5

5 6 7

8 9 10

I1 I2 12 13 14 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

13 30 2 14 3 8 10 15 31 2 I 2 2 2 5 16

2 17 28 18 1 3 7 19 20 1 18 22 3 3 1 7 23 -

1 25 3 6

Strain 629-6 1 732-61 666-54 523-56 228-56 108-56 432-55 189-53 1023-61 1159-61 126-56 6351-52 375-63 158-56 1901-54 161-56 163-56 338056 1301-61 7 15-56 567-61 425-63 194-55 1181-56 561-56 21 1-56 863-56 131-56 333-56 356-56 946-56 591-61 29-55 538-55 749-6 1 575-61 1171-61 1259-61

Arranged by 0-antigen numbers. Numbers italicized indicate reference antigens. Continued

185

186

R . SAKAZAKI

Appendix I11 -continued

Antigenic formula 0 H 34 34 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

26 32 34 24 29 24 29 3 3 21 3 4 12 4 1 24 -

1 6 12 1 32 26 7 3 3 -

24 27 12 23 24 -

11 7 33 9

Strain 122461 1268-61 45343 89141 343-61 43141 694-6 1 7 2 M1 130741 695-61 5 9 7 41 596-6 1 67141 67741 1256-61 475-6 1 114&61 931-61 117141 25241 1253-61 128341 1311-61 129461 410-61 134141 131461 389-63 42743 372-63 60241 376-61 4843 365-63 458-63 46063 466-63

Serology of Proteus mirabilis and Proteus vulgaris P. LARSSON Departments of Clinical Bacteriology and Clinical Immunology, University of Goteborg, Goteborg, Sweden ~~~

Introduction. . Taxonomy of Proteae . Previous serological systems . Present system for serotyping of P . mirabilis and P . vulgaris A. Surface structures . B. Serological techniques . C. Antigens . D. Antigenic variations . E. Antigenic cross-reactions . . F. Dienes phenomenon . V. Production of antisera . VI. Distribution of serotypes in clinical isolates . VII. Serotyping in epidemiological work. . References . Appendix .

I. 11. 111. IV.

187 188 189 191 191 194 198

202 203 205 206 207 208 209 213

I. Introduction Almost a century ago Hauser (1885) described a group of bacteria with a pleomorph morphology and an ability to swarm on solid media. This group was named Proteus (after a homeric sea deity) and could be divided into Proteus vulgaris, Proteus mirabilis and Proteus zenkeri. This division was later abandoned and several investigators have since proposed new classifications for the Proteus group of bacteria. Proteus is a Gram-negative rod belonging to the Enterobacteriaceae. Proteae are present in the normal faecal flora of man, although often in low numbers (Haenel and Dawidowski, 1961). Proteus is a common cause of urinary tract infection in young boys (Hallett et al., 1976) and in the elderly (Walkey et al., 1967). Nosocomial infections, localized mainly to the urinary tract, have been reported (Kippax, 1957; Edebo and Laurell, 1958; Larsson et al., 1978a). The Proteus group of bacteria also frequently gives rise to METHODS IN MICROBIOLOGY VOLUME 14

Copyright 0 1984 by Academic Press, London All rights of reproduction in any form reserved.

188

P. LARSSON

bacteraemia, especially in older patients (Adler et al., 1971; Larsson, 1980). P. mirabilis dominates in clinical isolates from man and constitutes roughly 75% of the Proteus group (Adler et al., 1971). For reviews on the ecology and importance of Proteus infections in man the reader is referred to Sedlak et al. (1959), Sedlak (1968) and Tomaschoff (1969). This chapter summarizes the different taxonomical considerations regarding Proteus, as they affect the design and build-up of the serological systems set up for Proteus. Only the serology of P . mirabilis and P. vulgaris, using the serological system presented by Kauffmann and Perch (1947, 1948; Perch, 1950; Kauffmann, 1969), is described in detail.

11. Taxonomy of Proteae

Hauser’s (1885) division of Proteus into three species, P . vulgaris, P. mirabilis, and P. zenkeri, was later challenged by Wenner and Rettger (1919), who suggested that the genus Proteus should consist of P . vulgaris, P . mirabilis and Bacillus proteus. They referred to Hauser’s Proteus zenkeri as the genus Zopjius. Bergey in 1923 (Krikler, 1953) recognized as many as six divisions: P . vulgaris, P. mirabilis, P . liquefaciens, P. asiaticus, P. valeriae and P. hydrophilus. In 1936 Rauss’ investigated 48 cultures of Morgan’s bacillus No. 1 and suggested that this organism belonged to the genus Proteus. The name P. morganii was later adopted. A new micro-organism, P. rettgeri, was added to the Proteus group by Rustigian and Stuart (1945), who divided the genus into four species: P. mirabilis, P. vulgaris, P. morganii and P. rettgeri. Kauffmann has suggested only three genera, Morganella, Rettgerella and Proteus, with P. mirabilis and P. vulgaris (Kauffmann, 1954). Belyavin et al. (195 1) and Krikler (1953) agreed with Kauffmann that P . mirabilis and P. vulgaris were biochemical varieties of the same species although they called it P. vulgaris with the subspecies variants mirabilis and vulgaris. The Providence group and its close resemblance to P . morganii and, P . rettgeri was described by Ewing et al. (1954). More recently Providencia has been included in the Proteus group as P. inconstans although Providencia has been referred to by other authors as a separate genus (Edwards and Ewing, 1972; Cowan, 1974; Lautrop, 1974). It is obvious that this brief review that the taxonomy of Proteae can be confusing. Although the question has not yet been settled classification of Proteus into P. mirabilis, P. vulgaris, P . morganii, P . rettgeri and P . inconstans has been widely accepted and is used in the eighth edition of “Bergey’s Manual

189

6 . PROTEUS MIRABILIS AND PROTEUS VULGARIS

of Determinative Bacteriology” (Lautrop, 1974). This classification has also been used in Table I to show the main biochemical characteristics. Recently, however, Brenner et al. (1978) proposed a new taxonomy for this group of bacteria: Proteus vulgaris, Proteus mirabilis, Providencia alcalifaciens, Providencia stuartii, Providencia rettgeri and Morganella morganii. Additional information on this classification is given in Chapter 1. TABLE I Differential characteristics of species within the genus Proteus P . vulgaris P . mirabilis P . morganii P . rettgeri P . inconstans

Gas from glucose Urease Indole production Gelatin liquefaction H,S. production Ornithine decarboxylase Growth requirement Nicotinic acid Pantothenic acid (G C) mole % DNA

*+

+ ++ +

+ + +-

+

+

+-

+-

39.3

39.3

+ +

1.2

1.4

+ + + + +

50.0 0.7

d

+

+-

39.0 1.5

41.5 0.6

From Lautrop (1974). Symbols: +, more than 90% have this characteristic; d, S e l l % positive; -, less than 10%positive.

111. Previous serological systems

The first major contribution to Proteus serology was made by Weil and Felix (1916, 1917), who investigated Proteus strains ( X strains) isolated from patients with typhus. Based on colony morphology on agar plates they described two distinct types of bacterial growth: the H-form (=mit Hauch) and the 0-form (= ohne Hauch). Immunization of rabbits with these variants produced 0-and H-antisera showing different agglutination patterns. The 0agglutination was granular ( = Korner), in contrast to the floccular (= Flocken) appearance of the H-form. They demonstrated in absorption experiments that the flagellar (H) and somatic (0)antigemreacted independently of each other. Furthermore, they found that the agglutination of sera from patients with typhus was related primarily to the 0-form (Weil and Felix, 1917).

190

P. LARSSON

On the basis of agglutination patterns in rabbit immune sera Wenner and Rettger ( 1 9 19) divided 73 P. vulgaris and P . mirabilis strains into seven groups, although no differentiation into somatic and flagellar antigens was made. Using the H- and 0-agglutination and absorption tests with six antisera Rustigian and Stuart (1945) divided 42 out of 57 P . vulgaris strains into five groups. Similarly, 138 P. mirabilis strains were divided into 38 groups with four antisera. They furthermore found 0-and H-antigens common to both P . mirabilis and P . vulgaris. H-antigens common to P . mirabilis, P . rettgeriand P. morganii and 0-and H-antigens common to P. vulgaris and P . morganii were also demonstrated. They did'not, however, consider the serotyping of Proteus to be of practical or taxonomical value. Winkle (1944/45) presented a serological typing system for P . hauseri ( = P. mirabilis and P . vulgaris) based on the analysis of a large number of strains. Eight serological types, designated A to H, were found among 961 Proteus isolates. The 4 X types, X2, X19, XL and XK, were also added to the system. A total of 13 0-antigens and eight H-antigens were described. A serological system for Proteus was also developed by MikolaSova in 1949 (as quoted by Sedlak, 1968). A comparison of the serological systems elaborated by Winkle, Perch and MikolaSova is shown in Table 11. TABLE I1 Comparison of the Proteus antigenic schemes of Winkle, Perch and Mikolsova Type

Winkle (194445) 0 H

X 19 XL

x2 XK

A F C D E B G

H

a, b, c

VII, VIII VIII, IX VIII, x v, VII XI1 XI11 From Sedlak (1968)

Symbols: . ,no information;

a, b, d a, b, d

f f g h

Perch (1950) 0 H la la, l b 2a 3a, 3b 3a, 3b 4a(. . .) l l a ... 12a 13a 23a 23a, 23b 23a 26a, 3b 21a 30a. . . 32a, 32c 42a

- , no antigens found.

la, lb, lc la, lb, Id la, lb, l c la, lc, le la, lc, le ? ?

la, lb, lc ?

Mikol&ova (1949) 0 H

XIV XXXIII XXXII

-

xv XI1

P gh

XI11

C

XXVIII IX

-

la, lc, le la, lc, le 2a, 2c, 2e 2a, 2c, 2e ? ?

18a, 18b

km

.

6. PROTEUS MIRABILIS A N D PROTEUS VULGARIS

191

Still another serological system developed by Belyavin et al. (195 1) was later used by Krikler (1953). Based on morphological phases found for Proteus on McConkey agar plates, 44 strains were divided into 18 0-groups, three of which were the X2, X19 and XK strains (Belyavin et al., 1951). Apart from the obvious identities of the X strains a few 0-antigens could be compared between the Belyavin et al. and Perch systems (Perch designation in parenthesis): 0-group 2 (0-group 30), 0-group 4 (0-group 23), 0-group 10 (0-group 4) and 0-group 3 (0-group 10). The serological systems for typing of P. mirabilis and P. vulgaris mentioned above have not been widely used with the exception of the one set up by Kauffmann and Perch (1947, 1948; Perch, 1950). Accordingly, this system is presented below in more detail.

IV. Present system for serotyping of P. mirabilis and P. vulgaris A. Surface structures

The well known swarming phenomenon is the main characteristic of Proteus growth on ordinary agar plates (Skirrow, 1969; Smith, 1972). The swarming, however, obscures the morphology of the separate colonies. Cultivation on agar plates containing 0.1% phenol revealed two different colony types“dull” and “bright”-according to Perch (1950). The dull forms showed long rods which swarmed rapidly with even dull surface growth. Living and formalin-treated cultures showed no or weak 0-agglutinability. The bright forms were short rods, with slow swarming and bright rippled agar surface growth. Living and formalin-treated cultures were 0-agglutinable. Colonies of the bright form were preferred for 0-antigen preparations but dull forms could also be selected provided that boiled washed cultures were used. Belyavin (1951) also reported different culture forms (A, B and C) and described form variations between these forms. The A-form consisted of uniformly bacillary rods swarming in a step-like manner on agar plates. This form was found in freshly isolated strains and A -+ B variation was suggested to be a shift from partial smooth (S)-+rough(R). A-C variation was also noted. The C-form consisted of uniformly filamentous rods often swarming in a uniform film on agar surfaces. No antigenic difference between the A- and Cforms was shown. The morphological variants of Perch and Belyavin have not been given consideration in more recent serological investigations. The cell wall composition of members of the Enterobacteriaceae has been studied extensively. Investigations have focused mainly on Escherichia, Salmonella and Klebsiella, which will not be discussed in this chapter. Excellent reviews on the biochemistry of the enterobacterial cell wall have

192

P. LARSSON

been published by Liideritz et al. (1966, 1968) and Jann and Westphal(l975). Comparatively little information is available on the surface structure of Proteus bacteria as a basis for understanding the Proteus serology. The lipopolysaccharides (LPS) found in the outer membrane have the same general structure for all enterobacteria with an 0-specific chain linked via a basal core to a lipid A component. Dmitriev et al. (1971) analysed the polysaccharides of several serotyped P. mirabilis and P. vulgaris strains. Three peaks could be demonstrated with gel filtration. Peak I contained high molecular weight polysaccharides with 0specificity, peak I1 corresponded to core polysaccharides and heptose and peak 111 was considered to be free KDO (2-keto-3-deoxyoctonate)and phosphate. Peak I was not found among rough Proteus strains. The chemical composition of LPS from 31 serotypes of P. mirabilis and 20 serotypes of P. vulgaris has been determined (Sidorczyk and Kotelko, 1973; Kotelko et al., 1975; Sidorczyk et al., 1975). These strains could be divided into 16 chemotypes (Table 111). P . mirabilis and P. vulgaris were found to contain galacturonic acid as well as glucuronic acid in the LPS, differing from other members of the Enterobacteriaceae. Also the presence of the two heptoses, L-glycero-D-mannoheptose and D-glycero-D-mannoheptose, located in the core region (Kotelko et al., 1977) has been found to be a characteristic for Proteus. By immunizing rats with boiled P. mirabilis antigens or LPS preparations Smith et al. (1970) demonstrated varying immune responses. Certain strains induced an IgM response, whereas others led to a rapid appearance of IgG antibodies. This difference could be correlated neither to the amount of antigen nor to the carbohydrate composition of the LPS. Alterations in the sequence of immunodominant sugars or differences in configuration were suggested as explanations. The bactericidal effect of normal human serum (SBS) on bacteria has been suggested to be a marker of bacterial virulence associated with the composition of the cell wall (Olling, 1977). Studies on Proteus have shown that spontaneously agglutinating (rough) P. mirabilis and P. vulgaris strains were significantly more sensitive to SBS than strains with an 0-antigen (Larsson and Olling, 1977). No differences were found, however, between strains of different 0-groups. Free lipid A from a P. mirabilis strain has been shown to consist of only two fatty acids: myristic acid and 3-hydroxymyristic acid (Sidorczyk et al., 1978). Lipid A preparations from smooth and rough forms have been suggested to be identical. A great similarity between lipid A from Proteus and Salmonella has been proposed on the basis of results from serological investigations (Sidorczyk et al., 1978). Similar to other members of the Enterobacteriaceae the outer membrane of

-1

IEX

000

z

N

0

0 0 0 0 0

00

Chemotype

Chemotype

Ribitol

Rhamnose

Galactose

a

?

2C

P

?

3C

Galactosamine

Glucosamine

Glucose

Galaturonic acid L-gIycero-D-mannoheptose D-glycero-D-mannoheptose KDO

Glucuronic acid

Chemotype

P . mirabilis

Z W

c.

21

0 000 000 0 0 0

00000

000000000

000

..... ......... ..... ..... .... ......... ..... ..... ..... ......... 00

$BEEX

TABLE III Chemotypes of Proteus mirabilis and Proteus vulgaris P . vulgaris

From Sidorczyk et a/. (1975) and Gmeiner et a/. (1977). Filled circles denote common components present in all the lipopolysaccharides.Open circles indicate components present in only some of the lipopolysaccharides.

194

P. LARSSON

P . mirabilis contains lipoprotein (Gruss et al., 1975; Katz et al., 1978; Rottem et al., 1979). A composition of 50-59 amino acids and a molecular weight of 5500-7000 has been proposed (Gruss et al., 1975; Katz et al., 1978). In contrast to other enteric bacteria the Proteus lipoprotein contains glycerine and phenylalanine. The lipoprotein has been suggested to be linked to the peptidoglycan by the C-terminal lysine residue (Gruss et al., 1975). Rottem et al. (1979) found that the outer membrane of a rough P . mirabilis strain had a similar polypeptide structure to that of the original wild type. The outer membrane polypeptides of the rough type were, however, more susceptible to proteolytic digestion than those of the smooth strain. This difference was suggested to depend on an increased availability of the polypeptides to the enzymes with shorter polysaccharide chains present in the rough strain (Rottem et al., 1979). Bub et al. (1980) recently from a P . mirabilis strain described two outer membrane proteins with molecular weights of 39 000 and 36 000. The flagella of Proteus bacteria are built up by flagellin protein. In the monomeric form the flagellin has a molecular weight of approximately 20 000 according to Erlander et al. (1960). Earlier observations (Gard et al., 1955) suggested a molecular weight of about 40 000. This difference could be due to the fact that flagellin exists in a monomeric form below pH 3.8 and as a dimer between ph 4.5 and 12. Izdebska-Szymona ( 1974a,b) compared the properties of flagella and flagellins of a smooth ( S ) P . mirabilis strain and its rough (R) mutant. The S type was motile, in contrast to the R mutant. Both forms had flagella as determined with electron microscopy although the R mutant had a lower number of flagella. Accordingly, the yield of R flagella was much less than of S flagella, but the flagellins had similar chemical compositions, molecular weights and were antigenically identical. In electrophoresis whole flagella moved to the anode, whereas flagellin moved towards the cathode. The fimbriae on the surface of P . mirabilis have been attributed a role in the attachment of Proteus bacteria to uroepithelial cells (Silverblatt and Ofek, 1978). Svanborg Eden et al. (1980) showed that the attachment properties of Proteus are different from that of E. coli. P . mirabilis attached only to squamous cells and not to transitional epithelial cells, whereas E. coli attached to both cell types.

B. Serological techniques I . Tube agglutination

In setting up the Proteus serological system Perch (1950) used both tube and slide agglutination. The ,tube agglutination for 0-antigen determination was

6 . P R O T E U S MIRABILIS AND P R O T E U S VULGARIS

195

performed with sera diluted to 1:10 or 1:20 up to 1:2560.Volumes of 0.2 ml of diluted serum and 0.2 ml of antigen were mixed. As 0-antigen was used a 20-h broth culture of the bacterial strain boiled for 1 h. The agglutination was read macroscopically after 20 h at 50°C. Determination of H-antigen was carried out in a similar manner (Perch, 1950). The serum dilutions for these determinations started from 1:25 or 150 and continued up to 1:25 600. H-antigens were prepared in the same way as Hantigens used for immunization (6-8 h highly motile broth culture with 0.5% formalin added). Agglutinations were read after 4-6 h at 50°C. For practical reasons typing of large numbers of Proteus strains requires simplified procedures. A routine typing technique for E. coli 0-antigens was described by Lincoln et al. (1970). This method has been modified for 0grouping of P . mirabilis and P . vulgaris (Larsson and Olling, 1977). In short, an overnight broth culture of each strain to be tested is autoclaved for 0.5 h or boiled for 2.5 h. One drop of this suspension is mixed in Perspex agglutination trays with one drop of Proteus 0-antiserum, properly diluted in each well (final dilution 1:800-1: 1600). Agglutinations are read after overnight incubation at 50°C using a plate microscope ( x 12.5). Care must be taken to determine the appropriate dilution for each antiserum used. This should be done using serial dilutions of the antiserum together with 0-antigen prepared from the homologous standard strain. To ensure accuracy bacterial strains with known strong cross-reactions should also be included. Some 20-30% of all strains are not typable with the 0-antisera. Such nonagglutinating strains are designated ONa and strains showing spontaneous (auto-)agglutination (Sa). 2. Slide agglutination

Slide agglutination is a very simple method, which is useful for serotyping of bacteria. Briefly described, one to two droplets of diluted antiserum and a small amount of bacterial culture are mixed on a microscopic slide. The slide is agitated and the agglutination read after 10-20 s. Perch (1950) recommended slide agglutination for the determination of H-antigens. By using specific Hantisera (0;H-antisera absorbed with homologous boiled culture) at a dilution of 1:100 unspecific and weak cross-reactions could be eliminated. Perch did not favour the slide technique for 0-agglutination since some strains show no or weak agglutination using this technique. Lanyi (1956) found that the 0-inagglutinability of living Proteus cultures could be eliminated by desoxycholate citrate (DC) agar plates. Growth on such plates would lead to underdevelopment of the flagellar antigens. No interference in the agglutination of the 0-antigen would thus take place. In

196

P. LARSSON

more recent studies, the slide agglutination technique has also been used to determine 0-and H-antigens (de Louvois, 1969; Larsson etal., 1978a; Penner and Hennessy, 1980). 3. Indirect haemagglutination The indirect or passive haemagglutination technique was developed by Neter et al. (1952). It has not been widely used for serotyping of the Enterobacteriaceae, but Penner and Hennessy ( 1980) recently used this technique to determine P . mirabilis and P . vulgaris 0-groups. The procedure involves centrifugation of autoclaved bacterial suspensions. The supernatants are then removed and diluted to 1:10 in phosphate-buffered saline and incubated for 1 h at 37°C with 1% suspension of washed sheep erythrocytes. The sensitized (coated) erythrocytes are, after washing and resuspension in buffer, used as antigen and added to two-fold dilutions of antiserum in micro-titration plates. Agglutination is read after incubation at 37°C for 1 h followed by storage overnight at 4°C. The indirect haemagglutination technique has also been used for serological diagnosis of Proteus urinary infections in patients (Bremner et al., 1969; Clark etal., 1971; Decker and Hirsch, 1971; Fairley etal., 1971; Larsson etal., 1978b). Renal infections give an increase in serum antibody levels to the infecting strain, in contrast to bladder infections where the antibody levels remain low. The patient’s own infecting strain has most commonly been used as antigen, but a pool of ten common Proteus 0-antigens has also been used (Bergman, 1978). By pretreating the serum samples with /I-mercaptoethanol (ME) a rough estimation can be made of antibodies of the IgM class (ME sensitive) and of the IgG class (ME resistant), as ME treatment disturbs antibodies of the IgM class and so lowers the titre of the serum sample. 4 . Gel precipitation techniques

Antigen-antibody reactions can be analysed with immunodiffusion techniques. Several different methods have been developed which permit determination and identification of bacterial antigenic structures. Only the techniques used for P . mirabilis and P . vulgaris will be presented here. For general information on immunodiffusion methods the reader is referred to handbooks (Ouchterlony and Nilsson, 1978). ( a ) Double diflusion-in-gel. This method has been useful in qualitative analyses of P . mirabilis and P . vulgaris antigens (Gard et al., 1955;Urbach and Schabinski, 1960; Larsson et al., 1973). Different variants of this method have

6 . PROTEUS MIRABILIS AND PROTEUS VULGARIS

197

been developed. The micro-technique of Wadsworth (1957) requires only small amounts of antigens and antisera and has proven to be sensitive and reliable. In this method a plexiglass cover is placed on a glass plate and a small slit (about 0.6 mm) formed between the plexiglass cover and the glass plate by strips of waterproof tape attached to the lower surface of the plexiglass cover. The space is filled with agar (1% agar and 0.85%NaCI). Small holes (diameter 2-3 mm) previously drilled in the plexiglass cover about 1 cm apart are then filled with the antigens or the antisera to be tested. The antigens and antibodies diffuse in the agar gel for 24-48 h and precipitates form between homologous components. ( b ) Immunoelectrophoresis. Several investigators have analysed the antigenic structure of P . mirabilis and P. vulgaris with immunoelectrophoretic techniques (Urbach and Schabinski, 1960; Imperato et al., 1968; Larsson et al., 1973; Sidorczyk and Kotelko, 1973). The macro-technique of Wadsworth and Hanson (1960) and the micro-technique of Scheidegger (1955) are both based upon the same principle: holes are cut in an agar gel supported by a glass plate and then filled with the antigen to be analysed. Electrophoresis is then performed, normally using veronal buffer pH 8.2-8.6 at 5-6 V/cm for 45-90 min. A long basin parallel to the electrical current is cut and filled with antiserum. 'The precipitation pattern can be analysed after 12-24 h. The Proteus surface antigens move primarily towards the anode and so it can be advantageous to punch the holes for the antigens towards the cathode. ( c ) Crossed immunoelectrophoresis. The crossed immunoelectrophoretic technique (Axelsen et al., 1973) has proven valuable in analyses of bacterial antigens. In particular, the serology of Pseudomonas aeruginosa has been extensively studied (Hsiby, 1975a,b).This technique has also been used in the serology of Bordetella pertussis and Salmonella typhi (Hsiby et al., 1976; Espersen et al., 1980). One P. mirabilis strain was analysed together with some 20-30 other bacterial species in these studies, which included cross-reactivity between strain. Larsson et al. ( 1973) used crossed immunoelectrophoresis together with other techniques in the analysis of P. mirabilis 0:3;H:1.

5 . Immobilization test

The addition of an antiserum to live Proteus bacteria will immobilize the normally motile bacteria. This phenomenon can be studied by phase contrast or dark field microscopy. The antibody titres can be determined by testing at different antiserum dilutions. This technique is laborious, however, and has not been widely used (Pazin and Braude, 1969, 1974).

198

P. LARSSON

6 . Enzyme-linked immunosorbent assay ( E L I S A )

The ELISA technique was originally developed by Engvall and Perlmann (1972). This method is very sensitive and permits differentiation of the antibody response into the various immunoglobulin classes. Antibodies to P . mirabilis 0- and H-antigens have also been determined with the ELISA technique (Larsson et al., 1978b). Serum samples were taken from patients with urinary tract infections caused by P . mirabilis. Boiled bacteria were used as 0-antigens and flagellar preparations as H-antigens.

C. Antigens I . 0-antigens A total of 49 different 0-antigens were recognized in the serological system of P . vulgaris and P . mirabilis set up by Perch (1950). These were numbered 0:1 to 0:49 (Table IV). A more detailed analysis was performed for the first 30 0antigens. The reference strains X 19, XL, X 2 and XK were included in the system and given the 0-antigens 1,2 and 3. The agglutination techniques used by Perch (1950) permitted differentiation into subdeterminants for the 0antigens 1,3,4,5,7,11,14-17,19,23,26 and 30 (Table IV). Immunoelectrophoretic studies of P . vulgaris and P . mirabilis carried out by Larsson et al. (1973) and Sidorczyk and Kotelko (1973) have shown that Proteus 0-antigens move towards the anode. This has so far been shown for the 0-antigens 3,5-7,10,13,14,16,23,24,26-30,41,43,48 and 49. The motility of the Proteus 0-antigens towards the anode contrasts with the 0-antigens of the clinically most commonly encountered E. coli strains, where the 0-antigens move towards the cathode (Orskov et al., 1977). The partial 0-antigens 3a and 3b of the 0:3a,3b; H:la,lc,le (XK) strain could not be identified using an immunodiffusion technique (Larsson et al., 1973). In the studies of Larsson et al. (1973) and Sidorczyk and Kotelko (1973) analyses of lipopolysaccharide antigens with immunoelectrophoretjc techniques using homologous antisera showed only one precipitate. Biochemical analyses of some P . mirabilis strains have, however, revealed a heterogeneity of the lipopolysaccharide (Gmeiner, 1975; Krajewska and Gromska, 1981). Not all P . vulgaris and P . mirabilis strains can be typed according to the 49 0-antigens of Perch. Larsson et al. (1978a) used provisional serotypes designated O:A to O:E to decrease the frequency of non-typable strains in an epidemiological study. Additional serotypes have also been suggested by Penner and Hennessy (1980), who included 11 new provisional 0-groups: 0:lOO to 0:104 for P . vulgaris and 0:200 to 0:205 for P . mirabilis. No capsular polysaccharide (K-antigen) has been found among P . vulgaris

199

6 . PROTEUS MIRABILIS AND PROTEUS VULGARIS

TABLE IV Diagnostic antigenic scheme of Proteus vulguris and Proteus mirubilis according to Perch (1950) (the extended antigenic formula is given for strains of 0-groups 1-30 and the simplified formula for 0:31-49) Antigens Reference 0-group strain

-

H

0

Subgenus"

-

1

X 19

2 3

x 2 XK F 403 F 248 U8 F 407 F 394 F 16 F 196 F 267 F 181 F 78 F 116 F 27

4' 5

6 7 8 9 10

11

12 13

14 15

XL

u 144 F 387 F 30 F 62 F 75 F 39 F2 F 73 F 506 F 280 F 47 F 67 F1 P 81 F 322 F 65 F 358 F 95 F 427 F 151 F 219 F 120 S 127 F 121

la la, l b 2a 3a, 3b 3a, 3b 3a, 3b 4a, 4b 4a, 4c 4a, 4c 5a, 5b 5a, 5c 5a, 5c 6a 6a 6a 7a, 7b 7a, 7c 7a, 7b 8a 9a 9a 1Oa 1Oa 1Oa 1Oa 1Oa lla, l l b lla, llb, llc lla, lld lla, lld lla, llb 12a 12a 13a 13a 13a 13a 14a, 14b 14a, 14c 15a

la, lb, lc la, lb, Id la, lb, lc la, lc, le 2a, 2b, 2e, 2f 2a, 2c, 2e la, lb, Id 8a 16a la, lc, le la, lc, le 3a, 3 b . . . la, lc, le 2a, 2b, 2e, 2f 3a, 3b.. . la, Id, le, If 3a, 3 b . . . 4a, 4b, 4c la, lb, lc la, lc, le 2a, 2c, 2e la, lc, le 2a, 2c, 2e 3a, 3 b . . . 4a, 4b, 4c 5a la, lc, le 2a, 2b, 2e, 2f 2a, 2b, 2e, 2f 3a, 3 b . . . 6a la, lb, lc 2a, 2c, 2e la, lc, le 2a, 2c, 2e 3a ... 4a, 4c,4d la, lc, le 3a, 3b.. . la, lb, I d . . .

1 1 1 2 2 2 1 1 1 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 1

P. LARSSON

200

TABLE IV continued ~

Antigens Reference 0-group strain 16 17 18 19

20 21 22 23

24

25 26 27 28 29 30

31 32

F 295a F 55 P 206 F 485 F 92 F 119 F 136 F 313 F 434 F311 u 349 F 475 F 382 M 205 F 233 F 162 F 431 F 63 F 45 F 296 F 288 F 103 F 90 F 330 F 276 F 58 F 458 P 372 F 25 U 501 U 509 F 87 F 10 F 384 F 29 F 321 F 152 U 96 F 49 F 110 F 125 F 139 F 388 F 53

H

0 15a, 15b 16a 16a, 16b 16a, 16b 17a, 17b 17a, l?c 18a 19a, 19b, 19c 19a, 19c, 19d 19a, 19e 19a, 19b, 19c 20a 20a 21a 22a 23a, 23b 23a, 23c, 23d 23a, 23c 23a, 23c, 23d 23a, 23c 24a 24a 24a 24a 25a 26a, 3b 26a, 3b 26a, 3b 27a 27a 28a 28a 29a 30a 30a 30a, 30b 30a 30a 30a 31 31 32 32 32

7a la, lc, l e 9a 14a la, lc, le 1Oa la, lc, le la, lb, lc la, lc, le 3a, 3b.. . 1 la la, lc, le 2a, 2c, 2e la, lb, lc la, lb, lc la, lc, le 2a, 2b, 2e, 2f 2a, 2d, 2f 3a, 3b.. . 12a la, lc, le 3a, 3b.. . 4a, 4b, 4e 13a la, lb, lc 2a, 2c 3a ... 6a 2a, 2c, 2e 3a, 3b.. . 2a, 2c, 2e 3a, 3b.. . 13a la, lc, le 2a, 2b, 2e, 2f 2a, 2c, 2e 4a, 4b, 4e 13a 15a 1 2 1 3 5

Subgenus" 1 2 2 2 2 1 2 1 2 2 1 2 2 1 1 2 2 1 2 1 2 2 2 2 1 2 2 1 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 1

20 1

6 . PROTEUS MIRABILIS AND PROTEUS VULGARIS

TABLE IV -continued Antigens 0-group 33 34 35 36 37 38 39

40 41 42 43

44 45 46 47 48 49

Reference strain

U 510 F 72 F 335 F 305 F 398 F lOOb F 420 F 158 F 105b F 386 F409 P 522 F 163 F 433 F 108 F 179 F 171 F 223 F 285 P 368 F 389

Subgenus"

0

H

33 34 35 36 36 37 38 38 39

3 6 2 3 7 17 1 2 18 4 1 2 1 2 11 19 11 17 1 1 2

40 41 41 42 43

44 44 45 46 47 48 49

2 1 2 2 1 1 2 2 1 2 2 2 1 2 1 1 1 1 1 2 2

The majority of the strains were isolated from faeces (F), urine (U) and pus (P). A few strains were isolated from autopsy (S) and one strain was from the heart of a mouse (M). Symbols: . . .. further.antigensmay be present but not investigated. Subgenus 1 is P . vulgaris, subgenus 2 is P . mirabilis.

and P . mirabilis strains (Perch, 1950; Dmitriev et al., 1971; Larsson et al., 1973; Sidorczyk and Kotelko, 1973). Using agglutination studies as a basis, however, Namioka and Sakazaki (1959) described an antigen in some P . mirabilis, P . rettgeri and P. vulgaris strains. This antigen had properties differing from regular K-antigens and was hence given the term C-antigen. 2. H-antigens

Perch (1950) divided the flagellar antigens into 19 H-types on the basis of agglutination tests. The H:l-antigen could be further subdivided into la, lb, lc, Id, le and I f with combinations of three or more subdeterminants for each H:l strain as seen in Table IV. The H:Zantigen consisted of 2a, 2b, 2c, 2d, 2e and 2f with several combinations. The H:3-antigen could be separated into 3a

202

P. LARSSON

and 3b. The H:Cantigen was divided into 4a, 4b, 4c,4d and 4e;combinations of three subdeterminants were also found for H:4. No such flagellar antigenic subdivision was reported for H-antigens 5 to 19 (Perch, 1950). Using immunodiffusion technique Gard et al. (1955) showed that purified flagella from ProteusX 19(Perch designation: 0 : l a ; H:la,lb,lc) formed three precipitation lines with rabbit antisera. One of these lines was considered to be the H-antigen; a second line was probably the 0-antigen and was regarded as an impurity. The third line could not be identified. By means of double diffusion-in-gel, immunoelectrophoresis and crossed immunoelectrophoresis Larsson et al. (1973) found only one flagellar precipitate when analysing an 0:3;H: 1 (XK) strain. Izdebska-Szymona (1974a,b) studied the antigenic properties of flagella and flagellin of P. mirabifis strain 1959 with similar techniques. Flagella were shown to have two antigenic determinants, one of which was also found on flagellin. 3. Common antigens

The members of the Enterobacteriaceae are closely related and, not surprisingly, common antigens have been identified. The enterobacterial common antigen (ECA) originally described for E. coli by Kunin (1963) has also been found in other bacteria including Proteus. This has been reviewed by Makela and Mayer (1976). Using immunodiffusion techniques Kaijser (1975) identified an antigen with a high electrophoretic mobility which was different from the ECA. This antigen was found in strains belonging to E. coli, Proteus and Pseudomonas, and antibodies to this antigen were also present in anti-meningococcal antiserum. Sompolinsky et al. (1980) have suggested it to have a total molecular weight of more than 400 000-600 000 with subunits of 62 0 0 0 4 5 000 and 59 000-62 000.

D. Antigenic variations Quantitative variations of the 0-antigens were reported by Kauffmann and Perch (1947). When analysing five colonies of Proreus in faecal samples P. Larsson (unpublished data) also observed an occasional variation in agglutinability to a specific antiserum for colonies reacting with an 0-antiserum. Some strains showed a tendency towards spontaneous agglutination. It has been known for several decades that Proteus strains stored on agar slants may show spontaneous agglutination (Felix and Rhodes, 1931). This variation from smooth to rough is natural, and occurs in all genera of the Enterobacteriaceae. Mutations in the synthesis of the 0-polysaccharide chain or in the basal core will give rise to variants with no or incomplete 0-antigen.

6 . PROTEUS MIRABILIS AND P R O T E V S VULGARIS

203

Quantitative variation of the H-antigens have been reported for the H: Ic antigen of the X 19 strain and the H:ld antigen of the XL strain (Kauffmann and Perch, 1947). Similarly, Perch (1950) reported on such variations of the Hantigens 3b and 4b. Natural form variation similar to that of Salmonella has not been found for P . mirabilis and P . vulgaris. By growing Proteus strains on media containing H-antisera to prevent swarming Perch (1950), however, could induce R forms. These R forms did not revert into the ordinary S forms, and were also shown to contain new H-antigens. Their antigenic determinants were given Roman numerals I-VI. The R forms were not considered to be of practical importance except when media containing H-antisera were used (Perch, 1950). E. Antigenic cross-reactions 1. Cross-reactions within the Proteus serological system

There are several 0-antigen cross-reactions within the system of P. mirabilis and P . vulgaris as described by Perch (1950) (Table V). In addition, Lanyi (1956) reported reactions between 0-groups 0:41 and 0:3 1,0:48 and 0:21. A cross-reactivity between 0:13 and 0:30 has also been noted (Larsson and Olling, 1977). Using haemagglutination technique Penner and Hennessy (1 980) were able to describe numerous cross-reactions, many of which were weak and were suggested to be due to antibodies against rough components. No cross-reactions of practical importance have been found between the Hantigens within the Proteus serology system (Perch, 1950).

2. Cross-reactions between Proteus and other bacteria Several serological cross-reactions have been noted between Proteus and other members of the Enterobacteriaceae. Kauffmann and Perch (1948) reported a reaction between Proteus 0:2 and E. coli 0:12. Later, Perch (1950) noted cross-reactions between: Proteus 0:1 1 and Salmonella 0:38; Proteus 0 : 2 5 and Salmonella 0:29 and 0:1,6,14,25; and Proteus 0:21 and 0:25 and Salmonella 0:1,3,14. Reactions between Proteus and Arizona were also found, namely between Proteus 0 : 5 and Arizona 0:14; Proteus 0:7 and Arizona 0:19; Proteus 0 : 8 and Arizona 0:13 and Proteus 0:1 1 and Arizona 0:16. A relation between Proteus 0:13 and a P . morganii strain was demonstrated as well (Perch, 1950). Frantzkn (1950) described cross-reactions between the following Proteus and E. coli bacteria: Proteus 0 : 2 and E. coli 0:12; Proteus 0:12 and E. coli

204

P. LARSSON

TABLE V 0-antigen cross-reactions within the Proteus mirabilis and Proteus vulgaris system

0-antigen

cross-reacting with

2 2 3 3 4 5 6 7 7 15 16 17 33 35 36 37 41 42 43 46 47 49

0-antigen 12 19 26 13" 9 9

8"

11 34

40 47 35 41 45 11" 5" 14, 16, 33" 38" 5, 49" 37" 7" 7"

From Perch (1950). Unilateral cross-reaction

0:29; Proteus 0:29 and E. coli 0:67; Proteus 0:32 and E. coli 0:48 and 0:54 and Proteus 0:47 and E. coli 0:76. Espersen et al. (1980), using cross-line immunoelectrophoretic techniques, recently demonstrated cross-reactions between Salmonella typhi and 24 other bacterial species, one of which was a P. mirabilis strain. The cross-reactivity between Proteus and other bacteria based on common antigens has already been mentioned (Section IV.C.3). 3. Other cross-reactions

The best known cross-reaction is that between certain Proteus strains and Rickettsia, which has been used in the Weil-Felix test for serological diagnosis of epidemic typhus. Weil and Felix (1916) observed that a Proteus strain isolated from the urine of a patient with typhus could be agglutinated with this patient's serum and also with sera from other cases of typhus. Additional

205

6. PROTEUS MIRABILIS A N D PROTEUS VULGARIS

Proteus strains with a similar ability were also found (Felix, 1916; Felix and Rhodes, 1931). These Profeus strains, X 19, X 2 and XK,were later found to show different agglutination patterns in patient sera, depending on the type of rickettsia1 disease (Table VI). Castaneda (1934) showed that the Proteus X 19 strain possessed an acid-stable and moderately alkali-stable polysaccharide antigen, which was responsible for the cross-reaction with Rickettsia. Immunoelectrophoretic techniques have also been used to demonstrate the antigenic relationship between Proteus X strains, R. prowazeki and C. burnetii (Urbach and Schabinski, 1960). Although specific rickettsia1 serological tests are available the Weil-Felix reaction is still widely in use for presumptive diagnosis of rickettsioses, probably due to the accessibility of antigens and simplicity of performance (Vinson, 1976). Cross-reactivity between human blood groups and Proteus strains have been reported. The presence of galactose, N-acetylglucosamine, Nacetylgalactosamine and fucose has been proposed to be responsible for blood group A and B activity in the lipopolysaccharide (Pardoe et al., 1968; Springer, 1971).

TABLE VI

Weil-Felix reaction in rickettsioses Agglutination of Proteus Disease Epidemic typhus Murine typhus Brill-Zinsser disease Spotted fever

Scrub typhus Rickettsia1 pox Q fever

Trench fever

Aetiological agent Rickettsia prowazeki R . mooseri R . prowazeki R . rickettsii R . conorii R . sibirica R . australis R . tsutsugamushi R . akari Coxiella burnetii Rochalimea quintana

0 : X 19 (0:ly 0 : X 2 (0:2) 0 : X K ( 0 : 3 )

++++

++++

+ +

-b

-b

++++ +

+ ++++

0 0 0 0

0 0 0 0

0 0

0 0 0

++ 0 0 0

From Vinson (1976). 0-antigen according to Perch (1950). Usually negative.

F. Dienes phenomenon The Dienes culture test (Dienes, 1946; Skirrow, 1969) is based on the ability of

206

P. LARSSON

most P. mirabilis and P . vulgaris strains to swarm on ordinary agar plates. When two or more strains of Proteus are grown on the same agar plate the spreading growth of the adjacent strains will sometimes form a line of demarcation, where growth of the strains meet. An antagonistic effect would indicate different strains, whereas a merging growth would point to identity between the isolates. Comparisons of the Dienes' phenomenon and serological characteristics have shown the Dienes' test to be unreliable sometimes (Krikler, 1953; de Louvois, 1969). Strains with identical 0- and H-serotypes show no line of demarcation. Strains with different 0-antigens and the same H-antigen also often show no antagonism. The outcome of the Dienes phenomenon seems mainly to depend upon the H-antigens (Krikler, 1953; de Louvois, 1969). The Dienes inhibition phenomenon has been suggested to be due to toxic products, possibly bacteriocins (Grabow, 1972; Smith, 1972; Senior, 1977a). The formation of such products has not been correlated to specific antigenic types. The Dienes phenomenon might serve to further distinguish strains within the same 0- and H-serotypes.

V. Production of antisera

For production of 0-antisera, Perch (1950) recommended a 20-h broth culture, which was boiled for 2.5 h to destroy the H-antigen. Formalin (0.5%) could be added as a preservative. Rabbits were injected intravenously every fifth day with doses of 0.25, 0.5, 1.0, 1.5 and 2.0ml. The animals were exsanguinated eight days after the last injection. Boiling for 2.5 h will sometimes not be sufficient to destroy the H-antigen completely. According to Perch (1950) this is often found if dull colonies are used for preparation of the antigen. The problem is eliminated, however, by washing the boiled bacterial culture and resuspending the cells in saline before injecting the rabbits (Perch, 1950). When producing 0-antisera care should be taken to ensure that the antigen culture is smooth (S). It is often difficult, due to the swarming phenomenon, to identify Proteus colonies as rough on ordinary agar plates. A rough strain (R) will, however, be autoagglutinable after boiling. Slide agglutination of an agar plate culture with saline may also disclose rough forms. Using purified lipopolysaccharide antigens allows more specific 0-antisera to be obtained. H-antisera are produced by vaccines prepared from 6-8 h broth cultures. Formalin (0.5%) is added and the culture left at 37°C overnight. To ensure an optimal H-antigen actively motile bacterial cells should be used for antigen. Repeated passages on semi-solid or 1% agar is recommended. The same scheme for injections is used as for 0-antiserum production (Perch, 1950).

207

6. PROTEUS M I R A B I L I S AND PROTEUS VULGARIS

The H-antisera produced according to this procedure contain antibodies against both 0- and H-antigens, and should be called 0;H-antisera. The presence of 0-antibodies is of minor importance when the sera are used in agglutination tests, as 0-and H-agglutinations have different appearances. 0agglutinations have a granular appearance, in contrast to the floccular Hagglutination. Absorption with boiled washed bacteria of the same strain or a strain with the same 0-antigen remove homologous 0-antibodies from the 0;H-antisera. Repeated absorptions with sufficient numbers of antigen cells must usually be used to produce good monospecific (factor) typing sera. Purified Proteus flagella or flagellin prepared according to Ada et al. (1964) can be used to immunize rabbits for H-antiserum production (P.Larsson and A. Soh1 Akerlund, unpublished data). VI. Distribution of serotypes in clinical isolates Relatively few studies have been performed on clinical isolates of P. mirabilis and P . vulgaris with regard to the presence and distribution of 0-antigens as a possible virulence factor. 0-antigen distributions of various studies are given in Fig. 1. The origin of the samples investigated varies widely between authors. This makes comparisons difficult. The strains collected by Perch (1950) were isolated primarily from faeces. Lanyi (1956) described faecal strains, mostly from children with diarrhoea. de Louvois (1969) and Larsson and Olling (1977) studied mostly strains from urinary tract infections. Sedlak et al. (1959) x 01

0 lypabk slrains

n 10

11

13

23

24

26

27

28

28

30 Oantlgen

Fig. 1. The 0-antigen frequency of P. mirabilis and P. vulgaris among 0-typable strains in different studies [Perch (1950) 538 strains, Lanyi (1956) 971 strains, Sedlak et al. ( 1959) 939 strains, de Louvois (1 969) 244 strains and Larsson and Olling (1 977) 299 strains].

208

P. LARSSON

did not present clinical data making the origin of the Proteus strains unknown. Figure 1 shows that 0:3 was common in all studies and that 0:10,0: 13,0:26, 0:28 and 0:30 were frequent. Among strains isolated from two hospitals in Canada the most frequent 0-groups among 194 P. mirabilis were 0:3, 0:6, 0:10,0:28 and 0:30, and among 208 P. vulgaris were 0:1,0:4,0: 12,0:32 and 0:100 (Penner and Hennessy, 1980). The clinical origins of these strains were not given, but they were isolated over extended periods of time. Sedlak et al. (1959) described two cases of Proteus bacteraemia with the antigens 0 : 3 and 0:13. One patient with bacteraemia caused by an 0:26a,3b strain was also later reported (Sedlak, 1968). A total of 23% of strains with the 0:3-antigen was found by Larsson and Olling (1977), who analysed 100 P. mirabilis and P. vulgaris strains from blood cultures. This could be taken to indicate that the 0:3-antigen might be associated with a high virulence. In a more recent study, however, Larsson (1980) did not find such a domination of the 0:3-antigen among 172 P . mirabilis and 17 P. vulgaris strains isolated from blood cultures over a nine-year period. The 0:3-, 0:23-, 0:lO-, 0:30- and 0:24-antigens were common, but their frequencies in blood isolates did not differ significantly from that of strains isolated from urines or faeces. Thus, specific 0-antigens do not seem to be associated with higher virulence. Information concerning the distribution of H-antigens among clinical isolates of Proteus is scarce. The H-antigens H: 1, H:2 and H:3 dominate and comprise 79% of all P. mirabilis and P. vulgaris strains, as reported by Kauffmann and Perch (1947). From the study of Lanyi (1956) a similar occurrence, 77.9%, can be calculated for the same antigens.

VII. Serotyping in epidemiological work Proteus mirabilis is a common cause of urinary tract bacteriuria in hospitals, especially in urological and geriatric wards, occasionally due to nosocomial spread. Biochemical and antibiotic resistance patterns have often been of limited value as epidemiological markers, inasmuch as rather few different types can generally be discerned. Consequently, several techniques have been developed to find better epidemiological markers, such as for Proteus the Dienes phenomenon (Story, 1954; Edebo and Laurell, 1958; Burke et al. 1970), bacteriocin (Cradock-Watson, 1965; Senior, 1977b), bacteriophage (France and Markham, 1968) and resistogram typings (Kashbur et al., 1974). 0-serotyping of P . mirabilis and P. vulgaris has been used in only a few epidemiological studies. de Louvois (1969) serologically studied P . mirabilis from hospital infections. Larsson et al. (1978a) analysed 3519 Proteus strains from a geriatric ward using both 0-and H-serotyping. Nosocomial spreading of faecal and urinary strains occurred between patients, probably as the result

6 . PROTEUS MIRABILIS AND PROTEUS VULGARIS

209

of inadequate nursing techniques. The serotyping technique proved to be reliable and allowed large numbers of strains to be handled. Some strains, however, were non-typable according to 0-and H-antigens. This indicates the need for additional typing techniques or further elaboration of the serological typing schemata. A combination of 0-serotyping, proticine production, proticine sensitivity and Dienes typing has recently been shown to provide a highly discriminating typing scheme for P. mirabilis and P . vulgaris (Senior and Larsson, 1983). 0-groups 1,2,4,8,15,21,22,25,34,37,39,42 and 4 4 4 7 of Perch are only found in P. vulgaris (subgenus 1) (Table IV). Only few 0-antigens (12,17,19,23,26,31,32 and 36) are found both in P. mirabilis and P . vulgaris. The remaining 0-groups are found only in P. mirabilis (subgenus 2). This difference in the distribution of 0-antigens has been further stressed by Penner and Hennessy (1980), who found that only 3.4% of their P . vulgaris isolates agglutinated in P. mirabilis antisera and 1.5% of the P. mirabilis strains in P . vulgaris antisera. The use of separate typing schemes for P. mirabilis and P . vulgaris in epidemiological studies was recommended.

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Dienes, L. (1946). Proc. Soc. ESP. B i d . Med. 63, 265-270. Dmitriev, B. A,, Hinton, N. A., Lowe, R. W. and Jones, J. K. N. (1971). Can. J . Microbiol. 17, 1385- 1394. Edebo, L. and Laurell, G. (1958). Acta Pathol. Microbiol. Scand. 43, 93-105. Edwards, P. R. and Ewing, W. H. (1972). “The Identification of Enterobacteriaceae”, 3rd edn., pp. 324336. Burgess, Minneapolis, Minnesota. Engvall, E. and Perlmann, P. (1972). J. Immunol. 109, 129-135. Erlander, S., Koffler, H. and Foster, J. F. (1960). Arch. Biochem. 90, 139-153. Espersen, F., Hsiby, N. and Hertz, J. B. (1980). Acta Pafhol. Microbiol. Scand. Sect. B 88, 243-248. Ewing, W. H., Tanner, K. E. and Dennard, D. A. (1954). J . Infkct. Dis.94, 134140. Fairley, K. F., Grounds, A. D., Carson, N. E., Laird, E. C., Gutch, R. C., McCullum, P. H. G., Leighton, P., Sleeman, R. L. and O’Keefe, C. M. (1971). Lancet 2, 6 1 5-6 18. Felix, A. (1916). Wien. Klin. Wochenschr. 29, 873-877. Felix, A. and Rhodes, M. (1931). J. Hyg. 31, 225-246. France, D. R. and Markham, N. P. (1968). J. Clin.Pathol. 21, 97-102. Frantzen, A. ( 1950). “Kulturelle og serologiske underssgelser over nogle grupper af Enterobacteriaceae”, pp. 1- 139. Thesis, Munksgaard, Copenhagen. Gard, S., Heller, L. and Weibull, C. (1955). Acta Pathol. Microbiol. Scand. 36, 30-38. Gmeiner, J. (1975). Eur. J. Biochem. 58, 621-626. Gmeiner, J., Mayer, H., Fromme, I., Kotelko, K. and Zych, K. (1977). Eur. J. Biochem. 72, 35540. Grabow, W. 0. K. (1972). J. Med. Microbiol. 5, 191-196. Gruss, P., Gmeiner, J. and Martin, H. H. (1975). Eur. J. Biochem. 57, 41 1414. Haenel, H. and Dawidowski, B. (1961). Zentralbl. Bakteriol. Parasitenkd. In,fektionskr. Hyg. Abt. 1 Orig. 182, 183-190. Hallett, R. J., Pead, L. and Maskell, R. (1976). Lancet 2, 1107-1 110. Hauser, G. (1885). “Uber Faulnissbacterien und deren Beziehungen zur Septicamie”, pp. 1-94. F. C. W. Vogel, Leipzig. Hsiby, N. (1975a). Scand. J. Immunol. 4, Suppl. 2, 187-196. Hsiby, N. (1975b). Acta Pathol. Microbiol. Scand. Sect. B 83, 433442. Hsiby, N., Hertz, J. B. and Andersen, V. (1 976). Acta Pathol. Microbiol. Scand. Sect. B 84, 395400. Imperato, S., Martinetto, P. and Gabriel, L. (1968). Riv. Ist. sieroter. Ital. 43, 91-98. Izdebska-Szymona, K. (1974a). Acta Microbiol. Pol. Ser. A 6, 163-172. Izdebska-Szymona, K. (1974b). Acta Microbiol. Pol. Ser. A 6, 241-251. Jann, K. and Westphal, 0. (1975). In “The Antigens” (M. Sela, Ed.), Vol. 3, pp. 290-340. Academic Press, New York and London. Kaijser, B. (1975). Int. Arch. Allergy Appl. Immunol. 48, 72-81. Kashbur, I. M., George, R.H. and Ayliffe, G. A. J. (1974). J. Clin. Pathol. 27,572-577. Katz, E., Loring, D., Inouye, S. and Inouye, M. (1978). J. Bacteriol. 134, 674676. Kauffmann, F. (1954). “Enterobacteriaceae”, 2nd edn., pp. 288-289. Munksgaard, Copenhagen. Kauffmann, F. (1969). “The bacteriology of Enterobacteriaceae”, 2nd edn., pp. 333-360. Munksgaard, Copenhagen. Kauffmann, F. and Perch, B. (1947). Acta Pathol. Microbiol. Scand. 24, 135-149. Kauffmann, F. and Perch, B. (1948). Acta Pathol. Microbiol. Scand. 25, 608-610. Kippax, P. W. (1957). J. Clin. Pathol. 10, 211-214. Kotelko, K., Fromme, I. and Sidorczyk, Z . (1975). Bull. Acad. Pol. Sci. Ser. Sci. Biol. 23, 249-256.

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21 1

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213

6 . PROTEUS MIRABILIS AND PROTEUS VULGARIS

Appendix Proteus serotype strains All strains listed below are deposited with the culture collection (CCUG) Department of Clinical Bacteriology, Guldhedsgatan 10, S-713 46 Goteborg, Sweden.

B. Perch 1

3 8 11 14 20 21 23 25 29 32 34 38 39

40 42 47 52 54 56 59 62 63 67 68 70 72 14 80 82 84 86 89

90

92 98 100 102 104 106

Strain No. X 19H3137 NC XL X23307NC XK F403 F407 F394 F 196 F181 F27 F30 F62 F73 P506 F280 F67 F65 F219 S 127 F295a F485 F 119 F 136 U349 F475 M 205 F233 F431 F90 F276 F458 F25 F87 F 10 F384 F 118 F 125 F388 U510 F72

0-antigen

H-antigen

la la, lb, Ic la, l b la, lb, Id 2a la, lb, lc 3a, 3b la, lc, le 3a, 3b 2a, 2b, 2e, 2f 4a, 4c 8a 4a, 4c 16a 5a, 5c la, lc, le 6a la, Ic, le la, Id, le, If 7a, 7b 8a la, lb, Ic 9a la, lc, l e 1Oa 3a, 3 b . . . 1Oa 4a, 4b, 4c 1Oa 5a 1 la, 1 lb, 1 lc 2a, 2b, 2e, 2f 12a la, lb, lc 13a 4a, 4c,4d 14a, 14c 3a, 3 b . . . 15a, 15b 7a 16a, 16b 14a 17a, 17c 10a 18a la, Ic, le 19a, 19b, 19c l l a 20a la, lc, le 21a la, Ib, lc 22a la, Ib, lc 23a, 23c, 23d 2a, 2b, 2e, 2f 24a 4a, 4b, 4e 25a la, Ib, Ic 26a, 3b 3a.. ... 27a 2a, 2c, 2e 28a 3a, 3 b . . . 29a 13a 30a la, Ic, le 30 15 31 2 32 3 33 3 34 6

Species P . vulgaris P . vulgaris P . vulgaris P . mirabilis P . mirabilis P . vulgaris P . vulgaris P . mirabilis P . mirabilis P . mirabilis P . vulgaris P . mirabilis P . mirabilis P . mirabilis P . mirabilis P . mirabilis P . vulgaris P. mirabilis P . mirabilis P . vulgaris P. mirabilis P . vulgaris P . mirabilis P. vulgaris P . mirabilis P. vulgaris P . vulgaris P . mirabilis P . mirabilis P . vulgaris P . mirabilis P . mirabilis P. mirabilis P . mirabilis P . mirabilis P . mirabilis P . mirabilis P . mirabilis P . mirabilis P . vulgaris

EF Culture Collection No. 4634 4635 4636 4637 4638 8785 4639 4640 4641 4642 4643 4644 4645 5082 8786 4646 4647 4648 4649 4650 465 1 4652 4653

4654 4655 4656 4657 4658 4659 4660 4661 4662 4663

4664 4665 8787 4666 4667 4668 4669

P. LARSSON

214

Appendix I1 -continued B. Perch 108 109

111 112 114 115 118 120 122 124 125 126 127 129 130

Strain No. F335 F305 Fl00b F420 F 105b F386 F409 F295b PSI3 F 179 F171 F223 F264 P368 F 389

0-antigen 35 36 37 38 39

40 41 42 43

44 45 46 47 48 49

H-antigen 2 3 17 1 18

4 1 1 2 19 11 17 1 1 2

Species

E F Culture Collection No.

P . mirabilis P . mirabilis P . vulgaris P . mirabilis P . vulgaris P . mirabilis P . mirabilis P . vulgaris P . vulgaris P . vulgaris P . vulgaris P . vulgaris P . vulgaris P . mirabilis P . mirabilis

4670 467 1 4672 4673 4674 4675 4676 4677 4678 4679 4680 468 1 4682 4683 4684

The following isolates examined by P. Larsson (Goteborg) may represent new serotypes : 1653 . . . . . O A . . . . . . . . . P . mirabilis . . 10700 1721 . . . . . OB . . . . . . . . . P . mirabilis . . 10701 3039 . . . . . o c . . ' ' . . . . . P . mirabilis . . 10702 3284 . . . . . O D ' ' ' ' ' ' . ' . P . mirabilis ' ' 10703 4855 . . . . . O E . . . . . . . . . P . mirabilis . . 10704 5485 . . . . . O F . . . . . . . . . P . mirabilis . . 10705

Index “Aerobacter” cloacae, 165, 166, 175-6 Alkalescens-Dispar, 7 Ankylosing spondylitis, 160-1 Arizona himshawii, 24; see also Salmonella arizonae Bacillus paratyphi-alvei of Bahr, 166 Bacterial agglutination, 70-3 automated typing methods, 73 slide agglutination, 71-2 tube agglutination, 72-3 macroscopic appearance, 73 prozone phenomenon, 73 titration, 72-3 Bacterial clone concept, 1 0 3 4 Cedecea, 37 Cedecea davisae, 37 biochemical-cultural characteristics, 36 (table) Cedecea lapagei, 37 biochemicalcultural characteristics, 36 (table) Citrobacter, 19 biochemicalcultural characteristics, 20-1 (table) Citrobacter amalonaticus, 19 biochemicalcultural characteristics, 20-1 (table) Citrobacter diversus, 19 biochemical-cultural characteristics, 20-1 (table) Citrobacter freundii, 19, 166 biochemical-cultural characteristics, 20-1 (table) Citrobacter intermedius, 19 Citrobacter koseri, 19 biochemical-cultural characteristics, 20-1 (table) “Cloaca” cloacae, 165

Clone concept, bacterial, 1 0 3 4 Colanic acid, 51, 88 Colominic acid, 51 Deoxyribonucleic acid (DNA) associated approaches, biochemically aberrant strains, 2-3 homology, 2-3 hybridization, 1-2, 3 Dienes phenomenon, 2 0 5 4 Edwardsiella, 32 Edwardsiella amylovora, 32 Edwardsiella anguillimortiferum, 32 biochemical-cultural characteristics, 34 (table) Edwardsiella hoshinae, 32, 34 Edwardsiella quercina, 32 Edwardsiella rubrifaciens, 32 Edwardsiella tarda, 32 biochemical-cultural characteristics, 34 (table) Enterobacter biochemical-cultural characteristics, 16-17 (table), 167 biochemistry of cell wall, 191-2 differentiation: from Hafnia, 18 (table), 166 from Klebsiella, 12 enteropathogenicity, 181 serology, 165-84 Enterobacter aerogenes, 15, 165 biochemical-cultural characteristics, 16-17 (table), 18 (table) from E. cloacae, 14 (table), 15 differentiation: from Klebsiella sp., 14 (table), 15 subsp. Hafnia, 166 Enterobacter agglomerans, 3, 15, 29, 165, 165-6

216

INDEX

Enterobacter agglomerans-cont. biochemical-cultural characteristics, 16-17 (table), 18 (table) Enterobacter alvei, 166 Enterobacter amnigerus, biochemicalcultural characteristics, 16-17 (table) Enterobacter cloacae, 165-180-1 antigens: intergeneric/extrageneric relationships, 1 7 5 4 schema, 173-5, 175 (table) antiserum: absorption, 171 H-antiserum preparation, 170-1 0-antiserum preparation, 170 preservation, 171 biochemical-cultural characteristics, 16-17 (table), 167 differentiation: from E. aerogenes, 14 (table), 15 from Klebsiella, 14 (table), 15 H-antigens, 167, 168 determination, 173 relationships, 174 (table) K-antigen, 167 M-antigen (slime), 170 0-antigens, 167, 168 determination, 171-3 relationships, 172 (table) Enterobacter culneris, biochemicalcultural characteristics, 16-1 7 (table), 18 (table) Enterobacter gergoviae, 165 biochemicalcultural characteristics, 16-17 (table), 18 (table) Enterobacter hafniae, 166 Enterobacter intermedium, 165 Enterobacter sakazakii, 165 biochemicalcultural characteristics, 16-17 (table), 18 (table) Enterobacteriaceae, 1 4 - 1 capsular polysaccharides (K-antigens), 50-1, 52 (table), 53 (table) computer taxonomy and identification, 2 DNA-DNA homology above 70% breakpoint, 2 genera differentiation, 6 (table) membrane proteins, 54 nomenclature changes, 4-5 (table) phenetic relationships, 2 surface structure, 4 5 4

taxonomy, 3-37 Enterobacterial common antigen (ECA), 202 Erwinia, 32 Erwinia adecarboxylata, 15 Erwinia herbicola, 15, 29 Erwinia proteomaculans, 25, 28 Erwinia stewartii, I5 Erwinia uredova, 15 Escherichia, 7 cross-reactions with Shigella, 124, 125 (table) subdivision, 45 Escherichia adecarboxylata, 7 biochemicalcultural characteristics, 8-9 (table) Escherichia blattae, 7 biochemical-cultural characteristics, 8-9 (table) Escherichia coli agglutination, see Bacterial agglutination anodic thermolabile antigen (ATA), 65

antibiotic resistance patterns, typing by, 68 a-antigen, 65 /3-antigen, 65 antigenic scheme, 554, 58-9 (table) antisera, 88-9 1 absorbed, 91-2 F, 90 H, 90 0, 88 0;K, 89-90 pooled/polyvalent, 90-1 bacterial clone concept, 10biochemical-cultural characteristics, 8-9 (table) biotyping, 66-8 cattle, 101-2, co-agglutination, 74 colicin typing, 66 cross-reactions, 92-7 diarrhoea-associated strains, 66, 67 (table), 81, 99-101 differentiation in Enterobacteriaceae family, 44 direct haemagglutination, typing by, 68-9

distinct species, 13

INDEX

DNA relatedness with Shigella, 7, 114 enteroinvasive (EIEC) strains, 100 (table), 101 enteropathogenic (EPEC) strains, 66, 67 (table), 81, 99, 100 (table) bacterial clone concept, 1 0 3 4 enterotoxigenic (ETEC) strains, 100 (table), 101 bacterial clone concept, 104 F-antigens (fimbrial proteins), 54, 6 1 4 , 65, 82 colonization factors, 62 cross-reactions, 96-7 mannose-resistant, 63, 86-7 type 1 fimbriae, 6 3 4 , 87 variational phenomena, 86-7 gel precipitation, 75-8 counter-current immunoelectrophoresis, 78-9 crossed immunoelectrophoresis, 78 immunoelectrophoresis, 76 two-dimensional, 7 5 4 genetics of antigens, 55 H-antigens (flagellar proteins), 54, 64, 82-3 cross-reactions, 96 variational phenomena, 87-8 homopolysaccharides, 47 human sources, 97-101 prevalence theory, 99 special pathogenicity theory, 98 urinary tract, 98 immunofluorescence, 74-5 indirect haemagglutination, 74 K-antigens (capsular polysaccarides), 50-1, 53 (table), 56-61, 81 acidic polysaccharide, 60 cross-reactions, 9 4 6 restricted use of term, 61 variational phenomena, 8 5 4 Kunin antigen, 64 L-antigen, 57 M-antigens, 51, 6 4 6 variational phenomena, 88 0-antigens, 56, 80 (table) acidic, 49 (table) cross-reactions, 9 2 4 geographical distribution, 99 neutral, 48 (table) variational phenomena, 84-5 0-inagglutinability, 45, 56, 65

217

outer membrane protein pattern, typing by, 68 phage typing, 6 5 4 pigs, 102-3, 1 0 3 4 polysaccharide antigens, 51, 52 (table), 53 (table), 64 poultry, 103 serogroup/serotype association with pathological conditions, 97-103 serotype: definition, 55 stability, 69 serotyping, 43-1 12 history of procedures, 56-69 variational phenomena, 83-8 S-forms, 46 thermolabile antigens (proteins), 65 Escherichia ewing, 7 Escherichia fergusonii, 7 Escherichia hermanni, 7 Escherichia vulneris, 7, 18 (table) Escherichieae, 113, 114-15 differentiation within tribe, 115, 116 (table) Galactosamine, chemotyping of P. mirabilis and P. vulgaris, 193 (table) Galactose, chemotyping of P. mirabilis and P . vulgaris, 193 (table) Galacturonic acid, chemotyping of P. mirabilis and P . vulgaris, 192, 193 (table) Glucosamine, chemotyping of P. mirabilis and P. vulgaris, 193 (table) Glucose, chemotyping of P . mirabilis and P . vulgaris, 193 (table) Glucoronic acid, chemotyping of P. mirabilis and P . vulgaris, 192, 193 (table) D-Glycero-D-mannoheptose, chemotyping of P. mirabilis and P. vulgaris, 192, 193 (table) L-Glycero-D-mannoheptose, chemotyping of P. mirabilis and P . vulgaris, 192, 193 (table) Hafnia, 15-19 biochemical-cultural characteristics, 20-3 (table), 167 differentiation from Enterobacter, 18 (table), 166

218

INDEX

Hafnia-cont . enteropathogenicity, 181 serology, 165-84 Hafnia alvei, 15- 19, 166 alpha-antigen, 170 antigens: cross-relations, 180 extrageneric relationships, 178-9 schema, 177, 179-80 (table) antiserum: absorption, 171 H-antiserum preparation, 170-1 0-antiserum preparation, 170 preservation, 171 biochemical-cultural characteristics, 18 (table), 167, 168-9 (table) enteropathogenecity, 181 H-antigens, 170 determination, 176-7 relationships, 178 (table) K-antigens, 170 lactose fermentation in isolates, 7 0-antigens, 170 ’ determination, 176 relationships, 177 (table) Hafnia protea, 15, 19 Group 1, biochemical-cultural characteristics, 22-3 (table) Groups 2, biochemical-cultural characteristics, 22-3 (table) /I-Hydroxymyristic acid, 46 Infantile diarrhoea, 66, 67 (table), 81, 99, 100 (table) bacterial clone concept, 1 0 3 4 Kauffmann-White scheme, 50, 70 2-Keto-3-deoxy-mannulosoctonic acid (KDO), 46 Klebsiella, 7-1 5 antigenic scheme, 147-50 antiserum production: K-antiserum,

153-4

0-antiserum, 154 differentiation, 14 (table), 15 from Enterobacter, 12 Group D, 12 (table), 14 (table) Group J, 12 (table), 14 (table) hospital infections, 158 K-antigens, 149 (table) agglutination, 155 capsular swelling reaction, I 5 5 4 capsular swelling reactions, 155

chemistry, 146, 147 (table) counter-current immunoelectrophoresis, 156 cross-reactions, 150-1 antigens of other bacteria, 159-60 eucaryotic cells, 160-1 indirect immunofluorescence, 156 serological determination, 155-7, 157-8 type stability, 157 0-antigens, 149 (table) chemistry, 146-7, 148 (table) cross-reactions, 151 serological determination, agglutination, 157 pathogenicity correlation, 158-9 serotyping, 143-64 serotyping procedures, 15 1-3 bacteriocin typing, 152 biochemical typing, 152-3 counter-current immunoelectrophoresis, 15 1-2 phage typing, 152 serological methods, 151-2 surface structures, 145-7 K-antigens chemistry, 146, 147 (table) morphology, 1 4 5 4 0-antigens chemistry, 146-7 taxonomy, 1 4 3 4 upper respiratory tract infections, 159 Klebsiella aerogenes, 13-1 5 , 144 biochemical-cultural characteristics, 10-1 1 (table) Klebsiella atlantae, 13 Klebsiella edwardsii, 13, 144 Klebsiella mobilis, 15 Klebsiella oxytoca, 13, 144, 145 biochemical-cultural characteristics, 10-1 1 differentiation from E. aerogenes and E. cloacae, 14 (table) DNS homology less than 70%, 13 Klebsiella ozaenae, 13, 144 biochemicalcultural characteristics, 10-1 1 (table) differentiation from E. aerogenes and E. cloacae, 14 (table) mobile strain, high DNA relatedness, 12

INDEX

ozaena, 159 Klebsiella planticola, 13, 145 biochemical-cultural characteristics, 10-1 1 (table) Klebsiella pneumoniae, 13, 144, 145 Group D/Group J klebsiella differentiation, 12 (table) motile strain, high DNA relatedness, 12 Klebsiella pneumoniae (sensu lato), 12, 13 biochemical-cultural characteristics, 10-1 1 (table), 18 (table) differentiation from E. aerogenes and E. cloacae, 14 (table) Klebsiella pneumoniae (sensu stricto), 12, 13, 144 biochemical-cultural characteristics, 10-1 1 (table) serotype-3, 13-15, 14 (table) differentiation from E. aerogenes and E. cloacae, 14 (table) Klebsiella rhinoscleromatis, 13, 144 biochemical-cultural characteristics, 10-1 1 (table) differentiation from E. aerogenes and E. cloacae, 14 (table) motile strain, high DNA relatedness, 12 rhinoscleroma, 159 Klebsiella terrigena, 13, 145 biochemical-cultural characteristics, 10-1 1 (table) differentiation from E. aerogenes and E. cloacae, 14 (table) Kluyvera, 32-5 Levinea, 19 Levinea amalonatica, 19 Livinea malonatica, 19 Lipid A, 46, 192 Lipopolysaccharides (LPS), 45, 46-7 acidic, 47 chemical analysis, 126 chemotypes, 47-50 neutral, 47 R-LPS, 47 unsubstituted core, 46 Lipoproteins, bacterial cell surface, 45 Metritis, in mares, 159

219

Morganella, 28-9, 188 Morganella morganii, 29, I89 biochemical-cultural characteristics, 30-1 (table) lactose fermentation in isolates, 7 Obesumbacterium proteus, 15 0-antigens serological determinants, 45 specificity of Gram-negative bacteria, 46 see also specific organisms Oligosaccharides, core, 46 0-specific polysaccharide, 46, 47 Oxytocum, 13 Ozaena, 159 Paracolobactrum anguillimortiferum, 32 “Paracolon Aerobacter”, 166 “Paracolon bacteria”, 176 Pasteurella multocida, 6 (table) Pectobacterium, 32 Pectobacterium carotovorum, 32 Pectobacterium chrysanthemi, 32 Plesiomonas shigelloides, 125 (table), 132 Proteus antigenic cross-reactions, 203-5 human blood groups, 205 other bacteria, 2 0 3 4 Rickettsia, 204-5 within Proteus serological system, 203 antigenic schemes, comparison of, 190 (table) antigenic variations, 202-3 antisera production: H, 206-7 antisera production: 0, 206-7 bacteraemia, 188 biochemical-cultural characteristics, 30-1 (table) differential characteristics of species within genus, 189 (table) epidemiological markers, 208 flagellin protein, 194 lipopolysaccharides of outer membrane, 192 R forms, 203 serological systems: current, 191-206 previous, 189-9 1

220

INDEX

Proteus-cont. serological techniques, 194-8 crossed immunoelectrophoresis, 197 double diffusion-in-gel, 196-7 enzyme-linked immunosorbent assay (ELISA), 198 gel precipitation techniques, 196-7 immobilization test, 197 immunoelectrophoresis, 197 indirect haemagglutination, 196 slide agglutination, 195-6 tube agglutination, 1 9 6 5 serotype strains, 213-14 spontaneous agglutination, 202 surface structures, 1 9 1 4 swarming phenomenon, 191 urinary tract infections, 187 serological diagnosis, 196 Proteus asiaticus, 188 Proteus hydrophilus, 188 Proteus inconstans, 188 biotypes A and B, 29 differential characteristics, 189 (table) Proteus liquefaciens, 188 Proteus mirabilis, 29, 188, 189 bactericidal effect of normal human serum (SBS), 192 bacteriuria in hospitals, 208 biochemical-cultural characteristics, 30-1 (table), 189 (table) C-antigen, 201 chemotypes, 192, 193 (table) diagnostic antigenic scheme (Perch), 198, 199-201 (table) Diene’s phenomenon, 205-6 flagella/flagellins, properties of, 194 H-antigen, 199-201 (table), 201-2 distribution in clinical isolates, 208 immune responses to antigens, 192 K-antigen, 198-201 lipid A, 192 0-antigen, 198-20 1 cross-reactions within Proteus serology system, 203, 204 (table) distribution in clinical isolates, 207-8 serotyping in epidemiological work, 208-9 outer membrane lipoprotein, 1 9 3 4 outer membrane proteins, 194 serotype strains, 213-14 see also Proteus

Proteus morganii, 29, 188 differential characteristics, 189 (table) Proteus myxofaciens, 29 biochemical-cultural characteristics, 30-1 (table) Proteus rettgeri, 29, 30, 188 biochemical-cultural characteristics, 30-1 (table) C-antigen, 201 differential characteristics, 189 (table) Proteus vulgaris, 29, 188, 189 bactericidal effect on normal human serum (SBS), 192 biochemical-cultural characteristics, 30-1 (table), 189 (table) C-antigen, 201 chemotypes, 192, 193 (table) diagnostic antigenic scheme (Perch), 198, 199-201 (table) Dienes phenomenon, 2 0 5 4 H-antigen, 199-201 (table), 201-2 K-antigen, 198 0-antigen, 198-20 1 cross-reactions within Proteus serology system, 203, 204 (table) distribution in chemical isolates, 207-8 serotyping in epidemiology, 208-9 serotype strains, 213-14 see also Proteus Proteus X-19, 202, 205 Proteus zenkeri, 188 Providencia, mistaken for shigellae, 115 Providencia alcalifaciens, 29, 189 biochemical-cultural characteristics, 30-1 (table) Providencia rettgeri, see Proteus rettgeri Providencia stuartii, 29, 189 biochemical-cultural characteristics, 30-1 (table) Prozone phenomenon, 73 Pseudomonas proteomaculans, 25

R-antigens, 47 Rahnella, 35 biochemical-cultural characteristics, 26-7 (table) Rahnella aquatilis, 35 biochemical-cultural characteristics, 26-7 (table) Rettgerella, 188

INDEX

Rhamnose, chemotyping of P . mirabilis and P . vulgaris, 193 (table) Rhinoscleroma, 159 Ribitol, chemotyping of P. mirabilis and P . vulgaris, 193 (table) Salmonella, 23-5 biochemical-cultural characteristics, 20-3 (table) subgenera differentiation, 2 3 4 Salmonella arizonae, 23, 24 biochemicalcultural characteristics, 20-1 (table) Salmonella cholerae-suis, 24 biochemical-cultural characteristics, 20-1 (table) Salmonella enteritidis, 24 biochemicalcultural characteristics, 22-3 (table) Salmonella houtenae, 23 biochemicalcultural characteristics, 20-1 (table) Salmonella kauffmanii, 23 Salmonella paratyphi A , biochemicalcultural characteristics, 20-1 (table) Salmonella salamae, 23 biochemicalcultural characteristics, 20-1 (table) Salmonella typhi, 24 biochemicalcultural characteristics, 20-1 (table) enterobacterial serotype, 103 lactose fermentation in isolates, 7 Serratia, 25-8 biochemical-cultural characteristics, 26-7 (table) SerratiaJicaria, 28 biochemicalcultural characteristics, 26-7 (table) Serratia fonticola, 25, 28 biochemicalcultural characteristics, 26-7 (table) Serratia grimesii, 25, 28 biochemicalcultural characteristics, 26-7 (table) Serratia liquefaciens, 25, 28 Serratia liquefaciens-like biotypes, 28 Serratia marcescens, biochemicalcultural characteristics, 26-7 (table)

22 1

Serratia marinorubra, 25 Serratia odorifera, 28 biochemicalcultural characteristics, 26-7 (table) Serratia plymuthica, 28 biochemical-cultural characteristics, 26-7 (table) Serratia proteomaculans, 25, 28 var. proteomaculans, 28 biochemical-cultural characteristics, 26-7 (table) var. quinovora, 28 biochemicalcultural characteristics, 26-7 (table) Serratia rubidaea, 25, 28 biochemicalcultural characteristics, 26-7 (table) Shigella, 7 antigenic analysis of cultures, 1 15-25 antisera production for typing, 13240 absorption of antisera techniques, 135-9 antisera for type determination, 135 bacterial strains, 132-3 immunization procedures, 1 3 3 4 polyvalent grouping antisera, 134, 136 (table) slide agglutination for type determination, 139-40 biochemicalcultural characteristics, 8-9 (table) cross-reactions with Escherichia, 124, 125 (table) differentiation, 11415, 117 (table) DNA relatedness to E. coli DNA, 7, 114 nomenclature, 120-1 (table) 0-antigens, 1 13 chemical structure, 126-32 relationship to E. coli 0-antigens, 125 (table) serology, 1 1 3 4 2 taxonomy, 114-15, 120-1 (table) Shigella boydii, 115, 121 (table) antigenic analysis of cultures, 119 biochemical-cultural characteristics, 8-9 (table) 0-antigens: chemical structure, 131-2 relationships with E. coli 0-antigens, 125 (table) preparation of absorbed antisera for

222

INDEX

Shigella boydii-conr. serotype identification, 124 (table) serogroup C strains, 116 Shigella dysenteriae, 120 (table) antigenic analysis of cultures, 118 antisera absorption, 122 (table) biochemicalcultural characteristics, 8-9 (table) 0-antigens: chemical structure, 1.26, 128-9 (table) relationship with E. coli 0-antigens, 125 (table), 126 serogroup A strains, 116 as type species, 1 14 Shigellaflexneri, 120 (table) agglutination in typing antisera, 123 (table) antigenic analysis of cultures, 118-19 antisera for group factors, 124 (table) biochemicalcultural characteristics, 7-8 (table) biotypes, 118 (table) gas fermentation from carbohydrates, 114 0-antigens: chemical structure, 127-3 1 relationship with E. coli 0-antigens, 125 (table) 0 : 6 ( S . newcastle), 115, 131 preparation of specific absorbed an tisera, 122 (table) serogroup B strains, 116 serotype/serogroup antigens, chemical structure, 127, 130 (table) Shigefla newcastle, 1 15, 131 Shigella sonnei, 121 (table)

antigenic analysis of cultures, 119 arginine dihydrolase system, 114 biochemicalcultural characteristics, 8-9 (table) lactose fermentation, 114 0-antigens: chemical structure, 132 relationship with E. coli 0-antigens, 125 (table) ornithine decarboxylation, 114 serogroup D strains, 116 serotype, 116 Threonine. 5 1 Weil-Felix reaction in rickettsiosis, 205 Yersinia, 29-32 biochemical-cultural characteristics, 33 (table) Yersinia enterocolitica, 3 biochemicalcultural characteristics, 33 (table) Yersinia frederiksenii, 3 biochemicalcultural characteristics, 33 Yersinia intermedia, biochemicalcultural characteristics, 33 (table) Yersinia kristensenii, biochemicalcultural characteristics, 33 (table) Yersinia pestis, biochemical-cultural characteristics, 33 (table) Yersinia pseudotuberculosis, biochemical-cultural characteristics, 33 (table) Yersinia ruckeri, biochemicalcultural characteristics, 33 (table)

Contents of published volumes Volume 1 E. C. Elliott and D. L . Georgala. Sources, handling and storage of media and equipment. R. Brookes. Properties of materials suitable for the cultivation and handling of microorganisms. G. Sykes. Methods and equipment for sterilization of laboratory apparatus and media. R. Elsworth. Treatment of process air for deep culture. J. J. McDade, G. B. Phillips, H. D. Sivinski and W . J. Whitjield. Principles and applications of laminar-flow devices. H . M . Darlow. Safety in the microbiological laboratory J . G. Mulvany. Membrane filter techniques in microbiology C. T. Calam. The culture of micro-organisms in liquid medium Charles E. Helmsretrer. Methods for studying the microbial division cycle Louis B. Quesnal. Methods of microculture R. C. Codner. Solid and solidified growth media in microbiology K. I. Johnstone. The isolation and cultivation of single organisms N. Blakebrough. Design of laboratory fermenters K. Sargeant. The deep culture of bacteriophage M . F. Mallette. Evaluation of growth by physical and chemical means C. T. Calam. The evaluation of mycelial growth H . E. Kubitschek. Counting and sizing micro-organisms with the Coulter counter J. R. Postgate. Viable counts and viability A. H. Stouthamer. Determination and significance of molar growth yields

Volume 2 D. G. MacLennan. Principles of automatic measurement and control of fermentation growth parameters J. W . Patching and A. H. Rose. The effects and control of temperature A. L. S. Munro. Measurement and control of pH values H.-E. Jacob. Redox potential D. E. Brown. Aeration in the submerged culture of micro-organisms D. Freedman. The shaker in bioengineering J. Bryant. Anti-foam agents N.G. Carr. Production and measurement of photosynthetically usable light evolution in stirred deep cultures G. A. Platon. Flow measurement and control Richard Y. Morita. Application of hydrostatic pressure to microbial cultures D. W . Tempest. The continuous cultivation of micro-organisms: 1. Theory of the chemostat C. G. T. Evans, D. Herbert and D. W . Tempest. The continuous cultivation of microorganisms: 2. Construction of a chemostat J. Ritica. Multi-stage systems R. J. Munson. Turbidostats R. 0. Thomson and W . H. Foster. Harvesting and clarification of cultures-storage of harvest

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Volume 3A S. P. Lapage, Jean E. Shelton and T. G. Mitchell. Media for the maintenance and preservation of bacteria S. P. Lapage, Jean E. Shelton, T. G. Mitchell and A. R. Mackenzie. Culture collections and the preservation of bacteria E. Y.Bridson and A. Brecker. Design and formulation of microbial culture media D. W. Ribbons. Quantitative relationships between growth media constituents and cellular yields and composition H. Veldkamp. Enrichment cultures of prokaryotic organisms David A. Hopwood. The isolation of mutants C. T. Calam. Improvement of micro-organisms by mutation, hybridization and selection Volume 3B Vera G. Collins. Isolation, cultivation and maintenance of autotrophs N. G. Carr. Growth of phototrophic bacteria and blue-green algae A. T. Willis. Techniques for the study of anaerobic, spore-forming bacteria R. E. Hungate. A roll tube method for cultivation of strict anaerobes P. N. Hobson. Rumen bacteria Ella M. Barnes. Methods for the gram-negative non-sporing anaerobes T. D. Brock and A. H. Rose. Psychrophiles and thermophiles 'N.E. Gibbons. Isolation, growth and requirements of halophilic bacteria John E. Peterson. Isolation, cultivation and maintenance of the myxobacteria R. J. Fallon and P. Whittlestone. Isolation, cultivation and maintenance of mycoplasmas M. R. Droop. Algae Eve Billing. Isolation, growth and preservation of bacteriophages Volume 4 C. Booth. Introduction to general methods C. Booth. Fungal culture media D. M . Dring. Techniques for microscopic preparation Agnes H. S. Onions. Preservation of fungi F. W . Beech and R. R. Davenport. Isolation, purification and maintenance of yeasts G. M . Waterhouse. Phycomycetes E. Punithalingham. Basidiomycetes: Heterobasidiomycetidae Roy Watling. Basidiomycetes: Homobasidiomycetidae M . J. Carlile. Myxomycetes and other slime moulds D. H. S. Richardson. Lichens S. T. Williams and T. Cross. Actinomycetes E. B. Gareth Jones. Aquatic fungi R. R. Davies. Air sampling for fungi, pollens and bacteria George L. Barron. Soil fungi Phyllis M. Stockdale. Fungi pathogenic for man and animals: 1. Diseases of the keratinized tissues Helen R. Buckley. Fungi pathogenic for man and animals: 2. The subcutaneous and deep-seated mycoses J. L. Jinks and J. Croft. Methods used for genetical studies in mycology R. L. Lucas. Autoradiographic techniques in mycology T, F. Preece. Fluorescent techniques in mycology

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225

G. N. Greenhalgh and L. V. Evans. Electron microscopy Roy Watling. Chemical tests in agaricology T. F. Preece. Immunological techniques in mycology Charles M. Leach. A practical guide to the effects of visible and ultraviolet light on fungi Julio R. Villanuevaand Isabel Garcia Acha. Production and use of fungal protoplasts Volume 5A L. B. Quesnel. Microscopy and micrometry J. R. Norris and Helen Swain. Staining bacteria A . M, Paton and Susan M . Jones. Techniques involving optical brightening agents T. Iino and M . Enomoto. Motility R. W. Smith and H. Kofler. Production and isolation of flagella C. L. Oakley. Antigen-antibody reactions in microbiology P. D. Walker,Irene Batty and R. 0. Thomson. The localization of bacterial antigens by the use of fluorescent and ferritin labelled antibody techniques Irene Batty. Toxin-antitoxin assay W. H. Kingham. Techniques for handling animals J . De Ley. The determination of the molecular weight of DNA per bacterial nucloid J. De Ley. Hybridization of DNA J. E. M. Midgley. Hybridization of microbial RNA and DNA Elizabeth Work. Cell walls. Volume 5B D. E. Hughes, J. W. T. Wirnpennyand D. Lloyd. The disintegration of microorganisms J. Sykes. Centrifugal techniques for the isolation and characterization of subcellular components from bacteria D. Herbert, P. J. Phipps and R. E. Strange. Chemical analysis of microbial cells I. W . Sutherland and J. F. Wilkinson. Chemical extraction methods of microbial cells Per-Ake-Albertsson. Biphasic separation of microbial particles Mitsuhiro Nozaki and Osarnu Hayaishi. Separation and purification of proteins J. R. Sargent. Zone electrophoresis for the separation of microbial cell components K. Hannig. Free-flow electrophoresis W . Manson. Preparative zonal electrophoresis K. E. Cooksey. Disc electrophoresis 0. Vesterberg. Isolectric focusing and separation of proteins F. J. Moss, Pamela A. D. Rickard and G. H. Roper. Reflectance spectrophotcnnetry W . D. Skidmore and E. L. Duggan. Base composition of nucleic acids Volume 6A A. J. Holding and J. G. Collee. Routine biochemical tests K. Kersters and J. de Ley. Enzymic tests with resting cells and cell-free extracts E. A. Dawes, D. J. McGill and M. Midgley. Analysis of fermentation products S. Dagley and P. J. Chapman. Evaluation of methods to determine metabolic pathways Patricia H. Clarke. Methods for studying enzyme regulation G. W. Could. Methods for studying bacterial spores W. Heinen. Inhibitors of electron transport and oxidative phosphorylation Elizabeth Work. Some applications and uses of metabolite analogues in microbiology W. A. Wood. Assay of enzymes representative of metabolic pathways

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CONTENTS OF PUBLISHED VOLUMES

H. C. Reeves, R. Rabin, W. S. Wegener and S. J. Ajl. Assays of enzymes of the tricarboxylic acid and glyoxylate cycles D. T. Gibson. Assay of enzymes of aromatic metabolism Michael C. Scrutton. Assay of enzymes of CO, metabolism

Volume 6B J. L. Peel. The use of electron acceptors, donors and carriers R. B. Beechley and D. W . Ribbons. Oxygen electrode measurements D. G. Nicholls and P. B. Garland. Electrode measurements of carbon dioxide G. W.Crosbie. Ionization methods of counting radio-isotopes J. H. Hash. Liquid scintillation counting in microbiology J. R. Quayle. The use of isotopes in tracing metabolic pathways C. H. Wang. Radiorespirometric methods N. R. Eaton. Pulse labelling of micro-organisms M. J. Allen. Cellular electrophysiology W. W. Forrest. Microcalorimetry J. Marten. Automatic and continuous assessment of fermentation parameters A. Ferrari and J. Marten. Automated microbiological assay J. R. Postgate. The acetylene reduction test for nitrogen fixation Volume 7A ‘G. C. Ware. Computer use in microbiology P. H. A. Sneath. Computer taxonomy H. F. Dammers. Data handling and information retrieval by computer M. Roberts and C. B. C. Boyce. Principles of biological assay D. Kay. Methods for studying the infectious properties and multiplication of bacteriophage D. Kay. Methods for the determination of the chemical and physical structure of bacteriophages Anna Mayr-Hurting, A. J. Hedges and R. C. W. Berkeley. Methods for studying bacteriocins W. R. Maxted. Specific procedures and requirements for the isolation, growth and maintenance of the L-phase of some microbial groups Volume 7B M . T. Parker. Phage-typing of Staphylococcus aureus D. A. Hopwood. Genetic analysis in micro-organisms J. Meyrath and Gerda Suchanek. Inoculation techniques-effects due to quality and quantity of inoculum D. F. Spooner and G. Sykes. Laboratory assessment of antibacterial activity L. B. Quesnel. Photomicrography and macrophotography Volume 8 L. A. Bulla Jr, G. St Julian, C. W . Hesseltine and F. L. Baker. Scanning electron microscopy H. H. Topiwala. Mathematical models in microbiology E. Canale-Parola. Isolation, growth and maintenance of anaerobic free-living spirochetes 0. Felsenfeld. Borrelia

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A. D. Russell, A. Morris and M . C. Allwood. Methods for assessing damage to bacteria induced by chemical and physical agents P. J. Wyatt. Differential light scattering techniques for microbiology

Volume 9 R. R. Watson. Substrate specificities of aminopeptidases: a specific method for microbial differentiation . C.-G. Heden, T. Illeni and I. Kuhn. Mechanized identification of micro-organisms D. B. Drucker. Gas-liquid chromatographic chemotaxonomy K. G. Lickfield. Transmission electron microscopy of bacteria P. Kay. Electron microscopy of small particles, macromolecular structures and nucleic acids M. P. Starr and H. Stolp. Bdellovibrio methodology Volume 10 T. Meitert and Eugenia Meitert. Usefulness, applications and limitations of epidemiological typing methods to elucidate nosocomial infections and the spread of communicable diseases G. A. J. Ayltfle. The application of typing methods to nosocomial infections J. R. W. Govan. Pyocin typing of Pseudomonas aeruginosa B. G n y i and T. Bergan. Serological characterization of Pseudomonas aeruginosa T. Bergan. Phage typing of Pseudomonas aeruginosa N. B. McCullough. Identification of the species and biotypes within the genus Brucella Karl-Axel Karlsson. Identification o f Francisella tularensis T. Omland. Serotyping of Haemophilus inpuenzae E. L. Biberstein. Biotyping and serotyping of Pasteurella haemolytica Shigeo Namioka. Pasteurella multocida -Biochemical characteristics and serotypes Nevlan A. Vedros. Serology of the meningococcus Dan Danielsson and Johan Maeland, Serotyping and antigenic studies of Neisseria gonorrhoeae B. Wesley Catlin. Characteristics and auxotyping of Neisseria gonorrhoeae Volume 11 Frits 0rskov and Ida 0rskov. Serotyping o f Enterobacteriaceae, with special emphasis on K-antigen determination R. R. Gillies. Bacteriocin typing of Enterobacteriaceae H. Milch. Phage typing of Escherichia coli P. A. M. Guinke and W. J. van Leeuwen. Phage typing of Salmonella S.Slopek. Phage typing of Klebsiella W . H, Traub. Bacteriocin typing of clinical isolates of Serratia marcescens T. Bergan. Phage typing of Proteus H. Dikken and E. Kmety. Serological typing methods of leptospires Volume 12 S. D. Henriksen. Serotyping of bacteria E. Thal. The identification of Yersinia pseudotuberculosis T. Bergan. Bacteriophage typing of Yersinia enterocolitica S. Winblad. Yersinia enterocolitica (synonyms “Pasteurella X ’ , Bacterium enterocoliticum for serotype 0 - 8 )

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S. Mukerjee. Principles and practice of typing Vibrio cholerae H. Brandis. Vibriocin typing P. Oeding. Genus Staphylococcus J. Rotta. Group and type (groups A and B) identification of haemolytic streptococci H. Brandis. Bacteriocins of streptococci and bacteriocin typing Erna Lund and J. Henricksen. Laboratory diagnosis, serology and epidemiology of Streptococcus pneumoniae

Volume 13 D. E. Mahony. Bacteriocin, bacteriophage, and other epidemiological typing methods for the genus Clostridium H. P. R. Seeliger and K. Hohne. Serotyping of Listeria monocytogenes and related species I. Stoev. Methods of typing Erysipelothrix insidiosa A. Saragea, P.Maximescu and E. Meitert. Corynebacterium diphtheriae. Microbiological methods used in clinical and epidemiological investigations T. Bergan. Bacteriophage typing of Shigella M . A. Gerencser. The application of fluorescent antibody techniques to the identification of Actinomyces and Arachnia W. B. Schaefer. Serological identification of atypical mycobacteria W. B. Redmond, J. H. Bates and H. W. Engel. Methods of bacteriophage typing of mycobacteria E. A. Freund, H. Erne and R. M . Lemecke. Identification of mycoplasmas U. Ullmann. Methods in Campylobacter