Microbial Stress Adaptation and Food Safety Edited by
Ahmed E.Yousef Vijay K. Juneja
CRC PR E S S Boca Raton London New York Washington, D.C. © 2003 by CRC Press LLC
Library of Congress Cataloging-in-Publication Data Microbial stress adaptation and food safety / editors, Ahmed E. Yousef and Vijay K. Juneja. p. cm. Includes bibliographical references and index. ISBN 1-56676-912-4 1. Food—Microbiology. 2. Adaptation (Biology 3. Stress (Physiology) I. Yousef, Ahmed Elmeleigy. II. Juneja, Vijay K., 1956QR115 .M4585 2002 664′.001′579—dc21
2002031435
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Preface Microorganisms, like all living beings, react when exposed to stress. When humans are subjected to limited stress, their reaction varies from fatigue to endurance. Interestingly, microorganisms behave similarly. When the stress cannot be tolerated, both humans and microorganisms may suffer injury. Given time and rest, both humans and microorganisms may recover from this injury. Severe injuries, however, may lead to death. Mild stresses, on the other hand, may be beneficial to both microorganisms and humans. These stresses activate the body’s natural defenses and test its ability to protect itself against incoming danger. When living beings respond appropriately to stress, they emerge from this experience better “adapted” and prepared for future stressful situations. It appears that the saying, “what doesn’t kill me only makes me stronger,” applies equally well to humans and microgorganisms. The words “stress,” “adaptation,” “injury,” “recovery,” and “death” describe human experiences and these expressions have been ported to the world of microbiology. Keeping the analogies between human and microbial experience in mind, the reader may quickly become familiar with the advanced terminologies used in this book. This book presents essential and advanced knowledge about microbial adaptation to stress, and relevance of this phenomenon to food safety. The book should serve readers who have basic understanding of microbiology and prior knowledge of food processing and preservation. The first chapter introduces the concept of stress adaptation in microorganisms. This contribution defines the terms, and briefly describes the stress adaptive response phenomenon and its implications to the safety of food processed by novel unconventional technologies. Responses of pathogens to physical and chemical stresses encountered during food processing are addressed in Chapters 2 and 3, respectively. Physical preservation factors discussed include heat, pressure, electric pulses, dehydration, freezing and irradiation. Food preservatives such as added organic acids and naturally occurring antimicrobials are also stresses of interest. Adaptation of food microbiota to stress as a survival strategy is the topic of Chapter 4. This covers important categories of foodborne microorganisms and how these develop different schemes to combat deleterious factors in food and during food processing. The food-processing environment occasionally harbors pathogens that eventually gain access to food. These environments may be hospitable enough to support not only growth of these pathogens, but also building communities called biofilms. Pathogens in food processing environments face a broad spectrum of stresses that may increase their tenacity and resistance to processing. This topic is addressed in detail in Chapter 5. The beneficial aspects of stress adaptation are the subject of Chapter 6. Survival of lactic acid bacteria in food and the human intestine may have a positive effect on food safety and human health. The authors of this chapter describe
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in detail how these beneficial bacteria adapt to stresses such as heat, cold, acid, salt, and oxidation. The physiological and molecular basis of stress adaptation response in this group of bacteria is also presented. Pathogens face stresses not only in food and processing environments, but also during the infection process. Resistance or adaptation to these stresses is an essential element of pathogens’ ability to cause diseases. Chapter 7 attends to this matter in depth. There are many physiological and molecular mechanisms that microorganisms use to sense the stress and respond to it. How foodborne microorganisms implement these mechanisms to adapt to stress is discussed in Chapter 8. Finally, strategies to overcome stress adaptation in foodborne pathogens are proposed in Chapter 9. The authors suggest practical control measures and emphasize the need for future research to counteract the stress adaptation phenomenon. We hope this book raises awareness about the microbial stress adaptation phenomenon and its consequences for food safety and human health and welfare. We are also hopeful that the topics covered in the book stimulate interest in research, leading to a better characterization of stress adaptive responses in foodborne microorganisms. Ahmed E. Yousef Vijay K. Juneja
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Acknowledgment We would like to thank all the contributing authors for their fine chapters and the staff members of CRC Press for their help and guidance. We also appreciate the patience of the members of our families who endured with us the stress of compiling this work. We are grateful to our co-workers who contributed significantly to our knowledge on this subject.
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Editors Ahmed E. Yousef, a professor of food microbiology at the Ohio State University (OSU), earned his Ph. D. degree from the University of Wisconsin-Madison in 1984. He teaches food microbiology at the Department of Food Science and Technology and the Department of Microbiology, OSU. His book, Food Microbiology, A Laboratory Manual, represents the laboratory component of the course he teaches. Since he joined OSU in 1991, Dr. Yousef has investigated methods to control foodborne pathogens and to determine causes of resistance of these pathogens to preservation factors. He is actively researching biopreservation, high pressure processing and pulsed electric field technologies. Pathogens targeted include Listeria monocytogenes, Salmonella spp., Escherichia coli O157:H7, and Clostridium botulinum. Adaptation of these pathogens to environmental and processing stresses is an ongoing investigation. He has published more than 70 research papers, review articles, books and book chapters. Dr. Yousef served on the editorial boards of the Journal of Food Protection and Journal of Food Science. He won several awards in recognition of his accomplishments in teaching and research. Vijay K. Juneja is supervisory microbiologist and lead scientist in the Microbial Food Safety Research Unit at the Eastern Regional Research Center (ERRC) of the Agricultural Research Service (ARS) branch of the United States Department of Agriculture (USDA) in Wyndmoor, Pennsylvania. Dr. Juneja received his Ph.D. degree in food technology and science from the University of Tennessee in 1991, then was appointed as a microbiologist at the ERRC-USDA. Dr. Juneja has developed a nationally and internationally recognized research program on foodborne pathogens, with emphasis on microbiological safety of minimally processed foods, and predictive microbiology. He is a co-editor of the book Control of Foodborne Microorganisms and serves on the editorial boards of the Journal of Food Protection and Journal of Food Science. Dr. Juneja is a recipient of several awards, including the Agricultural Research Service Senior Research Scientist; North Atlantic Area Scientist of the Year, 2000; Gold Medalist “Technical Accomplishment,” Federal Executive Board (FEB) 1998, 2000; ARS-FSIS Cooperative Research Award, 1998; USDA-ARS Certificate of Merit for Outstanding Performance, 2002, among others. His research interests include intervention strategies for control of foodborne pathogens and predictive modeling. Dr. Juneja’s research program has been highly productive, generating more than 180 research articles, book chapters, and abstracts, primarily in the area of food safety and predictive microbiology.
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Contributors Polly D. Courtney Department of Food Science & Technology The Ohio State University Columbus, Ohio P. Michael Davidson Department of Food Science and Technology University of Tennessee Knoxville, Tennessee Cormac G. M. Gahan Department of Microbiology and National Food Biotechnology Centre University College Cork Cork, Ireland Hany S. Girgis Department of Food Science Southeast Dairy Foods Research Center North Carolina State University Raleigh, North Carolina Mark A. Harrison Department of Food Science and Technology University of Georgia Athens, Georgia Colin Hill Department of Microbiology and National Food Biotechnology Centre University College Cork Cork, Ireland Eric A. Johnson Food Research Institute Department of Food Microbiology and Toxicology University of Wisconsin Madison, Wisconsin
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Vijay K. Juneja U.S. Department of Agriculture Agricultural Research Service Eastern Regional Research Center Wyndmoor, Pennsylvania Todd R. Klaenhammer Department of Food Science Southeast Dairy Foods Research Center North Carolina State University Raleigh, North Carolina John B. Luchansky USDA Eastern Regional Research Center Microbial Food Safety Research Unit Wyndmoor, Pennsylvania John S. Novak U.S. Department of Agriculture Agricultural Research Service Eastern Regional Research Center Wyndmoor, Pennsylvania Sadhana Ravishankar National Center for Food Safety and Technology Summit-Argo, Illinois Robin J. Rowbury Biology Department University College London London, United Kingdom John Samelis National Agricultural Research Foundation Dairy Research Institute Ioánnina, Greece
James Smith USDA Eastern Regional Research Center Microbial Food Safety Research Unit Wyndmoor, Pennsylvania
John N. Sofos Department of Animal Sciences, Colorado State University Fort Collins, Colorado
Ahmed E. Yousef Department of Food Science & Technology The Ohio State University Columbus, Ohio
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Table of Contents Chapter 1 Basics of Stress Adaptation and Implications in New-Generation Foods Ahmed E. Yousef and Polly D. Courtney Chapter 2 Adaptation of Foodborne Pathogens to Stress from Exposure to Physical Intervention Strategies Vijay K. Juneja and John S. Novak Chapter 3 Microbial Adaptation to Stresses by Food Preservatives P. Michael Davidson and Mark A. Harrison Chapter 4 Microbial Adaptation and Survival in Foods Eric A. Johnson Chapter 5 Adaptation or Resistance Responses of Microorganisms to Stresses in the Food-Processing Environment Sadhana Ravishankar and Vijay K. Juneja Chapter 6 Stress Adaptations of Lactic Acid Bacteria Hany S. Girgis, James Smith, John B. Luchansky, and Todd R. Klaenhammer Chapter 7 Relationship between Stress Adaptation and Virulence in Foodborne Pathogenic Bacteria Cormac G. M. Gahan and Colin Hill Chapter 8 Physiology and Molecular Basis of Stress Adaptation, with Particular Reference to the Subversion of Stress Adaptation and to the Involvement of Extracellular Components in Adaptation Robin J. Rowbury
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Chapter 9 Strategies to Control Stress-Adapted Pathogens John Samelis and John N. Sofos
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1
Basics of Stress Adaptation and Implications in New-Generation Foods Ahmed E. Yousef and Polly D. Courtney
CONTENTS Introduction Definitions Stress Stress Response Adaptation Tolerance Injury Stress, Adaptation and Food Safety Emerging Processing Technologies and Stress Adaptation High Pressure Processing Process Mechanism Potential Stress Adaptation Radiation Process Mechanism Potential Stress Adaptation Pulsed Electric Field Process Mechanism Potential Stress Adaptation Mechanism of Stress Adaptive Response Stress Sensing Regulation of Stress-Related Protein Synthesis General Stress Response Specific Stress Responses Heat Cold
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Acid Osmotic Stress Oxidative Stress Monitoring Stress Response Induction of Stress Adaptive Response: Practical Considerations Heat Acid Acid Shock during Exponential Phase Gradual Acid Stress Detecting and Quantifying Stress Response Detection of Stress Response Gene mRNA Analysis Detection of Stress Proteins Biosensors Measuring Increased Tolerance Perspectives and Areas for Future Work References
INTRODUCTION For many decades, researchers have noticed that microorganisms that endure a stressful environment subsequently survive conditions presumed lethal. Fay (1934), for example, noticed that exposing bacteria to osmotic stress increases tolerance to heat. Increase of an organism’s resistance to deleterious factors following exposure to mild stress is commonly described as stress adaptation. Stress adaptation in foodborne microorganisms was overlooked in the past, but now the significance of this phenomenon is becoming recognized. In 1987, Mackey and Derrick showed that heat shocking Salmonella enterica serovar Thompson increased its thermal resistance in food. Enhanced thermal tolerance was also observed by Farber and Brown (1990) when they heat shocked Listeria monocytogenes in sausage batter at 48°C for 120 min before the inoculated mix was heated at 64°C. Leyer and Johnson (1992) inoculated acid-adapted (pH 5.8) and non-adapted Salmonella typhimurium into fermenting milk. The researchers noticed that acid adaptation of the pathogen enhanced its survival during milk fermentation. Acid adaptation also enhanced survival in cheeses that were inoculated with the pathogen. Subsequent studies provided additional evidence of the stress adaptation phenomenon and its consequences during food processing. This chapter covers the basic aspects of stress adaptation and the relevance of this phenomenon to food safety, particularly products processed by emerging technologies.
DEFINITIONS Some terms describing stress adaptation are used loosely in scientific literature, so we will describe the way terms are used throughout this chapter. The interrelations among some of these terms are depicted in Figure 1.1.
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Stress-adapted
Relative Stress Tolerance
ve
Healthy (Steady state)
Mi Re
ld
co
St ve
res
ry
s
ry
Stress Adaptive Response
R
o ec
Stressed Mod St era res te Re
co
s
ve
ry
Injured
Se Str vere es s
Dead Physiological State FIGURE 1.1 Proposed interrelations among physiological states of microbial cell subjected to different stresses.
Stress Stress has different meanings depending on the context of usage. In physics, for example, stress is the force applied per unit area. When used in the field of biology, stress refers to the imposition of detrimental nutritional conditions, toxic chemicals and suboptimal physical conditions (Neidhardt and VanBogelen, 2000). Stress, as used in this chapter, refers to any deleterious factor or condition that adversely affects microbial growth or survival. According to this practical definition, many food processing treatments are considered stresses. Stresses encountered by microorganisms vary in magnitude and outcome. We use the word “mild” to describe sublethal stress levels that do not result in viability loss, but reduce or arrest growth rate. “Moderate” stress not only arrests microbial growth but also causes some loss in cell viability. “Extreme” or “severe” describes a stress level that is normally lethal to the cells, resulting in death of the majority of the population. Stresses that food microbiota encounter include uncontrollable pre-harvest environmental factors (e.g., radiation and dry air) and the deliberate postharvest application of preservation factors. Stresses to these microorganisms during food production and processing include: 1. Physical treatments such as heat, pressure, electric pulses, ultrasonic waves, light/radiation, and osmotic shock 2. Addition of chemicals such as acids, salts, and oxidants 3. Biological stresses, e.g., competition, microbial metabolites and antagonism © 2003 by CRC Press LLC
Foodborne microorganisms may experience stress gradually or abruptly, the latter being referred to as shock. For example, a bacterium may experience a drastic change in pH, or acid shock, when moving from the food medium into the stomach. On the other hand, microorganisms experience a gradual pH decrease during food fermentations. Stress Response Once microorganisms sense a stress, the cells respond in various ways. Bacteria sense stresses that change membrane fluidity (e.g., cold shock), alter cell protein structure or disrupt ribosomes (e.g., heat), or affect nucleic acids (e.g., γ radiation). At the molecular level, stress response includes transcription leading to the synthesis of regulatory proteins. The resulting regulation may lead to the synthesis of other proteins that cope with the imposed stress. Microbial response to stress may produce these outcomes: 1. Production of proteins that repair damage, maintain the cell, or eliminate the stress agent 2. Transient increase in resistance or tolerance to deleterious factors 3. Cell transformation to a dormant state, i.e., spore formation or passage to the viable-but-not-culturable state 4. Evasion of host organism defenses 5. Adaptive mutations Adaptation When microorganisms are stressed, an adaptive or protective response may follow. Response to stress, in this case, increases the organism’s tolerance to the same or to a different type of stress. This phenomenon is occasionally described as adaptive response, induced tolerance, habituation, acclimatization or stress hardening. Stress adaptation and stress adaptive response will be used interchangeably in this chapter. Tolerance Tolerance to a deleterious factor (e.g., low pH) refers to a microorganism’s ability to survive a stress. Each microorganism has an inherent tolerance level to a particular stress, but a transient or adaptive tolerance may also be induced. For example, lactic acid bacteria are inherently more acid tolerant than many other bacteria, yet they can become even more acid tolerant after acid adaptation. Resistance and tolerance have similar meanings; these terms will be used interchangeably in this chapter. Injury Damage to cellular components by stresses may impair the ability of microorganisms to multiply or may sensitize the cells to mildly deleterious factors. These changes are commonly described as injury. Injury is most noticeable when stress-exposed cells become sensitive to selective agents that healthy cells readily survive. The relationship between cell injury and stress adaptation has not been well characterized,
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but injury may result from a cell’s inability to respond to stress or a delayed or inadequate adaptive response. Injured cells may recover or die. Leistner (2000) indicated that simultaneous exposure of bacteria to different stress factors requires increased energy consumption and leads bacteria to cellular death through metabolic exhaustion and disturbed homeostasis.
STRESS, ADAPTATION
AND
FOOD SAFETY
Bacteria are exposed to stress in all links of the food chain, from production to digestion (Table 1.1). In the food production environment, sunlight, which contains ultraviolet radiation, may stress, injure or kill bacteria. Heat generated by sunlight may lead to microbial stress. Acidity of fermented vegetation, salinity of seawater, and dryness of arid climates are examples of other stresses that bacteria may encounter in the environment. Additionally, bacteria live in an environment that carries their own excretions (metabolites). Some of these metabolites constitute unique stresses to bacteria. Lack of essential nutrients for growth or survival (i.e., starvation) stresses, injures or kills bacteria, depending on the severity and duration of starvation. In summary, bacteria in the environment are frequently exposed to physical, chemical and nutritional stresses of varying magnitudes. Bacteria in food also are exposed to stresses including heat, acid, freezing, osmotic shocks, desiccation, oxidation, and starvation. Further information about environmental and processing stresses may be found in Chapters 4 and 5. Stress factors induce cellular responses that vary with the type, magnitude, and method of stress application. Although there are multiple outcomes, microorganisms’ adaptive response to stress is of paramount significance in food safety (Figure 1.2). Stress-adapted bacteria are capable of resisting similar (homologous) or different (heterologous) stresses and, in many cases, survive normally injurious or lethal conditions. For example, when bacteria are subjected to a heat shock, cells respond by becoming resistant to lethal heat treatments (Bunning et al., 1990). When Listeria monocytogenes was stressed by mild heat (45°C for 60 min), it became significantly more resistant to lethal doses of ethanol, hydrogen peroxide, and sodium chloride (Lou and Yousef, 1997). There are indications that adaptation of bacterial pathogens to stress may increase their ability to cause diseases. Data about increased virulence in stress-adapted cells are still limited, but if this relationship is confirmed in food applications, these results will have far reaching implications (see Chapter 7). During traditional food processing (e.g., pasteurization and retorting), bacterial cells are more likely to be killed than injured or stressed. However, there are processing conditions that constitute a mild stress and thus induce adaptive response in bacteria. Adaptation of Salmonella to acid stress, for example, increased the survival of this pathogen in cheese (Leyer and Johnson, 1992). Farber and Brown (1990) noticed that when L. monocytogenes was heat-shocked at 48°C for 120 min, the adapted cells exhibited increased tolerance to heat in sausage batter. Acid adaptation enhanced the survival of L. monocytogenes in acid foods such as yogurt, orange juice and salad dressing (Gahan et al., 1996). One may similarly hypothesize that certain processing conditions cause stress adaptation, which affects the safety of numerous foods. For example, acidity developed during sausage fermentation and the presence of salt in the formulation of this product may induce an acid adaptive
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TABLE 1.1 Deleterious Factors Likely to Provoke Stress Response in Foodborne Microorganisms at Various Links of the Food Chain, Including Production, Processing, Storage, Distribution, Consumption, and Digestion Factor
Stage in the Food Chain Pre-Harvest (Environmental)
Storage & Distribution
Processing
Consumption Site
In Host
Heat shock
Weather-related Composting
Mild processing
Temperature control failure
Cooking Reheating
Fever
Cold shock
Weather-related
Refrigeration
Refrigeration
Refrigeration fluctuation
—
Acidity
Acid rain Irrigation water Fermentation (e.g., silage production) Spoilage and decay (vegetation or product) Muscle stress Plant saps-fruit juices
Food fermentations Additives (e.g., acidulents, organic acids, acidic salts)
Spoilage by acid producers
Acidic additives during food preparation (e.g., vinegar and lemon juice)
Stomach Macrophages
Osmotic shock
Soil salinity Irrigation water
Additives (e.g., salt) Concentration Dehydration
Starvation
Non-nutritious environment
—
—
—
Iron starvation in macrophages
Oxidation
Air exposure of anaerobic microbiota
Exposure to air Oxidative sanitizers
Exposure to air Oxidative sanitizers
Exposure to air
Macrophages
Metal ions
Irrigation water
Equipment
—
Equipment
—
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Additives in food preparation
Non-Stressed
Process survivors
(relevant to preservation factors) *
Raw food
Pre-processing
Stressed
Mildly-processed Food
Response
Processing
Fully-processed Food
Stress-adapted
FIGURE 1.2 Potential hazards associated with stress adaptation of pathogens during food processing. *These cells may have been exposed to various environmental stresses during food production, but not to stresses specific to food preservation, e.g., high pressure.
response and osmotic shock response in pathogenic bacteria. Pathogens, adapted to acid and osmotic stress during sausage fermentation, may resist the heating and smoking steps or persist during storage of the product. Similarly, bacteria in milk that is heated at sub-pasteurization temperatures (e.g., for making certain varieties of cheese) may only suffer a mild heat shock (i.e., heat stress). These bacteria may become resistant to subsequent severe processing (e.g., cooking the product into processed cheese). Minimally processed foods are produced using mild treatments that may elicit stress adaptive responses in microbial contaminants including pathogens. Increasing use of alternative processing technologies (also referred to as “nonthermal,” “novel,” or “emerging” technologies) is arousing curiosity about the potential stress adaptation of foodborne pathogens. There are, however, some positive aspects to the adaptation of foodborne bacteria to stress. Probiotic bacteria (e.g., Bifidobacterium spp. and Lactobacillus acidophilus) are desirable supplements to some fermented products like yogurt. Viability of these bacteria, however, may decline rapidly during storage of such an acid food. Preadaptation to acid stress enhances survivability of probiotic bacteria in yogurt-like products (Shah, 2000). Fermentation starter cultures must also endure the stress of preservation by freezing or freeze-drying prior to use in food processing. Kim and Dunn (1997) demonstrated that cold shocking various starter cultures prior to freezing dramatically improved their cryotolerance compared to bacteria that were not cold shocked. Readers are advised to review Chapter 6 for details about the implications of stress adaptation in beneficial bacteria. In conclusion, microorganisms encounter a variety of sublethal stresses in food and environment. These stresses may induce stress adaptive responses that make foodborne pathogens resistant to subsequent lethal preservation factors (see Figure 1.2). Adaptation of pathogens to these stresses, therefore, constitutes potential health hazards to consumers.
EMERGING PROCESSING TECHNOLOGIES AND STRESS ADAPTATION Food processors currently rely on a variety of methods for preserving food. Conventional methods include heating, drying, freezing, and the addition of approved © 2003 by CRC Press LLC
preservatives. Heat is the most commonly used preservation method and heat-treated foods generally have a good safety record. When properly applied, heat can eliminate bacteria, fungi, viruses, parasites, and enzymes, which are the biological agents that spoil or compromise the safety of food. The applied dosage of conventional preservation factors can be varied to accomplish almost any degree of microbial inactivation, ranging from limited reductions of microbial load to complete sterilization. When heat is applied to milk, for example, at 71.6°C for 15 sec, a 5 to 6 log kill of non-spore-forming bacterial pathogens occurs, and the resulting product is considered pasteurized. Heating milk at 145°C for a few seconds produces a commercially sterile ultra high temperature-treated product, and the treatment is presumed to be a 12-D process when targeting Clostridium botulinum spores. Conventional technologies produce safe food but the product has lesser nutritional and sensory quality and consumer acceptability compared with its fresh counterpart (e.g., canned vegetables and fruits compared with fresh). Interest in alternative food processing technologies has been driven by consumer demand for food with fresh-like taste, crisp texture, high nutrient content, and natural color. Alternative technologies have been advanced by both industry and academia in an attempt to meet the challenge of producing safe processed food of a high quality. These emerging technologies include high pressure processing (HPP), pulsed electric field (PEF), pulsed light, and irradiation. The safety and microbiological quality of food processed using these technologies, however, needs to be affirmed. Alternative technologies cannot achieve the broad microbial lethalities that are currently attainable by conventional preservation factors, particularly heat. Current HPP and PEF technologies can only accomplish the equivalent of pasteurization when applied at their maximum lethal doses. The achievement of commercial sterility by these alternative technologies is not currently feasible. When food is treated with alternative processing technologies, the microbial load may become stressed, injured, or killed. Response of foodborne pathogens to the stress caused by these technologies is a concern and the adaptation of cells to such stress may constitute a microbial hazard. Alternative processing technologies introduce new challenges, and thus warrant the implementation of new safety strategies. The following is an overview of selected alternative processing technologies, structural and functional alterations in microbial cells by these technologies, and adaptive responses to these stresses. For additional details about these technologies, readers may seek relevant review articles, e.g., Barbosa-Canovas et al., 2000; Lado and Yousef, 2002; Farkas and Hoover, 2000.
HIGH PRESSURE PROCESSING Process Processing food with high pressure involves applying hydrostatic pressure in the range of 100 to 1000 MPa (equivalent to 14,500 to 145,000 psi). Equipment required to apply this intense treatment includes a thick-walled pressure vessel and a pressuregenerating device (Figure 1.3). Food, in flexible packages, is loaded into the vessel and the top is closed. The pressurizing medium, which is usually a water-based fluid,
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Vessel closure Pressurization fluid
Food in a Flexible package
Vessel
Valve Pressure
FIGURE 1.3 High pressure processing equipment: basic components.
is pumped into the vessel from the bottom. Since the applied pressure is uniform throughout the pressure medium and the food, the product retains its original shape, with minimal or no distortion. Once the desired pressure is attained, fluid pumping is stopped and the product is kept “at pressure” for a predetermined treatment period. Pressure is released after the treatment and the processed product is removed from the vessel. A pressure treatment cycle is normally completed in 5 to 20 min, depending on the pressure applied and equipment design. In lieu of this batch mode, semicontinuous or continuous HPP systems are now being developed. Mechanism Timson and Short (1965) suggested that ultrahigh pressure destroys biological systems because of protein precipitation. According to these authors, high pressure increases the solvation of ions and enhances the formation of ionic bonds. This decreases the number of the hydrophilic groups on the protein molecules and thus decreases the solubility of these proteins. On the contrary, Suzuki and Taniguchi (1972) suggested that high pressure damages biological systems because the treatment enhances protein–protein hydrophobic interactions. According to LeChatelier’s principle, pressure enhances reactions which lead to a decrease in volume and inhibits reactions which result in an increase in volume. Hydrophobic interactions among protein molecules under high pressure cause a decrease in volume, thus these reactions are favored during HPP. More recently, membrane damage was proposed as a mechanism of cell death by high pressure. Benito et al. (1999) found that the uptake of fluorescent stains (ethidium bromide and propidium iodide) was greater
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in pressure-sensitive than in pressure-resistant strains of Escherichia coli O157. Since these stains enter bacterial cells having damaged membranes, it follows that membrane damage occurs during the high-pressure treatment. Change in ribosomal conformation, as detected by differential scanning calorimetry, was proposed as a mechanism of microbial inactivation by HPP (Niven et al., 1999). Potential Stress Adaptation Mild pressure treatments may induce a stress response. When Welch et al. (1993) exposed exponentially growing E. coli to a pressure of 55 MPa, synthesis of several proteins was induced, particularly a 15.6 kDa protein. Most of the induction occurred after 60 to 90 min of pressure treatment. Many of these proteins were also induced by heat shock or cold shock. Wemekamp-Kamphuis et al. (2002) used two-dimensional gel electrophoresis, combined with western blotting, to demonstrate that cold shock or HPP elevated the levels of cold shock proteins (CSPs) in L. monocytogenes. When cold-shocked L. monocytogenes was pressure treated, the level of survival was 100-fold higher than that of cells grown exponentially at 37°C before the pressure treatment. The authors concluded that cold shock protects L. monocytogenes against HPP. Lucore et al. (2002) provided evidence of pressure adaptive response in E. coli O157:H7. When E. coli O157:H7 was subjected to sublethal pressure stress at 100 MPa and 37°C for 30 min, cells developed resistance to lethal pressures (at 300 MPa) and heat (57°C). Heat shocking the pathogen at 46°C for 15 min protected the cells against lethal heat and pressure treatments.
RADIATION The spectrum of electromagnetic radiation includes regions that are useful in food applications. Although some of these technologies were considered seriously by mid-20th century, interest in use as alternative processing methods increased only recently. Emerging radiation technologies in food preservation include gamma (γ), x-ray, ultraviolet (UV), microwave and radio frequency. Pulsed light and pulsed UV energy are beneficial technologies with great prospects in food applications. In this chapter, γ and UV radiation technologies only will be addressed. Process Treatment with γ radiation involves placing the food in proximity of a radiation source in a specially designed treatment chamber. The sources commonly used are 60Co and 137Cs. Ultraviolet radiation is generated from lamps that are placed in close proximity to the treated food. Short-wave UV, particularly of wave lengths 250 to 260 nm, has strong microbicidal properties. These can be generated from mercury lamps. Mechanism The short wavelengths of UV light inactivate microorganisms through alteration of DNA structure (Bintsis et al., 2000). Interaction of UV with DNA results in dimer
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formation, mainly cyclobutane-pyrimidine dimers, and DNA-protein cross linking. These alterations interfere with the cell’s ability to multiply, and thus lead to microbial demise. Pulsed light includes wavelengths that range from the ultraviolet (UV) to the infrared regions (Clark, 1995). It is therefore plausible to assume that the UV component of pulsed light contributes significantly to microbial lethality. Contrary to this hypothesis, some researchers believe that the thermal effect of pulsed light is the cause of microbial lethality (Corry et al., 1995). Gamma radiation generates hydroxyl radicals, which interact with cellular components and result in microbial inactivation. These radicals react with DNA and cause base modifications, single-strand or double-strand breaks, and DNA protein cross linkages (Von Sonntag, 1987). Kim and Thayer (1996) found that presence of air increases the lethality of γ radiation. Potential Stress Adaptation Sinha and Hader (2002) reviewed strategies to repair damage caused by UV radiation stress. Exposure of organisms to UV radiation induces mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers and 6-4 photoproducts. To overcome this stress, cells have developed repair mechanisms to counteract this type of DNA damage, regardless of the causative factor. One of the most common repair mechanisms involves photoreactivation with the help of the enzyme photolyase. Glycosylases and polymerases also help many organisms repair base and nucleotide excisions, respectively. Activation of these repair mechanisms by sublethal UV radiation likely protects cells against subsequent exposure to lethal doses of UV. Gamma-radiation resistant E. coli mutants have been recovered and studied (Verbenko and Kalinin, 1995), illustrating the ability of bacteria to change genetically to resist this stress.
PULSED ELECTRIC FIELD Process Pulsed electric field processing involves the application of pulses of high voltage (typically 20 to 80 kV/cm) to foods placed between two electrodes (Figure 1.4). When high electric voltage is applied, electrical current flows through liquid food materials. Liquid foods are commonly electrical conductors due to the presence of electrically charged ions. Because of the very short period of discharge time (i.e., microseconds or nanoseconds), heating of foods is minimized. Food treated with PEF has a better retention of natural flavor, color, taste, nutrients, and texture compared to that treated with heat (Dunn and Pearlman, 1987; Jia et al., 1999; Knorr et al., 1994). Mechanism Loss of cell membrane function is believed to cause microbial death during the PEF treatment (Tsong, 1991; Unal et al., 2002; Zimmermann, 1986). The cell membrane may be considered as a capacitor filled with a dielectric substance, with free charges accumulating on the inner and outer surfaces of the membrane. The normal resting
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Liquid Food
Electricity source (110 or 220 V)
Electric Pulses (µ s range )
Stepped-up Voltage (ca. 10 kV)
Power Supply
Pulser
Treatment Chamber (20-80 kV/cm)
PEF-treated product
FIGURE 1.4 Pulsed electric field (PEF) processing equipment: basic components.
potential difference across the membrane is 10 mV. The application of an electric field pulse causes an increase in the transmembrane potential. Since the charges at the two membrane surfaces are opposite, attraction between these charges reduces membrane thickness. This electric compressive force may reach a magnitude that causes a local breakdown of membrane (Zimmermann, 1986). The breakdown and pore formation occur when the PEF treatment induces a membrane potential greater than 1.0 V. Tsong (1991) suggested that electroporation of the cell membrane is a mechanism of microbial inactivation by PEF. When an external electric field is applied, electroporation occurs at protein channels due to protein denaturation caused by heating or electric modification of their functional groups. Electroporation leads to an osmotic imbalance of the cell, which may lead to death. Recently, Unal et al. (2002) stained PEF treated cells with fluorescent dyes and provided evidence of membrane permeation at lethal and sublethal electric fields. The growth region of yeast cells during budding was found particularly sensitive to PEF treatment (Castro et al., 1993). Potential Stress Adaptation Russell et al. (2000) treated L. monocytogenes and Salmonella typhimurium with PEF and plated the survivors on selective and nonselective agar media. These authors observed that mildly lethal PEF treatments did not result in any detectable cell injury. They concluded that PEF causes an “all or nothing” effect against foodborne pathogens. Unal et al. (2001) also observed no injury when foodborne bacteria were processed with PEF and the treated cells were grown on selective and nonselective media. Processes that result in no detectable cell injury usually do not induce a stress adaptive response. However, when bacterial cells were processed with sublethal levels of PEF and treated with fluorescent stains, leaky membranes were detected indicating cell injury (Unal et al., 2002). The authors concluded that PEF causes cell injury detectable only by the fluorescence staining technique. Evidence of stress adaptation due to PEF treatment is yet to be investigated.
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MECHANISM OF STRESS ADAPTIVE RESPONSE Response of microorganisms to stress includes immediate emergency responses (e.g., those produced in response to shock) and longer-term adaptation. In some cases, the same proteins are involved in both rapid and long-term responses. In addition to a general stress response that helps protect cells from a variety of stresses, cells have self-protective mechanisms against specific stresses. Overlap exists between the proteins involved in the general stress response and some specific stress responses. This section will focus on molecular mechanisms of stress adaptation in bacteria. Stress adaptation is a complex phenomenon that differs depending on the type of stress and the bacterial species. Adaptation results from induction of various stressrelated proteins that protect the cell from stress. Many stress-induced proteins have been identified. This chapter does not intend to provide a comprehensive review of stress-induced proteins in bacteria, but will introduce the variety of molecular mechanisms by which cells respond to stress and provide a general overview of how those mechanisms are regulated. Examples of a few well-characterized systems will be provided. For reviews of the molecular basis for stress response, the reader is referred to Chapter 8 of this book, the review by Abee and Wouters (1999), and the comprehensive book edited by Storz and Hengge-Aronis (2000).
STRESS SENSING For the cell’s metabolism to respond to a stress, the stress must somehow be sensed. In general, bacterial sensing of environmental changes is not well understood. Some stresses may affect folding of mRNA or change a protein’s half-life, resulting in changes in gene expression (Yura and Nakahigashi, 1999). Other stresses may affect protein structure. For example, OxyR senses reactive oxygen species via cysteine residues that are oxidized to form a disulphide bridge. The resulting oxidized protein positively regulates oxidative stress response (Mongkolsuk and Helmann, 2002). Levels of certain cellular metabolites, such as guanosine phosphate, guanosine tetra(ppGpp) and pentaphosphates (pppGpp) and phosphate, may also trigger the synthesis of stress-related proteins (Chatterji and Ojha, 2001; Rallu et al., 2000; Rao and Kornberg, 1999). Ribosomes were suggested as sensors for temperature shocks because of the sensitivity of these cellular components to heat (Duncan and Hershey, 1989). In addition, changes in the membrane structure or fluidity may trigger a signal to synthesize proteins to counteract a stress (Bremer and Krämer, 2000). Two-component signal transduction systems, consisting of a membrane-associated sensor kinase and an intracellular response regulator, have been implicated in the sensing of and response to some stresses. For example, in Bacillus subtilis, a two-component system is involved in expression of cold-inducible genes. In this system, a membrane-bound histidine kinase (DesK) that may sense changes in membrane fluidity transduces the signal to a response regulator (DesR) that putatively activates the transcription of fatty acid desaturase gene, des (Sakamoto and Murata, 2002).
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Molecular factors involved in sensing and controlling stress response
DNA
Transcription
mRNA
Ribosome
Translation
Stress- related protein
Methods to measure stress response
• Alternative σ factors • Anti σ factors • Transcription repressors
• Molecular probes to detect genes involved in stress response
• mRNA stability
• Northern blotting • Microarray • RT-PCR
• mRNA secondary structure • Ribosome stability
• Measurements of ribosome integrity (e.g., DSC methods)
• Protein stability • Protein modifications
• 2-D gel electrophoresis • Immunodetection
Changes in cell physiology to increase stress tolerance
• Relative stress resistance
FIGURE 1.5 A simplified representation of general cellular processes involved in stress response, molecular factors involved in sensing and controlling stress response, and methods used to measure some of these responses. The stress sensor is not depicted, but this includes a lipid, protein, or nucleic acid component that senses the stress and ultimately causes a change in transcription or translation. DSC: differential scanning calorimetry; RT-PCR: reverse transcription-polymerase chain reaction.
REGULATION
OF
STRESS-RELATED PROTEIN SYNTHESIS
Regulation of stress response is essential for the synthesis of appropriate stressrelated proteins only when necessary for protection of the cell. Regulation of stress responses occurs at different levels depending on the stress and the bacterium. Control may occur at the transcriptional or translational levels or by adjusting the stability of the mRNA or protein (Figure 1.5). Regulatory strategies vary considerably among bacteria and stresses. To add to the complexity, one stress response factor may be regulated at one or more levels. Transcriptional control of stress-induced genes and operons is a frequently encountered mechanism to control stress responses. One type of transcriptional control employs alternative sigma factors. The sigma subunit of RNA polymerase determines the specificity of promoter binding. Under non-stress conditions the constitutive sigma factor (σ70 in E. coli and σA in B. subtilis) directs expression of “housekeeping” genes. Binding of an alternative sigma subunit to the RNA polymerase core enzyme changes its specificity, directing it to transcribe a different group of genes and operons. Several stress-related regulons (coordinately regulated operons) are positively controlled by the synthesis of an alternative sigma factor. For example, the presence of active σS causes transcription of genes involved in the general stress response and stationary phase in E. coli. © 2003 by CRC Press LLC
A strategy to negatively control transcription of stress-related genes involves anti-sigma factors. Anti-sigma factors bind to a specific sigma factor forming a complex that prevents the sigma factor from binding to the RNA polymerase core enzyme (Hughes and Mathee, 1998). In E. coli, the RssB protein has anti-sigma factor properties; it inhibits the expression of σS-dependent genes in the presence of high σS levels (Becker et al., 2000). A stress sensor may trigger release of the sigma factor from the anti-sigma factor complex, resulting in transcription of stressrelated genes. A sigma factor may be released from the anti-sigma factor by an antianti-sigma factor that binds to the anti-sigma factor. For example, σB, required for general stress response in B. subtilis, is bound by an anti-sigma factor. An anti-antisigma factor is present in a phosphorylated form in the absence of stress. Stress increases the level of non-phosphorylated anti-anti-sigma factor, which is then able to bind to the anti-sigma factor, releasing σB (Hecker and Volker, 1998). Other transcriptional control mechanisms utilize repressor proteins that bind to the promoter region of a specific gene or operon, preventing transcription until conditions are appropriate, at which time the repressor protein is released from the DNA allowing transcription to proceed. The heat stress operons, dnaK and groE, are controlled in this manner in B. subtilis. They are under the negative regulation by the HrcA repressor protein binding to the CIRCE (controlling inverted repeat of chaperone expression) operator (Narberhaus, 1999). Synthesis of stress-related proteins can also be controlled at the translational level. Messenger RNA secondary structure near the ribosome binding site or translation start site can inhibit ribosome binding and translation of mRNA until stress conditions are experienced (Takayama and Kjelleberg, 2000). Translation of mRNA for the heat shock sigma factor (σ32) is regulated in this manner. Heat disrupts the hydrogen bonds holding the mRNA secondary structure together allowing the translation of the transcript under hot conditions (Yura and Nakahigashi, 1999). Changes in mRNA and protein stability provide another method of controlling the activity of stress-related proteins. The half-life of some molecules can be increased or decreased in response to stress. For example, the CspA mRNA involved in cold tolerance is extremely unstable at 37°C and dramatically stabilized at lower temperatures (Phadtare et al., 1999). Proteolytic degradation of stress-related proteins is also observed as a control mechanism. The ClpXP protease degrades σS under non-stress conditions (Hengge-Aronis, 1999).
GENERAL STRESS RESPONSE A general stress response system can be activated by several different stresses and protects against multiple stresses. Activation of the general stress response usually results in reduced growth rate or entry into stationary phase (Hengge-Aronis, 1999). The best-characterized general stress response systems are controlled by alternative sigma factors, σS, in E. coli and other Gram-negative bacteria and σB in B. subtilis and other Gram-positive bacteria. The general stress response induces multiple physiological changes in the cell including “multiple stress resistance, the accumulation of storage compounds, changes in cell envelope composition and altered overall morphology” (Hengge-Aronis, 1999).
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Genes induced by σS and σB include those for catalase, DNA repair, and osmoprotectant importation, suggesting that the cell is preparing for oxidative and osmotic stress (Hecker and Volker, 1998; Petersohn et al., 2001). Stress adaptive response in E. coli is coordinated by σS. Very little if any σS is detectable in non-stressed E. coli cells. When cells are exposed to stress, σS is induced, activating the σs-controlled promoters. Expression of these genes is necessary for survival under stress conditions. σS is regulated by transcriptional and translational control as well as by proteolysis (by ClpXP protease) in E. coli (Hengge-Aronis, 1999). Different stresses differentially affect these various levels of control. In B. subtilis, the activity of σB is modulated by an anti-sigma factor and an anti-anti-sigma factor as described in the previous section.
SPECIFIC STRESS RESPONSES Heat Foodborne bacteria commonly encounter heat stress during food preservation and processing. Heat causes damage to macromolecular cell components; thus the main function of heat-induced stress proteins is to repair or destroy these damaged components so they do not disrupt cellular metabolism. Many heat-induced stress proteins are protein chaperones that assist in folding and assembly of heat-damaged proteins (e.g., GroEL and DnaK) or are ATP-dependent proteases that degrade damaged proteins (e.g., Lon and ClpAP) (Arsène et al., 2000; Hecker et al., 1996). In addition to these changes, some bacteria also alter their cell membrane in response to heat by increasing the ratio of trans to cis fatty acids in the membrane. This structural change is thought to decrease fluidity caused by increasing temperatures (Cronan, 2002). In E. coli, the major heat-induced genes are controlled by the alternative sigma factor, σ32. Approximately 50 genes are induced by σ32 when denatured proteins are detected in the cytoplasm (Yura and Nakahigashi, 1999). σ32 is present at low levels under non-heat-stress conditions. This low level is governed by the short mRNA half-life and the low translation rate resulting from secondary structure at the 5′ end of the mRNA. After a temperature increase, the secondary structure is destabilized allowing translation to proceed. The half-life of σ32 also increases dramatically upon exposure to heat (Arsène et al., 2000; Yura and Nakahigashi, 1999). Two other alternative sigma factors, σE and σ54, control other regulons induced by heat. σE, an extracytoplasmic function (ECF) sigma factor, responds to the appearance of non-native proteins within the periplasm by means of an inner membrane-bound anti-sigma factor (Raivio and Silhavy, 2001). Release of σ E from the anti-sigma factor activates transcription of about 10 genes involved in proper assembly of outer membrane proteins (Raivio and Silhavy, 2001). How non-native proteins are sensed resulting in release of σE is not understood. σ54 controls one operon and is activated by disturbances in the cytoplasmic membrane by an unknown mechanism (Kuczynska-Wisnik et al., 2001). Gram-positive bacteria differ markedly in their regulation of heat shock response. In B. subtilis, several classes of heat shock genes have been identified. Class I consists
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of the chaperone-encoding dnaK and groE operons. These operons have σA-dependent promoters that are under the negative regulation of the HrcA repressor protein binding to the CIRCE operator. This regulatory system is widespread and conserved within the bacterial kingdom and has been described in more than 40 different species (Hecker et al., 1996). The σB regulon constitutes the Class II genes, the largest group of heat-induced genes in B. subtilis. These genes are not only induced by heat, but also by other stresses, as discussed above (Hecker and Volker 1998). Class III heatinduced genes are negatively controlled at the transcriptional level by a repressor protein, CtsR. CtsR binds to a specific sequence in the promoter region upstream of clp genes, clpP, clpE and clpC. These three genes are components of the Clp protease system which degrades damaged proteins (Derre et al., 1999). It is not clear how CtsR activity is changed after an increase in temperature. Other heat-induced genes, not controlled by the above mechanisms, are yet to be classified. Cold Physiological changes in response to cold include changes in the membrane fatty acid composition to promote optimum membrane fluidity (Russell et al., 1995), synthesis of DNA- and RNA-binding proteins that counteract the stabilizing effect of cold temperatures on nucleic acid secondary structures (Phadtare et al., 1999), and importation of compatible solutes (Ko et al., 1994; Angelidis et al., 2002). Proteins synthesized in response to cold can be classified as Csps (cold shock proteins) or Caps (cold-shock acclimation proteins). Csps are rapidly, but transiently overexpressed in response to cold. Caps are synthesized during continuous growth at cold temperatures; they are rapidly induced, but remain overexpressed several hours after the temperature downshift. A slow temperature downshift results in synthesis of some Csps and Caps (Phadtare et al., 1999). Upon decrease in temperature, the phospholipid bilayer membranes of all cells decrease in fluidity. To maintain optimum fluidity, cells increase the unsaturation or decrease the chain length of the membrane fatty acids, resulting in increased fluidity at lower temperatures (Russell et al., 1995). After cold shock in B. subtilis and cyanobacteria, synthesis and stability of a fatty acid desaturase increase as controlled by a two-component signaling system (Aguilar et al., 1998; Sakamoto and Murata, 2002). Cold shock also causes stabilization of the hydrogen bonds in nucleic acid secondary structures resulting in reduced efficiency of translation, transcription and DNA replication. These deleterious effects are overcome by induction of cold-shock proteins that serve as nucleic acid chaperones. CspA, the major cold-shock protein of E. coli, is proposed to regulate gene expression by functioning as an RNA chaperone at low temperatures. CspA-like proteins contain two conserved RNA binding sequences. CspA is regulated at the transcriptional and translational levels and by increased mRNA stability at low temperatures (Phadtare et al., 1999). In E. coli, Csps have been grouped into two classes. Class I proteins consist of RNA/DNA chaperones (including CspA), ribosome-associated proteins, a ribonuclease, and a protein involved in termination of transcription. Class I genes are barely expressed at 37°C, but dramatically increase after a shift to lower temperatures.
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Class II genes are involved in DNA stability and structure and include the DNAbinding protein, H-NS, and a subunit of DNA gyrase. Class II proteins are present at 37°C; after shift to colder temperatures, their transcription is only slightly higher (<10-fold) (Phadtare et al., 1999). Transport or synthesis of compatible solutes (see osmotic stress section) was reported to confer cold shock tolerance. In E. coli, the σS-dependent synthesis of trehalose by the otsAB gene products is cold-inducible. An additional level of regulation is provided by the instability of otsAB mRNA at higher temperatures (Kandror et al., 2002). Listeria monocytogenes transports the compatible solutes, betaine (Ko et al., 1994) and carnitine (Angelidis et al., 2002), in response to cold temperatures. Regulation of this system has not been reported. Acid Foodborne bacteria encounter organic and inorganic acids in foods or in the gastrointestinal tract and cells of the host. Bacteria respond to acid stress in many ways including changes in membrane composition, increase in proton efflux, increase in amino acid catabolism, and induction of DNA repair enzymes. Observed in most bacteria, the acid tolerance response (ATR) is a phenomenon whereby exposure to moderately low pH induces the synthesis of proteins that promote survival at extremely low pHs. ATR differs in exponential and stationary phase cells. This response also differs dramatically among different bacterial species. An overview of strategies which bacteria employ to combat acid stress is described in this section. The reader is referred to Chapter 8 of this book for more details. The signal for induction of acid shock or adaptation proteins may be intracellular or extracellular pH. External or periplasmic pH may be sensed by membrane bound proteins (Foster, 1999). Internal pH may affect gene expression directly or may alter a cellular component involved in gene expression. Exponential phase ATR in Salmonella typhimurium involves several regulatory proteins that each control a subset of acid-induced proteins. These regulatory proteins include σS, the two-component signaling system PhoPQ, and the iron regulator, Fur (Foster, 1999, 2000). The σS-dependent ATR genes that have been identified consist of several proteins of unknown function and a superoxide dismutase. Most of the PhoPQ-controlled genes are of unknown function, though Adams et al. (2001) reported decreased flagellin expression and cell motility upon activation of the PhoPQ pathway by acid. The authors suggest that “flagellar repression at low pH conserves ATP for survival processes and helps to limit the influx of protons into the cytosol.” The Fur-controlled acid-induced genes in Salmonella have not been identified (Foster, 2000), but Fur modulates urease expression in enterohemorrhagic E. coli, and thus, may be involved in acid tolerance of this organism (Heimer et al. 2002). Urease hydrolyzes urea into ammonia and carbon dioxide. The resulting ammonium ions may accumulate and modify internal and/or external pH. Stationary phase ATR in Salmonella involves stationary phase induction of σS resulting in a general stress tolerance and induction of acid stress proteins by OmpA (Foster, 2000). A deletion in the gene encoding σB in L. monocytogenes renders stationary phase cells acid sensitive (Gahan and Hill, 1999).
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Cyclopropane fatty acid (CFA) synthase catalyzes the synthesis of CFAs from unsaturated fatty acids in the bacterial membrane. In E. coli, CFA synthase gene expression increases with a decrease in pH to 5. Transcriptional activation is σSdependent. The increase in cfa gene expression results in increased survival to the lethal challenge of pH 3 (Chang and Cronan, 1999). The investigators suggest that the resulting changes may affect proton permeability through the membrane or the activity of a membrane-bound protein involved in acid stress. Limited information is available about the association of extracellular cell-tocell signaling and stress adaptation. Acid adapted E. coli is believed to secrete an extracellular protein that causes unadapted cells to become acid tolerant without acid adaptation (Rowbury and Goodson, 1999; Chapter 8 of this book). Gram-positive bacteria, which regulate internal pH with an F0F1 ATPase, can increase synthesis or activity of the ATPase upon pH decrease, providing the cell with a higher capacity for proton efflux (Foster, 2000). The F0F1ATPase is acidinducible at the transcriptional level in Lactobacillus acidophilus (Kullen and Klaenhammer, 1999), whereas in Streptococcus spp. or Enterococcus spp., enzyme activity is controlled at the subunit assembly stage (Foster, 2000). Low cytoplasmic pH can cause DNA damage. An acid-inducible DNA repair enzyme was identified in Streptococcus mutans (Hahn et al., 1999). The importance of DNA repair in acid stressed cells is supported by data revealing that mutations in the ada gene, involved in DNA repair, cause acid sensitivity in Salmonella (Foster, 2000). Amino acid catabolism can also help cells to fight a proton influx. Some Grampositive bacteria use the arginine deiminase system to alkalinize the cytoplasm (Foster, 1999). Arginine is broken down into ornithine, carbon dioxide and ammonia. The glutamate decarboxylase/GadC antiporter system (E. coli, Shigella, Lactococcus, [Foster, 2000], and Listeria [Gahan and Hill, 1999]) requires extracelluar glutamate which is imported via the GadC antiporter and decarboxylated within the cell, a reaction that consumes a proton. The resulting gamma amino butyric acid is exported via GadC. This system is induced by stationary phase or by acid in the exponential phase. A similar system involving arginine decarboxylase also protects E. coli from pH 2 (Foster, 2000). Osmotic Stress Bacteria may encounter osmotic stresses in foods that are high in salt or sugar or in a dried state. Under such conditions, it is essential for the cell to maintain turgor pressure and hydration. The mechanisms described refer to bacteria that reside in environments with moderate or occasional hyperosmotic conditions. The best-characterized mechanism by which bacterial cells respond to hyperosmotic conditions involves intracellular accumulation of compatible solutes. This accumulation can be accomplished by synthesis or import from the environment. Compatible solutes are polar, highly soluble compounds that counteract osmotic pressure without affecting normal cellular functions, even at very high concentrations. Glycine betaine, proline, ectoine, carnitine, choline, and trehalose, among others, are common compatible solutes. Accumulation of these compounds is regulated at the
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gene transcription level or by modifying enzyme activity directly (Bremer and Krämer, 2000). σS (E. coli) and σB (B. subtilis) control synthesis of some proteins required for osmoprotectant synthesis or transport. Sensing of osmotic stresses is poorly understood (Culham et al., 2001; Mellies et al., 1995; von Blohn et al., 1997). Additional changes in cell metabolism in response to osmotic stress involve the cell membrane. An increase in the ratio of trans to cis unsaturated fatty acids is observed in cells exposed to high salt concentrations (Cronan, 2002). In addition, the proportion of anionic phospholipid and/or glycolipids is increased in saltstressed, compared with unstressed, cells (Russell et al., 1995). In addition to σS, the σ32 and σE regulons are activated when E. coli experiences hyperosmotic conditions. Both regulons encode protein chaperones and proteases that assure proper assembly of proteins in the stressed cell (Bianchi and Baneyx, 1999). Hyperosmotic stress not only activates the σB regulon in B. subtilis, but also induces the extracytoplasmic function (ECF) sigma factor σW (Petersohn et al., 2001). This sigma factor controls expression of >30 genes, many encoding membrane proteins of unknown function (Huang et al., 1999). Oxidative Stress In foods, bacteria may be exposed to increased levels of reactive oxygen species such as hydrogen peroxide, hydroxyl radicals and superoxide. Such oxidants cause damage to cellular proteins, lipids and nucleic acids. Many of the known proteins induced by oxidative stress have antioxidant roles. Others are involved in repair of oxidative damage, particularly damage to nucleic acids. In E. coli, most oxidative stress-induced genes are part of the oxyR and soxRS regulons induced by hydrogen peroxide and superoxide, respectively (Storz and Zheng, 2000). OxyR senses oxidative damage via cysteine residues that are oxidized to form a disulphide bridge, altering the protein structure into the active form (Mongkolsuk and Helmann, 2002). There is significant overlap between the oxidative stress-induced proteins and those induced by σS, suggesting that oxidative damage is significant in stationary phase or stressed cells.
MONITORING STRESS RESPONSE Microorganisms in food or environment are often exposed to stresses and some of these evoke measurable responses (see Figure 1.5). The response varies mainly with the type and magnitude of stress and the microorganism’s physiological state. Under some stress conditions, microbial response is a protective effect, i.e., an adaptive response. Food microbiologists and processors are interested in the stress adaptive response since it alters the microorganism’s resistance to processing and preservation factors. Higher levels of stress may injure the cells. Injured cells probably become energy-exhausted by multiple responses which decrease their capacity to react to additional insults. Additional stress usually kills injured cells (see Figure 1.1). Injury is evident by the sensitization of treated cells to selective agents, antibiotics and other deleterious factors, or the impairment of cells’ ability to multiply.
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Detecting and measuring stress response have many beneficial applications. Food processors may learn about the consequences of mild treatments and the causes of resistance of pathogens to processes that are presumed lethal to these microorganisms. On the contrary, stresses that sensitize pathogens to processing may have beneficial applications in food preservation. Using stress response to sense undesirable agents (stressors) in the food processing environment is another area of potential interest to food processors, but this has not been explored. To determine the conditions likely to lead to adaptive responses, researchers may vary stress level and apply stress at various physiological states of the targeted microorganism. Based on experience and a large amount of published literature, microbial adaptive response is most apparent at sublethal levels of stress and when the microorganism is in an active metabolic state, i.e., the exponential phase of growth. Many researchers, however, have demonstrated appreciable stationary-phase inducible adaptive responses (e.g., Buchanan and Edelson, 1999). Similarly, lethal doses of stress may trigger considerable adaptive responses in the fraction of the population that survives the treatment. After applying the stress under investigation, procedures to detect or quantify the response should be followed. Stress responses measured include changes in gene expression products (RNA and proteins) and stress tolerance (see Figure 1.5). Although detection of stress adaptive response is generally laborious, distinction of injury is relatively simple. Stress-sensitized cells (i.e., injured) demonstrate reduced growth rate (e.g., reduced colony size on agar media), impaired growth in the presence of selective agents such as NaCl and bile salts, increased sensitivity to antibiotics, and loss of aerotolerance. Details about adaptive responses are included in this contribution, but sensitization by stress will not be addressed.
INDUCTION
OF
STRESS ADAPTIVE RESPONSE: PRACTICAL CONSIDERATIONS
The following are examples of the most commonly investigated stresses, heat and acid. Included is a brief description of methods of applying theses stresses for inducing adaptive responses. Once the stress response is developed, cells should be handled in a way to preserve the response. Active metabolism and multiplication of stress-adapted cells deteriorate the adaptation and thus it becomes difficult to detect. Heat Heat induces a universal protective response that is relatively easy to detect. Temperatures conducive to growth normally do not constitute stress to cells and thus are not used commonly in developing a stress response. Severe thermal stress may eliminate sizable proportion of the cell population and the adaptive response in the small fraction of the population that survives the treatment may not be measurable. Response to a mild heat shock is readily detectable when cells are treated at sublethal or minimally lethal temperatures. According to our experience, heat shock response is demonstrated best when L. monocytogenes exponential-phase culture is heated at 45°C for 1 h (Lou and Yousef, 1997). By comparison, injury of L. monocytogenes is most apparent at 55 to 60°C (El-Shenawy et al., 1989) and neither stress response
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nor injury can be reliably detected at 70°C. Heat shocking E. coli O157:H7 at 45 to 46°C for 15 to 30 min produces appreciable thermal adaptation (Juneja et al., 1998; Lucore et al., 2002). Heat may be applied rapidly, i.e., as a heat shock (Lou and Yousef, 1997) or gradually (Stephens et al., 1994), since both procedures produce significant adaptive response. Acid Acid Shock during Exponential Phase Actively growing microbial cells, in their mid-exponential phase, are treated with sublethal levels of an acid, i.e., cells are acid shocked. Incubation is continued to allow one to two doublings under the acid stress. During this additional incubation period, cells normally develop an acid adaptive response. Since the adaptive response is a transient phenomenon, further processing of these cells (e.g., centrifugation and washing) should be done promptly and under refrigeration conditions in order to preserve the developed response. This technique produces a strikingly different response from that observed in the non-treated culture and thus the adaptation is relatively easy to track. Response of these cells, however, is transient and the adaptation may degrade quickly before it can be measured, particularly if treated cells are mishandled. Additionally, collecting cells from mid-exponential phase can be tricky since cell density at this stage is normally low. Phase of growth should be determined in advance by plating the culture after different incubation periods and constructing a growth curve. Correlation of microbial counts with culture turbidity (measured spectrophotometrically) allows estimation of growth phase prior to the experiment. Researchers who successfully applied acid stress to mid-exponential phase cultures include Foster and Hall (1990), Leyer and Johnson (1992), and Lou and Yousef (1997). Gradual Acid Stress Microorganisms that produce acid as a byproduct of carbohydrate metabolism experience a gradual decrease in pH during culturing. This gradual acidification induces a stationary-phase acid resistance response (Buchanan and Edelson, 1999). Gradual acid exposure is a simple and practical method of producing acid-adapted cells. Most of the adaptation, however, occurs during the stationary phase when cells generally develop resistance to various deleterious factors (Watson, 1990). Consequently, the intrinsic stationary phase acid resistance may overshadow induction of acid resistance by carbohydrate fermentation. The non-acid adapted cells (control culture) are grown in the absence of a fermentable carbohydrate and thus produce energy through alternative metabolic ways. Unfortunately, these control cells may inadvertently be sensitized to acid or develop a starvation response during growth in the carbohydrate-free medium. Gradual application of acid stress may also be accomplished by manual incremental addition of acid to a growing culture. Alternatively, a chemostat may be used to gradually apply acid stress to a growing culture in a controlled manner. This latter procedure is most useful when the test microorganism does not produce acid during growth.
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DETECTING
AND
QUANTIFYING STRESS RESPONSE
Methods to detect and measure stress response vary depending on the response measured (see Figure 1.5). Evidence of stress response includes presence of genes involved in stress response mechanisms, elevated level of gene products such as mRNA, de novo protein synthesis in response to stress, and increased tolerance to lethal levels of the stress. Detection of Stress Response Genes Presence of genes encoding stress response proteins may indicate that the microorganism is capable of responding to a stress in a predictable fashion. Comparing the genomes of resistant and sensitive strains may reveal these genes involved in stress response (Koonin et al., 2000). Researchers have developed probes for detecting genes that contribute to stress response; these are useful tools to determine potential response to stress by an isolate. mRNA Analysis While presence of the gene is a prerequisite for a response, expression of this gene is needed for the ultimate manifestation of the response. Therefore, interest in detecting stress response at the transcriptional level is increasing. Synthesis of proteins that protect cells against stress is sometimes preceded by increased transcription of the relevant mRNA. Measuring these mRNAs demonstrates, or even quantifies, the stress response. Methods to measure mRNA include Northern analysis, microarray-genome-wide expression monitoring (also known as microarray analysis) and reverse transcription polymerase chain reaction (RT-PCR). Detection of Stress Proteins Synthesis of stress proteins provides yet more direct evidence of the microorganism’s response to stress. Proteins synthesized in response to stress include regulatory proteins (e.g., σ32 in E. coli and σB in L. monocytogenes), chaperones (e.g., GroEL), ATP-dependent proteases (e.g., Lon), and DNA repair proteins (e.g., UspA) (Duncan et al., 2000; Diez et al., 2000; Rosen et al., 2002). Many of these proteins have been successfully detected using a two-dimensional electrophoresis (e.g., Rince et al., 2002). Antibodies specific to some of the well-characterized stress proteins are commercially available to detect a stress response by immunodetection methods such as Western blotting (Duncan et al., 2000). If the corresponding antibodies are not commercially available, the gene of a specific stress protein can be cloned. The recombinant protein is then amplified, purified and used to generate the corresponding specific antibodies (Jayaraman and Burne, 1995). Biosensors Microorganisms have been genetically engineered for easy detection of stress response (LaRossa and Van Dyk, 2000). Reporter genes (e.g., lacZ which encodes
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for β-galactosidase) were fused to promoters of genes involved in adaptive response. Other useful reporter genes include luxAB, which encodes bacterial luciferase, luc, encoding insect luciferase, and gfp, for green fluorescence protein. When these fusion strains respond to stress, the reporter gene is expressed and fluorescent or luminescent products are produced. Gene fusion strains (biosensors) for detecting DNA damage, heat shock, oxidative stress, and starvation have been developed for basic research and are potentially useful in the field of food microbiology. Measuring Increased Tolerance Adaptive responses may be measured by comparing stress tolerance of cells that have been pre-exposed to sublethal stress to those that have not. Measurement of inactivation by stress uses simple plating techniques. A greater degree of survivability of the cells exposed to sublethal stress may indicate that the stress induced an adaptive response. Quantifying the stress by the cultural technique may require measuring changes in death rates as a result of pre-exposure to stress. Determining D-value (time required to decrease the population under stress by one log CFU unit) is a useful quantitative measure of resistance. Culture techniques provide direct evidence of stress adaptive response and the results of the analysis have great practical value to food processors. These techniques, however, are time-consuming and the results may be compromised by experimental artifacts.
PERSPECTIVES AND AREAS FOR FUTURE WORK Some researchers question the relevance of stress adaptation to food safety. This argument is based on these observations: • Stress adaptation is best demonstrated at the exponential, rather than at the stationary, phase of growth. Since pathogens in food are rarely in the exponential phase, significant adaptation to stress under most processing and production practices may be unlikely. • Direct determination of the degree of adaptation of microbiota in food is not currently feasible. Therefore, there is no knowledge on how much of processing resistance that these microorganisms experience is attributed to stress adaptive response. • Although the number of reports linking stress adaptation and virulence is rising (see Chapter 7), there is no evidence that directly links stress adaptation of pathogens to foodborne disease outbreaks. While these arguments have some merits, we believe that the stress adaptation phenomenon has a profound effect on the safety of food: • Although stress adaptation is remarkable in actively metabolizing cultures, microorganisms at all phases of growth do adapt to stress. Induction of stress adaptive response in stationary-phase cultures is well documented.
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Nevertheless, demonstration and quantification of these adaptive responses, under real processing conditions, need to be carefully investigated. • Lack of direct evidence is not a proof of the absence of the relationship between stress adaptation and food safety. With the continuous improvements in analytical tools and protocols, researchers may soon be able to verify these associations. Rapid methods to differentiate between transient and inherent resistance, and to quantify these traits in the food microbiota, are urgently needed. Availability of these methods will not only reveal the risks associated with stress adaptation, but processors may also use these techniques to gauge processing severity with the anticipated tolerance of the microbiota in food. Many researchers agree that there is a considerable potential risk of disease as a result of stress adaptation, particularly in food produced by minimal-processing or novel, alternative processing technologies (Abee and Wouters, 1999; Archer, 1996; Rowan, 1999; Yousef, 2000). Interest in these technologies has increased appreciably in the past decade. These technologies promise to maintain the critical balance between safety and marketability of a new generation of foods. It is of concern that processing conditions may be conducive to stress adaptive response in foodborne pathogens. Currently, stress adaptive responses of microorganisms in food processed by these technologies are poorly understood. As these novel food processing technologies become commercialized or used more widely, it is essential that researchers understand the adaptive responses that are induced by these treatments.
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Kandror, O., A. DeLeon, and A.L. Goldberg. 2002. Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures, Proc. Nat. Acad. Sci., 88:9727–9732. Kim, A.Y. and D.W. Thayer. 1996. Mechanism by which gamma irradiation increases the sensitivity of Salmonella typhimurium ATCC 14028 to heat, Appl. Environ. Microbiol., 62:1759–1763. Kim, W.S. and N.W. Dunn. 1997. Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance, Curr. Microbiol., 35:59–63. Knorr, D., M. Geulen, T. Grahl, and W. Sitzman. 1994. Food application of high electric field pulses, Trends Food Sci. Technol., 5:71–75. Ko, R., L.T. Smith, and G.M. Smith. 1994. Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes, J. Bacteriol., 176:426–431. Koonin, E.V., L. Aravind, and M.Y. Galperin. 2000. A comparative genomic view of the microbial stress response, in Bacterial Stress Responses. G. Storz and R. HenggeAronis, Eds. Washington, D.C.: American Society for Microbiology, pp. 417–446. Kuczynska-Wisnik, D., E. Laskowska, and A. Taylor. 2001. Transcription of the ibpB heatshock gene is under control of σ32- and σ54-promoters, a third regulon of heat-shock response, Biochem. Biophys. Res. Commun., 284:57–64. Kullen, M.J. and T.R. Klaenhammer. 1999. Identification of the pH-inducible, proton-translocating F1F0-ATPase (atpBEFHAGDC) operon of Lactobacillus acidophilus by differential display: gene structure, cloning and characterization, Mol. Microbiol., 33:1152–61. Lado, B.H. and A.E. Yousef. 2002. Alternative food preservation technologies: efficacy and mechanisms, Microbes Infection, 4: 433–440 LaRossa, R.A. and T.K. Van Dyk. 2000. Application of stress responses for environmental monitoring and molecular toxicity, in Bacterial Stress Responses. G. Storz and R. Hengge-Aronis, Eds. Washington, D.C.:American Society for Microbiology Press, pp. 453–467. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology, Int. J. Food Microbiol., 55:181–186. Leyer, G.J. and E.A. Johnson. 1992. Acid adaptation promotes survival of Salmonella spp. in cheese, Appl. Environ. Microbiol., 58: 2075–2080. Lou, Y. and A.E. Yousef. 1996. Resistance of Listeria monocytogenes to heat after adaptation to environmental stresses, J. Food Protect., 59:465–471. Lou, Y. and A.E. Yousef. 1997. Adaptation to sublethal environmental stress protects Listeria monocytogenes against lethal preservation factors, Appl. Env. Microbiol., 63:1252–1255. Lucht, L., G. Blank, and J. Borsa. 1997. Recovery of Escherichia coli from potentially lethal radiation damage: characterization of a recovery phenomenon, J. Food Safety, 17:261–271. Lucore, L.A., A.E. Yousef and T.H. Shellhammer. 2002. Stress induced resistance of Escherichia coli O157:H7 to high pressure processing, J. Food Prot. (submitted). Mackey, B.M. and C.M. Derrick. 1987. The effect of prior heat shock on the thermoresistance of Salmonella thompson in foods, Lett. Appl. Microbiol., 5:115–118 Mellies, J., A. Wise, and M. Villarejo. 1995. Two different Escherichia coli proP promoters respond to osmotic and growth phase signals, J. Bacteriol., 177:144–151. Mongkolsuk, S. and J.D. Helmann. 2002. Regulation of inducible peroxide stress responses, Mol. Microbiol., 45:9–15. Narberhaus, F. 1999. Negative regulation of bacterial heat shock genes, Mol. Microbiol., 31:1–8.
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2
Adaptation of Foodborne Pathogens to Stress from Exposure to Physical Intervention Strategies Vijay K. Juneja and John S. Novak
CONTENTS Introduction Sublethal Heat Stress Heat-Shock Response Synthesis of Heat-Shock Proteins Factors Affecting Heat-Shock Response Cell Membrane Adaptations Cross Protection Management Strategies High Hydrostatic Pressure Dehydration Restricting Water Activity Freezing Pulsed Electric Field Irradiation Ultraviolet Irradiation Gamma Irradiation Concluding Remarks References
INTRODUCTION The growth or survival of potentially life-threatening pathogens is a significant food safety hazard. The ability of low numbers of these pathogens to survive or proliferate Note: Mention of a brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over other products or companies of a similar nature.
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even when stored under refrigeration or in reduced oxygen atmospheres constitutes a potential public health hazard. It is estimated that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year (Mead et al., 1999). Known pathogens cause 14 million illnesses, 60,000 hospitalizations, and 1,800 deaths (Mead et al., 1999). Only a small portion of the foodborne illness episodes are reported and investigated annually, and the pathogens identified comprise an even smaller portion (Mead et al., 1999). Annual economic losses attributed to foodborne diseases associated with medical costs, productivity losses, and business losses due to legal problems may be as large as $5 billion to $6 billion (CAST, 1996). These food safety concerns are magnified because of consumer preference for convenient meals, processed using mild heat treatment, that require minimal preparation time prior to consumption. Accordingly, it is important to emphasize development and application of physical control processes for microorganisms with the objective of improving the safety of our food supply. A variety of established physical intervention strategies for control of foodborne pathogens include heat treatment, refrigeration, modified gaseous atmosphere, and ionizing irradiation. Microorganisms can also be controlled by novel nonthermal treatment methods, such as application of high hydrostatic pressure, high intensity pulsed electric field, oscillating magnetic field, or combinations of physical processes. These physical treatments used in food processing are designed to kill or decrease the number of pathogenic and spoilage microorganisms. If the treatment is not severe enough to ensure lethality, the surviving organisms are injured and may recover under the proper conditions (Iandolo and Ordal, 1966).
SUBLETHAL HEAT STRESS HEAT-SHOCK RESPONSE The microbial safety of thermally processed foods depends upon the assurance that foodborne pathogens, likely to be present in foods, are killed during heating. While thermal processing guidelines are generally adequate for destruction of pathogens in foods, there may be conditions when the microorganisms become more heat resistant. Sublethal heat stress (heat shock) or prior exposure to low heat renders the organism more resistant to subsequent heat treatment which would otherwise be lethal (Murano and Pierson, 1992; Lou and Yousef, 1996; Juneja et al., 1997). Microbial cells synthesize heat-shock proteins, coincident with sublethal heat stress acquired, which appear to render the cell resistant to a second elevated challenge normally considered to be lethal. Typically, “heat-shocked” cells need to be heated twice as long as “nonheat-shocked” cells in order to achieve the same extent of lethality (Farber and Brown, 1990). This phenomenon of a heat shock response and induced thermotolerance is of substantial practical importance to food processors for products normally heated at temperatures below 65°C. Thermotolerance may become a concern in meat products kept on warming trays before a final heating or reheating step, or when there is an interrupted cooking cycle during processing due to equipment failure. Thus,
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increased heat resistance due to prior heat shock must be considered while designing thermal processes to assure the microbiological safety of thermally processed foods. The heat treatment should be sufficient to inactivate the thermotolerant foodborne pathogens and spoilage organisms. Potential known pathogens which have been adapted to stress should be preferentially chosen for challenge tests as opposed to general foodborne pathogens during the simulation of process conditions. The heat shock response and induced thermotolerance has been reported in a wide range of bacteria, including Actinobacillus actinomycetemcomitans (Paju et al., 2000), Agrobacterium tumefaciens (Nakahigashi et al., 1999), Escherichia coli (Tsuchido et al., 1984), Salmonella typhimurium (Mackey and Derrick, 1986, 1990), Salmonella thompson (Mackey and Derrick, 1987), Salmonella enteritidis phage type 4 (Humphrey et al., 1993), Staphylococcus aureus (Hurst et al., 1974), Streptococcus thermophilus (Auffray et al., 1995), Lactococcus lactis (Kilstrup et al., 1997), Listeria monocytogenes Scott A (Fedio and Jackson, 1989; Linton et al., 1990), Leuconostoc eonos (Guzzo et al., 1997), E. coli O157:H7 (Murano and Pierson, 1992; Jorgensen et al., 1996; Juneja et al., 1997), and Yersinia enterocolitica (Shenoy and Murano, 1996). An increase in heat resistance of spores following heat shock has also been reported in spore-forming organisms such as Bacillus anthracis, B. cereus, B. megaterium, B. subtilis (Streips and Polio, 1985), B. stearothermophilus (Etoa and Michiels, 1988; Beaman et al., 1988), Clostridium acetobutylicum (Bahl et al., 1995), C. botulinum (Appleyard and Gaze, 1993), C. perfringens (Heredia et al., 1997, 1998), and C. sporogenes (Alcock, 1994). A direct relationship has been shown to exist between C. perfringens spore heat resistance and the temperature at which the spores are produced (Garcia-Alvarado et al., 1992). In addition to being studied extensively in broth systems, heat shock response and induced thermal tolerance have been shown to have significance in foods (Shenoy and Murano, 1996; Farber and Brown, 1990; Mackey and Derrick, 1987; Juneja et al., 1997). The extent to which cells become more thermotolerant after a heat shock has been found to depend on the physiological state of the cell (Lindquist, 1986; Linton et al., 1992), the time/temperature combination used in the sublethal heat treatment (Bunning et al., 1990; Farber and Brown, 1990), the media (Mackey and Derrick, 1990) and the method of recovery (Knabel et al., 1990; Linton et al., 1990). Synthesis of Heat-Shock Proteins Heat shock triggers a physiological response that leads to the synthesis of a specific set of proteins known as heat-shock proteins (HSPs) (Schlesinger, 1990; Lindquist, 1986). The increased synthesis of these HSPs usually occurs 5 to 60 min after heat shock and declines with the onset of normal protein synthesis 60 to 90 min after return to normal temperatures (Watson, 1990). These HSPs are highly conserved among prokaryotic and eukaryotic organisms (Lindquist, 1986) and increase the potential of bacteria to withstand severe subsequent stresses. HSPs may enhance the survival of pathogens in foods during exposure to high temperatures. Although the scientific literature provides some evidence regarding the cause and effect relationship
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between the synthesis of HSPs and the induced thermotolerance response, this evidence is largely indirect and insufficient. Researchers have suggested that HSPs are not necessarily the major contributory agents in the development of thermotolerance but are required for recovery from heat stress (Susek and Lindquist, 1989; Barnes et al., 1990; Smith and Yaffe, 1991). Their apparent role is to protect the cells against heat damage and to help the cells return to their normal physiological state following the stressful event. Schlesinger (1990) and Sanchez and Lindquist (1990) suggested that the role of HSPs in thermotolerance may be to act as chaperones to remove denatured proteins. Molecular chaperones constitute 15 to 20% of the total cellular protein in response to elevated (46°C) temperatures (Arsene et al., 2000). The primary function of classic chaperones, E. coli DnaK (HSP 70), DnaJ, GrpE, GroEL (HSP60), and GroES is to bind to and stabilize polypeptides already present in cells, modulate protein folding pathways to prevent misfolding or aggregation of proteins, and promote refolding and proper assembly (Georgopoulos and Welch, 1993). Some E. coli HSPs are proteases that are able to digest irreversibly damaged polypeptides for removal and assist in nucleic acid synthesis, cell division, and motility (Morris, 1993). In E. coli, regulation of stress responses through the transcriptional control of alternate sigma factors encoded by rpoS and rpoH in response to general stress and heat, respectively, has been studied in greater detail (HenggeAronis, 1993; Yura et al., 1984). The sigma factors direct RNA polymerase in the transcription of select subsets of genes including those associated with virulence (Aldsworth et al., 1998). In E. coli, about 17 heat-shock proteins are found which are diverse with respect to size, net charge, and levels or extent of induction in response to heat shock (Neidhardt et al., 1984). Ten of these Hsps are the products of known genes and have been characterized (Neidhardt and VanBogelen, 1987). It has been reported that two stress proteins of 60 and 69 kDa correspond to the GroEL and DnaK proteins of E. coli (Georgopoulos et al., 1990). The 60 kDa (GroEL) protein is involved in the morphogenesis of coliphage and is also essential for E. coli growth (Friedman et al., 1984; Fayet et al., 1989). GroEL has been shown to protect RNA polymerase (RNAP) from heat inactivation and “resurrect” heatinactivated, aggregated RNAP (Georgopoulos et al., 1994). Juneja et al. (1997) observed that the level of the 60 kDa GroEL protein in E. coli O157:H7 increased significantly following heat shock (46C/15 min), whereas the increase in the level of the 69-kDa DnaK protein was not as high. Murano and Pierson (1992) described stress proteins with molecular masses of 71 and 84 kDa in E. coli O157:H7 and found that the concentration of these proteins in heat-shocked cells depended upon the gaseous growth atmosphere prior to heat shock. Xavier and Ingham (1997) found an overexpression of seven proteins in S. enteritidis with apparent molecular weights of 14, 16, 21, 23, 60, 75, and 89 kDa, respectively, after application of a heat-shock treatment of 42°C for 60 min. Mackey and Derrick (1990) reported the induction of four major S. typhimurium heat-shock proteins with approximate molecular weights of 25, 64, 72, and 83 kDa. The authors thought that these heat-shock proteins observed in Salmonella spp. may be homologous with highly conserved heat-shock proteins such as DnaK and GroEL
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of E. coli. These proteins are examples of the extensively studied Hsp70 and Cpn60 groups (Landry et al., 1992). Morgan et al. (1986) observed that S. typhimurium produces certain stress proteins during anaerobic growth, which are similar in size to proteins produced as a result of heat shock. One of these proteins has a molecular weight of 85,000 Da and is produced in S. typhimurium also as a result of heat stress, as well as anaerobic stress. Anaerobic growth of E. coli as well as heat shocking of aerobically grown cells leads to the production of proteins of 84,000 and 71,000 Da (Murano and Pierson, 1992). Western blot analysis using monoclonal antibodies specific for the sigma subunit indicated that this protein was immunologically similar to the sigma 32 subunit of RNA polymerase, which is considered to be a stress protein (Grossman et al., 1987). Factors Affecting Heat-Shock Response According to Lindquist (1986), the persistence of heat shock-induced thermotolerance appears to be a function of many factors including the temperature at which heat shocking is done, previous incubation temperature of the cell, and the metabolic state of the cell. As temperature fluctuations are a common occurrence in food processing environments as well as during transportation, distribution, and storage or handling in supermarkets or by consumers, bacterial cells in meats are likely to encounter temperature shifts. Therefore, temperature plays a significant role and must be considered when determining the persistence of heat shock-induced thermotolerance. Juneja et al. (1997) suggested that guidelines be established so that prophylactic measures are adopted and environmental stresses (such as heat) do not render bacteria able to survive thermal processing procedures better than normally would be considered adequate. The time–temperature combination that produces the maximum thermotolerance and the persistence of the induced thermotolerance response after heat shock has been investigated. Lindquist (1986) has pointed out that for mesophilic bacteria, temperatures between 45 and 50°C are optimum for development of the heat-shock response and the demonstration of the increased thermotolerance. In a study by Farber and Brown (1990), where a sausage mix inoculated with 7 log10 CFU/g of L. monocytogenes was heat shocked at 48°C for 30, 60, and 120 min before being heated to a final temperature of 64°C, cells heat shocked for 30 or 60 min did not show a significant increase in thermotolerance. However, cells preheated for 120 min exhibited increased thermal tolerance when compared to non-heat-shocked cells. A 2.4-fold increase in D-value at 64°C was observed for heat-shocked compared to non-heat-shocked cells. After heat shocking, inoculated meat samples were held at 4°C for 24 h and then the bacteria were tested for heat resistance at 64°C. The authors reported that the heat-shocked cells retained their increased heat resistance. Mackey and Derrick (1986) increased the heat resistance of S. typhimurium grown at 37°C in tryptone soya broth by exposing the cultures to sublethal heat shock at 42, 45, or 48°C before exposing the organism to 55°C for 25 min. In that
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study, the heat resistance increased rapidly following the temperature increase and the degree of resistance as well as the rapidity of its onset increased with increasing temperature of heat shock, reaching near maximum levels within 30 min and persisting for 10 h. Pre-incubating cells at 48°C for 30 min increased their resistance to a range of lethal temperatures (52 to 59°C). The authors demonstrated a similar effect with S. thompson when the organism was preheated at 48°C and then heated to 54 or 60°C in tryptone soya broth, liquid whole egg, 10% (w/v) or 40% (w/v) reconstituted dried milk, or minced beef (Mackey and Derrick, 1987). Shenoy and Murano (1996) heat shocked Yersinia enterocolitica in brain heart infusion broth at 45°C for 5, 10, 15, 30, 45, or 60 min and reported that 60 min consistently resulted in an increased number of survivors following a subsequent treatment at 55 or 60°C when compared with non-heat-shocked cells. In a study by Pagan et al. (1997), the thermotolerance of L. monocytogenes at 65°C increased with the increase in the duration of heat shock for up to 120 min, regardless of the heat shock temperature of 40, 43, or 46°C. In contrast to these studies demonstrating a parallel increase in heat resistance with the increase in the time of heat shocking, Murano and Pierson (1992) heat shocked E. coli O157:H7 in trypticase soy broth (TSB) at 30, 34, 42, or 45°C for 0, 5, 10, or 15 min and reported that heat shocking at 42°C for 5 min resulted in the greatest log number of survivors to the subsequent heat treatment at 55°C compared to non-heat-shocked controls, regardless of the growth atmosphere. In another study, Linton et al. (1990) heat shocked log phase cells of L. monocytogenes Scott A in trypticase soy broth supplemented with 0.6% yeast extract (TSYE) at 40, 44, and 48°C for 3, 10, and 20 min, followed by heating at 55°C for 50 min. The optimum heat shock condition for increasing the heat resistance was 48°C for 10 min where D-values at 55°C increased 2.3-fold in nonselective agar (TSYE) and 1.6-fold in selective agar (McBride Listeria). Cells which were heat shocked at 48°C for 10 min were consistently more resistant to heating at 50, 55, 60, and 65°C as compared with non-heat-shocked cells. Although D-values increased due to heat shocking, z-values were not significantly affected regardless of the plating medium or heat shock. Juneja et al. (1997), used the submerged coil heating apparatus to determine the effect of heat shocking E. coli O157:H7, inoculated in a model beef gravy, on the persistence of the thermotolerance at 4, 15, and 28°C after heat shock. When beef gravy samples inoculated with a cell culture suspension of a four strain cocktail of E. coli O157:H7 were subjected to sublethal heating at 46°C for 15 to 30 min, followed by cooking to a final internal temperature of 60°C, the organism survived longer than non-heat-shocked cells and the “time to a 4-D (time to inactivate 99.99% of the population) inactivation” value at 60°C increased 1.56-fold. In this study, a linear decline in the log number of survivors with time was observed. The induction of thermotolerance by heat shock was maintained for at least 48 h at 4, 15, or 28°C (Table 2.1). However, when a similar study was conducted in bags of ground beef heated using a water bath, the primary thermotolerance response of E. coli O157:H7 switched to non-linear inactivation kinetics resulting in the presence of a shoulder (Juneja et al. 1997). The T4D values at 60°C increased 1.50-fold in ground beef.
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TABLE 2.1 The Effect of Prior Exposure of Escherichia coli 0157:H7 in Beef Gravy to 46°C Followed by Storage at 4, 15, or 28°C for 6 to 48 h on the Microorganism’s Time to a 4-D Inactivation (T4D)ab at 60°C Storage Time after Heating (h) 6 14 24 30 48
T4D (min) at Temperatures (°C)c 4
15
28
3.91 3.14 3.06 3.48 3.15
3.44 3.11 3.17 3.01 3.29
3.78 3.39 4.07 3.67 3.24
a
The T4D of non-heat-shocked cells was 2.38 ± 0.2 and was not significantly altered (p < 0.05) after storage at 4, 15, or 28°C for up to 48 h. b The T 4D of cells immediately after heat shocking was 3.73 ± 0.22 min. c Mean of two replications, each performed in duplicate. (Adapted from Juneja, V.K. et al., J. Appl. Microbiol., 84, 677, 1997.)
Unlike the beef gravy, it was interesting to note that E. coli O157:H7 cells in beef lost their thermotolerance after 14 h at 4°C and after 24 h in beef held at 15 or 28°C. Bunning et al. (1990) heat shocked stationary phase cells of L. monocytogenes grown at 35°C (control), at 42, 48, and 52°C for 5 to 60 min prior to heating at 57.8°C. Although heat shocking at 42 to 48°C for 5 to 60 min increased D-values at 57.8°C by 1.1- to1.4-fold, these data were not statistically different from nonheat-shocked cells. When similar experiments were conducted with S. typhimurium, D-values increased by 1.1- to 3.0-fold and were significantly different from those of non-heat-shocked cells. When L. monocytogenes cells were held at 42°C, thermotolerance remained at a maximum level for at least 4 h. However, in preheated cells incubated at 35°C the increased thermal tolerance lasted less than 1 h. Heat stress interacts with growth atmosphere in increasing the heat resistance of E. coli O157:H7. In the previously mentioned study by Murano and Pierson (1992), when log phase cells of E. coli O157:H7 grown either aerobically or anaerobically in trypticase soy broth (TSB) at 30°C were subjected to a heat shocking at 42°C for 5, 10, or 15 min before final heating at 55°C, D-values increased by more than 2-fold for aerobically grown cells, and 1.5-fold when grown under anaerobic conditions. Interestingly, the D-values at 55°C of anaerobically grown non-heatshocked control were significantly higher than those of aerobically grown controls. It has been reported that anaerobiosis is considered a form of stress to bacterial cells. Jorgensen et al. (1996) used the submerged coil apparatus, set at 58°C, to assess the effect of growth temperature and post heat shock incubation temperature on heatshock-induced thermotolerance and the persistence of this thermotolerance in L. monocytogenes. The authors reported that cells grown at 10 or 30°C showed no
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TABLE 2.2 Influence of Incubation Temperature and Time after Heat Shock on the Inactivation of Listeria monocytogenes Cells Grown at 4, 10, or 30°C Growth Temperature of Cells, °C
Heat Shock (30 min at 46°C)
Temperature of Post-Heat Shock Incubation (°C)
Time after Heat Shock (h)
Time to 4 log cfu/ml Reductions (min)
4 10 30 4 10 30 4 10 4 10 30
No No No Yes Yes Yes Yes Yes Yes Yes Yes
— — — — — — 4 10 30 30 30
— — — 0 0 0 48 24 4 4 4
3.4 5.4 5.1 19.8 15.0 14.8 7.1 6.6 7.6 6.5 7.1
(Adapted from Jorgensen, F. et al., J. Appl. Bacteriol., 79, 274, 1996.)
differences in thermotolerance but were significantly (p < 0.001) more heat resistant (1.5-fold) than cells grown at 4°C (Table 2.2). In this study, exposing cells grown at 10 and 30°C to a heat shock resulted in similar increases in thermotolerance, but this increase was significantly (p < 0.001) higher when cells were grown at 4°C prior to the heat shock. The effect of growth temperature prior to inactivation had negligible effects on the persistence of heat-shock-induced thermotolerance. For example, cells grown at 4, 10, or 30°C showed the same amount of reduction when held at 30°C after the heat shock. The degree to which E. coli O157:H7 heat-shocked and non-heat-shocked cells are injured following a heat process and the ability of injured cells to repair themselves under aerobic and anaerobic conditions have been described by Murano and Pierson (1993). It is known that bacteria encounter stress due to both excess oxygen and oxygen deprivation (Potter et al., 2000). In the prior study, not only was the D-value at 55°C of heat shocked cells (42°C/5 min) significantly increased, but the number of injured cells was also higher in heat-shocked cells than in controls (Murano and Pierson, 1993). Furthermore, when cells were recovered under anaerobic conditions, a higher recovery of injured cells was observed and thus a significantly higher D-value as compared with cells recovered aerobically. Interestingly, this phenomenon was observed regardless of whether the cells were previously heat shocked or not. This is probably attributable to the spontaneous formation of toxic oxygen radicals in aerobic media, which heated cells are unable to deactivate due to the heat inactivation of detoxifying enzymes like catalase and superoxide dismutase. Since anaerobic storage is a practice which is prevalent in the food industry for shelf-life extension of processed meats, the microbiological safety of such foods should be of concern because of the enhanced recovery of injured pathogens following
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heat treatment. The concern is further magnified if the cells are exposed to heat stress before heat treatment as the recovery can be enhanced even more. Linton et al. (1992) assessed the effect of recovery medium in conjunction with atmosphere (aerobic vs. anaerobic) during recovery on the heat resistance of L. monocytogenes. These researchers heat shocked log phase cells of L. monocytogenes Scott A in TSB supplemented with 0.6% (w/v) yeast extract (TSYE) at 48°C for 10 min, followed by heating at 55°C for up to 50 min. D-values at 55°C for heat-shocked cells were 2.1- fold higher than for non-heat-shocked cells on nonselective agar (TSYE) incubated aerobically and similarly 2.2- fold higher for cells enumerated anaerobically on TSYE agar. On selective medium (McBride ListeriaML), the values were 1.4-fold higher as compared with non-heat-shocked cells. Interestingly, no growth was observed on ML agar incubated anaerobically. Fedio and Jackson (1989) exposed stationary-phase cells of L. monocytogenes Scott A to a preheating treatment of 48°C for 1 h in TSYE broth followed by heating at 60°C for 20 min. Preheating rendered the pathogen more resistant, and a 4 log10 higher number of cells were recovered as compared to non-heat-shocked cells regardless of the recovery medium (selective or nonselective). Increases in D-values (up to 22% compared to the control) for S. enteritidis following heat shock (42°C for 60 min) were reported by Xavier and Ingham (1997). This study suggested that: (1) short-term temperature abuse of foods containing S. enteritidis may render the cells more resistant to subsequent heat treatments; (2) anaerobic microenvironments may enhance survival of heat-stressed cells (i.e., increases in D-values up to 28% compared to the aerobic value); and (3) heat shock results in the overexpression of proteins that may be related to increased thermotolerance. Heat-shock-induced thermal resistance conditions may be encountered in minimally processed, cook-chill processed foods of extended durability such as sousvide foods, in which there is a current increased interest. Slow heating rate/long come-up times and low heating temperatures employed in the production of sousvide cooked foods expose the microbial cells to conditions similar to heat shock, with the possibility of rendering these cells more thermal resistant. Stephens et al. (1994) and Kim et al. (1994) have shown that slowly raising the cooking temperature enhanced the heat resistance of L. monocytogenes in broth and pork, respectively. Hansen and Knochel (1996) found no significant difference between slow (0.3 to 0.6°C/min) and rapid (>10°C/min) heating and the heat resistance of L. monocytogenes in low pH (<5.8) sous-vide cooked beef prepared at a mild processing temperature. However, the latter authors did observe an increase in the heat resistance of L. monocytogenes in higher pH (6.2) sous-vide beef. Tsuchido et al. (1982 and 1984) increased the thermotolerance of E. coli by raising the temperature of the cell suspension from 0 to 50°C at various rates prior to holding at 50°C. Also, Thompson et al. (1979) increased the thermotolerance of S. typhimurium in beef under realistic conditions of constantly rising temperature. Subsequently, Mackey and Derrick (1987) reported that the heat resistance of S. typhimurium, measured as survival following a final heating at 55°C for 25 min, increased progressively as cells were heated during linearly rising temperatures. In that study, cells were heated at a rate of 0.6 or 10°C per min from 20 to 55°C, and then subjected to a heat challenge at 55°C for 25 min. The authors reported that the
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extent of induced thermotolerance was inversely related to the rate of heating, i.e., the slower the temperature rise, the greater the increase in resistance. Quintavala and Campanini (1991) determined the heat resistance of L. monocytogenes 5S heated at 60, 63, and 66°C in a meat emulsion at a rate of 5°C/min compared to instantaneous heating. D-values of cells heated slowly were two-fold higher than the cells heated instantaneously at all heating temperatures. Cell Membrane Adaptations Cellular targets for heat damage are ribosomes, nucleic acids, enzymes, and proteins (Abee and Wouters, 1999). Mild heat treatment can lead to modification of the cell membrane by increasing the saturation and length of the fatty acids in order to maintain optimal fluidity of the membrane as well as activity of intrinsic proteins (Russell and Fukanaga, 1990). In a study on the physiological state of cell membranes from Gram-negative bacteria, the total saturated fatty acids (SFA) and total unsaturated fatty acids (UFA) were highly influenced by temperature (Dubois-Brissonnet et al., 2000). When the temperature was increased from 15 to 40°C, SFA increased from 25 to 39%, whereas UFA decreased from 66.5 to 51% (Dubois-Brissonnet et al., 2000). An increase in unsaturation would be expected to contribute to membrane fluidity, especially at lower temperatures (Russell et al., 1995). Cross Protection Stress-adapted bacteria are capable of resisting similar (homologous) or different (heterologous) stresses. Termed “cross protection,” exposure to one stress alters resistance to another. This cross protection has been shown to be mediated by the rpoS gene, alluded to earlier. For example, heat shocking renders bacteria more resistant to treatments other than heat. Wang and Doyle (1998) reported that sublethal heat treatment of E. coli O157:H7 cells substantially increased their tolerance to acidity. Lou and Yousef (1996) examined the effect of sublethal heat (45°C/1 h) on the resistance of exponential phase cultures of L. monocytogenes to certain environmental stresses and found that this greatly increased resistance of the pathogen to normally lethal doses of hydrogen peroxide, ethanol and NaCl, etc. As a consequence of stress-induced cross-protection, Lou and Yousef (1996) introduced the “stress hardening” concept as a component of the “hurdle” concept, stating that stress hardening may counterbalance the benefits of the hurdle approach. In L. monocytogenes, stress induced with heat does not lead to acid tolerance, but cells induced with low pH do become more tolerant of heat, salt concentration, and antimicrobial peptide (Hill and Gahan, 2000). All of the different cross protection adaptations of specific pathogens of concern must be taken into account when assessing the microbial safety of different combinations of minimal food processing technologies. Komatsu et al. (1990) showed that exposure of yeast cells to a heat shock conferred protection against freezing in liquid nitrogen. Additionally, it was found that carbon starvation in E. coli elicits an essential need for dnaK expression in order to acquire heat and oxidation resistance
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(Rockabrand et al., 1998). Further research is needed to determine whether stresses elicit universal or specific adaptations in the different foodborne pathogens. Management Strategies Strategies for control of microorganisms are effective when they overcome, temporarily or permanently, the various homeostatic reactions that microorganisms have evolved to resist stress (Gould et al., 1995). Researchers have reported that food handling conditions should be optimized for maximum effectiveness of cooking treatments. For example, storage of foods at low temperatures may affect the response of pathogens such as E. coli O157:H7 to sublethal stresses. E. coli O157:H7 has been reported to be resistant to freezing in ground beef (Pandhye and Doyle, 1992) and chicken meat (Conner and Hall, 1996). Jackson et al. (1996) reported that the heat resistance of E coli O157:H7 in a nutrient medium and in ground beef patties was influenced by storage and holding temperatures. Cultures stored frozen (–18°C) without holding at elevated temperatures had greater heat resistance than those stored under refrigeration (3°C) or at 15°C, perhaps due to physiological changes within the bacterial cell as a result of freezing (Jackson et al., 1996). Another study (Katsui et al., 1982) showed that the exposure of non-heat-shocked E. coli to 0°C before heating significantly increased the heat sensitivity of the exposed cells. Juneja et al. (1997) reported that the heat resistance of non-heat-shocked cells of E. coli O157:H7 inoculated in ground beef was not altered after storage at 4°C for 48 h. The heat-shock response and exhibition of increased thermotolerance is rapidly lost upon chilling and rewarming of cells. Williams and Ingham (1997) refuted the hypothesis that short-term temperature abuse significantly increased the heat resistance of E. coli O157:H7 in ground beef.
HIGH HYDROSTATIC PRESSURE Elevated pressure manifests a variety of detrimental effects on microbial physiology and viability, including inhibition of protein and DNA synthesis, membrane-associated processes, and disruption of macromolecular quaternary structure (Somero, 1992; Yayanos and Pollard, 1969). Growth of microorganisms is generally inhibited at pressures in the range of 20 to 130 MPa, whereas higher pressures between 130 and 800 MPa could result in cell death (Abee and Wouters, 1999). Welch et al. (1993) found that the stress responses of E. coli to high hydrostatic pressure and cold temperature share some common features. In that study, the authors monitored the proteins induced by high pressure in E. coli by analyzing the biochemical and physiological responses to elevated pressure. They reported that exposure of E. coli to high hydrostatsic pressure induces a unique stress response which results in high levels of cold-shock proteins (CSPs) and heat-shock proteins (HSPs), as well as other proteins which appear only in response to high pressure. Increased pressure has been shown to result in enhancement of proteolytic activity by extracellular proteases and fatty acid desaturases in membrane systems (Lanciotti et al., 1997). Pressure-induced rates of elevated protein synthesis may result from the induction of ribosomes and the state of the ribosome at the time of exposure to high pressure.
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Both high pressure and high temperature can destabilize the quarternary structure of proteins (Jaenicke, 1981). It is presumed that an increased proportion of dissociated subunits could induce a δ32 factor-dependent heat-shock response (Craig and Gross, 1991). Alternatively, high pressure may affect the phosphorylation state or ATPase activity of the HSP, DnaK, which in turn could also modulate the heat shock response (McCarty and Walker, 1991). It would be interesting to examine cross tolerances to determine if a bacterial stress response to high pressure or cold temperature confers resistance to the other, and whether such cross-protective resistance would be relevant to the cold temperature storage or the hydrostatic pressure processing of foods. The food industry requires a better understanding of the kinetics and mechanism of pressure inactivation before adoption of pressure-based preservation processes. E. coli can acquire high levels of resistance to pressure killing by spontaneous mutation (Hauben et al., 1997). The authors used alternating cycles of exposure to high pressure and outgrowth of surviving populations to select for highly pressure-resistant mutants of E. coli MG1655. Three barotolerant mutants (LMM1010, LMM1020 and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42°C. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain decreased from 15% at 220 MPa to <0.1% at 700 MPa. Two of the three mutants (LMM1020 and LMM1030) also exhibited higher heat resistance, expressed as increased D-values at 58 and 60°C, and lower z-values compared to those for the parent strain. Interestingly, the ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37°C, suggesting that resistance to pressure inactivation in these mutants is unrelated to barotolerant growth. The generation of increased pressure-resistant mutants questions the safety of high pressure food processing, and may have significant implications for the successful application of high pressure processing in food preservation. Another investigation which examined the potential for high pressure-resistant mutants of E. coli to survive high pressure treatment (500 MPa) of fruit juices and low-pH buffers also indicated increased pressure resistance in mutants when compared to the parent strain (Garcia-Graells et al., 1998). However, surviving population densities declined considerably during subsequent storage of the pressure-treated juices at 8°C. This finding suggests exposure to high pressure sublethally injures the bacterial cells, thereby increasing sensitivity to low pH. It can be argued that ultrahigh pressure can inactivate microorganisms without compromising the quality of the food (Bower and Daeschel, 1999). However, sporeformers are known to exhibit enhanced pressure resistance; therefore it is recommended that high pressure technologies be used in combination with other treatments to be truly effective (Bower and Daeschel, 1999).
DEHYDRATION RESTRICTING WATER ACTIVITY Water activity (aw) is a measure of the free unbound water molecules available for metabolic reactions. Microorganisms are capable of growth within a very limited
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TABLE 2.3 Effect of NaCl Treatment during Growth and Heating on the Heat Resistance of Listeria monocytogenes at 60°C Treatment to NaCl Control Up-shock
Adaptation
NaCl Content (moles/ml) Growth Medium
Heating Menstruum
Time to 4-D Inactivation
0.09 0.09 0.09 0.09 0.50 1.00 1.00 1.50 1.50
0.09 0.50 1.00 1.50 0.50 1.00 1.00 1.50 0.09
1.6 2.0 4.6 13.2 2.5 7.4 7.4 38.1 3.8
(Adapted from Jorgensen, F. et al. J. Appl. Bacteriol., 79, 274, 1995.)
range of aw values specific for that microbe. Solutes such as NaCl or sugars are frequently used to lower or control aw levels to prevent pathogen growth in processed foods. Another available method is the physical freeze-drying of foods in combination with the use of preservatives. Jorgensen et al. (1995) used the submerged coil heating apparatus to determine the effect of osmotic up-shock and down-shock, and osmotic adaptation using different levels of NaCl on the corresponding changes in thermotolerance of L. monocytogenes. In this study, subjecting cells to an osmotic down-shift (1.5 to 0.09 mol/ml) caused a rapid loss of thermotolerance rendering cells ten-fold more heat sensitive than cells grown and heated in TPB containing 1.5 mol/ml NaCl (Table 2.3). Subjecting cells grown in media containing 0.9 mol/ml NaCl to a short osmotic up-shock in media containing 0.5, 1.0 or 1.5 mol/ml NaCl resulted in 1.3, 2.5 and 8-fold increases in thermotolerance, respectively. When cells were allowed to adapt to high salinities, an additional two- to three-fold increase in thermotolerance occurred compared to cells subjected to an osmotic up-shock at the equivalent level of NaCl. Thus, varying degrees of physical dehydration would lead to enhanced thermotolerance of the foodborne pathogen. The increased thermotolerance observed during the extended exposure to high salinities might be associated with the degree to which the cells have undergone deplasmolysis and accumulated compatible solutes, i.e., the concentration and composition of intracellular solutes. According to Piper (1993), increased thermotolerance could be a result of increased structurization of the intracellular water. This mechanism could be linked with the enhanced thermostability of ribosomal contents known to occur by both osmotic dehydration and heat shock in L. monocytogenes (Stephens and Jones, 1993). Foods contain a broad range of osmoprotectants, such as glycine betaine and carnitine, which L. monocytogenes can scavenge and use to regulate osmotic stress
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(Gutierrez et al., 1995; Smith, 1996). It may very well be possible to control growth of pathogens in foods by creating environments free from osmoprotectants provided that the physical act of dehydration does not cross-protect the microorganism against other types of food processing physical stresses.
FREEZING The increased use of freezing temperatures to minimize growth of spoilage organisms and to control pathogens has resulted in increased recent attention to microbial adaptations to freeze conditions. Bacterial adaptation to low temperature (cold shock) is thought to involve modification of membrane lipid composition for the purpose of maintaining optimum membrane fluidity in a process called homeoviscous adaptation (Hazel and Williams, 1990). Survival of the foodborne pathogen L. monocytogenes in low temperature environments and high salt concentrations is attributed to the accumulation of the osmoprotectants glycine betaine and carnitine (Sleator et al., 2001). Low temperature growth requires, in addition to membrane fluidity, mechanisms for regulating the uptake or synthesis of solutes and the maintenance of macromolecular structural integrity of ribosomes and other components important for gene expression and metabolism (Wouters et al., 2000). Proteins (7 kDa) produced in response to temperature downshock or sudden decrease in temperature are known as cold-shock proteins (CSPs). These proteins share greater than 45% amino acid similarity in a variety of foodborne pathogens including E. coli (Goldstein et al., 1990), B. subtilis (Willimsky et al., 1992), B. cereus (Mayr et al., 1996), S. enteritidis (Jeffreys et al., 1998), and S. typhimurium (Craig et al., 1998). In order to examine the adaptive response to cold shock in Vibrio vulnificus, a culture of the microorganism was shifted from 35 to 6°C with the resultant transition of the bacterium to a viable, but non-culturable state (Bryan et al., 1999). Cultures which were first adapted to 15°C prior to the 6°C downshift cold shock remained viable and culturable (Bryan et al., 1999). The exposure to 15°C was found to be necessary for the cold adaptation. Additionally, iron was found to be necessary for the stress adaptation to chill temperatures as addition of the iron chelator, 2,2′dipyridyl, to the culture prior to cold stress resulted in a decrease in culture viability by 2 log10 following cold adaptation (Bryan et al., 1999). Goldstein et al. (1990) reported that when E. coli cells grown at 37°C were frozen and thawed following preincubation at 10°C for 6 h, there was a 70-fold increase in survival compared to frozen and thawed cells that were not preincubated at 10°C. Willimsky et al. (1992) replaced a functional cspB gene on the B. subtilis chromosome with an interrupted copy of the gene, and then compared the effects of freezing on cell viability of the parent and mutant strain. In this study, freezing at –80°C for 24 h after incubation at 37°C resulted in the survival of 27% of the parent cells and only 2% of the mutant cells. However, preincubation at 10°C for 2 h prior to freezing increased the survival of both the parent and mutant, thus partially compensating for the lack of CspB in the mutant strain. These results suggest that bacteria that are cold shocked, or adapted to cold temperatures, prior to freezing, are more likely to survive.
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TABLE 2.4 Cold Shock Effects on Thermal Inactivation at 60°C of Listeria Strains Grown to Stationary Phase at 37°C Strain D-values (min)
L. innocua
L. monocytogenes (Scott A)
L. monocytogenes (V7)
Control Cold shock (0°C/3 h) % D-value decrease
1.44 ± 0.06 1.08 ± 0.03 25
1.27 ± 0.09 0.76 ± 0.05 40
1.31 ± 0.03 0.88 ± 0.02 33
(Adapted from Miller, A.J. and Eblen, B.S., Proc. 43rd Int. Congr. Meat Sci. Technol., Auckland, NZ, 1997.)
TABLE 2.5 Post-Cold-Shock Thermotolerance, Expressed as D-Values, of Listeria monocytogenes Grown at 37°C to Different Growth Phases Growth Phase of 37°C Cells Lag Exponential Stationary
Cold Shock Temperature Control
15°C
0°C
0.83 ± 0.05 0.79 ± 0.043 1.27 ± 0.09
0.60 ± 0.08 0.74 ± 0.04 0.75 ± 0.05
0.58 ± 0.09 0.75 ± 0.05 0.74 ± 0.00
(Adapted from Miller, A.J. and Eblen, B.S., Proc. 43rd Int. Congr. Meat Sci. Technol., Auckland, NZ, 1997.)
In a study by Miller and Eblen (1997), the submerged coil heating apparatus was used to determine if L. monocytogenes is more vulnerable to heating after a cold shock. In the model system, cultures were cold shocked by a temperature down-shift from 37 to 15 or 0°C for 0, 1, and 3 h. Cold-shocked and control samples were then evaluated for thermal resistance at 60°C. Heated samples were collected in 1 ml portions, plated onto a non-selective medium (brain–heart infusion agar) to allow recovery of both heat-injured and non-injured cells, then enumerated after a 36 h incubation at 37°C. The results indicated that the cells grown at 37°C to stationary phase, cold shocked at 0°C for 3 h, then heated at 60°C, exhibited lower D-values as compared to control cells that were not cold shocked (Table 2.4). The decrease in D-values at 60°C ranged from 25 to 40% for two L. monocytogenes strains and a strain of L. innocua. In a second experimental series by Miller and Eblen (1997), the effect of cold shock on thermal resistance (D60-values) of cells grown at 37°C to lag, exponential, or stationary phase was determined (Table 2.5). Stationary cells were over 50% more thermally resistant (D60 = 1.27 min), compared to lag and exponential cells, which had D60-values of 0.83 and 0.79, respectively. When these cells were cold shocked
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at 15 or 0°C prior to heating at 60°C, D-values were lowered by 42, 30, and 8%, compared to non-shocked controls for stationary, lag, and exponential cells, respectively. The authors pointed out that the maximum effect was in stationary phase cells, which most closely simulates cells contaminating food.
PULSED ELECTRIC FIELD Pulsed electric field is a technique that uses short electric field pulses (ns–ms) to permeabilize microbial membranes and can lead to death from an inability to maintain pH gradients necessary for cellular functions (Sale and Hamilton, 1967). This process is effective in creating pores in the cytoplasmic membrane dependent upon the intensity of the electric field and the number of pulses applied (Castro et al., 1993). The ability to inactivate L. monocytogenes has been measured, along with loss of cellular membrane integrity by leakage of UV absorbing materials into culture supernatants (Russell et al., 2000). Pulsed electric field may have limited application with regard to spore-formers; it may be effective against non-spore-formers when combined with other physical or chemical treatments. Cross protection is still being evaluated with this novel technology, as are the detrimental effects on food products.
IRRADIATION ULTRAVIOLET IRRADIATION Some physical preservation treatments may be best when applied in combination with other technologies. Ultraviolet irradiation (254 nm) can cause cumulative damage to microbial DNA (Bower and Daeschel, 1999). Sublethal UV irradiation leads to the induction of numerous proteins as well as increased protection against heat (Duwat et al., 2000). Strains of E. coli have been found to become UV resistant. Although the methodology is effective in decreasing cell numbers, it does not result in complete sterilization and therefore cannot be recommended as a definitive process to sanitize foods by itself (Bower and Daeschel, 1999).
GAMMA IRRADIATION It is known that Gram-negative bacteria are more sensitive to gamma irradiation than Gram-positive bacteria, such as lactobacilli (Tiwari and Maxcy, 1971). Lactobacillus sake exhibits gamma irradiation resistance with enhanced effects under nitrogen packaging (Bower and Daeschel, 1999). Medium doses of gamma irradiation (1.0 to 10.0 kGy or 100 to 1000 krad) reduced or eliminated non-spore-forming pathogens (Tarkowski et al., 1984). Ionizing radiation is known to damage pathogen DNA. At temperatures above freezing, cellular inactivation by DNA disruption and production of hydroxyl radicals occurs (Buchanan et al., 1999). At freezing temperatures, DNA damage was the cause of irradiation inactivation and not cellular membrane disruption (Kim and Thayer, 1996). Detrimental effects of ionizing radiation on food products include
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oxidative rancidity of lipids, which can be prevented by vacuum packaging, and a loss of some minor vitamin components (Farkas, 1987). Although bacterial spores are more resistant, synergistic effects of gamma irradiation and heat may be used to control spore-formers since the heat sensitization of irradiated spores is not readily repaired (Gombas and Gomez, 1978). Unfortunately, as with other food preservation methods, there is some indication of an acid (low pH)-induced cross protection against gamma radiation sensitivity in enterohemorrhagic E. coli (Buchanan et al., 1999). It is important to be mindful that the limitations of radiation that may be applied to a particular product are determined by the organoleptic changes that occur (Grant and Patterson, 1991).
CONCLUDING REMARKS Strategies for control of foodborne pathogens include physical microbiocidal treatments such as heat, ionizing radiation, cold, dehydration, high hydrostatic pressure, and pulsed electric field. Heat inactivation of pathogens is the oldest and most effective food-processing technology in use today. Due to the negative impact on the quality of certain foods, such as fruits and vegetables, or minimally processed, refrigerated, ready-to-eat foods, these are subjected to non-thermal cold pasteurization alternatives. As these techniques must be applied in intensities and durations that retain the organoleptic attributes of foods, there is the likelihood that sublethal doses are used on contaminating pathogenic microorganisms. Consequently, the pathogens may be stressed and acquire resistance and increased tolerance to subsequent homologous or heterologous treatments, thereby compromising the microbiological safety of foods. Researchers have provided sufficient evidence to document the stress adaptations and increased survival capabilities of the foodborne pathogens, even though they are transient in duration. Microorganisms have evolved various homeostatic mechanisms in order to withstand the stress imposed by a variety of physically based strategies used for their control. Interference with homeostasis by selective and logical application of physical preservation factors remains an attractive area of future research. Combinations of successive physical food processing technologies may provide the most effective solutions for processing high quality, microbiologically safe foods. Studies aimed at providing insight into the physiological and molecular basis of stress response mechanisms that underlie the physically based control strategies are expected to shed light on the phenomenon termed “stress hardening.” This area continues to challenge the efficiency and efficacy of emerging techniques in ensuring the microbiological safety of refrigerated foods. Researchers can use the available information from gene sequence analyses to identify target genes or encoded proteins perceived to play a role in microbial stress responses. Certainly, an increased understanding of mechanism and regulation of the way in which the “stress adaptation” protective mechanisms are triggered and proceed in response to physical control strategies and other preservation regimes will offer methodical solutions to pathogen control and increase effective design of novel physical intervention technologies. Thus, the knowledge will provide information and leads that are essential in guarding against the pathogens and in the development of microbiologically safe foods. © 2003 by CRC Press LLC
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Neidhardt, F.C. and VanBogelen, R.A. 1987. Heat shock response, in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Eds. F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter, and H.E. Umbarger, Washington, D.C.: Am. Soc. Microbiol., pp. 1334–1345. Pagan, R., S. Codon, and F.J. Sala. 1997. Effects of several factors on the heat-shock-induced thermotolerance of L. monocytogenes, Appl. Environ. Microbiol., 63:3225–3232. Paju, S., F. Goulhen, S. Asikainen, D. Grenier, D. Maryrand, and V.-J. Uitto. 2000. Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation, FEMS Microbiol. Lett., 182:231–235. Pandhye, N.V. and M.P. Doyle. 1992. Escherichia coli O157:H7: epidemiology, pathogenesis, and methods for detection in food, J. Food Prot., 55:55–565. Piper, P.W. 1993. Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae, FEMS Microbiol. Rev., 11:339–356. Potter, L., P. Millington, L. Griffiths, and J. Cole. 2000. Survival of bacteria during oxygen limitation, Int. J. Food Microbiol., 55:11–18. Quintavala, S. and M. Campanini. 1991. Effect of rising temperature on the heat resistance of Listeria monocytogenes in meat emulsion, Lett. Appl. Microbiol., 12:184–187. Rockabrand, D., K. Livers, T. Austin, R. Kaiser, D. Jensen, R. Burgess, and P. Blum. 1998. Roles of DnaK and RpoS in starvation-induced thermotolerance of Escherichia coli, J. Bacteriol., 180:846–854. Russell, N.J., M. Colley, R.K. Simpson, A.J. Trivett, and R.I. Evans. 2000. Mechanism of action of pulsed high electric field (PHEF) on the membranes of food-poisoning bacteria is an “all-or-nothing” effect, Int. J. Food Microbiol., 55:133–136. Russell, N.J., R.I. Evans, P.F. ter Steeg, J. Hellemons, A. Verheul, and T. Abee. 1995. Membranes as a target for stress adaptation, Int. J. Food Microbiol., 28:255–261. Russell, N.J. and N. Fukanaga. 1990. A comparison of thermal adaptation of membrane lipids in psychrophilic and thermophilic bacteria, FEMS Microbiol. Rev., 75:171–182. Sale, A.J.H. and W.A. Hamilton. 1967. Effect of high electric fields on microorganisms, I. Killing of bacteria and yeast, Biochim. Biophys. Acta, 148:781–788. Sanchez, Y. and S.L. Lindquist. 1990. HSPIO4 required for induced thermotolerance, Science, 248:1112–1115. Schlesinger, M.J. 1990. Heat shock proteins, J. Biol. Chem., 265:12111–12114. Shenoy, K. and E.A. Murano. 1996. Effect of heat shock on the thermotolerance and protein composition of Yersinia enterocolitica in brain heart infusion broth and ground beef, J. Food Prot., 59:360–364. Sleator, R.D., J. Wouters, C.G.M. Gahan, T. Abee, and C. Hill. 2001. Analysis of the role of OpuC, an osmolyte transport system, in salt tolerance and virulence potential of Listeria monocytogenes, Appl. Environ. Microbiol., 67:2692–2698. Smith, L.T. 1996. Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces, Appl. Environ. Microbiol., 62:3088–3093. Smith, B.J. and M.P. Yaffe. 1991. Uncoupling thermotolerance from the induction of heat shock proteins, Proc. Natl. Acad. Sci. USA, 88, 11091–11094. Somero, G.N. 1992. Adaptations to high hydrostatic pressure, Annu. Rev. Physiol., 54:557–577. Stephens, P.J. and M.V. Jones. 1993. Reduced ribosomal thermal denaturation in Listeria monocytogenes following osmotic and heat shocks, FEMS Microbiol. Letts., 106:177–182. Stephens, P.J., M.B. Cole and M.V. Jones. 1994. Effect of heating rate on thermal inactivation of Listeria monocytogenes, J. Appl. Bacteriol., 77:702–708. Streips, U.N. and F.W. Polio. 1985. J. Bacteriol., 162, 434.
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Susek, R.E. and S. Lindquist. 1989. HSP26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without demonstrable function, Mol. Cell Biol., 9, 5265–5271. Tarkowski, J.A., R.R. Baumer, and E.H. Kampelmacher. 1984. Low gamma irradiation of raw meat. I. Bacteriological and sensory quality effects in artificially contaminated sample, Int. J. Food Microbiol., 1:13. Thompson, W.S., F.F. Busta, D.R. Thompson, and C.E. Allen. 1979. Inactivation of Salmonellae in autoclaved ground beef exposed to constantly rising temperatures, J. Food Prot., 42:410–415. Tiwari, N.P. and R.B. Maxcy. 1971. Impact of low doses of gamma radiation and storage on the microflora of ground red meat, J. Food Sci., 36:833. Tsuchido, T., M. Takano, and I. Shibasaki. 1984. Effect of temperature-elevating process on the subsequent isothermal death of Escherichia coli K-12, J. Ferm. Tech., 52:788–792. Tsuchido, T., Hayashi, M., M. Takano, and I. Shibasaki. 1982. Alteration of thermal resistance of microorganisms in a non-isothermal heating process, J. Antibact. Antifung. Agents., 10:105–109. Wang, G. and M.P. Doyle. 1998. Heat shock response enhances acid tolerance of Escherichia coli O157:H7, Lett. Appl. Microbiol., 26:31–34. Watson, K. 1990. Microbial stress proteins, Adv. Microbial Physiol., 31:183–223. Welch, T.J., A. Farewell, F.C. Neidhardt, and D.H. Bartlett. 1993. Stress response of Escherichia coli to elevated hydrostatic pressure, J. Bacteriol., 175:7170–7177. Williams, N.C. and S.C. Ingham. 1997. Changes in heat resistance of Escherichia coli O157:H7 following heat shock, J. Food Protect., 60:1128–1131. Willimsky, G., H. Bang, G. Fischer, and M.A. Marahiel. 1992. Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures, J. Bacteriol., 174:6326–6335. Wouters, J.A., F.M. Rombouts, O.P. Kuipers, W.M. DeVos, and T. Abee. 2000. The role of cold-shock proteins in low temperature adaptation of food-related bacteria, System. Appl. Microbiol., 23:165–173. Xavier, I.J. and S.C. Ingham. 1997. Increased D-values for Salmonella enteritidis following heat shock, J. Food Prot., 60:181–184. Yayanos, A.A. and E.C. Pollard. 1969. A study of the effects of hydrostatic pressure on macromolecular synthesis in Escherichia coli, J. Biophys., 9:1464–1482. Yura, T., T. Tobe, K. Ito, and T. Osawa. 1984. Heat shock regulatory gene (htpR) of Escherichia coli is required for growth at high temperature but is dispensable at low temperature, Proc. Natl. Acad. Sci. USA, 81:6803–6807.
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3
Microbial Adaptation to Stresses by Food Preservatives P. Michael Davidson and Mark A. Harrison
CONTENTS Introduction Resistance Mechanisms Traditional Antimicrobials Short Chain Organic Acids Benzoic Acid Sorbic Acid Alkyl Esters of p-Hydroxybenzoic Acid (Parabens) Resistance to Naturally Occurring Antimicrobials Summary References
INTRODUCTION Food preservatives are chemical compounds added directly to food for the purpose of extending shelf life and improving food safety. The group of compounds known as food preservatives includes antioxidants and antibrowning agents in addition to antimicrobials. Therefore, a more precise term for those compounds used to control microorganisms is food antimicrobials. Food antimicrobials may be arbitrarily classified into two groups: traditional or “regulatory approved” and naturally occurring (Davidson, 2001). The former includes acetic acid and acetates, alkyl esters of p-hydroxybenzoic acids (parabens), benzoic acid and benzoates, dimethyl dicarbonate, lactic acid and lactates, nitrites and nitrates, sorbic acid and sorbates and sulfites. The latter includes compounds from microbial, plant and animal sources that are, for the most part, only proposed for use in foods as antimicrobials. A few, including lactoferrin (FDA, 2001), lysozyme (Federal Register, 1998), nisin (21CFR 184.1538), and natamycin (21CFR 172.155) are approved in the United States and certain other countries for use in selected foods.
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Throughout the ages, food antimicrobials have been used primarily to prolong shelf life and preserve quality of foods through inhibition of spoilage microorganisms. In the past 10 to 15 years, antimicrobials have been increasingly relied upon to inhibit or inactivate pathogenic microorganisms in foods. While food antimicrobials have been in use since ancient times, few are used exclusively to control the growth of specific foodborne pathogens. An exception is nitrite, which has been used for hundreds of years to inhibit growth and toxin production of Clostridium botulinum in cured meats in association with salt, ascorbate and erythorbate, and low pH. More recently, other antimicrobials have been applied to foods against foodborne pathogens. For instance, organic acids (e.g., lactic acid, acetic acid) have been employed as spray sanitizers against pathogens on beef carcasses. Organic acid salts (e.g., sodium lactate, sodium diacetate) have been added to processed meats to inactivate pathogens (primarily Listeria monocytogenes) (9CFR 424.21). Finally, nisin and lysozyme are approved for use in pasteurized process cheese as a safeguard against growth and toxin production by C. botulinum. In most instances, the antimicrobial is part of a multiple intervention system that involves the chemical along with environmental (extrinsic) and food related (intrinsic) stresses and processing steps. This has been termed “hurdle technology” or multiple interventions (Leistner, 2000; Leistner and Gorris, 1995). Some food antimicrobials have been used for thousands of years (e.g., sulfites, some organic acids) while most have been added to foods for the purpose of preservation for 100 years or less. Surprisingly, there are few data in the literature concerning developed resistance by microorganisms to these chemical compounds. Considering the length of time these compounds have been in use, that could indicate that resistance development has not been (and therefore is not) a major problem. However, there is concern about use of food antimicrobials for two major reasons. First is the increasing development and spread of therapeutic antibiotic-resistant microorganisms in the environment. Second is the new paradigm of using food antimicrobials as exclusive or primary methods for protection of foods against growth of, or presence of, foodborne pathogens. If traditional and natural food antimicrobials are to be a major part of the control system for inhibiting foodborne pathogens, we need to be well aware of the resistance factors that these microorganisms might possess.
RESISTANCE MECHANISMS Resistance responses of microorganisms to antimicrobials may be classified as either innate or acquired (Russell, 1991). Innate resistance is a chromosomally controlled property associated with the microorganism. Innate resistance is demonstrated by differences in resistance among related genera, species or strains of microorganisms under identical conditions of exposure. Because food antimicrobials are generally broad spectrum, they do not trigger specific microbial responses and, therefore, resistance is most likely due to unspecified reduced uptake controlled primarily by innate characteristics (Russell et al., 1997). Mechanisms may include barriers such as outer membrane of Gram-negative bacteria, teichoic acids of Gram-positive bacteria, efflux or pumping of the compounds and inactivation via enzymes. In food © 2003 by CRC Press LLC
application of antimicrobials, innate resistance may be influenced by environment, food component interactions, processing interactions or presence of antagonistic inhibitors. All of these factors may contribute to a microorganism’s resistance profile. Resistance caused by these factors may be more properly termed “apparent.” Acquired resistance results from genetic changes in the microbial cell through mutation or acquisition of genetic material. For example, resistance to therapeutic antibiotics can include enzymatic inactivation or modification, absence of enzyme or metabolic step, impaired uptake, efflux, modification of target site, bypass of a sensitive step, or overproduction of the target (Russell et al., 1997). Acquired resistance is of greatest concern for use of food antimicrobials. While acquired resistance to antimicrobials is rare (Russell et al., 1997), investigations into the potential for such resistance are of utmost importance to the future use of traditional and naturally occurring antimicrobials
TRADITIONAL ANTIMICROBIALS SHORT CHAIN ORGANIC ACIDS Short chain organic acids, including acetic acid and its salts (acetates and diacetates), lactic acid and lactates, propionic acid and propionates and, to a lesser extent, citric acid and citrates, are commonly utilized in a variety of foods as antimicrobial preservatives or acidulants (Doores, 1993). They may be added directly to foods or, in the case of acetic and lactic acids, as sprays or dips for surface decontamination of fresh meat and poultry. In the undissociated or protonated form, organic acids, which are weak acids, can diffuse across the cell membrane lipid bilayer. Once inside the cell, the acid dissociates because the cell interior (pHi) has a higher pH than the exterior (pHo). Microorganisms maintain pHi near neutrality to prevent conformational changes to the cell structural proteins, enzymes, nucleic acids and phospholipids. Protons generated from intracellular dissociation of the organic acid acidify the cytoplasm and must be extruded to the exterior using energy in the form of ATP. The lower the pHo, the greater the influx of organic acids. This constant influx of protons will eventually deplete cellular energy (Bearson et al., 1997). Resistance by microorganisms to organic acids and/or low pH must be a response to this mechanism. Some foodborne pathogens, when exposed to low pH via short chain organic acids or inorganic acids (e.g., HCl) may undergo changes that provide them with varying degrees of resistance to subsequent exposure to normally lethal acidic conditions. This increased resistance to low pH and/or organic acids through preexposure to acidic conditions has no universally accepted terminology but has been called acid habituation, tolerance and shock. Reviews on the mechanisms of acid stress and stationary phase responses of foodborne bacterial pathogens were published by Rees et al. (1995), Bearson et al. (1997), Abee and Wouters (1999) and Foster (1999). Acid habituation may be defined as extended exposure to moderately acid conditions (pH 4.5-6.0) leading to resistance at low pH (ð 2.5) (Buchanan and Edelson, 1999; Rowbury, 1995). Acid tolerance response (ATR) is generally defined
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as enhanced survival of a microorganism to pH of 2.5 to 4.0 following brief exposure to moderate (ca. pH 5.0 to 5.5) conditions (Buchanan and Edelson, 1999). ATR may be divided into stationary phase ATR and log phase ATR (Rowbury, 1995; Bearson et al., 1997; Foster, 1999). Stationary phase ATR has been attributed to rpoS-regulated and inducible pH-dependent acid resistance responses (Abee and Wouters, 1999; Rowbury, 1995). rpoS controls expression of several genes involved in the general stress response of Escherichia coli, Salmonella and Shigella, including acid stress (Abee and Wouters, 1999). Log phase ATR in Salmonella has two phases, pre-acid-shock and post-acidshock. Pre-acid shock involves an “emergency” pH homeostasis system in which amino acid decarboxylases are induced to consume intracellular protons (Bearson et al., 1997). Post-acid-shock is controlled by rpoS and includes production of a number of acid shock proteins that protect and repair cellular macromolecules (Bearson et al., 1997). Other regulators, including PhoPQ, a two component signal transduction system, and Fur, a ferric uptake regulator, may also control expression of several acid shock proteins (Foster, 1999; Bearson et al., 1997). Escherichia coli stationary phase ATR is apparently distinct from Salmonella and has three inducible acid resistance systems (Foster, 1999). The glutamate-dependent system which utilizes glutamate decarboxylase to neutralize protons is the dominant system. It is important to note that Foster (1999) also reports a difference in resistance responses of Salmonella to inorganic and organic acids. For example, the PhoP system affected tolerance to inorganic — but not organic — acids in Salmonella Typhimurium. Therefore, resistance characteristics may differ depending upon whether inorganic or organic acids are used to induce tolerance responses. There is no question that tolerance or adaptation to short chain organic acids exists among foodborne pathogens (Tables 3.1 through 3.3). However, is this important to food safety? To have an impact on food safety, acid adaptation or tolerance has to be induced in foodborne pathogens by conditions present in the current food processing system; any acid adapted or tolerant foodborne pathogens would have to possess enhanced survival in foods or food processing systems in which they are normally inactivated. As to the first point, there are a number of processing systems that could cause acid adaptation among foodborne pathogens. Direct acidification of a food or food ingredient may shock microflora so they become more acid resistant. For example, lactic acid is added to improve flavor and quality in cheese curd, unsalted butter, egg whites, egg yolks, beer, bread dough, olives, pickles, relishes, and infant foods containing dried milk (Shelef, 1994). Many other organic acids are utilized for improved sensory properties and as antimicrobials in a variety of foods (Doores, 1993). In fermented foods, the situation can be somewhat different. Lactic acid bacteria lower the pH of a substrate gradually over time so a pH gradient is more likely, rather than a sharp alteration in the pH as would be expected with direct acidification. The use of organic acid sprays as a sanitizer for meat carcasses (Dickson, 1995) could cause acid shock or tolerance of the meat microflora. Several research studies have demonstrated that acid adaptation or tolerance may produce pathogens with enhanced survival in fermented foods or foods to which organic acids have been added. Leyer et al. (1995) found that acid adapted (pH 5.0)
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TABLE 3.1 Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock Applied to Escherichia coli O157:H7 and Their Subsequent Organic Acid, Food Antimicrobial or Acid Food Tolerance Responses Acid Adaptation Conditions pH 5.0 (HCl adjusted), 37°C, 4–5 h
3 strains plus Salmonella, 3 serovars 4 h at pH 5.0, 37°C
1 pathogenic and 1 nonpathogenic strain (1) acetic acid, pH 5.0, 1 doubling time (2) acetic acid, pH 2.5 2 strains and 1 nonpathogenic strain lactic acid, pH 5.5, stationary phase cells lactic acid, pH 4.0, stationary phase cells Stationary phase, acid resistance response through growth in TSB + 1% glucose
TSB + glucose (1%) for 18 h 3 strains TSB + glucose (1 or 1.25%) for 18 h Strains: ATCC 43895, ATCC 43889, ATCC 43890 TSB + glucose (1%) for 18 h
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Exposure Conditions and Response
Reference
Increased resistance to pH 3.85, 125 mM lactic acid, 60 min of three strains Extent of resistance increase varied by strain from 1 to 5 logs Enhanced survival in acid foods, including sausage fermentation (+2 log at 16 h), salami (pH 5.0) and apple cider (+4 log at 30 h) Increased survival of all strains in ketchup at pH 3.6 No difference in mustard (pH 3.1) or sweet relish (pH 2.8) Increased resistance to acetic acid, pH 3.5 or 4.0 Extent of resistance dependent upon temperature of exposure, time and strain
Leyer et al. (1995)
Resistance to sodium lactate up to 30% (w/w) at pH 4.0, depending on strain Increased resistance to sodium chloride, depending upon strain
Garren et al. (1998)
Increased resistance to 0.5% acetic, lactic, malic, and citric acids at pH 3.0 Extent variable depending upon strain Lactic acid caused greatest decrease for all treatments Little difference in resistance to lactic, malic, acetic, or citric acids at pH 3.9 to 5.4 Little or no difference in resistance to acetic, citric or malic acids at pH 3.9 to 7.2 12 strains tested: 6 strains acid resistant (ATCC strains: 43895, 43894, 35150, 2886-75; 86-24; NADC 5570) to TSB, pH 2.5 (HCl), 6 h, i.e., non-adapted cells resistant; 2 strains adaptable to acid resistance (ATCC 43889, NADC 4477), to TSB, pH 2.5 (HCl), 6 h, i.e., non-adapted cells sensitive Acid resistant strain (43895) and acid adaptable strain (43889) showed increased resistance to 2% acetic acid spray treatment on pre-rigor beef carcass tissue compared to unadapted cells
Buchanan and Edelson (1999)
Tsai and Ingham (1997) Brudzinski and Harrison (1998)
Ryu et al. (1999) Deng et al., (1999) Berry and Cutter (2000)
TABLE 3.1 (continued) Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock Applied to Escherichia coli O157:H7 and Their Subsequent Organic Acid, Food Antimicrobial or Acid Food Tolerance Responses Acid Adaptation Conditions Strains: ATCC 43889, ATCC 43895 pH 5.0 (HCl), 4 h at 37°C
Exposure Conditions and Response Increased resistance in mango juice (pH 3.2) at 25°C but not at 7°C Increased resistance in asparagus juice (pH 3.6) at end of 25°C storage period (6–14 d) for both strains, and at end of storage period (12–20 d) at 7°C for strain 43889 only Adapted cells less susceptible than unadapted cells inoculated into yakult (fermented milk drink, pH 3.6) or low-fat yogurt (pH 3.9)
Reference Cheng and Chou (2001)
E. coli O157:H7 had greater survival than unadapted cells in acid foods including salami and apple cider (Table 3.1). Similarly, Tsai and Ingham found enhanced survival of E. coli O157:H7 and three Salmonella serovars in ketchup, but not in mustard or sweet relish. Interestingly, Cheng and Chou (2001) found that, while acid adapted cells of E. coli O157:H7 (pH 5.0) ATCC 43889 and 43895 were more tolerant to acidic conditions of mango juice and asparagus juice, both were actually less tolerant than unadapted cells in yakult (fermented milk drink) and yogurt. They also found that resistance was dependent upon temperature (lower storage temperature, less difference in resistance) and strain. Salmonella serovars Typhimurium, Enteritidis, Heidelberg and Javiana that were pre-exposed to pH 5.8 (HCl) demonstrated increased resistance to the food antimicrobials lactic, propionic and acetic acid (Leyer and Johnson, 1992) (Table 3.2). In addition, they had greater survival than unadapted strains in a milk fermentation and cheddar, Swiss and mozzarella cheeses. Gahan et al. (1996) demonstrated that L. monocytogenes LO28 acid adapted by exposing the microorganism to lactic acid at pH 5.5 for 60 min had enhanced survival in yogurt, cottage cheese, orange juice and salad dressing (Table 3.3). Little or no enhancement was found with higher pH foods such as cheddar cheese (pH 5.16) or mozzarella cheese (pH 5.6). Ravishankar and Harrison (1999) found that L. monocytogenes exhibited an acid tolerance response (ATR) when it was acid adapted to pH of 5.5 with lactic acid, then challenged in acidified skim milk at pH 3.5 and 4.0 (Table 3.3). However, when the challenge pH of 4.5 was used, there was no adaptive ATR. Since pH 4.5 is more closely related to pH levels that might be attained in fermented products made from skim milk, results based on this pH should be the most meaningful. In summary, induced acid tolerance may theoretically cause some pathogens to have enhanced survival in certain fermented or acidified foods. In food, numerous product and antimicrobial combinations are possible. Under certain scenarios, there may be reason for concern when considering whether or not
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TABLE 3.2 Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock Applied to Salmonella and Shigella and Their Subsequent Organic Acid, Food Antimicrobial or Acid Food Tolerance Responses Microorganism
Acid Adaptation Conditions
Salmonella Salmonella Salmonella Salmonella
HCl, pH 5.8, 1–2 doublings
Typhimurium Enteritidis Heidelberg Javiana
Salmonella Typhimurium
Salmonella Typhimurium, 2 strains Salmonella Dublin Salmonella Heidelberg
Salmonella Typhimurium S1 Staphylococcus aureus 147A Escherichia coli O157:H7 Campylobacter jejuni C186 Salmonella Typhimurium
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Exposure Conditions and Responses
Increased resistance to lactic, propionic, acetic acids Increased survival in milk fermentation, and cheddar, Swiss, and mozzarella cheeses pH 5.8 (HCl), 1–2 doublings Increased resistance to activated lactoperoxidase and NaCl Increased cell surface hydrophobicity Growth in TSB acidified with Inoculated onto beef and lactic acid at pH 5.0 for 24 h treated with 1.5 or 3.0% lactic acid No difference in susceptibility to lactic acid by acid tolerant or unexposed cells Acid adapted cells more heat sensitive than unadapted cells 4 transfers, 48–72 h at 30°C, No significant increase in reduced pH from 6.4 to 5.8 resistance to 2% lactic acid with 10% lactic acid decontamination step for 2 min on pork skin (1) Short chain fatty acid Mixture (2) and propionate mixture 1: acetate 8mM, (3) significantly increased butyrate 3 mM, lactate resistance (>3–4 logs) to 14 mM, propionate 2 mM, pH 3.0 for up to 3 h at 37°C in TSB medium succinate 9 mM (2) Short chain fatty acid Mixture (1) increased mixture 2: acetate 70 mM, resistance to pH 3.0 butyrate 26 mM, lactate (ca. 2 logs) for 1 h only in TSB medium 5 mM, propionate 25 mM, succinate 1 mM, valeate No acid specified for pH 5 mM adjustment (3) 100 mM propionate All at pH 7.0, exposure for 1 h at 37°C
Reference Leyer and Johnson (1992)
Leyer and Johnson (1993)
Dickson and Kunduru (1995)
Van Netten et al. (1998) Kwon et al. (2000)
TABLE 3.2 (continued) Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock Applied to Salmonella and Shigella and Their Subsequent Organic Acid, Food Antimicrobial or Acid Food Tolerance Responses Exposure Conditions and Responses
Microorganism
Acid Adaptation Conditions
Reference
Shigella flexneri, 3 strains
TSB + 1% glucose, 37°C, 18 h Increased resistance to lactic Tetteh and and acetic acid at pH 3.5 for Beuchat Exposed to pH 5.05 lactic acid 2 h and 30 min, respectively (2001) after growth for 16 h in TSB No increase in resistance to without glucose at 37°C propionic acid at pH 3.5 Very slight increase in resistance to propionic acid at pH 4.5 Acid adapted slightly more resistant than acid shocked
bacteria can acquire some degree of resistance toward a particular antimicrobial. For example, Kwon et al. (2000) found that S. Typhimurium cells adapted by exposure to short chain fatty acid mixtures or propionic acid at pH 7.0 had significantly increased resistance to low pH, compared to unadapted cells (Table 3.2). Pickett and Murano (1996) exposed L. monocytogenes to sublethal levels of lactic acid, citric acid, and propionic acid before challenging the cells to minimum inhibitory concentrations of each compound under various conditions (Table 3.3). There was no difference in susceptibility with cells pre-exposed to sublethal levels. While citric acid did not produce resistant cells at the test pH of 2.8, when the pre-exposure pH was raised to 5.1, the dissociated form of the acid yielded cells that were able to survive exposure to lethal levels. Pickett and Murano (1996) suggested that preexposure of the cells to the dissociated form of the acid enabled the cells to survive a lethal dose. This is supported by the reported conditions required to induce the acid tolerance response as outlined by Buchanan and Edelson (1999). Use of organic acid sprays as a sanitizing treatment of meat carcasses has become very common (Dickson, 1995). Could pathogens on the meat surface become more acid resistant when exposed to weak acid (e.g., <3%) solutions? One study by Van Netten et al. (1998) demonstrated a lack of increased resistance to lactic acid for E. coli O157:H7, S. Typhimurium, Staphylococcus aureus, Campylobacter jejuni when acid-adapted cells were inoculated on pork bellies and treated with 2% lactic acid as a sanitizer (Table 3.1). Similarly, Dickson and Kunduru (1995) found no difference in acid resistance between unadapted cells of S. Typhimurium, Dublin or Heidelberg and those adapted by exposure to pH 5.0 (lactic acid) on the surface of beef treated with 1.5 or 3.0% lactic acid (Table 3.2). They also found a lower heat resistance of adapted cells compared to unadapted cells. In contrast, Berry and Cutter
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TABLE 3.3 Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock Applied to Listeria monocytogenes and their Subsequent Organic Acid, Food Antimicrobial or Acid Food Tolerance Responses Acid Adaptation Conditions
Exposure Conditions and Response
Scott A
Incubation at pH 5.4 (citric acid) Acid tolerance response not inducible at 5.0, 5.8 or 6.0
Okereke and Thompson (1996)
Scott A
(1) Sublethal citric (0.2%), lactic (0.2%), propionic (0.1%) acids at pH 2.8 for up to 60 min at 37°C (2) Sublethal citric acid (0.2%) at pH 5.0 Lactic acid, pH 5.5, 37°C for 60 min (early log phase cells)
Significant resistance to pH 3.35 (no acid specified) for 4 h at 35°C No resistance increase to pH 4.35, for 4 h at 35°C; constitutive acid resistance proposed Little or no cross protection for 0.3–1.5 µg/ml (12-60 IU/ml) nisin at pH 6.0, 35°C for 90 min (1) No increased resistance in cells exposed to sublethal concentrations of organic acids at pH 2.8 (2) Increased resistance to lethal concentration (0.3%) of citric acid at pH 2.8 Increased resistance to lactic acid at pH 3.5, 2 h at 37°C Hydrochloric acid less effective at inducing ATR Growth to stationary phase induced lactic acid tolerance Enhanced survival in cottage cheese (pH 4.71) and yogurt (pH 3.90) Enhanced survival during milk fermentation by lactic acid bacteria Enhanced survival in orange juice (pH 3.76) and salad dressing (pH 3.0) Marginal or no enhanced survival in whole-fat cheddar cheese (pH 5.16), low-fat cheddar (pH 5.25) and mozzarella cheese (pH 5.6) Increased resistance to lactic acid, pH 3.5 for 2 h at 37°C Increased resistance to 100-200 IU/ml nisin and 650 AU/ml lactacin 3147, pH 5.5 (lactic acid) Increased C14:0, C16:0 and decreased C18:0 fatty acids
O’Driscoll et al. (1996)
Strain(s)
LO28 (serotype 1/2c)
LO28 (serotype 1/2c)
Lactic acid, pH 5.5, 37°C for 60 min (early log phase cells)
Scott A
pH 5.5, lactic acid
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Reference
Pickett and Murano (1996)
Gahan et al. (1996)
Van Schaik et al. (1999)
TABLE 3.3 (continued) Conditions of Organic Acid or Mineral Acid Adaptation, Tolerance or Shock Applied to Listeria monocytogenes and their Subsequent Organic Acid, Food Antimicrobial or Acid Food Tolerance Responses Strain(s) V7, V37, CA
N-7155 (1/2b) N-7144 (1/2b)
Acid Adaptation Conditions
Exposure Conditions and Response
(1) Lactic acid in skim milk at pH 3.5 or 4.0 (2) Skim milk acidified to 5.5, transfer to skim milk with lactic acid at pH 3.5 or 4.0 Stationary phase growth, 24 h at 35°C
Skim milk with lactic acid at: pH 3.0 — acid-adapted had greater survival (0.5–1.0 log); pH 4.0 — acid-adapted had greater survival (3–4 logs); pH 4.5 — no difference Lactoperoxidase system at pH 4.5 — no difference Exposure to acetic or lactic acid at pH 2.5 or 3.5 No resistance to lactic or acetic acid at pH 2.5 or acetic acid at pH 3.5 Increased resistance of all strains to lactic acid at pH 3.5 Increased resistance of most acid tolerant strains (N-7155, N-7144Sm+) to lactic acid (pH 3.5), and 2% acetic acid spray wash water (pH 3.2) and lactic acid spray wash water (pH 2.5) from beef decontamination Increased acid tolerance by Listeria monocytogenes in the presence of viable natural meat microflora was transient
Reference Ravishankar and Harrison (1999)
Samelis et al. (2001)
(2000) showed increased resistance to a 2% acetic acid spray treatment on beef carcasses by acid-resistant (ATCC 43895) and acid-adapted (ATCC 43889) E. coli O157:H7 strains (Table 3.1). The differences in these studies are most likely due to differences in experimental conditions, especially those used to create acid tolerant cells.
BENZOIC ACID Benzoic acid and its salts were the first antimicrobials approved for use in foods, about 1900. Their primary use is as antifungal agents in high acid foods. There are differences in resistance to benzoates among microorganisms due to innate tolerance. Since these are antifungal agents, innate resistance to benzoates would be more of a concern with the target microorganisms, yeasts and molds. Warth (1985) identified a number of yeasts that grew in the presence of ca. 500 µg/ml benzoic acid including Schizosaccharomyces pombe and Zygosaccharomyces bailii. Other yeasts, including © 2003 by CRC Press LLC
Pichia membranefaciens and Byssochlamys nivea, have also been shown to be naturally resistant to benzoates (Chipley, 1993). Warth (1988) suggested that the resistance mechanism of yeasts to weak-acid type preservatives, including benzoic and propionic acids, was related to membrane permeability and the ability of the cells to continuously remove the preservative. Some microorganisms, including the bacteria Bacillus, Pseudomonas, Corynebacterium, Micrococcus, and the mold Aspergillus, are able to metabolize benzoic acid utilizing the β-ketoadipate pathway to succinic acid and acetyl CoA (Chipley, 1993). A few studies have examined the potential for acquired resistance to benzoic acid. Warth (1988) incubated strains of yeast, including Candida krusei, Hansenula anomala, Kluyveromyces fragilis, Kloeckera apiculata, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Schizosaccharomyces pombe, and Z. bailii, overnight in the presence of either 0.25 mM (31 µg/ml) or 2 mM (244 µg/ml) of benzoic acid. The minimum inhibitory concentration for unexposed cells was significantly lower than cells exposed to subinhibitory levels of benzoic acid. A 1.4 to 2.2-fold increase in minimum inhibitory concentration was achieved with pre-exposure. Zygosaccharomyces bailii and S. pombe showed the greatest increases. The resistance mechanism proposed was an increased efflux by the adapted cells. There is little or no evidence of acquired resistance to benzoic acid by bacteria, including acid adapted cells.
SORBIC ACID Sorbic acid has been used as an antimicrobial in foods since the 1940s. Innate resistance to the compound has been demonstrated by bacteria, including catalasenegative lactic acid bacteria, Sporolactobacillus and some Pseudomonas, yeasts, including Z. bailii, Saccharomyces, Torulopsis, Brettanomyces and Candida, and molds, including Aspergillus, Penicillium, Fusarium, Geotrichum, and Mucor (Sofos and Busta, 1993). As with benzoic acid, some microorganisms can metabolize sorbic acid. Molds isolated from cheese, including seven Penicillium species, were shown to grow in the presence of and degrade 0.3 to 1.2% sorbate (Finol et al., 1982). Penicillium puberulum and P. cyclopium were the most resistant species evaluated. Marth et al. (1966) demonstrated that Penicillium species isolated from cheese degraded sorbic acid and produced 1,3 pentadiene, which was volatile and had a kerosene off-odor. Sorbic acid has been shown to be degraded by Mucor species to 4-hexenol and Geotrichum species to 4-hexenoic acid and ethyl sorbate (Liewen and Marth, 1985). High numbers of lactic acid bacteria can produce compounds such as ethyl sorbate, 2,4-hexadien-1-ol, 1-ethoxyhexa-2,4 diene, 5-hexadien-1-ol, and 2-ethoxyhexa-3,5 diene in sorbic acid-treated red wine (Liewen and Marth, 1985). This can result in geranium off-odors in wines and fermented vegetables which have been attributed to the 2,4 hexadien-1-ol (Liewen and Marth, 1985; Sofos and Busta, 1993). There is evidence that certain yeasts may acquire resistance to sorbic acid. Warth (1977) found that Z. bailii grown in the presence of 224.2 µg/ml acquired resistance to sorbic acid. Bills et al. (1982) investigated acquired resistance with an osmotolerant yeast, Saccharomyces rouxii. The yeast was pre-conditioned by growth in the
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presence of 0.1% sorbic acid for four transfers. Pre-exposure significantly increased resistance (shorter lag times, shorter time to stationary phase) of the cells to subsequent exposures of 0.1% sorbic acid in microbiological medium and chocolate sauce. The mechanism proposed for the resistance acquired by the yeasts is an inducible, energy-requiring system that increases efflux of the sorbic acid (Warth, 1977; Bills et al., 1982). Yeast resistance to sorbic acid and other weak acids probably involves several components (Brul and Coote, 1999). First, a H+-ATPase utilizes energy to remove excess protons from the cell. A membrane protein may also be induced, which can decrease the activity of the ATPase to conserve cellular energy pools. Finally, Piper et al. (1998) found that exposure of Saccharomyces cerevisiae to sorbic acid strongly induces the membrane protein ATP-binding cassette transporter Pdr12, a multi-drug resistance pump. Mutants without the transporter are hypersensitive to sorbic, benzoic, and propionic acids. Further, Piper et al. (1998) stated that Pdr12 conferred resistance by mediating energy-dependent extrusion of anions. To prevent a futile cycle of allowing the acid back into the cell, adapted yeasts reduce diffusion of weak acids, most likely, by altering cell membrane structure to reduce passage of the acids into the cell (Brul and Coote, 1999). There is little information on acquired resistance to sorbic acid among bacteria or molds. Schroeder and Bullerman (1985) found little or no increase in the resistance of Penicillium digitatum or P. italicum when exposed to increasing levels of sorbic acid. Considering the length of time that sorbic acids (and benzoic acid) have been applied to food products would indicate that the development of acquired resistance by spoilage or pathogenic microorganisms is virtually non-existent.
ALKYL ESTERS
OF P-HYDROXYBENZOIC
ACID (PARABENS)
Moir and Eyles (1992) compared the effectiveness of methyl paraben and potassium sorbate on the growth of four psychrotrophic foodborne bacteria, Aeromonas hydrophila, L. monocytogenes, Pseudomonas putida and Yersinia enterocolitica. Little or no adaptation was found to occur when cells were exposed to subinhibitory concentrations of antimicrobials. Bargiota et al. (1987) examined the relationship between lipid composition of S. aureus and resistance to parabens. Differences were found for total lipid, phospholipids and fatty acids between S. aureus strains which were relatively resistant and sensitive to parabens. The paraben-resistant strain was shown to have a higher percentage total lipid, higher relative percentage of phosphatidyl glycerol and decreased cyclopropane fatty acids than sensitive strains. It was suggested that these changes could influence membrane fluidity and, therefore, adsorption of the parabens to the membrane. Juneja and Davidson (1993) altered the lipid composition of L. monocytogenes by growth in the presence of added fatty acids (C14:0, C18:0 or C18:1). Growth of L. monocytogenes in the presence of exogenously added C14:0 or C18:0 fatty acids increased the resistance of the cells to parabens. However, growth in the presence of C18:1 led to increased sensitivity to the antimicrobial agents. Results indicated that, for L. monocytogenes, a correlation existed between lipid composition of the cell membrane and susceptibility to antimicrobial compounds.
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RESISTANCE TO NATURALLY OCCURRING ANTIMICROBIALS Naturally occurring antimicrobials are being studied extensively as alternatives to the so-called “synthetic” traditional antimicrobials. Naturally occurring antimicrobials come primarily from three sources: animals, plants and microorganisms. Animal sources include milk (lactoperoxidase system and lactoferrin) and eggs (lysozyme). Plant sources include herbs and spices (extracts and essential oils), onions and garlic (sulfur compounds), and the mustard family (isothiocyanates). Microbial products with antimicrobial activity include bacteriocins from Lactococcus, Pediococcus, Lactobacillus, Leuconostoc, Carnobacterium, Propionibacterium and fermentation products. There are innate differences in resistance among microorganisms to plant and animal antimicrobials. For example, lysozyme (1,4-β-N-acetylmuramidase) is an enzyme present in biological secretions that catalyzes hydrolysis of the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan of bacterial cell walls. Lysozyme is most active against Gram-positive bacteria, most likely because of the exposed peptidoglycan in the cell wall. Lysozyme is less effective against Gram-negative bacteria due to a reduced peptidoglycan content and presence of outer membrane of lipopolysaccharide and lipoprotein. However, there seem to be few studies on directly acquired resistance to natural antimicrobials from animal and plant sources. Some researchers have investigated the potential for cross-protection to natural antimicrobials afforded foodborne pathogens through acid adaptation or acid shock. Ravishankar and Harrison (1999) conducted experiments to determine if acid adaptation of L. monocytogenes enhanced survival in the presence of an activated lactoperoxidase system by means of cross-protection (Table 3.3). The lactoperoxidase system occurs in raw milk and involves the enzyme lactoperoxidase, thiocyanate and hydrogen peroxide. The survival rates were similar for the acid adapted and nonadapted cells at pH 4.5, both in the presence and absence of an activated lactoperoxidase system, indicating no cross-protection. In contrast, Leyer and Johnson (1993) reported cross-protection against an activated lactoperoxidase system with acid adapted S. Typhimurium when tested in a laboratory culture medium (Table 3.2). Since the activity of the lactoperoxidase system can vary depending on the medium, this may explain the contrasting result. Another factor could be related to the greater degree of acid tolerance exhibited by Salmonella compared to Listeria. The lactoperoxidase system may also be less effective as an antimicrobial toward Salmonella. In contrast to plant and animal sources, there has been significant research on microbially derived antimicrobials. The probable reason for this is that these compounds have distant similarities to therapeutic antibiotics, about which there is much concern due to resistance development. However, they generally have a much narrower spectrum and often have different mechanisms, reducing chances for resistance development. Two microbially derived antimicrobials that have been evaluated for development of acquired resistance are natamycin and nisin. Natamycin (formerly called
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pimaricin) is a polyene macrolide antibiotic produced by Streptomyces natalensis that is effective against nearly all molds and yeasts but has little or no effect on bacteria. Its primary use is as an antifungal agent on cheese. De Boer and StolkHorsthuis (1977) investigated the potential for development of resistance to natamycin among fungi. They found no evidence of resistant fungi in cheese warehouses in which natamycin had been used for various periods of time, up to several years. They also attempted to induce tolerance in 26 strains of fungi by transferring each culture 25 to 31 times in media containing concentrations of natamycin equal to and higher than the minimum inhibitory concentration (MIC). The MIC following multiple transfers increased in only 8 of 26 strains and by a maximum of 4 µg/ml. They concluded that the lack of increased resistance was due to strongly fungicidal activity of the compound along with its environmental instability. In contrast to natamycin, nisin resistance is known. Nisin is a peptide produced by a Lactococcus lactis ssp. lactis strain. Nisin has a narrow spectrum affecting primarily only Gram-positive bacteria and their spores, including lactic acid bacteria, Bacillus, Clostridium, Listeria, and Streptococcus. The compound alone generally does not inhibit Gram-negative bacteria, yeasts, or molds. Streptococcus thermophilus, Lactobacillus plantarum, other lactic acid bacteria, and certain Bacillus species produce the enzyme, nisinase, which neutralizes the antimicrobial activity of the peptide. More important, however, is the fact that spontaneous nisin resistant mutants, including L. monocytogenes and C. botulinum, could occur due to exposure of wild-type strains to high concentrations of nisin or transfer in increasing concentrations of nisin (Harris et al., 1991; Ming and Daeschel, 1993; Mazzotta et al., 1997). Listeria monocytogenes resistant mutants may occur at a rate of 1 in 106 to 108 (Harris et al., 1991; Ming and Daeschel, 1993) or even lower (Schillinger et al., 1998). These have been shown to be stable mutants. The mechanism of action of nisin against vegetative cells includes binding to the anionic phospholipids of the cell membrane, insertion into the membrane and pore formation. This disruption of cytoplasmic membrane causes efflux of intracellular components and eventual depletion of the proton motive force (PMF; Crandall and Montville, 1998). As might be expected, resistance of the cell involves adjusting to prevent these actions. Crandall and Montville (1998) did an extensive study on the potential mechanism of nisin resistance. They found that nisin resistant strains of L. monocytogenes (NisR) had altered phospholipid composition, including decreased anionic phospholipid (cardiolipin and phosphatidylglycerol) and increased phosphatidylethanolamine. This resulted in a decreased net negative charge, which would hinder binding of cationic compounds such as nisin. In addition, NisR strains have increased long chain fatty acids and reduced ratios of C15/C17 fatty acids, resulting in reduced fluidity and stabilization due to reduced effect on PMF (Ming and Daeschel, 1993; Mazzotta and Montville, 1997). Other cellular changes include cell wall alterations as evidenced by increased lysozyme resistance, decreased resistance to ampicillin and benzylpenicillin and increased resistance to Gramicidin S and Gentamicin. These changes suggested an alteration of cytoplasmic membrane to prevent access by nisin. NisR cells required divalent cations for resistance. This might be due to reduced binding or stabilization of the cytoplasmic membrane.
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Cross-protection of foodborne pathogens to bacteriocins may be induced by acid adaptation or acid tolerance. Van Schaik et al. (1999) demonstrated that acid adapted (pH 5.5, lactic acid) cells of L. monocytogenes Scott A had increased resistance to nisin and lacticin in addition to lactic acid at pH 3.5. These cells had altered fatty acid composition with increased C14:0, C16:0 and decreased C18:0. In contrast, Okereke and Thompson (1996) found no increase in resistance to nisin with acid adapted cells of L. monocytogenes. Van Schaik et al. (1999) suggested this was caused by a lower concentration of nisin used by the latter researchers. The obvious implication of the emergence of bacteriocin-resistant pathogenic microorganisms would be potential hazards in foods preserved exclusively by the compounds. To overcome the potential hazard, researchers have suggested use of combinations of bacteriocins or combinations of bacteriocins and other preservation methods or antimicrobials (Mulet-Powell et al., 1998; Schillinger et al., 1998). Cross resistance between various classes of bacteriocins has been demonstrated (Crandall and Montville, 1998). However, this cross resistance appears to be variable as Rasch and Knøchel (1998) found none between nisin- and pediocin-resistant strains of L. monocytogenes. Dykes and Hastings (1998) demonstrated that leucocin- and sakacin-resistant L. monocytogenes had a reduced growth rate in BHI broth without bacteriocin compared to bacteriocin-sensitive strains. In addition, resistant strains failed to compete with sensitive strains when grown in mixed populations even at frequencies of 1:1. The researchers concluded that the bacteriocin-resistant phenotype of L. monocytogenes was not likely to become stable in natural populations. In addition, nisin resistant C. botulinum spores have similar heat resistance to wild type spores, and nisin-resistant strains of L. monocytogenes and C. botulinum were not as resistant as wild-type strains to other traditional food antimicrobials including sodium chloride, sodium nitrite or potassium sorbate (Mazzotta and Montville, 1999; Mazzotta et al., 2000). All of these studies suggest that acquired resistance to bacteriocins may not confer resistance to other antimicrobials or preservative treatments or any natural advantage over susceptible populations in the absence of the inhibitor.
SUMMARY There is little evidence that microorganisms can directly acquire resistance or tolerance to most traditional food antimicrobials. The possible exception is microbial tolerance to short chain organic acids through acid adaptation. Little research has been done on acquired resistance to naturally occurring antimicrobials with the exception of microbially derived bacteriocins. The latter have definitively been shown to induce acquired resistance to microorganisms exposed to the compounds. Acquired resistance may be important in the microbiological safety of food products if the resistance is induced in foodborne pathogens by conditions present in the current food processing system and if that resistance enhances survival of those pathogens in foods or food processing systems in which they are normally inactivated. Both acid adaptation and acquired resistance to bacteriocins have the potential to induce resistance in foodborne pathogens under conditions currently present in the food processing system. Bacteriocin resistance development is of less concern
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because foodborne pathogens are not often repeatedly exposed to such compounds. In contrast, there are multiple steps in the food production and processing system in which a microorganism may be exposed to organic acids. While resistance theoretically enhances survival of foodborne pathogens to exposure to lethal treatments, cells that are resistant to bacteriocins have been found to be less able to compete than non-resistant cells in the environment. There is limited evidence that acid tolerance may increase resistance of pathogens to lethal organic acid treatments or conditions in foods. More evidence is needed to confirm that this is a real problem. If antimicrobials are to be used for exclusive control of foodborne pathogens, then potential for development of resistant cells should be evaluated. Development of resistance to processing and handling treatments could occur at numerous points in the food processing system and could influence the treatment efficacy. Evaluation of this type of response is probably more meaningful when conditions most like those in actual products or situations are included in the experimental plan. As was noted by Leyer and Johnson (1993), the physiological state of foodborne pathogens used in challenge studies in food and in evaluating hazard analysis critical control point (HACCP) programs is an important consideration.
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Crandall, A.D. and Montville, T.J. 1998. Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype. Appl. Environ. Microbiol. 64:231–237. Davidson, P.M. 2001. Chemical preservatives and natural antimicrobial compounds, p. 593–627, in Food Microbiology: Fundamentals and Frontiers, 2nd ed. M.P. Doyle, L.R. Beuchat, and T.J. Montville (Eds.). American Society for Microbiology, Washington, D.C. De Boer, E. and Stolk-Horsthuis, M. 1977. Sensitivity to natamycin (pimaricin) of fungi isolated in cheese warehouses. J. Food Prot. 40:533–536. Deng, Y., Ryu, J.-H., and Beuchat, L.R. 1999. Tolerance of acid-adapted and non-adapted Escherichia coli O157:H7 cells to reduced pH as affected by type of acidulant. J. Appl. Microbiol. 86:203–210. Dickson, J.S. and Kunduru, M.R. 1995. Resistance of acid-adapted salmonellae to organic acid rinses on beef. J. Food Prot. 58:973–976. Dickson, J.S. 1995. Susceptibility of preevisceration washed beef carcasses to contamination by Escherichia coli O157:H7 and Salmonellae. J. Food Prot. 58:1060–1068. Doores, S. 1993. Organic acids, p. 95–136, in P.M. Davidson and A.L. Branen (Eds.), Antimicrobials in Foods, 2nd ed. Marcel Dekker, Inc. New York. Dykes, G.A. and Hastings, J.W. 1998. Fitness costs associated with class IIa bacteriocin resistance in Listeria monocytogenes B73. Lett. Appl. Micro. 26:5–8. FDA. 2001. GRAS Notice No. GRN 000077. U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Food Additive Safety. August 14, 2001. Federal Register. 1998. Direct Food Substances Affirmed as Generally Recognized as Safe; Egg White Lysozyme. 63(49):12421–12425 (Friday, March 13, 1998). Finol, M.L., Marth, E.H., and Lindsay, R.C. 1982. Depletion of sorbate from different media during growth of Penicillium species. J. Food Prot. 45:398–404. Foster, J.W. 1999. When protons attack: microbial strategies of acid adaptation. Curr. Opinion Microbiol. 2:170–174. Gahan, C.G., O’Driscoll, B., and Hill, C. 1996. Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. Appl. Environ. Microbiol. 62:3128–3132. Garren, D.M., Harrison, M.A., and Russell, S.M. 1998. Acid tolerance and acid shock response of Escherichia coli O157:H7 and non-O157:H7 isolates provide cross protection to sodium lactate and sodium chloride. J. Food Prot. 61:158–161. Harris, L.J., Fleming, H.P., and Klaenhammer, T.R. 1991. Sensitivity and resistance of Listeria monocytogenes ATCC 19115, Scott A and UAL500 to nisin. J. Food Prot. 54:836–840. Juneja, V.K. and Davidson, P.M. 1993. Influence of altered fatty acid composition on resistance of Listeria monocytogenes to antimicrobials. J. Food Prot. 56:302–305. Kwon, Y.M., Park, S.Y., Birkhold, S.G., and Ricke, S.C. 2000. Induction of resistance of Salmonella typhimurium to environmental stresses by exposure to short-chain fatty acids. J. Food Sci. 65:1037–1040. Leistner, L. and Gorris, L.G.M. 1995. Food preservation by hurdle technology. Trends Food Sci. Technol. 6:41–46. Leistner, L. 2000. Basic aspects of food preservation by hurdle technology. Intl. J. Food Microbiol. 55:181–186. Leyer, G.J. and Johnson, E.A. 1993. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbiol. 59:1842–1847. Leyer, G.J. and Johnson, E.A. 1992. Acid adaptation promotes survival of Salmonella spp. in cheese. Appl. Environ. Microbiol. 58:2075–2080. Leyer, G.J., Wang, L.-L., and Johnson, E.A. 1995. Acid adaptation of Escherichia coli O157:H7 increases survival in acidic foods. Appl. Environ. Microbiol. 61:3752–3755.
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Liewen, M.B. and Marth, E.H. 1985. Growth and inhibition of microorganisms in the presence of sorbic acid: a review. J. Food Prot. 48:364–375. Marth, E.H., Capp, C.M., Hasenzahl, L., Jackson, H.W., and Hussong, R.V. 1966. Degradation of potassium sorbate by Penicillium species. J. Dairy Sci. 49:1197–1205. Mazzotta, A.S. and Montville, T.J. 1997. Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 10°C and 30°C. J. Appl. Microbiol. 82:32–38. Mazzotta, A.S. and Montville, T.J. 1999. Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Appl. Environ. Microbiol. 65:659–664. Mazzotta, A.S., Crandall, A.D., and Montville, T.J. 1997. Nisin resistance in Clostridium botulinum spores and vegetative cells. Appl. Environ. Microbiol. 63:2654–2659. Mazzotta, A.S., Modi, K.D., Chikindas, M.L., and Montville, T.J. 2000. Nisin-resistant (NisR) Listeria monocytogenes and NisR Clostridium botulinum are not resistant to common food preservatives. J. Food Sci. 65:888–890. Moir, C.J. and Eyles, M.J. 1992. Inhibition, injury and inactivation of four psychrotrophic foodborne bacteria by the preservatives methyl p-hydroxybenzoate and potassium sorbate. J. Food Prot. 55:360. Mulet-Powell, N., Lacoste-Armynot, A.M., Vinas, M., and Simeon de Buochberg, M. 1998. Interactions between pairs of bacteriocins from lactic bacteria. J. Food Prot. 61:1210–1212. O’Driscoll, B., Gahan, C.G.M., and Hill, C. 1996. Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl. Environ. Microbiol. 62:1693–1698. Okereke A. and Thompson S.S. 1996. Induced acid-tolerance response confers limited nisin resistance on Listeria monocytogenes Scott A. J. Food Prot. 59:1003–1006. Pickett, E.L. and Murano, E.A. 1996. Sensitivity of Listeria-monocytogenes to sanitizers after exposure to a chemical shock. J. Food Prot. 59:374–378. Piper, P., Mahe, Y., Thompson, S., Pandjaitan, R., Holyoak, C., Egner, R., Muehlbauer, M., Coote, P., and Kuchler, K. 1998. The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. EMBO J. 17:4257–4265. Rasch, M. and Knøchel, S. 1998. Variations in tolerance of Listeria monocytogenes to nisin, pediocin PA-1 and bavaricin A. Lett. Appl. Microbiol. 27:275–278. Ravishankar, S. and Harrison, M.A. 1999. Acid adaptation of Listeria monocytogenes strains does not offer cross-protection aganist an activated lactoperoxidase system. J. Food Prot. 62:670–673. Rees, C.E.D., Dodd, C.E.R., Gibson, P.T., Booth, I.R., and Stewart, G.S.A.B. 1995. The significance of bacteria in stationary phase to food microbiology. Intl. J. Food Microbiol. 28:263–275. Rowbury, R.J. 1995. An assessment of environmental factors influencing acid tolerance and sensitivity in Escherichia coli, Salmonella spp. and other enterobacteria. Lett. Appl. Microbiol. 20:333–337. Russell, A.D. 1991. Mechanisms of bacterial resistance to non-antibiotics: food additives and food and pharmaceutical preservatives. J. Appl. Bacteriol. 71:191–201. Russell, A.D., Furr, J.R., and Maillard J.-Y. 1997. Microbial susceptibility and resistance to biocides. ASM News 63:481–487. Russell, A.D. 1997. Plasmids and bacterial resistance to biocides. J. Appl. Microbiol. 83:155–165. Ryu, J.H., Deng, Y., and Beuchat, L.R. 1999. Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids. J. Food Prot. 62:451–455.
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Samelis, J., Sofos, J.N., Kendall, P.A., and Smith, G.C. 2001. Influence of the natural microbial flora on the acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination fluids. Appl. Environ. Microbiol. 67:2410–2420. Schillinger, U., Chung, H.-S., Keppler, K., and Holzapfel, W.H. 1998. Use of bacteriocinogenic lactic acid bacteria to inhibit spontaneous nisin-resistant mutants of Listeria monocytogenes Scott A. J. Appl. Microbiol. 85:657–663. Schroeder, L.L. and Bullerman, L.B. 1985. Potential for development of tolerance by Penicillium digitatum and Penicillium italicum after repeated exposure to potassium sorbate. Appl. Environ. Microbiol. 50:919–923. Shelef, L.A. 1994. Antimicrobial effects of lactates: a review. J. Food Prot. 57:445–450. Sofos, J.N. and Busta, F.F. 1993. Sorbic acid and sorbates, p. 49–94, in P.M. Davidson and A.L. Branen (Eds.), Antimicrobials in Foods, 2nd ed. Marcel Dekker, New York. Tetteh, G.L. and Beuchat, L.R. 2001. Sensitivity of acid-adapted and acid-shocked Shigella flexneri to reduced pH achieved with acetic, lactic and propionic acids. J. Food Prot. 64:975–981. Tsai, Y.-W. and Ingham, S.C. 1997. Survival of Escherichia coli O157:H7 and Salmonella spp. in acidic condiments. J. Food Prot. 60:751–755. Van Netten, P., Valentijn, A., Mossel, D.A.A., and Huis in’t Veld, J.H.J. 1998. The survival and growth of acid-adapted mesophilic pathogens that contaminate meat after lactic acid decontamination. J. Appl. Microbiol. 84: 559–567. Van Schaik, W., Gahan, C.G.M., and Hill, C. 1999. Acid-adapted Listeria monocytogenes displays enhanced tolerance against the lantibiotics nisin and lacticin 3147. J. Food Prot. 62:536–539. Warth, A.D. 1977. Mechanism of resistance of Saccharomyces bailii to benzoic, sorbic and other weak acids used as food preservatives. J. Appl. Bacteriol. 43:215–230. Warth, A.D. 1985. Resistance of yeast species to benzoic and sorbic acids and to sulfur dioxide. J. Food Prot. 48:564–569. Warth, A.D. 1988. Effect of benzoic acid on growth yield of yeasts differing in their resistance to preservatives. Appl. Environ. Microbiol. 54:2091–2095.
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4
Microbial Adaptation and Survival in Foods Eric A. Johnson
CONTENTS Introduction The Spectrum of Stress Responses in Microorganisms Importance of Microbial Stress Responses to the Safety and Quality of Foods Impact of Stress Responses on Preharvest Survival of Foodborne Pathogens Stress Responses and Their Impact on Survival on Gram-Negative Foodborne Pathogens and Spoilage Bacteria Salmonella Escherichia coli O157:H7 Shigella spp. Yersinia enterocolitica Campylobacter jejuni Vibrio parahaemolyticus and Vibrio cholerae Pseudomonas aeruginosa Gram-Positive Bacteria Staphylococcus aureus Listeria monocytogenes Bacillus spp. Clostridium spp. Impact of Stress Adaptation on the Performance of Beneficial Microorganisms in Food Fermentations Cross-Protection among Microbial Stress Responses Conclusions and Perspectives References
INTRODUCTION Microorganisms can induce adaptation responses to environmental stresses by expressing specific sets of genes on exposure to acid, salt, heat, cold, reactive oxygen species (ROS), nutrient starvation, and other stresses (reviewed in Abee and Wouters, 1999; Costa and Moradas-Ferreira, 2001; Hecker and Völker, 2001; Hohmann and Mager, 1997; Jennings, 1993; Lin and Lynch, 1996; Storz and Hengge-Aronis, 2000; Welch, 1993; Young and Elliott, 1989).
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Adaptation enhances tolerance to environmental, chemical, and biological stresses and may promote survival or growth in adverse environments. Adaptation to stresses is mediated by changes in physiology of the organism, including alterations in metabolism and structural changes. These changes can have profound effects on the ability of food-spoilage and pathogenic organisms to survive food processing operations and to survive or even grow in normally adverse or harsh food environments. Adaptation of stress responses also affects the performance of beneficial organisms in carrying out food fermentations and other desired transformations of foods. The stressed cellular state probably reflects the actual physiology of many bacteria and fungi in foods and in food processing environments since microorganisms in these conditions are often periodically or continually exposed to adverse stresses. Exposure to stresses has been widely shown to induce a spectrum of adaptive responses, ranging from relatively minor physiological adaptations to extreme changes in cellular structure such as alterations in the cell surface or formation of endospores, as well as changes in population structure, including entry into stationary phase and the formation of macrocellular structures such as biofilms. It is surprising that microbial stress responses have not, until recently, been integrally considered in various disciplines of food microbiology including resistance to unit food processing operations, formulation design of foods, challenge studies and shelf-life evaluations, contributions to virulence, formation of endospores and biofilms, and inactivation by sanitizing procedures. This chapter focuses on practical aspects and industrial relevance of stress responses of various microorganisms of importance in the food industry, with an emphasis on foodborne bacterial pathogens and beneficial fermentation organisms. This discussion makes a unique contribution since the majority of research on stress responses has focused on the molecular aspects of the various responses, particularly in Salmonella enterica serovar Typhimurium and Escherichia coli, and in the yeast Saccharomyces cerevisiae and a few other fungal species (reviews are cited above). Many of the practical consequences of stress that are of importance to the food industry have only begun to be studied in depth. The investigation of stress responses as applied to food-related microorganisms and food systems would appear to provide a plethora of opportunities and is anticipated to yield valuable information to enhance the safety and quality of many foods. In this chapter, various stages in food production are considered, including preharvest survival, resistance to food processing operations and sanitation procedures, and survival and growth during a shelf-life of a food. The impact of stress responses on beneficial aspects of microorganisms in food fermentations is also discussed, followed by newer approaches to study stress responses, and conclusions and perspectives.
THE SPECTRUM OF STRESS RESPONSES IN MICROORGANISMS The myriad of stress-associated phenomena in microorganisms can be classified into distinct and yet interrelated classes, as shown in Table 4.1 (Storz and Hengge-Aronis,
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TABLE 4.1 Classes of Microbial Stress Responses Specific Stress Responses and Causative Phenotypic Triggers Heat shock Cold shock Sensing envelope stress Acids and acidity Reactive oxygen species (ROS) Reduced osmolarity Sodium Metals, possibly other ions DNA damage General Stress Responses Bacterial sporulation and resistance Survival in the stationary phase Pathogenic responses Stress-induced mutations Drug resistance Stress-induced competence in Bacillus and possibly other organisms Cell-to-cell communication and quorum sensing Stress-induced biofilm formation (Adapted from Storz, G. and Hengge-Aronis, R., Eds., Bacterial Stress Responses, ASM Press, Washington, D.C., 2000.)
2000). In addition to these categories, it seems plausible to include populationinduced stress responses including quorum sensing (Hardman et al., 1998), programmed cell death and cell aging (Beckman and Ames, 1998; Jazwinski, 1999; Lewis, 2000; Nature Insight, 2000), and formation of multicellular or multispecies biofilms (Costerton et al., 1999). Of interest and potential importance in food microbiology is the finding that Gram-negative bacteria (e.g., E. coli, Salmonella serovars), Gram-positive eubacteria (e.g., Bacillus, Staphylococcus, Listeria, Clostridium) and fungi (S. cerevisiae) appear to have evolved different physiological, genetic, and structural mechanisms to cope with various stresses (reviewed in Hecker and Völker, 2001; Hohmann and Mager, 1997; Jennings, 1993; Storz and Hengge-Aronis, 2000). It is beyond the scope here to describe the molecular mechanisms of the different responses among the various microbial groups; these are described elsewhere in this book. In the literature on microbial stress, various descriptors have been used to describe stress responses, including “adaptation,” “tolerance,” “habituation,” “shock,” and other terms. These descriptors are used in various laboratories and may very well reflect different physiological responses. As the overall theme of this chapter is to describe the practical impact of stress responses on microorganisms of importance in foods, the terms are collectively grouped as “stress responses” and the reader is referred to the original papers for the specific experimental details and interpretations of the responses. © 2003 by CRC Press LLC
IMPORTANCE OF MICROBIAL STRESS RESPONSES TO THE SAFETY AND QUALITY OF FOODS Stress responses would be expected to affect the resistance and survival of pathogens and spoilage organisms through the entire food production chain, from preharvest activities, processing operations, and storage of foods during their shelf life. The stress responses elicited by food-related microorganisms would also vary according to the food commodity, its processing steps, and its respective microbial ecology and species associated with the different commodities (Roberts et al., 1998). It is useful to consider stress responses of food-related bacteria and fungi in the context of environmental factors that have systematically been demonstrated to affect growth and survival in food systems. For most species of food-related bacteria and fungi, survival has long been known to be affected by various chemical, processing and environmental factors, commonly categorized as intrinsic factors, processing factors, extrinsic factors, and implicit factors (Table 4.2) (Cole, 2001; Gould, 2000; Mossell and Ingram, 1955). These preservative factors and processing technologies impose stresses upon microorganisms in foods, potentially eliciting stress responses that would affect growth and survival in many instances. The application of multiple factors (hurdle technology) involving exposure to combinations of sublethal conditions in foods could also result in the promulgation of adaptive stress responses (Archer, 1996; Knøchel and Gould, 1995; Leistner, 1995; Rowan, 1999). Stress responses elicited in response to traditional food preservatives such as sorbate, benzoate, lactate, sulfite, nitrite, nisin, smoke, and other preservatives have not yet been established. Newer processing technologies such as treatment of foods with high pressure, pulsed electric fields, light, sound, and others (reviewed in Rahman, 1999) would also be expected to induce stress responses, including novel or unexpected responses, but more research is needed in this area. As with most foodborne pathogens and spoilage organisms discussed in this chapter, the vast majority of studies on stress responses have been conducted in vitro in media or in buffer systems, and very few studies have investigated the impacts of stress responses on growth or survival in food products. Secondly, many investigations have demonstrated that considerable variation in resistance to environmental conditions exists among different serovars and strains of pathogens, e.g., Salmonella (Humphrey et al., 1995; reviewed in Roberts et al., 1996). Although less well documented for other species, strain variation is also observed for other bacterial and fungal species, and the selection of standard universally accepted strains should be used to enable interlaboratory comparisons. Care must be exercised in extrapolating the resistance data and ability to induce stress responses among different species, serovars, and strains of related organisms. Lastly, several reports have been published in recent years on stress responses of microbes and their potential significance in foods, and it is not possible in this chapter to discuss each of these studies. To develop a comprehensive understanding for a single organism, the reader can refer to references in recent publications or conduct literature searches using available electronic databases. The interactions of factors affecting survival (Table 4.2) have long been known to affect the growth and survival of food-related organisms and their resistance to
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TABLE 4.2 Classification of Preservation Factors Affecting the Microbiological Safety and Quality of Foods Intrinsic Factors Chemical Nutrients Acidity and organic acids Oxidation-reduction potential Antimicrobial substances
Physical Water activity, relative humidity Ice and freeze concentration Structure of food Microstructure (e.g., emulsification)
Processing Factors Thermal heating and cooling Cold storage and freezing High pressure Irradiation Pulsed electric field treatment Ohmic heating Light treatment Sonic treatment Drying, reduction in water activity Modified atmosphere packaging Extrinsic Factors Change in relative humidity during storage Temperature during storage Gas levels (oxygen, carbon dioxide, other gases) Implicit Factors Microbial growth rates Synergistic effects derived from food components and intereactions among microorganisms Antagonistic effects derived from food components and intereactions among microorganisms (Modified from Gould, G.W., in The Microbiological Safety and Quality of Foods, Lund, B.M. et al., Eds., Aspen Publishers, Gaithersburg, MD, 2000).
processing procedures (Cole, 2001; Gould, 2000; Mossel and Ingram, 1955). Some early studies showed that exposure of salmonellae to low pH increased their acid tolerance (Baird-Parker et al., 1970; Huhtanen, 1975). It was also known that “injured” cells could be more readily recovered on rich media lacking antimicrobials (Baird-Parker and Davenport, 1965; reviewed in Hurst, 1977; Ray, 1986). Furthermore, cross protection induced by one antimicrobial resulting in resistance against other agents was also shown quite early (Szybalski and Bryson, 1952). Surprisingly, the concept of stress responses and their impact on virulence and survival of pathogens was not articulated until much later, particularly during the past two decades. The origin in understanding of stress response systems emanated
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from an increased understanding of fundamental microbial metabolism and physiology (Gottschalk, 1979; Ingraham et al., 1983), emerging to studies of basic physiological responses to specific stresses (Booth, 1985; Csonka, 1989). Experimental strategies and techniques that enabled the elucidation of multigene systems and regulons included the advent of two-dimensional polyacrylamide gel electrophoresis (2-D gels) for analysis of the proteome (Neidhardt, 1987; Neidhardt and Van Bogelen, 2000; O’Farrell, 1975), advances in molecular genetics (Beckwith and Zipser, 1970; Stent, 1978), and development of the fields of genomics and metabolomics. The importance of stress responses for survival of pathogens and spoilage microorganisms in foods and during food processing was not widely studied until recently, mainly during the past decade. Although studies of specific stress responses have only recently been applied to evaluation of food safety and quality, survival studies of pathogens in foods containing a variety of “barriers” have long suggested that microbial stress responses are important in food safety and quality. The barrier or “hurdle” (Leistner, 1995) approach has become popular for design of preservation and safety systems for foods. However, sublethal exposure of microorganisms to multiple stresses could also lead to adaptive responses and increased survival, as well as possible induced adaptive mutations conferring resistance to the barriers (Archer, 1996; Cairns et al., 1988; Rosche and Foster, 2000). These adaptive responses could permit growth under adverse conditions and actually be detrimental to the safety and quality of certain foods.
IMPACT OF STRESS RESPONSES ON PREHARVEST SURVIVAL OF FOODBORNE PATHOGENS Very little information is available regarding the impact of microbial stress responses on survival of microbes in preharvest plant and animal tissues. Plants are well known to induce responses to surface infections by bacterial and fungal pathogens and saprophytes (Hammerschmidt, 1999; Kuc, 1995). These responses include the induced synthesis of various classes of small molecular weight microbial inhibitors, including organic acids, phenolic compounds, terpenoids, flavonoids, alkaloids, and other classes of compounds (Billing and Sherman, 1998; Cowan, 1998; Hammerschmidt, 1999). In response to microbial infection, plants also induce the synthesis of peptides and enzymes, particularly cell wall hydrolases (Asselin, 1993; Lebeda et al., 2001), enzymes that generate reactive oxygen species (ROS) such as peroxidases (Baker and Orlandi, 1995), and defense peptides (Fritig et al., 1998). Pathogen virulence and ability to colonize plant tissues correlates with phytoalexin tolerance, mediated by detoxification of plant inhibitors and other mechanisms (Hammerschmidt, 1999). Bacteria and fungi could adapt to other stresses including organic acids such as salicylic and jasmonic acids (Verberne et al., 2000), small molecular weight phytoalexins (Hammerschmidt, 1999), lytic enzymes and defense peptides (Fritig et al., 1998; Lebeda et al., 2001), and ROS produced during the plant oxidative burst (Low and Merida, 1996), but little is known regarding microbial stress responses on preharvest tissues. Biological control by bacterial or fungal antagonistic organisms
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has been evaluated for prevention of growth of E.coli O157:H7 in apple wounds (Janisiewicz et al., 1999; Riordan et al., 2000), but this research is in its early phases and substantive conclusions on the efficacy of this intriguing approach cannot be made at this time. Mammals and their secretions (such as milk) also possess a diverse array of inhibitory peptides and enzymes, including defensins, lysozyme, lactoferrin, and peroxidases (Johnson et al., 1990; Kolb, 2001; Khush and Lemaitre, 2000; Low and Merida, 1996). The ability of food-related organisms to induce resistance to these agents has not been well studied, although some foodborne pathogens, including certain species of staphylococci and bacilli, are known to be naturally resistant to lysozymes (Hughey and Johnson, 1987). Salmonella enterica serovar Typhimurium has been shown to acquire resistance to organic acids during passage in the rumen (Kwon et al., 2000). These authors suggested that short chain fatty acids (SCFA) in the gastrointestinal tract (particularly the large intestine) of animals may increase the persistence of S. enterica serovar Typhimurium in the food animal and during pre- and post-harvesting handling of the animal. S. enterica serovar Typhimurium exposed to SCFA mixtures were more resistant to various stresses including acid (pH 3.0), increased osmolarity (2.5 M NaCl), and ROS (20 mM H2O2) than were cells exposed to SCFA from the small intestine. In contrast, hay-fed or grain-fed steers yielded E. coli O157:H7 populations with equal acid resistances (Hovde et al., 1999). Bacteria and fungi have also been demonstrated to induce resistance responses to ROS, antimicrobial peptides, and other antimicrobials associated with plants and animals in preharvest or during harvesting or slaughter (Brul and Coote, 1999). Very little is known about resistance to most naturally occurring antimicrobial compounds (Brul and Coote, 1999). More research is needed to elucidate the frequency and mechanisms of adaptation and resistance to natural antimicrobial agents.
STRESS RESPONSES AND THEIR IMPACT ON SURVIVAL OF GRAM-NEGATIVE FOODBORNE PATHOGENS AND SPOILAGE BACTERIA As discussed above, a variety of stress responses have been demonstrated or postulated to affect the resistance of foodborne pathogens to processing operations and growth and survival during food storage. Since stress responses have been shown to differ in various genera and species of pathogens, the responses of different species are discussed separately.
SALMONELLA Most studies of the influence of stress responses on survival of salmonellae and other pathogens have been conducted in media. Salmonella enterica serovar Enteritidis were more resistant to heat and acid when grown to stationary phase cells in the presence of glucose compared to cells grown in the absence of an added carbon source (Wilde et al., 2000). The presence of the sugar promoted production of acid
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with consequent acid habituation. The habituated cells were not more resistant to inactivation during air drying. Bunning et al. (1990) showed that S. enterica serovar Typhimurium subjected to heat-shock had substantially increased heat resistance in trypticase-soy/yeast extract broth compared to non-shocked cell populations, and they noted a complementary effect of anaerobiosis and heat shock on thermal resistance. Mattick et al. (2000) showed that S. enterica serovars Enteritidis PT4 and Typhimurium DT104 survived at low aw for long periods, and that rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. Incubation at an increased temperature of 37°C led to a greater rate of inactivation by the solutes. Interestingly, when subjected to osmotic stress, the two serovars formed long filaments, supporting that structural changes are involved in the stress response. The formation of filaments would have significant implications for methods of enumeration and microbiological monitoring. The authors concluded that the variable survival of Salmonella strains should be considered in predictive growth/survival modeling and in conducting risk assessments. In addition to studies in media, the induction of adaptive responses has been demonstrated to enhance the survival of Salmonella in adverse food environments. Acid adaptation of S. enterica serovar Typhimurium at a pH of 5.0 to 5.8 for one to two doublings enhanced survival compared to non-adapted cells during a milk fermentation and on incubation in various cheeses including cheddar, Swiss, and mozzarella cheeses kept at 5°C (Leyer and Johnson, 1992). These results supported the theory that acid adaptation is an important survival mechanism enabling Salmonella spp. to persist in fermented food products. Acid-adapted salmonellae were reported to have equal or greater acid and thermal sensitivity than control cells on lean beef tissue, suggesting that acid-adaptation did not result in bacteria resistant to organic acid rinses on beef (Dickson and Kunduru, 1995). Acid-shocked cells of multidrug-resistant S. enterica serovar Typhimurium DT104 did not show markedly different rates of thermal inactivation in various egg products (Jung and Beuchat, 2000). Other studies indicated that resistance to acid of salmonellae was not influenced by the pH of the food (Weissinger et al., 2000). It was concluded that the acid stress may affect certain strains of salmonellae to a greater extent than others, but the small differences in heat resistance could also be due to differentially expressed stress responses in the various strains. The variations in these studies suggest that the food matrix and composition influences stress responses and subsequent resistance properties of adapted cells. The studies also indicate the need to develop appropriate biomarkers such as expression of proteins or structural changes to clearly demonstrate the expression of a stress response and to distinguish stressed cells from injured cells. Lastly, it is apparent that the use of standard strains is necessary to accurately compare results from different laboratories. In addition to acid, other stress responses have been shown to affect resistance properties of Salmonella spp in vitro and in foods. Exposure of various Salmonella spp. to reduced aw increased subsequent heat tolerance (Bunning et al., 1990; Mattick et al., 2000). Reduced water activity has long been known to increase the thermal tolerance of various pathogens (Baird-Parker et al., 1970; Fay, 1934), although the mechanisms were not known at the time of these early reports. Recent work indicates
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that induction of stress response proteins (heat-shock proteins), including chaperones and ATP-dependent proteases, contributes to increased heat tolerance (Yura et al., 2000). In an interesting study, it was demonstrated that S. enterica serovar Enteriditis strains with greater thermal or hydrogen peroxide tolerance also survived longer on surfaces (Humphrey et al., 1995). In the same study, growth of the culture to the stationary phase increased resistance to heat, acid and hydrogen peroxide. Our laboratory made the unexpected observation that acid adaptation resulted in marked sensitization of S. enterica serovar Typhimurium to halogen-based sanitizers including chlorine (hypochlorous acid) and iodine (Leyer and Johnson, 1997). On the other hand, acid-adapted S. enterica serovar Typhimurium was more resistant to certain other classes of sanitizers. The sensitization of S. typhimurium to chlorine was attributed to alteration in structure and permeabilization of the outer membrane, reaction with essential sulfhydryl groups in proteins, and disruption of energy metabolism (Leyer and Johnson, 1997), but other mechanisms such as activation of signal transduction pathways by oxidation could also have contributed to sensitization. This study on enhancement of sanitizer action could provide a basis for innovative intervention strategies and technologies to inactivate Salmonella, and implies that acid pretreatment of food plant environments may increase the efficacy of halogen sanitizers.
ESCHERICHIA
COLI
O157:H7
Stress responses and cross-protection have been studied extensively in nonpathogenic E. coli (reviewed in Finkel et al., 2000; Lin and Lynch, 1996; Storz and Hengge-Aronis, 2000; Matin et al., 1989; Rowbury, 1995). These studies with traditional laboratory strains showed that acid “habituation,” nutrient starvation, and growth into the stationary phase yielded populations of cells that were more resistant to various stresses than were control populations. Although studies of nonpathogenic E. coli provided many basic insights into the physiology of stress responses, the discussion in this chapter focuses on E. coli O157:H7 because of its importance as a foodborne pathogen. Due to its significance as a pathogen, many studies of stress responses in E. coli O157:H7 and their potential impact on survival in foods have been performed, and representative studies are presented here. As with other tested foodborne pathogens, different isolates of enterohemorrhagic E. coli (EHEC) vary in their resistance to acid (Benjamin and Datta, 1995; Duffy et al., 2000). In general, this group of E. coli has relatively high acid tolerance similar to that of Shigella flexneri (Benjamin and Datta, 1995; Gorden and Small, 1993). Outbreak strains of EHEC were reported to have greater acid resistance than natural isolates (McKellar and Knight, 1999). Natural isolates of E. coli O157 also varied in resistance to hydrostatic pressure, heat, salt, hydrogen peroxide, and compounds causing membrane damage (Benito et al., 1999). As is the case with most other foodborne pathogens, EHEC cells grown to stationary phase had higher resistance to heat and acid than cells harvested during exponential phase. These variations in resistance properties depended on the strain and growth conditions. These results reinforce the importance of using resistant strains in evaluating the efficacy of food preservation treatments and in developing process criteria.
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Several researchers have evaluated the impact of stress responses on the resistance of E. coli O157:H7 in buffer and media systems. Exposure to acidic environments significantly increased the heat tolerance of various strains of E. coli O157:H7 (Ryu and Beuchat, 1999). D-values of acid-adapted cells were significantly higher than were those of acid-shocked or nonadapted cells. In concordance with these results, E. coli O157:H7 exhibited a pH-dependent, stationary phase acid resistance that increased the pathogen’s tolerance to heat (Buchanan and Edelson, 1999a). In adapted cells, the time needed to obtain a five-log reduction of viable cells in brain–heart infusion broth was increased two- to four-fold compared to nonadapted cells, depending on the pH. Increased heat-resistance was also observed in milk and chicken broth, but not with apple juice, indicating that the intrinsic parameters of a food affected the resistance properties of the adapted cells. The authors indicated that stress responses must be considered to accurately determine thermal tolerance of E. coli O157:H7 in various foods. The authors also evaluated the pH-dependent stationary-phase resistance of E. coli O157:H7 to various acidulants (Buchanan and Edelson, 1999b). Nine strains of E. coli O157:H7 differed markedly in their resistance to hydrochloric acid or various organic acids. The variation in resistance differed with the medium for recovery, and fewer numbers of cells were recovered on MacConkey agar compared to BHI agar. Hydrochloric acid was the least damaging to the cells, and lactic acid was most damaging, while acetic, malic and citric acids showed intermediate effectiveness. These results are surprising, as acetic acid has been shown in many other studies to be more effective than lactic acid against many foodborne pathogens (e.g., Roberts et al., 1996). The authors concluded that the accurate determination of survival of enterohemorrhagic E. coli in acidic foods must consider the strain and its ability to induce stress responses. The strain, type of organic acid, and acid adaptation or acid shocking were also shown to affect the acid resistance of E. coli O157:H7 in trypticase soy broth and in orange juice and apple cider (Ryu and Beuchat, 1998). Combinations of lactate, ethanol, and low-pH conditions were demonstrated to enhance the killing of E. coli O157:H7 (Jordan et al., 1999). In addition to enhancing acid and heat resistance, acid adaptation also increased resistance to other detrimental treatments. Radiation resistance of E. coli O157:H7 was dependent upon strain and the induction of acid-resistance (Buchanan et al., 1999). While pH during exposure had little effect on survival during irradiation by Cs-137, acid-resistance consistently enhanced radiation resistance. The results indicated that induction of a pH-dependent stationary phase response afforded crossprotection against irradiation, and such a response must be considered in determining irradiation D values in foods. Various studies have demonstrated that acid adaptation of E. coli O157:H7 enhanced survival in acidic foods including fermented dairy products, fermented meats such as shredded hard salami (Leyer et al., 1995), and in acidic fruit juices, particularly apple cider (Leyer et al., 1995; Miller and Kaspar, 1994). In contrast, acid adaptation was reported to decrease resistance of E. coli O157:H7 to 2% acetic acid spray in washing of carcasses (Berry and Cutter, 2000). Survival of E. coli O157:H7 in dried beef powder was also not significantly enhanced by acid adaptation, suggesting that this stress response did not afford cross protection against
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dehydration or osmotic stresses (Ryu et al., 1999a). As with most foods, dried beef powder has a complex composition and it is possible that other factors present in the food also significantly affected survival. This study supports that it is difficult to predict survival in many foods based on in vitro growth and survival responses, and that challenge studies in the food systems are required to adequately assess growth and survival of pathogens. Collectively, these various studies indicate that it is very important to precisely define the strain, its growth conditions, and the food environment to determine survival. It would be valuable for food microbiologists to reach a consensus on the use of strains, and to show their uniformity among labs by discriminating techniques such as pulsed field gel electrophoresis (Swaminathan et al., 2001) or other comparative typing methods. One aspect of stress adaptation that has received limited attention is the measurement of in vivo expression of stress-related genes in food systems. A method of RNA isolation and RT-PCR to detect E. coli O157:H7 was applied to detecting the pathogen on beef carcass surfaces (Berry, 2000). The method used a selectively inducible green fluorescent protein (GFP) reporter gene in a plasmid-transformed strain of E. coli O157:H7 inoculated onto beef carcass surfaces. Expression of stressrelated genes could be evaluated by quantitation of fluorescence in cells on the food surface. Although this procedure does not reflect naturally contaminated beef, it did show the probable locations of the bacteria on the meat. This approach using reporter gene technology should be useful in studying the genetic responses of the bacteria when exposed to stresses and antimicrobial interventions in various food systems.
SHIGELLA
SPP.
Since Shigella spp. are closely related to E. coli, they would be expected to show analogous stress responses. Indeed, the acid resistance response of Shigella has been considered to be analogous to that of E. coli (Gorden and Small, 1993), and a homologous alternative sigma factor, encoded by an rpoS allele, has been demonstrated in E. coli and S. dysenteriae (Small, 1994). An important stress response contributing to virulence in S. dysenteriae is acid tolerance (Small, 1994). Since the infectious dose of Shigella is low (10-200 CFU) in humans and is considerably less than that of E. coli and most other Enterobacteriaceae (Small, 1994), it is likely that other factors for Shigella virulence are involved in addition to acid resistance, but these determinants have not yet been identified. In reviewing the literature, published studies were not found on the impact of stress responses on growth and survival of Shigella in foods.
YERSINIA
ENTEROCOLITICA
Stationary phase cells of Y. enterocolitica showed increased resistance to acid, but the resistance was dependent on the presence of urea in the medium and an active urease enzyme (Koning-Ward and Robins-Browne, 1995). The catabolism of urea to form ammonia and carbon dioxide through urease activity probably provided a buffering effect that promoted gastric passage. Thus, Y. enterocolitica appears to have a unique mechanism of induced acid tolerance compared to most other Enterobacteriaceae.
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CAMPYLOBACTER
JEJUNI
Members of the genus Campylobacter are among the major causes of food-related bacterial gastroenteritis, and their incidence is increasing in several countries (Mead et al., 1999). Due to the success of Campylobacter spp. in eliciting gastrointestinal symptoms, it would be expected that Campylobacter spp. would elicit stress responses that promoted survival on food vectors and during passage in animal and human gastrointestinal tracts. However, available evidence indicates that Campylobacter spp. lack a stationary phase stress response analogous to most other Gramnegative foodborne pathogens (Kelly et al., 2001). Unexpectedly, resistance of Campylobacter jejuni to thermal stress (50°C) or aeration was greatest in the exponential phase of growth and declined in early stationary phase. Analysis of the recently available genomic sequence supports that C. jejuni NCTC 11351(Parkhill et al., 2000) lacks rpoS homologues that have been clearly demonstrated to be involved in various stress responses in Salmonella and E. coli (Hengge-Aronis, 2000). It is surprising that more research has not been published on C. jejuni, considering the importance of the organism in foodborne disease.
VIBRIO
PARAHAEMOLYTICUS AND
VIBRIO
CHOLERAE
Acid adaptation promoted resistance to various stresses in V. parahaemolyticus (Koga et al., 1999). Induction in vitro of an acid tolerance response (ATR) was demonstrated to increase infectivity of V. cholerae in a mouse model, but the ATR apparently was not induced during passage in humans (Merrell et al., 2002). The impact of stress responses on the survival of V. parahaemolyticus, V. cholerae, or other vibrios in foods appears to not have been studied. Again, such studies would be valuable in food and water microbiology since many vibrios are extremely important causes of food and waterborne disease.
PSEUDOMONAS
AERUGINOSA
An rpoS-dependent stress response has been demonstrated in the important spoilage organisms, P. aeruginosa, P. putida, and P. fluorescens (Ramos-González and Molin, 1998; Sarniguet et al., 1995; Suh et al., 1999). Mutations in rpoS affected heat resistance and virulence of P. aeruginosa in an animal model (Suh et al., 1999). The importance of stress responses for infection of plant substrates or in growth and survival in foods has not been reported in the pseudomonads.
GRAM-POSITIVE BACTERIA Much less is known about the molecular biology and practical food aspects of stress responses in Gram-positive bacteria than in their Gram-negative counterparts (Hecker and Völker, 2001; Price, 2000; Storz and Hengge-Aronis, 2000). The alternative sigma factor σB is mainly responsible for the induction of genes encoding stress proteins, although anti-sigma factors and other molecular mechanisms are operative in certain Gram-positive species.
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In general, Gram-positive bacteria are more resistant than Gram-negatives to environmental stresses including heat, irradiation, osmotic stress, and acid (Roberts et al., 1996) and thus the possibility that they can induce stress responses and further increase their resistance is a concern for food microbiologists. Several genera of Gram-positive bacteria including Bacillus and Clostridium are able to form endospores, which can impart extraordinary physical and chemical resistance properties to the organisms (Setlow and Johnson, 2001). Although sporulation can be considered a specialized and complex form of stress response, its coverage is beyond the scope of this chapter. The mechanisms of resistance and importance of spores in food microbiology has recently been reviewed (Setlow, 2000; Setlow and Johnson, 2001). The following discussion focuses on stress responses in important foodrelated Gram-positive vegetative organisms.
STAPHYLOCOCCUS
AUREUS
S. aureus has long been recognized as among the most osmotolerant foodborne bacterial pathogens, surviving or even growing in foods in the aw range of 0.88 to 0.91. Most strains of S. aureus only produce their characteristic enterotoxins at slightly elevated water activities (aw ~0.9) compared to the lower minimal aw that supports growth. Several investigators have shown that environmental and growth parameters influence the resistance properties of S. aureus. Shebuski et al. (2000) demonstrated that the growth medium had a marked effect on heat resistance of S. aureus. Growth of S. aureus at an aw value of 0.94 increased its thermal tolerance at 60°C. The authors also provided evidence for stress-induced heat-shock proteins as well as the accumulation of compatible solutes that contributed to enhanced thermal tolerance of S. aureus. The σB regulon has been identified and contributes to survival of S. aureus during harsh conditions including food processing and during food storage (Chan et al., 1998; Hecker and Völker, 2001). Staphylococcal thermonuclease, lipase, and α-hemolysin are hyperproduced in certain σB mutants (Hecker and Völker, 2001). Interestingly, overexpression of the σB operon led to thickening of the cell wall and resistance to beta-lactams (Morikawa et al., 2001), which implies that this response — characterized by thickening of the cell wall — could result in enhanced resistance to food processing procedures and to antimicrobials in foods. The hyper mutant acquired resistance to the lytic activity of lysostaphin, and had increased yields of carotenoids, which can protect microorganisms against ROS (Johnson and Schroeder, 1996). A chromosomal locus encoding several ORFs in S. aureus also contributed to oxidative defense and is induced by the cell-wall antibiotic oxacillin (Singh et al., 2001). Biofilm formation in S. aureus and S. epidermidis appears to be induced by stress responses. In S. epidermidis, formation of biofilms was influenced by ethanol and salt stress (Knobloch et al., 2001; Rachid et al., 2000). Salt stress resulted in biofilm formation on food packaging material (Le Magrex-Bebar et al., 2000). These studies clearly show that stress responses in food-related staphylococci can influence food safety and spoilage by increasing resistance properties and by inducing the formation of biofilms.
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LISTERIA
MONOCYTOGENES
Due to the importance of L. monocytogenes in foods, several studies have addressed its stress responses and associated resistance properties, though few studies have identified the roles that stress responses exhibit for survival in actual food systems. Induced acid tolerance has been demonstrated for L. monocytogenes (Davis et al., 1996; Hill et al., 1995; Kroll and Patchett, 1992; Phan-Thanh et al., 2000). As with other foodborne pathogens and spoilage organisms, expression of acid tolerance was growth phase dependent and varied according to type of organic acid exposure and the strain of the pathogen. Molecular studies showed that L. monocytogenes contains a σB controlled stress response (Becker et al., 1998) that is analogous to B. subtilis, and that induction of gene expression by σB contributes to enhanced osmotolerance, acid resistance and virulence (Becker et al., 1998; Hecker and Völker, 2001; Wiedmann et al., 1998). Adaptation of L. monocytogenes to sublethal stresses has been demonstrated to protect the pathogen on exposure to a variety of normally lethal conditions present in certain foods (Lou and Yousef, 1997). Sublethal exposure to ethanol (5% v/v), acid (HCl, pH 4.5 to 5.0) H2O2 (500 ppm) or NaCl (7% w/v) added to cultures during the exponential phase for 1 h protected against subsequent exposure to ethanol (17.5% v/v), NaCl (25% w/v), H2O2 (0.1%), or acid (pH 3.5). The authors emphasized that “stress hardening” should be considered in design and evaluation of food processing technologies for control of L. monocytogenes and other foodborne pathogens. In contrast to certain other bacterial species, acid adaptation of L. monocytogenes was initially reported not to provide cross-protection against an activated lactoperoxidase system (activated-LPS) (Ravishankar and Harrison, 1999). Later, it was reported that acid adaptation of L. monocytogenes with lactic acid did result in crossprotection against an activated-LPS in tryptic soy broth (Ravishankar et al., 2000). The enhanced survival of acid-adapted cells was greater at the lower pHs tested (3.5 and 4.0 compared to 4.5). These authors also provided evidence of the altered expression of several proteins in the acid-adapted cells. This latter study supports that stress responses of L. monocytogenes would enhance its survival in various dairy products and probably other foods in which ROS are formed. Although the role of acid-adaptation and other stress responses has been investigated in vitro, relatively few studies have addressed the impact of stress responses on survival of L. monocytogenes in foods. Acid adaptation increased survival of L. monocytogenes in acidic foods and during milk fermentation (Gahan et al., 1996; Hill et al., 1995). Heat shock of L. monocytogenes in log or stationary growth phases and in broth or minced meat affected thermotolerance of the pathogen (Farber and Brown, 1990; Jorgensen et al., 1999). The heat resistance following heat shock was dependent on growth phase, pH and lactic acid concentration. In particular, the concentration of lactic acid appeared to most strongly influence the heat resistance. The D-values at 60°C were two to six-fold higher in minced beef than in TPB, emphasizing the importance of food constituents on heat resistance. Heat resistance was found to be dependent on various conditions of culture including pH, acidulant, and growth temperature (Juneja et al., 1998). Our laboratory found evidence that pre-growth of L. monocytogenes in salt-enriched media increased its salt resistance in buffer systems and in commercial cheese brines (Larson et al., 1993, 1999).
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Since L. monocytogenes is more resistant than many foodborne pathogens to organic acids and other inhibitors, it can be difficult to control or eradicate in foods and in food processing facilities. Methods to sensitize the organism to inhibitors and sanitizers could be valuable for its control. In an interesting study, exposure of L. monocytogenes to ethanol sensitized the pathogen to low pH, organic acids, and osmotic stress (Barker and Park, 2001). The combination of organic acids, low pH and ethanol was effective as a listericidal treatment (Barker and Park, 2001). Our laboratory has shown that certain lipophilic terpenoids sensitized L. monocytogenes to various inhibitors compatible with foods (B. Brehm-Stecher and E. A. Johnson, 2001, unpublished data). In both of these studies, damage to the cytoplasmic membrane and increased leakiness appeared to initiate the killing. Other membrane active agents including glycerol monolaurate and hop beta acids are also strongly inhibitory to L. monocytogenes (Larson et al., 1996; Wang et al., 1992, 1993; Oh and Marshall, 1993). The cytoplasmic membrane has been suggested as an important target for cell inactivation in stressed cells of various food-related bacteria (Russell et al., 1995). Food-compatible agents that damage the cytoplasmic membrane of L. monocytogenes could lead to novel technologies for inactivation of this recalcitrant and persistent pathogen. On the other hand, alteration of the cell envelope could also promote resistance to various antimicrobial agents such as nisin (Davies et al., 1996). A simple method for evaluating the adaptive response of L. monocytogenes in foods has been proposed (Bolton and Frank, 1999) that, if verified for different strains of L. monocytogenes, could be quite valuable for assessing the physiological state of cell populations. A fertile research area should be the development of methods to quantitatively detect individual cells of various species of foodborne pathogens and spoilage organisms exhibiting in vivo stress responses in foods.
BACILLUS
SPP.
The σB regulon in Bacillus subtilis has served as a paradigm for the expression and control of stress-related genes in many Gram-positive organisms (Hecker and Völker, 2001; Price, 2000). Although such a regulon also would be expected to occur in food-related Bacillus species such as Bacillus cereus, very little information is available on the presence of these genes in food-related bacilli, and the role of such a stress system in food processing and during food storage has not been investigated. As mentioned above, the importance of endospores formed by bacilli, clostridia, and other endospore-formers on food safety and spoilage has recently been reviewed (Setlow and Johnson, 2001).
CLOSTRIDIUM
SPP.
Clostridia produce resistant endospores and are able to survive many food processing procedures. Several species can grow under low pH conditions and either spoil foods or produce harmful protein toxins such as C. perfringens enterotoxin and C. botulinum neurotoxins (Bahl and Dürre, 2001; Johnson, 2000; Rood et al., 1997). Despite the importance of clostridia to food spoilage and human disease, our understanding of stress responses is very rudimentary. Stress responses have mainly been studied
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at the physiological level and in nonpathogenic species (Woods and Jones, 1986). Very little published information is available related to the contribution of stress responses to clostridia in food systems. Acid shock markedly increased the thermal tolerance of C. perfringens (Villarreal et al., 2000). Heat adaptation at 46°C increased the heat resistance of C. perfringens, and Western blots provided evidence for the expression of at least four major putative heat shock proteins (Novak et al., 2001). Prior heat shock also increased the thermal resistance of ten strains of C. perfringens in a beef gravy system (Juneja et al., 2001). Heat-shock genes and chaperones have been shown to be expressed in the nonpathogen Clostridium acetobutylicum (Bahl et al., 1995). The involvement of protein phosphorylation has been detected in response to stress (Balidimos et al., 1990), possibly indicating a two-component kinase sensing and signalling system involved in the stress response of C. acetobutylicum. Two-component signalling systems have been implicated in stress responses and sensing of the environment in a large number of organisms. Considering the importance of toxigenic clostridia to food safety, it would be valuable for researchers to investigate stress responses in relation to food processing and survival during storage in this group of organisms. The genomic sequences that are becoming available for various clostridia including C. perfringens and C. botulinum should facilitate a genomic approach to identifying stress-related genes in these organisms. Our laboratory has found that a σB homolog is present in the C. botulinum type A genome based on the sequence posted on the Sanger web site (www.sanger.ac.uk). Further analysis should provide insight into the role of σB in protection against environmental stresses and in toxin production in C. botulinum.
IMPACT OF STRESS ADAPTATION ON THE PERFORMANCE OF BENEFICIAL MICROORGANISMS IN FOOD FERMENTATIONS The benefit of certain groups of microorganisms in food fermentations and preservation has long been appreciated (Adams and Nout, 2001; Pasteur, 1866; Pederson, 1976; Steinkraus, 1996). The major beneficial groups of microorganisms important in food fermentations are yeasts, particularly Saccharomyces cerevisiae and related fermentative ascomycetous yeasts, and lactic acid bacteria (LAB). Stress adaptation and tolerance responses have been observed in a variety of fungi (Jennings, 1993), but have been most extensively studied in laboratory and industrial strains of S. cerevisiae (Brul and Coote, 1999; Hohmann and Mager, 1997). The ability of S. cerevisiae to tolerate environmental stresses has been deemed a key to optimal performance in certain industrial fermentations such as baking, brewing, winemaking, and distiller’s fermentations (Attfield, 1997). During these fermentations, yeasts are exposed to a variety of environmental stresses including elevated temperatures, oxygen radicals and other oxidants, hyperosmolarity, dessication/rehydration, and freezing/thawing. S. cerevisiae has evolved to rapidly respond to environmental stresses (Attfield, 1997). Compared to bacterial responses, yeasts and fungi have different molecular mechanisms leading to stress adaptations, particularly the maintenance of intracellular © 2003 by CRC Press LLC
levels of ATP for repair of cellular damage and for expulsion of H+ by the activity of plasma membrane ATPase. S. cerevisiae also rapidly activates various signalling pathways to the cytoplasm and nucleus in stressful situations including those involving RAS-adenylate cyclase, protein kinase A, and MAP-kinase (Brul and Coote, 1999; Hohmann and Mager, 1997; Mager and De Kruijff, 1995; Ruiz and Schüller, 1995). Exposure to heat and oxidative stresses also leads to molecular damage in fungi, and ROS appear to play a central role in stress-induced injury in yeast, particularly in conditions of low water activity and RH, dehydration, freezing, heat, and presence of organic acids and alcohols (Attfield, 1997; Costa et al., 1997; Davidson et al., 1996; Moradas-Ferreira et al., 1996). Industrial strains of baker’s yeast are much more resistant to ROS compared to most laboratory strains (Attfield, 1997). The molecular mechanisms of high levels of resistance to ROS have provided experimental strategies for rational development of improved yeast strains for industrial fermentations (Attfield, 1997). The response of fungi to osmotic stress is one of the better understood adaptive responses (Attfield, 1997; Hohmann and Mager, 1997). Exposure of yeast to hyperosmotic stress involves expression of genes governing glycerol/polyol biosyntheses, efflux of cations, as well as a general stress response (Attfield, 1997). Accumulation of glycerol and certain other polyols is essential for tolerance of hyperosmotic stress. Lastly, the accumulation of high levels of trehalose (10 to 15%) leads to robustness and enhanced ability of yeast to withstand dehydration and freezing in industrial preparation of starter strains, and for rapid availability of energy during inoculation of new fermentations. Lactic acid bacteria (LAB) are used for a variety of food fermentations, such as in the production of fermented dairy products, where they may encounter a variety of stresses such as heat, acid, salt, low oxygen tension, and antimicrobial agents. The molecular aspects of stress responses in LAB are covered elsewhere (Duwat et al., 2000; Klaenhammer et al. in Chapter 6 of this book; Sanders et al., 1999). The study of stress responses in Lactococcus lactis is increasing in order to optimize its fermentation performance (Kim et al., 1999; Sanders et al., 1999). Various beneficial phenotypic traits of LAB in food fermentations such as rapid acidification, selective proteolysis, tolerance of osmotic and stresses, resistance to ROS, and ability to thrive in nutrient poor conditions and at low temperatures are influenced by stress responses in various species of LABs (O’Sullivan and Condon, 1997; Sanders et al., 1999). Furthermore, adaptation to a particular stress can lead to tolerance of other stresses (Sanders et al., 1999). In Lactococcus lactis, the ability to withstand freezing and lyophilization is an important attribute of lactic acid bacteria developed as starter cultures. The ability of commercial L. lactis ssp. lactis and L. lactis ssp. cremoris to withstand freezing at –60°C for 24 h was significantly improved by a prior 25 min heat shock at ~40°C or by a 2 h cold shock at 10°C (Broadbent and Lin, 1999). Thermal stress treatments also enhanced the resistance of several LAB strains to lyophilization. Analysis of membrane fatty acid composition suggested that enhanced resistance to freezing and lyophilization may be related to cell membrane lipid composition, but that other factors were involved as well in the enhanced resistance properties. Other bacterial fermentation or spoilage LAB including Leuconostoc mesenteroides (McDonald et al., 1990), Lactobacillus delbrueckii subsp. bulgaricus (Lim et al.,
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2000) and Lactobacillus alimentarius (Lemay et al., 2000) elicit stress responses. It will be valuable to determine which stress responses contribute most importantly to desired fermentation parameters in actual food systems, as this will facilitate the development of strains with optimal fermentation characteristics.
CROSS-PROTECTION AMONG MICROBIAL STRESS RESPONSES Cells adapted to a particular stress such as acid often show enhanced survival on subsequent exposure to a different stress such as heat or salt. This toleration of distinct stresses after adaptation to an individual stress has been termed cross-protection. A variety of phenotypic properties have been shown to be afforded by cross-protection including virulence, cell morphology, antibiotic and food antimicrobial resistance, resistances to heat, acid, salt, surface active agents, and nutritional requirements (Archer, 1996; Leyer and Johnson, 1993; Rowan, 1999). Acid-adaptation induced cross-protection against heat, salt, an activated lactoperoxidase system (generating ROS), and surface active agents in Salmonella enterica serovar Typhimurium (Leyer and Johnson, 1993). Acid adaptation also increased cell-surface hydrophobicity, which could affect the ability of Salmonella to bind to surfaces and to form biofilms. Similarly, in Vibrio parahaemolyticus, acid adaptation induced cross-protection against heat, crystal violet, bile and deoxycholic acid (Koga et al., 1999). Acidadapted salmonellae showed increased resistance to organic acid rinses on beef surfaces (Dickson and Kunduru, 1995). Since general stress responses are actively being elucidated in many species of Gram-negative and Gram-positive bacteria, it is expected that many more species will be found to induce cross-protection against chemical and environmental stresses. It is anticipated that cross-protection will markedly affect the survival of pathogens in foods containing multiple barriers. Rowe and Kirk (1999) emphasized that understanding the food conditions leading to stress and the kinetics of stress responses would enable more accurate estimates of risk assessment and development of adequate safety systems in food processing operations. A long-standing and debatable phenomenon is whether certain classes of mutations in microorganisms occur randomly as initially proposed by Luria and Delbrück (1943), or whether mutations are induced by selections encountered in stressful environments (see Cairns et al., 1998; Rosche and Foster, 2000). Cairns et al. (1998) showed that Lac+ mutations accumulated with time when Lac- cells were grown in selective conditions for lactose utilization. Archer (1996) and Rowan (1999) have emphasized that adaptive mutations induced by stress could have considerable importance in food safety.
CONCLUSIONS AND PERSPECTIVES Study of stress responses in foodborne pathogens and in organisms involved in beneficial fermentation organisms is a relatively new field. There is little doubt that stress responses in these organisms have a marked impact on food safety and quality. Unfortunately, there is a paucity of studies on the practical importance of stress
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responses of microorganisms in actual food systems, and much more research is needed. For these studies, standard strains and growth conditions should be used among laboratories for concise comparison of the results. Stress responses probably have a much greater impact on food microbiology than is currently appreciated. For example, many food processes and formulations are tested for safety by inoculation with cells harvested from exponential phase. However, cells grown to the stationary phase or adapted to various stresses will have greater resistance than exponential cells. Our studies support the hypothesis that it is important in laboratory food challenge studies and in testing of food processing procedures to use stressed or adapted cells, since the use of healthy exponentially growing cultures may inaccurately represent their survival state in the actual food environment (Leyer and Johnson, 1992, 1993). The survival of foodborne pathogens in certain acidic food products such as mayonnaise, salad dressings and sauces (Smittle, 2000) may warrant re-evaluation using acid-adapted populations of cells. Similar considerations should be given to foods stabilized by brining or by many other food preservation processes that could trigger stress responses. The physiological stress state of the pathogens may also affect current trends in risk assessment, predictive modeling, HACCP programs, and in reaching a food safety objective. The practical research of stress responses in foods could be complemented by the elegant and currently more extensive studies of the molecular biology of these responses. For example, it is becoming possible with the development of genetic tools in many foodborne pathogens to create isogenic mutants, and strains can be constructed that possess mutations in single genes involved in specific and general stress responses. These mutants could then be evaluated for their survival of food processing operations and for survival during storage of foods. Newer applications of stress response analysis include comparative genomic and proteomic views of stress responses in microbial species (Koonin et al., 2000; Neidhardt and VanBogelen, 2000) and the utilization of stress responses for environmental monitoring and molecular toxicology (LaRossa and van Dyck, 2000). The rapidly increasing availability of complete genome nucleotide sequences (Koonin et al., 2000; Wodicka et al., 1997) and amino acid sequences of expressed open reading frames (ORFs) (Neidhardt and VanBogelen, 2000) in food-related organisms will enable the identification of genes and proteins involved in stress responses and evaluation of their importance in the physiology of food-related organisms. For example, the complete genome of a strain of S. cerevisiae has been sequenced with the identification of over 6000 ORFs and 5800 predicted protein coding regions (Dujon, 1996). About one-third of these ORFs have completely unknown function (Attfield, 1997; Dujon, 1996; Goffeau, 2000). As emphasized by Attfield (1997), much more research is needed in physiological research for industrial strains of yeast within the context of industrially relevant conditions. An understanding of physiological and genetic determinants of stress responses and industrial performance will provide rational methods using classical breeding approaches or recombinant DNA technology for improvement of microbial strains. The use of genome-wide expression monitoring (Wodicka et al., 1997) will also be useful for following the temporal expression of food microorganisms as they encounter stresses and induce adaptive responses during industrial food fermentations.
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The food industry has long applied combinations of sublethal intrinsic, extrinsic, and implicit inhibitory factors for the control of undesirable organisms in foods and to promote the growth of desirable microbes. As the public demands foods with enhanced freshness and appeal, the industry is using milder processes and reducing levels of antimicrobials in many food products. In turn, this results in more frequent exposure of pathogens and spoilage organisms to sublethal stresses, which could induce resistance responses and compromise the safety and shelf-life of these food products. This trend further supports the need for practical studies of stress in food microbiology, risk assessment, and preventive programs. Although the study of stress responses and their impact on food safety and quality is in its gestation phase for most food-related organisms, increased study should result in our understanding of the microbiology of food systems and enhancement of the safety and quality of our food supply.
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5
Adaptation or Resistance Responses of Microorganisms to Stresses in the Food Processing Environment Sadhana Ravishankar and Vijay K. Juneja
CONTENTS Introduction Biofilms Bacterial Attachment Stages in Biofilm Formation Methods of Studying Biofilms Control of Biofilms Chemical Control Biological Control Chemical Stress Acidic and Alkaline Treatments Phosphates and Other Chemicals Ozone Sanitizer Stress Chlorine and Chlorinated Compounds Nonchlorinated Compounds Sanitizer Stress Adaptation and Cross-Protection Metal Ion Stress Links to Antibiotic Resistance Adaptation to Heavy Metal Ions and Cross-Protection Antibiotic Stress Cross-Resistance The authors wish to acknowledge Dr. Ravishankar Palanivelu for his assistance with the figures. The authors are also grateful to Drs. Sizer, Slade, and Palumbo for critical review of the manuscript, and to Ms. Vasuhi Rasanayagam for her assistance with the citations.
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Other Stresses Adaptation to Starvation Stress Adaptation to Osmotic Stress Cross-Protection Adaptation to Oxidative Stress Cross-Protection Conclusions References
INTRODUCTION Foodborne bacteria are exposed to a variety of stresses in the environment. Oftentimes, they are able to tolerate such stresses, survive and/or grow in food and cause spoilage as well as illness. If the stress is mild, it causes injury to the bacteria and if it is severe, it causes inactivation. Injured bacteria in food are of concern, since they can revive when favorable conditions are encountered, as well as multiply and grow in food. Such mild stresses are very often encountered by the bacteria in food as well as in the food processing environment. For instance, with the present day consumer demand for fresh-like, preservative-free food products with good nutritional quality, minimal processing is done in which mild treatments are given to the food product. The bacteria once exposed to a mild stress are able to tolerate further severe stresses. This ability of the bacteria is called stress adaptive response (SAR) or stress hardening, which enables the bacterium to resist further homologous as well as heterologous stresses (Yousef, 2000). In the food processing environment, several treatments are given to the food to preserve its quality as well as shelf-life. The environment and the equipment used for processing in a plant handling wet processes are regularly or periodically cleaned to keep them, as well as the processed food, contamination free. Under such conditions bacteria are exposed to a variety of chemicals, sanitizers, heavy metal ions, antibiotics, etc. If these treatments are not severe enough, the bacteria survive and are able to adapt to even harsher treatments. These bacteria can form microcolonies on the equipment surfaces or other areas of the plant which, in course of time, form biofilms. Also, there are certain areas either in the equipment or other places in a plant that are inaccessible or hard to reach for cleaning, and the bacteria escape treatment. These areas are also most vulnerable for biofilm formation. Once biofilms have been formed it becomes very difficult to eradicate them. For instance, Listeria monocytogenes is a foodborne pathogen well known for its presence in processing plants (Smoot and Pierson, 1998; Cox et al., 1989) and for the formation of biofilms, due to which the food industry has incurred heavy losses especially in dairy (Mafu et al., 1990) and processed ready-to-eat meat products. This bacterium is easily disseminated by aerosols and contaminated food products in the processing plants (Cox, 1996) and can survive in aerosols (Spurlock and Zottola, 1991). When this bacterium forms biofilms it has an enhanced resistance to sanitizers (Frank and Koffi, 1990). Listeria monocyotgenes was isolated from domestic, retail and industrial refrigerators in Greece (Sergelidis et al., 1997). Pathogens
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such as Listeria, Staphylococcus aureus and Salmonella were isolated from a poultry abattoir in South Africa (Geornaras et al., 1997) and Salmonella from healthy swine and from abattoirs in Brazil (Lázaro et al., 1997). In a poultry slaughtering facility the main airborne contaminants (bioaerosols) were bacteria and the highest count was found in the shackling area and decreased toward the packaging area (Lutgring et al., 1997). The microbial load of the floor after cleaning in different types of food processing facilities was assessed and it was determined that a milk site had the lowest load followed by the pastry site where the load decreased initially after cleaning and then increased, while a meat site had the highest load with a rapid increase (Mettler and Carpentier, 1998). Salmonella serovars were isolated from a citrus processing plant both inside the premise (in the juice as well as surface of the fruits) and outside the premise, from amphibians captured outside the plant (Parish, 1998). In plants handling wet processes, enough moisture and other favorable conditions to promote microbial growth are found. In plants where processing takes place under dry conditions, bacteria are able to enter the plant through air, raw ingredients, worker traffic and other means. They are exposed to dehydration and are able to adapt to such conditions, survive and contaminate food products. For instance, Salmonella has been a problem due to its contamination from the plant environment in dry dairy products, grain products, chocolate products and others, causing several recalls (Gabis and Faust, 1988). Aerobic spore-formers such as Bacillus species are the predominant microflora in the food packaging material such as paper and board and these bacteria were found to produce enzymes which degrade papermaking chemicals and were resistant to industrial biocides (Väisänen et al., 1989). In dry processing plants, bacteria may encounter areas where there is lack of nutrients. In such conditions bacteria are exposed to starvation stress (Figure 5.1). Some other stresses that a bacterium may encounter in a processing environment during exposure to chemicals, sanitizers, or otherwise, include oxidative stress, osmotic stress, acidic/alkaline stress, etc. (Figure 5.1). According to Bower and Daeschel (1999), the resistance responses of bacteria in food environments are conferred by various factors including innate structures such as impermeable outer membrane of bacterial cell, mechanisms for antibiotic inactivation, and biofilm formation on food processing surfaces as an adaptive response to prevent the hazardous effects of cleaners and sanitizers. Antibiotic resistant bacteria present in food animals, exhibiting cross resistance to biocides, entering a food processing plant may pose a contamination risk to the processed product. Antibiotic residues present in meat and milk have contributed to the development of resistant bacteria (Brady and Katz, 1992; Brady et al., 1993). In this chapter, various aspects dealing with the adaptation as well as resistance responses of bacteria to various stresses in the food processing environment such as chemicals, sanitizers, metal ions, starvation and antibiotics will be discussed.
BIOFILMS In food processing plants, microorganisms are able to attach to solid surfaces and form microcolonies. These, along with various nutrients, minerals and organic matter, deposit together forming biofilms. Biofilms are defined as bacterial populations © 2003 by CRC Press LLC
FIGURE 5.1 Various stresses encountered by bacteria in a food processing environment.
adhering to a surface or to each other in aggregates enclosed by a matrix of polysaccharides (Poulsen, 1999). The organisms become sessile in a biofilm and the matrix forms a protective barrier against the effect of antimicrobial agents. Biofilms serve two purposes for the forming microorganisms: protection from hostile environment as well as acting as a trap for nutrient acquisition (Mattila-Sandholm and Wirtanen, 1992). Bacteria in a biofilm are exposed to starvation, dehydration and oxidative stresses. As a result of these exposures bacteria adapt to these stresses. In addition this exposure may cross-protect the bacteria against other stresses. Stress signaling and cell to cell communication occur in a biofilm community. These could be possible reasons for the enhanced resistance of biofilm bacteria compared to those that are in a planktonic state. Hence biofilms and their resistances are discussed in this chapter. Biofilms have been described as self-regulating in that they give rise to other biofilms (Hood and Zottola, 1995). Once the biofilm grows in size, it breaks into pieces allowing for more cells to attach to each piece of the matrix, which then grows in size. This breaking up process could be attributable to possible alteration in the properties of cell surface or attached substratum (Marshall, 1992). Food processing plants have abundant exposed surfaces available and accessible for biofilm formation and a typical biofilm can contain in excess of 107 cells/cm2 (Holah and Kearney, 1992). Persistent L. monocytogenes isolated from poultry and ice cream plants were able to show enhanced adherence capabilities to stainless steel even with a short contact time (1 h) (Lunden et al., 2000). Thus they can become a real threat to the processing industry. Biofilms have created problems in the industry in everything from corroding water pipes to computer-chip malfunctions (Potera, 1996).
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BACTERIAL ATTACHMENT All spoilage and pathogenic microorganisms can form biofilms under suitable conditions. However, bacteria belonging to the genera Pseudomonas, Enterobacter, Flavobacterium, Alcaligenes, Staphylococcus and Bacillus have a tendency to form biofilms more than others (Mattila-Sandholm and Wirtanen, 1992). It may take several hours to days for the microorganisms to form a biofilm on a surface. Bacteria adhere at different rates to different substrates as described by Marshall (1992). Some bacteria possess the needed structures such as fimbriae or pili as well as other extracellular polymeric substances and are able to attach quickly to surfaces. This is called passive adhesion involving a physico-chemical reaction. Others require prolonged exposure to attach to a surface, whereby a physiological response is needed and this is called active adhesion. Initially at this stage the bacteria are not very firmly attached, still showing some Brownian movement, and can be removed by moderate shear. A transition from loose to firm attachment occurs causing irreversible adhesion. Some bacteria attach more efficiently when starved, while others attach efficiently when nutrients are available. Listeria monocytogenes was able to compete and form biofilms with 8 other species at low nutrient levels at 10°C on stainless steel coupons, and a Flavobacterium species stimulated the biofilm formation of L. monocytogenes (Jeong and Frank, 1994). Listeria monocytogenes was also able to form biofilms under nutrient limiting conditions involving a chemically defined minimal medium with certain nutrients such as mannose, trehalose and tryptone enhancing biofilm development, and alteration of phosphate to levels other than those present in minimal medium reduced biofilm development (Kim and Frank, 1995). Pseudomonas putida cells that grew very slowly in a flow chamber biofilm were able to grow faster when provided with a readily metabolizable carbon source (Sternberg et al., 1999). A particular strain of Pseudomonas attaches immediately on high energy surfaces but slowly on low energy surfaces (Marshall, 1992). Bacteria have their own approaches of attachment initiation to surfaces and involve specific microbial surface structures such as flagella. Flagella may be directly required for attachment and initiation of biofilm formation or indirectly required through enabling mobile organisms to reach a surface as well as move along a surface in a developing biofilm (Pratt and Kolter, 1998). Escherichia coli cells lacking flagella or having paralyzed flagella were unable to initiate the initial biofilm formation (Pratt and Kolter, 1998). It has been observed that biofilms have capillary water channels through which water and nutrients are transported and distributed to various areas (Costerton et al., 1995). These capillary water channels also serve to transport oxygen to the inner areas of the biofilm (Poulsen, 1999). The inner areas of the biofilm, however, may receive low oxygen due to the diffusion limitations and exhaustion of oxygen by the outer areas and thus it is possible that both aerobic and anaerobic species of microorganisms can thrive in a biofilm (Poulsen, 1999). Also, the properties of the substratum surface such as surface free energy, charge, roughness or toxicity towards a specific microbial species influence the types or species of microorganisms colonizing a surface (Marshall, 1992). In case of microorganisms with different mor-
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phological structures, variants with rough morphology were able to attach better and form biofilms than variants with smooth morphology (Pringle et al., 1983; Herald and Zottola, 1988; Sasahara and Zottola, 1993). There is either competition or cooperation among certain bacterial species as well as coaggregation with certain species in mixed biofilm populations (Geesey et al., 1992). Allison et al. (1998) found that cell to cell signaling mechanisms involving homoserine lactones promote surface attachment and biofilm formation and that enzymatic degradation of extracellular polymeric substance is involved in detachment and dispersal of biofilms under conditions of starvation. Chemical signals that are freely diffusible and employ N-acyl homoserine lactones involved in biofilm development and maintenance also have been found in the case of other Gram-negative bacteria (Davies et al., 1998; Heys et al., 1997). Listeria monocytogenes grown in a medium with lactic acid showed enhanced adhesion to stainless steel and cells appeared more hydrophobic when attached to stainless steel at 4°C (Briandet et al., 1999). Sasahara and Zottola (1993) found that L. monocytogenes alone was not able to attach to glass while P. fragi could. When mixed with P. fragi culture, L. monocytogenes was able to attach to glass and form biofilms, and the authors suggested that L. monocytogenes, a non-exopolymer producer, needs an exopolymer producing organism such as P. fragi for attachment and this is more important than other requirements such as hydrophobicity, surface charge or flagellar mobility, particularly for attachment to glass. Stainless steel surfaces (reactive) were found to be colonized and attached by 10-fold more coliforms and heterotrophic bacterial cells when compared to an inert surface such as polycarbonate (Camper et al., 1996). However, if stainless steel slides were replaced with polycarbonate slides, these could harbor the same amount of bacteria as did stainless steel. Growth media and conditioning of the attaching surface were found to be contributing factors in the attachment of P. fragi, S. typhimurium and L. monocytogenes to stainless steel (Hood and Zottola, 1997a). Environmental factors such as temperature and pH affected the attachment of L. monocytogenes to stainless steel and Buna-N rubber, with rate of adhesion being slow at alkaline conditions (Smoot and Pierson, 1998a). The addition of trypsin to attachment medium decreased the adhesion by 99.9%, implying that proteins are involved in the initial attachment of this organism to surfaces (Smoot and Pierson, 1998b). Aeromonas hydrophila was able to attach to stainless steel and form biofilms at 28°C, but not at 42 or 4°C and this could be a problem in seafood and aquaculture processing plants if effective sanitation measures are not applied (Farid et al., 1998). In a poultry processing plant, bacteria from the rinse of whole broiler carcasses were found to attach to stainless steel and form biofilms (Arnold, 1998). In such a biofilm, mixed populations of E. coli and S. aureus were found. When grown as a pure culture separately, E. coli cells formed extracellular fibrils while S. aureus did not (Figure 5.3). However, in mixed culture, the formation of extracelluar fibrils was different from what was seen in pure culture. Thus the physiological behavior of the bacterial species changes in a mixed population biofilm and each species is able to adapt and grow in a competitive environment. Gram-negative bacteria were better able to adhere to glass and form biofilms with a higher population than Gram positives (Sommer et al., 1999). E. coli cells
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FIGURE 5.2 Initial steps in biofilm formation.
attached and formed biofilms on stainless steel and the formation was affected by the nutrient conditions of the medium used (Dewanti and Wong, 1995). These authors found that in a nutrient limiting medium, the cells were shorter and more hydrophobic due to starvation stress, but an extensive and thicker extracellular matrix was produced when compared to those grown in nutrient rich medium. On chlorinated polyvinyl chloride pipe and glass surfaces, Klebsiella pneumoniae, Salmonella enteritidis and E. coli attached and formed biofilms, with K. pneumoniae forming the most populated and metabolically active biofilm followed by S. enteritidis and then E. coli, respectively (Jones and Bradshaw, 1996). L. monocytogenes produced copious amounts of attachment fibrils, while E. coli did not, on stainless steel surfaces after one week incubation (Mustapha and Liewen, 1989). Biofilm formation in meat processing plants was studied by fixing stainless steel chips adjacent to food contact surfaces and cast iron chips in floor drains, and Pseudomonas, Klebsiella, Aeromonas and Hafnia were found to produce biofilms (Hood and Zottola, 1997b). Microorganisms attached to vegetable surfaces are not completely removed by washing and minimal processing and thus can grow and form biofilms during storage (Carmichael et al., 1999). Bacteria were able to attach and form biofilm on the surface of lettuce with pseudomonads being the dominating microflora (Carmichael et al., 1999). On apples, E. coli O157:H7 was observed at depths up to 70 µm below the skin, with greater numbers on puncture wounds and greater attachment levels on the intact skin, lenticels, russet areas and floral tubes (Burnett et al., 2000).
STAGES
IN
BIOFILM FORMATION
The process of biofilm formation occurs in several stages. The initial steps in biofilm formation are depicted in Figure 5.2. The various stages involved in biofilm formation explained in the proposed theories are shown in Table 5.1. Marshall et al. (1971) explained it to be a two stage process. The first stage is called the reversible stage, in which the bacterial cells are in close proximity, but not yet in actual contact with the substrate or surface, and they are held close together by electrostatic and hydrophobic interactions and van der Waals forces. The second stage is called the irreversible stage, in which the cells attach to the surface by producing complex polysac-
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FIGURE 5.3 Escherichia coli and Staphylococcus aureus in pure (A and B) and mixed culture (C) biofilm attached to stainless steel. The products of extracellular fibrils in single culture biofilm are different from that of mixed culture biofilm. (From Arnold, J.W. 1998. Poultry Avian Biol. Rev., 9(1):1–9. With permission.)
TABLE 5.1 Various Stages in Proposed Biofilm Formation Theories
Stages Reversible adhesion Irreversible adhesion Transport of material to surface Adsorption Consolidation Adhesion Co-adhesion Adaptation Growth and biofilm formation Colonization and biofilm formation Detachment and dispersal
Marshall et al., 1971
Busscher & Weerkamp, 1987; Notermans et al., 1991
Characklis & Cooksey, 1983; Characklis, 1984; Ganesh Kumar & Anand, 1998
Gilbert et al., 1993
Bos et al., 1999
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Characklis, 1981
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charides and the attachment involves hydrophobic, dipole–dipole, ion–ion, covalent bonds and hydrogen interactions. The cells multiply forming microcolonies and eventually form biofilm with cells embedded within the polysaccharide matrix. Many of the chemical and physical characteristics of the matrix, such as binding to metal ions and viscoelastic behavior, depend on the type of polysaccharide (Christensen, 1989). A three stage process has been proposed by Busscher and Weerkamp (1987) and Notermans et al. (1991), which involves 1) adsorption of cells to the surface, 2) consolidation involving electrostatic interactions, van der Waals forces and other interactions, and, finally, 3) colonization.
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Characklis (1981) described a four stage process of biofilm formation, involving 1) transport of the molecules to the surface by diffusion or turbulent flow, 2) adsorption of the molecules at the surface, 3) adhesion of microorganisms to the surface using van der Waals, electrostatic and hydrophobic interactions, and 4) colonization resulting in biofilm formation. A five stage process of biofilm formation has also been described (Characklis and Cooksey, 1983; Characklis, 1984; Ganesh Kumar and Anand, 1998). The five stages include 1) transport of organic and inorganic material to the surface and conditioning of the surface, 2) adsorption of conditioning film and adhesion of microorganisms, 3) attachment of cells, growth and formation of microcolonies 4) formation of biofilm and bacterial metabolism within the film and 5) detachment and dispersal of biofilms. Gilbert et al. (1993) describe the process of surface colonization in six steps as follows: 1) arrival of bacteria in close proximity to the surface, 2) reversible adhesion to the surface, 3) irreversible adhesion to the surface, 4) adaptation to an attached phenotype, 5) growth and division, microcolony and biofilm formation, and 6) dispersion. Bos et al. (1999) provide an excellent review of the physico-chemistry of microbial adhesive interactions in which they describe the initial steps in biofilm formation in six stages: 1) adsorption of conditioning film components, 2) microbial transport and aggregation, 3) adhesion of single organisms and microbial coaggregation, 4) coadhesion between microbial pairs, 5) exopolymer production and establishment of firm irreversible adhesion, and 6) growth. All these models explain a similar phenomenon but in different ways.
METHODS
OF
STUDYING BIOFILMS
Bacteria in the biofilms are quantitated by different methods such as swabbing, rinsing, agar flooding, agar contact (Ganesh Kumar and Anand, 1998), microscopic beads (Oh and Marshall, 1995), scraping (Frank and Koffi, 1990; Costerton and Lappin-Scott, 1989) and vortexing (Mustapha and Liewen, 1989; Oh and Marshall, 1995). Another method utilizing Robbins device has been described and used in which biofilms are formed on small coupons (metal or plastic studs) simulating a surface, by exposing them to large amounts of liquid for about 3 to 4 weeks, after which the studs are removed and the amount of biofilm formed is estimated (Costerton and Lappin-Scott, 1989; Jass et al., 1995). The Robbins sampler also could be used to determine the concentrations of antimicrobial agents needed to inactivate biofilm bacteria and hence can be useful in designing such treatments (Costerton and Lappin-Scott, 1989). Other techniques of monitoring biofilms include epifluorescence microscopy (Wirtanen and Mattila-Sandholm, 1993; Blackman and Frank, 1996), scanning electron microscopy (Blackman and Frank, 1996; Farid et al., 1998), environmental scanning electron microscopy (Little et al., 1991), scanning confocal laser microscopy (Caldwell et al., 1992; Debeer et al., 1997; Carmichael et al., 1999; Sternberg et al., 1999), light section microscopy (Marshall et al., 1989) atomic force microscopy
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(Beech, 1996), interference reflection microscopy (Marshall et al., 1989), nuclear magnetic resonance (Blenkinsopp and Costerton, 1991), Fourier transformation infrared spectrometry (Nichols et al., 1985; Bremer and Geesey, 1991; Cheung et al., 2000), quartz crystal microbalance (Nivens et al., 1993) and cellular automation (Wimpenny and Colasanti, 1997). Some researchers have used the direct viable count methods for enumeration of biofilms and assessing the efficacy of disinfection (Lytle et al., 1989; Feipeng et al., 1993; Leriche and Carpentier, 1995). The impedance method was the most effective enumeration method for biofilm bacteria due to its ability to quantitate both reversibly and irreversibly attached cells while some other methods require additional steps to remove reversibly attached cells (Mosteller and Bishop, 1993). Control of the growth rate of biofilms has been achieved using the Perfused biofilm fermenter (Gilbert et al., 1989) and Sorbarod filters (Hodgson et al., 1995). Methods for studying biofilms have been reviewed by Ladd and Costerton (1990) and Nivens et al. (1995).
CONTROL
OF
BIOFILMS
In the food processing industry biofilms are a source of pre- or post-processing contamination and hence care should be taken to avoid the formation of biofilms. Proper cleaning and sanitation are needed to avoid formation or to eradicate the formed biofilm. The areas of the processing equipment most prone to biofilms include gaskets made of Buna-n rubber or Teflon, pipe elbows, caps in dead-end areas, the vacuum breaker near the pasteurizer, backplates of pumps, conveyer belts, as well as drains, floors and other stainless steel surfaces (Czechowski, 1991). Food processing surfaces such as stainless steel, nylon, Teflon and polyester floor sealant support the growth of L. monocytogenes biofilms, with polyester floor sealant and stainless steel allowing the most, Teflon allowing an intermediate and nylon allowing the least formation when incubated at 21°C in tryptic soy broth (Blackman and Frank, 1996). Chemical Control If the process conditions are different from those required for optimum growth of microorganisms, biofilm formation can be prevented (Poulsen, 1999). However, in many processing conditions, this is not the case and so other methods such as maintaining proper hygiene or frequent cleaning and sanitizing are required. Frequent cleaning with a gap of about 8 h was found to remove the attached organisms easily and prevented biofilm formation (Zottola, 1994). The best way to control biofilms is to effectively clean and then sanitize the surfaces to which they are attached. Initial cleaning with a detergent should be done in such a way that it dissolves the biofilm and removes it from its attachment site. A sanitizing step following this will inactivate the surface attached microorganisms. Factors affecting the efficacy of a sanitizing agent include the type of the sanitizer, concentration used, cleaning temperature and time, flow rate of the sanitizing solution, hardness of water for diluting the sanitizer, age of the biofilm, and the type of surface to be cleaned (Czechowski, 1991). Disinfection of K. pneumoniae attached to glass surfaces by chlorine depended on the surface, biofilm age, encapsulation and nutrients, while disinfection © 2003 by CRC Press LLC
by monochloramine was only affected by surfaces (LeChevallier, 1988a). It is a generally accepted fact that inactivation of biofilms is difficult since the sanitizer or the toxic compound has to penetrate the exopolysaccharide barrier. However, Nichols (1989) proposed that the cells deeply embedded in a biofilm may have a different cellular physiology compared to the cells at the surface and hence their sensitivity to a particular sanitizer or toxic substance may be different from those at the surface. A number of researchers have investigated the efficacy of various disinfectants and sanitizers in controlling biofilms. Chlorine and monochloramine were tested for their efficacy against planktonic and attached organisms (Yu et al., 1993). During formation of biofilms there was no change in the susceptibility of the planktonic cells to these disinfectants. There was no difference between the resistance of attached and planktonic cells. Monochloramine proved to be more effective in eliminating the attached organisms than free chlorine. Monochloramine was more effective in penetrating the biofilm and inactivating bacteria while free chlorine reacted with a number of compounds before it penetrated into the biofilms (LeChevallier et al., 1988b). In another study with Enterobacter cloacae, about 50% of the cells survived exposure to 0.5 mg chlorine per liter and multiple exposures to chlorine did not increase resistance of the cells (Lytle et al., 1989). In the same study it was found that 25 mg chlorine was better in inactivation than 0.5 or 2 mg. Maintaining 1 mg/L free chlorine residual was not sufficient to control coliform biofilms in water distribution pipelines (LeChevallier et al., 1987). Chlorine (0.5 mg/L) was ineffective in removing the total bacterial population in the biofilm formed in a water distribution system consisting of coliforms, K. pneumoniae and other bacteria (Morin et al., 1996). Monochloramine at 2 mg/L was ineffective in controlling K. pneumoniae and P. aeruginosa biofilms (Huang et al., 1995). These authors also studied the respiratory activity of these biofilms during disinfection with monochloramine and they found that there was spatially nonuniform loss of respiratory activity with less activity near the biofilm–bulk fluid interface and more activity near the substratum or in the center microcolonies. The reason was attributed to transport limitation and the depletion of the sanitizer near the substratum or center of microcolonies as compared to the surface. Wirtanen and Mattila-Sandholm (1992) studied biofilm formation of P. fluorescens, L. monocytogenes and B. subtilis and found that at least 2 h are needed for biofilm formation and that the resistance of the formed biofilms to chlorine was higher when formed in milk when compared to meat medium. Older biofilms of Pseudomonas were more resistant to chlorine and this resistance was attributed to changes in the metabolic activity of bacteria in the biofilms or production of extracellular compounds that can react with chlorine, preventing its diffusion (Sommer et al., 1999). In the same study the authors also found that both large and small biofilms had similar resistance to chlorine, and they attributed the reason to the formation of microcolonies having similar resistances. Mechanically scrubbing and cleaning the surface along with disinfectants is one effective way of eliminating biofilms. In a study by Exner et al. (1987) various chemical disinfectants were tested for their efficacy in controlling biofilms against mechanical cleaning. Aldehydes and chlorine were effective at 10% concentration, peracetic acid at 0.5% and hydrogen peroxide at 1.5% with 30 min to 1 h exposure
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times. Mechanical cleaning was the most effective with scanning electron micrographs of treated biofilms showing clean surfaces and no microorganisms being isolated after the treatment. In a food factory environment, biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa were most effectively eliminated by mechanical cleaning using a high pressure spray and mechanical floor scrubber than by chemical methods involving detergents (Gibson et al., 1999). In the same study applying an alkaline, acidic or neutral detergent prior to water spray did not prove effective in removing biofilms. The acidic detergent was most bactericidal towards S. aureus causing about a six-log reduction, while alkaline detergent was bactericidal towards P. aeruginosa, causing four- to five-log reductions. The authors concluded that since mechanical spray and scrub have potential to disseminate bacteria through aerosols, it might be more effective to utilize a mechanical treatment followed by chemical methods such as using bactericidal detergents to eliminate the contamination. Bacteria in a biofilm are more resistant to the effect of sanitizers than the free living cells and, the older the biofilm, the greater the resistance to sanitizer treatments. Oh and Marshall (1995) found that a combination of monolaurin at 50 µg/ml and heating at 65°C for 5 min effectively controlled L. monocytogenes adhered on to stainless steel. They also found that planktonic cells were more sensitive to the treatment than attached cells; 1-day-old biofilms were more sensitive than 7-day-old biofilms; and resistance in a nutrient rich environment was better than that in a nutrient lacking environment. Attached cells of L. monocytogenes and S. typhimurium were found to be more resistant to trisodium phosphate than the planktonic cells (Somers et al., 1994). Attached cells of B. subtilis and P. aeruginosa on stainless steel and polyurethane surfaces were more resistant to iodophor, peracetic acid and hydrogen peroxide mixture, and chlorhexidine gluconate than the planktonic cells (Lindsay and von Holy, 1999). Cells of S. aureus, E. coli, S. enteritidis and L. monocytogenes attached to PVC, Teflon, Plexiglas, wood, rubber and stainless steel were more resistant to QAC (quaternary ammonium compound) sanitizer than non-attached ones (Dhaliwal et al., 1992). A non-foaming acidic sanitizer and a hypochlorite sanitizer were not effective on P. putida biofilms formed on rubber surfaces in that they caused merely injury; resuscitation of the samples had growth equal to those of untreated controls (Chumkhunthod et al., 1998). E. coli O157:H7 was able to attach to meat grinder surface from contaminated meat and the surfaces that came in contact with the processed meat having the lowest fat level were the hardest to clean; chlorine and peroxyacetic acid sanitizer effectively reduced the bacterial count but following enrichment, injured cells were recovered (Farrell et al., 1998). Rinsing with superheated water was not effective in removing Bacillus biofilms from Teflon and stainless steel; however increasing the alkaline phase wash of alkaline-acid treatment and addition of EDTA to the alkaline detergent proved effective (Wirtanen et al., 1996). The effects of chemical treatments including electrolytes, dimethyl sulfoxide and Tween 20 on microbial adhesives and biofilm matrix polymer using P. aeruginosa were investigated using interferon reflection microscopy and light section microscopy (Marshall et al., 1989). There was contraction of the microbial polymers and adhesives during treatment with electrolytes and expansion during treatment with Tween 20. The dimethyl sulfoxide treatment caused contraction on the adhesive
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but had no effect on biofilm polymer. It was concluded that both adhesive and polymer have acidic groups and undergo hydrophobic reactions. However, it was unclear as to the difference in the behavior of the adhesive and polymer to dimethyl sulfoxide reaction, whether it was due to differences in their structure or due to their proximity to glass surface. Both pure and mixed culture biofilms of P. flourescens and B. cereus were equally affected by sanitizers such as chlorine, iodophor, peracetic acid, acid anionic and fatty acid sanitizers; however, pure culture biofilms of Y. enterocolitica were more resistant to these sanitizers than when they were in a mixed culture (Mosteller, 1993). Sodium periodate, cetyltrimethylammonium bromide, and sodium hydroxide inhibited attachment of P. fragi to stainless steel, and sodium dodecylsulfate, sodium periodate and sodium hydroxide effectively removed attached cells (Herald and Zottola, 1989). Aeromonas hydrophila biofilms on stainless steel surfaces were inactivated by chlorine with exposure to 25 ppm for 1 min for 8-h biofilms, and 75 ppm for 1 min for 8-day-old biofilms (Farid et al., 1999). Peroctanoic acid (Cords, 1993) was more effective on binary species biofilms of P. aeruginosa and L. monocytogenes than peracetic acid and chlorine on stainless steel coupons (Fatemi and Frank, 1999). Ozone was suggested as a potential disinfectant against P. flourescens biofilms, where the ozone treated cells appeared non-intact and shriveled (Bott, 1991). The effect of 58 chemical compounds including antibiotics, detergents, surfactants, nutrients and inorganic and organic acid salts on swarming (movement of elongated swarm cells across a surface) of Bacillus species was studied and it was found that most of the compounds tested inhibited swarming through their action on flagellar mechanisms and motility (Thampuran and Surendran, 1996). Since motility is an important feature facilitating bacterial biofilm formation, these chemical compounds could play a role in impeding motility and thus biofilms. A buffered organic acid anionic acid surfactant (containing citric acid, EDTA, sodium lauryl sulfate and an anionic detergent) exhibited sanitizing efficiency by inactivating S. aureus, S. typhimurium, L. monocytogenes and P. aeruginosa on formica countertop surfaces, both in the presence and absence of protein (Restaino et al., 1994). QACs and chlorine sanitizer effectively reduced S. aureus populations on abraded and smooth stainless steel and polycarbonate surfaces but not on mineral resin (Frank and Chmielewski, 1997). A number of sanitizers (iodophors, amphoterics, QAC, chlorine, Biguanide) were assessed for efficacy using a surface disinfectant test against attached cells of P. aeruginosa, S. aureus and Proteus mirabilis and attached cells were found to be 10 to 100 times more resistant than free cells (Holah et al., 1990). Chlorine (25 ppm), QAC (20 ppm) and iodine(12.5 ppm) were effective against P. fluorescens, Staphylococcus haemolyticus and Bacillus in a simulated water coolant system at 25 and 4°C (Overdahl and Zottola, 1991). Attachment of P. aeruginosa and Staphylococcus epidermidis to glass and stainless steel coupons was loosened by treatment with disinfectants such as sodium hypochlorite, Dodigen™, and sodium dodecylsulfate, while Tween-80 strengthened the attachment of S. epidermidis to stainless steel (Eginton et al., 1998). Some lactic acid bacteria are capable of producing ropy slime on the surface of cooked meat products and vacuum packaged meats. The efficacy of sanitizers was tested against the ropy slime forming lactobacilli, and QACs, peracetic acid sanitizers
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and hydrogen peroxide were found effective, while chlorine sanitizers and polyhexamethylene biguanide chloride were ineffective (Mäkelä et al., 1991). Biological Control Apart from detergents and sanitizers, the use of enzymes for biofilm control has been investigated. Johansen et al. (1997) tried a combination of enzymes to break up Staphylococcus and Pseudomonas biofilms on steel and polypropylene. A combination of oxidoreductases and polysaccharide hydrolyzing enzymes was found to be the most effective in removing biofilm as well as being bactericidal. A combination of glucose oxidase and lactoperoxidase was bactericidal, but did not remove biofilms, and a mixture of polysaccharide hydrolyzing enzymes removed the biofilm but did not prove to be bactericidal. Other enzymes that have been used for controlling biofilms include proteases (Aldridge et al., 1994), cellulase (Wiatr, 1990), polysaccharide lyases (Sutherland, 1995) and lactoperoxidase (Thomas et al., 1983). The antimicrobial peptides magainins and defensins were found to be bactericidal toward rough strains of S. typhimurium compared to smooth ones (Rana and Blazyk, 1989). Bacterial cells respond to an antimicrobial peptide by altering their membrane composition (Brul and Coote, 1999). L. monocytogenes cells showing resistance to nisin demonstrated enhanced levels of zwitterionic phosphatidylethonalamine and lowered levels of anionic phosphatidylglycerol and cradiolipin (Crandall and Montville, 1998). The phospholipid membrane composition of nisin resistant L. monocytogenes was different from that of nisin susceptible strains (Verheul et al., 1997). Treatment of stainless steel coupons with skim milk before inoculation of microorganisms reduced the attachment of the organisms, and individual milk proteins such as α-casein, β-casein, κ-casein and α-lactalbumin inhibited the adhesion of S. aureus and L. monocytogenes (Barnes et al., 1999). Concanavalin A inhibited the attachment of P. fragi to stainless steel, and trypsin was effective in removal of attached cells (Herald and Zottola, 1989). Nisin films adsorbed onto silica surfaces prevented the growth of L. monocytogenes while the organism was able to grow on a non-nisin silica surface (Bower et al., 1995). Nisin spray was effective in reducing populations of Gram-positive bacteria such as Brochothrix thermosphacta, Carnobacterium divergens and Listeria innocua on the surface of beef carcass tissue (Cutter and Siragusa, 1994a). Pratt and Kolter (1998) found that α-methyl-d-mannoside inhibited biofilm development on polycarbonate, polystyrene and borosillicate glass, and hence mannose could be used in antimicrobial treatments to treat and prevent biofilms in the food processing plants. Bdellovibrios, predatory microorganisms that can grow within the periplasm of Gram-negative bacteria and prey upon them, have been found to be capable of removing E. coli O157:H7 and Salmonella attached on the surfaces of food processing equipment (Fratamico and Cook, 1996).
CHEMICAL STRESS Bacteria are exposed to a variety of chemicals in the food processing plant environment. They may adapt to the chemical stresses during this exposure and exhibit
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enhanced resistance to the chemicals they are further exposed to. In E. coli and other enterobacteria extracellular induction components called alarmones present in the environment warn the organisms of any chemical stresses in the environment by cross talk leading to induction of tolerance responses or adaptation in the organisms (Rowbury, 2001). The response of the bacteria to chemicals depends on various factors: the type of the chemical and the bacteria, the concentration of the chemical, exposure type, and other physical, chemical and biological environmental factors (Levine and Case, 1997). The efficacy of acetic acid spray wash against E. coli O157:H7 on beef surface depended on the initial inoculum level and the inoculation menstrum but not on the spray temperature or tissue type (Cutter et al., 1997). Any chemical compound causing microbial inactivation is also referred to as biocide (Russell and Russell, 1995). Biocides have enhanced activity at elevated temperatures, and other factors influencing biocidal activity include: presence of non-ionic surfactants, materials such as polyvinyl chloride or nylon to which a preservative agent may bind, or rubber in which the agent may partition, thereby resulting in reduced effect (Russell, 1992). The Gram-positive cocci are generally more susceptible to biocidal action than Gram-negative cocci. Excellent literature reviews on effects of chemicals and biocides on microorganisms have been published (Levine and Case, 1997; Levine and Black, 1996; Levine and Rachakornkij, 1994; Russell and Rusell, 1995). Some of those specific to foodborne microorganisms and their actions are summarized in Table 5.2. Nirmalakhandan et al. (1994) studied the toxic effects of a variety of chemicals alone and in combinations and found that the effect was additive. In their study they also developed an approach to analyze and predict chemical toxicity using the molecular structural features of the constituent chemicals. The toxicities of selected industrial chemicals to microorganisms and aquatic organisms were compared and it was found that aquatic organisms were more sensitive to these chemicals than microorganisms (Vaishnav and Korthalis, 1990). Some possible reasons for the low sensitivity of microorganisms were 1) the presence of cell wall and capsule barriers through which the chemical has to travel to reach the cell membrane or the target component, 2) endospore formation under adverse conditions, 3) spontaneous mutations, and 4) ability of microbial cells to detoxify certain chemicals. These are also some possible reasons for the adaptation and resistance of bacteria to chemical stresses in the food processing plants. When Leuconostoc mesenteroides was exposed to ethanol and sodium arsenite, there was an increase in the expression of 70 and 60 kDa proteins while the total protein synthesis was reduced (Salotra et al., 1995).
ACIDIC
AND
ALKALINE TREATMENTS
Some of the bacterial control methods used in the food industry include acidic and alkaline treatments. Organic acid sprays and rinses are used to reduce or eliminate surface contamination. Oftentimes, bacteria develop resistance to these treatments which could arise from adaptation. Hot acid sprays of 1.5% acetic, citric and lactic acids were ineffective in reducing E. coli O157:H7 populations on beef surface (Brackett et al., 1994). Lactic acid was found to be ineffective in reducing E. coli and Salmonella typhimurium populations on beef (Anderson and Marshall, 1990).
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TABLE 5.2 Mechanism/Action of Various Chemical/Biocidal Compounds on Microorganisms Compound Alcohols
Antimicrobial peptides, proteins and enzymes Carbanyl cyanide m-chlorophenyl hydrazone Chlorhexidine diacetate
Chlorine
Cycloserine D Dinitrophenol
Ethidium bromide Ethylenediamine-tetraaceticacid Ethylene oxide Glutaraldehyde
Hexachlorophane
Hydrogen peroxide Iodine compounds Long chain polyphosphates Mercury compounds Naturally occurring compounds from plants A. Spice essential oils B. Isothiocyanates C. Thymol P-aminobenzoic acid Parabens Peracetic acid Phenols
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Mechanism/Action Leakage of cytoplasmic constituents, disruption of membrane, inhibition of membrane ATPase and transport proteins Cell wall hydrolysis, inhibition of protein synthesis, cell membrane disruption Inhibition of electron transport and oxidative phosphorylation Cell wall disruption, protoplast/spheroplast lysis, leakage of cytoplasmic constituents, inhibition of membrane ATPase, coagulation of DNA, RNA, protein Inhibition of mRNA, protein synthesis and oxidative phosphorylation, release of dipicolinic acid from spores, spore coat and cortex degradation Inhibition of cell wall protein synthesis Dissipation of membrane proton motive force (pmf), inhibition of transport and enzymes in the cytoplasm and membranes Inhibition of transcription Cell wall disruption Alkylation of proteins and nucleic acids Binding to cell wall components and inhibition of essential functions, cross linking of proteins, RNA and DNA, reaction with spore outer layer Protoplast/spheroplast lysis, leakage of cytoplasmic constituents, cytoplasmic coagulation, interference in electron transport Oxidation of DNA, RNA, proteins and lipids, removal of coat protein, lysis of spore protoplast Oxidation of sulfhydryl groups of proteins and disruption of protein structure Cell wall binding and chelation of metal ions Reaction with enzyme thiol groups Membrane perturbation, membrane rupture Oxidative cleavage of disulfide bonds and inactivation of extracellular enzymes, formation of reactive thiocyanate radicals aiding in oxidation of biomolecules Plasma membrane disruption Inhibition or interference with peptidoglycan layer synthesis Dissipation of proton motive force, inhibition of transport and enzymes in cytoplasm and membrane Disruption of thiol groups Leakage of cytoplasmic constituents
TABLE 5.2 (continued) Mechanism/Action of Various Chemical/Biocidal Compounds on Microorganisms Compound Quaternary ammonium compounds Silver salts Weak organic acids
Mechanism/Action Disruption of cell wall and leakage of cytoplasmic constituents, coagulation of DNA, RNA, protein Reaction with sulfhydryl groups of proteins to form mercaptides Membrane disruption, inhibition of metabolic reactions, accumulation of anions and protons inside the cell, thereby affecting the intracellular pH homeostasis, dissipation of pmf
(Adapted from Brul, S. and Coote, P., Int. J. Food Microbiol., 50, 1, 1999; Lee, R.M. et al., 1994; Russell, A.D. and Russell, N.J., Symp. Soc. General Microbiol., 53, 327, 1995.)
E. coli was found to be resistant to 3% acetic acid on beef (Greer and Dilts, 1992). E. coli O157:H7 was more resistant to acetic acid compared to S. typhimurium and L. monocytogenes on beef (Dickson, 1991). Acetic and lactic acids were ineffective while fumaric acid was effective in reducing populations of E. coli O157:H7, S. typhimurium and L. monocytogenes on beef (Podolak et al., 1995). However, acid adapted Salmonella strains were sensitive to lactic acid rinse on beef (Dickson and Kunduru, 1995). Lactic acid (2 to 5%) when used to decontaminate meat increased the generation times of Yersinia enterocolitica and L. monocytogenes by up to twofold while at 1% there was no effect (van Netten et al., 1997). Acetic, lactic and citric acids at 1, 3 and 5% concentrations brought about one- to two-log reductions in E. coli O157:H7 populations but did not inactivate the pathogen completely on beef tissue, while greater reductions were achieved for P. fluorescens with the same treatments (Cutter and Siragusa, 1994b). Acetic and lactic acids and trisodium phosphate reduced the counts of E. coli O157:H7, L. innocua and C. sporogenes on beef surface to less than 1.3 logs (Dorsa et al., 1997). A combination of sodium hydroxide and acetic acid was effective in eliminating L. monocytogenes biofilms attached to glass, while a combination of sodium chloride with acids was not effective (Arizcun et al., 1998). Catfish fillets inoculated with L. monocytogenes were dip treated with various acids and such treatments resulted in 16-, 7.5-, 4.3-, 3.7- and 3.4-fold reductions in the population of the organism with tartaric, succinic, acetic, malic and propionic acids, respectively, while no effect from tannic acid treatment was seen (Marshall and Bal’a, 1995). Fumigation of mung bean seeds with gaseous acetic acid (242 µl acid/l of air for 12 h at 45°C) inactivated E. coli O157:H7 and S. typhimurium, but not L. monocytogenes (Delaquis et al., 1999). Microorganisms grown in mild acid or alkalinity in broth were able to resist stronger acidic or alkaline conditions and habituation to acid in broth can occur in as small a duration as 15 min and to alkalinity in 30 to 60 min (Rowbury et al., 1989). This shows that the time required for bacteria to adapt to stresses could be very © 2003 by CRC Press LLC
short. Tolerance to alkaline (pH 10 to 11.5) conditions was induced in E. coli by adapting at pH 8.5 to 9.5 and this induction required protein synthesis, which was inhibited by glucose and amiloride but not by L-leucine, FeCl3 or FeSO4 (Rowbury et al., 1996).
PHOSPHATES
AND
OTHER CHEMICALS
A variety of chemical treatments were used to inactivate pathogens and spoilage organisms on chicken skin (Hwang and Beuchat, 1995). Trisodium phosphate (1%) and 1% lactic acid were effective in reducing populations of Salmonella species, L. monocytogenes and psychrotrophs from chicken skin. Addition of 5% Tween 80 and 0.05% sodium hydroxide to trisodium phosphate effectively reduced Salmonella species and psychrotrophs but not L. monocytogenes, due to its resistance to alkalinity of sodium hydroxide. Ten percent solutions of sodium tripolyphosphate, monosodium phosphate, sodium acid pyrophosphate or sodium hexametaphosphate were not effective against Salmonella species or L. monocytogenes. Trisodium phosphate and cetylpyridinium chloride with a high pressure spraying action reduced S. typhimurium on chicken skin by 1.5 to 2.5 logs (Wang et al., 1997). Trisodium phosphate, cetylpyridinium chloride, acetic acid and grapefruit seed extract brought about 1.5- to 2.0-log reduction in S. typhimurium populations on chicken skin (Xiong et al., 1998). Cetylpyridinium chloride was effective against E. coli O157:H7, S. typhimurium and mesophilic bacteria on beef lean and adipose tissue surfaces after cleaning and during storage (Cutter et al., 2000) and against S. typhimurium on chicken skin (Breen et al., 1997). Trisodium orthophosphate alone and in combination with KCl at 3% level and 60 sec contact time caused 99.6% reduction in the surviving populations of P. aeruginosa, B. cereus and Moraxella osloensis in fish and shell fish (Bender and Brotsky, 1993). Trisodium phosphate was more effective than sodium tripolyphosphate and sodium metaphosphate in reducing total coliform and aerobic plate counts on catfish frames (Marshall and Jindal, 1997). Trisodium orthophosphate at 4 to 12% concentrations and alkaline pH (>11.5) was also effective in reducing or retarding growth of Salmonella and Campylobacter on poultry (Bender and Brotsky, 1994). Trisodium phosphate was effective in reducing planktonic as well as biofilm populations of E. coli O157:H7, Campylobacter jejuni and S. typhmurium but not L. monocytogenes on stainless steel and Buna-N rubber (Somers et al., 1994). In the same study it was found that biofilms of S. typhimurium and L. monocytogenes formed on stainless steel were more resistant to trisodium phosphate treatment than the ones formed on Buna-N rubber. Trisodium phosphate was better than acetic acid and phosphate buffered saline rinses in reducing levels of E. coli O157:H7 and K12 on beef tissue (Fratamico et al., 1996). Trisodium phosphate was effective on 48-h-old S. enteritidis biofilms on glass flow cells, but not on 72-h-old biofilms; bacteria within small crevices that were artificially created on the substrate escaped treatment (Korber et al., 1997). Long chain polyphosphates (0.5 to 1%) were found to inhibit Clostridium tyrobutyricum growth in processed cheese spreads (Loessner et al., 1997). The antibacterial
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mechanism of long chain polyphosphates was studied using S. aureus as the test organism and it was found that these compounds act by binding to the cell wall and chelate metals and remain in a bound state (Lee et al., 1994b). Sodium chlorate was found to be bactericidal towards E. coli O157:H7 and S. typhimurium DT104 in rumen contents in vitro (Anderson et al., 2000). These organisms possess respiratory nitrate reductase, which can reduce chlorate to toxic chlorite and hence, this compound may have antimicrobial action against other bacteria possessing these enzymes. A water wash followed by acidified sodium chlorite effectively reduced the populations of E. coli O157:H7 and S. typhimurium on beef carcasses; however, bacteria were still detected after treatment in 30 to 50% of the carcasses (Castillo et al., 1999). A combination of hot water wash and lactic acid spray was effective in removing E. coli O157:H7, S. typhimurium and other Enterobacteriaceae on beef carcasses (Castillo et al., 1998). Triclosan (an antimicrobial agent used in hand soaps and other toiletries) incorporated plastic showed antimicrobial activity against a number of bacteria including E. coli, Bacillus species, S. typhimurium, S. aureus and Shigella flexneri, but not on meat surfaces, probably due to the presence of fatty acids (Cutter, 1999). Sodium hexametaphosphate combined with hop resins inhibited the growth of E. coli in laboratory medium and mashed potatoes, and the mechanism might be due to damage to the cell membrane; however, complete inactivation was not achieved (Fukao et al., 2000)
OZONE Recently, ozone has gained popularity as a disinfectant due to its several advantages over chlorine: 1) the reaction of ozone with organic compounds does not produce toxic or carcinogenic compounds, unlike chlorine; 2) ozone is unstable and so it does not persist in the environment after use; 3) the cost of ozone generation unit and its maintenance is comparable to or less than cost of chlorine compounds; and 4) ozone does not require heat thereby saving the cost of power consumption (Greene et al., 1993). Compared to chlorine, lower concentrations and shorter contact times are required for inactivating microorganisms by ozone (Kim et al., 1999). The potential applications of ozone as a disinfectant in a processing plant include: controlling microbial growth in water recirculating systems such as cooling, washing, and product fluming operations; its role in clean in place systems to control microbial growth; and controlling microbial growth on surfaces exposed to the environment (Bott, 1991). Ozone acts on the bacterial cell membrane causing cell lysis and on sulphydryl groups of bacterial enzymes as well as on bacterial nuclear region. Gram-negative bacteria were more sensitive to ozone than Gram-positive ones; however effectiveness of ozone was reduced in food based systems such as milk or meat broths (Moore et al., 2000). Ozonated water was compared to chlorine for its effectiveness towards inactivating organisms in milk biofilms and ozone was found to be as effective as chlorine (Greene et al., 1993). Ozone caused a 5.6 and 4.4 log reduction, while chlorine brought about 4.6 and 4.2 log reductions in populations of P. fluorescens and Alcaligenes faecalis, respectively. The various applications of ozone in food processing are discussed by Kim et al. (1999).
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SANITIZER STRESS In a processing plant, sanitizers are used daily to reduce microbial load, minimize access to microbes and prevent contamination. However, we still hear about the source of contamination causing outbreaks arising from the processing plant. One possible reason is bacteria escaping or surviving the sanitizer treatment and contaminating the food. Surviving the sanitizer treatment may arise from adaptation to sanitizer stress and becoming more resistant to the treatment. Non-psychrotrophic pathogens present in food industry premises are probably the most resistant to disinfectants though they might be present in few numbers, and this resistance could be due to their exposure to adverse conditions in such an environment (Leriche and Carpentier, 1995).
CHLORINE
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CHLORINATED COMPOUNDS
There are several sanitizers available for use in the food industry of which chlorine and chlorinated compounds form the active ingredient. The inactivation by chlorine has been described to involve inhibition of mRNA and protein synthesis, and oxidative phosphorylation in bacterial cells (Bunduki et al., 1995). Dychdala (1991) provides more information on the mechanisms of action of chlorine on bacteria. Chlorinated water is often used in the food industry for washing fresh produce and other commodities. Dipping of fresh cut watercress, onions and potatoes in chlorine solution increased the microbial counts during storage and this could have been due to chlorine damaging the produce cut tissues, which could cause cellular fluid release that can promote microbial growth (Park and Lee, 1995; Gunes et al., 1997). Lettuce inoculated with E. coli O157:H7 from bovine feces was washed with chlorinated water and it was found that washing with 200 ppm chlorinated water was not more effective than a water wash with 1 and 5 min hold time (Beuchat, 1999). Neither tap water nor hypochlorite was effective in removing Pseudomonas and other Enterobacteriaceae from lettuce leaf surface (Adams et al., 1989). It is possible that the organisms lodge in inaccessible areas such as stomata and cut edges and escape treatment. The efficacy of chlorinated water in inactivating E. coli O157:H7, Salmonella or L. monocytogenes on whole apples, tomatoes and lettuce was studied (Beuchat et al., 1998). Chlorine at 2000 ppm was effective in inactivating the pathogens on these vegetables. There was a difference in the inactivation of Salmonella and L. monocytogenes using 200 and 2000 ppm chlorine on apples while the two treatments were similar in inactivating E. coli O157:H7 on both apples and tomatoes. On lettuce leaves chlorine treatment was not as effective as on other produce. There was about a log difference between water wash and chlorine wash (200 ppm chlorine). Chlorine at 60 and 110 ppm was effective in removing Salmonella montevideo on raw tomatoes (Zhuang et al., 1995). Chlorine at 200 ppm reduced the population of E. coli O157:H7 on fresh and cut edges of lettuce by about one log, but total inactivation was not achieved (Takeuchi and Frank, 2000). When fresh cut lettuce and cabbage were washed with 200 ppm chlorine, there were about 1.7- and 1.2-log reductions, respectively, in the population
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of L. monocytogenes (Zhang and Farber, 1996). In this study no more than a onelog reduction was achieved with the other sanitizers and disinfectants (chlorine dioxide, trisodium phosphate, lactic and acetic acids). Vinegar (5.2% acetic acid) and chlorine (200 ppm) were effective against Shigella sonnei on whole and chopped parsley (Wu et al., 2000). Chlorine at 200 mg/L was not very effective on L. monocyotgenes in that it reduced the population of the organism by 2 logs on Brussels sprouts compared to a water wash which brought about a 1.5-log reduction (Brackett, 1987). Chlorine at 1040 ppm did not effectively eliminate 102 to 103 Salmonella stanley cells on alfalfa seed, but when alfalfa seed with 101 to 102 CFU/g of the organism was treated with 2040 ppm of chlorine the organism was reduced to undetectable levels (Jaquette et al., 1996). Chlorine in the form of Ca(OCl)2 (Š2000 ppm), acidified ClO2 (Š100 ppm), acidified NaClO2 (Š500 ppm) and also hydrogen peroxide (Š0.2%) and trisodium phosphate (Š1%) was effective in reducing E. coli O157:H7 from alfalfa seeds; however, the few surviving cells could grow during sprouting and germination (Taormina and Beuchat, 1999) and hence alternative measures may be needed to prevent growth. This survival and growth could be due to enhanced resistance arising out of stress adaptation. Hypochlorous acid, the active form of chlorine, loses its activity when encountering organic material and hence, the efficacy of chlorine could be limited on fresh produce which has organic matter on the surface (Beuchat et al., 1998). Alfalfa sprouts have been vehicles of salmonellosis in recent years (Ponka et al., 1995). Copious amounts of biofilms of naturally occurring microflora were present on alfalfa, broccoli, clover and sunflower sprouts, which were more abundant on the cotyledon region, and these can afford protection for the colonizing pathogenic organisms (Fett, 2000). Effectiveness of chemicals in reducing Salmonella populations on the surfaces of alfalfa sprouts has been investigated. Ethanol (80%), hydrogen peroxide (6%) and sodium hypochlorite or calcium hypochlorite at chlorine concentrations of 1800 to 2000 µg/ml effectively reduced Salmonella populations on alfalfa seeds by more than 1000-fold (Beuchat, 1997). However, in this study viable Salmonella cells were still detected on seeds soaked in these chemical solutions for 10 min. The reason was that Salmonella in hard to reach areas such as crevices and area between the cotyledon and testa of the seed could have escaped the treatment or the severity of the treatment. When the bacterial cells escape the severity of the treatment, there is a possibility of becoming adapted to that chemical treatment and hence, surviving the treatment. The effect of chlorine on sanitizing poultry products and fish has been the subject of investigation by many researchers. Chlorination after sonication (aids in detaching bacterial cells) was effective on inactivating S. typhimurium cells attached to broiler skin, while either treatment alone was not effective (Lillard, 1993). Peroxidase catalyzed sanitizer and chlorinated water were equally effective on inactivating S. typhimurium and S. enteritidis on chicken egg shells (Kuo et al., 1997) and Enterococcus faecalis and P. aeruginosa on inert carriers and egg shell surfaces (Kwon et al., 1997); peroxidase catalyzed sanitizer was also effective against S. typhimurium on chicken breast skin and broiler carcasses (Bianchi et al., 1994), and on egg shell surfaces of hatchery eggs (Kuo et al., 1996). Chlorine at 100 and 200 mg/L was more effective in inactivating Yersinia enterocolitica and natural
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microflora on the shell of eggs than 1 and 3% acetic and lactic acids (Favier et al., 2000). Washing eggs with QAC and sodium hypochlorite prevented S. enteritidis penetration and no damage to the egg shell surface occurred, while sodium carbonate wash facilitated the penetration of organisms into the shell and resulted in recontamination (Wang and Slavik, 1998). S. aureus isolated from defeathering machinery in poultry processing plants were eight times more resistant to chlorine than the natural chicken skin microflora, and this may be due to their growth in clumps as well as to the production of extracellular slime (Bolton et al., 1988). Chlorinated water was effective in removing L. monocytogenes from fish (King Salmon) surface; however, a Listeria free product could not be achieved (Bremer and Osborne, 1998). Chlorine dioxide is an oxidizing agent and a sanitizer which has a good potential for sanitizing surfaces in the food industry. It can be used either as a gas or liquid, with the liquid form being more powerful than the gaseous form. Chlorine dioxide has advantages over chlorine because it does not react with ammonia or other nitrogenous compounds, is not affected by pH and is less reactive with organic matter than chlorine. The efficacy of chlorine dioxide in sanitizing epoxy surfaces in aseptic juice storage tanks was studied and it was found that 10 mg/L chlorine dioxide gas with a 30 min exposure time effectively inactivated the inoculated juice spoilage organisms, Lactobacillus buchneri, Leuconostoc mesenteroides, Saccharomyces cerevisiae, Candida sp., Eurotium sp. and Penicillium sp., leaving a clean epoxy surface (Han et al., 1999). Chlorine dioxide (1.3 ppm) effectively reduced the bacterial load of cucumber hydrocooling water (Reina et al., 1995) and poultry chiller water (Tsai et al., 1995). Chlorine dioxide was more effective than aqueous chlorine on inactivating streptomycin resistant L. monocytogenes on fish cubes and in the rinse solutions from these cubes (Lin et al., 1996). However chlorine dioxide spray (20 ppm) was not effective for removing fecal contamination on beef carcass tissue (Cutter and Dorsa, 1995).
NONCHLORINATED COMPOUNDS Apart from chlorine, quaternary ammonium compounds (QAC), iodine compounds, biguanides and acid anionic sanitizers are also commonly used in the food industry. QACs are hydrophilic cations, which can adsorb easily to negatively charged bacterial surface, and into the cell wall, thereby causing disruption of the cytoplasmic membrane causing leakage of cytoplasmic contents (Merianos, 1991). Gram-negative bacteria are more resistant to the action of QACs than Gram-positive bacteria. The resistance of P. aeruginosa to benzalkonium chloride was attributed to an increase in the content of cellular fatty acids (phospholipids and fatty and neutral lipids), thus resulting in a reduction in the permeation of the sanitizer through the cell wall (Sakagami et al., 1989). QAC at 50 ppm for 1 min was effective in inactivating L. monocytogenes (more than a four-log reduction) on smooth as well as porous stainless steel surfaces, while 200 ppm of sodium hypochlorite for 2 min and 400 ppm of the sanitizer for 2 min were required to inactivate the organism on smooth and porous surfaces, respectively, and QACs were found to be more effective between the two (Mustapha and Liewen, 1989). In the same study cells incubated for 1 h on stainless steel surface were more
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resistant to sodium hypochlorite treatment than those incubated for 24 h, and the reason was attributable to the presence of more moisture after 1 h than after 24 h. QACs (25 ppm), acid anionic sanitizer (25 ppm) and chlorine containing sanitizers (50 ppm) were effective in inactivating Listeria species, with QACs being the most effective sanitizing agent, although L. monocytogenes was the most resistant to QACs and acid anionic sanitizers and L. innocua the most resistant to chlorine sanitizers (Sallam and Donnelly, 1992). Adherent cells of L. monocytogenes on glass slides when treated with Benzalkonium chloride and acid anionic sanitizer with or without heat (55°C) were reduced by two to three logs in the first 30 sec, followed by a resistant population still remaining viable for at least 20 min, while planktonic cells grown in broth were rapidly inactivated (Frank and Koffi, 1990). L. monocyotgenes cells attached to stainless steel for 8 days were more resistant to hypochlorite and heat treatment (65°C for 3 min) than those attached for 4 h (Lee and Frank, 1991). These studies show that L. monocytogenes survives treatments with sanitizers especially when adhered to surface and, hence, proper cleaning and sanitizing measures to inactivate adherent cells are needed. The efficacy of a variety of chemicals and sanitizers on inactivating L. monocytogenes attached to a variety of equipment contact surfaces was studied (Krysinski et al., 1992). The most resistance was found on polyester/polyurethane surfaces, followed by solid polyester and etched stainless steel. Chlorine and iodophor were ineffective on stainless steel, while none of the biocides were effective on polyester/polyurethane. The most effective sanitizers were acidic quaternary ammonia, peracetic acid and chlorine dioxide, moderately effective were mixed halogens, acid anionics and fatty acid sanitizers and least effective were chlorine, iodophor and neutral quaternary ammonia. Some of these least effective ones are commonly used in the food industry. QACs were found more effective on reducing L. monocytogenes attached to chitin (contains organic matter) than chlorine and iodine sanitizers (McCarthy, 1992). QAC (100 and 200 ppm), chlorine and acid anionic sanitizers were more effective than iodine and quaternary ammonium detergent sanitizers in removing extracellular material from L. monocytogenes and S. typhimurium biofilms on various surfaces (Ronner and Wong, 1993). Chlorine, Zepamine A (QAC), and Ultra-Kleen (peroxide-based powder) were effective in removing L. monocytogenes (suspended in phosphate buffered saline) attached to gloves, while Zep-i-dine (iodine based sanitizer) and Zep Instant Hand Sanitizer (60% ethanol) were ineffective. However, in the presence of crab cook water that had organic matter, only UltraKleen was effective and all other sanitizers failed (McCarthy, 1996). The efficacy of hand sanitizers was tested using real soil involved in the food processing, and hand washing with a mild soap and water for 20 sec was better than 70% alcohol hand sanitizer (Charbonneau et al., 2000). Hydrogen peroxide is a commonly used disinfectant for sterilizing packaging material for aseptic filling of foods. The ability of hydrogen peroxide to sanitize eggs has been studied (Sheldon and Brake, 1991; Padron, 1995). Hydrogen peroxide vapor was effective for surface decontamination and prevention of decay in cantaloupes (Sapers and Simmons, 1998). The antibacterial action of hydrogen peroxide is believed to be due to its breakdown mediated by transition metal ions into cytotoxic radicals such as hydroxyl radicals, which can then initiate oxidation of biomolecules
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such as DNA, RNA, proteins and lipids; this breakdown can be initiated by reducing agents and peroxidases (Juven and Pierson, 1996; Halliwell and Gutteridge, 1992). The effect of hydrogen peroxide on spores is stronger at high temperatures due to radical formation. Spores of Bacillus species were inactivated by hydrogen peroxide at elevated (sublethal) temperatures and neutral pH, and extended exposures or higher concentrations of the disinfectant caused damage to the spore coat, cortex and protoplast (Shin et al., 1994). In this study it was found that some transition metal ions protected spores against the action of hydrogen peroxide while no protective effect was seen in the case of vegetative cells and, hence, the authors concluded that antibacterial mechanism of hydrogen peroxide towards spores is different from that of vegetative cells. Hydrogen peroxide was found to have a synergistic effect with peracetic acid towards inactivating bacteria isolated from water (P. aeruginosa, E. coli and S. aureus) (Alasri et al., 1992) and for disinfecting ultrafiltration membranes by inactivating spores of Bacillus species (Alasri et al., 1993). This synergy was maintained or increased with increasing contact time and it allowed for usage of less concentration of each biocide. Fresh fruits are surface washed with sanitizing agents and the efficacy of various agents in inactivating microorganisms on the surface has been studied. E. coli O157:H7, Salmonella and other natural microflora on surfaces of cantaloupes, honeydew melons and asparagus were effectively inactivated by chlorine (200 to 2000 ppm), acidified sodium hypochlorite, Tsunami™ (contains peracetic acid) and hydrogen peroxide, with the first three chemicals being more effective than hydrogen peroxide and water on the pathogens (Park and Beuchat, 1999). Hydrogen peroxide was less effective than chlorine in inactivating E. coli on cantaloupe surfaces at 4°C, while both sanitizers were ineffective at 20°C after 24 to 72 h storage (Ukuku et al., 2000). Acetic acid (5%) and 80 ppm peroxyacetic acid were more effective than hypochlorite or phosphoric acid treatments for sanitizing cider apples bringing about three-log reductions in E. coli O157:H7 populations and causing no injury (Wright et al., 2000). Salmonella chester on apple disks was found to be resistant to hydrogen peroxide, trisodium phosphate, calcium hypochlorite and sodium hypochlorite, and the reasons could be attributed to the firm attachment of the organism to the stem and calyx region as well as partial resistance of the bacteria to these sanitizers (Liao and Sapers, 2000). Peroxyacetic acid, chlorine dioxide or a chlorine-phosphate buffer solution were studied for their efficacy on removing non-pathogenic E. coli O157:H7 on apple surface and none of these sanitizers could bring a five-log reduction of the organism at the manufacturer’s recommended concentration (Wisniewsky et al., 2000). Immersing fresh Valencia oranges in hot water at 80°C for 1 min or 70°C for 2 min was more effective in causing five-log reductions than chemical sanitizers such as chlorine, chlorine dioxide, mixture of peroxyacetic acid and hydrogen peroxide, acid anionic sanitizer and trisodium phosphate treatments for 8 min, which caused about two- to three-log reductions in the populations of E. coli and other natural microflora on the surfaces of oranges (Pao and Davis, 1999). When orange fruits were washed with various cleaners followed by potable water rinse, an alkaline sodium orthophenylphenate was more effective than others in reducing populations of E. coli; however, adding sanitizers to the washing solutions did not improve the
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effect (Pao et al., 2000). A number of sanitizers were tested for their efficacy in inhibiting citrus spoilage organisms and organism on citrus fruit surface, and QACs, chlorine dioxide and iodophor were very effective, requiring low concentrations for inhibition, while peracetic acid, hypochlorite, dimethyldicarbonate and a phosphoric acid anionic sanitizer required higher concentrations to inhibit (Winniczuk and Parish, 1997). Chlorine, acidified sodium chlorite, trisodium phosphate, Tsunami, and hydrogen peroxide were ineffective in inactivating Alicyclobacillus acidoterrestris spores on apple surfaces (Orr and Beuchat, 2000). A sanitizer named Oxonia active (a mixture of hydrogen peroxide, peracetic acid, acetic acid and 1-hydroxyethylydene-1,1-diphosphonic acid) was more effective on Clostridium spores than on Bacillus spores, with B. cereus being the least sensitive to the sanitizer (Blackistone et al., 1999).
SANITIZER STRESS ADAPTATION
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Previous exposure to one stress is known to offer cross-protection against certain other stresses in bacteria. Starved planktonic cells of L. monocytogenes exhibited greater resistance to benzalkonium chloride than non-starved cells (Ren and Frank, 1993). Cold temperature reduced the efficacy of iodophor and QACs against L. monocytogenes strains especially at lower concentrations and shorter exposure times, while low temperature did not affect the efficacy of chlorine (Tuncan, 1993). Using higher concentrations and longer exposure times may overcome these limitations. L. monocytogenes cells, when exposed to sublethal levels of various disinfectants and sanitizers, chlorine, iodophor, QACs, citric, lactic and propionic acids (chemical shock), were not resistant to lethal levels of these compounds (Pickett and Murano, 1996). However, exposure to sublethal levels of acid anionic sanitizer, increased the resistance of L. monocytogenes cells to further exposure at minimum inhibitory concentrations or lethal levels of the same sanitizer. When the acids were pH adjusted to result in dissociation of the acid and the organism was exposed, increased resistance was observed. When E. coli O157:H7 was exposed to sublethal levels of peroxyacetic acid, it exhibited enhanced tolerance to peroxidative stress in hydrogen peroxide (Zook et al., 2001). However, this exposure did not cross-protect the bacterium against thermal stress as well as acetic acid stress. An increase in cell density enhanced the resistance of P. aeruginosa biofilm to iodine (Brown and Gautier, 1993). Nutrient starvation, increased cell density and increased production of exopolymeric substance increased the resistance of S. typhimurium in binary species biofilm with P. aeruginosa to chlorine sanitizer (Leriche and Carpentier, 1995). Exposure of E. coli cells to free chlorine for less than 1 sec activated the heat shock and soxRS regulons, but not the oxyR or SOS regulons or universal stress protein (uspA) (Dukan et al., 1996). Exposure of E. coli O157:H7 to chlorine prior to heat treatment resulted in an increase in the heat shock proteins (Dukan et al., 1996). However, pre-exposure to chlorine did not result in an increase in the D-value of the organism at 58°C in apple juice (Folsom and Frank, 2000). Acid adaptation of S. typhimurium increased the resistance of the organism towards an acid anionic sanitizer, but sensitized the cells to hypochlorous acid (due to oxidation of essential
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cell components, energy depletion, and changes in cell membrane permeability) and iodine (Leyer and Johnson, 1997). Exposure to sublethal levels of trisodium phosphate increased the sensitivity of C. jejuni, E. coli, P. flourescens and S. enteritidis to lysozyme and nisin (De Melo et al., 1998). Antibiotic resistant as well as sensitive strains of L. monocytogenes and S. typhimurium were equally sensitive to acid anionic sanitizers (Lopes, 1998). Pre-exposure of L. monocytogenes cells to starvation, ethanol, acid and hydrogen peroxide enhanced its thermal resistance (Lou and Yousef, 1996).
METAL ION STRESS Another stress that a bacterium can be exposed to in a food processing plant is metal ions. Heavy metals are a group of 65 metallic elements with varying physical, chemical and biological properties and are able to exert toxic effects on microorganisms (Gadd, 1992). However, some bacteria are able to tolerate these effects due to intrinsic properties, metabolic changes during interaction with a metal or environmental factors, while some others can resist the effects by certain detoxification mechanisms. Some detoxification mechanisms include: metal chelation by iron chelating compounds such as siderophores, and other chelators such as ethylenediaminetetriaceticacid (EDTA) or nitrilotriaceticacid (Schets and Medema, 1993); presence of metal binding extracellular polymers or metal binding proteins such as metallothioneins and phytochelatins; methylation of certain compounds such as mercury, lead, or tin, which become volatile and are lost in the environment or by removal of alkyl or aryl groups (Gadd, 1992). A particular protein (PsMTA) was expressed in E. coli as a carboxyterminal extension of glutathione-s-transferase and this protein exhibited metal binding properties (Tommey et al., 1991). Transcription of the fliC gene encoding flagellin, the protein of flagellae in E. coli was found to be regulated by heavy metal ions such as aluminum, copper, iron and nickel (Guzzo et al., 1991). Gram-negative bacteria are generally more resistant to the action of heavy metals than Gram-positive bacteria. Bacteria are able to adapt to the stress from metal ions and are able to survive. The molecular mechanisms of genetic adaptation to xenobiotic compounds including heavy metals are discussed by van der Meer et al. (1992). According to the authors, the various molecular and biochemical processes leading to such an adaptive response in bacteria include 1) induction of specific enzymes to degrade the heavy metal ions or other pollutants, 2) ability to grow and metabolize the substrate, and 3) selection of mutants possessing altered enzymatic capabilities or unique/novel metabolic activities. The inhibitory effect of various electroplated coatings of cobalt, zinc, copper, silver, chromium and cobalt-containing alloys of nickel, zinc and chromium etc. on Enterococcus faecalis, methicillin-resistant S. aureus, E. coli, P. aeruginosa and K. pneumoniae was tested and depended on the amount of hydrogen peroxide produced from the surface of the coating, with the greatest inhibition occurring from the highest amounts of hydrogen peroxide (10–6 mmol/cm2) producing coating surfaces
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(Zhao et al., 1998). Bacteria when exposed to heavy metal ions respond by reducing their physiological activity (Scrader and Cardamone, 1985). The ability of Aeromonas hydrophila to attach to various surfaces such as stainless steel, copper and polybutylene was studied and the fewest number of attached cells were found on copper; these cells had altered surfaces compared to the cells attached on other surfaces (Assanta et al., 1998). This was attributed to the antimicrobial effect of copper on the bacterium affecting its ability to attach, and this may increase the susceptibility of the attached cells to sanitizers. Cuprous oxide can react with adsorbed cells on the surface and the resulting product involving free cupric ions could inhibit or resist adhesion (Jonas, 1991). A similar finding was reported with Legionella pneumophila in which fewer cells of the organism were found attached on copper surfaces compared to polybutylene (Lee and West, 1991). When copper resistant E. coli was grown in a copper supplemented medium, an increased production of exopolymer was seen (Chao and Chen, 1991). The copper binding abilities from exopolymers of copper resistant as well as copper sensitive Pseudomonas strains were studied and both bound the same amount of copper ion; however, the sensitive strain could not grow in media with copper while the others did and it was found that the low pH of the medium inhibited the growth (Chao and Chen, 1991). Thus the exopolymer production and acid tolerance of the organism may play a role in survival of the microorganism in environments with high concentrations of heavy metals. Microorganisms require certain metal ions in low amounts for their growth; for instance, L. monocytogenes requires magnesium and iron for growth in a minimal medium (Premaratne et al., 1991). Supplementing the growth medium with metal ions such as zinc, magnesium, manganese, cobalt and calcium pantothenate increased the propionibacteria biomass yield, while there was no influence on the production of volatile fatty acids (Kujawski et al., 1992). Growth inhibition of L. monocytogenes (in brain–heart infusion broth at 19°C, pH 6.0) by sodium polyphosphate was reversed by low concentrations of polyvalent metal ions such as manganese, calcium, magnesium and zinc, but not by copper, cobalt, nickel and aluminum; however, growth inhibition of the organism did not occur in mineral rich foods such as pureed beef, green beans and sweet potatoes (Zaika et al., 1997). The antibacterial effects of certain food grade phosphates such as sodium ultraphosphate, sodium polyphosphate glassy and tetrasodium pyrophosphate on S. aureus were reversed by polyvalent metal ions such as calcium, magnesium and iron, when these metal ions were added to the media containing polyphosphates either before or after inoculation of the organism (Lee et al., 1994a). The lost enzymatic activity by EDTA of a 52-kDa metalloproteinase was restored by metal ions such as zinc, calcium, manganese, magnesium and iron (Kim and Kim, 1993). These studies show the protective effects of low concentrations of metal ions towards microorganisms.
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Often resistance to metal ions is related to antibiotic resistance and common plasmids seem to be involved. An E. coli strain carrying the robA plasmid from a cyclohexane
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tolerant mutant strain exhibited increased tolerance to solvents and increased resistance to antibiotics and heavy metal ions such as silver, mercury and cadmium (Nakajima et al., 1995). Staphylococcus aureus strains that were resistant to mercury also carried penicillinase plasmids (Silver and Misra, 1988). Marine Vibrio species isolated from yellowtail fishes in Japan were found to be resistant to lead acetate, cobalt chloride, sodium arsenate and nickel sulfate as well as to the antibiotic aminobenzylpenicillin (Hayashi et al., 1993). Some Gram-negative bacteria carry plasmids exhibiting resistances to antibiotics as well as to heavy metal ions such as arsenic, cadmium, cobalt and nickel (Lyon and Skurray, 1987).
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CROSS-PROTECTION
Inducible resistance to heavy metal ions and its cross-protection against other stresses have been observed in bacteria. Bacillus subtilis exhibited growth at lethal levels of zinc when previously exposed to mild concentrations of the metal ion and this was a chromosomally determined resistance (Podlesek et al., 1993) as opposed to a plasmid determined resistance (Nies, 1992). This inducible response involved a change in protein expression of the organism with the induction of a 150 kDa protein and suppression of a 127 kDa protein. No inducible cadmium resistance was observed in the organism and adaptation to zinc did not offer any cross-protection against cadmium and vice versa (Podlesek et al., 1993). Cadmium tolerance to lethal concentrations was induced in E. coli pretreated with mild concentrations of the metal ion as well as with mild heat; however, adaptation to mild concentrations of cadmium did not induce thermotolerance in the organism (Inbar and Ron, 1993). In another study, pre-exposure of E. coli cells to a mild concentration of cadmium offered cross protection against lethal temperatures (VanBogelen et al., 1987). Cadmium tolerance was found to induce 40 stress proteins (Blom et al., 1992). Listeria monocytogenes strains from various sources were tested for the presence of plasmids and 40% of food isolates did possess plasmids; more than 95% of plasmid positive strains were cadmium resistant, implying that cadmium resistance in L. monocytogenes is plasmid determined (Lebrun et al., 1992). Cadmium resistance in S. aureus was found to be chromosomally determined (Lyon and Skurray, 1987; Witte et al., 1986).
ANTIBIOTIC STRESS In recent years several antibiotic resistant strains of bacteria have emerged, a source of concern. One such example of a foodborne pathogen that has emerged due to its resistance to multiple antibiotics is Salmonella typhimurium DT104. In 1985, pasteurized milk from a contaminated dairy plant was implicated in an outbreak of S. typhimurium (involving 180,000 cases) which was resistant to five antibiotics (Ryan et al., 1987). New antibiotics are manufactured to combat resistant organisms by altering their chemical structure either to escape bacterial defenses or weaken them. Bacteria are able to resist those new antibiotics as well. Some resistant bacteria are able to transfer this resistance to sensitive cells (Russell and Chopra, 1996). A number of mechanisms play a role in the development of these resistances. The
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resistance could be either intrinsic or acquired. The intrinsic resistance is due to some inherent property of the organism such as virulence. Acquired resistance usually involves a mutation and sometimes mutation can occur in genes that regulate multiple functions within a cell (Shlaes, 1993); and this single mutation could confer resistance to multiple antibiotics (Murray, 1991). According to Russell and Chopra (1996), some mechanisms of antibiotic resistance include antibiotic alteration, insensitive or modified target site, impaired antibiotic uptake, enhanced efflux of antibiotic, and absence of an enzyme and metabolic pathway. In addition, access to the target site may be prevented (Neu, 1992) or there is overproduction of the target site (Miller and Sulavik, 1996). A mutation in a target enzyme could occur, resulting in decreased binding to the site, or an alternative reaction bypassing the antibiotic-sensitive step could occur (Volk et al., 1996). Efflux of the antibiotic is usually by cell membrane proteins that are plasmid or transposon encoded and they bind the antibiotic and push it out of the cell (Service, 1995; Russell and Day, 1996). Resistance in some Gram-negative bacteria occurs through alteration in the permeability of the outer membrane proteins called porins, with these proteins having restrictive channels than the normally expressed protein (Volk et al., 1996). It has been shown in studies with E. coli that the antibiotic resistant systems show similarities at the amino acid sequences and among their genetic regulators (Miller and Sulavik, 1996). Some antibiotics such as aminoglycosides, β lactams and chloramphenicols are inactivated by hydrolysis or by formation of inactive derivatives by the bacterial cell (Davies, 1994). Starvation or nutrient deprivation and growth rate contribute towards the resistance of biofilm bacteria to the action of antibiotics (Brown et al., 1988). The major groups of antibiotics that have been used against microorganisms include β lactams, fluoroquinolones, aminoglycosides, chloramphenicols, tetracyclines, MLS group (macrolides, lincosamides, streptogramins) and antibiotics that inhibit cell wall precursor biosynthesis. The mechanism of action of some antibiotics and bacterial resistance mechanisms against these are listed in Table 5.3. Russell and Day (1996) and Volk et al. (1996) have given detailed descriptions of actions of various classes of antibiotics. Several researchers have investigated the effect of antibiotics on foodborne bacteria. Older biofilm cells of S. aureus were resistant to tobramycin and cephalexin under iron limited conditions, while the planktonic cells were susceptible, and the younger biofilm cells which were more resistant than planktonic cells could be eradicated by the combinations of the two antibiotics (Anwar et al., 1992). A similar phenomenon was seen with P. aeruginosa, where the older biofilms grown under iron limited conditions were resistant to tobramycin and piperacillin (Anwar and Costerton, 1990). The exopolysaccharides in the biofilms may bind the antibiotics and prevent penetration into the cell (Anwar et al., 1992) or embedded biofilm cells may be able to produce enzymes that can degrade the antibiotic (Giwarcman et al., 1991). Moreover, the old biofilm cells are under starvation stress which may confer protection against the action of antibiotics. The resistance of S. aureus isolated from fish processing plant workers to various antibiotics was studied and the highest percent resistance was seen for ampicillin followed by penicillin and then tetracycline, polymyxin-B, erythromycin, kanamycin, neomycin, chloramphenicol and streptomycin in decreasing order (Sanjeev and
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TABLE 5.3 Antibiotic Action on Microorganisms and Mechanisms of Resistance Antibiotic
Mode of Action
Aminoglycosides
Inhibition of protein synthesis
Bacitracin, Ristocetin Chloramphenicol Cycloserine
Blocks cell wall peptidoglycan synthesis Inhibition of protein synthesis Inhibition of cell wall peptidoglycan synthesis Inhibition of protein synthesis
Erythromycin Fosfomycin Fusidic acid
Interference with bacteria cell wall synthesis, enzyme inactivation Inhibition of protein synthesis
Mupirocin
Interference with protein synthesis
Penicillin, Cephalosporin Polymyxin Quinolones
Blockage of cell wall synthesis
Rifampin Rifampicin Streptomycin Sulfonamides Tetracyclines Trimethoprim Vancomycin, Teicoplanin
Distortion of outer membrane Inhibition of synthesis or metabolism of nucleic acids Inhibits DNA-directed RNA polymerase activity Inhibition of mRNA synthesis Inhibition of protein synthesis Modification of energy metabolism Inhibition of protein synthesis Modified energy metabolism Inhibition of cell wall peptidoglycan synthesis
Resistance Mechanism Aminoglycoside modifying enzymes, reduced ribosomal binding Unknown Modification of the target (ribosomal 50S subunit) Altered transport, production of elevated enzyme levels Modification of target by producing methylating enzymes Altered transport due to chromosomal mutation Target site, factor G involved in translocation is modified Altered target (second resistant enzyme Isoleucyl tRNA synthetase) Altered penicillin binding proteins or absence of target enzyme Unknown Modified target (DNA Gyrase) Reduced DNA polymerase binding Modification of RNA polymerase Modification of target (S30 ribosomal unit) Modified target (dihydroperoate synthetase) Active efflux from the cell Excessive production of dihydrofolate reductase Modified target
(Adapted from Davies, J., Science, 264, 375, 1994; Neu, H.C., Science, 257, 1064, 1992; Russell, A.D. and Day, M.J., Microbios, 85, 45, 1996; Service, R.F., Science, 270, 724, 1995; Volk, W.A. et al., in Essentials of Medical Microbiology, 5th ed., Lippincott-Raven, Philadelphia, 1996, 253.)
Mahadeva Iyer, 1988). S. aureus and E. cloacae were exposed to single or combinations of antibiotics for a period of time and minimum inhibitory concentrations (MIC) determined, and an increase in the MIC for S. aureus was seen, indicating that a “safe” level of antibiotics appearing in foods can select for antibiotic resistant population of the organism (Brady and Katz, 1992; Brady et al., 1993). The antibiotic resistance of Salmonella is believed to come from animal sources where animals are fed antibiotics to increase feeding efficiency and thereby weight gain, and bacteria gains resistance to the drug. Outbreaks of salmonellosis are linked to the consumption of foods from animal origin (Epling and Carpenter, 1990).
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S. typhimurium DT 104 isolated from poultry sources were found resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, penicillin and spectinomycin (Rajashekara et al., 2000) and the resistance was found to be chromosomally integrated (Threlfall et al., 1994). Salmonella serotypes isolated from different parts of slaughtered pigs were found resistant to tetracycline (Bhattacharyya et al., 1991). In another study Salmonella isolated from pork carcasses were found to be resistant to penicillin, trimethoprim, ampicillin and tetracycline, and moderately resistant to streptomycin, kanamycin and chloramphenicol (Epling and Carpenter, 1990). Use of antibiotics in the feed for pigs undergoing asymptomatic S. typhimurium infection could cause an increase in antibiotic resistant organisms (Ebner and Mathew, 2000). Salmonella isolated from fish and crustaceans were found resistant to single or multiple antibiotics such as bacitracin, penicillin and novobiocin, and the reason could be attributed to human wastewater mixing in aquaculture ponds in Third World countries as well as use of antibiotics in aquaculture ponds (Hatha and Lakshmanaperumalsamy, 1995). In Salmonella wein, a 74 kDa iron repressible outer membrane protein and a plasmid were related to multiple antibiotic resistance, bacteriocin resistance and aerobactin production (Visca et al., 1991). Salmonella, Staphylococcus, and some other members of the Enterobacteriaceae family isolated from slaughterhouse and retail chicken samples exhibited multiple antibiotic resistance, with slaughterhouse samples showing higher resistance than retail samples, since the animals are fed subtherapeutic doses in chicken farms (Manie et al., 1998). Use of tetracycline and penicillin in feed should be avoided since the resistance from these is easily transferred causing multiple resistance. Coliforms (E. coli, Enterobacter, Citrobacter, Klebsiella and Serratia) isolated from slaughtered chickens showed multiple antibiotic resistance; the highest resistance is seen for tetracycline, followed by cephalotine, cotrimoxazole and nalidixic acid (Turtura et al., 1990). Aeromonas hydrophila isolated from chicken during various processing stages were found to be multiple antibiotic resistant with the greatest resistance observed for ampicillin and cephalothin and most resistant isolates were recovered from carcasses immediately after evisceration and from chill water samples (Barnhart and Pancorbo, 1992). Campylobacter strains isolated from poultry abattoir effluent and two sewage purification plants (one receiving mixed sewage including that of poultry abattoir and the other one not receiving any sewage from meat processing) were resistant to quinolones, with the resistance of isolates from the sewage purification plant receiving mixed sewage being higher than that of those from plant receiving no meat processing sewage (Koenraad et al., 1995). Campylobacter strains isolated from poultry and humans were found resistant to fluoroquinolones, and cross resistance to nalidixic acid was also seen, and the transmission route was suggested to be from chicken to man and not vice versa (Endtz et al., 1991). Antibiotic resistance of Enterococcus isolated from turkeys fed virginiamycin was studied and E. faecium was found resistant to ampicillin, gentamicin and quinupristin-dalfopristin antibiotics but not to virginiamycin, with strains from older turkeys showing greater ampicillin and quinupristine-dalfopristine resistance (Welton et al., 1998). Enterobacteriaceae isolated from minced meat were resistant to cephalothin followed by sulfisoxazole, ampicillin and tetracycline with 48% of the isolates showing multiple resistance; however, no resistance to a disinfectant was seen
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(Stecchini et al., 1992). Antibiotic resistance of enterococcal isolates from pork, water and clinical sources was compared and pork isolates showed lower antibiotic resistance in general than water or clinical isolates except in case of tetracycline, and with cefazolin and imipenem the resistance was higher than clinical isolates, while water isolates showed greater resistance in case of cephalosporins, amikacin, gentamicin, imipenem and rifampin (Knudtson and Hartman, 1993). Enterococci isolated from raw meat from Italy were found resistant to vancomycin and this resistance was associated with resistance to methicillin, teicoplanin, erythromycin, tetracycline and chloramphenicol; however, susceptibility to imipenem, rifampin and ampicillin in some isolates was observed (Pavia et al., 2000). Bacteria in noncarbonated mineral water were resistant to nalidixic acid and other antibiotics with 51% of the isolates showing multiple resistance; this is of public health concern, since mineral water is widely consumed (Massa et al., 1995). Plesiomonas shigelloides strains isolated from blue crab were susceptible to gentamicin, nalidixic acid and tetracycline and resistant to ampicillin, carbenicillin, kanamycin and streptomycin, with streptomycin resistance being linked to small size plasmids (Marshall et al., 1996). Gram-negative psychrotrophs isolated from vegetables showed multiple antibiotic resistance patterns, with high resistance to chloramphenicol; however, less than 10% of the isolates showed resistance to mezlocillin-ticarcillingentamicin or ceftizoxime-norfoxacin and although 50% of the strains were QACs tolerant, sensitive strains were inactivated by QACs (Fernandez-Astorga et al., 1995). Listeria monocytogenes strains isolated from Italian meat products were resistant to antibiotics such as tetracycline, co-trimoxazole and erythromycin and the resistance was not plasmid mediated (Barbuti et al., 1992). However, antibiotic resistance (to chloramphenicol, erythromycin, streptomycin and tetracycline) in L. monocytogenes was found to be mediated by a 37 kb plasmid, which was also self transferable to other organisms such as Enterococcus faecalis, Streptococcus agalactiae and S. aureus (Poyart-Salmeron et al., 1990). Erythromycin resistance in Listeria species was associated with the presence of ermC genes encoding rRNA methylases and this was found transferrable to L. monocytogenes, L. innocua and E. faecalis (Roberts et al., 1996). These studies showed the transfer potential of antibiotic resistance genes from one organism to the other. Listeria species isolated from raw milk were tested for their antibiotic resistance and most isolates were resistant to sulfisoxazole; only two L. innocua and none of L. monocytogenes were resistant to tetracycline and the resistance in L. innocua was not plasmid mediated (Slade, 1991). Salmonella and Listeria species were isolated from sausage samples in Greece and some Salmonella strains were resistant to ampicillin, chloramphenicol and tetracycline, while all Listeria isolates were sensitive to penicillins and aminoglycosides, but resistant to cephalosporins (Abrahim et al., 1998).
CROSS-RESISTANCE The aquisition of a plasmid in a bacteria that confers antibiotic resistance could alter the cell envelope which in turn could render the bacteria susceptible to a subsequent biocidal treatment (Russell, 1991). This might suggest a cross-resistance existing between antibiotics and biocides (Russell and Day, 1996). There is evidence in the
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literature that antibiotic resistance, especially if plasmid mediated, could offer protection against biocides. For instance, antibiotic resistant strains of S. aureus and S. epidermidis exhibited resistance towards chlorhexidine and quaternary ammonium compounds (Russell, 1997). Antibiotic resistance in E. coli offered cross-protection against some household disinfectants such as pine oil (Moken et al., 1997).
OTHER STRESSES Some other stresses that a bacterium may undergo in a food processing environment include starvation, osmotic and oxidative stresses. The oxidative and osmotic stresses may also be encountered when the organism is exposed to a chemical, sanitizer or other compounds. Bacteria in a biofilm formed on any equipment or other surfaces in a food processing environment may undergo starvation and oxidative stresses.
ADAPTATION
TO
STARVATION STRESS
Starvation in general makes a bacterium more resistant in the environment and protects against several subsequent stresses. Stationary phase cells in general are able to resist harsh conditions better than log phase cells. The mechanism by which a cell regulates its metabolism under starvation conditions is described as the stringent response, which involves induction of stationary phase specific genes as well as stimulation of certain biosynthetic pathways (Nystrom, 1994). In E. coli the sigma factor, σS, regulating general stress response is induced during the stationary phase (Loewen and Hengge-Aronis, 1994). The response to starvation in Vibrio species has been characterized by Kjelleberg et al. (1993) in which the starved cells of the organism undergo morphological differentiation into ultra-micro sized (smaller) cells, which undergo cell division with no major increase in cell biomass; these cells can regain their normal size under conditions of sufficient nutrient availability. This ability is seen in carbon starved cells, but not in nitrogen or phosphorus starved cells and only carbon starvation can offer cross-protection against other stresses. The authors describe three adaptive phases of the nutrient starved Vibrio cells; the first phase involves some physiological changes causing a decrease in the rate of macromolecular synthesis, an increase in the rate of protein degradation and temporary accumulation of ppGpp. The second phase causes changes in membrane fatty acid composition, degradation of reserve material and resistance development to other stresses. The third phase involves changes in the metabolic activities causing reduced rate of respiration and synthesis of RNA, protein and peptidoglycan; however, the cell does not cease to develop, recover and grow. Also there are induction and repression of a number of stress proteins. The chaperones DnaK and GroEL have been found to be induced in Vibrio under conditions of starvation (Holmquist et al., 1993). Under conditions of starvation, cells are able to retain ribosomes in excess of that required for protein translation (Flärdh et al., 1992; Kjelleberg et al., 1993) and degradation of ribosomes occurs very slowly with the half life of ribosomes estimated to be 80 h during the first 72 h of starvation (Flärdh et al., 1992). Bacterial α-glucan phosphorylases are involved in the regulation of endogenous glycogen metabolism
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during starvation, sporulation and other stresses (Schinzel and Nidetzky, 1999). Phosphorylases provide energy to the bacterial cells and help them to adapt to the quickly changing environment (Böck and Schinzel, 1998). In E. coli, the glycogen phosphorylase is found most active during the stationary phase and under substrate deprived conditions (Yang et al., 1996). Nutrient starvation in S. aureus cells caused accumulation of higher levels of ppGpp and ppGp nucleotide (Crosse et al., 2000). Listeria monocytogenes undergoes stress in the absence of energy yielding substrates. Growth of L. monocytogenes in different solutions lacking all the required nutrients for growth, for instance, tap water, deionized water, normal saline and different dilutions of phosphate buffered saline, was studied (Lee, 1995). None of these promoted the growth of the organism with complete death occurring in deionized water in less than 5 days. The population decline in 0.3 mM phosphate buffered saline was more rapid compared to normal saline, and the number of survivors were more in tap water after 25 days. Starved cells were prevented from lysis in 200 mM phosphate buffered saline. When starved in salts solution the population increased initially and then declined more than 3 logs after 30 days and then remained constant for 120 days. Escherichia coli O157:H7 was able to survive in water at cold temperatures and there were changes in the outer membrane proteins (Wang and Doyle, 1998). Escherichia coli, Shigella and S. typhimurium cells incubated in seawater, distilled water, phosphate buffer and phosphate buffered saline exhibited increased acid resistance, the greatest resistance being induced with exposure to seawater and in stationary phase cells and this resistance partly depended upon rpoS sigma factor and de novo protein synthesis (Gauthier and Clément, 1994).
ADAPTATION
TO
OSMOTIC STRESS
Microorganisms respond to osmotic stress in three phases: the cytoplasm shrinks or swells due to efflux or influx of water from hyper- or hypo-osmolarity, the cell then undergoes biochemical changes to restore volumes compatible with growth and then growth occurs (Brown and Edgley, 1980). Bacteria are able to accumulate proline and glycinebetaine either by increased synthesis or uptake under conditions of osmotic stress (Csonka, 1989). Escherichia coli K12 has the ability to convert choline to glycinebetaine under conditions of osmotic stress which can act as an osmoprotectant (Landfald and Strøm, 1986). Accumulation or increase in some other solutes such as potassium (Meury et al., 1985), glutamate (Botsford, 1984), trehalose (Dupray et al., 1995; Larson et al., 1987) and γ-aminobutyrate (Measures, 1975) has been observed in bacteria during osmotic stress. Two-dimensional gel electrophoretic analysis of E. coli exposed to osmotic stress showed three proteins to be synthesized (Clark and Parker, 1984). The transcription of the genes kdp, proU proP, ompF and ompC is osmotically regulated (Csonka, 1989). Cross-Protection Salmonella manhattan exhibited enhanced survival in seawater with prior exposure to wastewater, which caused synthesis of trehalose that can act as an osmoprotectant,
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and there was accumulation of trehalose and repression of two degradative cytoplasmic enzymes during exposure to seawater (Dupray et al., 1995). Pre-exposure to heat and ethanol enhanced the survival of L. monocytogenes cells in 25% sodium chloride (Lou and Yousef, 1997). Acid adapted cells of S. typhimurium had enhanced tolerance towards salt (Leyer and Johnson, 1993). In B. subtilis pre-exposure to mild salt or mild heat offered protection against lethal salt levels, but pre-exposure to mild salt did not induce thermotolerance as did mild heat shock (Volker et al., 1992). Compared to other stresses, thermal induction seems to offer global protection against several other lethal stresses in bacteria.
ADAPTATION
TO
OXIDATIVE STRESS
When bacteria are exposed to reactive oxygen species such as superoxide and hydrogen peroxide, they undergo oxidative stress. The oxidant radicals can react with amino acids and the resulting derivatives can inactivate enzymes (Wolff et al., 1986). The oxidative stress in bacteria is regulated by the oxyR system for hydrogen peroxide and SoxRS system for superoxide (Farr and Kogoma, 1991). Hydrogen peroxide treatment in E. coli induced the expression of katG, dps, ahpCF and gorA genes, but not oxyR and this expression was oxyR dependent and σS independent, and exposure to sodium chloride induced the expression of the same genes including oxyR, which implies that the two stresses involve overlapping genetic control (Michan et al., 1999). E. coli, when grown anaerobically, required superoxide dismutase for a smooth transition to growth in an aerobic environment, and a sudden transfer to aeration (superoxide presence) in the absence of superoxide dismutase causing oxidative stress, blocked amino acid biosynthesis and affected growth (Kargalioglu and Imlay, 1994). Exposure of E. coli cells to ozone induced catalase and superoxide dismutase (Whiteside and Hassan, 1987). Some of the proteins induced during oxidative stress and regulated by oxyR have been identified as antioxidant enzymes such as catalase, alkyl hydroperoxide reductase and glutathione reductase (Christman et al., 1985). Cross-Protection Oxidation reduction potentials of chemically modified water (with added chlorine, bromine and acids) and electrolyzed oxidizing water were found to enhance inactivation of E. coli O157:H7 (Kim et al., 2000). Aeromonas hydrophila exhibited sensitivity to hydrogen peroxide at 1 mmol/L; however, tolerance to the sanitizer was developed when the cells pre-exposed to a sublethal level of hydrogen peroxide exhibited tolerance to lethal levels (Landre et al., 2000). This tolerance involved the synthesis of a set of protective polypeptides and also was dependent on the growth phase and stock culture age, with stationary phase and older cells exhibiting higher resistance to hydrogen peroxide (Landre et al., 2000). Starvation of E. coli cells induced tolerance to heat or hydrogen peroxide (Jenkins et al., 1988). In this study, the cells starved for 4 h afforded the maximum and better protection compared to cells exposed to sublethal stresses of heat, hydrogen peroxide or ethanol, and this
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response involved synthesis of protective proteins, with three proteins found common to heat, hydrogen peroxide and ethanol stresses In this study adaptation of E. coli to hydrogen peroxide or ethanol did not result in enhanced heat resistance. However, S. typhimurium cells when exposed to sublethal levels of hydrogen peroxide produced some heat shock proteins and exhibited enhanced thermal tolerance (Christman et al., 1985). It is possible that different sets of stress proteins are produced in the two organisms with different stresses and hence there is difference in the crossprotection responses. Pre-exposure of Enterococcus faecalis cells to mild concentrations of hydrogen peroxide enhanced their survival under lethal concentrations of the peroxide. An acid pretreatment at large and, to a limited extent sodium chloride and heat but not ethanol and alkalinity, afforded protection against hydrogen peroxide (Flahaut et al., 1998). Some of these responses involved de novo protein synthesis, especially those induced by hydrogen peroxide and sodium chloride. However, in E. coli, alkalinity was found to enhance oxidative stress (Rowbury, 1997) and, in B. subtilis, ethanol but not heat enhanced protection against hydrogen peroxide (Dowds, 1994). Adaptation of L. monocytogenes to various stresses such as acid, ethanol, hydrogen peroxide, heat or sodium chloride enhanced its survival in higher concentration of hydrogen peroxide (Lou and Yousef, 1997). Acid adaptation of L. monocytogenes strains offered protection against an activated lactoperoxidase system involving hydrogen peroxide in tryptic soy broth (Ravishankar et al., 2000) but not in skim milk (Ravishankar and Harrison, 1999). E. coli O157:H7 cells exposed to heat were sensitive to oxygen in that they did not recover as well on aerobic media as they did on anaerobic media (Bromberg et al., 1998). Hence, recovering thermally stressed E. coli O157:H7 on aerobic media may give misleading results. Moreover, a hurdle approach involving thermal treatment followed by oxidative stress may prove useful in inactivating this pathogen. When cells of E. coli O157:H7, S. enteritidis and L. monocytogenes were grown, heated and recovered anaerobically, the heat resistance of these organisms was higher than when they were grown, heated and recovered aerobically (George et al., 1998). These results may have serious implications on the safety of sous vide food products as well as foods with a low redox potential.
CONCLUSIONS The stress response of bacteria and the physiological and molecular mechanisms behind these responses are emerging areas of research. We have gained some understanding of these responses and further investigation is still needed. The availability of modern biotechnological tools has made it feasible to understand the responses of the bacterium to an external environmental stress; we need to identify the important ones under given conditions (Brul and Coote, 1999) so that proper control strategies can be devised and implemented. Control of resistant microorganisms can be done through changing current practices of antibiotic use, developing newer antibiotics, utilizing alternative practices such as competitive exclusion, preventing bacterial adhesion and biofilm formation, applying a multiple hurdle approach, etc. (Bower and Daeschel, 1999). © 2003 by CRC Press LLC
In food processing plants, maintaining a clean environment is very important, and this can be achieved through an effective sanitation program. Chemical cleaning with detergents, accompanied by manual scrubbing of food contact surfaces and followed by sanitizer application, is an effective way to control biofilms. When designing food contact equipment, care should be taken to select the appropriate type of material to prevent microbial adhesion and importance should be given to the cleanability of the equipment. Employees working in a food processing plant should be properly educated about the importance of maintaining a hygienic environment. A better understanding of the physiology of microbial adaptive responses and thereby devising appropriate novel approaches to combat them can help us achieve the goal of increasing the safety of minimally processed foods and a safer food supply in the world.
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6
Stress Adaptations of Lactic Acid Bacteria Hany S. Girgis, James Smith, John B. Luchansky, and Todd R. Klaenhammer
CONTENTS Introduction Heat Shock Response and Thermotolerance Thermotolerance Heat Shock Genes Classification groE Operon: groES and groEL dnaK Operon: hrcA, grpE, dnaK, and dnaJ clp Family of Genes: clpB, clpC, clpE, clpP, and clpX Cold Stress Response and Cryoprotection Physiological Response and Adaptation Cold-Stress Genes and Gene Products Acid Adaptation Tolerance and Adaptation to Low pH Proton Movement: H+-ATPase Arginine Deaminase (ADI) Pathway Degradative Amino Acid Decarboxylases Citrate Transport System Alkaline Stress Response Osmotic Stress Compatible Solute Protein Synthesis during Osmotic Shock Oxidative Stress Tolerance and Adaptation to Oxidative Stress Regulation and Function of Oxidative Stress Response Proteins NADH Oxidase/NADH Peroxidase Glutaredoxin and Thioredoxin Superoxide Dismutase recA, fpg, and DNA Damage Starvation Overlapping Regulatory Networks and Cross-Protection
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The Future Conclusions References
INTRODUCTION Lactic acid bacteria (LAB) are widely used in dairy and other food fermentations for their ability to impart desirable flavor and rheological attributes to food, and for their ability to inhibit unwanted bacteria (Gilliland, 1985). During a successful fermentation, LAB must be able to tolerate the accumulation of toxic byproducts of their growth such as lactic acid and hydrogen peroxide, withstand antimicrobial agents produced by neighboring microorganisms, and endure deleterious environmental conditions required for the proper fermentation of a raw food item. Furthermore, LAB used as probiotics must be able to adapt to harsh conditions such as the acidic environment of the stomach during ingestion and the low temperatures associated with freezing prior to their storage and distribution. The LAB are equipped with complex stress response mechanisms that provide a selective advantage in compromising environments. The last few years have seen a tremendous increased interest in these stress response systems in LAB. The impetus for this heightened awareness is sparked by the industrial goal of minimizing losses associated with reduced cell viability upon inoculation in food and during passage through the gastrointestinal tract. Also, with the increased incidence of food poisoning, researchers are looking to enhance tolerance to environmental stress in LAB as a way to improve food safety. This chapter discusses the literature investigating adaptation to many of the harmful factors that LAB encounter in food systems and in the environment. Recently, a review article was published discussing the stress response mechanisms in Lactococcus lactis (Sanders et al., 1999). This chapter presents an extensive review of the existing knowledge on many of the species of LAB comprising six genera — Streptococcus, Lactococcus, Enterococcus, Leuconostoc, Oenococcus, and Lactobacillus. Occasionally, references will also be made to systems that have been thoroughly investigated in model organisms such as Escherichia coli and Bacillus subtilis.
HEAT SHOCK RESPONSE AND THERMOTOLERANCE THERMOTOLERANCE The heat shock response refers to an abrupt increase in temperature causing the induction of a small group of proteins called heat shock proteins. Heat shock proteins play essential physiological roles as molecular chaperones in protecting cells against damage due to thermal stress by binding to cellular proteins in a manner that maintains their native conformation and minimizes denaturation (Martin et al., 1992; Craig et al., 1993). The heat shock response is involved with a variety of other challenges and conditions such as ethanol (Strauss et al., 1987), osmotic (Vachova et al., 1994), acid (Heyde and Portalier, 1990), alkaline (Taglicht et al., 1987), oxidative (Morgan et al., 1986; Ericsson et al., 1994) stresses, and DNA damage (Lage and Menezes, © 2003 by CRC Press LLC
1994). See “overlapping regulatory networks and cross-protection section” below. The regulation of the heat shock response has been studied extensively in E. coli (Bukau, 1993; Yura et al., 1993; Gross, 1996) and B. subtilis (Hecker and Völker, 1990; Völker et al., 1994; Hecker et al., 1996). The heat shock response in E. coli is regulated by the rpoH-encoded alternative sigma factor σ32. The heat shock response is mediated through a variety of mechanisms in B. subtilis and other Gram-positive microorganisms. See “classification” below for a description of the regulatory mechanisms. Microorganisms exposed to a sublethal heat treatment acquire the transient ability to withstand subsequent lethal heat challenges and this phenomenon is called acquired thermotolerance (Lindquist, 1986; Hahn and Li, 1990; Mackey and Derrick, 1990; Boutibonnes et al., 1991). Heat-induced thermotolerance has been achieved in numerous LAB such as Lactococcus lactis (Boutibonnes et al., 1991; Whitaker and Batt, 1991), Lactobacillus bulgaricus (Teixeira et al., 1994), Enterococcus faecalis (Boutibonnes et al., 1993; Flahaut et al., 1996), Streptococcus thermophilus (Auffray et al., 1995), Lactobacillus acidophilus (Broadbent et al., 1997), Lactobacillus casei (Broadbent et al., 1997), Lactobacillus helveticus (Broadbent et al., 1997), and Lactobacillus collinoides (Laplace et al., 1999). The induction of heat shock proteins in response to thermal stress has been analyzed by SDS-PAGE in E. faecalis (Boutibonnes et al., 1993; Flahaut et al., 1996), L. lactis (Auffray et al., 1992; Kilstrup et al., 1997; Broadbent and Lin, 1999), S. thermophilus (Auffray et al., 1995), Oenococcus oeni (Guzzo et al., 1994, 1997), Lb. acidophilus, Lb. casei (Broadbent et al., 1997), Lb. helveticus (Broadbent et al., 1997), and Lb. collinoides (Laplace et al., 1999). The induction of heat shock proteins and the acquisition of thermotolerance has also been observed in organisms exposed to a variety of growth-limiting hazards such as chemicals, UV irradiation, viral infection, and pH shifts. Although heat-induced thermotolerance is accompanied by the induction of heat shock proteins, the contribution of heat shock protein synthesis to the development of acquired thermotolerance remains controversial (Yamamori and Yura, 1982; VanBogelen et al., 1987; Hahn and Li, 1990; Sanchez and Lindquist, 1990; Smith and Yaffe, 1991; Boutibonnes et al., 1992; Weber, 1992).
HEAT SHOCK GENES Classification Heat shock genes are classified according to the mode of regulation and fall within four general classes as described for the Gram-positive model microorganism, B. subtilis. Class I genes are organized in two operons, the groE operon and the dnaK operon. The CIRCE (controlling inverted repeat of chaperone expression) operator sequence serves as a cis-acting regulatory element and a binding site for a repressor protein named HrcA (for heat regulation at CIRCE [Schulz and Schumann, 1996]), the first gene product of the dnaK operon and a negative regulator of class I heat shock genes (Yuan and Wong, 1995; Schulz and Schumann, 1996). In B. subtilis, expression of class II genes is dependent on an alternative sigma factor named sigma B; the synthesis and activity of sigma B increase under stress conditions. No such regulator of class II heat shock genes has been identified in LAB.
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Class III genes are defined as those lacking a CIRCE element that are sigma B independent. Members of this class include clpC, clpE, and clpP. Class III genes are negatively regulated by a repressor protein called CtsR (for class three stress gene repressor [Derre et al., 1999]), the product of the first gene in the clpC operon (Krüger and Hecker, 1998; Derre et al., 1999; Nair et al., 2000). Class IV stress response genes are expressed independent of HrcA, sigma B, and CtsR and the regulatory mechanisms remain to be identified (Hecker et al., 1996). Examples of class IV stress response genes are ftsH, lonA, and htpG. groE Operon: groES and groEL The gene products of the groE (or groESL) operon are the widespread and highly conserved classical heat shock chaperone proteins, GroES and GroEL. As chaperone proteins, GroES and GroEL function to protect the cells against heat shock by binding to cellular proteins in a manner that maintains their native conformation and minimizes denaturation (Craig et al., 1993). Understanding the manner in which these proteins function is facilitated through the description of the three-dimensional molecular structure of GroES and GroEL. The GroEL protein is tetradecameric, consisting of two stacked rings with seven subunits in each ring forming a barrelshaped structure. The GroES protein is heptameric, resembling a dome-shaped structure (Hartl, 1996). After partially denatured proteins enter the GroEL hydrophobic chamber, GroES forms a dome enclosing the chamber, and thereby creating a protected environment wherein proteins can fold into native structures (Houry et al., 1999). Amino acid alignment data suggest that the bicistronic groE operon is highly conserved, containing only two genes and always in the same order: groES followed by groEL (Segal and Ron, 1996). A defining characteristic is the presence of a highly conserved CIRCE operator sequence (TTAGCACTC-N9-GAGTGCTAA), which preceeds the first structural gene in both the groE and dnaK operons (Zuber and Schumann, 1994; Yuan and Wong, 1995; Mogk et al., 1997) and the dnaJ gene in L. lactis (van Asseldonk et al., 1993). The CIRCE operator sequence serves as a binding site for HrcA, the first gene product of the dnaK operon and a negative regulator of class I heat shock genes (Yuan and Wong, 1995; Schulz and Schumann, 1996). In most bacteria, the CIRCE is transcribed with the corresponding genes and participates in the regulation of expression at both the DNA and mRNA levels (Zuber and Schumann, 1994; Yuan and Wong, 1995). Among the LAB in which the groE operon has been cloned and characterized are L. lactis (Kim and Batt, 1993), Lb. helveticus (Broadbent et al., 1998), and Lactobacillus johnsonii (Walker et al., 1999). Two sets of CIRCE elements were found flanking the promoter region of the groE operon in Lb. helveticus and Lb. johnsonii, whereas one CIRCE element was found downstream of the promoter in L. lactis (see Table 6.1). The presence of two copies of CIRCE elements rather than one could provide a stronger level of negative regulation. This stronger level of negative regulation may be manifested in a higher temperature required for heat shock induction. Although the results of northern hybridization showed increased expression of the groE operon in these three organisms after heat shock, the induction
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TABLE 6.1 Structure and Location of CIRCE Elements in LAB Gene/Operon groE
dnaK
dnaJ
a
Organism (References) Bacillus subtilis (Li and Wong, 1992) Lactococcus lactis (Kim and Batt, 1993) Lactobacillus helveticus (Broadbent et al., 1998) Lactobacillus johnsonii (Walker et al., 1999) Bacillus subtilis (Wetzstein et al., 1992) Lactococcus lactis (Eaton et al., 1993) Lactobacillus sakei (Schmidt et al., 1999) Streptococcus mutans (Jayaraman et al., 1997) Lactococcus lactis (van Asseldonk et al., 1993)
Upstreama Inverted Repeatb None
TTAGCACTC-N9-GAGTGCTAA
None
TTAGCACTC-N9-GAGTGCTAA
TTAGCACTA-N9-AAGTGCTAA
TTAGCACTT-N9-GAGTGCTAA
TTAGCACTC-N9-AAGTGCTAA
TTAGCACTT-N9-GAGTGCTAA
None
TTAGCACTC-N9-GAGTGCTAA
AAATTAGCACTC-N9-GAGTGCTAATTT
TTAGCACTT-N9-GAGTGCTAA
None
TTAGCACTC-N9-AAGTGCTAA
None
TTAGCAGTC-N9-GAGTGCTAA
AATTAGCACTCTT-N5-AAGAGTGCTAATT
None
Orientation of “upstream” and “downstream” is in reference to the transcriptional start site. Underlined sequences are the conserved motifs in a number of Gram-positive heat shock genes.
b
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Downstreama Inverted Repeatb
temperature was higher, 52 and 55°C, for Lb. helveticus and Lb. johnsonii (which contain two CIRCE elements), respectively, compared to 42°C for L. lactis (Kilstrup et al., 1997). Other forms of environmental stress can elicit a heat shock response (Lindquist, 1986; Georgopoulos, 1992), as shown by the induction of GroES in L. lactis by acid and UV254nm irradiation (Hartke et al., 1997). Furthermore, induction of heat shock proteins GroEL, GroES, and DnaK was also induced in response to salt stress in L. lactis (Kilstrup et al. 1997), which was not the case when salt was added to the growth medium of E. coli and B. subtilis (Clark and Parker, 1984; Hecker et al., 1988; Hecker and Völker, 1990; Völker et al., 1994). The DnaK and GroEL homologs in Leuconostoc mesenteroides were overexpressed in response to heat shock, cold shock, and ethanol treatment (Salotra et al., 1995). The relationship between the expression of heat shock proteins and enhanced tolerance to environmental stress has been investigated in some LAB. In E. faecalis, the contribution of GroEL and DnaK synthesis to thermotolerance was inconclusive, whereas de novo protein synthesis was required (Flahaut et al., 1997). Heat shock induction of the groESL operon in Lb. johnsonii provided some cross-protection against freeze injury (Walker et al., 1999), demonstrating the potential to improve tolerance to environmental stress by increasing chaperone concentration at opportune times. dnaK Operon: hrcA, grpE, dnaK, and dnaJ The DnaK or HSP70 (70 kDa) family of proteins are among the most well-known heat shock proteins. These proteins are ubiquitous and have been found in all prokaryotic and eukaryotic organisms examined to date. The DnaK heat shock protein has been studied extensively in E. coli and is essential for viability at high temperatures (Itikawa and Ryu, 1979; Paek and Walker, 1987). At optimum temperatures for growth, DnaK is involved in the synthesis of RNA and DNA and in cell division (Paek and Walker, 1987; Sakakibara, 1988). Whereas the groE operon has maintained a highly conserved organization, the number and order of genes within the dnaK operon have not been highly conserved (Segal and Ron, 1996). The most common sequence of genes in the dnaK operon is hrcA-grpE-dnaK-dnaJ; this organization has been found, for example, in Gram-positive bacteria such as B. subtilis (Wetzstein et al., 1992), Staphylococcus aureus (Ohta et al., 1994), and Clostridium acetobutylicum (Narberhaus et al., 1992; Behrens et al., 1993). In addition to these genes, the dnaK operon in S. aureus and C. acetobutylicum contains a fifth gene located downstream of dnaJ. In B. subtilis, three additional genes are transcribed (Homuth et al., 1997). In E. coli, grpE is not linked to dnaK (Lipinska et al., 1988) and no hrcA homologue has been identified. The dnaK operon has been identified in a number of LAB. In Lactobacillus sakei and Streptococcus mutans, the dnaK operon consists of four heat shock genes with the organization hrcA-grpE-dnaK-dnaJ (Jayaraman et al., 1997; Schmidt et al., 1999). Results of northern hybridization showed induction of these genes by heat shock, salt, and ethanol in Lb. sakei (Schmidt et al., 1999), whereas in S. mutans, these genes were induced by heat, acid, and alkali shock (Jayaraman et al., 1997). The induction of the dnaK operon by heat and salt in Lb. sakei is in agreement with
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the results of similar experiments performed on L. lactis (Kilstrup et al., 1997). Degenerate primers have been used to clone the dnaK operon from L. lactis (Eaton et al., 1993). In L. lactis, the operon contains three heat shock genes with the arrangement hrcA-grpE-dnaK followed by a fourth open reading frame (Eaton et al., 1993). Unlike the genetic organization of the dnaK operon in Lb. sakei and S. mutans and in most prokaryotic microorganisms, the L. lactis dnaK operon does not contain a dnaJ gene; rather, the dnaJ gene is a separate transcriptional unit and is located downstream of the dnaK operon (van Asseldonk et al., 1993). Another unusual aspect of the L. lactis dnaJ gene is the presence of its own CIRCE element and its location upstream of the transcriptional start site (van Asseldonk et al., 1993). Normally, CIRCE elements are found downstream of the site of transcription initiation. Also, the inverted repeat comprising the CIRCE element in the L. lactis dnaJ gene is longer than the consensus sequence (van Asseldonk et al., 1993), which may have an impact on the temperature required for heat shock induction. Deletion of this inverted repeated resulted in a higher transcriptional level at lower temperatures, in comparison to the wild-type gene, confirming its role in the regulation of the heat shock response (van Asseldonk et al., 1993). The CIRCE element preceding hrcA in the L. lactis dnaK operon was identical to the consensus sequence (Eaton et al., 1993). In Lb. sakei and S. mutans, a CIRCE element which differed by only one base from the consensus sequence (Hecker et al., 1996) was found upstream of hrcA between the transcriptional and translational start sites (see Table 6.1) (Jayaraman et al., 1997; Schmidt et al., 1999). clp Family of Genes: clpB, clpC, clpE, clpP, and clpX Prokaryotic and eukaryotic cells respond to harsh environmental conditions by synthesizing a group of chaperone proteins and proteases, which together serve to maintain quality control of intracellular proteins. As stated earlier, chaperone proteins are responsible for promoting proper assembly of proteins and preventing misfolding and aggregation (Craig et al., 1993). Proteases, on the other hand, degrade permanently damaged proteins. A large family of proteins named Clp contains members that exhibit both proteolytic and chaperone activities. Constituents of this large family of proteins include ClpA, ClpB, ClpC, ClpD, ClpE, ClpP, ClpX, and ClpY (Schirmer et al., 1996). The proteins comprising the Clp family are classified according to structural features and sequence similarities. In E. coli, the ClpP protein resembles the structure of the eukaryotic 26S proteasome (Kessel et al., 1995) and contains a central barrel that can be flanked at both ends by associating with either ClpA or ClpX ATPases (Kessel et al., 1995; Wang et al., 1997) to form ClpAP or ClpXP, respectively. When not associated with one of these ATPases, ClpP functions as a serine protease (Maurizi et al., 1990) and degrades peptides less than seven amino acids long (Woo et al., 1989). In the absence of ClpP, the ClpA and ClpX ATPases function as molecular chaperones (Wickner et al., 1994; Wawrzynow et al., 1995). However, when associated, the ClpAP or ClpXP complex exhibits protease activity against substrates with specificity determined by the associated ATPase subunit. The first substrate degraded by the protease complex was casein, thus the designation Clp for caseinolytic protease (Katayama et al., 1988).
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In B. subtilis and other Gram-positive microorganisms, the gene encoding ClpP (clpP) is among the class III heat shock genes (Msadek et al., 1998) which also encode the ClpC (Krüger et al., 1994; Msadek et al., 1994), ClpX (Gerth et al., 1996), and ClpE (Derre et al., 1999) ATPases. As stated above, class III genes are negatively regulated by a repressor protein called CtsR (for class three stress gene repressor (Derre et al., 1999)), the product of the first gene in the clpC operon (Krüger and Hecker, 1998; Derre et al., 1999; Nair et al., 2000). Recently, the clpP gene was identified in L. lactis (Frees and Ingmer, 1999) and was found to be induced by salt (Kilstrup et al., 1997)) heat shock, low pH, and puromycin (Frees and Ingmer, 1999), which is a tRNA analogue that prematurely terminates translation resulting in the synthesis of truncated, misfolded peptides. In E. coli and L. lactis, puromycin induces a heat shock response (VanBogelen et al., 1987; Frees and Ingmer, 1999). The L. lactis clpP mutant degraded puromycyl-containing peptides at a reduced rate and extent, relative to the wild type cells, suggesting that ClpP in L. lactis degrades misfolded proteins caused by environmental stress (Frees and Ingmer, 1999). Furthermore, L. lactis clpP mutants failed to grow at an elevated temperature (37°C) and in the presence of puromycin. The CtsR binding sequence was found overlapping the –35°C region of the L. lactis clpP promoter, suggesting that clpP expression might be negatively regulated by a CtsR homologue in L. lactis. The genes encoding ClpC, ClpE, and ClpB have also been identified in L. lactis, with ClpE being part of a new Clp protein family. Northern blot analysis showed that L. lactis clpB and clpE were strongly induced at the transcriptional level by heat shock, whereas the clpC protein was only mildly induced (Ingmer et al., 1999). Although clpC, clpE, and clpB are labeled as heat shock genes, mutants containing disruptions in these genes responded as wild-type cells to heat and salt treatments. However, clpE mutants showed increased sensitivity to puromycin, relative to the wild type, suggesting that ClpE may play a role similar to ClpP in the degradation of randomly folded proteins (Ingmer et al., 1999). The clpX gene was cloned from O. oeni using degenerate primers based on conserved regions in the amino acid sequence (Jobin et al., 1999). In the Gramnegative microoganisms E. coli and Haemophilus influenzae, clpX is found in a gene cluster with tig and clpP (Gottesman et al., 1993; Fleischmann et al., 1995). However, in O. oeni, as in B. subtilis, tig is located next to clpX but clpP is found at a different location on the chromosome and is transcribed as a monocistronic gene (Gerth et al., 1996). Although expression of the B. subtilis clpX gene is independent of CtsR (Gerth et al., 1996), a region resembling the ctsR consensus recognition sequence (Derre et al., 1999) was found overlapping the transcriptional start site of the O. oeni clpX gene; however, it was not determined if this sequence had any impact on the regulation of O. oeni clpX. The clpX mRNA transcript showed increased expression after a temperature shift from 30 to 42°C. Expression was relatively high during the exponential phase of growth and gradually declined to undetectable levels as cells entered stationary phase. Although O. oeni mutants were not constructed, B. subitilis clpX mutants exhibited impaired growth in response to salt, ethanol, and heat (Gerth et al., 1998).
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COLD STRESS RESPONSE AND CRYOPROTECTION PHYSIOLOGICAL RESPONSE
AND
ADAPTATION
Whereas high temperatures diminish protein stability, low temperatures present a cell with a wide array of challenges, such as decreased rate of enzymatic reactions, lower affinity for substrate uptake (Nedwell and Rutter, 1994), decreased fluidity of the cellular membrane (Wada et al., 1990), impaired activity of RNA polymerase (Grau et al., 1994), and increased intracellular solute concentration which can invoke osmotic injury on proteins (Franks, 1995). Death associated with freezing and thawing is primarily attributed to membrane damage and DNA denaturation (Alur and Grecz, 1975; Calcott and MacLeod, 1975; El-Kest and Marth, 1992). Bacterial adaptation to low temperatures is an active process resulting in increased fatty acid unsaturation (Murata and Wada, 1995) and polypeptide synthesis (Jones et al., 1987). Many comprehensive reviews of the microbial cold shock response have been published (Jones and Inouye, 1994; Wolska, 1994; Graumann and Marahiel, 1996; Panoff et al., 1998). The study of the cold shock response in LAB is particularly important because these microorganisms are routinely exposed to a variety of stresses, including low temperature conditions, during the production of fermented food products (Rallu et al., 1996). For example, fermentations normally begin with the addition of a frozen “starter” culture to “raw” food material. Therefore, understanding the cold shock response in these organisms may contribute to the development of starter cultures with a greater capacity for freeze tolerance. The physiological response to suboptimal growth temperatures has been investigated in a number of LAB including L. lactis (Panoff et al., 1994, 1995; Kim and Dunn, 1997), S. thermophilus CNRZ302 (Wouters et al., 1999), S. thermophilus TS2 (Kim and Dunn, 1997), Lb. acidophilus CRL 639 (Lorca and de Valdez, 1999), E. faecalis JH2-2 (Thammavongs et al., 1996; Panoff et al., 1997), Pediococcus pentosaceus PO2 (Kim and Dunn, 1997), and Lb. helveticus LB1 (Kim and Dunn, 1997). Increased capacity towards survival in extreme cold temperatures after preconditioning at low positive temperatures, a phenomenon termed cryotolerance, was achieved for S. thermophilus CNRZ 302 (Wouters et al., 1999), E. faecalis JH2-2 (Thammavongs et al., 1996), L. lactis subsp. lactis (Panoff et al., 1995; Kim and Dunn, 1997), and P. pentosaceus, but not for L. lactis subsp. cremoris, Lb. helveticus LB1, and S. thermophilus TS2 (Kim and Dunn, 1997). The addition of chloramphenicol to the growth medium, thereby inhibiting protein synthesis, during a cold treatment abolished cryotolerance in L. lactis (Wouters et al., 1999). The preconditioning treatment also imparted L. lactis subsp. lactis with an improved survival capacity against freezing temperature (–20°C) and heat (52°C) challenge (Panoff et al., 1995). Furthermore, exponential-phase L. acidophilus cells growing at 25°C displayed a greater resistance to variety of environmental stresses including exposures to ethanol, peroxide, lactic acid, and osmotic stress relative to cells growing at the optimal temperature of 37°C (Lorca and de Valdez, 1999).
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COLD-STRESS GENES
AND
GENE PRODUCTS
Similar to the heat shock response, bacteria respond to low temperatures by expressing a number of cold shock proteins (Jones et al., 1987) regulated at both transcriptional (La Teana et al., 1991; Jones et al., 1992) and translational (Brandi et al., 1996; Goldenberg et al., 1996; Jones et al., 1996; Panoff and Lucas, 1996) levels. However, in contrast to heat shock proteins, which include chaperones and proteases required for protein folding and degradation, respectively, cold shock proteins perform a variety of different functions in bacterial cells. One of the major outcomes of low temperature exposures is the formation of stable DNA and RNA secondary structures which interfere with efficient DNA replication and mRNA transcription and translation. Cold shock proteins comprise a family of small (7 kDa) transiently expressed proteins that function as RNA chaperones to facilitate translation of mRNA by blocking the formation of secondary structures (Jiang et al., 1997). Study of the cold shock response has defined a nomenclature for the specific types of proteins expressed in response to low temperature. Low-temperature stress proteins are generally labeled as cold shock proteins (CSPs), whereas cold acclimation proteins (CAPs) or cold-induced proteins (CIPs) are labeled as such according to the size of the protein and the method by which the organisms were transferred to low temperatures. The CSPs are immediately and transiently induced upon an abrupt shift to a low temperature (Jones et al., 1987; Lottering and Streips, 1995), whereas CAPs are synthesized during continuous growth at low temperatures (Roberts and Inniss, 1992; Whyte and Inniss, 1992; Berger et al., 1996). The CIPs are defined as CSPs larger than 10 kDa (Graumann and Marahiel, 1996). Aside from a single study that differentiates between CSPs and CAPs in E. faecalis (Panoff et al., 1997), research on microbial response to low temperature at the protein level in LAB has focused entirely on CSPs. Much of the current understanding of the cold shock response has come from investigations on E. coli and B. subtilis. E. coli contains a large family of CSPs, consisting of nine proteins from CspA to CspI (Lee et al., 1994). Three proteins (CspB, CspC, and CspD) have been identified in B. subtilis (Graumann et al., 1996). All CSPs share high sequence similarity (over 40%) to eukaryotic Y-box proteins (Wolffe et al., 1992) and both CspA of E. coli and CspB of B. subtilis recognize the highly conserved Y-box sequence ATTGG (La Teana et al., 1991; Jones et al., 1992; Graumann and Marahiel, 1994), which is present in regulatory regions of major histocompatibility complex II genes (Sommerville and Ladomery, 1996). The Y-box proteins serve regulatory functions at the transcriptional and translational level. Accordingly, the CSP-homologous domain was shown to confer sequence-specific binding to single-stranded DNA and RNA (Sommerville and Ladomery, 1996). Both NMR and x-ray crystallography showed that the three-dimensional structures of CspB (B. subtilis) and CspA (E. coli), the major cold shock proteins (Goldstein et al., 1990; Graumann and Marahiel, 1994), displayed very similar five-stranded βbarrel structures with outward-facing residues for ssDNA binding (Schindelin et al., 1993; Schnuchel et al., 1993; Newkirk et al., 1994; Schindelin et al., 1994). Physiological investigations have confirmed CspA and CspB as ssDNA-binding proteins (Graumann and Marahiel, 1994; Newkirk et al., 1994). Furthermore, CspA and CspB
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exhibit mRNA binding capacity because both have RNA-binding motifs, RNP-1 (ribonucleoprotein) and RNP-2 (Schindelin et al., 1993; Jones and Inouye, 1994; Jiang et al., 1997). Taken together, this information suggests that CSPs exhibit regulatory effects at the transcriptional and translational level through protein–nucleic acid interactions. Indeed, CspA enhanced the transcription of two cold-inducible genes, gyrA and hns (La Teana et al., 1991; Jones et al., 1992), suggesting that CspA may function as a transcriptional activator of other cold shock genes. Furthermore, CspA functions as an RNA chaperone by binding to mRNA and preventing the formation of secondary structures (Jiang et al., 1997). Although, the physiological contribution of CSPs to low temperature adaptation is not well understood, cold shock causes a 200-fold induction of CspA in E. coli (Jones et al., 1987; Goldstein et al., 1990). Additionally, disruption of CspB in B. subtilis resulted in increased sensitivity to freezing (Willimsky et al., 1992) and deletion of the three known CSPs in B. subtilis was lethal (Graumann et al., 1997). Only recently has the study of cold shock genes and gene products been focused on LAB, but it is now progressing quickly. The proteins expressed in response to a shift from optimal to suboptimal temperatures in Lb. acidophilus (Lorca and de Valdez, 1999), E. faecalis (Panoff et al., 1997), L. lactis (Panoff et al., 1994; Broadbent and Lin, 1999; Wouters et al., 1999), and S. thermophilus (Wouters et al., 1999) were extracted and viewed by two-dimensional SDS-PAGE gel electrophoresis. A cold shock gene showing high sequence similarity with the major cold shock protein of E. coli (68%) and B. subtilis (70%) was cloned from L. lactis subsp. lactis using a PCR-based approach (Kim and Dunn, 1997). This approach was taken a step further to amplify the major cold shock protein from 11 other LAB strains (Kim et al., 1998), suggesting that the major cold shock protein is highly conserved in LAB. The first cold shock gene identified in LAB, cspB, was cloned in L. lactis (Chapot-Chartier et al., 1997). Results of northern hybridization (Chapot-Chartier et al., 1997) and cspB-directed B-galactosidase assays (Chapot-Chartier et al., 1997; Wouters et al., 1998) indicate that the L. lactis cspB is cold-shock inducible (Wouters et al., 1998). Two additional cold-shock genes, cspL and cspP, were cloned from Lb. plantarum (Mayo et al., 1997). The identification of five cold shock genes, cspA, cspB, cspC, cspD, and cspE (Wouters et al., 1998), in L. lactis quickly followed. Four of these genes were found to be clustered, for the first time, in two tandem groups (cspA/cspB and cspC/cspD), whereas cspE was found as a single gene. These cold shock genes can be divided, on the protein level, into two groups based on isoelectric point (pI) and homology: CspA and CspC share 80% identical residues and have a pI of 9, whereas CspB, CspD, and CspE share 85% identical residues and have a pI of 5 (Wouters et al., 1998). Transcriptional analyses showed that cspA, cspB, cspC, and cspD were cold-shock inducible, whereas cspE was not (Wouters et al., 1998). The RNA-binding motifs (RNP-1 and RNP-2) (Schindelin et al., 1993; Jones and Inouye, 1994; Schroder et al., 1995) were found within the L. lactis gene products, including a putative β-barrel structure formed by five β-strands (Wouters, 2000). Expression levels of these CSPs, in response to low temperature treatment, coincide with freeze survival (Wouters et al., 1999).
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Perhaps the single greatest contribution to the understanding of the physiological and regulatory roles of CSPs in LAB was made very recently by Wouters et al. (2000), where attempts were made to overproduce these CSPs in L. lactis using the nisin controlled expression (NICE) system (Kuipers et al., 1998). Using the NICE system, CspB, CspD, and CspE were overproduced to high levels, whereas the concentrations of CspA and CspC were limited due to low protein and mRNA stability, respectively. The reduced stability of CspA is attributed to the presence of an Arg residue at position 58 rather than a Pro residue, which are known to reduce the entropy of unfolded proteins (Schindler et al., 1999). Replacing the Arg residue at position 58 for a Pro residue increased the concentration of CspA* 20-fold upon induction with nisin. Overproduction of CspA* resulted in the induction of CspE and several CIPs. Likewise, overproducing CspC resulted in the induction of CspB and the putative CspF and CspG proteins, in addition to several CIPs. This suggests that CspA* and CspC may be transcriptional activators acting on Y-box motifs (La Teana et al., 1991; Jones et al., 1992; Brandi et al., 1994) observed in the upstream regions of the lactococcal csp genes (Wouters et al., 1998). Overproduction of CspA, CspB, CspD, or CspE did not affect the level of any of the other CSPs; however, overproduction of CspB and CspD increased the synthesis of several CIPs, suggesting a regulatory role for these proteins. With respect to adaptation to freeze-survival, overproduction of CspB and CspE resulted in approximately a ten- and five-fold increased survival, respectively, compared to that of non-induced cells after four repetitive freeze–thaw cycles. In a previous report, overexpression of CspD in L. lactis cells enhanced survival after freezing approximately two- to ten-fold compared to the control cells (Wouters et al., 1999). Overproduction of CspA, CspC, or CspA* provided no additional freeze-protective effects compared to control cells (Wouters et al., 2000). These results indicate that CspB, CspE, and CspD are directly involved in the protection against freezing (Wouters et al., 1999, 2000).
ACID ADAPTATION TOLERANCE
AND
ADAPTATION
TO
LOW PH
The understanding of acid tolerance and adaptation in LAB is expected to contribute to enhancement of probiotic survival through the gastrointestinal tract. Furthermore, this understanding is important with regard to starter culture performance during fermentation since cell growth is always accompanied by lactic acid accumulation. Lactic acid poses a significant threat to the cell because, in a low pH environment, organic acids remain protonated and uncharged and can thereby pass easily into the cell through the cell membrane. At a similar extracellular pH, a strong inorganic acid, such as HCl, is likely to be in a disassociated state and will not passively diffuse through the cell membrane (Kashket, 1987). Accordingly, reducing the intracellular pH of L. lactis and S. bovis was more effective when the extracellular pH was adjusted with lactic acid than with HCl acid (Poolman et al., 1987; Cook and Russel, 1994). Bacteria are equipped with a number of mechanisms that confer acid tolerance. Among the mechanisms that will be reviewed in this chapter are proton translocation,
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arginine deaminase (ADI) pathway, amino acid decarboxylation-antiporter reactions, and the citrate transport system. The activation of these mechanisms is the result of an altered pattern of gene expression when bacteria are confronted with a change in the extracellular pH (reviewed by Olson, 1993). Gene expression-dependent adaptation is evident upon analysis of whole-cell protein extracts separated by twodimensional gel electrophoresis. Changes in protein patterns have been observed during acid adaptation in E. coli (Heyde and Portalier, 1990; Hickey and Hirschfeld, 1990), Salmonella typhimurium (Hickey and Hirschfeld, 1990; Foster, 1991, 1993), Aeromonas hydrophila (Karem et al., 1994), and Listeria monocytogenes (Davis et al., 1996). The adapted cells showed enhanced survival capacity against lethal acid challenge relative to unadapted cells (Goodson and Rowbury, 1989; Foster and Hall, 1990; Davis et al., 1996; O’Driscoll et al., 1996). This inducible adaptation to acid is termed the acid tolerance response (ATR; Foster and Hall, 1990) and is dependent on protein synthesis (Foster, 1991; Raja et al., 1991; Karem et al., 1994; O’Hara and Glenn, 1994; Davis et al., 1996). The ATR has been observed in several LAB such as Lc. mesenteroides (McDonald et al., 1990), Lb. plantarum (McDonald et al., 1990), S. mutans (Belli and Marquis, 1991), Enterococcus hirae (Belli and Marquis, 1991), and L. lactis (Hartke et al., 1996; Rallu et al., 1996; O’Sullivan and Condon, 1997). The ATR was displayed in L. lactis in response to sublethal exposures to lactic acid (Hartke et al., 1996), HCl (Rallu et al., 1996), and UV radiation (Hartke et al., 1995). Furthermore, resting cells were much better adapted to low pH than actively dividing cells (Hartke et al., 1994). Although the ATR was achievable in L. lactis, the requirement for protein synthesis is controversial: L. lactis subsp. lactis IL1403 showed acid adaptation in the presence of chloramphenicol (Hartke et al., 1996), whereas L. lactis subsp. cremoris MG1363 (Rallu et al., 1996) and L. lactis subsp. cremoris 712 (O’Sullivan and Condon, 1997) did not. The ATR in L. lactis subsp. cremoris 712 also conferred enhanced resistance to lethal doses of heat, ethanol, sodium chloride, and hydrogen peroxide; however, with the exception of heat, mild treatments of other environmental stresses did not induce tolerance to acid (O’Sullivan and Condon, 1997). Cross-protection induced by acid has also been observed in S. typhimurium (Leyer and Johnson, 1993; Lee et al., 1995) and L. monocytogenes (O’Driscoll et al., 1996). To identify some of the genes associated with acid tolerance, insertional mutants of L. lactis were prepared using the pG+host9:ISS1 plasmid (Maguin et al., 1996). Twenty-one mutants were isolated based on their ability to grow under high temperature and low pH, conditions under which wild-type L. lactis does not grow well (Rallu et al., 2000). All of the mutants were acid tolerant and 11 were resistant to a variety of stresses. Many of the insertions in acid-tolerant mutants took place in genes implicated with glutamate/glutamine transporters, high-affinity phosphate transporters, and purine metabolism. These results suggest an intimate relationship between stress response mechanisms and cellular metabolic pathways in L. lactis (Rallu et al., 2000).
PROTON MOVEMENT: H+-ATPASE The F0F1 ATPase functions to maintain a favorable intracellular pH and protect cells during exposures to acidic environments by translocating protons to the environment at the expense of ATP. The activity and number of proton-translocating ATPases
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increases in several LAB as the extracellular pH is adjusted from neutral to pH 5.0 (Kobayashi et al., 1984; Belli and Marquis, 1991; Nannen and Hutkins, 1991). Whereas the atp operon, encoding various subunits of the H+-ATPase, is minimally affected by pH in E. coli (Kasimoglu et al., 1996) and constitutively expressed in B. subtilis (Santana et al., 1994), acidification of the growth medium leads to elevated gene expression in Lb. acidophilus (Kullen and Klaenhammer, 1999). Proton-translocating ATPase is an important mechanism in maintaining cytoplasmic pH in L. lactis subsp. lactis and cremoris (Nannen and Hutkins, 1991), Lb. casei (Bender and Marquis, 1987; Nannen and Hutkins, 1991), E. faecalis (Kobayashi et al., 1984, 1986), E. hirae (Belli and Marquis, 1991), S. mutans (Bender et al., 1986; Belli and Marquis, 1991), and Lb. acidophilus (Kullen and Klaenhammer, 1999). An acid-sensitive isolate of L. lactis contained a mutation in the ATPase structural gene and was unable to maintain a neutral intracellular pH in an acidic environment (Amachi et al., 1998). Furthermore, mutants of S. typhimurium lacking the proton-translocating ATPase are extremely acid sensitive and do not display an ATR (Foster and Hall, 1991).
ARGININE DEAMINASE (ADI) PATHWAY Bacteria metabolize arginine by the arginine deaminase (ADI) pathway (Cunnin et al., 1986). This pathway consists of three enzymes: arginine deaminase, ornithine carbamoyltransferase, and carbamate kinase. A fourth component, identified in L. lactis (Poolman et al., 1987), Lb. sake (Zuniga et al., 1998), and Pseudomonas aeruginosa (Verhoogt et al., 1992), is a membrane-bound antiport protein that catalyzes the exchange between arginine and ornithine. These enzymes catalyze the conversion of arginine to ornithine, ammonia, and carbon dioxide and generate 1 mol of ATP per mole of arginine consumed (Figure 6.1). By generating ammonia, the ADI pathway is a mechanism for survival in acidic environments (Marquis et al., 1987). HOOC-CH(-NH2)-(CH2)3-NH-C(=NH)-NH2 (arginine) arginine deiminase NH4+ HOOC-CH(-NH2)-(CH2)3-NH-CO-NH2 (citrulline) ornithine carbamoyltransferase
HOOC-CH(-NH2)-(CH2)3-NH2 (ornithine)
Pi
H2N-CO- P (carbamoyl phosphate) ADP
carbamate kinase ATP HCO3, NH4+
FIGURE 6.1 The ADI pathway. (From Cunin, R.N., Microbiol. Rev., 50: 314–352, 1986. With permission.)
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The development of acid tolerance depends on the rise in pH associated with ammonia production (Marquis et al., 1987). The enzymatic properties of the ADI pathway are well documented in a variety of bacteria (Stalon, 1972; Stalon et al., 1972; Fenske and Kenny, 1976; Crow and Thomas, 1982; Cunnin et al., 1986). The enzymes in the ADI pathway are inherently acid tolerant and are activated in response to low pH (pH 2 to 3) in several species of Streptococcus. As such, these enzymes allow bacteria to recover from acid stress severe enough to prevent the cell membrane from functioning normally (Casiano-Colon and Marquis 1988). In most LAB, the ADI pathway is repressed by glucose and induced by arginine (Simon et al., 1982; Hiraoka et al., 1986; Manca de Nadra et al., 1986; Poolman et al., 1987). The ADI pathway imparts LAB with enhanced tolerance to acid, primarily through the continuous production of acid-neutralizing ammonia from arginine.
DEGRADATIVE AMINO ACID DECARBOXYLASES Another strategy bacteria employ to maintain a favorable intracellular pH depends on amino acid decarboxylation-antiporter reactions. These reactions involve transporting an amino acid into the cell where it is decarboxylated. A proton is consumed in the reaction, and the product is exported from the cell via an antiporter. The result of this reaction is a decrease in intracellular acidity (Molenaar et al., 1993). A gadC-encoded glutamate-γ-aminobutyrate antiporter and a gadB-encoded glutatmate decarboxylase have been identified in L. lactis (Sanders et al., 1998). The two genes are located in a bicistronic gadCB operon and show increased expression during growth and acidification of unbuffered media supplemented with glutamate (Sanders et al., 1998). According to the model proposed by Waterman and Small (1996) for Shigella flexneri, the putative membrane protein, GadC, is involved in the antiport of glutamate, while glutamate decarboxylase, GadB, converts the internalized glutamate to γ-aminobutyrate with the simultaneous consumption of a proton and production of one molecule of CO2. The net result is the removal of a proton from the cytosol, which increases the intracellular pH. Support for the amino acid decarboxylation-antiport model is displayed in a histidine decarboxylase mutant of Lactobacillus 30a that was unable to alkanize its environment in the presence of histidine (Recsie and Snell, 1972). Another function for GadCB may be production of a proton motive force and generation of energy in the presence of glutamate, as shown for a strain of Lactobacillus (Higuchi et al., 1997). A similar mechanism was described for a different Lactobacillus strain, where the action of amino acid antiport and decarboxylation are combined for pH regulation and energy production. The aspartate–alanine antiporter generated ATP in Lactobacillus strain M3 (Abe et al., 1996), and histidine decarboxylation coupled with electrogenic histidine–histamine antiport contributed to energy production and intracellular acid reduction in Lb. buchneri (Molenaar et al., 1993).
CITRATE TRANSPORT SYSTEM Citrate is present in milk at low concentrations and is co-metabolized with glucose by many strains of LAB (Cocaign-Bousquet et al., 1996). Citrate fermentation has been studied in detail in Lc. mesenteroides (Marty-Teysset et al., 1995, 1996) and © 2003 by CRC Press LLC
L. lactis (Hugenholtz, 1993). The citrate fermentation pathway is induced by citrate in Lc. mesenteroides (Marty-Teysset et al., 1996). Alternatively, citrate utilization in L. lactis subsp. lactis biovar diacetylactis is dependent on the rate of uptake, catalyzed by the product of the citP gene (David et al., 1990), and expression of citP is influenced by extracellular pH (Garcia-Quintans et al., 1998). Activity of CitP is also dependent on extracellular pH, with the highest uptake rates observed at pH 4.5 (Magni et al., 1996). Accordingly, cell growth accompanied by the natural acidification of the medium results in increased synthesis of CitP, higher citrate transport activity, and greater flux through the citrate fermentation pathway. Upon entering the cell, citrate is cleaved by citrate lyase, which yields acetate and oxaloacetate. Decarboxylation of oxaloacetate yields carbon dioxide and pyruvate, consumes a proton, and results in alkalinization of the cytoplasm (Ramos et al., 1994; Lolkema et al., 1995; Marty-Teysset et al., 1996). Pyruvate is converted to the end product lactate, which leaves the cell through the CitP transporter in exchange for citrate. Together, the consumption of a proton during oxaloacetate decarboxylation and the excretion of lactate, in exchange for citrate, provide citrate-fermenting LAB with a resistance mechanism against acid toxicity. This acid resistance mechanism was demonstrated by the undiminished growth of L. lactis in a medium at pH 4.5 containing both glucose and citrate, whereas growth was poor in the absence of either glucose, citrate, or CitP (Garcia-Quintans et al., 1998); glucose is required to produce lactate that drives the exchange for citrate via the CitP transporter. The CitP transporter is purported to be among the proteins that are synthesized de novo during inducible adaptation to acid (e.g., acid tolerance response), shedding some light on the poorly understood mechanisms involved in acid resistance in bacteria.
ALKALINE STRESS RESPONSE The alkaline response is the least studied of the stress responses. Exposure to sublethal alkaline conditions results in increased resistance to lethal alkalinization (pH 10.0 to 10.5) in E. coli (Goodson and Rowbury, 1990) and thermotolerance at 55°C in Salmonella enteritidis (Humphrey et al., 1991). However, sublethal alkaline conditions sensitized E. coli to acid (Rowbury et al., 1993) and, conversely, sublethal acid treatment sensitized E. coli to alkaline pH (Rowbury and Hussain, 1996). With regard to the LAB, the alkaline stress response has been investigated only for Enterococcus species. As early as 1934, Sherman and Stark, identified E. faecalis by its ability to grow at pH 9.6. A neutral cytoplasmic pH is not required to withstand high alkaline pH in Enterococcus hirae (Mugikura et al., 1990); however, the alkaline treatment amplifies the Na+-ATPase (Kakinuma and Igarashi, 1990), suggesting modification in gene expression. This hypothesis was confirmed when whole-cell proteins extracted from E. faecalis, viewed on two-dimensional gel electrophoresis, showed amplification of 37 polypeptides after a 30-minute alkaline treatment at pH 10.5 (Flahaut et al., 1997). Furthermore, cells adapted to pH 10.5 were tolerant to pH 11.9. The addition of chloramphenicol to the culture at pH 10.5 resulted in a minor decrease in alkaline tolerance. Acquisition of acid tolerance of acid-exposed cells treated with chloramphenicol was similar to untreated cells (Flahaut et al., 1997). These observations suggest that protein synthesis in E. faecalis is not a © 2003 by CRC Press LLC
prerequisite to developing tolerance to lethal extremes in pH, similar to results found with L. lactis subsp. lactis IL1403 (Hartke et al., 1996). Furthermore, as found in E. coli (Rowbury et al., 1993; Rowbury and Hussain, 1996), acid-exposed E. faecalis cultures were sensitized to alklaline pH, and alkaline-treated cells acquired sensitivity to acid damage (Flahaut et al., 1997). The next section will highlight what has been published to date for select genera and species of LAB relative to osmoregulation.
OSMOTIC STRESS COMPATIBLE SOLUTE Organisms, both eukaryotic and prokaryotic, respond to osmotic stress in essentially the same way: by accumulating non-toxic low molecular weight compounds. These compounds, called compatible solutes, which include sugars, polyols, amino acids and amine derivatives, do not inhibit vital cellular functions even when present in very high concentrations. Compatible solutes have at least three functions: 1) allow the cell to retain positive turgor pressure which contributes to osmotic balance with the extracellular environment; 2) enhance enzyme stability at low aw; and 3) maintain the integrity of the cellular membrane during desiccation (Kets and de Bont, 1994). A review on the role of compatible solutes in osmoregulation in bacteria has recently appeared (Bremer and Kramer, 2000). Lactobacillus acidophilus IFO 3532 is tolerant of osmotic pressures from electrolytes or non-electrolytes up to an osmolality of 2.8 M. Glycine betaine was identified some years ago as the intracellular osmolyte which protected Lb. acidophilus from osmotic stress (Hutkins et al., 1987). Glycine betaine is a constituent of the yeast extract present in MRS medium and upon the addition of NaCl (1 M), glycine betaine is transported into the cells by a specific transport system. The rate of glycine betaine transport is proportional to the osmolality of the medium. Energy in the form of a fermentable sugar was necessary for glycine betaine transport. Transport of glycine betaine in Lb. acidophilus appears to be activated (stimulated) rather than induced by osmotic stress. Chloramphenicol did not inhibit glycine betaine transport indicating that the induction of new protein synthesis was not necessary for transport (Hutkins et al., 1987). In a defined medium, the growth of Lb. plantarum strain P743 in 0.6 M NaCl decreased seven-fold; however, the addition of 2 mM glycine betaine permitted growth almost to the level of the control treatment that was lacking salt. In addition, glycine betaine addition allowed growth at higher NaCl levels (Kets and de Bont, 1994). Survival of dried cells increased significantly when Lb. plantarum was grown in the presence 2 mM betaine and 0.6 or 1.0 M NaCl as compared to cells grown in the absence or presence of glycine betaine or salt (Kets and de Bont, 1994). Similar results were obtained with Enterococcus faecium strain URL-EF1 and Lb. halotolerans ATCC 35410 (Kets et al., 1996). Cells grown under osmotic stress (NaCl) in the presence of glycine betaine survived drying at higher levels than did unconditioned cells. However, Linders et al. (1997) found that Lb. plantarum strain P743 grown in a complex medium with 1 or 1.25 M NaCl prior to drying had reduced survival as compared to controls lacking salt. In addition, Lb. plantarum strain P743
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grown in the absence of salt produced dried cells with higher levels of residual glucose fermenting ability than cells grown in a salt-containing medium (Linders et al., 1997). This resulted from the fact that lower cell numbers were recovered from the NaCl-containing medium. At 0.11 g dried cells/g sample, the residual glucose fermenting activity of Lb. plantarum strain P743 dried cells harvested from a synthetic medium lacking salt was approximately 12 times that of the same amount of dried cells obtained from a salt-containing medium (Linders et al., 1998). In dried cells of Lb. plantarum strain P743, residual glucose-fermenting activity was a function of the cell density before drying. At the highest cell density (0.23 g dried cells/g sample), the glucosefermenting activity was eight times that of the lowest cell density (0.025 g dried cells/g sample). To obtain enzymatically active dried cells of Lb. plantarum, Linders et al. (1998) recommended growing cells in an osmotically unstressed medium. They also suggested that harvested and washed cells should not be diluted before drying. Lactobacillus plantarum strain P743, when grown in a complex medium (MRS broth) with NaCl, accumulated glycine betaine and another compatible solute which was identified as L-carnitine. No accumulation of carnitine occurred when beef extract was eliminated from the formulation for MRS broth; similarly, glycine betaine was not accumulated by Lb. plantarum when yeast extract was omitted (Kets and de Bont, 1994). Addition of 0.5 mM L-carnitine to a chemically defined medium containing 0.4 M NaCl led to a doubling of the growth rate as compared to a saltcontaining medium lacking carnitine. Thus, carnitine acts as a compatible solute making Lb. plantarum more tolerant to salt stress (Kets et al., 1994). In Lb. plantarum ATCC 14917 growing in a chemically defined medium containing high levels of KCl, the preferred osmoprotectant was glycine betaine. However, it was necessary to add glycine betaine to the chemically defined medium, indicating that the organism could not synthesize the compound (Glaasker et al., 1996b). While K+ accumulates intracellularly under KCl stress, it does not act as a osmoprotectant. In a medium in which the growth of Lb. plantarum is inhibited by KCl or NaCl, the addition of glycine betaine or proline increased the specific growth rate. Moreover, transport and accumulation of glycine betaine increased rapidly with an increase in osmolarity even in the presence of chloramphenicol (Glaasker et al., 1996b). Iso-osmolar sucrose, lactulose, NaCl, or KCl had similar effects on transport rates and accumulation of glycine betaine. The final levels of accumulated glycine betaine were proportional to the increase in the final medium osmolarity, but the initial rate of uptake was similar regardless of osmolarity. Glycine betaine transport rates (activated transport) increase when the difference between internal and external osmolarity reaches a certain threshhold. When the osmolarities are balanced, then net glycine betaine uptake ceases. Osmotic downshock caused an efflux of glycine betaine, which was rapid and greatly exceeded the uptake rates (Glaasker et al., 1996b). Efflux, too, depended on osmolarity. Metabolic energy is not necessary for efflux, whereas uptake required ATP synthesis (Glaasker et al., 1996b). Glycine betaine, accumulated at high osmolarity by Lb. plantarum ATCC 14917, was released from the cell upon osmotic downshock. Efflux of glycine betaine was biphasic, with a rapid release phase and a slower release phase (Glaasker et al., 1996a). The rapid efflux phase was mechanosensitive channel-mediated, whereas
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the slow efflux of glycine betaine was mediated through a carrier (uniporter) transport system (Glaasker et al., 1996a). Addition of equimolar amounts of KCl or lactose to a chemically-defined medium used to grow Lb. plantarum ATCC 14917 enhanced the rate of glycine betaine uptake to the same extent. Facilitated influx of lactose to the equilibration level combined with uptake of glycine betaine resulted in hyperosmolarity of the cytoplasm. As a compensatory mechanism to decrease the resultant hyperosmolarity, there was a net exit of glycine betaine (Glaasker et al., 1998b). Cells stressed by KCl behaved differently than cells stressed by lactose. While there was uptake of KCl into Lb. plantarum cells, the increased cellular level of KCl did not compensate for a decrease of turgor. Therefore, glycine betaine uptake was necessary to reverse the cell turgor decrease imposed by the salt stress, and efflux of glycine betaine did not occur (Glaasker et al., 1998b). The intracellular accumulation of quaternary ammonium compatible solutes in Lb. plantarum ATCC 14917 is mediated via a single transport system, QacT (quaternary ammonium compound transporter), which has a high affinity for glycine betaine or carnitine and a low affinity for proline (Glaasker et al., 1998a). Transport uptake rates were inhibited by internal glycine betaine or proline; however, with an increase in osmolarity, the inhibition by the internal osmolyte was relieved with the rapid activation of the QacT system. Upon osmotic downshock, there was release of glycine betaine via a system resembling that of a mechanosensitive ion channel (Glaasker et al., 1998a). At least in Lb. plantarum, both uptake and efflux of compatible solutes utilize osmoregulated systems. Therefore, in Lb. plantarum ATCC 14917, compatible solutes are taken up via a single system, QacT, which is activated by osmotic shock. The QacT uptake system is turgor-regulated; however, when cell turgor is restored, solute uptake is diminished. Inhibition of QacT by an internal compatible solute such as glycine betaine also acts to control excessive accumulation of compatible solute. On hypo-osmotic shock, compatible solutes are released from the cell in order to maintain cell turgor. Efflux occurs via two mechanisms: 1) a rapid almost instantaneous release of solute mediated by a channel system, followed by 2) a slow release of solute via an efflux carrier system (Glaasker et al., 1996a, 1996b, 1998a, 1998b). Osmoregulation in Lb. plantarum ATCC 14917 has been envisioned by Poolman and Glassker (1998) as follows: under osmostasis, there is a basal level of glycine betaine or other compatible solute which is maintained by the combined action of efflux via the specific efflux carrier system and uptake by QacT; the efflux channel system does not play a role in osmostasis. During hyperosmotic shock, QacT is activated and glycine betaine enters the cell; both efflux systems are inhibited. Under hypo-osmotic shock, QacT is inhibited but both efflux systems are activated (Poolman and Glaasker, 1998). Therefore, maintenance of cell turgor is tightly regulated in Lb. plantarum. Subjecting Lb. plantarum strain L-73 to glycerol or NaCl in a solute/water medium at –5.6 MPa had little or no effect on the subsequent viability of the cells when the solute was added rapidly (within 1 s) to simulate shock conditions or added slowly, over 20 min (Poirier et al., 1998). As another example, a commercial culture of Lactobacillus alimentarius has been used as a bioprotectant in certain meat products (Andersen, 1997). The culture
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is generally used as a “biohurdle” in conjunction with chemicals such as NaCl, organic acids, and/or sulfite. The growth of Lb. alimentarius strain BJ33 in MRS broth was not inhibited by 8% NaCl, 50 mM citric acid or 100 mM gluconic acid. However, when washed cells from a medium containing one of the organic acids were inoculated into MRS broth supplemented with 8% NaCl, there was limited growth (OD600nm = 0.2 to 0.3 within 7 days) (Lemay et al., 2000). When the reverse experiment was conducted, growth in 8% NaCl followed by inoculation of washed cells into either citric acid or gluconic acid containing MRS broth, an OD600nm of 0.6 to 0.8 was reached in 3 to 4 days. Thus, Lemay et al. (2000) demonstrated that Lb. alimentarius grown in the presence of sublethal levels of organic acids grew poorly when subsequently inoculated into a medium containing a sublethal concentration of NaCl. It is not clear why cells grown in organic acids are stressed by sublethal concentrations of salt. It is clear, however, that if the above mentioned strain of Lb. alimentarius is used as a biohurdle in foods, the influence of chemical hurdles on the growth of the organism must be determined. In the presence of 500 mM KCl, glycine betaine uptake was stimulated fivefold in cells of L. lactis subsp. cremoris MG1363 or L. lactis subsp. lactis IL1403 (van der Heide and Poolman, 2000). In contrast to the finding that proline inhibits uptake of glycine betaine in Lb. plantarum (Glaasker et al., 1998a), van der Heide and Poolman (2000) found that proline did not inhibit the uptake of glycine betaine in these two subspecies of L. lactis. Molenaar et al. (1993) previously demonstrated that the proline uptake system in L. lactis subsp. lactis ML3 had a higher affinity for uptake of glycine betaine than for proline; therefore, it is not surprising that proline did not inhibit uptake of glycine betaine (van der Heide and Poolman, 2000). Osmotic downshock in the L. lactis subspecies led to an almost instantaneous efflux of glycine betaine from the cells. The efflux on downshock was proportional to the decrease in osmolarity of the downshock. As such, efflux is not due to an activation of a transport system, but rather it is mediated by a channel-like activity. An NaCl level of 0.4 M present in a chemically-defined medium reduced the growth rate of the bacteriocin-producing strain of L. lactis subsp. lactis ADRIA 85LO30 by about 70%, but it did not inhibit the production of lacticin 481 (Uguen et al., 1999). Addition of 1 mM glycine betaine to the medium containing 0.4 M salt increased the growth of L. lactis to a level comparable to the control lacking NaCl and betaine glycine. However, glycine betaine completely eliminated production of lacticin 481. The relief of osmolarity stress was detrimental to the production of the bacteriocin. Growth of eight L. lactis strains in a complex medium supplemented with NaCl or with glucose at the same aw indicated that there were two classes of L. lactis: a salt-tolerant group and a salt-sensitive group. L. lactis subsp. lactis strains C10, BA1 and BA2 and L. lactis subsp. cremoris NCDO 712 (and its plasmid-free derivative, MG1363) were capable of growth at 4% NaCl, whereas the growth of strains BK5, HP and US3 of L. lactis subsp. cremoris was inhibited by >2% NaCl (O’Callaghan and Condon, 2000). In a chemically defined medium, the growth of the salt-tolerant strains of L. lactis was stimulated by the addition of glycine betaine when NaCl was present. However, glycine betaine did not stimulate the growth of the salt-sensitive strains in the presence of NaCl. In addition, the salt-sensitive strains accumulated
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very little glycine betaine, indicating that the strains had little or no glycine betaine transport activity (O’Callaghan and Condon, 2000). The moderately halophilic lactic acid bacterium, Tetragenococcus halophila (formerly known as Pediococcus halophilus), is associated with the production of soy sauce and cured anchovies. When grown in a complex medium, T. halophila accumulated glycine betaine and carnitine even in the absence of NaCl (Robert et al., 2000). Addition of glycine betaine or carnitine to T. halophila growing in a chemicallydefined medium increased the growth rate and final yield of cells in the absence of NaCl, as well as in saline concentrations up to 2.5 M. Unlike other LAB that have been studied, T. halophila can oxidize choline to produce glycine betaine; thus, choline is an osmoprotectant, since it can be enzymatically converted to glycine betaine (Robert et al., 2000). There are two systems for the uptake of glycine betaine. One system transports only glycine betaine, whereas the other system transports glycine betaine, carnitine and choline. De novo synthesis of protein is not necessary for the uptake of the osmoprotectants, since uptake was not inhibited by chloramphenicol. In the absence of glycine betaine or carnitine, T. halophila cannot control intracellular Na+ levels. In the presence of osmoprotectants, the intracellular Na+ level is maintained at 150 to 320 nmol/mg (dry weight of cells) in media with salinities ranging from 0 to 2 M. Glycine betaine or carnitine, therefore, can maintain the intracellular sodium level of T. halophila within narrow limits, regardless of the external sodium level (Robert et al., 2000). Glycine betaine and carnitine not only act as osmotic stabilizers in T. halophila but also act as stabilizers of intracellular Na+ levels.
PROTEIN SYNTHESIS
DURING
OSMOTIC SHOCK
Exponentially growing cells of E. faecalis ATCC 19433 subjected to 6.5% NaCl or 52% sucrose for 2 h were resistant to heat (62°C for 15 min), ethanol (22%), H2O2 (45 mM), bile salts (0.3%) and SDS (0.017%) (Flahaut et al., 1996). Salt stress led to the induction of at least 96 proteins; approximately half of these proteins were induced 2- to 4-fold in concentration as compared to unstressed cells, and 20 of the proteins increased at least 10-fold. Addition of chloramphenicol during the 2-hour adaptation period to sugar or salt led to the inhibition of heat resistance, but blockage of protein synthesis did not inhibit tolerance to the other stresses (Flahaut et al., 1996). Thus, de novo protein synthesis is necessary for resistance to heat by osmotically adapted cells, but cross-protection against ethanol, bile salts, H2O2, and SDS does not require de novo protein synthesis. L. lactis subsp. cremoris strain MG1363 subjected to a temperature shift of 30 to 43°C produced 17 heat shock proteins (HSPs) including GroES, GroEL and DnaK. Eleven of these proteins (including GroES, GroEL and DnaK) also were produced by cells stressed with 2.5% NaCl. However, seven of the eleven proteins were produced at higher levels under heat stress. In addition, a salt shock protein (SSP; Ssp21), produced at high levels by salt stress, was produced at low levels with heat stress (Kilstrup et al., 1997). The data presented by Kilstrup et al. (1997) indicated that there is an overlap in the type of stress proteins produced by heat- and saltshocked cells.
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Smeds et al. (1998) cloned, sequenced and characterized a stress-inducible gene, htrA, from Lb. helveticus strain CNRZ 32. The addition of NaCl (4%) to the growth medium induced an eight-fold increase in the level of htrA transcription. Exposure of growing Lb. helveticus cells to ethanol (5%) or puromycin (100 µg/mL) resulted in approximately a five-fold induction of the transcription of htrA. Upshift of growing cells from 37 to 52°C led to a doubling of htrA mRNA. The protein encoded by Lb. helveticus htrA is a serine protease; however, its role and how its expression is regulated are unknown (Smeds et al., 1998). Lactobacillus sakei strain LTH681, a commercial starter culture for fermented sausages, has a dnaK operon that consists of four heat shock genes in the order, hrcA-grpE-dnaK-dnaJ (Schmidt et al., 1999). Transcription of the genes is induced by heat shock (42°C), NaCl (6%) and ethanol (10%). Analysis of the transcription start site revealed that the dnaK operon was preceded by an sA-type promoter (P2); the transcription starting site varied depending on the type of stress. Transcription induced with either heat or ethanol had a different start site than transcription induced by salt (Schmidt et al., 1999). A CIRCE element was located between the transcription and translation start sites (Schmidt et al., 1999). Schmidt et al. suggest that under non-stress conditions, HrcA represses the expression of heat shock genes or operons by binding to the cis-element CIRCE.
OXIDATIVE STRESS TOLERANCE
AND
ADAPTATION
TO
OXIDATIVE STRESS
LAB are facultative anaerobes that metabolize carbohydrates via fermentation. Although they lack a functional electron transport chain, LAB perform several oxidation and reduction reactions during the catabolism of carbohydrates. Some of these reactions (Table 6.2) use molecular oxygen (O2) as a substrate. The presence of oxygen can generate partially reduced toxic intermediates of O2 such as superoxide anion (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) (McCord et al., 1971; Repine et al., 1981). These intermediates are also formed through a variety of intracellular reactions. For example, H2O2 is formed through the activity of H2O2forming flavoprotein oxidases (Whittenbury, 1964), such as NADH oxidase and pyruvate oxidase (see Table 6.2), and during the dismutation of O2– by superoxide dismutase (SOD) (Britton et al., 1978). The simultaneous presence of hydrogen peroxide and superoxide anions can lead further to the formation of hydroxyl radicals (O2– + H2O2 → OH- + •OH + O2 [Gregory and Fridovich, 1974]), which are particularly harmful in Lactobacillus since members of this genus lack SOD and are unable to eliminate superoxide anions (Gregory and Fridovich, 1974). Together, these reactive oxygen intermediates can cause severe oxidative damage such as strand breaks in DNA (Storz et al., 1987; Teebor et al., 1988; Piard and Desmazeaud, 1991), oxidation of membrane lipids (Meads, 1976), and inactivation of enzymes (Wolff et al., 1986). To counter oxidative stress, LAB maintain an inducible defense system to detoxify the oxidants and repair the damage. The dismutation of reactive oxygen intermediates in LAB depends on the activities of NADH oxidase, NADH peroxidase, glutathione, and thioredoxin. With the excep-
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TABLE 6.2 Reactions Involving Oxygen and Oxygen Metabolites Enzyme
Reaction
Gene
Organism
NADH:H2O2 NADH + H+ + O2 → NAD+ + H2O2 nox-1 oxidase NADH:H2O oxidase 2 NADH + H+ + O2 → 2NAD+ + H2O nox
S. mutans
nox-2
S. mutans
E. faecalis
NADH:peroxidase
NADH + H+ + H2O2 → NAD+ + H2O npr
E. faecalis
Glutathione reductase
NADPH+ + H+ + GSSG → NADP+ + gor 2 GSH 2 GSH + H2O2 → GSSG + 2 H2O 2 H2O2 → 2 H2O + O2 katA
S. thermophilus
O2– + 2H+ → H2O2
sod
L. lactis
pyruvate + phosphate + O2 + FAD + TPP → acetylphosphate + CO2 + H2O2
poxB
Lb. plantarum
Haem-dependent catalase Superoxide dismutase Pyruvate oxidase
spxB Oxidase α-glycerophosphate + O2 → — α-glycerophosphate dihydroxyacetone phosphate + H2O2
S. thermophilus Lb. sake
S. pneumoniae
References (Higuchi et al., 1994) (Ross and Claiborne, 1992) (Higuchi et al., 1993) (Ross and Claiborne, 1991) (Pébay et al., 1995) (Knauf et al., 1992) (Sanders et al., 1995)
(Murphy and Condon, 1984) (Spellerberg et al., 1996)
S. faecium (Koditchek and Umbreit,1969)
TPP: thiamine pyrophosphate; FAD: flavine adenine dinucleotide; NAD: nicotinamide adenine dinucleotide (Adapted from Condon, S., FEMS Microbol. Rev., 46, 269, 1987, and de Vos, W.M., Antonie Van Leeuwenhoek, 70, 223, 1996.)
tion of certain strains of Lactobacillus sake (Knauf et al., 1992), Lb. plantarum (Kono and Fridovich, 1983), Lactobacillus pentosus, and Pediococcus acidilactici (Wolf et al., 1991), LAB are notable for their inability to produce catalase. LAB exhibiting this rare property are summarized by Hammes et al. (1990). Enhanced tolerance to H2O2 after a sublethal treatment of H2O2 has been described in Gram-negative bacteria such as E. coli and S. typhimurium (Demple and Halbrook, 1983; Christman et al., 1985) and in Gram-positive bacteria such as B. subtilis (Murphy et al., 1987; Dowds, 1994), other Gram-positive bacteria such as E. faecalis (Flahaut et al., 1998) and L. lactis (Condon, 1987) exhibited an inducible oxidative stress response when exposed to sublethal concentrations of H2O2. The induced response provided enhanced protection against normally lethal levels of H2O2. Inhibition of protein synthesis by rifampin during H2O2 pretreatment blocked the acquisition of resistance, suggesting that de novo protein synthesis is required (Flahaut et al., 1998). © 2003 by CRC Press LLC
REGULATION
AND
FUNCTION
OF
OXIDATIVE STRESS RESPONSE PROTEINS
NADH Oxidase/NADH Peroxidase Some LAB have NADH oxidases (Anders et al., 1970; Lucey and Condon, 1986; Condon, 1987; Smart and Thomas, 1987) that use molecular oxygen to oxidize NADH. The NADH oxidases are thought to detoxify molecular oxygen by catalyzing its reduction via NADH into either H2O or H2O2 (Higuchi, 1992). The H2O-forming NADH oxidase has been proposed to function as a defense against oxidative stress, based on the production of large amounts of H2O-forming NADH oxidase to reduce O2 relative to smaller amounts of H2O2-forming NADH oxidase in S. mutans (Higuchi, 1992). Streptococcus mutans has two distinct NADH oxidases, Nox-1 catalyzing the formation of H2O2 and Nox-2 producing H2O (Higuchi et al., 1993). The two enzymes reveal different characteristics (Higuchi et al., 1993): Nox-1 catalyzes the two-electron reduction of O2 by NADH, whereas Nox-2 catalyzes the four-electron reduction of O2 by NADH (see Table 6.2). Furthermore, antibodies raised against Nox-1 or Nox-2 reacted with the corresponding antigens but did not cross-react (Higuchi et al., 1993). Working with E. faecalis, Ross and Claiborne (1992) were the first to identify the nox gene encoding NADH:H2O oxidase. This was followed by the isolation of the homlogous gene for NADH:H2O oxidase from S. mutans NCIB 11723 (Matsumoto et al., 1996). The gene encoding NADH:H2O2 oxidase has also been identified and characterized from S. mutans NCIB 11723 (Higuchi et al., 1994). Since the genes encoding two distinct NADH oxidases were characterized from the same S. mutans strain (NCIB 11723), the NADH:H2O2 oxidase gene was named nox-1 and the NADH:H2O oxidase gene was designated nox-2 (see Table 6.2) (Higuchi et al., 1994). Also, nox-1 and nox-2 were located at different positions on the genome and the deduced amino acid sequence of each gene showed little homology between these enzymes (Higuchi et al., 1994; Matsumoto et al., 1996). Recently, the NADH oxidase gene (nox) was identified in Streptococcus pneumoniae (Auzat et al., 1999). The growth rate of a nox mutant was similar to the wild type under aerobic and anaerobic conditions, suggesting that NADH oxidase in this strain does not provide resistance to oxidative stress. However, the nox mutant strain showed decreased competence and attenuated virulence (Auzat et al., 1999). Based on these results, the researchers concluded that Nox provides protection against oxidative stress in two ways. First, the reduction of oxygen to water evades the formation of any toxic intermediates (Higuchi, 1992). Second, the development of competence through NADH oxidase activity provides an extracellular source of DNA to aid in repairing damage to the chromosome caused by oxygen radicals (Auzat et al., 1999). The production of a reactive oxygen species such as H2O2 by Nox-1 to counter oxidative damage is illogical. However, located directly upstream of the nox-1 gene on the S. mutans chromosome is an ahpC gene encoding an enzyme homologous with the non-flavoprotein component (AhpC) of S. typhimurium alkyl hydroperoxide reductase. This enzyme system functions to defend cells against oxidative damage (Jacobson et al., 1989). Because nox-1 is linked to ahpC, AhpC can reduce the H2O2
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produced by nox-1 to H2O. The combined reactions of Nox-1 and AhpC are as follows: 2NADH + 2H+ + O2 → 2 NAD+ + 2H2O (Higuchi et al., 1999). Therefore, Nox-1 functions in combination with AhpC to form an alkyl hydroperoxide reductase system in S. mutans (Poole et al., 1997). In S. typhimurium, alkyl hydroperoxide reductase is composed of AhpC and AhpF and defends against oxidative damage by reducing organic hydroperoxides and hydrogen peroxide (Jacobson et al., 1989; Poole and Ellis, 1996). S. typhimurium and E. coli ahpCF-defective mutants showed increased sensitivity to cumene hydroperoxide (Storz et al., 1989). Transforming an ahpCF-defective E. coli mutant with both nox-1 and ahpC genes from S. mutans not only restored, but also actually enhanced resistance to cumene hydroperoxide relative to the E. coli parent strain (Higuchi et al., 1999). Surprisingly, S. mutans lacking Nox-1 and/or AhpC did not show increased sensitivity to cumene hydroperoxide and H2O2, suggesting that an alternative antioxidant defense system is functioning in response to oxidative damage (Higuchi et al., 1999). Another flavoprotein oxidase that protects cells against the threat of H2O2induced oxidative stress is NADH peroxidase. In converting H2O2 to water, NADH peroxidase plays an analogous role to Nox-2 and alkyl hydroperoxidase reductase (Table 6.2). The gene encoding NADH peroxidase (npr) has been identified and characterized in E. faecalis (Ross and Claiborne, 1991), and like nox-2 and ahpC in S. mutans (Higuchi et al., 1999), the npr gene is induced in E. faecalis upon exposure to oxygen (Rothschild et al., 1991). The upstream region of the npr gene shares homology with the OxyR-binding site of the ahpC gene from S. typhimurium (Ross and Claiborne, 1991). In S. typhimurium and E. coli, OxyR is a transcriptional activator and global regulator protein (Christman et al., 1985) that mediates the H2O2induced oxidative stress response (Tartaglia et al., 1989; Storz et al., 1990; Storz and Altuvia, 1994). More specifically, it controls the expression of a set of antioxidants that detoxify reactive oxygen species and repair the damage caused by oxidative stress (Storz and Imlay, 1999). Upon a shift in the intracellular redox potential, OxyR binds to a specific sequence located just upstream from the promoter region of the corresponding structural genes (Tartaglia et al., 1989). No homologue of OxyR has been found in LAB; however, OxyR purified from E. coli binds to and retards DNA fragments containing npr from E. faecalis in gel shift assays, suggesting that npr from E. faecalis may be regulated by OxyR (Ross and Claiborne, 1997). The activity of catalases that detoxify H2O2 have been detected in a limited number of LAB consisting mostly of lactobacilli and pediococci (Hammes et al., 1990). To date, the katA gene encoding catalase has been characterized only from L. sake LTH677. This gene was cloned and phenotypically expressed in L. casei (Knauf et al., 1992), which is catalase negative, illustrating the potential to enhance adaptation to oxidative stress in LAB (de Vos, 1996). Glutaredoxin and Thioredoxin Glutaredoxin and thioredoxin are structurally similar, particularly in the region of the active site (Holmgren and Aslund, 1995). The active site of these proteins contains two conserved cysteine residues that form a disulfide when oxidized and a dithiol when reduced (Holmgren, 1989). Aside from participating in the reduction
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Thioredoxin System thioredoxin reductase (trxB)
thioredoxin (trxA)
glutathione reductase (gor)
glutathione (gshA gshB)
NADPH Protein
glutaredoxin (grxA grxB grxC) Glutaredoxin System
FIGURE 6.2 E. coli components of the thioredoxin system (top) and glutaredoxin system (bottom) and the corresponding genes. (Adapted from Prinz, W.A. et al., J. Biol. Chem., 272, 15661, 1997.)
of essential enzymes, such as ribonucleotide reductase and a number of metabolic enzymes that form a disulfide as part of the catalytic cycle (Holmgren, 1989; Rietsch and Beckwith, 1998), glutaredoxin and thioredoxin function to repair oxidatively damaged proteins (Holmgren, 1989; Wells et al., 1993) and maintain a favorable intracellular redox potential by reducing disulfide bonds (Prinz et al., 1997). To return to the functional state, these proteins must be reduced. Thioredoxin reductase and glutathione reductase are flavoenzymes that use NADPH to reduce thioredoxin and glutathione, respectively, and glutathione then reduces glutaredoxin. These reactions are well studied in E. coli. Figure 6.2 diagrams the E. coli thioredoxin system (consisting of thioredoxin reductase and thioredoxin) and the glutaredoxin system (consisting of glutathione reductase, glutathione, and three glutaredoxins) (Gleason and Holmgren, 1988; Holmgren, 1989; Prinz et al., 1997). Many recent reviews discuss these systems in more detail (Holmgren, 1985, 1989; Holmgren and Aslund, 1995; Aslund and Beckwith, 1999; Ferrari and Soling, 1999; Mustacich and Powis, 2000). Since these systems are highly conserved, it is reasonable to assume that they are present in most LAB. Thioredoxin is a ubiquitous protein isolated and characterized from bacteria, yeast, plants, and animals (Holmgren, 1985). The active site of thioredoxin contains two cysteine residues that form a disulfide when the protein is oxidized or a dithiol when reduced. The disulfide bond of oxidized thioredoxin is reduced by NADPH and an enzyme called thioredoxin reductase (Moore et al., 1964). In B. subtilis, thioredoxin is an essential protein (Scharf et al., 1998), whereas in E. coli it is nonessential (Holmgren et al., 1978). However, E. coli mutants lacking both proteins are non-viable (Prinz et al., 1997). In B. subtilis, thioredoxin was induced by a variety of stresses including heat, salt, and ethanol (Scharf et al., 1998). The gene encoding thioredoxin (trxA) identified in O. oeni was induced by hydrogen peroxide and heat © 2003 by CRC Press LLC
shock (Jobin et al., 1999), which is also the case in B. subtilis (Scharf et al., 1998). In E. coli and S. typhimurium, trxA genes are not known to be regulated (Farr and Kogoma, 1991). Although trxA in O. oeni is induced by heat, no CIRCE element or CtsR consensus sequence was found in the promoter region. Glutathione can provide intracellular reducing capacity and accumulation of glutathione in LAB is dependent on the type of medium (Fernándes and Steele, 1993) and transport from the environment (Wiederholt and Steele, 1994). In some LAB, such as L. lactis, glutathione is present in high concentrations (Fahey et al., 1978). Glutathione reductase is involved in the oxidative stress response in S. typhimurium with expression regulated by OxyR (Christman et al., 1985). The glutathione reductase gene (gor) was identified in S. mutans (Yamamoto et al., 1999), S. thermophilus CNRZ368 (Pébay et al., 1995), and L. acidophilus NCFM (Girgis et al., 2000). S. thermophilus CNRZ 368 growing aerobically showed increased gor expression according to Northern blot analysis and glutathione reductase enzyme activity (Pébay et al., 1995). In the presence of 2 mM diamide, a thiol-specific oxidant, an S. mutans gor mutant failed to grow, whereas proliferation of the wildtype strain was not significantly inhibited (Yamamoto et al., 1999). Expression of the glutathione reductase gene in Lb. acidophilus NCFM increased in response to 500 µM H2O2 after 15 minutes or during the transition from the exponential to stationary phase of growth (Girgis et al., 2000). Superoxide Dismutase Superoxide dismutase (SOD) converts superoxide anions (O2–) to molecular oxygen (O2) and hydrogen peroxide (H2O2) (see Table 6.2) (Bannister et al., 1987). Therefore, this enzyme provides defense against oxygen toxicity and a direct correlation has been found between the concentration of SOD in an organism and its level of tolerance to oxygen (Tally et al., 1977). Many LAB eliminate oxygen radicals by superoxide dismutase or a high internal Mn2+ concentration (Archibald and Fridovich, 1981). LAB that lack SOD use Mn2+ to scavenge O2–, as demonstrated in Lb. plantarum and many other lactobacilli and streptococci strains (Archibald and Fridovich, 1981). However, organisms possessing SOD were more oxygen tolerant than organisms dependent upon Mn2+ for scavenging O2– (Archibald and Fridovich, 1981). Using degenerate primers, internal regions of the gene encoding superoxide dismutase (sod) were amplified and sequenced from L. lactis, E. faecalis, E. faecium, Streptococcus agalactiae, S. pneumoniae, and Streptococcus pyogenes, in addition to C. perfringens and S. aureus (Poyart et al., 1995). The entire gene was also identified, cloned, and sequenced from S. mutans through the complementation of a sod-deficient E. coli strain (Nakayama, 1992). Based on N-terminal amino acid sequence, SOD was also identified in L. lactis during a search for proteins expressed at higher levels in a medium with a low pH (Sanders et al., 1995). Aeration was effective in increasing the transcriptional expression (Sanders et al., 1995) and enzyme activity of SOD in L. lactis (Hansson and Häggström, 1984; Smart and Thomas, 1987). Oxygen radicals formed during aeration inhibited the growth of sod-deficient strains of S. mutans (Nakayama, 1992) and L. lactis (Sanders et al., 1995). A similar effect was observed in E. coli (Carlioz and Touati,
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1986). The E. coli superoxide dismutase gene was cloned and expressed in L. lactis and Lb. gasseri (Roy et al., 1993); since Lb. gasseri lacks SOD, this study demonstrates the potential to increase tolerance to oxidative stress in LAB (de Vos, 1996). recA, fpg, and DNA Damage The recA gene is ubiquitous among bacteria and responds to DNA damage caused by oxidative stress. In the absence of oxidative stress, RecA initiates recombination between homologous strands of DNA (Cassuto et al., 1980) (for reviews, see Miller and Kokjohn, 1990, and Roca and Cox, 1990). When DNA is damaged, the RecA protein is activated upon binding to single-stranded DNA (Roberts and Devoret, 1982). The activated RecA protein induces expression of several DNA-repair genes in the SOS pathway (Walker, 1984). Therefore, RecA serves a regulatory function in response to oxidatively damaged DNA (Walker, 1984; Miller and Kokjohn, 1990). Using degenerate primers, internal regions of the recA gene were amplified, cloned, and sequenced from L. lactis subsp. lactis ML3 and IL 1403 and L. lactis subsp. cremoris IL 736, Lb. bulgaricus, Lb. helveticus, Lc. mesenteroides, and Streptococcus salivarius subsp. thermophilus, in addition to B. subtilis, Clostridium acetobutylicum, L. monocytogenes, and S. aureus (Duwat et al., 1992). An L. lactis mutant with a reduced capacity for recombination showed increased sensitivity to UV (Anderson and McKay, 1983); however, the location of the mutation has not been identified. Another L. lactis recA mutant exhibited a recombination frequency about 104-fold lower than wild type and increased sensitivity to DNA damage caused by UV light, mitomycin C, ethyl methane sulphonate, and methyl methane sulphonate (Duwat et al., 1995). These compounds were effective in increasing recA expression by three- to five-fold (Duwat et al., 1995). A number of genes associated with DNA repair have been identified in a study in which UV-sensitive mutants of L. lactis strain MG1363 were obtained by ISS1 mutagenesis. Of the 18 mutants sensitive to mitomycin and UV, DNA sequence analysis identified 11 insertions of ISS1 within genes associated with DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), and various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA) (Duwat et al., 1997). The polA, hexB, and deoB mutants were more sensitive to low doses of UV treatment than the other mutants and homologous recombination was reduced by 10- to 300-fold in the gidA, polA, and uvs-75 mutants. These seemingly unrelated sets of affected genes suggest that UV resistance involves several interactive mechanisms in L. lactis. In addition to DNA damaging agents, expression of recA was also induced in aerated cultures. An L. lactis recA mutant was highly sensitive to aeration, as evidenced by a lower growth rate and reduced viability during stationary phase (Duwat et al., 1995). As L. lactis produces hydrogen peroxide and acid in the presence of iron, hydroxyl radicals are formed. Hydroxyl radicals can be produced by the Fenton reaction: H2O2 + Fe2+ + H+ → •OH +H2O + Fe3+ (Fenton, 1984; Lesko et al., 1980). It is believed that hydroxyl radical formation is the leading cause of the poor growth of the recA aerated culture because the addition of catalase to the recA aerated growth medium restored growth, such that the doubling time was the same as in the nonaerated culture (Duwat et al., 1995). Furthermore, the removal of Fe2+, by adding
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the Fe2+-specific chelating agent ferrozine (Artiss et al., 1981), also restored the doubling time of the aerated recA cultures to that of non-aerated cultures (Duwat et al., 1995). The L. lactis recA mutant had three-fold higher levels of HflB (which down regulates expression of heat shock proteins in E. coli [Herman et al., 1995]), and decreased levels of heat shock proteins, and it showed poor growth at 37ºC relative to the wild type strain. These observations suggested that recA may also be involved in the regulation of the heat shock response (Duwat et al., 1995). To further investigate the role recA plays in the heat shock response, insertional mutants of a thermosensitive recA-deficient strain of L. lactis were isolated based on their ability to withstand high temperatures (Duwat et al., 1999). Eighteen trm (for thermoresistant mutant) mutants were characterized that contained insertions of six genes implicated in purine metabolism (deoB, guaA, tktA), phosphate uptake (pstB and pstS), and mRNA stability (pnpA), and in one uncharacterized gene (trmA). A deoB insertional mutant conferring UV sensitivity was isolated previously (Duwat et al., 1997). Furthermore, insertional mutations in four genes — deoB, guaA, pstB and pstS — were obtained by Rallu et al. (2000), using a similar procedure was followed to isolate acid-tolerant insertional mutants of L. lactis. See section on “acid adaptation.” The inability to synthesize purines and import phosphate from the extracellular environment simulated a starvation-like physiology in the corresponding mutants that conferred resistance to multiple forms of stress. This finding suggests that stress response mechanisms in L. lactis are interactive and are intimately associated with metabolic pathways (Duwat et al., 1999; Rallu et al., 2000). Upstream of the L. lactis recA gene is a region of DNA with strong homology with the gene encoding the DNA repair enzyme formamidopyrimidine DNA glycosylase (fpg), found in E. coli and Bacillus firmus (Boiteux et al., 1987; Boiteux and Huisman, 1989). In E. coli, the fpg gene product is involved with DNA repair associated with oxidative stress (Czeczot et al., 1991) and is unlinked to recA (Boiteux and Huisman, 1989). L. lactis fpg is co-transcribed with recA, and Duwat et al. (1992) suggest the proximity of recA and fpg in L. lactis may indicate overlapping regulation linking recombination and DNA repair. The L. lactis Fpg protein is structurally and functionally similar to the Fpg protein in E. coli. In both organisms, Fpg protects DNA against the mutagenic action of 8-oxoguanine (Michaels and Miller, 1992; Grollman and Moriya, 1993; Duwat et al., 1995).
STARVATION Bacterial cells enter the stationary phase upon depletion of essential nutrients from the growth medium. During nutrient starvation, there is a gradual decrease in the growth rate which eventually approaches zero. To survive, bacteria must make an orderly transition into the stationary phase in such a manner that DNA replication is not terminated prematurely, that viability is maintained, and that cells can return to exponential growth when starvation is relieved. In non-sporulating bacteria during starvation, there occur a number of changes in cellular protein composition that are characterized by degradation of some previously synthesized proteins, increased synthesis of some proteins common to exponential phase growth and de novo protein
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synthesis. Starvation also induces resistance to a number of environmental stresses without prior exposure to those stresses (Kolter et al., 1993). The proteins synthesized during starvation are probably involved in maintenance of cell viability and in resistance to numerous stresses. When the synthesis of starvation proteins is completed, metabolic activity is greatly diminished; however, the cells are not dormant like bacterial spores. The starved cells do maintain some level of metabolic activity (Kolter et al., 1993), because when fresh nutrients are added, the cells respond rapidly. Synthesis of RNA starts almost immediately, but protein synthesis lags for a short period. Increases in cell mass, in rate of DNA synthesis, and in cell number follow the reinstating of RNA and protein synthesis, but there is a progressive loss of the enhanced resistance to environmental stresses that was induced during starvation (Kolter et al., 1993). The responses to starvation of some LAB are described in this section. Enterococcus faecalis JH2-2 cells from the exponential growth phase are less tolerant to a number of stresses than cells from the stationary growth phase (i.e., starved cells). Glucose-starved cells of E. faecalis are more resistant to ethanol (17%), acid (pH 3.7; adjusted with lactic acid), H2O2 (20mM) and heat (62°C for 30 min) than cells from the exponential phase (Giard et al., 1996). In general, stress resistance increased up to 24 h after entrance into the stationary phase. However, ethanol resistance was established early in the stationary phase and did not increase over time during starvation. Starvation did not increase the resistance of E. faecalis to UV irradiation (Giard et al., 1996). Utilizing chloramphenicol as a protein synthesis inhibitor, Giard et al. (1997) found that early stationary-phase protein synthesis was necessary for the acquisition of resistance against heat, acid, and oxidative stresses, but not against ethanol stress. After 24 h of starvation, approximately onethird of the proteins typically observed in exponential cells were reduced in concentration or were completely absent, indicating that protein degradation had taken place. Glucose starvation induced significant increases in the synthesis of 42 proteins (Giard et al., 1996, 1997). The synthesis of these proteins was time dependent and different proteins were seen at different stages of starvation. Proteins synthesized between 6 and 24 h into the stationary phase were crucial for the development of maximal resistance to heat, H2O2 and acid. Some of these proteins may be involved in the development of alternate pathways of energy production that permit survival of E. faecalis under stress conditions (Giard et al., 1996, 1997). Lactococcus lactis subsp. lactis IL1403, upon glucose-starvation, demonstrated augmented resistance to heat (52°C for 30 min), ethanol (20%), pH 4.0 (adjusted with lactic acid), NaCl (3.5 M), and H2O2 (1.5 mM) in the absence of prior exposure to these stresses (Hartke et al., 1994). The starvation-induced cross-protection to stress was evident at the beginning of the stationary stage. The acquisition of these stress resistances was initiated during the period of transition from growth to non-growth, and reached a maximum upon entrance into stationary phase. Interestingly, addition of chloramphenicol or rifamycin during the transitional growth phase did not inhibit acquisition of stress resistance, but rather it increased resistance to a level comparable to that seen in cells in the stationary stage (Hartke et al., 1994). When the authors compared stress adapted, exponentially grown L. lactis subsp. lactis IL1403 cells with starved cells, they found that adapted cells had comparable resistance to heat,
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acid or osmotic stresses as starved cells. However, adapted cells were not as resistant as starved cells to ethanol, but were more resistant than starved cells to H2O2 stress. During glucose starvation of L. lactis subsp. lactis ML3 cells, at least 45 polypeptides which are present in exponentially growing cells were no longer synthesized (Kunji et al., 1993). In addition, there was significant degradation of protein during the first 60 to 90 min of starvation, and protein degradation during starvation was non-selective (Kunji et al., 1993). After the initial degradation period, the polypeptide pool remained stable for up to 40 h. During the first hour of starvation, the synthesis of at least two proteins was induced and as starvation continued, an additional 14 or 15 proteins were synthesized. It is likely that the amino acids produced by degradation of exponential growth proteins were used to synthesize the starvation-specific proteins. These newly synthesized proteins probably play a role in the increased stress resistance shown by the starving bacteria. Unlike the wild type, recA mutants of L. lactis subsp. cremoris MG1363 do not grow at 39.3°C. By subjecting the recA strain to insertional mutagenesis and selecting those mutants that grew at 39.3°C, Duwat et al. (1999) isolated several double mutants that could grow at that temperature. These double mutants also displayed resistance to heat shock (55°C for 15 min), H2O2 (1 mM) and carbon starvation. The following are the mutations in the recA double mutant strains: deoB (involved in purine and pyrimidine salvage and nucleoside degradation), guaA (involved in synthesis of GMP from XMP), tktA (involved in transformation of xylose-5-P to ribose-5-P), or pstB and pstS (involved in phosphate transport) induced multiple stress resistance during both the exponential and stationary stages (Duwat et al., 1999). Expression of multiple stress resistances during the exponential stage by these double mutants suggests that mutations leading to the reduction of the guanine nucleotide pool or phosphate pool can induce a starvation-like physiology in the cells (Duwat et al., 1999). In a similar fashion, Rallu et al. (2000), using insertional mutagenesis to isolate acid-resistant mutants of L. lactis subsp. cremoris MG1363, found that mutations in pstS and pstB, guaA, deoB or RelA (involved in both synthesis and degradation of [(p)ppGpp]) not only induced increased resistance to acid (pH 3.7 or 3.0 adjusted with HCl), but also increased resistance to hydrogen peroxide (1 mM for 30 min) and heat shock (55°C for 15 min). When GMP synthase (coded for by guaA) of the wild type strain of L. lactis subsp. cremoris MG1363 was inhibited, there was induction of the stress-resistance phenotype (Duwat et al., 2000). Addition of guanine to the growth medium of the RecA-guaA double mutant abolished the stress-resistant phenotype. Similarly, addition of phosphate to the RecA-pstS double mutant eliminated stress resistance (Duwat et al., 2000). The work of Duwat et al. (1999 and 2000) and Rallu et al. (2000) suggests that a decreased internal phosphate concentration, a decreased guanine nucleotide pool, and/or an increased (p)ppGpp concentration may be perceived by cells of L. lactis subsp. cremoris as intracellular stress signals leading to tolerance to a number of stresses. Stationary phase cells of L. lactis subsp. lactis strains LL-40-1, LL-41-1 and LL43-1 were resistant to pH 2.5 (adjusted with HCl), bile salts (0.1%) and freezing at –20°C for 24 h (Kim et al., 1999). Cells of L. lactis subsp. cremoris strains LC-10-1, LC-11-1 and LC-12-1 from the stationary phase were resistant to pH 3.0 (adjusted
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with HCl) and 0.04% bile salts. Strains LC-10-1 and LC-11-1 were also resistant to freezing, but strain LC-11-1 was not. Subjecting the L. lactis subsp. cremoris strains to pH 2.5 or 0.1% bile salts resulted in death of the cells (Kim et al., 1999). Thus, L. lactis subsp. cremoris strains are typically less stress tolerant than L. lactis subsp. lactis strains.
OVERLAPPING REGULATORY NETWORKS AND CROSS-PROTECTION Aside from synthesizing a specific set of proteins in response to an individual stress, many microorganisms induce a stress regulon consisting of an overlapping set of general stress response proteins which may confer general protection to a variety of deleterious conditions. The universal induction of many of the same stress proteins following exposure to a variety of different mild stresses has been demonstrated in E. coli (Jenkins et al., 1991), B. subtilis (Hecker and Völker, 1990; Völker et al., 1992), E. faecalis (Flahaut et al., 1996), and L. lactis (Hartke et al., 1994, 1995, 1997). For example, a significant overlap between acid- and heat-inducible polypeptides was observed in L. lactis subsp. lactis. Of the 12 heat shock proteins detected in this strain, 9 were also induced by acid treatment, including DnaK and GroEL (Hartke et al., 1996). This suggests a relationship between the mechanisms responsible for the heat shock response and the acid tolerance response. In E. faecalis, a considerable number of heat shock proteins were also expressed in response to ethanol (Boutibonnes et al., 1993) and bile (Flahaut et al., 1996). Similar results have been found in E. coli (Heyde and Portalier, 1990) and S. typhimurium (Foster, 1991) in which heat shock proteins were among the proteins induced by acid adaptation. Furthermore, acid was effective in inducing members of the SOS and H2O2 stimulons of L. lactis (Hartke et al., 1995). This production of overlapping stress response proteins due to a variety of different environmental stresses may be responsible for the phenomenon known as cross-protection, which is observed when cells survive an otherwise lethal exposure to one form of stress after adapting to a different sublethal condition. Cross-protection has been demonstrated in E. coli (Jenkins et al., 1990), S. typhimurium (Leyer and Johnson, 1993), and B. subtilis (Völker et al., 1992). Among the LAB, cross-protection has been described in L. lactis, E. faecalis, and Lactobacillus collinoides. For example, carbohydrate-starved cultures of L. lactis are significantly more resistant to heat, ethanol, acid, and osmotic stress than nourished, exponential-phase cells (Hartke et al., 1994). Thermotolerance in L. lactis was developed after exposures to ethanol (Boutibonnes et al., 1991), puromycin (Boutibonnes et al., 1992), or chemicals such as cadmium chloride, mercury chloride, sodium azide, and β-mercaptoethanol (Boutibonnes et al., 1995). Furthermore, heat-induced cross-protection against freezing and lyophilization was achieved in L. lactis. Cross-protection was abolished in the presence of erythromycin, indicating that protein synthesis is required for tolerance (Broadbent and Lin, 1999). Enterococcus faecalis showed enhanced tolerance to lethal doses of hydrogen peroxide after a 30-minute incubation in acid or NaCl, or after thermal treatment
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(Flahaut et al., 1998). Heat-adapted cells of E. faecalis showed significant crossprotection against bile (Flahaut et al., 1996) and ethanol (Boutibonnes et al., 1993), and bile-adapted cells provided resistance against heat challenge (Flahaut et al., 1996). As stated above, the overlap in the number of heat shock proteins expressed during exposure to heat, bile, and ethanol, suggesting similar mechanisms of response, may be the basis for cross-protection. Preconditioning E. faecalis with heat or bile failed to induce acid tolerance, and acid-adapted cells displayed slight resistance to heat and no resistance to bile challenge (Flahaut et al., 1996). Apparently, the response of E. faecalis to acid treatment is distinct from the method of adaptation to bile or heat. Adaptation to ethanol and heat in Lb. collinoides conferred homologous resistance and enhanced tolerance to acid. However, adaptation to acid did not provide protection from ethanol or heat (Foster and Hall, 1990). As observed in E. faecalis (Flahaut et al., 1996), acid treatment elicits a specific response relative to treatments with heat or ethanol.
THE FUTURE LAB used as starter cultures are normally stored and distributed in liquid, spraydried, frozen, or lyophilized forms (Porubcan and Sellars, 1979; Sandine, 1996). Such preparations drastically reduce population numbers and severely damage their capacity for growth, fermentation, or survival upon passage through the gastrointestinal tract. Furthermore, during the production of fermented food products, lactic starter cultures are typically subjected to extremes in temperature, pH, and osmolarity. As knowledge regarding stress response systems of LAB accumulates, methods will inevitably be developed to engineer strains that are more resistant to routine industrial practices. An enormous volume of knowledge will be provided through the sequencing of microbial genomes. In 1999, L. lactis IL1403 became the first LAB to have its entire genome sequenced and published (Bolotin et al., 1999). Within the year, the genomes of another 23 industrially important LAB will be sequenced. This group includes Lb. acidophilus, Lb. plantarum, Lb. johnsonii, L. lactis subsp. cremoris, Lb. delbrueckii subsp. bulgaricus, Lb. sakei, Lb. casei, Lb. helveticus, Lb. rhamnosus, S. thermophilus, O. oeni, Lb. gasseri, Lactobacillus brevis, L. lactis subsp. cremoris, Lc. mesenteroides, and P. pentosaceus. The information gathered from whole-genome sequencing combined with new technologies designed to analyze genomic data, such as microarrays, will inevitably provide a global view of the genetic mechanisms which contribute to the observed physiological responses of the LAB to environmental stress.
CONCLUSIONS A microorganism’s ability to grow and survive depends largely on its capacity to adapt to changing environments. LAB are constantly subjected to harsh conditions that can affect their performance in food fermentations. Adaptation to adverse environments is usually associated with the induction of a large number of genes, the synthesis of stress response proteins, and the development of cross resistance to
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a variety of stresses. The regulation of these responses is complex, interactive, and sometimes intimately related to metabolic pathways. The further elucidation of molecular genetic mechanisms involved in the regulation of the stress responses of these organisms will provide fundamental information regarding the development of stress adaptation and tolerance. Information gathered on stress adaptation will ultimately raise the possibility of enhancing tolerance to adverse environmental conditions and consequently improve viability and performance of these organisms in food systems.
TABLE 6.3 Genes Induced by Environmental Stress in LAB Stress Heat
Gene groEL
Function of Protein chaperone
Organisms Lb. johnsonii Lb. acidophilus L. lactis
groES
chaperone
E. faecalis Lc. mesenteroides Lb. helveticus Lb. johnsonii Lb. acidophilus L. lactis
hrcA
heat shock regulator
dnaK
chaperone
grpE
chaperone
dnaJ
chaperone
ctsR
heat shock regulator
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Lb. helveticus S. mutans Lb. sakei Lb. acidophilus L. lactis L. lactis S. mutans E. faecalis Lc .mesenteroides Lb. sakei Lb. acidophilus L. lactis S. mutans Lb. sakei Lb. acidophilus L. lactis Lb. sakei L. lactis S. salivarius
References (Walker et al., 1999) (Girgis et al., 1999; Girgis et al., 2000) (Kim and Batt, 1993; Hartke et al., 1997) (Flahaut et al., 1997) (Salotra et al., 1995) (Broadbent et al., 1998) (Walker et al., 1999) (Girgis et al., 1999; Girgis et al., 2000) (Kim and Batt, 1993; Hartke et al., 1997) (Broadbent et al., 1998) (Jayaraman et al., 1997) (Schmidt et al., 1999) (Girgis et al., 2000) (Eaton et al., 1993) (Eaton et al., 1993; Barril et al., 1994) (Jayaraman et al., 1997) (Flahaut et al., 1997) (Salotra et al., 1995) (Schmidt et al., 1999) (Girgis et al., 2000) (Eaton et al., 1993) (Jayaraman et al., 1997) (Schmidt et al., 1999) (Girgis et al., 2000) (van Asseldonk et al., 1993) (Schmidt et al., 1999) (Derre et al., 1999) (Derre et al., 1999)
TABLE 6.3 (continued) Genes Induced by Environmental Stress in LAB Stress
Gene
Function of Protein
Organisms S. pneumoniae S. pyogenes S. thermophilus E. faecalis Lc. oenos Lb. sake L. lactis L. lactis Lb. acidophilus L. lactis Lb. acidophilus Lb. sake O. oeni L. lactis S. salivarius L. lactis O. oeni L. lactis L. lactis
clpB clpC
protease protease
clpE
protease
clpX clpP
chaperone/protease protease
ftsH hsp18 cspA cspB
heat shock regulator membrane maintenance RNA stabilization RNA stabilization RNA stabilization RNA stabilization
Acid
cspC cspD cspE cspF cspG cspL cspP gadCB
RNA stabilization RNA stabilization acid stress protection
L. lactis L. lactis L. lactis L. lactis L. lactis Lb. plantarum Lb. plantarum L. lactis
Oxidative
citP atp arcABCTD sodA
acid stress protection acid stress protection acid stress protection O2– scavenging
L. lactis Lb. acidophilus Lb. sake L. lactis
Cold
recA
DNA repair
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E. faecalis E. faecium S. agalactiae S. pneumoniae S. pyogenes S. mutans L. lactis Lb. bulgaricus Lb. helveticus
References (Derre et al., 1999) (Derre et al., 1999) (Derre et al., 1999) (Derre et al., 1999) (Derre et al., 1999) (Derre et al., 1999) (Ingmer et al., 1999) (Ingmer et al., 1999) (Girgis et al., 2000) (Ingmer et al., 1999) (Girgis et al., 2000) (Stentz et al., 1997) (Jobin et al., 1999) (Frees and Ingmer, 1999) (Giffard et al., 1993) (Nilsson et al., 1994) (Jobin et al., 1997) (Wouters et al., 1998) (Chapot-Chartier et al., 1997; Wouters et al., 1998) (Wouters et al., 1998) (Wouters et al., 1998) (Wouters et al., 1998) (Wouters et al., 2000) (Repine et al., 1981) (Mayo et al., 1997) (Mayo et al., 1997) (Sanders et al., 1998; Small and Waterman, 1998) (Garcia-Quintans et al., 1998) (Kullen and Klaenhammer, 1999) (Zuniga et al., 1998) (Poyart et al., 1995; Sanders et al., 1995) (Poyart et al., 1995) (Poyart et al., 1995) (Poyart et al., 1995) (Poyart et al., 1995) (Poyart et al., 1995) (Nakayama, 1992) (Duwat et al., 1995) (Duwat et al., 1992) (Duwat et al., 1992)
TABLE 6.3 (continued) Genes Induced by Environmental Stress in LAB Stress
Osmotic
Gene
Function of Protein
Organisms
References (Duwat et al., 1992) (Duwat et al., 1992) (Duwat et al., 1992; Duwat et al., 1995) (Gostick et al., 1999) (Higuchi, 1992; Matsumoto et al., 1996; Higuchi et al., 1999) (Auzat, 1999) (Marty-Teysset et al., 2000) (Ross and Claiborne, 1991; Ross and Claiborne, 1992) (Knauf et al., 1992) (Pébay et al., 1995) (Yamamoto et al., 1999) (Girgis et al., 2000) (Jobin et al., 1999) (Smeds et al., 1998)
fpg
DNA repair
Lc. mesanteroides S. salivarus L. lactis
fnr nox
O2– scavenging H2O2 reducing
L. lactis S. mutans
npr
H2O2 reducing
S. pneumoniae Lb. delbreuckii E. faecalis
katA gor
H2O2 reducing H2O2 reducing
trxA htrA
H2O2 reducing stress protection
Lb. sake S. thermophilus S. mutans Lb. acidophilus O. oeni Lb. helveticus
Paper No. FSR-0043 of the Journal Series of the Department of Food Science, NCSU, Raleigh, NC 27695-7624. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service, or the US Department of Agriculture, of the products named nor criticism of similar ones not mentioned. Work on the stress response of lactobacilli, conducted at NCSU is supported by grants from the Southeast Dairy Foods Research Center, Dairy Management Inc., and Rhodia, Inc.
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7
Relationship between Stress Adaptation and Virulence in Foodborne Pathogenic Bacteria Cormac G. M. Gahan and Colin Hill
CONTENTS Introduction Infection and the Need for Environmental Sensing Infection with Salmonella spp. Infection with Listeria monocytogenes Two-Component Systems and Environmental Sensing Environmental Stresses Encountered by Bacteria during Infection Body Temperature, Heat-Shock and the General Stress Response Acid Tolerance and Virulence Oxidative Stress Response Osmotic Stress Starvation Stress Methods to Detect Genes Transcribed in Vivo In Vivo Expression Technology (IVET) Green Fluorescent Protein (GFP) Technology Signature-Tagged Mutagenesis Conclusion Acknowledgments References
INTRODUCTION Bacteria capable of causing foodborne infections must negotiate a long and tortuous passage from the environment to the site of infection in the susceptible host. Foodborne pathogens may encounter stressful environments during the production, preparation and storage of food. Following consumption they are exposed to the low pH of the stomach and survivors subsequently encounter volatile fatty acids, bile and
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low oxygen in the small intestine. Bacteria that survive to this point must compete with established gut flora for niches and nutrients and must overcome, among other insults, antimicrobial peptides produced by their competitors (Dunne et al., 1999). Those organisms capable of invasion subsequently penetrate the gut epithelium and are internalized within phagosomes, specialized organelles which prevent bacterial multiplication by means of acidic pH, and through the production of defensins (oxygen-independent mechanisms), hydrogen peroxide and superoxide radicals (oxygen-dependent mechanisms). To survive and grow in these inhospitable environments, foodborne pathogens possess mechanisms to overcome these stresses, and thus are capable of colonization resulting in either clinical or sub-clinical infection. In this chapter, the mechanisms employed by foodborne pathogens to adapt to the host environment and cause disease have been considered. Our primary focus is the pathogenesis of Listeria monocytogenes and Salmonella enterica serovar Typhimurium as examples of Gram-positive and Gram-negative foodborne pathogens capable of causing invasive disease in a mouse model of infection.
INFECTION AND THE NEED FOR ENVIRONMENTAL SENSING INFECTION
WITH
SALMONELLA
SPP.
The invasiveness of various Salmonella serovars varies greatly. S. enterica serovar Typhimurium (hereafter referred to as S. Typhimurium) causes a localized gastrointestinal infection in humans with symptoms of vomiting and diarrhea. S. Cholerasuis can cause more severe symptoms, while S. Typhi are responsible for the serious invasive disease typhoid fever. However, S. Typhimurium infection of mice results in a disease with similarities to human typhoid fever and, as a consequence, murine S. Typhimurium infection is extensively used as a model for invasive disease. The usefulness of this model and the ease with which S. Typhimurium can be manipulated at the genetic level have led to extensive studies of this pathogen and phenomena uncovered first in S. Typhimurium are now being investigated in other foodborne pathogens. Studies indicate that S. Typhimurium invade primarily via M cells of the small intestine (Jones et al., 1994). However, the pathogen can adhere to and invade numerous mammalian cell lines (Figure 7.1A). Adherent Salmonella induce a specific membrane ruffling effect at the surface of appropriate host cells that leads to rearrangement of host actin in the vicinity of the bacterial cell (Finlay and Ruschkowski, 1991; Hardt et al., 1998). Membrane ruffling is dependent upon bacterial expression of SopE, a protein which is required for efficient invasion of cultured cells (Hardt et al., 1998). SopE is a substrate of a protein secretion system (type III) that translocates bacterial proteins into the host cell. The genes encoding this type III secretion system and other invasion-associated loci are clustered on a pathogenicity island — Salmonella pathogenicity island 1 (SPI1) — and are expressed prior to invasion. Bacteria are subsequently engulfed by the host cell and are internalized within host cell phagosomes, whereupon SPI1 genes are repressed (Pegues et al., 1995; Bajaj
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B
A
6 1
1
2 2 3 5 4
FIGURE 7.1 (A) Intracellular pathogenesis of Salmonella Typhimurium. 1. Membrane ruffling and invasion predominately mediated through expression of genes in Salmonella pathogenicity island 1 (SPI1); 2. Existence within the phagosome requires expression of SPI2 genes with downregulation of SPI1 genes. SPI2 genes involved in preventing maturation of the phagosome to phagolysozome. Genes involved in sensing low pH, low Mg2+ and low osmolarity (PhoP-PhoQ and OmpR-EnvZ) are also implicated in promoting survival. (B) Genes involved in intracellular pathogenesis of Listeria monocytogenes. 1. invasion (invA/invB); 2. entrapment in the phagosome; 3. escape (hly/plcA); 4. growth; 5. actin polymerization (actA); 6. cell to cell spread (plcB).
et al., 1996; Cotter and Miller, 1998). S. Typhimurium can survive within this hostile environment by preventing the maturation of phagosomes to lethal phagolysosomes. Recent evidence suggests that S. Typhimurium may interfere with trafficking of oxidase-containing vesicles to the phagosome through expression of components of Salmonella pathogenicity island 2 (SPI2) (Vazquez-Torres et al., 2000). However, many other bacterial genes are involved in the survival of the bacterium in the host phagosome, including genes involved in acid tolerance and responses to low iron, carbon starvation, oxidative stress and high osmolarity.
INFECTION
WITH
LISTERIA
MONOCYTOGENES
The genus Listeria comprises both avirulent and virulent species. Although Listeria seeligeri and L. ivanovii are capable of causing human or animal disease, it is L. monocytogenes that is the most common cause of infection (listeriosis) in humans (Farber and Peterkin, 1991; Gahan and Collins, 1991). The potentially high mortality rates associated with outbreaks of listeriosis highlight the serious nature of L. monocytogenes infection and eliminating the organism from ready-to-eat foods remains an imperative for the food industry. Infection of mice with L. monocytogenes has long been accepted as a suitable model for the study of pathogenesis and resulting immunity to this Gram-positive organism. L. monocytogenes, like S. Typhimurium, invades tissue culture cells by inducing its own phagocytosis. However, it does so by using a so-called trigger mechanism rather than the membrane-ruffling (zipper mechanism) exhibited by Salmonella spp. (Isberg and Tran Van Nhieu, 1994; Cossart and Lecuit, 1998). Invasion is mediated
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by a number of genes including invA and invB, although other genes may also be necessary for adherence prior to invasion (Gaillard et al., 1991; Dramsi et al., 1995; Milohanic et al., 2000). During murine infection, L. monocytogenes does not seem to have a preference for invasion of a specific cell type and will invade a wide variety of cell types in vitro (Gaillard et al., 1987; Kathariou et al., 1990). The organism can penetrate the small intestine through either M cells or enterocytes and, immediately following invasion, the phagocytic cells of the Peyers patches provide a focus of bacterial accumulation (Pron et al., 1998). Subsequently the spleen and liver become foci of infection, possibly seeded by migrating macrophages containing internalized bacteria. In humans, bacterial infection of the meninges causes a potentially fatal meningitis; in pregnant women, infection of the fetus may result in spontaneous abortion. The genetic loci contributing to intracellular pathogenesis of L. monocytogenes have been well characterized (reviewed by Cossart and Lecuit, 1998). Survival of L. monocytogenes within host cells is mediated by production of virulence factors including a hemolysin (listeriolysin) encoded by hly, and a phospholipase encoded by plcA. These may act singly or in concert to lyse the phagosomal membrane releasing the bacterium into the host cell cytoplasm where bacterial division can occur (see Figure 7.1B). In this environment L. monocytogenes can mobilize host actin filaments for motility using the virulence factor actin polymerase (ActA), and can infect neighboring cells without an extracellular phase. Release from a doublemembrane bound vesicle into the neighboring cell is mediated in part by another virulence factor encoded by plcB. Coordinate regulation of virulence factors is mediated by the transcriptional activator, PrfA (positive regulatory factor A), together with a putative PrfA-binding factor (Bockmann et al., 1996). Expression of the PrfA regulon is in turn regulated by environmental signals encountered during the infectious cycle.
TWO-COMPONENT SYSTEMS
AND
ENVIRONMENTAL SENSING
It should be apparent, given the complexities of the pathogenic cycles of foodborne pathogens, that an ability to sense and react to extracellular stimuli is essential for adaptation to new host environments. Two-component regulatory systems provide a means to detect perturbations in growth conditions and to respond with the synthesis of gene products which facilitate adaptation. These systems involve a sensor molecule which is often present in the bacterial cell membrane to detect environmental signals, and a cytoplasmic response regulator which functions to effect intracellular changes in response to the initial stimulus (Parkinson and Kofoid, 1992; Russo and Silhavy, 1993). Two-component systems in various bacteria control diverse functions including chemotaxis, sporulation and responses to nitrogen, phosphate or carbon source availability. In pathogenic bacteria, two-component systems have emerged as mechanisms signalling host–pathogen interactions and play a dynamic role in bacterial adaptation to the host environment. In S. Typhimurium the membrane sensor EnvZ and its companion response regulator, OmpR, represent a two-component system which responds to changes in osmolarity. The EnvZ-OmpR system plays a major role in signalling entry of Salmonella
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into the host cell and in triggering bacterial acid adaptation (Lee et al., 2000; Bang et al., 2000). Similarly, the PhoP-PhoQ two-component system responds to acid stress and low Mg2+ conditions encountered in the macrophage phagosome and regulates genes involved in stress adaptation and virulence (García Véscovi et al., 1994; Bearson et al., 1998). In addition, a recent study has identified a two-component system in L. monocytogenes (LisR-LisK) which plays a role in pH homeostasis and is required for full virulence of the pathogen (Cotter et al., 1999). These roles of these sensor-regulators will be discussed in later sections.
ENVIRONMENTAL STRESSES ENCOUNTERED BY BACTERIA DURING INFECTION During infection, bacterial pathogens encounter stressful conditions that range from sub-optimal to potentially lethal. Non-lethal stresses often induce the expression of bacterial genes whose function is to protect against further stress. Gene products in this category include molecular chaperones and heat shock proteins as well as ATPases and other systems responsible for maintaining cellular homeostasis in adverse conditions. In addition, environmental stresses in host microenvironments can act as stimuli for the regulation of genes which play a specific role in colonization and pathogenesis. For instance, the production of cholera toxin by Vibrio cholera is activated by pH and temperature in the small intestine, a process mediated through the ToxR-ToxS two-component system (DiRita, 1992).
BODY TEMPERATURE, HEAT-SHOCK
AND THE
GENERAL STRESS RESPONSE
The sudden shift in temperature from environmental/ambient temperatures to body temperature at the outset of infection has the potential to directly influence regulation of genes involved in bacterial virulence. However, bacteria also possess the ability to react to even higher temperatures by inducing the expression of genes in the heatshock stimulon. The products of many of these genes are capable of protecting cells against numerous stresses and can be considered general stress proteins. S. Typhimurium demonstrates a classical heat-shock response when exposed to elevated temperature (Bunning et al., 1990). Components of this response include the chaperonins GroEL, GroES, DnaK and DnaJ, whose function is to maintain the integrity of cellular proteins under certain stress conditions (Langet et al., 1992). The temperature-dependent expression of the heat-shock response in Salmonella is dependent upon the alternate sigma factor σH (also referred to as RpoH or σ32) (Yura et al., 1993). Considerable overlap exists between oxidative stress responses and the heat-shock response in E. coli and S. Typhimurium. Deletions in rpoH render E. coli extremely sensitive to peroxide and superoxide stress as well as heat stress (Farr and Kogoma, 1991) while S. Typhimurium cells adapted to peroxide stress exhibit greatly increased resistance to heat-shock (Christman et al., 1985; Morgan et al., 1986). Since bacterial cells may be exposed to elevated temperatures as well as oxidative stress during infection, researchers have examined the expression of heat-shock proteins by bacteria during internalization by macrophages. Two separate studies
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have used two-dimensional gel electrophoresis to examine the expression of heatshock proteins by S. Typhimurium during infection of macrophage cell cultures. Infection of J774 macrophages resulted in the expression of 34 proteins at significantly increased levels relative to Salmonella grown in laboratory media (Buchmeier and Heffron, 1990). Many of these proteins seem to be specific for macrophages and were not induced in cultured epithelial cells. In one study, both DnaK and GroEL were shown to be synthesized by macrophage-internalized bacteria but were not produced by bacteria grown in epithelial cells (Buchmeier and Heffron, 1990). Interestingly a similar study examining infection of U937 macrophage cells failed to demonstrate induction of DnaK and GroEL by intracellular S. Typhimurium but did illustrate the synthesis of proteins from other stress stimulons (Abshire and Neidhardt, 1993). The use of different macrophage cell lines may explain the lack of correlation between studies, but both give an indication of the extent of bacterial adaptation which must take place as Salmonella cells struggle to maintain homeostasis during infection. Listeria monocytogenes has the capacity to elicit a heat-shock response and also induces synthesis of DnaK and GroEL homologues following sub-lethal heat shock (Bunning et al., 1990; Hanawa et al., 1995). An analysis of those proteins produced by Listeria grown within J774 macrophages revealed that none were involved in in vitro responses to heat shock or oxidative stress responses (Hanawa et al., 1995). Similarly, a separate study showed that synthesis of DnaK and GroEL actually decreases during growth in mouse phagocytes (Hevin et al., 1993). It has been suggested that rapid escape from the phagosome prevents expression of stress proteins by L. monocytogenes during macrophage infection (Hanawa et al., 1995). However, an increase in the expression of GroEL mRNA was detected recently in macrophage-internalized L. monocytogenes using both reverse transcriptase-polymerase chain reaction (RT-PCR) and green fluorescent protein technology (Figure 7.2), indicating that cells may respond with the synthesis of heat-shock proteins but at levels which may not be detectable on protein gels (Gahan et al., 2001). Recently the dnaK gene of L. monocytogenes has been cloned and sequenced (Hanawa et al., 1999). A deletion mutant in dnaK is not phagocytosed efficiently by macrophages, although the mutant grows as efficiently as the wild-type once internalized. The authors suggest a possible role for DnaK in the synthesis, folding and/or translocation of surface proteins involved in adhesion (Hanawa et al., 1999). Interestingly, exposure of the Gram-positive pathogen Enterococcus faecalis to bile salts has been shown to elicit expression of some heat-shock genes including GroEL and DnaK (Flahaut et al., 1996). In addition, heat shock of E. faecalis induced increased protection against bile salts in vitro (Flahaut et al., 1996). These findings suggest that heat-shock proteins may play an essential role in bacterial survival of bile salts encountered during colonization of the small intestine. In S. Typhimurium and L. monocytogenes, a number of other proteins associated with thermotolerance and general stress resistance may play a role in vivo. For example, in Salmonella and E. coli several genes involved in high-temperature resistance (htr) have been identified (Delaney et al., 1993). Of these the best characterized is htrA, encoding a serine protease (HtrA) required for survival of E. coli at high temperatures (>42°C) and for the resistance of S. Typhimurium to oxidative stress
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FIGURE 7.2 Use of green fluorescence protein (GFP) technology to determine gene expression in Listeria monocytogenes during infection of J774 mouse macrophage cells. (a) J774 cells infected with L. monocytogenes carrying promoterless copy of GFP (negative control). (b) J774 cells infected with L. monocytogenes expressing GFP from the promoter of the heatshock operon groESL.
(Strauch et al., 1989; Johnson et al., 1991). HtrA is thought to assist in the degradation of denatured proteins which may accumulate under stress conditions (Strauch et al., 1989). In Gram-negative organisms, the expression of htrA is not regulated by σH but by σE, a sigma factor synthesized under extreme stress conditions (Hiratsu et al., 1995; Humphreys et al., 1999). Deletion of htrA in S. Typhimurium greatly reduces ability to survive within macrophages and significantly attenuates virulence for mice (Chatfield et al., 1992; Baumier et al., 1994). Elimination of the σE regulon by deletion of rpoE has a greater effect on attenuation of virulence than deletion of the htrA gene alone (Humphreys et al., 1999). Since rpoE mutants are rapidly eliminated from host tissues they demonstrate reduced immunogenicity in mice and fail to work as effective vaccines. In contrast htrA mutants retain the ability to proliferate in host tissues and represent excellent vaccine candidates (Humphreys et al., 1999). Homologues of htrA have been identified in Gram-positive organisms including Bacillus subtilis (Noone et al., 2000) and play a role in stress resistance. To date no work has focused upon HtrA homologues in L. monocytogenes. Other heat-shock proteins involved in proteolysis include the family of Clp proteases which play a role in heat tolerance of both Gram-negative and Grampositive organisms (Squires and Squires, 1992; Hecker et al., 1996). In Listeria, this family of proteases is evidently of vital importance in governing stress responses during infection and subsequent survival in the host. The gene encoding ClpC in L. monocytogenes was identified in a transposon mutant displaying sensitivity to low iron conditions (Rouquette et al., 1995). Disruption of clpC results in reduced thermotolerance and increased sensitivity to high salt and low iron growth conditions. ClpC mutants also display significantly attenuated virulence for mice and reduced ability to grow in cultured macrophages (Rouquette et al., 1996, 1998). Examination of macrophages infected by ClpC mutant strains using electron microscopy suggests
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that the ability of these mutants to escape from the macrophage phagosome is impaired (Rouquette et al., 1998). Identification of clpE in L. monocytogenes has also been described (Nair et al., 1999). Mutation of clpE impaired prolonged survival at high temperatures and significantly reduced virulence for mice. Interestingly, clpE is not induced under stress conditions but is upregulated in a clpC mutant, indicating cross regulation of these Clp ATPases (Nair et al., 1999). Another member of the family of Clp proteases, ClpP, is required for growth under stress conditions and for virulence of L. monocytogenes. Evidence suggests that ClpP may be required for full activity of the essential hemolysin, listeriolysin (Gaillot et al., 2000). Regulation of Clp protease expression is mediated by CtsR, the product of the first gene in the ClpC operon (Nair et al., 2000). This regulator is homologous to the B subtilis CtsR repressor of stress responses; accordingly, deletion of the gene in L. monocytogenes results in increased stress tolerance. Constitutive expression of CtsR in L. monocytogenes results in significant attenuation of virulence for mice, most likely as a consequence of the repression of the stress response. In L. monocytogenes, the transcriptional regulator PrfA coordinates virulence factor expression in response to environmental changes experienced within the host. Virulence genes encoding internalins (inlA, inlB and inlC), hemolysin (hly), ActA (actA) and phopholipases (plcA and plcB) are all under PrfA regulation and numerous other genes uncovered by the Listeria genome sequencing project are postulated to be PrfA-regulated (Glaser et al., 2001). Increased temperature provides a key signal for the increased expression of certain PrfA-regulated genes and L. monocytogenes cells are hemolytic at temperatures of 37°C and higher but show reduced hemolysis below 30°C (Datta, 1994). Virulence genes are coordinately regulated in sequence at specific stages of intracellular pathogenesis, such that genes required for escape from the phagosome are expressed at an earlier stage than genes required for intracellular motility and cell-to-cell spread; see Figure 7.1B (Bubert et al., 1999; Freitag and Jacobs, 1999). The bacterium evidently has evolved the ability to sense specific aspects of the host cell environment and to respond with the synthesis of appropriate virulence factors. While PrfA undoubtedly plays an important role in coordinating this response, other factors must play a role in detecting extracellular signals and a putative PrfA-binding factor is thought to enhance the sensitivity of the system (Böckmann et al. 1996).
ACID TOLERANCE
AND
VIRULENCE
Invasive foodborne pathogens will encounter low pH and/or organic acids during passage through the stomach, during transient colonization of the small intestine and during residence within the host cell phagosome. In order to survive in these sub-optimal environments, bacteria have evolved mechanisms which allow adaptation to low pH. The adaptive response, termed the acid tolerance response (ATR), involves the acquisition of acid tolerance following a brief exposure to mildly acidic growth conditions and involves a significant shift in patterns of protein synthesis as bacteria are subjected to the reduction in pH of the growth media (Foster, 1991; O’Driscoll et al., 1997). This response is shared by a number of foodborne pathogens
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including S. Typhimurium (Foster and Hall, 1991) and L. monocytogenes (O’Driscoll et al., 1996; Davis et al., 1996) and contributes to the survival of these pathogens in low pH foods (Leyer and Johnson, 1992; Gahan et al., 1996). Studies have also been performed to determine whether the ability to survive at low pH can contribute to virulence potential. A number of studies have attempted to determine whether acid adaptation of pathogens prior to use in virulence studies can affect the outcome of infection. In S. enterica serotype Enteritidis PT4, acid tolerant bacterial cells (stationary phase) demonstrate similar virulence potential to acid sensitive, chilled log-phase cells (Humphrey et al., 1998). Similarly, an increase in acid tolerance following acid adaptation of L. monocytogenes fails to alter the virulence of the pathogen (Gahan and Hill, 1999). It was suggested that this may simply be a consequence of the ability of Listeria cells to naturally develop acid tolerance following uptake by macrophages, such that natural stress adaptation during infection eliminates any advantage of prior adaptation (Gahan and Hill, 1999). In support of this view, we have demonstrated that a mutant of L. monocytogenes which is incapable of inducing an adaptive acid tolerance response is significantly impaired in its virulence potential for mice, relative to the parent (Marron et al., 1997). Furthermore, a spontaneous acid tolerant mutant of L. monocytogenes displaying increased acid tolerance without prior adaptation, demonstrates increased survival potential in mice relative to the wild type (O’Driscoll et al. 1996). This may be a result of an increased ability to survive the initial exposure to low pH encountered by internalized Listeria cells. Indeed, a recent study demonstrates that both acid-adapted Listeria and a constitutively acid tolerant mutant are capable of increased survival and growth following uptake by macrophage (J774.A1) or enterocyte-like (Caco-2) cell lines (Conte et al., 2000). Similarly, an acid tolerant S. Typhimurium mutant displays moderately elevated virulence potential in the mouse typhoid model (Wilmes-Riesenberg et al., 1996) and an acid tolerant strain of S. Enteritidis is more virulent than a phenotypically normal reference strain (Humphrey et al., 1996). Collectively, these data provide evidence that acid tolerance plays a role in the virulence potential of these foodborne pathogens and that further molecular characterization of the response is warranted. In S. Typhimurium the development of acid tolerance is regulated in part by the starvation/stationary phase alternate sigma factor σs encoded by rpoS. The avirulent, acid-sensitive laboratory strain S. Typhimurium LT2 harbors a mutation in rpoS (Lee et al., 1995; Swords et al., 1997; Wilmes-Riesenberg et al., 1997). Introduction of wild-type rpoS from virulent S. Typhimurium strains into the LT2 strain restores both virulence potential and acid tolerance (Lee et al., 1995). Similarly, the live oral typhoid vaccine S. Typhi Ty21a, in common use, is an rpoS mutant which is susceptible to a variety of environmental stresses (Robbe-Saul et al., 1994). Expression of rpoS in S. Typhimurium is clearly induced following a shift to low pH (Lee et al., 1995) and during macrophage infection (Chen et al., 1996) and deletion mutants in rpoS are avirulent (Wilmes-Riesenberg et al., 1997). However, since σs regulates the virulence plasmid-associated spv operon (Kowarz et al., 1994) and is involved in oxidative stress and starvation stress responses, it is difficult to attribute loss of virulence simply to loss of acid tolerance. Indeed, one study
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demonstrated that in a plasmid-cured background both rpoS+ and rpoS- strains survive well during the first 5 days of infection, but that at 21 days postinfection the rpoS+ strain reaches significantly higher numbers than the mutant strain (Kowarz et al., 1994). In another study utilizing infection of mice by the oral route, a plasmidcured rpoS– strain demonstrated a reduced ability to colonize Peyers patches relative to its rpoS+ counterpart (Nickerson and Curtiss, 1997). The results suggest a role for σs in bacterial persistence in vivo, even in the absence of plasmid-borne spv genes (Kowartz et al., 1994; Nickerson and Curtiss, 1997). The acid induction of σs is controlled by the product of the mouse virulence gene mviA (Bearson et al., 1996; Benjamin et al., 1996). MviA has significant homology to bacterial response regulator proteins and is most likely involved in environmental sensing, in turn leading to induction of the σs regulon. This occurs through an mviA dependent reduction in proteolytic turnover of σs, resulting in increased levels of the protein (Foster, 1999). Therefore, both mviA and rpoS contribute to virulence potential of S. Typhimurium. While the σs regulon appears to respond primarily to organic acids, responses to low pH (elevated H+ concentration) are influenced by PhoP, the regulatory element of the PhoP-PhoQ two-component system (Bearson et al., 1998). This two-component system senses and responds to conditions encountered within the host cell phagosome including low Mg2+ (Soncini et al., 1996) and inorganic acid stress (Bearson et al., 1998). Deletion of this response mechanism significantly attenuates virulence of S. Typhimurium for mice (Miller et al., 1989) with mutant strains demonstrating an impaired ability to survive within macrophages (Fields et al., 1986), impaired growth in low Mg2+ media (Soncini et al., 1996) and reduced resistance to bile (van Velkinburgh and Gunn, 1999), low pH (Bearson et al., 1998) and host defense antimicrobial peptides (Groisman et al., 1992, 1997). Constitutive expression of the phoP regulon also attenuates virulence, suggesting that dynamic regulation of PhoP-activated genes (pag) and PhoP-repressed genes (prg) during infection is necessary for full virulence (Miller and Mekalanos, 1990). Examination of genes regulated by PhoP is ongoing. An important prg group encode elements of the Salmonella pathogenicity island 1 (SPI1) type III secretion system. This locus is required for entry of Salmonella into host cells but is repressed following internalization as a result of activation of the PhoP-PhoQ system (Pegues et al., 1995; Bajaj et al., 1996; Cotter and Miller, 1998). Many pag products show no similarity to proteins in the database, while others encode genes with diverse functions in many aspects of bacterial physiology (Gunn et al., 1998). One gene, pagC, shows homology to a Yersinia enterocolitica invasion protein (Miller et al., 1989) while another, pagO, encodes a product similar to that of the Yersinia virulence plasmid (Gunn et al., 1998). Other pags play a role in resistance of Salmonella to antimicrobial peptides present within the macrophage phagosome (Gunn and Miller, 1996; Soncini and Groisman, 1996). Interestingly, the important virulence locus, Salmonella pathogenicity island 2 (SPI2), contains genes that are necessary for virulence and are induced by low Mg2+ in a cascade that is modulated by PhoP-PhoQ (Deiwick et al., 1999). However, the regulation of SPI2 is further complicated by the fact that SsrAB, a two-component
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regulatory system encoded on SPI2 also plays a role in expression of other SPI2 genes (Deiwick et al. 1999). Furthermore, yet another two-component regulatory system, OmpR-EnvZ, is essential for SPI2 gene expression in response to low pH (Lee et al., 2000). The PhoP-PhoQ regulon is obviously extremely complex; however, it is clear that this two-component system plays a major role in coordinate regulation of virulence genes in response to environmental changes. Indeed, in a recent study of genetic loci induced during infection it was found that, out of seven genes subject to pH or Mg2+ mediated regulation, all were part of the PhoP-PhoQ regulon (Heithoff et al., 1999). In another study, eight out of fourteen S. Typhimurium genes identified as in vivo inducible were demonstrated to be regulated by PhoPPhoQ (Valdivia and Falkow, 1997). Finally, the iron regulator protein Fur also plays a role in acquisition of acid tolerance in S. Typhimurium (Foster, 1991). As a repressor of genes involved in iron acquisition, Fur down-regulates transcription from appropriate promoters when intracellular Fe(II) concentrations are high. However, Fur also influences the expression of several acid shock proteins in an iron-independent manner. It is low pH rather than iron concentration that influences Fur regulation of proteins involved in acid adaptation (Foster and Hall, 1992). In addition, a specific mutation in fur can block responses to iron but has no effect on acid tolerance (Hall and Foster, 1996). Knockout mutants in fur demonstrate an obvious virulence defect when analyzed in the mouse typhoid model (Wilmes-Riesenberg et al., 1996). However, this effect may be pleiotrophic and may affect iron metabolism as well as acid tolerance during infection. The recent construction of acid-blind/iron-sensing and iron-blind/acidsensing mutants in fur will allow a more precise determination of the in vivo requirement for iron and acid-regulated Fur-dependent proteins (Foster, 1999). In L. monocytogenes, relatively little is known of the circuits involved in regulation of stress responsive genes. The alternative sigma factor, σB, has been identified and sequenced in L. monocytogenes (Wiedmann et al., 1998; Becker et al., 1998). This sigma factor appears to regulate the synthesis of a number of stress responsive proteins. Mutation of the σB gene eliminates the ability to tolerate acid stress and reduces the ability to respond to high salt environments (Wiedmann et al., 1998; Becker et al., 1998). Elimination of sigma B in L. monocytogenes also reduces the starvation stress response (Herbert and Foster, 2001) and decreases resistance to bile salts (Begley, Hill and Gahan, manuscript in preparation). However, the mutation does not appear to influence the virulence potential of this strain (Wiedmann et al., 1998). Similarly, mutating the σB homologue in Staphylococcus aureus fails to influence virulence of the pathogen in a mouse abscess model of infection (Chan et al., 1998). To date, the data indicate that σB-influenced gene expression may not play a significant role in the in vivo survival of Gram-positive pathogens. Nonetheless, further studies will be required to investigate any potential role for σB in virulence. Another sigma factor, σH, is induced in L. monocytogenes following a shift to acidic growth conditions (Phan-Thanh and Mahouin, 1999; Phan-Thanh et al., 2000). In Bacillus subtilis, this sigma factor plays a major role in environmental adaptation; however, a mutant in σH is not yet available in L. monocytogenes. As mentioned previously, recent work has identified an operon in L. monocytogenes with significant sequence homology to two-component regulatory systems of
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A orfA
lisR
lisK
orfD
Region deleted in ∆lisK
Log CFU Listeria / spleen
B 7
Wild-type ∆lisK
6
5
4
3
0
1
2
3
4
Day FIGURE 7.3 (A) Genetic map of Listeria monocytogenes genes encoding the stress responsive two-component regulatory system LisR-LisK. (B) Survival of knockout mutant in LisR during infection of mice relative to the wild-type strain (LO28). (From Cotter, P.D. et al., J. Bacteriol., 181: 6840–6843, 1999. With permission.)
Group A streptococci, Lactococcus lactis and B. subtilis (Cotter et al., 1999). This two-component signal transduction system, designated LisR-LisK, appears to play a role in the regulation of acid resistance in L. monocytogenes. Mutation of either the histidine kinase component (lisK) or the response regulator (lisR) results in a significant attenuation of virulence potential, as evidenced by an inability to survive during the early stages of infection in the mouse model (Figure 7.3). Interestingly, a mutation in the Enterococcus faecalis homologue of lisR also results in a virulence defect, suggesting a general role for this two-component system in virulence of Gram-positive pathogens (Teng et al., 2002). In addition to environmental sensors and regulators, some information is available concerning effectors which play a direct role in maintaining intracellular pH homeostasis during shifts in external pH. A mutation in the major proton translocating ATPase (atp) in virulent S. Typhimurium increases acid sensitivity, eliminates the ability to induce an ATR and significantly decreases virulence in the mouse typhoid model (Garcia del-Portillo et al., 1993). As mentioned previously, virulent S. Typhimurium strains contain wild-type rpoS. Inactivation of single genes known
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to contribute to acid tolerance in the attenuated strain LT2 has only a marginal effect on acid tolerance in virulent Salmonella (Wilmes-Riesenberg et al., 1996). In a virulent S. Typhimurium background, mutation of two or more genes was required to eliminate acid resistance and the ability to induce an ATR. Double and triple mutants containing a polA (DNA polymerase I) mutation lacked an ATR and were highly attenuated in mouse and macrophage tissue culture models. A portion of the F0-F1 ATPase of L. monocytogenes has recently been characterized (Cotter et al., 2000). Disruption of ATPase activity eliminates the ability to induce an acid tolerance response. However, further work is required to establish the role, if any, of the F0-F1 ATPase in the virulence of the pathogen. The role of the glutamate decarboxylase system in L. monocytogenes acid tolerance has also been investigated (Cotter et al., 2001). In this system, cells grown in appropriate media accumulate glutamate via a specific antiporter (encoded by gadC). Glutamate is converted to γ-amino butyrate (GABA) by the enzyme glutamate decarboxylase (encoded by gadB) with the net consumption of a single proton, thereby reducing intracellular pH. GABA is then exported from the cell via the glutamate antiporter. Initial evidence suggested that L. monocytogenes possesses two glutamate decarboxylase genes, gadA and gadB, with different roles in regulating pH homeostasis. Mutation of gadB renders cells acid sensitive while deletion of gadA only marginally reduces acid tolerance (Cotter et al., 2001). The role of a third glutamate decarboxylase gene, uncovered as a result of the Listeria genome sequencing project (Glaser et al., 2001), remains unknown. Addition of glutamate to an in vitro model of gastric acid (pH 2.5) significantly improves survival of wild-type L. monocytogenes. In addition, a double mutant in gadA/gadBis exquisitely sensitive in the gastric acid model even when glutamate is added. The results suggest a role for glutamate in aiding gastric survival of the pathogen, a phenomenon mediated by the GAD system (Cotter et al., 2001). In a recent study, knock-out of the lysine decarboxylase system (cad) in V. cholerae greatly reduced acid tolerance but did not affect ability to colonize the small intestine in mice (Merrell and Camilli, 1999). However, in this study the authors did not add lysine to the inoculum prior to feeding. Collectively these studies suggest that these decarboxylase systems are essential for full acid tolerance in complex media but further studies are required to determine their roles in protecting against gastric acid.
OXIDATIVE STRESS RESPONSE Bacterial pathogens encounter oxidative stress as a result of normal aerobic metabolism but are exposed to severe oxidative stress following uptake by macrophages which generate phagosomal superoxide anion (O2–) and hydrogen peroxide (H2O2) as well as the reactive nitrogen intermediate, nitric oxide (NO). S. Typhimurium reacts to superoxide and peroxide with increased expression of two different, but overlapping, sets of proteins. As mentioned previously, some overlap also exists between responses to oxidative stress and heat shock, and the heat shock proteins GroES and GroEL are induced by both peroxide- and superoxide-mediated oxidative stresses (Farr and Kogoma, 1991). However, well defined regulons exist in S. Typhimurium which only react to peroxide stress (oxyR) or superoxide stress (soxRS).
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OxyR is a transcriptional regulator with similarity to the LysR family (Christman et al., 1989) and activates nine genes in E. coli in response to peroxide stress (Christman et al., 1985). These genes include one of two catalase genes (katG), a gene encoding glutathione reductase (gorA), and an NADPH-dependent alkyl hydroperoxidase (ahpFC) (Morgan et al., 1986; Tartaglia et al., 1990; Michan et al. 1999). An OxyR-regulated locus (ahpC) is clearly upregulated in Salmonella during macrophage infection (Francis et al., 1997). However, despite an obvious requirement for OxyR in resistance to in vitro peroxide stress, deletion of either oxyR or katG has no effect on ability of S. Typhimurium to resist neutrophil bactericidal activity (Papp-Szabo et al., 1994). Similarly, a double mutant lacking KatE and KatG activity is not affected in virulence for mice or sensitivity to macrophage killing (Mahan et al., 1996). SoxR and SoxS regulate the expression of a number of genes in response to superoxide stress. The SoxR-SoxS regulon includes genes encoding the manganesecontaining Mn-cofactored superoxide dismutase (sodA), glucose-6-phosphate dehydrogenase (zwf), oxidation resistant fumarase (fumC), ferredoxin-NADPH oxidoreductase (fpr), an inner membrane efflux pump (acrAB), and at least five other genes involved in superoxide resistance (Liochev and Fridovich, 1992; Liochev et al., 1994; Fang et al., 1997). SoxR is activated by exposure to superoxide and in turn activates expression of soxS, the product of which is a transcriptional regulator. SoxS is necessary for resistance of S. Typhimurium to redox-cycling agents such as paraquat, which generate intracellular superoxide (Fang et al., 1997). However, SoxS is not required for resistance to macrophage killing or for virulence in mice (Fang et al., 1997). Surprisingly, mutation of a SoxS-regulated gene, zwf, which encodes a glucose 6-phosphate dehydrogenase, significantly reduces virulence potential in the mouse model as well as compromising resistance to oxidative stress (Lundberg et al., 1999). However, this locus is not exclusively regulated by SoxR-SoxS and may be expressed at basal levels in the absence of SoxS (Fawcett and Wolf, 1995). Deletion of SoxS-regulated sodA in S. Typhimurium does not significantly attenuate virulence for mice, even though mutants do exhibit reduced resistance to macrophage killing in vitro (Tsolis et al., 1995). A distinct Cu/Zn-cofactored superoxide dismutase (SodC) exists outside of the SoxR-SoxS regulon and appears to have been acquired by S. Typhimurium through bacteriophage-mediated horizontal transfer. Curing of the prophage or deletion of the sodC gene dramatically reduces virulence potential for mice, suggesting that sodC plays a significant role in Salmonella pathogenesis (Farrant et al., 1997; De Groote et al., 1997; Figueroa-Bossi and Bossi, 1999). In addition to SoxR-SoxS and OxyR regulons, another transcriptional regulator, SlyA, has been implicated as a regulator of resistance to both peroxide and superoxide stress in S. Typhimurium. A slyA mutant is highly sensitive to both hydrogen peroxide and paraquat, is avirulent in mice and is unable to replicate in mouse macrophages (Libby et al., 1994; Buchmeier et al., 1997). In addition, transcription of this gene is significantly enhanced following uptake by macrophages, suggesting an important role in coordination of the bacterial response to oxidative stress during infection (Buchmeier et al., 1997).
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Iron-containing compounds are particularly prone to damage by oxidative stress (Farr and Kogoma, 1991). In addition, hydrogen peroxide reacts with iron to form the extremely reactive hydroxyl radical, which can cause further damage to cellular components. Under conditions of oxidative stress, uptake of ferric iron is therefore reduced. Recent evidence indicates that in E. coli both OxyR and SoxS activate the expression of Fur, the global repressor of iron uptake (Zheng et al., 1999). The data indicate that oxidative stress responses and iron metabolism are coordinately regulated. Activated macrophages are capable of producing toxic nitrogen intermediates (e.g., nitric oxide) in addition to peroxide and superoxide. Nitrosative stress, imposed by nitric oxide donors such as S-nitrosothiols, has the ability to activate OxyR at the transcriptional level in E. coli (Hausladen et al., 1996). In S. Typhimurium, a component of the SoxR-SoxS regulon, the zwf gene product, is involved in resistance to nitrosative stress as well as oxidative stress (Lundberg et al., 1999). In addition, SodC plays a significant role in protecting bacteria from both nitric oxide and superoxide both in vitro and during macrophage infection (De Groote et al., 1997). However, further pathways exist which are specific for nitrosative stress and not shared by oxidative responses. A gene encoding a hemoglobin homolog, flavohemoglobin (hmp) is required for resistance to S-nitrosothiols and acidified nitrite, but not resistance to oxidative stress (Crawford and Goldberg, 1998a). Transcriptional activation of hmp is independent of SoxS and OxyR but requires inactivation of Fur. Interestingly, other Fur-repressed genes were also activated by nitric oxide, suggesting that Fur plays a role not only in coordination of iron metabolism and responses to acid stress, but also responses to nitric oxide (Crawford and Goldberg, 1998b). Another gene contributing to nitric oxide resistance in S. Typhimurium is metL encoding a protein involved in homocysteine biosynthesis. Deletion of metL in S. Typhimurium dramatically reduces resistance to S-nitrosothiols and significantly attenuates virulence for mice (De Groote et al., 1997). The superoxide dismutase and catalase genes of L. seeligeri and the superoxide dismutase gene from L. monocytogenes have been cloned and sequenced (Haas et al., 1991; Hess et al., 1997). However, there is some evidence to suggest that neither catalase nor superoxide dismutase play a role in the virulence of L. monocytogenes. For instance, catalase negative strains of L. monocytogenes have been isolated from listeriosis patients, indicating that catalase production by the pathogen is not necessary to cause human disease (Swartz et al., 1991; Bubert et al., 1997). In addition, transposon-induced mutants lacking catalase activity do not exhibit a virulence defect (Leblond-Francillard et al., 1989). Also, unlike salmonellosis, listeriosis is not a common opportunistic infection of individuals with chronic granulomatous disease, a condition in which phagocytes fail to produce reactive oxygen intermediates (Gallin et al., 1983; Safe et al., 1991). Analyses of the mechanisms of bacterial killing by macrophages have failed to determine whether reactive oxygen intermediates are absolutely required. Studies have demonstrated that murine macrophages can kill L. monocytogenes by a mechanism which depends predominately upon nitric oxide, rather than superoxide or peroxide (Beckerman et al., 1993; Boockvar et al., 1994). However, a more recent study indicates that reactive oxygen intermediates may be most important for bactericidal
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activity (Ohya et al., 1998). Further studies are evidently required to determine the extent to which L. monocytogenes encounters reactive oxygen species during infection and the mechanisms used to counteract this potentially lethal stress.
OSMOTIC STRESS During the pathogenic cycle, foodborne pathogens encounter a range of environments with differing osmolarities. Following foodborne infection, pathogens are exposed to an osmolarity in the intestinal lumen equivalent to 0.3 M NaCl, while the osmolarity of blood is about 0.15 M NaCl (Chowdhury et al., 1996). S. Typhimurium and L. monocytogenes differ in their ability to naturally survive exposure to high salt environments with Listeria spp. capable of surviving salt concentrations as high as 30%, while the tolerance of Salmonella spp. for high salt environments is much lower. However, both organisms respond to increases in external osmolarity by synthesis and/or uptake of osmoprotectants, substances which counterbalance external pressure, prevent water loss from the cell and thereby maintain cell turgor. Osmoprotectants capable of being transported into the cell during periods of osmotic stress include glycine betaine, proline and carnitine; proline, glutamate and trehalose can be synthesized internally when required (Csonka and Hanson, 1991; Foster and Spector, 1995). In S. Typhimurium, the proU and proP systems govern uptake of both glycinebetaine and proline in response to shifts in external osmolarity (Csonka et al., 1994) while the putP system is a high affinity proline transporter (Liao et al., 1997). The proU uptake system is clearly regulated by environmental stresses, including osmotic stress and low pH stress, but no information is available concerning a possible role during infection (Foster and Spector, 1995). In E. coli, a strain capable of causing urinary tract infections and pyelonephritis demonstrates an abnormally high level of proP activity, while deletion of proP dramatically reduces ability of this strain to colonize mouse bladders (Culham et al., 1998). Similarly, inactivation of putP in S. aureus significantly reduces virulence in an experimental endocarditis model (Bayer et al., 1999). Studies analyzing the role of osmoprotectant uptake and synthesis systems in virulence of foodborne pathogens are awaited with interest. A glycine betaine uptake system (BetL) linked to the salt tolerance of L. monocytogenes has recently been characterized (Sleator et al., 1999). This system is one of three known mechanisms for the uptake of glycine betaine during periods of osmotic stress. Other systems comprise the gbuABC operon and a system homologous to OpuC in Bacillus subtilis (Ko and Smith, 1999; Sleator et al., 2001a). A betL mutant is significantly impaired in its ability to survive salt stress when glycine betaine is the most abundant osmoprotectant, but does not differ from the wild-type during intraperitoneal infection of mice (Sleator et al., 2000). The region upstream of betL contains a putative σB promoter binding site and transcription of betL appears to be up-regulated at high osmolarity (Sleator et al., 1999, 2000). Given that σB mutants are not attenuated for mice (Wiedmann et al., 1998), it is not surprising that mutation of this putatively σB-regulated gene does not result in a virulence defect. In L. monocytogenes a homologue of the B. subtilis carnitine transporter, OpuC plays a major role in carnitine transport (Fraser et al., 2000) and is also capable of
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glycine betaine transport in an ATP-dependent manner (Sleator et al., 2001a). Carnitine is predicted to be the most important osmolyte in foods of animal origin (Smith, 1996). Indeed, an OpuC mutant in L. monocytogenes demonstrates significantly impaired survival during manufacture of fermented sausage and may therefore play a significant role in facilitating growth of the pathogen to high levels in meat products (Sleator et al., unpublished data). Recent work has demonstrated that knockout of opuC in L. monocytogenes strain LO28 can reduce the virulence potential of this strain following intraperitoneal infection. Interestingly, this effect is strain specific and was not seen in a knockout mutant in L. monocytogenes ScottA. However, elimination of opuC in both strains significantly reduced the ability to colonize the upper small intestine in mice following peroral administration. This suggests that this uptake system plays an important role in growth and survival in the osmotically challenging environment of the gastrointestinal tract (Sleator et al., 2001a). Finally, it is possible that systems for synthesis of osmoprotectants may play a role in maintainance of homeostasis during infection, reducing the importance of uptake systems. Recent evidence indicates that L. monocytogenes posesses a glycine betaine synthesis system (Phan-Thanh and Mahouin, 1999; Glaser et al., 2001). However a role for this system in maintainance of cell turgor has not yet been examined. In addition, a Listeria proline synthesis system has been characterized which is homologous to a locus in Salmonella (proBA) known to play a role in salt tolerance (Sleator et al., 2001b). Knockout of this locus in L. monocytogenes reduces salt tolerance in complex broth but does not appear to affect virulence potential when administered to mice by the intraperitoneal or peroral routes. This finding supports an earlier study which suggested that proline auxotrophs do not demonstrate impaired virulence and suggests that host tissues contain a relatively abundant source of free proline (Marquis et al., 1993). Furthermore, manipulation of the system to induce overproduction of proline fails to alter the virulence potential in L. monocytogenes (Sleator et al., 2001c). In S. Typhimurium, a two-component system, OmpR-EnvZ which responds to perturbations in external osmolarity, has emerged as a global regulator of virulence potential. EnvZ is the inner membrane sensor component that signals changes in specific environmental signals via phosphorylation of OmpR, a transcriptional regulator. The system was originally identified as a regulator of the outer membrane proteins, OmpF and OmpC. Mutation of ompR dramatically reduces virulence of both Shigella flexineri and S. Typhimurium for mice, suggesting a major role in both pathogens (Dorman et al., 1989; Bernardini et al., 1990). In S. Typhimurium, OmpR mutants fail to lyse infected macrophages and so fail to induce a key step in pathogenesis (Lindgren et al., 1996). Other studies have demonstrated that double mutation of ompF and ompC also results in attenuation of virulence but not to the same extent as ompR deletion, indicating that other components of the OmpR regulon are important for infection (Chatfield et al., 1991). One theory is that the osmolarity of the intestine favors expression of OmpC, a porin with small pore size that should exclude harmful molecules such as bile salts. OmpF is most likely expressed outside of the host environment (Russo and Silhavy, 1993; Foster and Spector, 1995). A role for these proteins in intestinal survival is supported by the fact that double mutants in ompC and ompF are severely attenuated when administered to mice by
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the oral route but only marginally affected when administered intravenously (Chatfield et al., 1991). As indicated above, the influence of EnvZ-OmpR on virulence potential extends beyond the regulation of outer membrane proteins. Other genes regulated by OmpR include aas, a gene encoding 2-acylglycerolphosphoethanolamine acyltransferase, which is induced within macrophages and is influenced by low pH (Valdivia and Falkow, 1997). However, mutation of aas has no significant effect on virulence potential (Lee et al., 2000). In addition, a gene responsible for the formation of Salmonella-induced filaments within HeLa cells (sifA) is regulated by OmpR (Mills et al., 1998). Deletion of this gene results in partial attenuation of virulence indicating some requirement for filament formation during infection (Stein et al., 1996). Since mutations of individual components of the OmpR regulon have only a marginal effect on virulence potential, researchers have continued the search for the key component of the regulon. In this regard, the most interesting recent discovery is the fact that OmpR regulates the two-component system SsrA-SsrB in SPI2, which in turn regulates a type III secretion system required for replication in macrophages and infection of mice (Lee et al., 2000). DNA footprinting studies demonstrated that OmpR binds directly to the promoter region in ssrA. In addition, OmpR was necessary for SPI2 gene expression when cells were grown at low pH (Lee et al., 2000). Evidence suggests that EnvZ responds to the low pH and low osmolarity of the phagosome and activates OmpR, which in turn stimulates rapid expression of ssrA and ssrB. SsrA-SsrB then detects another signal (possibly mediated by PhoP-PhoQ), and in turn activates expression of the SPI2 type III secretion system (Deiwick et al., 1999; Lee et al., 2000). That OmpR is involved in low pH-dependent stimulation of SsrA-SsrB expression is interesting, given recent evidence implicating OmpR as the major regulator of stationary-phase acid tolerance responses (Bang et al., 2000). Mutants in OmpR are defective for an inducible stationary-phase acid tolerance response, yet can still induce acid tolerance in log phase. It appears that OmpR activation by low pH and/or low osmolarity following host cell invasion leads to induction of acid tolerance, as well as stimulation of SPI2 genes and other genes which may play a role in pathogenesis (e.g., sifA).
STARVATION STRESS During residence in the host cell phagosome and during colonization of the small intestine, invasive foodborne pathogens may struggle to accumulate adequate amounts of phosphate, carbon and nitrogen. Starvation for such nutrients represents a stress for the bacterium and results in a distinct physiological response. Moreover, in Salmonella the starvation stress response induces potent cross resistance against acid stress, heat stress, oxidative stress and osmotic challenge (Foster and Spector, 1995; Spector et al., 1999). Starvation stress may therefore represent a mechanism to induce resistance to a number of in vivo stresses. In S. Typhimurium, starvation resistance requires a number of genes including rpoS and the starvation survival genes, stiA, stiB and stiC (Spector and Cubitt, 1992;
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O’Neal et al., 1994). Mutants deleted in rpoS fail to exhibit carbon-starvationinduced cross-protection and stiA, stiB and stiC are also required for the development of cross-protection (Foster and Spector, 1995). Recent evidence suggests that stiA encodes a nitrate reductase and is required for carbon-starvation-inducible thermotolerance and acid tolerance (Spector et al., 1999). Interestingly, this locus is significantly induced during infection of Madin-Darby canine kidney epithelial cells suggesting responsiveness to intracellular conditions. In addition, a deletion mutant in stiA demonstrates reduced infectivity for mice by the oral route (Spector et al., 1999). Relatively little is known of starvation stress responses in L. monocytogenes. However, it is clear that synthesis of virulence factors, such as hemolysin and phospholipase, is repressed in the presence of rapidly metabolizable carbon sources, with the result that a shift from complex to minimal growth conditions may act as a trigger for the synthesis of virulence factors (Bohne et al., 1994, 1996; Milenbachs et al., 1997). It is likely that limitation of nutrients experienced in vivo serves to induce virulence factor expression. Furthermore, the global regulator of virulence gene expression, PrfA, is required for an efficient starvation stress response, suggesting a further role for this factor in maintaining homeostasis under starvation conditions (Herbert and Foster, 2001).
METHODS TO DETECT GENES TRANSCRIBED IN VIVO Traditionally, the analysis of the role of stress genes in virulence experiments has followed from in vitro studies. Using this approach, the physiological role of a gene (e.g., groEL) is well established before tests are performed to analyze its function, if any, in bacterial virulence. However, a new range of techniques have been developed to identify genes, many of them previously unknown, which are expressed during infection but not during growth on laboratory media. Many of these genes expressed as bacteria attempt to adapt to the stresses of the new host environment.
IN VIVO EXPRESSION TECHNOLOGY (IVET) Mahan et al., (1993) have developed an elaborate strategy for the selection of in vivo induced (ivi) genes in S. Typhimurium. This strategy, termed in vivo expression technology (IVET), was initially based on the use of an avirulent purine auxotroph (pur–) mutant of S. Typhimurium. Random integration of the pur gene back into the mutant resulted in a bank in which integration of pur downstream of properly positioned promoters resulted in expression of pur and complementation of virulence. Mutants of interest were those that expressed pur in vivo and therefore survived screening in a mouse model of infection, yet were Pur – in vitro. These represented fusions of pur to promoters that were induced exclusively during infection. Further IVET strategies in Salmonella utilized a promoterless cat gene encoding chloramphenicol resistance. Mice and/or tissue cultured macrophages inoculated with a bank of clones expressing cat from random promoters were subsequently treated with chloramphenicol to select for promoters that are active in vivo (Mahan et al., 1995).
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The use of these IVET approaches has led to an appreciation of the array of genes expressed during host infection, but not during growth in laboratory media. Some of these genes were already closely associated with virulence and included the regulator phoP and the plasmid-encoded virulence gene spvB. Other genes that are evidently expressed as bacteria adapt to intracellular stresses and include a catalase cofactor (hemA), a gene involved in acid tolerance (cadC), a gene involved in recombination/repair (recA) and genes involved in iron acquisition (entF, fhuA) and Mg2+ uptake (mgtA/mgtB) (Heithoff et al., 1997). The IVET system has been instrumental in determining members of the PhoP-PhoQ regulon as recent characterization of S. Typhimurium ivi genes has revealed many loci that are regulated by low pH and low Mg2+ in a PhoP-dependent manner (Heithoff et al., 1999). Other foodborne pathogens analyzed by IVET include V. cholerae and Y. enterocolitica. Analysis of ivi genes in V. cholerae revealed genes involved in amino acid and carbon metabolism, including a gene encoding a component of the TCA cycle enzyme succinase (sucA). In addition, genes involved in motility and a gene designated hlyC encoding a secreted lipase were also isolated (Camilli and Mekalanos, 1995). The Y. enterocolitica study revealed a number of ivi genes, including five loci involved in the iron starvation response, a gene involved in DNA repair (mutL), a gene encoding a stress response regulator (acrR) and the malate synthase gene (aceB) (Young and Miller, 1997). A recent IVET approach for L. monocytogenes utilizes the hemolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo (Gahan and Hill, 2000). Hemolysin functions in vivo to allow escape of the bacterium from the phagosome into the cytoplasm of host cells. Hemolysin negative mutants of L. monocytogenes are avirulent for mice and do not produce zones of hemolysis on blood agar plates. A hly- host was used to create a bank of clones in which the hemolysin gene is expressed from random promoter elements (Figure 7.4). Infection of mice with this bank allowed the selection of ivi clones that express hly in vivo but not on blood agar plates. Using this procedure it was determined that L. monocytogenes selectively induces a number of genes in response to the hostile host environment. These loci include a gene encoding the TCA cycle enzyme fumarate hydratase (fum), a gene involved in DNA supercoiling and putatively in gene regulation (DNA topoisomerase, topB), and a gene involved in transport of a cellobiose analogue (celB) (Gahan and Hill, 2000).
GREEN FLUORESCENT PROTEIN (GFP) TECHNOLOGY GFP from the jellyfish Aequorea victoria will cause bacteria to fluoresce if the gene is placed downstream of an active promoter. This fluorescence can be readily detected by fluorimetry, fluorescence microscopy or by flow-cytometry. Since no cofactors are required for fluorescence, this reporter system represents a useful means of monitoring expression of in vivo expressed genes. Recently, the system has been used to screen for ivi promoters in S. Typhimurium (Valdivia and Falkow, 1997). Integration of promoterless gfp was used to create a bank of S. Typhimurium cells in which expression of the protein is dependent on random promoter elements.
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A
or i
Em r
pCOR2 hly
MCS
Fragments of LO28 chromosomal DNA
B
hly
P
X
Y P
X
Y
LO28 ∆ hly
P
P
X Y hly
Emr
X Y
INFECTED MICE
C
FIGURE 7.4 An IVET system to detect in vivo induced genes in Listeria monocytogenes. (A) The pCOR2 IVET suicide vector comprises a promoterless copy of the hemolysin gene downstream of the multiple cloning site (MCS). A gene bank is created by cloning random fragments of Listeria DNA into the MCS. (B) The vector then integrates into the chromosome in a hemolysin negative (∆hly) L. monocytogenes host at the point of homology provided by the cloned DNA. The IVET bank represents clones expressing hly from random promoter elements (P). This bank is then used to infect mice. Survivors of murine infection represent clones expressing hly in vivo. (C) Plating of clones onto blood agar plates is used to determine in vitro expression. (Adapted from Gahan, C.G.M. and Hill, C., Mol. Microbiol., 36, 498, 2000.)
This bank was used to infect macrophages which were then sorted based on fluorescence intensity using a fluorescence-activated cell sorter (FACS). Clones of interest were fluorescence positive during infection but negative in laboratory media. Using this system eight members of the PhoP-PhoQ regulon were identified, as well as a gene encoded on SPI2 (Valdivia and Falkow, 1997). A benefit of this approach
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is that the GFP fusion strains can subsequently be used to analyze the kinetics of gene expression in response to environmental stimuli. Green fluorescent protein technology has been applied to L. monocytogenes to study the sequential expression of various virulence factors during host cell infection (Bubert et al., 1999; Freitag and Jacobs, 1999). Recently, the approach has been used to detect other genes induced during residence in macrophages (Wilson et al., 2001). A number of ivi genes were identified by the screening procedure, including a mannose phosphotransferase system, a xylose repressor and a hemolysin-like protein (yhdP). Deletion of the yhdP gene resulted in a virulence defect, suggesting a role for this locus in pathogenesis (Wilson et al., 2001).
SIGNATURE-TAGGED MUTAGENESIS Signature-tagged mutagenesis involves the creation of a transposon bank in which each inserted transposon is marked with a unique DNA sequence tag. This allows identification of transposon mutants in the bank which fail to survive mouse infection (Hensel et al., 1995). The system results in the detection of genes that are absolutely required for infection, as opposed to genes that are simply expressed in vivo, and thereby isolates ready-made mutants which can then be subjected to further analysis. This system resulted in the discovery of SPI2 and its type III secretion system (Shea et al., 1996; Hensel et al., 1997). Signature-tagged transposon mutagenesis has recently been applied to L. monocytogenes (Autret et al., 2001). The study identified ten distinct loci essential for murine infection, including genes involved in cell wall decoration, a transcriptional regulator and membrane proteins.
CONCLUSIONS It is becoming increasingly evident that, while some gene products can be classified as “true” virulence factors (those encoding toxins or invasins, for example), there exists a large class of proteins involved in stress management strategies which are necessary if a bacterium is to mount a successful infection. These “stress” proteins may be absolutely required or may only play a minor role in virulence, but collectively they are a necessary part of the arsenal of pathogenic bacteria. The dissection of the role of each protein within the complex orchestration of overlapping regulons is difficult, much more so than for the “true” virulence factors, and represents a significant challenge to researchers. The advent of elegant and imaginative techniques for detecting genes expressed in vivo, allied to the completion of entire genome sequences, offers the possibility that a more complete understanding of the relationship between stress and virulence is within reach. In particular, the use of gene chip technology and proteomics can be expected to reveal more of the strategies employed by pathogenic bacteria to overcome host defenses. A thorough understanding of these strategies can be expected to lead to the development of more effective control measures for the food industry, and in preventing or interrupting colonization of susceptible hosts.
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ACKNOWLEDGMENTS The authors wish to thank the Health Research Board (Ireland), The Irish Department of Agriculture, Food and Forestry, and BioResearch Ireland.
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Haas, A, Brehm, K., Kreft, J., and Goebel, W. 1991. Cloning, characterisation, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases, J. Bacteriol. 173:5159–5167. Hall, H.K. and Foster, J.W. 1996. The role of Fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically seperable from its role in iron acquisition, J. Bacteriol. 178:5683–5691. Hanawa, T., Yamamoto, T., and Kamiya, S. 1995. Listeria monocytogenes can grow in macrophages without the aid of proteins induced by environmental stresses, Infect. Immun. 63:4595–4599. Hanawa, T., Fukuda, M., Kawakami, H., Hirano, H., Kamiya, S., and Yamamoto, T. 1999. The Listeria monocytogenes DnaK chaperone is required for stress tolerance and efficient phagocytosis with macrophages, Cell Stress Chaperones 4:118–128. Hardt, W-F., Chen, L-M., Schuebel, K.E., Bustelo, X.R., and Galán, J.E. 1998. S. typhimurium encodes an activator of rho GTPases that induces membrane ruffling and nuclear responses in host cells, Cell 93:815–826. Hausladen, A., Privalle, C.T., Keng, T., DeAngelo, J., and Stamler, J.S. 1996. Nitrosative stress: activation of the transcription factor OxyR, Cell 86:719–729. Hecker, M., Schumann, W., and Völker, U. 1996. Heat-shock and general stress response in Bacillus subtilis, Mol. Microbiol. 19:417–428. Heithoff, D.M., Conner, C.P., Hanna, P.C., Julio, S.M., Hentschel, U., and Mahan, M.J. 1997. Bacterial infection as assessed by in vivo gene expression, Proc. Natl. Acad. Sci. USA 94:934–939. Heithoff, D.M., Conner, C.P., Hentschel, U., Govantes, F., Hanna, P.C., and Mahan, M.J. 1999. Coordinate intracellular expression of Salmonella genes induced during infection, J. Bacteriol. 181, 799–807. Hensel, M., Shea, J.E., Gleeson, C., Jones, M.D., Dalton, E., and Holden, D.W. 1995. Simultaneous identification of bacterial virulence genes by negative selection, Science 269:400–403. Hensel, M., Shea, J.E., Bäumler, A.J., Gleeson, C., Blattner, F., and Holden, D.W. 1997. Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12, J. Bacteriol. 179:1105–1111. Herbert, K.C. and Foster, S.J. 2001.Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components, Microbiology 147:2275–2284. Hess, J., Dietrich, G., Gentschev, I., Miko, D., Goebel, W., and Kaufmann, S.H. 1997. Protection against murine listeriosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the naturally somatic antigen superoxide dismutase, Infect. Immun. 65:1286–1292. Hevin, B., Morange, M., and Fauve, R.M. 1993. Absence of an early detectable increase in heat-shock protein synthesis by Listeria monocytogenes within mouse mononuclear phagocytes, Res. Microbiol. 144, 679–689. Hiratsu, K., Amemura, M., Nashimoto, H., Shinagawa, H., and Makino, K. 1995. The rpoE gene of Escherichia coli, which encodes σE, is essential for bacterial growth at high temperature, J. Bacteriol. 177:2918–2922. Humphrey, T.J., Williams, A., McAlpine, K., Lever, M.S., Guard-Petter, J., and Cox, J.M. 1996. Isolates of Salmonella enterica Enteritidis PT4 with enhanced heat and acid tolerance are more virulent in mice and more invasive in chickens, Epidemiol. Infect. 177: 79–88. Humphrey, T.J., Williams, A., McAlpine, K., Jorgensen, F., and O’Byrne, C. 1998. Pathogenicity in isolates of Salmonella enterica serotype Enteritidis PT4 which differ in RpoS expression: effects of growth phase and low temperature, Epidemiol. Infect. 121: 295–301.
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Mahan, M.J., Tobias, J.W., Slauch, J.M., Hanna, P.C., Collier, R.J., and Mekalanos, J.J. 1995. Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host, Proc. Natl. Acad. Sci. USA 92:669–673. Mahan, M.J., Slauch, J.M., and Mekalanos, J.J. 1996. Environmental regulation of virulence gene expression in Escherichia, Salmonella and Shigella spp., in Escherichia coli and Salmonella: Cellular and Molecular Biology, F.C. Neidhardt, R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger, Eds. Washington, D.C.: ASM Press, pp. 1075–1090. Marquis, H., Bouwer, H.G., Hinrichs, D.J., and Portnoy, D.A. 1993. Intracytoplasmic growth and virulence of Listeria monocytogenes auxotrophic mutants, Infect. Immun. 61:3756–3760. Marron, L., Emerson, N., Gahan, C.G.M., and Hill, C. 1997. A mutant of Listeria monocytogenes LO28 unable to induce an acid tolerance response displays diminished virulence in a murine model, Appl. Environ. Microbiol. 63, 4945–4947. Merrell, D.S. and Camilli, A. 1999. The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance, Mol. Microbiol. 34:836–849. Michan, C., Manchado, M., Dorado, G., and Pueyo, C. 1999. In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress, J. Bacteriol. 181:2759–2764. Milenbachs, A.A., Brown, D.P., Moors, M., and Youngman, P. 1997. Carbon source regulation of virulence gene expression in Listeria monocytogenes, Mol. Microbiol. 23:1075–1085. Miller, S.I., Kukral, A.M., and Mekalanos, J.J. 1989. A two-component regulatory system (phoP and phoQ) controls Salmonella typhimurium virulence, Proc. Natl. Acad. Sci. USA 86:5054–5058. Miller, S.I. and Mekalanos, J.J. 1990. Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages, J. Bacteriol. 172:2485–2490. Mills, S.D., Ruschowski, S.R., Stein, M.A., and Finlay, B.B. 1998. Trafficking of porindeficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments, Infect. Immun. 66:1806–1811. Milohanic, E., Pron, B., the European Listeria Genome Consortium, Berche, P., and Gaillard, J.L. 2000. Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells, Microbiology 146:731–739. Morgan, R.W., Christman, M.F., Jacobson, F.S., Sturz, G., and Ames, B. 1986. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins, Proc. Natl. Acad. Sci. USA 83:8059–8063. Nair, S., Frehel, C., Nguyen, L., Escuyer, I., and Berche, P. 1999. ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes, Mol. Microbiol. 31:185–196. Nair, S., Derre, I., Msadek, T., Gaillot, O., and Berche, P. 2000. CtsR controls class III heatshock gene expression in the human pathogen Listeria monocytogenes, Mol. Microbiol. 35:800–811. Nickerson, C.A. and Curtis, R. III. 1997. Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection, Infect. Immun. 65:1814–1823. Noone, D., Howell, A., and Devine, K.M. 2000. Expression of ykdA, encoding a Bacillus subtilis homologue of HtrA, is heat shock inducible and negatively autoregulated, J. Bacteriol. 182:1592–1599. O’Driscoll, B., Gahan, C.G.M., and Hill, C. 1996. Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid tolerant mutant which displays increased virulence, Appl. Environ. Microbiol. 62, 1693–1698.
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8
Physiology and Molecular Basis of Stress Adaptation, with Particular Reference to the Subversion of Stress Adaptation, and to the Involvement of Extracellular Components in Adaptation Robin J. Rowbury
CONTENTS Introduction Stresses Likely to Be Encountered by Bacteria in Food Preparation and Likely Responses Stresses Important in Food, Food Processing and Preparation, and in Cooking Stress Due to External Acidity Stress Due to Internal Acidity Heat Stress Cold Stress Osmotic Stress and Salt Stress Irradiation Stress Starvation Stress Enhancing Effects of Metabolites on the Levels of Lethality of Some Stresses Lethal Sites Affected by Stresses Gradual Build-Up of Stressing Agents and Relevance to Stress Tolerance in Foods Factors Influencing Stress Tolerance The Basis for Enhanced Inherent Stress Tolerance Growth Phase and Stress Tolerance The author’s research on the role of ESC/EIC pairs in stress tolerance induction is funded by the Royal Society, and he would like to express his thanks for this support.
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Filamentation as a Factor in Survival and as a Major Potential Problem in Food Microbiology Induced Tolerance to Stress Cross Tolerance and Sensitization Responses Methods of Studying Mechanisms of Inducible Stress Tolerance Studies of Proteins Synthesized de novo on Response Induction or in Increased Levels Isolation of Mutants Altered in Stress Tolerance Examination of the Role of Established Cellular Components in Tolerance Induction Studying Involvement of Regulatory Components Involvement of Specific Enzymes and Metabolites in Response Induction Predicting the Likely Components and Stages Involved in Stress Responses Stages in the Induction of Stress Responses Switching-On of Stress Responses The Likely Occurrence of Both Intracellular and Extracellular Stress Sensors for Some Responses Stress Sensors: Nature and Location Some Stress Responses Probably Have Intracellular Sensors Responses to Specific Stresses Responses to Acidity and Acid Tolerance Responses Induced by Other Conditions Inducible Acid Tolerance Responses Induced by Mild Acidity Mutants Altered in Acid Habituation Dissecting the Early Stages of the Process An Extracellular Acidity Sensor Formed at Neutral pH Stationary-Phase Acid Tolerance Responses Acid Tolerance Induced by Weak Acids at Neutral pH Acid Tolerance Induced by Amino Acids, Sugars and Salts at Neutral pH Heat-Induced Acid Tolerance Other Responses Which Affect the Level of Acid Tolerance Alkali Sensitization at Acidic pH Responses to Alkalinity Heat Tolerance Induced by an Alkaline Shift Responses to Heat in Food Preparation and Cooking On the Nature of the Thermal Sensor Ribosome Membrane Components The Medium DNA DnaK Chaperone
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Activation of the Thermotolerance ESC by Other Stresses On the Factors Governing the Heat-Shock Response and Its Possible Relation to Thermotolerance On the Involvement of Chaperones in Regulation Responses to Cold Sensing of Cold in Bacteria Proteins Synthesized after Triggering of the Cold-Shock Response by Sensor Activation Responses Induced by Changes in Osmotic Pressure of Media Do Both Intracellular and Extracellular Osmosensors Occur? Responses to Salt Stress Sensing of Salt Stress Regulation of Salt-Induced Responses Regulation of Responses to Irradiation Sensing of Irradiation Stress Stages in the Switching-On of Responses to Irradiation Following Sensor Activation Starvation Stress Stress Tolerance Induction by Killed Cultures Acid Tolerance Induction by Killed Cultures Other Stress Responses Induced by Killed Cultures Biochemical Changes Leading to Stress Tolerance Role of Chaperones in the Biochemistry of Stress Tolerance Biochemical Changes in Habituated Organisms Making Them Acid-Tolerant Biochemical and Physiological Changes Making Organisms Inducibly Alkali-Tolerant Biochemical Changes Leading to Heat Tolerance in Phenotypically Thermotolerant Organisms Physiological and Biochemical Changes in Organisms Inducibly Tolerant to Cold Osmotic Stress Tolerance: Physiological and Biochemical Changes Involved in Inducible Responses Biochemistry of Tolerance to Salt Biochemical Changes Following Starvation Counteracting the Induction of Stress Tolerance Responses Subverting Stress Responses Using Metabolites Subverting Other Stress Responses Alkali Tolerance Induction Acid-Induced Alkali Sensitization Cu2+-Induced Thermotolerance Extracellular Alarmones and Cell-to-Cell Communication Future Experiments and Conclusions Glossary References
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INTRODUCTION This chapter will first briefly list and review stresses likely to be faced by contaminating organisms in food, and in food production and preparation processes. Secondly, it will discuss the likely tolerances that can arise when organisms are exposed to those stresses. Thirdly, it considers the mechanisms involved in the induction of stress responses and, finally, the biochemical bases for the tolerances outlined.
STRESSES LIKELY TO BE ENCOUNTERED BY BACTERIA IN FOOD PREPARATION AND LIKELY RESPONSES The ability of contaminating organisms to survive in foods and food production and preparation procedures, during cooking and in domestic situations related to food, depends on whether or not such organisms show tolerance towards the significant number of stresses found in these situations. Table 8.1 shows the major stresses likely to be faced by contaminating bacteria in foods or in other situations where the exposed organisms may subsequently enter foods; with respect to all stresses, inherent tolerance levels will be important but the major factor governing survival will be whether or not inducible responses are put in place. Accordingly, Table 8.1 also shows responses likely to be induced by stress exposures.
STRESSES IMPORTANT AND IN COOKING
IN
FOOD, FOOD PROCESSING
AND
PREPARATION,
The stresses to be considered initially here are as follows: Stress Due to External Acidity Exposure to acidic pH is of major importance in food microbiology because contaminating organisms commonly face low external pH in many acidic or acidulated foods and at some stages in food production. Organisms may also face low pH in the aquatic environment and this will be significant for survival in food, if contaminated water from such acidified environments is subsequently used in food production or in the domestic environment. Stress Due to Internal Acidity Very commonly, organic acids are present in foods and often these foods are acidic in pH also. In this case, the stress is due both to external acidity and to the internal acidity arising because organic acids frequently collapse ∆pH (Salmond et al., 1984). Heat Stress Heat is not only involved in cooking but in numerous stages in food production also. It should be noted that cooking can involve a wide range of temperatures, especially with respect to the interior of the food, which will kill all organisms, down to those which will act merely to induce thermotolerance.
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TABLE 8.1 Stresses in the Environment, in Foods, in Food Preparation and Cooking and Responses to Such Stresses
Stress Acidity
Relevance to Food
Site of Stress Damage
Occurs in aquatic DNA, OM environment* and in food production
Acid pH + In acidic or acidulated weak acids foods Weak acids In certain foods at pH 7. 0 Alkalinity In egg-white
DNA, OM, and CM OM, CM DNA, OM, and CM
Stress Responses, Cross Tolerances Responses or Gene Products Induced on Stress Exposures Acid tolerance1; alkali sensitivity, UV resistance, salt tolerance, thermotolerance, H2O2 tolerance, resistance to polymixin B, induction of Hyd genes2, of RpoS, PhoP, HSPs and lysine3 or arginine decarboxylase, Acid tolerance1, induction of Fur, AhpC and HSPs Acid tolerance1 Alkali tolerance, thermotolerance, resistance to UV, acid sensitivity, AHP tolerance, AhpC, HSP and NhaA induction Thermotolerance; acid tolerance,1 alkali tolerance, UV tolerance, induction of HSPs Induction of CSPs Osmo-tolerance, tolerance to oxidative components, thermotolerance, induction of HSPs, ProP, ProU etc Acid sensitization, NhaA induction, PhoE induction
Heat
During food production, DNA, OM, preparation, and cooking ribosomes
Cold Osmotic stress
During refrigeration In many foods with high levels of sugars or salt
RNA OM, transport processes
Salt shock
Foods containing high [NaCl]
Irradiation
In foods irradiated for preservation
Starvation5
In contaminated waters, if used for food processing
Effects on enzyme activity and on protein synthesis DNA UV-tolerance, thermotolerance, acid tolerance4 alkali tolerance, induction of HSPs, induction of UvrA, B, C, RecA, LexA Proteins6 Thermotolerance, tolerance to acid, alkali, salt and H2O2, osmotolerance, induction of proteases, HSPs DNA, proteins Cu2+-tolerance, thermotolerance tolerance to acid, alkali and Cd2+
Exposure to In contaminated waters Cu2+
* Relevant to food if contaminated water is used in production or preparation. HSPs = heat shock proteins. AHP = alkylhydroperoxide. 1 Tolerance to inorganic acid. 2 Anaerobiosis and formate also needed. 3 Lysine and anaerobiosis needed. 4 Irradiation induces acid tolerance and UV can activate an acidity ESC. 5 For carbon. 6 A major effect of starvation is degradation of many proteins.
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TABLE 8.1 Stresses in the Environment, in Foods, in Food Preparation and Cooking and Responses to Such Stresses
Stress
Relevance to Food
Site of Stress Damage
Stress Responses, Cross Tolerances Responses or Gene Products Induced on Stress Exposures
Cold Stress There are also several types of cold that can be faced by contaminating organisms, from exposure to relatively mildly cold temperatures inducing acclimatization processes, down to conditions needing the formation of a whole range of cold-induced proteins to survive freezing and thawing. Osmotic Stress and Salt Stress Osmotic stress occurs in many foods due to the presence of very high concentrations of sugars or salts. It should be noted that at lower NaCl concentrations, which have no major osmotic effect, there can be a specific salt stress. Irradiation Stress This results from the use of some forms of irradiation, such as those used for food preservation; irradiation is also, on occasions, used to sterilize foods such as shellfish. Starvation Stress Organisms starved in natural waters can gain stress tolerance; if such organisms subsequently enter foods, they may resist stresses because they have induced crossprotection responses.
ENHANCING EFFECTS OF SOME STRESSES
OF
METABOLITES
ON THE
LEVELS
OF
LETHALITY
Frequently, if the effects of potentially lethal agents or conditions are incomplete, i.e., if a proportion of organisms survive these effects, then normally harmless metabolites or other molecules may have an enhancing effect on lethality, and if added with the stress, may greatly reduce or abolish survival of contaminating organisms. Studies on this effect have been made, particularly on cultures exposed to sublethal doses of heat stress, since reducing heating can in many situations improve the organoleptic properties of foods. First, bacteriocins like nisin and pediocin, although alone unable to kill organisms like E. coli and Salmonella spp, enhance killing by sublethal heat. The heating damages the outer membranes and allows the bacteriocin through to degrade the cytoplasmic membranes. Heat killing is also enhanced by polyphosphates, by cysteine and glutathione, by citrate and by
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some sugars (Doyle and Mazzotta, 2000). Interestingly, killing by heat can also be increased by low, normally sublethal, doses of other stresses, such as irradiation.
LETHAL SITES AFFECTED
BY
STRESSES
It is likely that there are at least four major kinds of damage to enteric bacteria like Escherichia coli and Salmonella spp caused by stresses and, strikingly, many of the stresses mentioned above cause several or all of these. Damage to DNA is almost certainly the most critical lethal effect of stresses and many chemical and physical stresses act primarily on this macromolecule, although they may damage other molecules or structures as well. Certainly, irradiation, thermal stress, stress by extreme acidity and alkalinity, and oxidative stress by, for example, hydrogen peroxide, all damage DNA, with this being the likely primary cause of death in each case. Second, the ribosome is often targeted by lethal agents, with many leading to inhibition of translation of m-RNA molecules by the ribosomes. Third, the outer membrane is damaged by heat, acidity, alkalinity and oxidative components, and even if the organisms are not killed by the damage, it allows other lethal agents (for example, nisin), which are normally unable to penetrate, to pass through to the cytoplasmic membrane and destroy the organism. Fourth, individual enzymes can be damaged by several stresses and, where these are essential, this can also be lethal.
GRADUAL BUILD-UP OF STRESSING AGENTS AND RELEVANCE TO STRESS TOLERANCE IN FOODS In many situations, levels of toxic chemicals vary depending on amounts entering the environment (Rowbury et al., 1989). There can be periodic rises in concentration, possibly to potentially lethal levels, followed by falls due to dilution by unpolluted medium, with such changes sometimes being repeated several times. The slow buildup of toxic stresses means that there will be times when levels are non-lethal, but induce tolerance responses which allow polluting or contaminating organisms to survive potentially lethal levels, which build up later. The locations where gradual build-ups of chemical stressing agents can occur include foods. In some foods, acidity builds up gradually, eventually leading to potentially lethal pH values. Because the pH falls gradually, organisms will habituate during early stages of H+ accumulation and, therefore, survive (Rowbury et al., 1989).
FACTORS INFLUENCING STRESS TOLERANCE There is a range of factors which influence stress tolerance. First, levels of inherent tolerance vary widely between natural isolates, e.g., in one study with S. enteritidis PT4, it was striking to observe that organisms isolated from clinical situations had higher levels of inherent tolerance to heat, acidity, oxidative stress and surface stress than non-clinical ones (Humphrey et al., 1995). Such increased tolerances probably make clinical isolates better able to resist host defenses, since the stress resistant isolates also show enhanced virulence (Humphrey et al., 1996).
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THE BASIS
FOR
ENHANCED INHERENT STRESS TOLERANCE
The differences in inherent stress tolerances between isolates depend, first, on RpoS differences. RpoS is a sigma factor and presumably allows induction of certain stressrelated components in tolerant (rpoS+) isolates which cannot form or form at lower levels in rpoS mutants. Second, envelope mutations alter acid tolerance (Bielicki et al., 1982), due to changes in proton penetration through the outer or cytoplasmic membranes (OMs or CMs). Since surface changes are known to occur during growth in vivo, these could lead to altered penetration of chemical stressing agents and changed stress tolerance.
TABLE 8.2 Resistance of Free and Attached Escherichia coli to Chemical and Biological Inhibitory Agents % Survival (or % Growth*) after Incubation of Attached or Free Organisms with Inhibitor
Inhibitory Agent (concentration, where applicable)
Attached Organisms
Free Organisms
Inorganic acid (pH 2.5)* Trans-cinnamic acid (30 mM)* Lactic acid (30 mM)* Sorbic acid (30 mM)* Citric acid (30 mM)* Propionic acid (30 mM)* Phage T4 Colicin V Acrylate (1 mgml–1) Chlorine (11 µgml–1)* H2O2 (42 mM)* Cu2+ (5 µgml–1)
82.0 75.9 95.5 117.0 81.5 100.0 50.9 74.0 87.1 94.9 65.4 90.3
0.0 0.8 7.5 17.7 36.4 69.4 0.1 4.0 0.04 3.8 6.0 0.5
Results are given for single representative experiments but each was repeated with consistent results. * Organisms were incubated with the inhibitor and, after removal of organisms from the surface, if required, and removal of inhibitor, growth in pH 7.0 broth followed, with results being compared to those of the control without inhibitor. Weak acids were tested at pH 3.5; at this pH without weak acid, there were only slight effects on either free organisms (9.8% inhibition of subsequent growth) or attached organisms (0.4% inhibition). Treatments with inorganic acid and weak acids were on E. coli strain P678-54ColV; all other treatments were with E. coli strain 1829ColV except that colicin V-sensitivity was tested with the sensitive strain P678-54. Some of these results are from the Ph.D. thesis of G.C. Whiting (University of London, 1990).
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Another factor affecting inherent stress tolerance is the finding that some isolates but not others attach to surfaces. This often results from the presence of specific surface appendages e.g., pili. Strikingly, attached but not free E. coli are resistant to many agents relevant to survival in the environment, in foods and food preparationproduction, in domestic, hospital and commercial situations and in the body. Thus, on attachment, E. coli becomes resistant to Cl2, fatty acids, acidity and alkalinity, to acrylate, metal ions, detergents and antibiotics (Table 8.2). For these, levels of agents which kill most free organisms allow survival of most attached ones (Hicks and Rowbury, 1986). Several of the above agents are relevant to survival in foods or in food preparation or production. Attached bacteria in shellfish may resist chemical and physical agents, e.g., weak fatty acids, acrylate and irradiation, in natural waters and agents (e.g., chlorine and UV irradiation) used to decontaminate the product after harvesting. It is not clear how attachment protects, but it may be because such chemicals cannot penetrate surface layers. Of interest is the finding that attachment protects organisms from weak acids at acidic external pH, pHo (see Table 8.2), e.g., trans-cinnamate is lethal at pH 3.5 on free but not on attached organisms. Attached organisms are also less affected than free ones by biological agents. Thus, attachment allows organisms to tolerate both bacteriophages and bacteriocins (see Table 8.2); some bacteriocins appear to play a role in protecting certain foods from potentially pathogenic bacteria, so attachment of the latter might allow them to avoid being killed. Attachment may also enhance resistance to physical conditions; e.g., it has recently been shown that attachment can protect Salmonella spp from heat (Humphrey et al., 1997), with organisms attached to muscle tissue being protected; this clearly has major implications for food safety.
GROWTH PHASE
AND
STRESS TOLERANCE
Another factor playing a major role in inherent stress tolerance levels and likely to influence survival in foods is the growth stage of the contaminating organism. It is known that stress tolerance is generally at its lowest in midlog phase and increases in late log phase and in the stationary phase. There is no doubt that processes induced via RpoS play a major role in this stationary phase tolerance response. Published studies show that RpoS acts in two ways, first by inducing transcription of systems which lead to increased levels of components which protect from stress damage, e.g., molecules which protect proteins or DNA-binding components which protect DNA, i.e., there is primarily induction of damage limitation components. Second, RpoS is likely to be involved in induction of systems which repair damage to both proteins and DNA and possibly other molecules. Another factor must be emphasized. During entry into stationary phase, rounds of chromosome replication in progress continue but no new rounds begin because protein synthesis and mass increase are needed for initiation of replication (Donachie, 1968). Accordingly, after a short time in stationary phase, rounds of replication have terminated and there are no replication forks. In this situation, organisms tolerate many stresses which damage DNA by acting at replication forks. For example, starvation and entry into stationary phase lead to tolerance to heat, H2O2 (Jenkins et al., 1988), osmotic stress (Jenkins et al., 1990), irradiation (Row-
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bury, 1972), acid (Matin, 1991) and alkali. Each of these damages DNA and part of the stress tolerance in stationary phase, therefore, is likely to result from chromosome termination; this has been overlooked. It is easy to prove whether completion of rounds of replication per se leads to stress tolerance by comparing effects of passage into stationary phase (or of starvation) with effects of completion of rounds of replication without starvation. Such a situation occurs if a strain with temperature-sensitive DNA synthesis initiation is shifted to restrictive temperature (Rowbury, 1972). After the shift, organisms rapidly produce completed chromosomes. Analysis of stress tolerances in this situation would be of interest as to whether such organisms show enhanced tolerance coinciding with completion of rounds; they do show increased tolerance to UV irradiation (Rowbury, 1972). In view of the above, one cannot state confidently the basis for inherent stationary phase stress tolerance but the above two factors are likely to play a role.
FILAMENTATION AS A FACTOR IN SURVIVAL PROBLEM IN FOOD MICROBIOLOGY
AND AS A
MAJOR POTENTIAL
Another factor in stress tolerance has been emphasized by pioneering work of Humphrey and his group (Phillips et al., 1998; Mattick et al., 2000), who have found that two stresses, low aw and low temperature, lead to filament formation in contaminating organisms. These filaments frequently survive for long periods, and are able, given changed conditions, to fully divide rapidly. Only these two stresses have been shown to lead to filament formation so far, but others could do so. What is important is that both stresses can be significant in foods. It is striking that the filaments can, under suitable conditions, divide to give as many as 100 organisms per filament. For this reason, foods that appear, on the basis of cfu, to have as few as 1 organism mL–1, may, within a short period have one hundred times as many, if conditions have ensued that allow division. Accordingly, it is critical that foods are entirely free of viable organisms. It is striking that for the organisms exposed to cold, filament formation was much more marked for S. enteritidis isolate E (rpoS+) than for isolate I (rpoS); the fact that E is more pathogenic (Humphrey et al., 1996), means that the possibility of filaments in cold medium dividing to give many cells on warming up, will be highly significant. The effects of high osmolality on cell size are also of interest although there was no enhanced filamentation in rpoS+ strains, compared to rpoS ones.
INDUCED TOLERANCE
TO
STRESS
A third major factor affecting tolerance is whether organisms have induced any specific log-phase tolerances, i.e., tolerances against a specific stress induced by low levels of the same stress. Exposure of E. coli to micro-molar levels of hydrogen peroxide, for example, leads to tolerance to milli-molar H2O2 levels (Demple and Halbrook, 1983) and mild heat treatment induces tolerance to potentially lethal heat stress (Mackey and Derrick, 1986). Such tolerance inductions usually occur very rapidly and are generally dependent on de novo protein synthesis.
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CROSS TOLERANCE
AND
SENSITIZATION RESPONSES
Cross-tolerance or sensitization responses are ones (Tables 8.1 and 8.3) where expo-
TABLE 8.3 Some Characteristics of Cross Responses Leading to Increased Sensitivity or Tolerance to Stresses: Role of Extracellular Components and Regulatory Molecules Involvement of Extracellular Involvement Induction Regulatory and of Extracellular Component Inhibitors of (EIC) Other Components Sensor (ESC) Response
Inducer of Cross Response
Response Induced
pH 9.0
Acid H-NS sensitivity Acid H-NS, RelA1, sensitivity cAMP2, PhoE
Yes
Yes; two EICs?
KCl, Amil, Fe3+ NaCl N.T. Yes, dialyzable Glucose, non-protein FeCl3, EIC acetate L-leucine Acid RelA*, H-NS, Fur, N.T. Yes; small Glucose, Fe3+, sensitivity CysB, OmpA protein EIC Fe2+ NaCl, Nal, Tet pH 5.5 Alkali IHF, H-NS, Lrp, Fur, Yes, small heat- Yes, small heat- Nal, sensitivity CysB, NhaA stable protein stable protein phosphate, ESC EIC NaCl, Fe2+ Shift up to 45°C Acid H-NS Yes, protein ESC Yes, protein EIC Nal tolerance N.T. = not tested * Appreciable effect. 1 envZ lesions reverse effect of relA on this response. 2 Glucose represses this response but cAMP reverses this repression and allows induction. The responses shown here were induced by pH 9.0, NaCl 300mM, L-leucine 50 µgml–1, pH 5.5 and a temperature shift up respectively. Nal = nalidixic acid; amil = amiloride; Tet = tetracycline. The salt-induced acid sensitization response was only partially inhibited by chloramphenicol, rifampicin and tetracycline whereas the L-leucine-induced acid sensitization response was abolished by tetracycline but only slightly inhibited by chloramphenicol and rifampicin.
sure to one stress induces tolerance or sensitivity to another; several are relevant to survival of contaminating organisms in food. For example, mild heat shock induces acid tolerance (Humphrey et al., 1993) in both S. enteritidis and E. coli. This response could allow contaminating organisms, which had survived heating during food production or preparation, to pass through the stomach because of their heatinduced acid tolerance. Another major cross response is the heat tolerance induced by alkaline pH (Humphrey et al., 1991). Organisms from egg-white, which has an alkaline reaction, would, therefore, survive normally lethal heat shocks, allowing © 2003 by CRC Press LLC
such heated organisms to go on to cause disease. Shifts to acidic pH also induce cross responses, organisms becoming tolerant to heat (some strains), salt, oxidative components (Leyer and Johnson, 1993) and irradiation (Goodson and Rowbury, 1991). Accordingly, contaminating organisms exposed to acid might later resist irradiation, heat or salt in foods or salt and oxidative components in the body. One interesting response is thermotolerance induced by Cu2+. Organisms exposed to copper in natural waters and then entering foods or food components might survive cooking because of this process; Cu2+-exposed organisms also gain acid and alkali tolerance. Cross responses can also induce stress sensitivity, e.g., E. coli on growth at alkaline external pH or plus salt or L-leucine (see Table 8.3) becomes acid sensitive (Rowbury, 1997) while incubation at pH 5.5 induces alkali sensitization (Rowbury, 1997). As stated above, several stresses induce cross tolerance against potentially lethal heat. It has been shown, in this laboratory, that glycerol (2 M) strongly induces thermotolerance and smaller effects occur with phosphate (10 mM), FeCl3 (1 mM)and FeSO4 (1 mM).
METHODS OF STUDYING MECHANISMS OF INDUCIBLE STRESS TOLERANCE STUDIES OF PROTEINS SYNTHESIZED OR IN INCREASED LEVELS
DE NOVO ON
RESPONSE INDUCTION
The primary method of studying regulatory mechanisms and biochemical bases for inducible stress tolerance has been to examine and analyze labelled proteins immediately induced on exposure to the stress. This technique has led to useful findings but two major factors have been overlooked. First, it is unlikely that information can be obtained by this method about the most important aspect of the process, namely the switching-on of the response. This is because the sensors which detect the stress must be present in unstressed cells or they cannot detect it. Accordingly, the sensor is unlikely to show enhanced synthesis on exposure to the stress. It is, therefore, unlikely that studying proteins labelled at high levels on stress exposure will throw light on stages needed to switch on the response. It is not that regulatory proteins are not induced by stress, only that those involved in the initiating or switching-on stages are unlikely to be. Secondly, studies of labelled proteins have thrown light on many of the components involved in the biochemistry of stress tolerance, e.g., temperature up-shifts lead to the enhanced synthesis of several chaperones which are involved in repair of heat-damaged proteins. Nonetheless, many of the proteins showing enhanced synthesis may be red herrings as they appear not to be directly related to the tolerance response.
ISOLATION
OF
MUTANTS ALTERED
IN
STRESS TOLERANCE
Mutants totally unable to induce a response are likely to have lesions in regulation, because mutants altered in the biochemistry of tolerance will probably show loss of only one aspect of tolerance, and this would usually lead to organisms with reduced
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but not abolished tolerance. Thus, most mutants which have lost a response entirely are probably regulatory ones, and useful results throwing light on how responses are switched on have been obtained by studying such mutants; e.g., work on ompB mutants led to elucidation of the processes governing OmpF/OmpC synthesis regulation, while studies of oxyR mutants altered in oxidative responses (Kullick et al., 1995) involving peroxides, and of soxRS mutants involved in oxidative responses relating to superoxide, have thrown light on these responses. Similarly, studies of fur mutants, which appear to be altered in switching-on of some acid tolerance responses in Salmonella spp, suggest that Fur may, by analogy with its iron sensing role, act as a proton sensor (Foster and Moreno, 1999), detecting intracellular H+, just as an extracellular sensing component (ESC) detects extracellular H+ (Rowbury and Goodson, 1999a). Analysis of mutants with reduced tolerance induction can throw light on biochemical bases for tolerance. This is because when organisms lose one tolerance component, analysis can indicate the nature of individual components involved e.g., if a DNA repair enzyme is absent from a mutant with low stress tolerance, DNA damage is a likely effect of the stress, and repair of this damage a feature of tolerance.
EXAMINATION OF THE ROLE IN TOLERANCE INDUCTION
OF
ESTABLISHED CELLULAR COMPONENTS
Studying Involvement of Regulatory Components Another approach to studying the molecular biology of stress responses has been to examine whether a response or individual components of it are aberrant if specific regulatory gene products are absent, or if specific regulatory metabolites are added. One study involved examining induction of the L-lysine and L-arginine decarboxylases. Bennett and his group have shown that CysB and IHF are essential for AdiA synthesis while H-NS interferes with induction (Shi et al., 1993; Shi and Bennett, 1994). A similar approach establishes that only CysB (of a range of components) is needed for acid tolerance induction at pH 5.0, and that cyclic AMP interferes with such induction (Rowbury and Goodson, 1997). Involvement of Specific Enzymes and Metabolites in Response Induction Stress tolerance studies in strains altered in enzymes or components known to be involved in specific protection or repair can throw light on tolerance responses; e.g., RecA, PolA and UvrA gene products are involved in DNA repair and Sinha (1986) found that E. coli mutants altered in these genes are acid-sensitive. This, however, applies to inherent acid tolerance and the levels of inducible tolerance are unaffected by lesions in these genes (Goodson and Rowbury, 1991), suggesting that a novel DNA repair process appears in acid-habituated organisms.
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PREDICTING THE LIKELY COMPONENTS IN STRESS RESPONSES
AND
STAGES INVOLVED
This is a new approach for studying stress responses induced by extracellular stresses, which involves predicting likely components involved, their properties and how cultural conditions may influence their structures and functioning. The first work was by Nikolaev (1996, 1997a, b) who argued that organisms exposed to lethal extracellular chemicals might secrete “protectants” to neutralize or inactivate the stress and demonstrated agents which had these properties. More comprehensive studies came from a proposal (Rowbury and Goodson, 1998) that in the presence of toxic extracellular chemical agents, E. coli might produce extracellular induction components (EICs) which induce stress tolerance, EIC production acting as an early warning against stress. These EICs have been found to function for most stress responses where the stressing agent is extracellular. It was then proposed that EICs are not secreted on exposure to stress, but arise from extracellular sensing components (ESCs), already present in the medium (Rowbury and Goodson, 1999a). The proposal is that these ESCs are produced in the absence of stress, and are activated by stress (i.e., the ESC is a stress sensor). The proposal was made because presence of the sensor in the medium would allow an immediate response to stress. Recent studies confirm that many stress responses have ESC–EIC pairs functioning. To function efficiently, these agents need to diffuse from the site of formation to influence organisms nearby. These agents are, therefore, usually small. This predictive approach has also been used to propose how stress sensors might anticipate changes in stress levels. Another useful prediction relates to killed cultures. It has been found that, although ESCs and EICs are highly sensitive to reversible activation or inactivation by very low levels of stress, they are insensitive to irreversible inactivation. Because of this, another major prediction can be made, namely that killed cultures can confer stress tolerance and this has been shown to occur (see “Stress Tolerance Induction by Killed Cultures,” later in this chapter).
STAGES IN THE INDUCTION OF STRESS RESPONSES The stages in induction of a stress response can be defined as 1) interaction of the stress with the stimulus (stress) sensor, and 2) production of a signal or component from the above interaction, which can set in train a series of reactions which generally lead to increased transcription.
SWITCHING-ON
OF
STRESS RESPONSES
The first stage of induction involves activation by the stress (stimulus) of a specific sensor. Sensors are defined as components which are produced in the presence or absence of the stress, and which are activated by the stress to an induction component, which sets in train the initial stages of induction. To be certain that a particular component is the sensor, and that the induction component produced by activation
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of this sensor by the stress leads to response induction, several other conditions need to be fulfilled: 1. That agents or mutations which stop synthesis of the proposed sensor ESC or its conversion to EIC also stop induction of the response. 2. Agents or conditions which destroy or remove the sensor or induction component block induction. 3. Where more than one stressing agent produces the same response, each of the stressing agents should interact with the proposed sensor to produce the same induction component. 4. If the proposed sensor and component produced by its activation are indeed involved in induction of the response, then addition of the induction component should induce the response. Some other conditions may apply if the sensor and induction component are extracellular. Where the sensor is intracellular, the product of sensor activation produces an internal signal which leads to a series of internal reactions which culminate in increased transcription of stress response genes. Sensors for many inducible and repressible non stress-related processes have been known for many years to be integral CM proteins, and when studies of osmotic stress showed that a sensor which detects osmotic shock (Igo and Silhavy, 1988) is a CM, it was assumed that all stress sensors would be intracellular components. The location of such sensors has become of interest, however, because it is now known that many, if not most, are extracellular.
THE LIKELY OCCURRENCE OF BOTH INTRACELLULAR STRESS SENSORS FOR SOME RESPONSES
AND
EXTRACELLULAR
Where organisms are exposed to stresses, it appears to be the rule that where the stressing agent is initially in the medium, it is sensed by an ESC, whereas when it is produced internally (e.g., as for some toxic electrophiles, Ferguson, 1999), it is sensed by an intracellular sensor. If the stressing agent is present in the medium and also produced intracellularly, then dual sensors are likely. It is important for an organism to immediately detect a rise in the level of a stressing agent, since any delay could lead to lethal effects. For a primarily external agent, the sensor would need to be extracellular, so that there would be no delay while the agent penetrated to an intracellular site. Conversely, if the agent were to be formed or released in the cell, an extracellular sensor might not be activated with the needful response not occurring at all. Since many lethal agents occur in media and can also be formed in the cell, for many responses there may be dual sensors for a stress. Stress Sensors: Nature and Location It is now well established that several reponses induced by osmotic stress involve sensing by CM proteins. Although the above sensors are intracellular, it can be argued that where the stress is by a chemical agent, intracellular sensing may delay
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response induction if the agent is in the medium. Such delay may make theoretically resistable levels of the chemical stress lethal in practice. It seems likely that extracellular stress sensors would have evolved to prevent delayed responses to external toxic chemicals. The first extracellular sensing component (ESC) reported was the acidity sensor involved in induction of acid tolerance at pH 5.0. This sensor is found in medium filtrates from cultures grown under a range of conditions, and is converted to an extracellular induction component (EIC) on exposure to acidity. The EIC then induces acid tolerance. This ESC/EIC pair exemplify a large group of extracellular response induction pairs. It should be noted that the EIC is not formed by the cells; it arises by chemical activation of ESC at acidic pH, organisms not being needed.
SOME STRESS RESPONSES PROBABLY HAVE INTRACELLULAR SENSORS Some toxic chemicals are produced intracellularly. For these, it is important that the sensor is also intracellular. For agents which can be present in the medium and formed in the cell, e.g., H+, OH–, hydrogen peroxide and electrophiles, there may be both intracellular and extracellular sensors. There is some evidence for such dual sensing systems. Thus, as stated above, extracellular acidity is sensed by an extracellular sensor (Rowbury and Goodson, 1999a). There is, however, evidence that the Fur gene product acts as an intracellular acidity sensor. Firstly, fur mutants of S. typhimurium fail to induce some acid habituation responses (Foster and Hall, 1992). The evidence that protons interact with Fur to switch on some responses is indirect but compelling. Thus, ferric ions are known to interact directly with Fur to switch off some responses to low iron, and various mutants are refractory to this Fe3+ effect. Foster and colleagues have now established (Hall and Foster, 1996; Foster and Moreno, 1999) that one class of mutants, unable to bind and respond to ferric iron, also fails to induce some responses by H+. Foster calls this group “iron-blind” and suggests that they fail to bind both H+ and Fe3+. Dual (intracellular and extracellular) sensing may also apply to hydrogen peroxide and other peroxides. Thus, OxyR is an intracellular component activated by peroxides (Kullick et al., 1995); it presumably has evolved to detect intracellularly produced peroxides, and after activation induces transcription. Recent studies have established that H2O2 oxidizes OxyR to form an intramolecular bond between Cys199 and Cys208 of this protein. This is both the sensing mechanism and the activating one, the OxyR form with the intramolecular bond enhancing transcription of the oxyR regulon. Peroxides can also be produced extracellularly and detection may then involve an ESC, since a non-protein extracellular sensor is known to be involved in alkylhydroperoxide tolerance induction (details are given below) and an ESC/EIC pair is used for tolerance induction by extracellular H2O2 (Rowbury, 2001). Almost certainly, the mode of O2– sensing will involve the functioning of the intracellular SoxR gene product, and detection of NO involves an intracellular sensor, if these lethal agents are produced intracellularly, although whether extracellular O2– or NO (e.g., from the phagolysosome) activates ESCs is not known.
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RESPONSES TO SPECIFIC STRESSES Many reviews have considered induction of stress responses, but where the switching-on of the process and the stages in induction have been considered, there has often been a failure to consider satisfactorily how the stress is sensed, and how activation of the sensor switches on the response. This section will redress the balance, with the main emphasis being on sensing of chemical and physical stresses.
RESPONSES TO ACIDITY AND ACID TOLERANCE RESPONSES INDUCED BY OTHER CONDITIONS There is a very wide range of conditions which lead to acid tolerance induction; these and several other responses related to the extent of acid tolerance will be considered here. Inducible Acid Tolerance Responses Induced by Mild Acidity On a shift to acidic pH, Escherichia coli and Salmonella spp gain acid tolerance (become acid habituated). One approach to the response has been to look for acid shock proteins (ASPs) induced at acidic pH, and attempt to establish how their synthesis is regulated and their identity and the basis for their synthesis. Many ASPs have been found (>50 in S. typhimurium) and some progress has been made in identification. The problem has been to understand why specific proteins are induced by acidity. For several, it is not possible to understand the value that induction of particular proteins has for acid-induced bacteria. Studies of regulation have been interesting even if most identified regulated proteins have no obvious relevance. Thus, in Salmonella typhimurium, synthesis of a group of eight proteins is regulated by RpoS — four also being controlled by Fur and four by PhoPQ (Foster and Moreno, 1999). It is likely that Fur has an acidity-sensing role. Obviously, if Fur does have such a sensing role, this would apply to internal sensing whereas, when enteric bacteria first detect acidity in the medium, the sensor is an extracellular protein ESC (Rowbury and Goodson, 1999a). If Fur acts as an intracellular sensor, it is likely that the proton-activated form would be a positive regulator of synthesis of one class of ASPs; since some fur mutants are acid-sensitive, it can be assumed that some of this group of ASPs (or other unidentified components) are essential for some aspects of acid tolerance, either acting as regulatory components governing acid tolerance component synthesis, or actually functioning in the tolerance processes themselves. Another set of ASPs is controlled by PhoPQ. One of these, namely ASP29, is PhoP itself; i.e., PhoP, like the acid-tolerance regulator RpoS, is acid-induced. However, whereas RpoS functions in tolerance to both organic and inorganic acids, the PhoPQ system is only involved in inorganic acid tolerance. Foster proposes that the PhoQ component senses acid (presumably protons), thus inducing PhoP; certainly PhoP-LacZ can be induced by high proton concentrations, so possibly PhoQ senses H+. In view of the role of ESCs in early warning against acidity (Rowbury and Goodson, 1999a), one must note that sensing of protons here relates to internal H+.
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As indicated above, mildly acidic pH, which induces an rpoS-dependent acid tolerance, also induces RpoS synthesis. It is proposed that an activated protease, in the absence of stress, degrades RpoS rapidly. In contrast, during stress exposure, the activator loses its ability to enhance protease and RpoS becomes more stable. Mutants Altered in Acid Habituation Another approach to studying habituation has been to attempt to isolate mutants altered in acid tolerance. These could be altered in response induction or in the components which actually make the cells tolerant. Studies have also been made of how mutations in known major regulatory components affect acid habituation. Dissecting the Early Stages of the Process One approach to studying induction of acid habituation has been to look for ASPs. The problems with this approach have already been indicated, namely that sensors and most other regulatory components might not be detected by this approach, as these components would all have to be present in unstressed cells, or the response could not be switched on. The above applies whether the sensor is extracellular or intracellular or whether regulation involves functioning of two components or more; if they are needed to switch on the response, all would need to be present when the stress appears, i.e., would need to have been present under non-stressing conditions. In view of the above, another approach has been to consider what are likely to be the early stages of acid tolerance induction, and look for components which appear likely to occur. We opined that, since most acidic challenges to bacteria are due to external acidity, it was likely that responses would be more rapid if early regulatory intermediates were in the medium and interacted there with organisms to produce tolerance. We therefore looked for components in media from acidified cultures, which were essential for tolerance of such cultures and induced acid tolerance in non-stressing conditions. Filtrates from neutralized pH 5.0-grown cultures contained such a component. Evidencing this were two initial findings: 1) procedures that removed or destroyed ECs, e.g., continuous filtration or protease treatment, prevented tolerance induction at pH 5.0, and 2) neutralized filtrate from pH 5.0 cultures induced acid tolerance in organisms at pH 7.0. The latter filtrate was inactivated if ECs were removed or if proteins in it were destroyed by protease (filtrate is not inactivated by RNase or DNase) or by incubation in a boiling water bath, but not at 75°C (Rowbury and Goodson, 1998). Other studies suggest that this extracellular induction component (EIC) is a fairly small protein. An Extracellular Acidity Sensor Formed at Neutral pH Accordingly, neutralized filtrates from pH 5.0-grown cultures contain an EIC, but, as stated earlier, if the acidity-detecting system is to function rapidly, there ought to be an extracellular acidity sensor, able to detect acidity and converted by it to an EIC. There is indeed such an extracellular sensing component (ESC), and it is present in culture filtrates from organisms grown at neutral or alkaline pH. The ESC can be removed from such filtrates, if the filtrate-containing vessel is immersed in a reservoir containing a large volume of the same medium without organisms, with the filtrate vessel being separated from the medium reservoir by a 0.2 µm pore filter. Because
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of the large volume in the reservoir, the ESC passes from the filtrate vessel, through the membrane, into the surrounding medium. This ESC is synthesized at pH values from 4.5 to 9.0 and is secreted to the medium. The properties and nature of the ESC synthesized at pH 4.5 to 6.0 are not readily studied as such sensor is immediately activated (chemically or enzymically) to EIC, leading to induction of tolerance. In contrast, the ESCs formed at pH values from 6.5 to 9.0 can be easily examined. Incubation of ESC-containing filtrates with several proteases and with RNase or DNase, establishes the protein nature of the sensor, while its ability to resist exposure to 75°C (but not to 100°C) establishes its heat stability (Table 8.4). This protein passes through 30 K nominal molecular weight
TABLE 8.4 Induction of Acid Tolerance by Acidity and at Neutral pH by Amino Acids, Salts and Other Components; Involvement of Extracellular Sensing Components and Extracellular Induction Components Extracellular Components and Switching-On of Acid Tolerance by Inducer
Acid Tolerance Inducer pH 4.5 to 6.0 L-glutamate L-aspartate L-proline L-glutamine Glucose Glucosamine FeCl3 KCl NH4Cl Glycerol
Involvement of ESC
Involvement of EIC
Yes; heat-stable non-dialyzable protein ESC Yes; protein ESC senses L-glutamate Yes; protein ESC senses L-aspartate Yes; non-protein ESC senses L-proline N.T. Yes, ESC senses glucose* N.T. Yes; ESC senses Fe3+ N.T. N.T. N.T.
Yes; heat-stable non-dialyzable protein EIC Yes; non-dialyzable protein EIC Yes; non-dialyzable protein EIC Yes; non-dialyzable, non-protein EIC Yes; non-protein EIC Yes, small (ca. 10 kda) EIC* N.T. Yes; non-protein EIC Yes, small (ca. 10 kda) non-protein EIC Yes, dialyzable non-protein EIC N.T.
N.T. = not tested. * Probably protein components.
(NMW) membranes but is retained by 5 NMW membranes and is not removed from filtrates by dialysis. This sensor formed at pH 7.0 is, therefore (like the EIC which it gives rise to), a rather small heat-stable protein. Incubation of the sensor formed at pH 7.0 under acidic conditions, i.e., at pH 4.5 to 6.0 (but not at 2.0) converts it to the EIC; organisms are not required for this conversion, but if the activated filtrate is neutralized and pH 7.0-grown organisms added, the EIC induces them to acid tolerance rapidly at pH 7.0, i.e., the EIC can induce acid tolerance in unstressed organisms. Thus, the ESC is converted in the medium at acidic pH to EIC. One possibility is that this is a chemical activation. The alternative is that acidity unmasks an auto-enzyme
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activity in the ESC which converts it to the EIC. The ESC can be activated by other stresses. Of particular interest is the finding (Rowbury and Goodson, 1999b) that transfer of ESC-containing filtrates from low temperature to 42 to 55°C also activates the ESC to EIC. This probably explains why organisms grown at >37°C are more acid-tolerant than those grown at 25 to 37°C (Humphrey et al., 1993). Stationary-Phase Acid Tolerance Responses Stationary-phase organisms are more acid tolerant than log-phase ones, and at least three tolerance responses are induced in stationary phase, with induction generally requiring acidification. The so-called oxidative system appears during aerobic growth to stationary phase, pH 5.5 being needed for induction. This is glucoserepressed and generally needs Cya and Crp; it also requires glutamate or glutamine for activation, these functioning by a protein synthesis-independent mechanism. If organisms grow without these acids, this system is non-functional, but brief exposure to glutamate or glutamine without protein synthesis activates the system, i.e., all components are formed in the absence of glutamate/glutamine but one component needs activation by one of these. How organisms induced for this system are protected from acid is not known (Castanie-Cornet et al., 1999). The other processes are fermentative with functioning of amino acid decarboxylases during acid challenge; one needs arginine (Arg) during challenge (but not added to induction media) and is AdiA+-dependent, the other needs L-glutamate during challenge, but not added during induction. The Arg-dependent system is RpoS-independent and an rpoS lesion has only a small effect on the glutamatedependent one (Castanie-Cornet et al., 1999). The AdiA-dependent process evidently uses Arg during challenge to produce agmatine, which is transported out of the cell in protonated form, keeping pHi from falling. This system is dependent on CysB, as expected, since AdiA synthesis needs this component. The final system needs glutamate during challenge; it is assumed that γ-aminobutyric acid (GABA) produced by decarboxylation leads to pHi rise on passage of protonated GABA to the outside. This system is absent from gadC mutants, since these cannot transport GABA out of the cell. Other mutant studies show that either of the glutamate decarboxylase isoforms (two are present, encoded by gadA and gadB) can function to produce GABA. Little is known of how the above are switched on. Presence of weak fatty acids in stationary-phase cultures suggests that intracellular sensors and induction components might be involved. Two processes need decarboxylases, however; by analogy with decarboxylase-dependent glutamate-induced acid tolerance (see below), some or all may show involvement of ESCs and EICs. Acid Tolerance Induced by Weak Acids at Neutral pH E. coli normally fails to show acid tolerance induction at pH values greater than 6.0 unless metabolites are present. Guilfoyle and Hirshfield (1996), however, induced tolerance at pH 6.5 in the presence of butyric and propionic acids, while recently Kwon and Ricke (1998) induced tolerance in S. typhimurium grown at neutral pH
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on addition of propionate. The latter induction process was protein synthesis-dependent and was enhanced in anaerobic conditions. No studies have been made on sensing of the weak acids but since EICs specific for such a process could give early warning of exposure to lethal weak acid concentrations, it is likely that an ESC detects the weak acids, and that its conversion to an EIC leads to acid tolerance induction. Acid Tolerance Induced by Amino Acids, Sugars and Salts at Neutral pH Three amino acids induce such tolerance without any pH change during induction. Induction by L-glutamate, L-aspartate and L-proline requires EICs (Rowbury, 1999). The EICs for the first two are proteins whereas that for the L-proline response is not. For each, an ESC present in media from cells grown without inducer senses inducer and is activated by it (Rowbury and Goodson, 1999b) to give an EIC (see Table 8.4); ESC closely resembles EIC in properties but cannot induce the response. A few other amino acids also induce acid tolerance at pH 7.0. Glucose induces acid tolerance at pH 7.0 in E. coli (see Table 8.4). On incubation of organisms with pH 7.0 broth, tolerance appears on addition of glucose, with no fall in pH, and an EIC (able to convert organisms in pH 7.0 broth to acid tolerance) is formed in the medium. This EIC is a protein and arises from an extracellular sensing component (ESC) which is activated by glucose (Rowbury and Goodson, 1999b). Such tolerance induction by glucose (it occurs with other sugars also) has probably evolved because medium acidification results from glucose degradation, and the response protects organisms from anticipated acidity. Several salts induce acid tolerance at pH 7.0 in E. coli (see Table 8.4). In each case, an EIC appears in media during induction, and the EIC (in filtrates dialyzed to remove the salt) induces tolerance in organisms in broth at pH 7.0. For the one salt tested further, FeCl3, EIC is formed by interaction of an ESC with Fe3+ (Table 8.4 and Rowbury and Goodson, 1999b). Heat-Induced Acid Tolerance E. coli and Salmonella spp transferred from low temperatures to, e.g., 45°C become more acid-tolerant by a protein synthesis-dependent process (Humphrey et al., 1993). These findings are of medical and applied importance since contaminating organisms in food which have survived cooking could, on ingestion, resist gastric acidity and go on to cause disease. The histone-like regulatory component H-NS may be involved in the control of this response since hns mutant organisms are acid-tolerant after growth at 25°C (Rowbury, 1997). Such acid tolerance arises on exposure of cultures to elevated temperatures, because the acid tolerance-related ESC is activated not only by acidity, but also by elevated temperatures. One germane finding is that, although salt normally reduces acid tolerance when added to media, organisms grown at low temperature and shifted to 44°C become much more acid-tolerant if salt is present in the medium. Thus organisms surviving in partially cooked salty foods might, on ingestion, survive gastric acidity (Rowbury, 1997).
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Other Responses Which Affect the Level of Acid Tolerance There are three responses which reduce acid tolerance. It is known that H+ passage across the OM is impeded and that some OM lesions cause acid sensitivity due to enhanced H+ penetration into the periplasm (Bielecki et al., 1982), while introduction or modification of certain OM pores directly or indirectly increases OM penetration of protons and acid sensitivity. This is true for two responses considered here and probably for a third. Acid sensitization induced by salt involves induction (Table 8.5)
TABLE 8.5 Conditions and Responses Inducing Acid Sensitization; Do These Result from Porin Derepression?
Response, Condition or Mutation Leading to Acid Sensitivity Phosphate starvation Mutation in phoS Mutation in phoR Mutation in phoT Introduction of F′128 into phoE mutant Growth at alkaline pH Growth with NaCl Growth with L-leucine
Is a Porin or Other OMP Derepressed or Modified by Response, Mutation or Culture Condition? Yes, Yes, Yes, Yes, Yes,
PhoE PhoE PhoE PhoE PhoE appears
None shown so far Yes, PhoE OmpA protein modified
Is Porin or OMP Change Responsible Directly or Indirectly for Acid Sensitivity? Yes, sensitization lost in phoE mutant Yes, sensitization lost in phoS, phoE strain Not proven so far Not proven so far Yes, loss of F′128 leads to loss of PhoE and acid resistance N.A. Yes, sensitization abolished by phoE lesion Yes, sensitization abolished by ompA lesion1
N.A. = not applicable. 1
ompA deletions and certain point mutations abolish response. (See “responses to specific stresses,” in this chapter, and Rowbury, R.J., Lett. Appl. Microbiol., 24, 319, 1997.)
of the PhoE OM pore (see “Regulation of Salt-Induced Responses” later in this chapter) and response induction involves an EIC (see Table 8.3). In addition, transfer of organisms to pH 9.0 induces sensitization (acid sensitivity induction, ASI), a process independent of alkali habituation which also occurs at this pH. No OM pore induction has been linked to this response, but one probably occurs. This response has two parts, one protein synthesis-dependent, the other protein synthesis-independent. Both sensitization components are switched on by EICs. It is likely that the first component of ASI is switched on by a protein EIC, the second by a non-protein EIC (Rowbury, 1999). The third sensitization response is switched on by L-leucine; an EIC is involved, although an ESC has not been tested for. The later stages of induction are not well studied, but several regulatory components are needed (see Table 8.3) and ompA deletion mutants lack this response; some ompA point mutants have the response, others do not. The finding that only loss of
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an acidic amino acid from a surface loop of the OmpA protein abolishes the response suggests that leucine modifies OmpAp to give it pore activity (Rowbury, 1999), thus directly or indirectly allowing H+ to cross the OM (see Table 8.5). Cells exposed to leucine become less sensitive to phage K3 (receptor is OmpAp), in accord with leucine altering OmpAp surface properties (Rowbury, 1997, 1999). Alkali Sensitization at Acidic pH Organisms transferred from pH 7.0 to pH 5.5-6.0 become alkali-sensitive by a process distinct from the acid tolerance response. Induction depends (Tables 8.3 and
TABLE 8.6 Regulatory Components and Extracellular Components Involved in Alkali Habituation at pH 9.0 and in Alkali Sensitization at pH 5.5 Regulatory or Induction Component
Needed for Alkali Tolerance Induction
Needed for Alkali Sensitization
PhoE NhaA NhaB Fur IHF H-NS CysB RelA TonB Extracellular sensing component (ESC) Extracellular induction component (EIC)
Yes Yes No Yes Yes No Yes No Yes Yes, possibly 2 ESCs Yes, possibly 2 EICs
Yes Yes Yes Yes1 Yes Yes Yes No No2 Yes, dialyzable heat-stable protein Yes, dialyzable heat-stable protein
1 2
fur mutants have reduced response tonB mutants are constitutively alkali-sensitive
8.6) on the functioning of regulatory components, antiporters and PhoE (Rowbury, 1997). Sensitization only occurs if an EIC is present (Rowbury, 1999). This EIC derives from an ESC which is synthesized at a range of pH values and activated to EIC at acidic pH in the absence of organisms. Formation of this ESC needs H-NS, IHF and Fur; but involvement of other components has not been tested. The EIC and ESC are very heat-stable proteins or peptides (survive exposure to boiling water for 15 min) of less than 5000 Da.
RESPONSES
TO
ALKALINITY
Switching-on of inducible alkali tolerance (alkali habituation) involves functioning of ECs. Thus, a sterile cell-free filtrate from a pH 9.0-grown culture can, after neutralization, induce alkali tolerance in organisms at pH 7.0, whereas a filtrate from a pH 7.0-grown culture cannot. Accordingly, the pH 9.0 culture contains an alkali-
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tolerance-inducing EIC. Although the pH 7.0 filtrate is ineffective in inducing the response, transfer to pH 9.0 in the absence of organisms rapidly activates it. This pH 7.0 → pH 9.0 process converts an alkali tolerance ESC to the corresponding EIC and the ESC behaves as an alkali sensor. The filtrates containing either EIC or ESC are only partially inactivated by proteases; possibly there are two EICs and two ESCs, one of each being a protein, the other being a non-protein component (see Table 8.6). Although it is not clear how the EIC induces the response, there is some information on which regulatory components function; NhaA, IHF, TonB and Fur (Rowbury, 1997) are needed, whereas NhaB and H-NS are not (see Table 8.6). These studies were with NaOH as inducer, but KOH was almost as effective in inducing alkali habituation. The alkali sensor above is an ESC. In contrast, Padan et al., (1999) consider NhaA an alkali sensor because its activity is greatly increased at alkaline pH. Heat Tolerance Induced by an Alkaline Shift In pioneering studies, Humphrey et al. (1991) showed that a shift from neutral to alkaline pH induces thermotolerance in S. enteritidis PT4. Accordingly, organisms grown in the alkaline egg-white will be thermo-tolerant and this will allow survival on cooking. It is essential to examine the regulation of this response, especially its switching-on. It is now known that alkaline pH functions by activating the thermotolerance-related ESC; exposure of this component to pH 9.0 at 30°C, in the absence of organisms, converts this ESC to the corresponding EIC, with concomitant thermotolerance induction. Activation can also occur at other alkaline pH values.
RESPONSES
TO
HEAT
IN
FOOD PREPARATION
AND
COOKING
When considering the survival of contaminating organisms during stresses likely to be encountered in food production, preparation or cooking, heat is likely to be the major stress challenge, and numerous studies have been made on the influence of prior exposure conditions on survival during heating, and on the effects that a wide range of components present during heating, have on survival (Doyle and Mazzotta, 2000). In contrast, until now, little has been known of how exposure to heat switcheson processes likely to aid survival. Clearly, survival of contaminating organisms in partially cooked foods will result partly from their ability to induce thermotolerance on temperature up-shift, with tolerant organisms surviving exposure to 50 to 55°C, and some resisting higher temperatures for short periods. It is important to know how thermotolerance is induced, and what biochemical changes occur as a result of induction, which make the organisms thermotolerant, especially as knowledge of the process could make it possible to subvert it. There are two major matters to be considered with respect to response of organisms to increased temperature: 1) the role of responses in allowing organisms to grow and multiply better at high temperatures, and 2) the role of responses in preventing killing by potentially lethal temperatures, i.e., their role in thermotolerance.
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It is essential first to establish how increased temperature is sensed in enterobacteria. Two approaches have been taken to establishing the sensor. First, the possible ways in which organisms change their major metabolic pathways at higher temperatures have been considered, as well as the way in which a sensor could function to ensure that a switch-over could be rapidly achieved. This has led to the proposal that the ribosome senses temperature. In contrast, when considering damage by heat and how this can be avoided, the approach has been to consider as likely sensors components damaged by heat; if these are altered in some way by the damage so as to lead to a response, this could give early warning of potential lethality, and allow organisms to prepare to avoid death. The major components damaged by heat appear to be membranes, DNA and ribosomes, so proposals on heat sensing for thermotolerance induction have usually suggested involvement of one of these components. On the Nature of the Thermal Sensor Five components have been proposed to function as the thermal sensor. Ribosome Van Bogelen and Neidhardt (1990) suggested that the ribosome senses temperature and switches on the heat-shock response. This proposal was based on the finding that so-called H (heat-shock response) antibiotics (e.g., kanamycin), which lead to empty ribosomal A sites, induce a heat-shock response. They argued that at high temperatures, there may initially be a fall in charged t-RNA, leading to some or all A-sites being empty; this will indirectly induce the heat-shock response and H antibiotics mimic this. It was proposed that the so-called H ribosomal state may induce the response by causing accumulation of (p)ppGpp, which is known to be associated with effects of H antibiotics and with high temperatures. Since organisms must change protein synthesis rates after temperature shift-up, the ribosome would appear to be an ideal thermal sensor. The above is rather indirect evidence, so we must ask whether the ribosome fits the criteria for stress sensors and whether the agent resulting from interaction of stress and proposed sensor ((p)ppGpp) fits the criteria for a heat-shock induction component. First, obviously ribosomes are present in both heat-stressed and unstressed cells. Second, sudden temperature rises do affect the ribosome but do they lead to empty A-sites? Whether they do or not, there is indeed a rise in (p)ppGpp levels as temperature increases (Pao and Dyess, 1981). Finally, does (p)ppGpp induce the heat-shock response? As to other criteria where the proposed sensor is an essential component, one cannot achieve inhibitory conditions, or mutations which stop sensor synthesis or conditions which remove or destroy it. It is, however, possible to inhibit chemically (or mutationally block) synthesis of (p)ppGpp; does this stop heat-shock response induction on thermal stress and does addition of (p)ppGpp at low temperatures induce? Additionally, where several stresses produce a response, each stress should interact with the proposed sensor to produce the same induction component and so one must ask whether ethanol exposures, alkaline shifts and amino acid analogue exposures (all these induce the response) lead to a fall in charged t-RNA levels, to
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empty A-sites and high (p)ppGpp levels, and whether lesions that stop (p)ppGpp synthesis stop response-inducing effects of ethanol, and the other stresses mentioned above. Results relating to some of the above questions support functioning of (p)ppGpp, whereas others do not. Membrane Components Secondly, it is possible that a membrane component acts as thermal sensor. I will mention two findings here. The first is the finding that thermal sensing for thermotaxis involves activation of a CM component (Nishiyama et al., 1999); although this response does not involve altered transcription and induction of a new pathway, it does involve thermal activation of a CM component, and similar interactions could have evolved to give response induction. The second is the finding that induction/repression of the OmpF/OmpC proteins, in response to altered osmolality, involves sensing of stress by a CM component, namely EnvZ (activation of this protein leading to altered auto-phosphorylation) and these changes affect the phosphorylation and activity of the DNA-binding protein OmpR. Clearly, therefore, a CM component can act as a stress sensor and switchon stress responses. The Medium Recently, there has been evidence suggesting that thermal sensing takes place in the medium. There would appear to be no advantage in having a sensor in the medium because if temperature increases, the inside of the cell and the outside will be at the same temperature. In fact, however, a thermal ESC can allow early warning of a likely heat shock. This is because an EIC arising from an ESC can diffuse to regions not yet facing raised temperature, and unstressed organisms can be given early warning and be prepared to face thermal damage; i.e., there would be “cross-talk” involving EICs leading to intercellular communication between heat-stressed and unstressed organisms. Several evidences now suggest that ECs function in thermal sensing. First, a thermotolerance-inducing EIC is present in filtrates from cultures grown at 45°C. This EIC, which induces thermotolerance at 25 or 30°C, is cleaved by protease, but passes only poorly through dialysis membranes, implying that the EIC is a protein of ca. 10,000 Da (Rowbury and Goodson, 2001). This EIC is now known to arise from an ESC, which has similar properties to the EIC, but cannot induce thermotolerance in unstressed cells, unless first activated to the EIC (Rowbury and Goodson, 2001). It is now clearly established that this ESC functions as a biological thermometer, detecting temperature rise and inducing thermotolerance. I believe that activation of this ESC may induce the heat-shock response also, and propose to examine synthesis of HSPs at 25 or 30°C in the presence of the corresponding EIC, following the induction of β-galactosidase and alkaline phosphatase from HSP–LacZ and HSP–PhoA fusions. A second finding relates to heat-induced acid tolerance. It is well proven that on transfer of E. coli or S. enteritidis from 25, 30 or 37°C to 42 or 45°C, acid tolerance induction occurs (Humphrey et al., 1993; Rowbury, 1997). The basis for this has now been established. Growth at 37°C produces an acid-sensing ESC. Although this ESC normally senses and is activated by acid, it can also be activated to the acid tolerance-inducing EIC at 40, 42, 45, 50 or 55°C, i.e., this ESC is a © 2003 by CRC Press LLC
thermal sensor as well as an acidity sensor (Rowbury and Goodson, 1999b), ESC activation leading to formation of an acid tolerance-inducing EIC. The extent of activation of the ESC increases with increasing temperature at least within the range 37 to 55°C and, accordingly, this component behaves like a thermometer, at least over a limited temperature range. The temperature for thermal activation of this sensor depends on the temperature during ESC synthesis; the ESC formed at 25°C is activated to EIC at 30 or 37°C, whereas that synthesized at 37°C is not; i.e., this sensing component occurs in more than one form, the form synthesized depending on the culture conditions. These different forms could be oligomers of the EIC that arises from them, or the EIC and various forms of the ESC could simply differ from each other in conformation. A third ESC can act as a thermometer, as the alkali tolerance ESC also shows gradual activation as the temperature is raised from 37°C to 50°C. DNA Some have considered DNA as a likely thermal sensor. Heat damages the DNA and so, by analogy with RecA/SS DNA as SOS sensor, an SS region (or some other exposed region) deriving from heat-damaged DNA could act with a cellular component in sensing; a similar lesion could also be detected following other stresses, since many of them damage DNA. An alternative is that damage alters the conformation of the DNA, releasing bound regulatory components and derepressing operons transcriptionally blocked by them. It is striking that H-NS represses many stress responses by binding to their operons. If the binding of H-NS to these operons were particularly weak, damaging stress treatments might lead to general stress response induction by altering DNA conformation in such a way as to release H-NS only from operons to which it is poorly bound. DnaK Chaperone A very exciting suggestion is that the HSP70 DnaK chaperone acts as a thermal sensor, detecting either rises in temperature or changes in protein structure which occur at high temperature. For example, McCarty and Walker (1991) suggested that DnaK can rather precisely sense increases in temperature in the range 30 to 53°C. This proposal was based on the finding that both the ATPase activity of DnaK and its autophosphorylation at threo-199 are massively enhanced in the temperature range responsible for induction of the heat shock response and thermotolerance. At low pH values, autophosphorylation was enhanced by several hundred-fold and ATPase activity by nearly two orders of magnitude. DnaK needs rapid ATPase functioning to efficiently repair damaged proteins and co-chaperones function to bring this about; the enhancement of DnaK activities at high temperatures could clearly also play a role. These authors also suggest that, since binding of inactive DnaK to σ32 reduces both its stability and activity, that activation (of the ATPase activity) of DnaK might lead to its release from σ32 and enhanced stability and activity of this σ-factor, leading to the heat-shock response. On this basis, DnaK would be a biological thermometer. It is also possible that DnaK might act indirectly as a thermometer by detecting thermal changes by their results, i.e., by detecting damage to proteins.
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Activation of the Thermotolerance ESC by Other Stresses As indicated above, thermotolerance is generally induced by temperature shifts, and induction follows the activation of a thermotolerance ESC to the corresponding EIC, which switches on the response. There are, however, several cross-tolerance responses, which switch on thermotolerance, and induction by alkalinity (Humphrey et al., 1991) or acidity (Leyer and Johnson, 1993) is well-known. It has now been shown, in my laboratory, that UV irradiation and exposure to metal ions, such as Cu2+, also induce thermotolerance, and that for all four stresses (exposures to acid, alkali, UV irradiation and Cu2+), induction follows activation of the thermotolerance ESC (by the stress) to the EIC. On the Factors Governing the Heat-Shock Response and Its Possible Relation to Thermotolerance The initial aim is to give an account of how the heat-shock response is switched on in E. coli. This response is not only switched on by thermal stress, but also appears after exposure of bacteria to ethanol, amino acid analogues and alkali. Some believe that this response does not relate to inducible thermotolerance, i.e., to responses that lead to survival in the face of potentially lethal thermal stress. This seems highly unlikely and I propose to outline how the major heat-shock response is induced,
TABLE 8.7 Induction of Stress Responses and Chaperone Synthesis Inducible Stress Response
Stimulus
Thermotolerance
Temperature up-shock1
Heat-shock response
Temperature up-shock1
Cold-shock response Acid shock response Weak acid shock Alkali shock response Osmotic shock response Starvation response Oxidative stress response SOS response or other response to DNA damage
Temperature down-shock Low pH, generally pH 2.0-5.5 Weak acid at pH 6.5 or 8.0 High pH (pH 8.5-9.0) High osmotic pressure Starvation for carbon compounds3 H2O2, O2– or AHP DNA damage, e.g., by irradiation
1
Chaperones Synthesised DnaJ, DnaK, GrpE, GroEL, GroES, HtpG DnaJ, DnaK, GrpE, GroEL, GroES, HtpG Hsc66, HscB, CsdA, CspA2 GroEL, DnaK, HtpG, HtpM GroEL, GroES, DnaK, GrpE, HtpG GroEL, DnaK GroEL, GroES, DnaK GroEL, GroES, GrpE, DnaK, HtpG GroEL, GroES, DnaK GroEL, DnaK, GroES?
The heat-shock response involves the de novo synthesis of several novel proteins and the increased synthesis of other proteins, which is induced by mild heat shocks (e.g., shift to 40°C) and by exposure to agents such as ethanol and nalidixic acid. The organisms sometimes, but not always, become thermotolerant. To induce thermotolerance, exposure is to higher temperatures (generally 45 to 50°C) and a novel second heat-shock response induced by σE generally occurs, in addition to the classical heat-shock response. 2 CspA and CsdA may act as RNA chaperones. 3 Other forms of starvation response can occur.
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with the production of a large group of heat-shock proteins (HSPs). The HSPs are components present at low levels in unstressed cells; amounts increase very rapidly, but often transiently, after a temperature shift up, maximum rates of synthesis being achieved within 5 min. Several HSPs are molecular chaperones (Table 8.7), which are also induced by other stresses (Table 8.8). Chaperones mediate proper assembly/folding of proteins during synthesis, as well as protecting proteins from damage and aiding repair during heat-shock. Where heat-shock temperature is potentially lethal, nearly all protein synthesis ceases on transfer, virtually all proteins synthesized after such transfer being HSPs. This behavior at lethal temperatures suggests HSP involvement in thermotolerance. The major response after a temperature shift up involves the initial activity of RpoH. This gene product is present in very low amounts in non heat-stressing conditions but on transfer, e.g., from 30 to 42°C, amounts increase within a few seconds and rise to a maximum within 5 min, followed by a rather sharp fall, i.e., the rise in RpoH is transient. The amount of RpoH rises for two reasons. The major reason lies in its stability/instability properties. Under non-stressed conditions, this component has a half-life of about 60 sec, whereas it becomes stabilized on transfer to 42°C, and this allows a rapid rise in its level. In addition, rate of synthesis of RpoH increases at 42°C and this also relates to a stabilization process. In this case, m-RNA for RpoH is stabilized at 42°C, allowing increasing RpoH formation, i.e., increased synthesis depends on increased m-RNA translation. It is RpoH (σ32) that induces synthesis of the major HSPs since a nonsense mutation in rpoH leads to markedly reduced induction of HSPs at, e.g., 42°C, and the strain with this mutation fails to grow at >20°C. This σ-factor binds to the RNA polymerase core enzyme and is needed for recognition of heat shock promoters, i.e., the RNA polymerase only binds to these if associated with σ32 (Grossman et al., 1984). HSPs induced by RpoH include the DnaJ, DnaK, GrpE, GroEL, GroES, Lon, ClpB, ClpP and HtpG gene products. Many of these are chaperones, e.g., Dna J, Dna K, GrpE, GroEL, GroES and HtpG, and are needed for numerous processes because of their role in protein processing and folding, and in protection against heat inactivation and in repair. A few minutes after the rise in the concentration of RpoH has occurred, its level falls off again. This appears to be due to the functioning of chaperones in regulation of RpoH stability and rpoH m-RNA stability. As indicated above, some researchers believe that the heat-shock response induced by σ32 is not involved in appearance of inducible thermotolerance. This proposal has been made because, if σ32 induction occurs at low temperatures, thermotolerance does not appear. One suggestion is that there is a second heat-shock response, induced at elevated temperatures, which is responsible for the thermotolerance response. There is the possibility that this response is σE-induced. This sigma factor induces transcription from the rpoH P3 promoter and also leads to transcription of other genes; this sigma factor appears to be particularly functional at lethal temperatures, in accord with its being involved in survival at such temperatures, rather than just being needed for accommodation. My view is that both regulons (i.e., that induced by σ32 and that induced by σE) are needed for induction of thermotolerance and resistance to potentially lethal temperatures, such as may occur during food production and preparation procedures
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TABLE 8.8 Components Involved in Induction of Responses, Especially Tolerance Responses to Heat Stress, Cold Stress, and to Other Selected Stresses and Components Involved in the Biochemistry of Stress Tolerance
Response
Sensor
Thermotolerance1 ESC Heat-shock response Cold-shock response
Ribosome? ESC? DNA, DnaK Ribosome? ESC? Membrane
Osmotic tolerance KdpD, ProP, ESC?3
Regulatory Components Involved EIC, RpoE, RpoH? RpoH, EIC? CspA, H-NS, RecA*
KdpE, cAMP, H-NS, RpoS
Salt shock responses Irradiation tolerance
NhaA, NhaR, ESC?5
NhaR, H-NS
RecA, ESC
RecA, LexA
Starvation stress for carbon
1. ESC?
RpoS, H-NS,
2. ESC?
cAMP-CAP AlgAC
Are Regulatory Components Stabilized or Destabilized, and Mechanism of These Changes RpoH destabilized by GrpE? GroEL? GroES? GrpE, GroEL, GroES destabilize RpoH 1. m-RNAs are stabilized 2. m-RNA translation is enhanced by DB enhancer Stress may stabilize RpoS by stopping proteolytic cleavage4 NhaR plus Na+ may stabilize NhaA m-RNA6 LexA becomes stabilized due to reversible loss of protease activity Stress stabilizes RpoS by protease deactivation
Enzymes and Other Proteins Involved in Biochemistry of Stress Tolerance Chaperones? IbpA, IbpB, OtsA,B2 Chaperones, Lon, Clp H-NS, GyrA, CspA, CsdA, RecA*
Kdp-ATPase, ProP, ProU, OtsA,B, TreA NhaA RecA, UvrA, B, C
ClpB, OtsAB, Dps, Catalase HPII
1
See Table 8.7 footnote for details of thermotolerance induction. OtsA and B function in stationary-phase thermotolerance, and may be involved in heat-induced thermotolerance, although this has been discounted by some. 3Also see Table 8.9. 4RpoS is needed, e.g., for osmotic induction of OtsA and B; there is evidence that some stresses prevent proteolytic breakdown of RpoS. 5An EIC functions in salt-induced acid sensitivity (see Table 8.3) and, therefore, an ESC probably acts as NaCl sensor. 6Suggested as an unlikely possibility by Dover, N. et al., J. Bacteriol., 178, 6508, 1996; for pex mutants and for cst mutants. * RecA may have a regulatory function, as well as simply functioning in repair. 2
and in cooking at low temperatures. This possibility is of particular interest, as is the possibility that different thermal sensors function to switch on the two heatshock responses. In addition, however, there is strong evidence for the involvement of trehalose in some thermotolerance responses and, accordingly, the heat-induced
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thermotolerance response could be governed by the sensor and regulatory components which control trehalose accumulation. However, there have been claims that trehalose is not needed for heat-induced thermotolerance. My view is that, since the heat-induced thermotolerance response will need repair processes as well as protection processes (trehalose may, if it functions at all, play a role in the latter), there is likely to be involvement of σ32 and σE-governed processes in this response. The role of H-NS in thermotolerance induction ought to be mentioned again here. There is no doubt that a lesion in hns leads to derepression of the thermotolerance response, i.e., to thermotolerance in organisms grown at low temperatures. It seems highly likely that there are groups of genes needed for thermotolerance and that some of these are repressed by H-NS binding. Presumably, H-NS is ejected from these regions at high temperatures, leading to thermotolerance induction, and absence of H-NS in hns mutants gives the same phenotype at low temperature. Mutants in himA show a similar phenotype, suggesting that IHF plays a role with H-NS in repressing the thermotolerance response. Sigma 32 is not essential for growth at low temperatures, since the rpoH deletion mutant can grow at below 20°C but not above. Some products controlled by RpoH are needed for growth at below 20°C, e.g., some chaperones, but there is presumably some transcription at their promoters, catalyzed by RNA polymerase activated by another sigma factor. On the Involvement of Chaperones in Regulation Chaperones synthesized in the presence of RpoH mainly function in normal protein assembly, folding and processing and in repair (Tables 8.7 and 8.8). Several, however, also play a major part in governing synthesis and stability of RpoH and, accordingly, are important in regulating the heat-shock response, including their own synthesis. Thus, DnaJ, DnaK and GrpE gene products destabilize RpoH by enhancing activity of proteases which degrade it, e.g., mutations in these chaperone-encoding genes lead to RpoH stabilization and, on a temperature shift up, HSP synthesis for an extended period. These chaperones also appear to inhibit RpoH activity under some conditions.
RESPONSES
TO
COLD
Contaminating organisms in food face three types of cold stress. First, at low temperature they need to acclimate to cold and there are a group of proteins involved in acclimatization. Secondly, transfer to low temperatures can involve a sudden drop in temperature, termed a cold shock. Finally, polluting organisms can also be exposed to freezing conditions and, for survivors, to thawing during recovery. Substantial studies have been made of cold shock recently, although the work has rarely involved consideration of the applied importance of findings, and experimental design has not generally borne in mind questions related to cold shock in food microbiology. There is evidence that the responses which aid growth and recovery after exposure to each of the above three processes are related and, in particular, that failure to
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form a regulator, CspB, required for induction of cold-shock proteins, leads to sensitivity to freezing/thawing in Bacillus (Graumann and Marahiel, 1996). Sensing of Cold in Bacteria First, one must ask how low temperature is sensed. One consideration is how organisms accommodate to reduced temperature; there are changes in protein synthesis with reduced translation, and since it is deficiencies in ribosomal function that reduce growth rate in the cold (Das and Goldstein, 1968), it has been proposed that ribosomes detect falling temperature. Studies of antibiotic effects on protein synthesis show that there are C (cold-shock) antibiotics which induce a cold-shock response (van Bogelen and Neidhardt, 1990). These agents block the ribosomal A-site, e.g., one C antibiotic, chloramphenicol, inhibits peptidyl transferase and the accumulating charged t-RNA blocks the A-site. It is proposed that a down-shift leads to reduced translation and associated blockage of the A-site, which induces the coldshock response, whereas making the A-site empty leads to the heat-shock response, so this ribosomal theory explains responses at high and low temperatures. The blocking of the A-site in the cold plus C antibiotics leads to a fall in (p)ppGpp. Evidence for the ribosome as cold sensor is better than for its functioning as heat sensor. First, the proposed sensor is present under stressed (cold-shock) and unstressed conditions. Second, interaction of cold with ribosomes leads to a fall in (p)ppGpp (Pao and Dyess, 1981) and third, a fall in (p)ppGpp (the proposed inducing condition) switches on cold-induced protein (CIP) synthesis; conversely, a rise in (p)ppGpp leads to reduced synthesis of CIPs following cold shock (Jones et al., 1992). In summary, the evidence appears quite good for the ribosome as cold sensor. A second possibility for a cold sensor, responsible for inducing the cold-shock response, is a membrane component; there is no evidence so far for this, although the work of Nishiyama et al. (1999) on thermotaxis shows that a CM protein can act as a cold sensor. A third possibility, as melting of DNA becomes a problem at low temperatures, is that an altered DNA configuration could be involved in sensing cold, or that low temperatures might lead to regulatory components being dislodged from some areas of DNA, with associated operon derepression. There is no evidence for this so far. In view of the involvement of ECs in thermotolerance, it seems likely that such components could function during temperature down-shifts. Although internal and external temperatures will be the same when there is a temperature down-shift, to have an extracellular cold sensor could lead to early warning of stress for unstressed cells. This is because the EIC, which would arise by cold activation of the proposed cold-sensing ESC, could diffuse away from the cold region and interact with unstressed cells before they face cold shock. This could be investigated as follows. The proposal would be to down-shift E. coli, prepare a cell-free filtrate from the down-shifted culture, expose organisms to the filtrate at 37°C and examine whether major cold-induced proteins appear at this temperature. Use could be made of fusions, of cold-induced genes to lacZ, so that induction by the EIC could be followed by studying β-galactosidase levels. If evidence were obtained for a cold-shock EIC, a study could be made of whether this EIC arises in the cold from an ESC formed
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at normal temperatures, and whether these ECs are involved in protection from freezing/thawing. Proteins Synthesized after Triggering of the Cold-Shock Response by Sensor Activation On cold shock to 10°C, growth stops in E. coli and synthesis of most cellular proteins is abolished. Some time after, synthesis of a group of some 20+ proteins begins, with one of the earliest appearing being the low MW CspA (Goldstein et al., 1990). In addition, regulatory proteins are induced, the most significant being H-NS and GyrA. Cold shock is distinct from heat shock, and not only are HSPs not induced on a transfer from 37 to 10°C, but their levels also fall substantially at the lower temperature. This applies, for example, to DnaK and GroE gene products, so there are tiny concentrations of the heat-shock chaperones present at 10°C. Components involved in protein folding and damage repair are, however, formed at low temperatures, since specific cold-shock chaperones probably occur; e.g., Hsc66 appears to be a classic Hsp70 homologue, induced at low temperatures in E. coli but not formed during heat shock (Lelivelt and Kawula, 1995). It is probably a chaperone and a DnaJ homologue, HscB, also appears. In addition, the CspA and CsdA proteins may function as RNA chaperones under some conditions. E. coli, on cold shock, shows very great induction of a group of small acidic proteins. Of these, CspA is the first and most markedly induced (Goldstein et al., 1990), forming as much as 10% of protein synthesized at 10°C. There may be no cold-shock sigma factor and for some CSPs, at least, derepression involves stabilization of m-RNA (e.g., for CspA) and increased translation of m-RNA (Etchegaray and Inouye, 1999) due to presence of a downstream box enhancer (for CspA and B). Strikingly, neither chloramphenicol nor kanamycin appreciably inhibits the synthesis of CspA, CspB or CspG at low temperatures and the only proteins synthesized in the presence of these antibiotics are the above three cold-shock proteins (Etchegaray and Inouye, 1999). All three are of very low molecular weight (70, 71 and 70 amino acids, respectively, for CspA, B and G), and it is possible that ribosomal translation of m-RNA for such small proteins is less affected by inhibitory antibiotics. The alternative is that some stress-related proteins are synthesized by a slightly modified synthetic pathway, since the synthesis process for the ESC for acid tolerance induction is also refractory to antibiotics that normally block ribosomal function (Rowbury and Goodson, 1999a).
RESPONSES INDUCED
BY
CHANGES
IN
OSMOTIC PRESSURE
OF
MEDIA
Whereas there may be multiple thermal sensors, there definitely are multiple osmosensors. As illustration, three responses will be considered. First, E. coli grown at low osmolality produces high levels of OmpF porin and low levels of OmpC, whereas growth in richer media leads to the reverse. These Omp proteins function in the OM to ensure passage across the OM of low (up to ca. 600 Da) molecular weight uncharged or positively charged hydrophilic molecules.
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It has been proposed that shift from high OmpF/low OmpC to low OmpF/high OmpC as the osmotic pressure is raised has evolved to ensure that the porin with the smaller pore size is predominant in rich medium, e.g., in the body; growth would be supported well by OmpC because of the high nutrient content, but inhibitory agents would permeate less well across the OM because of the smaller pore size of OmpC porin. In contrast, in very poor media, e.g., in natural waters, where osmolality is low, derepression of OmpF would ensure that the poor nutrient supply is used most effectively. One must note that the change from high OmpF/low OmpC to low OmpF/high OmpC occurs at a low osmotic pressure compared to other responses considered and that the OmpF/OmpC changes have not evolved to protect from very high osmotic pressure, as is the case for the other responses (Table 8.9). Two systems that have evolved for such a purpose are that involving the ProP transporter, which takes up proline to protect cells from osmotic damage, and that using the Kdp products to take up K+ for the same purpose. The ProU system is also used for such protection; it involves the functioning of ProV, W, X to import, for example, glycine betaine (see Table 8.9). Do Both Intracellular and Extracellular Osmosensors Occur? One osmotically controlled system is porin regulation. The intracellular sensor is well established; mutants in the ompB group are aberrant in control and one of the ompB genes, namely the envZ gene, controls synthesis of EnvZ gene product which is an integral CM protein. It has been shown in vivo that raising osmotic pressure leads to phosphorylation of EnvZ. The proposal is that EnvZ is the osmosensor and on sensing a rise in such pressure, the protein changes in conformation, leading to auto-phosphorylation. Sensor activation sends a signal to shift porin synthesis, by phosphorylating the gene product of the other OmpB gene, OmpR. The striking fact is that both the sensor and the component altered by the activated sensor are intracellular, by contrast with extracellular stress sensors, that on activation produce extracellular EICs. Increased osmolality also leads to autophosphorylation of the sensor kinase KdpD, which then phosphorylates KdpE. This phosphorylated component then interacts with the kdp promoter, leading to increased transcription (Wood, 1999) and induction of the kdpFABCDE operon. The KdpA, B, C and F components form the Kdp-ATPase which catalyses K+ uptake. The activity of this complex is also activated
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Physiological Response
Osmotic Shift Inducing Response
1. Alteration to porin levels
Slight upshift
2. Induced K+ uptake
Large upshift
3. Induced glycine-betaine uptake
Large upshift
4. Induced proline uptake
Large upshift
Function of Response To choose most suitable porin Accommodation to high OP Accommodation to high OP Accommodation to high OP
Osmosensor
Regulator
Gene Products Induced by Sensor Activation
Effect of High OP on Activity of Induced Components
EnvZ
OmpR
OmpC (OmpF repressed)
?
KdpD
KdpE
KdpA, B, C, D, E, F
Glutamate dehydrogenase* Glutamate dehydrogenase*
N.E.1
ProV, W, X (i.e., ProU system)
Activation of Kdp-ATPase Activation of ProU
N.E.2
ProP
Activation of ProP
OP = osmotic pressure. N.E. = not established. 1
H-NS inhibits proV,W,X transcription by binding downstream of its promoter; K glutamate reverses. CRP-cAMP binds to P1 promoter of proP and stops transcription; K glutamate reverses. * Glutamate dehydrogenase would be activated by the increased pHi which may occur at high OP. If so, it is acting as an osmosensor, in the sense that the rise in internal glutamate produced by its increased activity would induce the response by reversing the effect of H-NS (on proV,W,X transcription) or of CRP-cAMP (on proP transcription). Alternatively, other osmosensors may indirectly lead to proV,W,X or proP induction, e.g., those osmosensors which induce K+ uptake may act indirectly as transcription of the above operons may need K+. 2
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TX69124 ch08(280) frame55 Page 280 Thursday, October 31, 2002 2:01 PM
TABLE 8.9 Sensing, Induction and Biochemistry of Osmotic Responses
by high osmotic pressure (see Table 8.9). The factors governing induction of ProP and ProV, W, X by osmotic shock are not so clear (see Table 8.9). Transcription of the respective genetic regions is blocked (Xu and Johnson, 1997) at low osmotic pressure (OP) by CRP-cAMP (proP) and H-NS (proV, W, X); K glutamate reverses this and, accordingly, osmosensing may be indirect, with components which sense high OP and respond by raising internal K+ and/or internal glutamate, being indirectly responsible for inducing the above two genetic regions. Several other proteins have been proposed as osmosensors. Two — ProP and MscL — should be mentioned. They have been proposed as sensors because high OP activates ProP and low OP activates MscL. The above osmosensors are intracellular; extracellular ones have not been looked for. The rationale for evolution of intracellular osmosensors would be that they must detect differences between external and internal OP, and one would expect intracellular sensors to do this. However, it is likely that extracellular sensors would have evolved to give early warning of rising solute level in the medium with some responses being switched on, not by altered difference between internal and external OP, but by the sensing of a change in the concentration of extracellular solutes. Such sensing could alter a solute concentration-sensing ESC to an EIC and induce a response. This would ensure earlier induction than with a sensor which detects an internal versus external change. In addition, if EIC produced by a rise in external solute concentration were to diffuse away to unstressed cells, this would give early warning. Tests will be made for such an ESC, using exposure to increased solute © 2003 by CRC Press LLC
levels, such as induce OmpF; filtrates from such cultures and those from cultures not exposed to increased solute concentrations, but then exposed to such stress, will be compared to filtrates from unstressed cultures with respect to ability to induce OmpF-LacZ in unstressed ompF-lacZ+ strains.
RESPONSES
TO
SALT STRESS
Two responses will be considered here; inducible resistance to salt switched on by high Na+ and acid sensitization switched on by high salt. Sensing of Salt Stress There may be sensing of salt by an intracellular sensor, but it is also likely that an ESC functions to detect high levels of salt, since acid sensitization by salt depends on functioning of an EIC as inducer of the response (Rowbury, 1999), and all EICs studied in detail so far arise from ESCs, i.e., for all such responses examined, an ESC/EIC pair functions, and this is likely to be so for salt stress. Other enzymes, CM components and regulatory components are also induced by salt; for example, the NhaA antiporter is induced at high Na+ concentrations. It is proposed to examine whether ESCs and EICs play any role, e.g., by looking for NhaA-LacZ induction at low salt concentrations, by medium filtrates from cultures exposed to high Na+ or to filtrates from low Na+ cultures exposed to high Na+, followed by dialysis to remove Na+. If ESCs and EICs play any role, suitable filtrates should induce NhaA-LacZ at low salt levels. Also, NhaR senses Na+ intracellularly, high salt levels leading to induction of NhaA, induction being enhanced by the changes in NhaR binding to nhaA DNA caused by rise in Na+ (Padan et al., 1999). Regulation of Salt-Induced Responses Tolerance of high [NaCl] involves induction and activation of NhaA. This component is an integral CM protein which functions as an Na+/H+ antiporter with stoichiometry of 2H+/Na+. This component is the major protein determining NaCl tolerance and is induced by Na+; studies of NhaA-LacZ synthesis show that intracellular levels of Na+ are the signal for NhaA synthesis and that, at specific concentrations of Na+, alkaline pH enhances induction (Dover et al., 1996; Rowbury, 1997). NhaR is an activator required for NhaA synthesis and nhaR deletions are Na+-sensitive because of the greatly reduced levels of NhaA (Rahav-Manor et al., 1992). NhaR binds directly to the nhaA gene and Na+ specifically affects the interaction of NhaR with base –60 of nhaA (Padan et al., 1999). NhaR binds Na+ and such binding causes a conformational change which alters the footprint of NhaR on the DNA, altering attachment of NhaR to –60. Such altered binding is pH-dependent, occurring most favorably at alkaline pH. Salt induces acid sensitivity and this process is independent of both the NhaA and NhaR gene products (Rowbury, 1997). As with so many stress responses, ECs are required. As stated above, an EIC has been implicated in the response but there is no information on possible involvement of an Na+-sensing ESC, although its involvement
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is likely. Studies have been made of which regulatory components are needed for induction of this response and results (see Table 8.3), show that H-NS, RelA and cAMP are needed for induction (Rowbury, 1997). It had earlier been established that acid sensitivity depended on the level of the OM pore protein PhoE (Rowbury and Goodson, 1993) and it appears that it is increased PhoE synthesis which allows saltinduced sensitization (see Table 8.5). As stated above, the OM impedes protons from entering the periplasm and lesions in OM components (Bielecki et al., 1982) or induction of certain OMPs leads to easier permeation of H+ and acid sensitivity. As stated above, there is evidence that the PhoE pore is involved directly or indirectly in passage of protons from the outside across the OM. Salt induces both PhoE-LacZ and PhoE-PhoA and lesions or agents which block sensitization by salt usually prevent synthesis of PhoE-LacZ (Lazim et al., 1996), e.g., glucose stops sensitization by salt and stops induction of PhoE-LacZ while cAMP which reverses effects of glucose on sensitization induction also allows PhoE-LacZ synthesis in the presence of glucose.
REGULATION
OF
RESPONSES
TO IRRADIATION
Irradiation damages the DNA, and is lethal at appropriate levels. It is, therefore, important to consider both regulation of responses to irradiation and the biochemistry of inducible irradiation tolerance. Here, the regulation of the SOS response will be considered. Sensing of Irradiation Stress Although regulation of the SOS response has been studied in great detail, sensing is still not fully solved. It is generally considered that the RecA gene product, or a component associated with it, functions as the DNA damage sensor; DNA damage switches on expression of numerous SOS genes, because RecA, on activation by damage, gains protease activity. This activity destroys LexA and allows transcription for SOS genes to begin. Accordingly, the idea is that RecA interacts directly or indirectly with a stimulus produced by DNA damage, and this interaction unmasks the protease activity which allows the SOS response to be derepressed (Walker, 1984). The finding, in early studies, that oligonucleotides induced ϕ80 (Irbe et al., 1981) suggested that these nucleotides might arise from a damaged region of DNA and seemed ideal as an induction stimulus. This now seems unlikely, since some mutants that show little DNA degradation show a strong SOS response. The likelihood now is that SS DNA regions arise either directly following DNA damage, e.g., by nalidixic acid (the damaged regions being unwound to give SS DNA), or result from DNA replication following DNA damage (Sassanfar and Roberts, 1990). Such replication leaves gaps and SS regions arise from these. In accord with this idea, SS DNA plus NTP leads to in vitro activation of RecA gene product. Interestingly, there may be a second intracellular sensor of DNA damage, since some processes are switched on by DNA damage in recA mutants.
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It could be argued that ESCs would not be involved in sensing, because internal and external sensors would be equally affected by irradiation, with an internal sensor being as efficient as an ESC. This, however, overlooks the fact that EICs could diffuse away to an area not exposed to irradiation, and prepare cells there to resist, i.e., could give early warning of impending irradiation. One would expect such an ESC to be converted to an irradiation tolerance-inducing EIC. Such ESCs and EICs had not been looked for, but it seemed likely that they occurred, since we had shown that an acidity-sensing ESC is activated by irradiation (Rowbury and Goodson, 1999b). The same applies to a thermotolerance ESC; its irradiation converts it to the thermotolerance EIC, and to an alkali tolerance ESC, which is activated to the alkali tolerance EIC, by irradiation at pH 7.0. I have now taken filtrates from organisms grown with mild irradiation, from those grown without irradiation and the latter filtrates irradiated in the absence of organisms and have shown that the activated filtrates on incubation with unirradiated organisms induce UV tolerance, i.e., a UV tolerance ESC/EIC pair occurs. Stages in the Switching-On of Responses to Irradiation Following Sensor Activation The switching-on of the SOS response is rather well understood apart from sensing. Once the protease activity of the RecA gene product has been unmasked by interaction with SS DNA and NTP, RecA cleaves LexA. This protein normally (i.e., in the absence of DNA damage) binds to the so-called SOS box of numerous (at least 25) genes, preventing their expression; there is evidence that LexA dimerizes onto the operator region of these genes to block expression, with the strength of binding varying from gene to gene. After RecA activation, initially, when the LexA gene product level begins to fall appreciably, several genes that have weak binding of LexA become derepressed. Later, as LexA level falls further, other genes with stronger LexA binding are derepressed.
STARVATION STRESS Although other types of starvation occur (e.g., for N or P), this account will be of carbon starvation. Matin and his group (Jenkins et al., 1988, 1990; Matin, 1991) described two classes of genes governing the response to starvation stress. The first class are the cst genes, which are controlled by cAMP-CAP; in these studies, 19 cst loci were revealed. None of the lesions alter stress tolerance, e.g., there is no loss of stress tolerance in cya mutants. These genes are involved in aiding organisms to escape from starvation by inducing pathways that can degrade novel carbon compounds. It would be expected that these genes would show induction by derepression of cAMP synthesis due to carbon starvation, this being sensed as follows: assuming that carbon starvation is due to a fall in glucose level, this leads to a rise in the level of Protein IIIGlc-phos. Increased phosphorylation of this CM component leads to greater adenyl cyclase activity, increased cAMP synthesis, and cst induction. Accordingly, the fall in carbon, i.e., glucose is sensed by the CM, so there is intracellular sensing.
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The other genes defined by Matin were pex genes, governing tolerance to carbon starvation and cross tolerances. Matin showed that starvation induced not only starvation tolerance, but also thermotolerance, osmotic tolerance, oxidative tolerance and acid tolerance (salt tolerance also appears). Matin revealed that the general stationary-phase response showed different characteristics than specific tolerance responses, e.g., H2O2 tolerance induced by H2O2 is regulated by OxyR whereas the stationary-phase H2O2 tolerance is governed by RpoS. Many protection/repair systems are controlled by RpoS, although some are not. Also, during starvation or in stationary phase, organisms become spherical and show altered RNA polymerase structure, components are stored for protection or as reserves, and changes in DNA conformation occur (Hengge-Aronis, 1993). RpoS is the major component controlling starvation responses, with relation to tolerance (but see “growth phase and stress tolerance” for role of chromosome termination in tolerance responses), and it functions as a σ-factor, allowing RNA polymerase to bring about increased transcription of a huge number of genes. Stress enhances the translation of rpoS-mRNA and increases RpoS stability (see Table 8.8), stopping its cleavage by protease. The major interest here, namely, how carbon starvation leads to RpoS induction, is not well understood; the nature of neither the precise stimulus nor of the sensor is clear. It is assumed that both cst genes and pex genes are switched on by mechanisms involving intracellular sensors. However, since cAMP can function via extracellular components (Rowbury, 1999), it is important that the possibility of extracellular sensors functioning to detect starvation should be considered. During growth into stationary phase, the levels of RpoS increase, leading to the induction of numerous proteins. As indicated above, this increase in RpoS levels results partly from increased m-RNA translation and partly from increased stability of this sigma factor. This stabilization occurs due to changes in the ClpXP protease. Aside from degrading abnormal or damaged proteins, this complex specifically targets RpoS (and some other proteins) and destroys it. In log phase, this sigma factor is actively cleaved by the ClpXP complex and, accordingly, shows a very short half-life. This is because, in log phase, the complete clpP, clpX operon is transcribed and gives so-called long transcripts, which are translated into complete ClpXP complexes (Li et al., 2000), which have protease activity. On growth into stationary phase, however, there is premature termination of transcription, so that most of the transcripts (short transcripts) arise only from the proximal gene of the operon, clpP. These transcripts cannot be translated into complete ClpXP (i.e., active protease) complexes and so degradation of RpoS is greatly reduced in stationary phase, and the half-life of this σ-factor increases markedly. As the organisms leave the stationary phase, the longer transcript begins to form, the full ClpXP arises on its translation, and this enzyme functions proteolytically to cleave both RpoS and many of the numerous proteins which have been induced by RpoS in stationary phase (Li et al., 2000).
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STRESS TOLERANCE INDUCTION BY KILLED CULTURES Polluting and contaminating bacteria often face naturally occurring or man-produced conditions which are lethal to them. This can occur in the natural environment for organisms that go on to enter foods. It would be expected that killing cultures would prevent them from growing and further altering the properties of the environment; also that it would prevent them from going on to cause disease. They would also not be expected to influence behavior of any organisms which enter the environment later. Recent findings have established, however, that dead cultures killed by several means can confer stress tolerance legacies on living organisms appearing in the same environment later (Rowbury, 2000). As indicated above, organisms in natural waters or in other environmental situations can eventually enter foods and food materials and, therefore, the likely effects of killed organisms in a range of locations, including the natural environment, will be considered here.
ACID TOLERANCE INDUCTION
BY
KILLED CULTURES
Appropriate cultures killed by many means confer an acid tolerance legacy on other organisms entering their environment. This applies to cultures killed by acidity, alkalinity, heat, irradiation, metal ions and antibiotics. For example, exposure of appropriate cultures to levels of alkali which kill more than 99.9% of organisms leads to preparations which can induce acid tolerance in living cultures (Table 8.10). Similarly, cultures killed by heat have essentially no living organisms (<0.001%) but they induce acid tolerance in living cultures. The highest percentage survival is for cultures killed by novobiocin, where as many as 2% survive the lethal agent. This survival rate is too little to explain acid tolerance induced in the living culture, i.e., acid tolerance is not due to survivors from the antibiotic treatment. Also, use of genetically marked pairs of strains established that indeed tolerant organisms arose by induction of tolerance, by the killed culture, in the organisms of the added living culture; the acid-tolerant organisms are not survivors of the killing process. The killed preparations do not act by slowing the growth-rate of the living cultures, neither do they lower the pH of the mixture. Properties of the active agents in the killed cultures (protease sensitivity and non-dialysability) suggest that it is EICs in the killed cultures which induce acid tolerance in the living organisms. Appropriate cultures killed by other means — by exposure to irradiation, to metal ions and to acidity itself — also induce acid tolerance (Rowbury, 2000). Other Stress Responses Induced by Killed Cultures It seems highly likely that numerous other stress responses will be induced by killed cultures. So far, it has been established that alkali tolerance can be induced by appropriate killed cultures and alkali sensitivity by others. More recently, killed cultures have been shown to confer thermotolerance also (Rowbury and Goodson, 2001). It is essential that ability of killed cultures to induce tolerance responses to irradiation, starvation, oxidative components and metal ions be established.
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TABLE 8.10 Acid Tolerance Induction by Cultures Killed by Alkali, Heat, Acidity and Novobiocin Culture Induced to Acid Tolerance by Added Filtrate or Killed Culture
Filtrate or Killed Culture Used to Induce Acid Tolerance None None Filtrate from pH 7.0-grown culture Filtrate from pH 5.0-grown culture Alkali-killed pH 7.0-grown culture Alkali-killed pH 7.0 culture → pH 5.0 Alkali-killed pH 5.0-grown culture Heat-killed pH 7.0-grown culture Heat-killed pH 7.0-grown culture → pH 5.0 Heat-killed pH 5.0-grown culture Acid-killed pH 7.0-grown culture Acid-killed pH 7.0 culture → pH 5.0 Acid-killed pH 5.0-grown culture Novobiocin-killed pH 5.0-grown culture
pH pH pH pH pH pH pH pH pH pH pH pH pH pH
% Survival ( ± SEM) after Acid Challenge for Culture Incubated with or without Filtrate or Killed Culture
7.0-grown 5.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown 7.0-grown
0.6 45.7 0.93 19.0 2.5 11.9 11.3 6.9 42.0 11.15 4.9 16.8 16.8 31.2
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 3.1 0.07 1.96 0.22 0.7 1.8 1.1 2.0 0.6 0.23 0.7 1.1 1.4
Organisms of E. coli strain 1829 ColV were grown to log phase at the stated pH and cell-free filtrates were prepared. Some cultures were killed by alkali (pH 11.0 for 15 min), by heat (70°C, 15 min), by acid (pH 2.0, 15 min) or by novobiocin (5 µgml-1 for 120 min at pH 5.0). After activation at pH 5.0 (if required) and neutralization, the filtrates or killed cultures were incubated for 45 min at pH 7.0 with log-phase strain 1829 ColV cultures grown at pH 7.0. After incubation, mixtures were washed once with broth before acid challenge (pH 3.0, 7 min). Plating for survivors after challenge was on NA for 20 h at 37°C.
BIOCHEMICAL CHANGES LEADING TO STRESS TOLERANCE ROLE
OF
CHAPERONES
IN THE
BIOCHEMISTRY
OF
STRESS TOLERANCE
Chaperones and chaperonins are tiny intracellular biological “work horses” used for processing proteins under normal and stressed conditions, and function as essential components in many inducible tolerance responses (see Table 8.7). Under normal conditions, they function to fold, assemble and process newly synthesized proteins, aid secretion of proteins through membranes, de-oligomerize proteins and play a role in degradation of seriously abnormal proteins. On exposure to stress, these molecules play some role in protection of proteins from damage, but they work mainly on damaged proteins, converting them back to a native state. HSP70 chaperones like DnaK are proteins folded to give a structure with a lidded “pocket” into which polypeptides with non-native exposed hydrophobic regions are introduced. Entry is allowed by opening and closing of the “lid,” so the structure © 2003 by CRC Press LLC
can occur in an open state which has ATP bound. In this state, the polypeptide to be folded or repaired enters the pocket, associates by hydrogen bonding with the chaperone and the short hydrophobic region is refolded; following refolding, the lid opens, releasing folded polypeptide and the ADP and inorganic phosphate resulting from ATP cleavage. Association of non-native polypeptide and DnaK involves very short hydrophobic (leucine-rich) regions on the polypeptide, spanned by basic regions. Each entry into the pocket folds only a short region of the polypeptide, and so there must be rapid cycles, i.e., following release there must be rapid reattachment, entry into the pocket, folding and release with ATP being bound and cleaved at the appropriate time. This cycle may then have to be repeated many times for the same non-native or damaged protein. For this reason, each cycle must be rapid and this is ensured by the binding of co-chaperones; DnaJ binds both to the substrate and to DnaK, aiding their interaction, stimulating the ATPase activity and allowing more rapid functioning. GrpE also enhances DnaK functioning, probably by enhancing release of ADP and inorganic phosphate. DnaK functioning is increased in another way also. McCarty and Walker (1991) showed that ATPase of DnaK is greatly enhanced at high temperatures, allowing more rapid protein repair. The 60 kDA chaperones are also known as chaperonins (Ellis, 1996). They are used for the folding of some proteins during synthesis, with the further function of repairing stress-damaged polypeptides. In bacteria, the GroEL HSP60 forms cylinders with a channel through them. The bacterial situation is complicated, however, in that these tiny GroE machines also contain the ca. 10 kDA GroES protein (cochaperonin) which binds to the GroEL cylinder, forming a lid. It has other functions, however, in that on GroES and ATP binding, the interior of the channel is profoundly altered, so that the compartment becomes larger, its hydrophobicity is modified and, in the open form, the polypeptide can enter for folding or repair. Small cytoplasmic chaperones also play a role in repair of damaged components, often after oligomerization to form large complexes. The small HSPs IbpA and IbpB, for example, are believed to be chaperones, which function to confer resistance to heat and oxidative stresses (Kitagawa et al., 2000), presumably repairing damage caused by such challenges. Chaperones occur not only in the cytoplasm of the Gram-negative bacterium, but also in the periplasm. Chaperones in this compartment differ from cytoplasmic ones, especially in being ATP-independent. In this environment, these proteins function, first, to aid the maturation (e.g., by appropriate folding) of newly synthesized periplasmic and outer membrane proteins and, secondly, to refold abnormal proteins arising on exposure to stress. Accordingly, a role as a chaperone can be most readily established by implicating components in normal folding or by showing that they can stop aggregation following stress damage. On this basis, several proteins seem to function as chaperones in the periplasm, amongst them the SurA, Skp and DegP proteins. Skp, which is a small protein, can bring about the maturation of unfolded OmpF protein, while the larger (448 residue) DegP protein aids the folding of the periplasmic MalS protein (Speiss et al., 1999). The third protein of this group, the 46 kDA SurA protein, shows peptidyl prolyl isomerase activity, its functioning as a chaperone being revealed both by its ability to aid LamB folding and by its inhibiting thermal aggregation of proteins (Behrens et al., 2001). After considering
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mutants with lesions in one or more of these three components, Rizzitello et al. (2001) proposed that the Skp and DegP proteins function as chaperones in one pathway, and SurA in another. Strains altered in one pathway can survive, albeit with poor growth in some cases, whereas those altered in both cannot. One exciting recent finding in the chaperone field has been that some proteins or protein complexes can function both as proteases and chaperones. This dual functioning is of particular relevance when considering abnormal or damaged proteins. If these show major abnormalities or damage, then proteases degrade them, so that their components can be reused. In contrast, proteins with less damage or fewer abnormalities can be refolded by chaperones, to reconstitute the active molecule. In addition, it may be that growth conditions influence which pathway is chosen, namely, whether a damaged or abnormal protein is repaired or not. Such dual functioning applies to the cytoplasmic ClpXP and ClpAP complexes. These were originally believed to be proteases, with the P sub-unit having the enzyme activity, and the ClpX or A moieties simply determining the specificity of the proteins degraded. It is now known, however, that the X or A components can, as well as determining the above specificity, switch the complex to a chaperone mode. Such duality also applies to periplasmic components, with the DegP protein aiding folding of, for example, the periplasmic MalS at low temperatures, while degrading abnormal proteins at high temperatures (Spiess et al., 1999).
BIOCHEMICAL CHANGES ACID-TOLERANT
IN
HABITUATED ORGANISMS MAKING THEM
It has not been possible to establish the relevance of most ASPs to the biochemical changes that lead to acid tolerance but there appear to be three main biochemical mechanisms by which tolerance appears. First, many tolerant organisms, but not all, gain novel pH homeostasis. Enteric bacteria growing at neutral external pH (pHo) maintain their internal pH (pHi) slightly alkaline at ca. 7.8 and at neutral pHo there is, therefore, a significant ∆pH, and the inside is alkaline. As the medium is acidified with inorganic acid, pHi is maintained at or near neutrality down to a pHo value of ca. 4.5; in this situation ∆pH is ca. 2.0 to 2.5; thus a neutral internal milieu is maintained with pHi only falling ca. 0.8 units compared to a fall of 2.5 units in pHo. Once pHo falls appreciably below 4.5, however, the non-habituated organism cannot maintain pHi at close to neutrality, and by the time pHo 3.0 is reached, death ensues unless tolerance has been induced during challenge. For tolerant organisms, one process preventing death is the new pH homeostatic system which acts at lower pHo values and still maintains pHi at near to neutral. Early studies suggested atp involvement in this novel system of homeostasis (Foster and Hall, 1990) but atp involvement has not been mentioned recently. It has also been claimed that there is a lysine decarboxylase-dependent homeostasis system, which maintains pHi near neutrality at acidic pHo. The second biochemical change that occurs in inducibly acid-tolerant cells is that, independent of the pH homeostasis changes, tolerant cells gain ability to better resist acid damage, i.e., there is a damage limitation system induced, in habituated organisms. It is likely that DNA is the critical macromolecule, with respect to acid
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damage. Certainly, DNA is readily damaged by acid, as evidenced by the finding (Sinha, 1986) that mutants altered in DNA repair are acid-sensitive. Assuming the DNA damage is the major lethal change, then it is highly significant that acid-tolerant cells of E. coli are far more resistant to such damage (Raja et al., 1991); perhaps tolerant cells contain SS DNA-binding proteins which can protect DNA replication forks from low pH. Third, acid-tolerant cells are better able than non-habituated ones to repair any acid damage to DNA which occurs. Acid-habituated organisms repair acid-damaged plasmid better than non-habituated ones do; more plasmid-containing (p+) transformants are produced when acid-damaged plasmid is transformed into an habituated culture (Raja et al., 1991). The occurrence of novel DNA repair mechanisms in tolerant organisms is also established by the finding that acid-habituated organisms are better able than non-habituated ones to repair UV-damaged phage (Goodson and Rowbury, 1991). Foster has proposed that Ada plays a role in repair of acid damage to DNA, since ada mutants are acid-sensitive (Foster and Moreno, 1999). It should be noted that acid sensitivity is shown by polA, recA and uvrA mutants (Sinha, 1986; Goodson and Rowbury, 1991), suggesting that acid damage repair involves several repair systems. The new repair system induced at acidic pH (Goodson and Rowbury, 1991) was able to function in mutants altered in any of the above genes (polA, recA or uvrA), suggesting that it involves novel DNA repair processes, independent of the PolA, RecA and UvrA gene products.
BIOCHEMICAL AND PHYSIOLOGICAL CHANGES MAKING ORGANISMS INDUCIBLY ALKALI-TOLERANT Organisms transferred from pH 7.0 to pH 9.0 become alkali-tolerant (habituate to alkali; Goodson and Rowbury, 1990; Rowbury, 1997). There appear to be at least two biochemical components in the tolerance response. First, there is less alkali damage to DNA in tolerant organisms: NhaA, which is induced at high pHo, is needed for tolerance induction (Rowbury, 1997); it seems likely that high levels of this gene product are responsible for the ability of tolerant organisms to resist alkali damage. A second component of tolerance involves alkali-habituated organisms showing better repair of alkali damage to DNA (Rowbury, 1997); NhaA appears not to be involved. The new repair system seems independent of RecA and PolA.
BIOCHEMICAL CHANGES LEADING TO HEAT TOLERANCE IN PHENOTYPICALLY THERMOTOLERANT ORGANISMS Kusukawa and Yura (1988) established a key role of GroEL/GroES proteins of E. coli in governing normal maximum growth temperature. These workers took a strain deleted in rpoH which was unable to grow at higher than 20°C and isolated revertants able to grow at higher temperatures. They showed that a series of strains able to grow at temperatures above 20°C and up to 40°C produced increasing amounts of the HSPs GroEL and GroES. Inability of the deletion strain to grow at >20°C was due to low levels of GroE proteins being produced in the rpoH deletion strain, and derepression of these gene products allowed growth at increased temperatures. These strains with
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derepressed GroE were unable even with the highest levels to grow at 42°C but increased synthesis of DnaK protein allowed growth; similar changes might be needed as some component of inducible thermotolerance. It seems likely that both σE-induced responses (with DegP protease being one critical component induced) and σ32-induced responses are involved in thermotolerance induction by heat, although little is known of the biochemical changes, which are important. It is also possible, however, that trehalose is involved in heat-induced thermotolerance, since the ability to accumulate this sugar is linked to starvationinduced thermotolerance (Hengge-Aronis, 1993). It should also be reiterated that the apparent expulsion of H-NS and IHF from appropriate regions of the DNA also appears to be needed for the induction process.
PHYSIOLOGICAL AND BIOCHEMICAL CHANGES TOLERANT TO COLD
IN
ORGANISMS INDUCIBLY
One important example of how the cold-shock response aids tolerance to cold relates to the CsdA protein. At low temperatures, stable secondary structure forms in RNAs, especially in m-RNAs, and this acts to reduce m-RNA translation. The CsdA protein is a major cold-shock protein (Jones et al., 1996) associated with the ribosomes. It acts to destabilize the DS RNA helix, i.e., it functions to unwind stable secondary structure and, accordingly, allows increased translation at low temperatures. Strikingly, another major cold-shock protein, namely, CspA, may function with CsdA. CspA binds to SS RNA (or SS DNA) and hence may prevent re-annealing of unwound m-RNA. Accordingly, this mechanism allows a third means of increasing synthesis of Csp proteins at low temperatures, without increasing m-RNA synthesis, the other two being stabilization of Csp m-RNA at low temperatures and increased Csp m-RNA translation, due to the presence of the downstream box translational enhancer.
OSMOTIC STRESS TOLERANCE: PHYSIOLOGICAL AND BIOCHEMICAL CHANGES INVOLVED IN INDUCIBLE RESPONSES Increased osmotic pressure (OP), leads to numerous inducible responses, and some are shown in Table 8.9. On an increase in external OP, organisms are protected from growth inhibition, damage and death by increased synthesis or accumulation of compatible solutes such as K+, glutamate, proline, glycine betaine and trehalose. The responses that occur function to bring about such changes in synthesis and/or accumulation; for example, the changes in synthesis and activity of the ProP and ProU systems function to take up increased amounts of proline, glycine betaine and related compounds, and similar changes in Kdp-ATPase synthesis and activity aid K+ uptake, while changes in the activity of glutamate dehydrogenase (and possibly glutamate synthase) raise internal glutamate concentration.
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BIOCHEMISTRY
OF
TOLERANCE
TO
SALT
Factors governing the protection of organisms have been considered earlier, but one should note that a major factor influencing salt tolerance is the level of the NhaA sodium/hydrogen antiporter. Organisms which induce this protein (rises in intracellular Na+ appear to induce NhaA synthesis, NhaR regulating induction) can expel Na+ more effectively, but it is the massive increase in activity of the antiporter at alkaline pH which has the greatest influence on Na+ expulsion.
BIOCHEMICAL CHANGES FOLLOWING STARVATION Firstly, RpoS controls synthesis of many proteins involved in tolerance (HenggeAronis, 1993; but see “growth-phase and stress tolerance” in this chapter for role of chromosome termination in stress tolerance, induced by starvation). Among these are: 1) enzymes for trehalose synthesis and uptake; trehalose is needed for starvationinduced thermotolerance and for other tolerances, and has membrane and protein protection properties; 2) enzymes for H2O2 degradation, e.g., catalase HPII; 3) enzymes for DNA protection or repair, e.g., the Dps DNA-binding protein and exonuclease III; and 4) enzymes involved in glycogen synthesis (not all are RpoS-controlled). Many other proteins are induced by starvation but are not rpoS-controlled, e.g., GlgA and C glycogen synthetase proteins are controlled by cAMP. Starvation-induced thermotolerance needs RpoS-controlled OtsAB, implicating trehalose accumulation in thermotolerance, but starvation also induces HSPs like GroEL, GroES, DnaK, GrpE and HtpG. (see Table 8.7). In addition, of critical importance during starvation are proteases which break down dispensible proteins to give amino acids for synthesis of essential proteins. Mutants altered in the genes which encode such proteases show poor starvation survival. Another physiological change, which occurs in organisms as they enter stationary phase, may play a major role in inducing the stress tolerances that appear in this growth phase; it has been established that the phospholipids of the CM become enriched in cyclopropane fatty acids (CFAs), a change which may itself lead to some stress tolerances. For example, such a fatty acid change has been implicated in the acid tolerance that appears at acidic pH in E. coli (Brown et al., 1997), and may lead to other stress tolerances. Initially, it was believed that such CFA synthesis was controlled by RelA, since mutations in the gene which encodes this product lead to CFA deficiencies in stationary phase. In fact, ppGpp is needed for RpoS synthesis, and, accordingly, it is RpoS which controls CFA synthesis, one synthase promoter needing this stationary-phase sigma factor for functioning (Eichel et al., 1999).
COUNTERACTING THE INDUCTION OF STRESS TOLERANCE RESPONSES Very few efforts have been made to inhibit the induction of stress responses, although if a common metabolite were able to prevent or counteract a stress response of applied or medical importance, this could provide a means of preventing the response.
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SUBVERTING STRESS RESPONSES USING METABOLITES Efforts have been made to prevent induction of acid tolerance in E. coli. As Table 8.11 shows, many metabolites and other agents prevent tolerance induction at
TABLE 8.11 Counteracting Acid Tolerance Responses by Adding Metabolites During Induction Agent Used to Subvert Response cAMP NaCl Sucrose Ethanol Urea Phosphate SDS DOC N-acetyl serine L-leucine
Effect of Agent on Acid Tolerance Induced by: Acidic pH
Glucose
L-glutamate
L-aspartate
Tolerance abolished Tolerance abolished Tolerance abolished Tolerance abolished Very marked inhibition Tolerance abolished Tolerance abolished Tolerance abolished Tolerance abolished Tolerance abolished
Marked inhibition Tolerance abolished Tolerance abolished Slight inhibition No effect
Tolerance abolished Tolerance abolished Tolerance abolished Slight inhibition No effect
Tolerance abolished Tolerance abolished Tolerance abolished No effect
Marked inhibition Tolerance abolished Tolerance abolished N.T.
Marked inhibition Tolerance abolished Tolerance abolished N.T.
N.T.
N.T.
FeCl3
N.T.
Tolerance reduced Tolerance abolished Tolerance abolished Slight inhibition Slight inhibition Slight inhibition Tolerance abolished Slight inhibition N.T.
N.T.
N.T.
Marked inhibition Slight inhibition Marked inhibition No effect
N.T. = not tested.
pH 5.0, or by specific inducers at neutral pH. In the case of a few of the agents that act at pH 5.0, the possibility that they act on synthesis or action of the ECs has been examined. For example, phosphate inhibits synthesis of the ESC and cAMP acts both on ESC synthesis and on interaction of EIC with pH 7.0-grown organisms. The results also indicate that HCO3– can interfere with tolerance induction; HCO3– has not been tested on acid habituation, because it is decomposed at pH 5.0. Because both main biological stages in the functioning of ECs in acid habituation can be examined at pH 7.0 (ESC → EIC, which needs pH 5.0, is probably simply a chemical reaction), the potential role of bicarbonate can be studied; it is able to inhibit both ESC synthesis and interaction of EIC with organisms.
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The effects of urea and N-acetyl-L-serine on acid habituation at pH 5.0 are of interest, since CysB-dependent responses (acid habituation at pH 5.0 is CysBdependent) are often inhibited by these agents. The striking additional finding was that urea markedly inhibited L-aspartate-induced acid tolerance and this may indicate CysB-dependence of this response.
SUBVERTING OTHER STRESS RESPONSES Several of the pH responses described above can be abolished or strongly inhibited by metabolites and other agents, and a few effects are discussed next. Alkali Tolerance Induction First, induction is strongly inhibited by glucose, and of interest here is the finding that cyclic AMP does not reverse this inhibition; this nucleotide usually reverses inhibitory effects of glucose on inducible responses. Second, phosphate also inhibits alkali tolerance induction and the same is true for NaCl. Acid-Induced Alkali Sensitization Induction of this response is greatly reduced by phosphate, NaCl and FeSO4. Cu2+-Induced Thermotolerance Several tested metabolites reduced such tolerance induction by copper. The most significant effects were with glutathione (GSH), L-cysteine and urea, which all substantially reduced thermotolerance induction, and ethanol which virtually abolished the response. It is now known (Rowbury and Goodson, 2001) that copperinduced thermotolerance involves an ESC/EIC pair; accordingly, it would be of interest to know whether GSH, L-cysteine and urea act on ESC synthesis, on ESC → EIC, on interaction of EIC with sensitive organisms or on some other process.
EXTRACELLULAR ALARMONES AND CELL-TO-CELL COMMUNICATION In the early 1980s, Bruce Ames and his group (Bochner et al., 1984) proposed that cellular damage, by several chemical stresses, could lead to the production of nucleotide “alarmones,” the sensing of which could switch on damage limitation and damage repair processes. These alarmones were polyphosphates and, although they were able to leak poorly from the producing organisms, they would be virtually unable to permeate into other cells and, accordingly, only influence the producing organisms. For this reason, the success of these alarmones, in protecting organisms, depended on their being synthesized rapidly, and following low levels of damage. It was also essential that the sensing system was able to detect tiny concentrations of the alarmones. In contrast, the discovery of the ESC/EIC pairs demonstrated that chemical and physical stresses could produce extracellular alarmones, the stress activating the ESC
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to the EIC alarmone, with activation occurring in the medium. Since organisms have receptors which allow the uptake of the EICs, these alarmones are able to warn other organisms (including unstressed ones, and those which cannot produce the ESC), and trigger favorable responses. Table 8.12 lists the properties of the ESC/EIC pairs;
TABLE 8.12 Characteristics of the ESC/EIC Pairs Allowing Them to Provide Early Warning Systems against Chemical and Physical Stresses 1. The ESCs are extracellular stress sensors and are secreted into the media of stressed or unstressed cultures; accordingly, the stress is sensed in the medium with no delay, producing EIC and, therefore, inducing the response with no delay. 2. The ESCs are extremely sensitive to activation by chemical stress, so that a response can occur at very low levels of toxic agents. 3. The ESC occurs in several forms; the form synthesized under particular conditions is that which can most rapidly respond (Rowbury, R.J. and Goodson, M., FEMS Microbiol. Lett., 174, 49, 1999a). Sometimes, the ESC can be activated even before the level of the agent (e.g., protons) becomes stressing. 4. Formation of the EIC from the ESC is essential for response induction in the presence of the stress, but the EIC can also induce the response in unstressed cells. 5. The EIC can also induce its response in non-producers (cells which fail to form ESC and, therefore, EIC). 6. The EICs are small molecules and can, therefore, diffuse away to other regions, including those not subject to stress; this behavior of EICs makes them alarmones, and the system constitutes an early warning against stress, warning organisms of impending stress and preparing them to resist it. 7. The characteristics listed in 4, 5 and 6 allow cell-to-cell communication, with the EICs acting pheromonally. 8. The EIC receptors on the cell surface can occur in different forms, the form synthesized being that which can most favorably bind the EIC present in the medium. 9. The ESCs and EICs are highly resistant to irreversible inactivation by lethal conditions, making the ESC/EIC pair system highly robust, and allowing stress-killed cultures to induce responses.
these properties, especially three of them, namely, the diffusibility of these agents, their ability to act on unstressed organisms, and their cross-feeding characteristics, allow these EC pairs to influence other organisms and so these EC pairs are acting as pheromones. Accordingly, the functioning of the ESC/EIC pairs allows other organisms to be warned of impending stresses, with damage limitation and damage repair processes being triggered.
FUTURE EXPERIMENTS AND CONCLUSIONS The stresses relevant to the survival of organisms in food have been outlined here, and the responses affecting tolerance to such stresses listed. The regulation of such responses has then been considered in detail; in particular, the switching-on of such responses by both intracellular and extracellular stresses has been discussed. Although internal levels of some stresses are detected by classical means, i.e., by intracellular sensors with all other reactions and components related to induction
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being intracellular, many extracellular stresses are sensed by extracellular components (ECs), and interaction of the stress with an extracellular sensing component, ESC (which is synthesized in the absence of stress), activates the ESC to an extracellular induction component (EIC). The EICs are usually small diffusible proteins and can diffuse to and warn unstressed organisms, i.e., they are alarmones which allow cross-talk between stressed and unstressed organisms. Such EICs associate with receptors on organisms in the medium, leading to the stress response. Such ESCs and EICs occur for many stress responses and it appears that each ESC exists in several forms, depending on the culture conditions (see Table 8.12); the receptors on the cell surface which interact with the EIC can also be modified according to the culture conditions. Such ESC/EIC pairs are needed for numerous pH responses and for salt-stress responses. Most recently, it has been shown in my laboratory that UV tolerance induction involves a specific ESC/EIC pair, with increased temperature leading to increased UV tolerance, following the ESC to EIC conversion. The ribosome has been proposed as the thermal sensor involved in triggering the heat-shock and cold-shock responses, and DnaK is another proposed thermometer, but the evidence for these is mostly indirect. The finding that a heat-activated EIC is involved in thermotolerance induction suggests that the heat-shock and cold-shock responses may be switched-on by ESC/EIC pairs, and that these ECs are involved in induction of most other stress responses. Further support for a role for ESCs as biological thermometers comes from the demonstration that the triggering of acid tolerance, alkali tolerance and UV tolerance by temperature rises involves activation of the appropriate ESCs by the temperature shift. Further work on the role of ECs in the switching-on of responses to cold, irradiation, osmotic shock and to starvation should be urgently undertaken. Although the nature of the sensor(s) for induction of heat shock and cold shock is controversial, there is good information on how the later stages of these two responses are regulated and, for heat shock, on which biochemical changes in the organisms allow protection against heat and ability to repair heat damage. Substantial studies have also been made on the molecular biology of the regulation for several other stress responses, e.g., relating to killing and growth inhibition by irradiation, starvation, osmotic stress and oxidative stress (although not on the role of ECs) and this work has been reviewed. Additionally, a brief account has been given of some important biochemical changes which lead to tolerance, although only a few examples have been given of the changes occurring on induction of each stress response. It was pointed out that little has been done to subvert stress tolerance induction, except that many agents are known to prevent acid tolerance induction and, in a few cases, agents that subvert responses have been shown to inhibit ESC synthesis or EIC interaction with organisms or both processes. Recent work reveals that appropriate killed cultures induce stress tolerance in living organisms (Rowbury, 2000) and these findings are of public health significance and important in food microbiology; it is critical that the basis for these responses is established, although all the evidence so far suggests that it is stress response ECs in the killed cultures which induce the tolerance responses.
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GLOSSARY AdiA arginine decarboxylase ahpC gene encoding the smaller subunit of alkyl hydroperoxide reductase ASI acid sensitivity induction at pH 9.0 cAMP adenosine-3′-5′-cyclic phosphate CAP catabolite activator protein CFA cyclopropane fatty acid clpA, B, P, X structural and regulatory genes governing synthesis of heat-shock protease/chaperones CM cytoplasmic membrane CsdA cold-shock protein CspA, B cold-shock proteins cysB/CysB a gene and its gene product implicated in regulation of cysteine biosynthesis and other processes cyclic AMP adenosine-3′-5′-cyclic phosphate ⌬pH the difference between internal pH of a cell and the external milieu DOC deoxycholic acid DnaJ a chaperone protein which is induced by heat-shock and certain other stresses DnaK a chaperone protein which is induced by heat-shock and certain other stresses DNase deoxyribonuclease EC extracellular component EIC extracellular induction component EnvZ cytoplasmic membrane component involved in regulation of OmpC/F biosynthesis ESC extracellular sensing component fur/Fur ferric uptake regulator, gene and gene product GABA γ-amino-butyric acid gadA, B genes which encode two forms of glutamate decarboxylase GlgA/C glycogen synthetase components GroEL a heat-shock chaperone GroES a heat-shock chaperone GrpE a heat-shock chaperone GSH glutathione GyrA a DNA helicase himA/HimA the gene which encodes one subunit of integration host factor, and its gene product H-NS a histone-like regulatory protein, which affects numerous genetic regions after binding to the DNA HSP heat-shock protein hsp-lacZ a fusion of a heat-shock protein gene to the lacZ gene hsp-phoA a fusion of a heat-shock protein gene to the alkaline phosphatase gene HtpG a heat-shock protein HtpM a heat-shock protein
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Hyd components proteins needed for formic hydrogenlyase activity IHF integration host factor kdp genes a group of genes encoding or regulating synthesis of Kdp components Kdp components proteins involved in governing K+ uptake and osmotic pressure LexA regulator of the SOS response Lon a heat-shock protein with protease activity NhaA the major sodium/hydrogen antiporter of E. coli NhaB a secondary sodium/hydrogen antiporter of E. coli NhaR the regulator of NhaA induction NMW nominal molecular weight OM outer membrane ompA/OmpA a gene and its gene product involved in outer membrane stability and permeability OmpC/F porins for uptake of uncharged molecules and cations OmpR protein involved in regulation of OmpC/F biosynthesis O.P. osmotic pressure OtsA, B proteins involved in osmotic tolerance and thermotolerance induction oxyR/OxyR a gene and its gene product involved in regulation of H2O2 tolerance pHi internal pH pHo external pH PhoE porin for anion uptake PhoP/Q regulatory gene products which control some acid tolerance responses and other processes PhoS periplasmic phosphate-binding protein PolA the Kornberg polymerase ppGpp a guanosine tetraphosphate involved in controlling the stringent response pppGpp a guanosine pentaphosphate involved in controlling the stringent response (p)ppGpp refers to enzymes or processes using ppGpp or pppGpp pro genes genes involved in governing proline biosynthesis and some responses to high osmotic pressure RecA a protein which functions in recombination and in the regulation of the SOS response RelA (p)ppGpp synthetase RNase ribonuclease RpoH the major heat-shock sigma factor rpoS gene encoding the RpoS stationary-phase sigma factor E a heat-shock sigma factor 32 the major heat-shock sigma factor SDS sodium dodecyl sulfate TonB a cytoplasmic membrane protein, which aids entry of components across the OM and into the periplasm TreA protein involved in trehalose synthesis and in osmotic tolerance induction ts temperature-sensitive, referring to mutants unable to grow at the wild-type growth temperature UvrA, B and C gene products needed for repair of DNA damage
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Guilfoyle, D.E. and Hirshfield, I.N., 1996. The survival benefit of short-chain organic acids and the inducible arginine and lysine decarboxylase genes for Escherichia coli. Letts Appl. Microbiol. 22, 393–396. Hall, H.K. and Foster, J.W., 1996. The role of Fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition. J. Bacteriol. 178, 5683–5691. Hengge-Aronis, R., 1993. Survival of hunger and stress: the role of rpoS in early stationaryphase gene regulation in Escherichia coli. Cell 72, 165–168. Hicks, S.J. and Rowbury, R.J., 1986. Virulence plasmid-associated adhesion of Escherichia coli and its significance for chlorine resistance. J. Appl. Bacteriol. 61, 209–218. Humphrey, T.J., Richardson, N.P., Gawler, A.H.L. and Allen, M.A., 1991. Heat resistance in Salmonella enteritidis PT4 and the influence of prior exposure to alkaline conditions. Letts. Appl. Microbiol. 12, 258–260. Humphrey, T.J., Richardson, N.P., Statton, K.M. and Rowbury, R.J., 1993. Effects of temperature shifts on acid and heat tolerance in Salmonella enteritidis phage type 4. App. Environ. Microbiol. 59, 3120–3122. Humphrey, T.J., Slater, E., McAlpine, K., Rowbury, R.J. and Gilbert, R.J., 1995. Salmonella enteritidis phage type 4 isolates more tolerant of heat, acid or hydrogen peroxide also survive longer on surfaces. Appl. Environ. Microbiol. 61, 3161–3164. Humphrey, T.J., Williams, A., McAlpine, K., Lever, M.S., Guard-Petter, J. and Cox, J.M., 1996. Isolates of Salmonella enterica Enteritidis PT4 with enhanced heat and acid tolerance are more virulent in mice and more invasive in chickens. Epidemiol. Infect. 117, 79–88. Humphrey, T.J., Wilde, S.J. and Rowbury, R.J., 1997. Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue. Letts. Appl. Microbiol. 25, 265–268. Igo, M.M. and Silhavy, T.J., 1988. EnvZ, a transmembrane sensor of Escherichia coli is phosphorylated in vitro. J. Bacteriol. 170, 5971–5973. Irbe, R.M., Morin, L.M.E. and Oishi, M., 1981. Prophage ϕ80 induction in Escherichia coli by oligonucleotides. Proc. Nat. Acad. Sci. 78, 138–142. Jenkins, D.E., Schulz, J.E. and Matin, A., 1988. Starvation-induced cross-protection against heat or H2O2-challenge in Escherichia coli. J. Bacteriol. 170, 3910–3914. Jenkins, D.E., Chaisson, S.A. and Matin, A., 1990. Starvation-induced cross-protection against osmotic challenge in Escherichia coli. J. Bacteriol. 172, 2779–2781. Jones, P.G., Cashel, M., Glaser, G. and Neidhardt, F.C., 1992. Function of a relaxed-like state following temperature down-shifts in Escherichia coli. J. Bacteriol. 174, 3903–3914. Jones, P.G., Mitta, M., Kim, Y., Jiang, W. and Inouye, M., 1996. Cold-shock induces a major ribosomal-associated protein that unwinds ds RNA in Escherichia coli. Proc. Nat. Acad. Sci. 93, 76–80. Kitagawa, M., Matsumara, Y. and Tsuchido, T., 2000. Small heat-shock proteins, IbpA and IbpB, are involved in resistances to heat and O2– stress in Escherichia coli. FEMS Microbiol. Letts. 184, 165–171. Kullik, I., Toledano, M.B., Tartaglia, L.A. and Storz, G., 1995. Mutation analysis of the redoxsensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation. J. Bacteriol. 177, 1275–1284. Kusukawa, N. and Yura, T., 1988. Heat-shock protein GroE of Escherichia coli: key protective roles against thermal stress. Genes Dev. 2, 874–882. Kwon, Y.M. and Ricke, S.C., 1998. Induction of acid resistance of Salmonella typhimurium by exposure to short chain fatty acids. Appl. Environ. Microbiol. 64, 3458–3463. Lazim, Z., Humphrey, T.J. and Rowbury, R.J., 1996. Induction of the PhoE porin as the basis for salt-induced acid sensitivity in Escherichia coli. Letts. Appl. Microbiol. 23, 269–272.
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Lelivelt, M.J. and Kawula, T.H., 1995. Hsc66, an Hsp70 homolog in Escherichia coli, is induced by cold-shock but not by heat-shock. J. Bacteriol. 177, 4900–4907. Leyer, G.J. and Johnson, E.A., 1993. Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbiol. 59, 1842–1847. Li, C., Tao, Y.P. and Simon, L.D., 2000. Expression of different size transcripts from clpPclpX operon of Escherichia coli during carbon deprivation. J. Bacteriol. 182, 6630–6637. Mackey, B.M. and Derrick, C.M., 1986. Changes in the heat resistance of Salmonella typhimurium during heating at rising temperatures. Letts. Appl. Microbiol. 4, 13–16. Matin, A., 1991. The molecular basis of carbon starvation-induced general resistance in Escherichia coli. Molec. Microbiol. 5, 3–10. Mattick, K.L., Jorgensen, F., Legan, J.D., Cole, M.B., Porter, J., Lappin-Scott, H.M. and Humphrey, T.J., 2000. The survival and filamentation of Salmonella enteritidis PT4 and Salmonella typhimurium DT104 at low water activity. Appl. Environ. Microbiol. 66, 1274–1279. McCarty, J.S. and Walker, G.C., 1991. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc. Nat. Acad. Sci. 88, 9513–9517. Nikolaev, Y.A., 1996. General protective effect of exometabolite(s) produced by tetracyclinetreated Escherichia coli. Microbiology (Moscow) 65, 749–752. Nikolaev, Y.A., 1997a. Involvement of exometabolites in stress adaptation of Escherichia coli. Microbiology (Moscow) 66, 38–41. Nikolaev, Y.A., 1997b. Comparative study of two extracellular protectants secreted by Escherichia coli cells at elevated temperatures. Microbiology (Moscow) 66, 790–795. Nishiyama, S., Umemura, T., Nara, T., Homma, M. and Kawagishi, I., 1999. Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant. Molec. Microbiol. 32, 357–365. Padan, E., Gerchman, Y., Rimon, A., Rothman, A., Dover, N. and Carmel-Harel, O., 1999. The molecular mechanism of regulation of the NhaA Na+/H+ antiporter of Escherichia coli, a key transporter in the adaptation to Na+ and H+. Novartis Found. Symp. 221, 183–199. Pao, C.C. and Dyess, B.T., 1981. Stringent control of RNA synthesis in the absence of guanosine 5′-diphosphate-3′-diphosphate. J. Biol. Chem. 256, 2252–2257. Phillips, L.E., Humphrey, T.J. and Lappin-Scott, H.M., 1998. Chilling invokes different morphologies in two Salmonella enteritidis PT4 strains. J. Appl. Microbiol. 84, 820–826. Rahav-Manor, O., Carmel, O., Karpel, R., 1992. NhaR, a protein homologous to a family of bacterial regulatory proteins (LysR), regulates nhaA, the sodium proton antiporter gene in Escherichia coli. J. Biol. Chem. 267, 10433–10438. Raja, N., Goodson, M., Smith, D.G. and Rowbury, R.J., 1991. Decreased DNA damage and increased repair of acid-damaged DNA in acid-habituated Escherichia coli. J. Appl. Bacteriol. 70, 507–511. Rizzitello, A.E., Harper, J.R. and Silhavy, T.J., 2001. Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli. J. Bacteriol. 183, 6794–6800. Rowbury, R.J., 1972. Observations on starvation-induced resistance enhancement (SIRE) in Salmonella typhimurium. Int. J. Radiat.Biol. 21, 297–302. Rowbury, R.J., 1997. Regulatory components, including integration host factor, CysB and HNS, that influence pH responses in Escherichia coli. Letts. Appl. Microbiol. 24, 319–328.
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Rowbury, R.J., 1999. Acid tolerance induced by metabolites and secreted proteins, and how tolerance can be counteracted. Novartis Found. Symp. 221, 93–111. Rowbury, R.J., 2000. Killed cultures of Escherichia coli can protect living organisms from acid stress. Microbiology 146, 1759–1760. Rowbury, R.J., 2001. Cross-talk involving extracellular sensors and extracellular alarmones gives early warning to unstressed Escherichia coli of impending lethal chemical stress and leads to induction of tolerance responses. J. Appl. Microbiol. 90, 677–695. Rowbury, R.J. and Goodson, M., 1993. PhoE porin of Escherichia coli and phosphate reversal of acid damage and killing and of acid induction of the CadA gene product. J. Appl. Bacteriol. 74, 652–661. Rowbury, R.J., Goodson, M. and Whiting, G.C., 1989. Habituation of Escherichia coli to acid and alkaline pH and its relevance for bacterial survival in chemically-polluted natural waters. Chem. Ind. 1989, 685–686. Rowbury, R.J. and Goodson, M., 1998. Induction of acid tolerance at neutral pH in log-phase Escherichia coli by medium filtrates from organisms grown at acidic pH. Letts. Appl. Microbiol. 26, 447–451. Rowbury, R.J. and Goodson, M., 1999a. An extracellular acid stress-sensing protein needed for acid tolerance induction in Escherichia coli. FEMS Microbiol. Letts. 174, 49–55. Rowbury, R.J. and Goodson, M., 1999b. An extracellular stress-sensing protein is activated by heat and u.v. irradiation as well as by mild acidity, the activation producing an acid tolerance-inducing protein. Letts. Appl. Microbiol. 29, 10–14. Rowbury, R.J. and Goodson, M., 2001. Extracellular sensing and signalling pheromones switch-on thermotolerance and other stress responses in Escherichia coli. Sci. Prog. 84, 205–233. Salmond, C.V., Kroll, R.G. and Booth, I.R., 1984. The effect of food preservatives on pH homeostasis in Escherichia coli. J. Gen. Microbiol. 130, 2845–2850. Sassanfar, M. and Roberts, J.W., 1990. Nature of the SOS-inducing signal in Escherichia coli: the involvement of DNA replication. J. Molec. Biol. 212, 79–96. Shi, X. and Bennett, G.N., 1994. Effects of rpoA and cysB mutations on acid induction of biodegradative arginine decarboxylase in Escherichia coli. J. Bacteriol. 176, 7017–7023. Shi, X., Waasdorp, B.C. and Bennett, G.N., 1993. Modulation of acid-induced amino acid decarboxylase gene expression by hns in Escherichia coli. J. Bacteriol. 175, 1182–1186. Sinha, R.P., 1986. Toxicity of organic acids for repair-deficient strains of Escherichia coli. Appl. Environ. Microbiol. 51, 1364–1366. Spiess, C., Beil, A. and Ehrmann, M., 1999. A temperature-dependent switch from chaperone to protease in a widely conserved heat-shock protein. Cell 97, 339–347. van Bogelen, R.A. and Neidhardt, F.C., 1990. Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc. Nat. Acad. Sci. 87, 5589–5593. Walker, G.C., 1984. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol. Rev. 48, 60–93. Wood, J.M., 1999. Osmosensing by bacteria: signals and membrane-based sensors. Microbiol. Molec. Biol. Rev. 63, 230–262. Xu, J. and Johnson, R.C., 1997. Cyclic AMP receptor protein functions as a repressor of the osmotically-inducible promoter proP P1 in Escherichia coli. J. Bacteriol. 179, 2410–2417.
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9
Strategies to Control StressAdapted Pathogens John Samelis and John N. Sofos
CONTENTS Introduction Foods Involved in Bacterial Foodborne Outbreaks Potential Reasons for Pathogen Emergence Effects of Stress on Bacteria Effects of Food-Related Stresses on Bacteria Research Needed to Control Stressed Pathogens in Foods Research Approaches for Control of Stressed Pathogens Novel Pathogen Control Strategies Practical Application of Pathogen Control Strategies Conclusions References
INTRODUCTION Despite the extensive scientific progress and technological developments achieved in recent years, food safety problems continue to exist and may actually increase in the future. It is estimated that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year, most due to unknown causative agents (Mead et al., 1999). Among the known pathogens associated with foodborne illness, there is an increasing involvement of environmentally resistant and host-adapted species or strains, which may be difficult to inactivate or control with traditional food preservation methods (Alterkruse et al., 1997; Foster, 1997; Tauxe, 1997). Intensified research in recent years indicates continuous adaptation and development of resistance by pathogenic microorganisms to antibiotics (Threlfall et al., 2000) and to various food-related stresses, such as low pH or acidity, heat, cold temperature, dry or low water activity environments, and chemical preservatives (Abee and Wouters, 1999; Bower and Daeschel, 1999; Brul and Coote, 1999; Sheridan and McDowell, 1998). Prolonged exposure of adapted pathogens to antibiotics and other stresses may lead to the rise of new genotypes, as a result of bacterial evolution resulting in adaptive mutations (Lederberg, 1997, 1998). These
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mutants are capable of surviving and potentially multiplying under adverse conditions, while they may also be of enhanced virulence (Archer, 1996). Indeed, in vivo studies with animals indicate that stressed bacterial pathogens may increase their virulence and, accordingly, decrease their oral infectious dose (Bearson et al., 1997; Foster, 1995; Gahan and Hill, 1999; O’Driscoll et al., 1996; Robertson and Roop, 1999; Wong et al., 1998). Examples are the multidrug-resistant Salmonella Typhimurium DT104 (Davis et al., 1999; Glynn et al., 1998) and the acid tolerant Escherichia coli O157:H7 (Armstrong et al., 1996; Park et al., 1999), both associated with recent foodborne illness outbreaks (Alterkruse et al., 1997; Mead et al., 1999). Therefore, it seems that, as a response to exposure of bacteria to sublethal stresses, the microbial ecology of our food supply is undergoing changes toward an increasing occurrence of resistant pathogens of enhanced virulence (Archer, 1996; Lederberg et al., 1992; Lederberg, 1997, 1998; Sheridan and McDowell, 1998; Sofos, 2001). The genetic and associated biochemical mechanisms that bacteria possess or develop, and then express or activate, to enhance survival in stressful environments and during food processing, are discussed in previous chapters. The scope of this chapter is to present a brief overview of existing knowledge on pathogen stress responses in correlation with known or potential consequences in foods, and to discuss potential strategies for control of stress-adapted pathogens with the objective of enhancing the safety of our food supply.
FOODS INVOLVED IN BACTERIAL FOODBORNE OUTBREAKS Occurrence of foodborne disease outbreaks has increased in the past 15 to 25 years (Bean et al., 1997; Mead et al., 1999; Tauxe, 1997) and foodborne illness remains a global problem despite major scientific and technological developments in food science and technology. Foodborne illness episodes are still frequent even in countries or regions with advanced food chains, such as the U.S., Canada and Europe. In addition to the increasing number of pathogenic agents involved (Alterkruse et al., 1999; Beuchat, 1996b; Bryan and Doyle, 1995; CDC, 1999; Davis et al., 1999; Doores, 1999; Park et al., 1999; Sterling and Ortega, 1999), the number of the types of foods associated with foodborne illness has also increased (Alterkruse et al., 1997; Beuchat, 1996a, Doores, 1999; Keene et al., 1997; Tauxe et al., 1997; Tilden et al., 1996). Examples of food vehicles and associated pathogens involved in foodborne illness episodes include E. coli O157:H7 and other hemorrhagic E. coli serotypes from ground beef, apple juice and cider, other fruit juices, alfalfa, radish and other types of sprouts, jerky, mayonnaise, watermelon, other produce and dry fermented meats; Salmonella from ice cream, cantaloupes, watermelon, potatoes, alfalfa sprouts, tomatoes, and other produce; Salmonella Enteritidis from eggs and ice cream; Shigella from produce; Camplylobacter from poultry and garlic butter; Yersinia enterocolitica from chitterlings and tofu; Yersinia pseudotuberculosis from milk, pork and possibly fruit juice; Vibrio vulnificus from oysters; Clostridium botulinum from potato salad, garlic sauce, sauteed onions, eggplant, bean dip, clam
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chowder, olives, summer sausage, and canned bamboo shoots; Listeria monocytogenes from milk, cheeses, coleslaw, hot dogs and luncheon meats; Cryptosporidium parvum from water and fresh-pressed apple juice; Cyclospora cayetanensis from raspberries and basil; Hepatitis A virus from frozen strawberries; and Norwalk-like virus from oysters, salads and frostings (www.cdc.org). Considering the increasing number of pathogenic agents transmitted by an also increasing number of foods, including products traditionally considered as low-risk, an important question is why and how specific pathogens are transferred to, and established in, specific foods to cause foodborne illness. Several reviews (Armstrong et al., 1996; Beuchat, 1996a; Gill, 1998; Sofos, 1994, 2001) and recent studies (Elder et al., 2000; Keene et al., 1997; Samelis and Metaxopoulos, 1999; Sofos et al., 1999a) have dealt with the factors leading to cross contamination of a specific food with a specific pathogen. Knowledge of the sources and routes of contamination of bacterial pathogens from the environment to the food, and their mechanisms for transfer, attachment, distribution and survival in food processing environments is essential for pathogen control (Buchanan, 1997; Tauxe, 1997). In most cases, existing knowledge on the microbial ecology of different types of foods was adequate to explain the incidence and potential establishment of a pathogen in a specific food. For example, the prevalence of Salmonella and Campylobacter on live birds, their eggs and fresh poultry (Bryan and Doyle, 1995), or that of L. monocytogenes on fresh meat (Farber and Peterkin, 1999; Sofos, 1994) are well established. Therefore, safety problems caused by these pathogens, following their survival or post-processing contamination in such products, could be expected. For example, the survival of Salmonella (Mertens et al., 1999) and L. monocytogenes (Samelis and Metaxopoulos, 1999) in stuffed ham and ham-like products due to undercooking could be recognized. Also, the growth of L. monocytogenes that potentially occurred on post-processing contaminated frankfurters and luncheon meats to result in the fatal 1998–1999 multistate outbreak in the United States (CDC, 1999) is a known safety risk associated with such products (Farber and Peterkin, 1999). Likewise, the cross contamination of ground beef with E. coli O157:H7 due to bovine fecal contamination, and the hemorrhagic colitis outbreaks caused by consumption of undercooked hamburgers (Bell et al., 1994; Riley et al., 1983) could be expected considering the habitat of the pathogen (Armstrong et al., 1996). In all these situations, the occurrence of the pathogens in the final products and the associated outbreaks were due to faulty processes that allowed survival of the food poisoning agent. In several recent instances of food products serving as vehicles of bacterial foodborne outbreaks the route of contamination and the potential for survival of the pathogenic agent were unexpected, but not surprising. For example, contamination of apple juice and cider with E. coli O157:H7 may be due to its survival in wounds of fallen apples after contact with manure (Dingman, 2000), while experimental transmission in apples by fruit flies has also been demonstrated (Janisiewicz et al., 1999a). Also, E. coli O157:H7 and Salmonella may have caused foodborne illness (Ackers et al., 1998; Besser et al., 1993; CDC, 1995a,b,c; Keene et al., 1997; Sauer et al., 1997; Tauxe et al., 1997; Tilden et al., 1996; Wall et al., 1994; Wood et al., 1991) because of their ability to attach firmly and survive on fresh produce (Beuchat,
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1996a), in unprocessed fruit juices (Miller and Kaspar, 1994; Roering et al., 1999; Ryu and Beuchat, 1999b; Zhao et al., 1993) and vegetable salads (Abdul-Raouf et al., 1993), or on dried or fermented meats and fruits (Burnham et al., 2001; Calicioglu et al., 1997; Clavero and Beuchat, 1996; Glass et al., 1992; Harrison and Harrison, 1996; Hinkens et al., 1996; Ihnot et al., 1998; Nissen and Holck, 1998; Riordan et al., 1998), with their survival being potentially enhanced by refrigeration of stored products (Clavero and Beuchat, 1996; Faith et al., 1997; Tsai and Ingham, 1997; Zhao et al., 1993; Zhao and Doyle, 1994). Water contaminated with human waste, subsequently used to apply fungicide, was reported as the potential route of contamination of raspberries with Cyclospora (Sterling and Ortega, 1999). Based on new routes of transmission of emerging foodborne diseases, Tauxe (1997) stated that, while in the past prevention involved avoidance of contamination of human food with sewage or animal manure, in the future prevention will increasingly depend on controlling contamination of feed and water by the animals themselves. In other words, pre-harvest control measures to prevent or minimize transfer of contamination with pathogens from the field or the stable to the plant need to be established. Use of water of good microbiological quality for spraying or rinsing is an essential good agricultural or processing practice, while poor hygiene and sanitation practices and inadequate chilling or cooking should be avoided throughout food processing.
POTENTIAL REASONS FOR PATHOGEN EMERGENCE The reasons for the increasing numbers of foodborne disease outbreaks in recent years seem to be multiple. They are associated with changes in consumer lifestyles and food preferences, food production and distribution practices, consumer lack of proper food handling knowledge and habits, advances in microbiological detection methods, and, more importantly, the adaptive responses of microorganisms in the environment (Alterkruse et al., 1997; Lederberg et al., 1992). Thus, the factors that contribute to pathogen emergence, either singly or through their interactions, may be classified as biological, environmental, food-related, societal and consumer-associated. As indicated, the types of pathogens that may, expectedly or unexpectedly, be found in a food niche depend on natural selection of species, bacterial evolution (Lederberg, 1997, 1998) or environmental cross contamination. The responses of bacteria to stressful environments may also lead or contribute to the emergence of pathogenic strains or species. The recognition and documentation of E. coli O157:H7, S. Typhimurium DT104 and several other previously unknown or unrecognized pathogenic bacteria, as the causative agents of diseases transmitted by foods, have been based on advanced microbiological detection methods (de Boer and Beumer, 1999; Meng et al., 1994; Swaminathan and Feng, 1994; Vernozy-Rozand, 1997). Thus, advances in molecular microbiology, immunology and detection methods have contributed significantly to the recognition of agents classified as emerging foodborne pathogens. Environmental factors associated with variations in geographic location and climate, as well as natural stresses, may also induce biological changes and lead to new pathogens or enhanced virulence. This probability is supported by differences © 2003 by CRC Press LLC
found in the microbial ecology of similar foods harvested or processed in different geographical zones or countries with diverse climates and food preferences (Mossel et al., 1995). However, the increasing international food trade and centralized food production and processing in large volumes have the potential to transfer and distribute foodborne bacteria and other food-related pathogenic agents between distant continents or countries (Kaferstein et al., 1997). Food-related factors that may lead to pathogen emergence, increased resistance or enhanced virulence include changes in food production and harvesting, processing modifications, marketing developments, preparation practices and development of new food products to meet consumer demands (Lammerding and Paoli, 1997; Zink, 1997). As mentioned, establishment and transmission of pathogenic agents is enhanced in large-scale production of agricultural products or feeding of animals. Since animal and plant diseases may cause high economical loses in such large production units, the demand for and the use of new and more effective antibiotics and pesticides in agriculture is under continuous consideration. However, by releasing large amounts of chemicals in nature and altering or increasing the level of traditional food preservation methods, humans play a major role in the development of resistance and the adaptive, cross-protective responses of bacterial pathogens to stresses (Bower and Daeschel, 1999; McManus, 2000; Tollefson and Miller, 2000). The increasing use of decontamination interventions to reduce microbial contamination on harvested produce (Beuchat and Ryu, 1997), slaughtered animals (Siragusa, 1995; Smulders and Greer, 1998; Sofos and Smith, 1998) and during other food processing steps (Sofos, 1993) may lead to new or evolving bacterial pathogens. There is a potential risk for decontamination to alter the microbial ecology of a food by substantially reducing the numbers of the food-specific natural flora, and allowing the underlying, potentially new or more resistant, pathogenic species to grow (Jay, 1996, 1997). Also, food manufacturers may adopt processing modifications which are inadequately validated for their microbiological safety, while attempting to meet consumer demands for more convenient and “healthy” preservative-free foods (Zink, 1997). These changes may lead to new sublethal stresses exerted on microorganisms or reduction of existing food preservation hurdle intensities, leading to increased pathogen resistance or virulence, as well as failure of traditional hurdles to assure food safety (Sofos, 1993). For example, product formulations with reduced fat or salt may result in increased food safety risks. This is because reduced fat levels are usually replaced with added water in the product, which further dilutes the low level of salt and other preservatives in the water phase of the product; thus, microbial growth may become more prolific (Sofos, 1993, 2001). Marketing efforts undertaken to make food products more attractive to consumers may also have an impact on pathogen emergence if factors such as the packaging conditions (e.g., the type of container, film permeability, packaging atmospheres) of the product are altered without previous testing. Such alterations may change the hurdle effect and the microbial ecology of foods and may enhance outgrowth of certain pathogens, which otherwise would be inhibited. Therefore, processes for new food products need to be validated for their ability to lead to microbiologically safe products prior to their commercial application (Lammerding and Paoli, 1997).
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Societal factors may also contribute to pathogen emergence. Following urbanization in most countries, there is a need for transportation of large amounts of food products from centralized production and processing locations to distant markets at urban centers. In developed countries, large quantities of foods are manufactured in big factories at centralized locations (Tewari et al., 1999). Seasonal food products are now available throughout the year, while consumers may travel throughout the world with no need to change their dietary preferences. Thus, food products need to be of adequate shelf life for distribution, marketing and consumption in distant areas. Also, food product transportation from exporting to importing countries is steadily increasing. Accordingly, transmission of foodborne disease and infection of large populations is much easier than in the past (Kaferstein et al., 1997; Majkowski, 1997). Improper processing, handling and storage of foods during transportation and distribution may result in foodborne illness outbreaks affecting large numbers of consumers (Hall, 1997; McMeekin et al., 1997). Furthermore, it may be more difficult to recall faulty food prior to infecting large populations of consumers (Majkowski, 1997). The globalization of the food industry and the increased travelling of people enhance the transfer of pathogenic agents between countries (Kaferstein et al., 1997), and this may also lead to new pathogens following their adaptation to diverse environmental conditions. Nowadays, patients undergo more sophisticated medical treatments, which may prolong the life of immunocompromised individuals who are more susceptible to foodborne illness from pathogens at lower infectious doses (Morris and Potter, 1997). Outbreaks are more thoroughly investigated, and detailed surveillance data are available in developed countries (Bean et al., 1997; Mead et al., 1999), while news media undertake extensive, but sometimes uninformative, reports to address an outbreak and increase public awareness. The internet and telecommunications have dramatically increased the exchange of information, and have helped consumers to express increased interest in food safety issues (Bruhn, 1997). Consumer-associated factors include changing demographics and human lifestyles, increased life expectancy, modified eating habits and, most importantly, lack of adequate food handling education (Alterkruse et al., 1997; Collins, 1997; Hall, 1997; Lederberg, 1997). Human lifestyles have undergone major changes in the last 15 to 20 years, and so have consumer demands for all goods, particularly foods (Collins, 1997). People living in big cities are interested in maintaining fitness and having a healthy diet. Therefore, it is not surprising that most consumers and public media characterize foods as “healthy” or “unhealthy” based more on a compositional, rather than a microbiological safety, basis. Consumers demand foods that have reduced levels of calories, fat and additives, while being “natural” or “organic,” and potentially having properties that do not enhance incidence of cancer, heart disease and other illnesses. New generation foods are less processed, enriched in nutrients, pre-prepared and convenient for use to reduce the time required for preparation of meals (Zink, 1997). Moreover, following the major changes in demographics and consumer lifestyles (e.g., urbanization and reduction of human populations directly involved in agriculture), domestic or small-scale production and consumption of foods has shrunk. Increasing numbers of consumers who do not prepare their own meals at home but purchase ready-to-eat foods or meals from commercial suppliers
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or food service outlets, increase the potential for spread of foodborne illness outbreaks. Moreover, it is disappointing that in urban societies most consumers lack basic knowledge on how to prepare, handle and keep food safely to protect themselves from food poisoning; this was not the case in the past, especially with people living in the countryside and involved in agriculture. Since improper food handling in the kitchen appears to be increasing (Collins, 1997), educational material through public news media and detailed food labeling (specific warnings, expiry dates and/or directions for use) have been increasingly used or legislated to enhance food safety (Bruhn, 1997). Educating consumers on proper food handling practices should be a major priority for food scientists, regulators, industry and trade associations, and public health providers (www.foodsafety.gov).
EFFECTS OF STRESS ON BACTERIA Bacteria may encounter several environmental stresses or less than optimal conditions affecting their survival and growth, and bacteria that do not find ways to cope with stress may die (Foster, 1995; Hengge-Aronis, 1993). Stresses on bacteria may include starvation, cold, heat, acid, osmolarity, low moisture, high atmospheric pressure, low oxygen or anaerobic conditions, bactericidal gases (i.e., carbon dioxide, carbon monoxide, etc.), other antimicrobials occurring naturally or applied by humans (e.g., antibiotics, sanitizers, preservatives), and competing bacteria. Exposure of bacteria to environmental stresses may be continuous and of varying intensity, starting in the soil and water, and continuing in environments where bacteria establish niches, including animal hides, harvested plant products, slaughtered animals, or plant and food service surfaces (Abee and Wouters, 1999; Bower and Daeschel, 1999). Passage of bacteria through the host may be long and tortuous (Foster and Spector, 1995; Gahan and Hill, 1999). Exposure to acid excretion in the stomach and other multiple or sequential stresses in the small intestine, such as volatile fatty acids, bile, low oxygen and competition with the intestinal flora are among the primary defensive mechanisms of the host to inactivate pathogens and prevent infection. Following invasion through the intestinal epithelial cells, bacteria may be taken up by macrophages and internalized within phagosomes, where specialized organelles prevent their multiplication by means of acidic pH and/or production of defensins, hydrogen peroxide and superoxide radicals (Foster and Spector, 1995; Gahan and Hill, 1999). Consequently, a bacterial pathogen either finds ways to survive these multiple stresses, or it dies. According to Dorman (1994), “bacterial cells have a remarkable capacity to reinvent themselves as they endeavor to adapt to changing environmental conditions. The host-pathogen interaction represents a form of biological politics in which two contending parties seek to reconcile their competing interests. If the host prevails, the infection fails; if the bacterium wins an outright victory, the host suffers disease and the bacterium may find itself without a host.” Thus, the primary effect of stress on bacteria is the triggering of mechanisms, modulated by specific genes or gene groups, to adapt, develop resistance, survive and potentially multiply under stressful conditions (Archer, 1996; Hengge-Aronis, 1993, 1996). These responses may lead to unpredictable difficulties in controlling © 2003 by CRC Press LLC
bacterial pathogens in human and animal infections, and foods (Abee and Wouters, 1999; Bower and Daeschel, 1999). Overall, bacteria attempt to cope with environmental stress by two major types of responses, namely temporary changes and mutations (Archer, 1996; Sheridan and McDowell, 1998). Detailed information on these aspects in relation to food research is given in other chapters, while the fundamentals and the latest knowledge on bacterial stress responses can also be found in a publication of the American Society for Microbiology (Storz and Hengge-Aronis, 2000). Therefore, only some principles of bacterial stress responses are summarized below. • Bacteria appear to possess complex sensory systems that alert them to the presence of one or more stresses. Such systems are always activated, irrespective of stress, when a bacterium enters into its stationary phase of growth. • Trusting their sensors, bacteria develop adaptive stress tolerance responses, and activate various defensive mechanisms, to prevent irreversible injuries, develop resistance and eventually survive the stress. • Appropriate genes are induced to activate essential defensive mechanisms, in most cases temporarily. This gene expression does not result in permanent genomic changes (mutations), and when response to a stress is not required, the genes involved are switched off. • Although stresses and their target cell sites might be different, in several cases the same or related genes, such as rpoS, are involved in the adaptive processes to regulate the cell defense. As mentioned, this often results in cross-protection effects (e.g., a bacterium successfully adapted to one stress may develop resistance to other stresses). • Cells are more resistant to stress in their stationary phase compared to the exponential phase, as they develop/possess a generalized stress response (GSR) system, which is regulated mainly by RpoS to face upcoming starvation; the GSR is independent of specific-stress. • Upon an extended exposure to one or more stresses, mutant strains may arise to enhance bacterial survival, and some of these mutants may possess increased virulence. Mutations are of two major types: spontaneous and adaptive or directed. Spontaneous mutations occur mostly in exponentially growing cells, when all intracellular activities and the replication of the genome are at high speed to respond to sudden stress. Such mutations may yield large numbers of cells, which temporarily become resistant to the stress encountered, but the mutation is not permanent to benefit the bacterial population at later times. In contrast, mutations that occur in stationary phase cells, in the absence of growth (e.g., no genomic replication), are more stable than spontaneous mutations and may be advantageous to the bacterial population as they provide permanent increased resistance to one or more stresses. When a specific selective agent is present in the environment to induce bacterial adaptation and achieve its utilization or prevention from its lethal effects, these stationary-phase mutations are termed adaptive or directed. Overall, adaptive mutations of stationary-phase cells are more frequent and, accordingly, of greater sci-
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entific concern than spontaneous mutations of growing cells. This is because mutants of bacterial pathogens triggered by exposure to specific stresses may be significantly more stress-resistant and virulent than their parental strains. Escherichia coli O157:H7, which is reported to have originated from an O55:H7 ancestor through horizontal transfer and recombination (as cited by Park et al., 1999) and to have evolved in a way that is more a Shigella with a little cloak of E. coli antigens (Lederberg, 1998), appears to be a classic example. This E. coli O157:H7 evolution may have occurred in the bovine gastrointestinal tract, where it attained an exceptional acid resistance (Armstrong et al., 1996; Park et al., 1999). In several strains of E. coli O157:H7, acid resistance is permanent, while in other strains it may be increased by a pH-inducible adaptive acid tolerance (Benjamin and Datta, 1995; Buchanan and Edelson, 1999a; Conner and Kotrola, 1995; Lin et al., 1996). To survive and grow, bacterial cells must maintain their integrity and homeostatic balance within their surrounding environment. However, environmental stresses may cause disruption of cell homeostasis, while the cell attempts to prevent or minimize such disruption (Gould, 1995; Leistner, 2000). The membrane is the cell component that primarily protects the cell from external factors and, therefore, it is the first component that suffers damage and it is where most cellular changes occur to prevent or repair damage. Exposure of most bacteria to cold temperatures induces phospholipid and fatty acid alterations (e.g., increases in the proportion of unsaturated fatty acids in the cell membrane), resulting in increased membrane fluidity (Berry and Foegeding, 1997; Sofos, 1989). Also, in most bacteria, specific sets of cold shock proteins are induced upon abrupt shifts to cold temperatures, and functional enzymes become simpler in structure and more flexible. These changes enhance survival and potential growth in cold environments, but at much lower than optimal reaction rates (Berry and Foegeding, 1997). Opposite phenomena occur when cells are exposed to elevated temperatures. The concentration of saturated fatty acids in the membrane increases, and there is a heat shock response expressed by the synthesis of specific proteins, which results in increased thermotolerance. Changes in membrane lipid composition may also confer increased resistance to certain antimicrobials, which may attack the cell by binding on, creating pores and disrupting the proton motive force of the membrane (Berry and Foegeding, 1997; Sofos and Busta, 1999). Weak organic acids penetrate the cell membrane in their undissociated form and thereafter dissociate and acidify the cytoplasm, leading to cell death (Alakomi et al., 2000; Gould, 1995; Young and Foegeding, 1993). Bacteria respond to the lowering of the intracellular pH by expelling protons and by regulating the pH membrane gradients (Diez-Gonzalez and Russell, 1997; Dilworth and Glenn, 1999; Gould, 1995; Slonczewski and Foster, 1996; Sofos and Busta, 1999). Also the membrane cyclopropane fatty acid content is a major factor in acid resistance of E. coli (Brown et al., 1997; Chang and Cronan, 1999). Another example of bacterial response to stress is that associated with osmotic pressure. When the osmotic pressure in the surrounding environment increases, water efflux occurs from the cell; to prevent shrinkage and eventually plasmolysis, cells
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activate osmoregulation systems (i.e., mechanisms that provide equilibration of the intracellular with the environmental pressure) (Gould, 1995; Pichereau et al., 2000). Osmoregulation is achieved either by active passage of charged (e.g., K+ or glutamate) solutes, followed by passage of compatible solutes (e.g., trehalose, proline, glycine betaine, carnitine), or by de novo biosynthesis and accumulation of such osmoprotectants, when they are not available in the substrate (Pichereau et al., 2000). Bacterial stress responses become more complex when cross induction of protective mechanisms occurs, which confers cross-protection to several stresses regulated by the same gene or group of genes, such as rpoS (Bearson et al., 1997; HenggeAronis, 1993, 1996). As mentioned, significant progress has been made in recent years in elucidating the underlying genetic and biochemical mechanisms that protect bacteria from stress, but still these mechanisms need further elucidation (Storz and Hengge-Aronis, 2000).
EFFECTS OF FOOD-RELATED STRESSES ON BACTERIA Food-related stresses may occur naturally or may be applied purposefully or inadvertently during processing and storage of foods to inactivate or prevent growth of spoilage and/or pathogenic microorganisms (Archer, 1996; Bower and Daeschel, 1999). It is important to consider that there is a high degree of similarity between the stresses bacteria encounter in the host and those in foods and food processing environments (Sheridan and McDowell, 1998). For example, major stresses present in the human body, such as acid, osmolarity, anaerobiosis, and temperature shifts from the host to the environment at excretion, are also present in acidic, dried, cooked/refrigerated, and canned or frozen foods, respectively (Table 9.1). Thus, there
TABLE 9.1 Food-Related Stresses against Pathogenic Bacteria in Food Environments, Processes and Products Stress Acid Heat Cold Osmolarity Oxidation Anaerobiosis Starvation
Food Products or Processes Mayonnaise, juices, fermented foods Cooking, minimum thermal processing Refrigeration Brine, marinades, fish Hydrogen peroxide treatment Vacuum packaging, sous vide Food contact surfaces
(Modified from Sheridan, J.J. and McDowell, D.A., Meat Sci., 49, 5151, 1998.)
is a need to consider foods as potential stressful environments for bacteria, because “stresses introduced in foods by preservation or naturally present in foods have a © 2003 by CRC Press LLC
profound effect on gene expression in bacterial pathogens” (Archer, 1996). This indicates that the intensity of total stress that may be encountered by bacteria in a food is a function of the nature of the food and its “hurdle effect,” as affected by the type, number and severity of technologies applied to preserve it (Leistner, 2000). When the intrinsic, extrinsic, processing or implicit factors affecting the dynamics of microbial growth in foods (Mossel and Ingram, 1955; Mossel et al., 1995) move toward their upper or lower limits during food processing or storage, they act as hurdles which stress the bacteria (Gould, 1995; Leistner, 2000; Sofos, 1993). Hurdles in foods are dynamic as they continuously change during processing and storage (Sofos, 1993), while in most instances they are combined to exert a synergistic antimicrobial effect as multiple barriers (Leistner, 2000). As indicated, hurdle factors that may stress foodborne microorganisms include, among others, temperature (Palumbo, 1986; Berry and Foegeding, 1997), pH (Dilworth and Glenn, 1999; Rowbury, 1997), water activity (Gailani and Fung, 1986; Pichereau et al., 2000), redox potential and anaerobiosis (Potter et al., 2000), carbon dioxide (Genigeorgis, 1985; Hotchkiss and Banco, 1992), sodium chloride (Sofos, 1984), other chemical preservatives (Brul and Coote, 1999; Sofos, 1989; Sofos and Busta, 1999), natural antimicrobials (Sofos et al., 1998), nutrient limitation or starvation (Gill, 1976; Rees et al., 1995), and microbial competition (Fredrickson and Stephanopoulos, 1981; Hugas, 1998). How, and to what extent, these factors shift from optimal to stressing and, thus, cause variable effects on bacterial survival or growth depends on the compositional, processing and storage conditions of the food, as well as on the physiological and biochemical properties of the bacterial populations present in the food (Mossel et al., 1995; Sofos, 1993). Bacteria may respond to stressful food environments in the same manner they respond to any stress in nature (i.e., by developing respective tolerances, such as acid tolerance, osmotolerance, thermotolerance, cryotolerance and tolerance to oxidative stress). Expression of the induced tolerances, which may potentially shift to longer term resistances, depends on stress intensity and whether acid, heat, cold and other stresses are applied instantaneously or increase gradually over longer periods of time in the food and, more importantly, in the food plant environment. In most cases, the bacterial responses to stress involve complex mechanisms, which may be regulated by groups of genes (Archer, 1996; Sheridan and McDowell, 1998). Expression of these genes may be variable with bacterial species, and may be affected by the physiological state of the cells, the rate and severity of the stress applied and other environmental factors. For example, one of the most important and intensively investigated food-related resistances of bacterial pathogens is the acid tolerance response (ATR) (Davis et al., 1996; Foster, 1995; Rowbury, 1997; Slonczewski and Foster, 1996). The development of acid tolerance can be pH-dependent, pH-independent or a combination of both types, depending on the growth phase of the bacteria undergoing the acid stress (Lee et al., 1994; Lin et al., 1995). There are at least 11 different ATRs induced under various conditions during growth (Rowbury, 1997), which differ from the acid resistance (AR) responses that only occur in complex culture media during stationary phase (Lin et al., 1995, 1996). Overall, bacterial pathogens acquire increased resistance to pH upon exposure to sublethal pH while they are growing exponentially (Davis et al., 1996; Jordan
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TABLE 9.2 Effect of the Type of Acid on the Inactivation Rate of AcidAdapted (TSB+G) or Nonadapted (TSB-G) Enterohemorrhagic Escherichia coli Strains at pH 3.0 and 37ºC Cells Grown In Strain S2 S4 S8
TSB+G
TSB-G
HCl = malic
HCl<malic
TSB+G: Tryptic soy broth with 1% added glucose TSB-G: Tryptic soy broth without glucose (Modified from Buchanan, R.L. and Edelson, S.G., J. Food Prot., 62, 211, 1999a.)
et al., 1999; Lin et al., 1996; O’Driscoll et al., 1996). In addition, they possess a GSR that is expressed upon entry into stationary phase, results in a pH-independent acid tolerance and is regulated by RpoS (Arnold and Kaspar, 1995; Cheville et al., 1996; Hengge-Aronis, 1996). The complex mechanisms and regulation of expression of acid resistance, the description of which is out of the scope of this chapter, may overlap in a way in which more than one ATR mechanisms are simultaneously activated to protect the cells in concert (Dilworth and Glenn, 1999; Jordan et al., 1999; Lin et al., 1995, 1996). To what extent each of the concerted ATR contributes to acid resistance expressed under a given set of environmental conditions is difficult to determine. For example, stationary-phase cells may exhibit an induced pH-dependent acid resistance, which further increases their GSR (Buchanan and Edelson, 1996, 1999a; Foster, 1995; Lee et al., 1994), but this response varies greatly between different strains, and in relation to the type of acidulant (Table 9.2). Also, different bacterial genera or species, such as Salmonella, Shigella, and E. coli, possess different combinations of ATR (Lin et al., 1995). The complexity of various bacterial responses to acid has resulted in variation in the techniques and the terminology used to describe these phenomena in food studies. In general, shifting an exponentially growing culture from neutral to sublethally acid conditions (pH ≤ 5.5) may confer protection of pathogens to lethally acid conditions. When acid shocked cells are subsequently exposed to pH values below 4.0, they may exhibit an increased ATR and cross-protection to other stresses compared to non-shocked cells (Davis et al., 1996; Leyer and Johnson, 1993; Leyer et al., 1995; Lou and Yousef, 1996, 1997; O’Driscoll et al., 1996). This approach would be better described as acid shock (Ryu et al., 1999a), rather than acid adaptation (Leyer and Johnson, 1993; Leyer et al., 1995; Lou and Yousef, 1997; O’Driscoll et al., 1996). Another approach is to produce stationary-phase cells under conditions that result in acid adaptation, (e.g., culturing E. coli O157:H7 in glucose containing media to induce higher levels of ATR compared to cultures grown without glucose) (Buchanan and Edelson, 1996). When acid-adapted cells are subsequently © 2003 by CRC Press LLC
TABLE 9.3 D-Values (min) at Different Heating Temperatures of Previously Non-Heat-Shocked and Heat-Shocked Escherichia coli O157:H7 in Correlation with the Nature of the Heated Substrate Substrate Tryptic soy broth
Ground beef slurry
D-Value at Temperature
Non-Heat Shocked
Heat Shocked (45ºC, 30 min)
54 58 62 58
12.1 2.2 0.6 4.2
16.6 3.7 0.9 4.1
(Modified from Williams, N.C. and Ingham, S.C., J. Food Prot., 60, 1128, 1997.)
transferred to real or simulated food environments with low pH, they demonstrate an enhanced survival compared to the non-adapted cells (Berry and Cutter, 2000; Buchanan and Edelson, 1999a; Gahan et al., 1996; Leyer and Johnson, 1992; Ryu et al., 1999a). Also, acid adapted cells may be cross-protected against heat or other stresses (Buchanan and Edelson, 1999b; Ryu and Beuchat, 1999a,b). Acid adaptation may be considered more realistic than acid shock in food microbiology studies because microorganisms in foods are more often likely to occur in stationary phase under nutrient deprivation (Rees et al., 1995), thus, with an activated GSR. In addition, acid conditions in many foods are built up slowly by fermentation (Caplice and Fitzgerald, 1999; Lucke, 2000; Samelis et al., 1998), enabling bacterial acid adaptation by exposure to a gradual decrease in pH, rather than an immediate exposure to low pH. There are, however, food processes or interventions, which cause acid shock to bacteria as, for example, processing of acidified foods and decontamination of meat or fresh produce with organic acids (Beuchat and Ryu, 1997; Smulders and Greer, 1998). In addition to acid, other stresses (e.g., heat, cold) may also be applied either instantaneously (shock) or gradually (adaptation) during food processing. Heat shock has been shown to increase the D values of E. coli O157:H7 (Table 9.3) and Salmonella Enteritidis (Table 9.4), especially in culture broth and under anaerobic conditions of growth. On the other hand, the tempering or slow heating rates of bulk foods, such as cooked ham and other cured meat products permit gradual bacterial exposure and adaptation to heat and may result in survivors of increased thermal resistance in the finished product (Carlier et al., 1996; Mackey et al., 1994; Samelis and Metaxopoulos, 1999). Likewise, slow cooling rates after cooking and prolonged periods of drying or refrigerated storage of foods enhance adaptation to cold environments or low water activity (Mackey et al., 1994). In contrast, the spraying of carcasses with hot water (Sofos and Smith, 1998), the rinsing of fresh produce with nonacid disinfectants (Beuchat and Ryu, 1997), the spray-drying of liquids to form powders, the blast freezing of small food pieces (e.g., patties, sticks), or the cleaning of food equipment surfaces with sanitizers (Kumar and Anand, 1998; Mah and O’Toole, 2001) are short-term shocking treatments. Some of these treatments, how© 2003 by CRC Press LLC
TABLE 9.4 D-Values (min) of Heat-Shocked and Non-Heat-Shocked Salmonella Enteritidis in Culture Broth Heat Shock (42ºC, 60 min) Control Heat shocked Heat shocked
D-Value at Temperature (ºC) Incubation Atmosphere
52
58
Aerobic Aerobic Anaerobic
5.3 16.9 20.0
0.9 1.3 1.8
(Modified from Xavier, I.J. and Ingham, S.C., J. Food Prot., 60, 181, 1997.)
ever, may be followed by extended times of exposure to the residual activity of the stress applied, resulting in adaptation also. Starvation is probably the most common stress on bacteria in foods (Rees et al., 1995). Although starvation does not induce a specific stress resistance per se or trigger mutations (Archer, 1996) it induces multiresistances as it accelerates bacterial entry into the stationary phase to enhance survival (Hengge-Aronis, 1993; Pichereau et al., 2000). As a consequence, starvation has been associated with cross-protection to several stresses (Arnold and Kaspar, 1995; Jenkins et al., 1990; Nystrom et al., 1992), while, as mentioned, any stress can confer cross-protection to another stress sharing regulatory systems (Rowe and Kirk, 1999). For example, starvation and acid adaptation (pH 4 to 7) increased thermotolerance (56°C) of L. monocytogenes (Lou and Yousef, 1996), whereas starvation induced cross protection against osmotic challenge in E. coli (Jenkins et al., 1990). Acid adaptation (pH 5.5, 1 h) protected L. monocytogenes against thermal (54°C) and osmotic (2.5 M NaCl) stresses (O’Driscoll et al., 1996). An increased thermotolerance at 50°C was induced by high osmolarity (0.15 to 0.3 M NaCl) in Salmonella (Fletcher and Csonka, 1998). Acid adaptation (pH 4.5 to 5.0) protected L. monocytogenes against lethal doses of ethanol (17.5% vol/vol) and hydrogen peroxide (0.1% wt/vol) (Lou and Yousef, 1997), while it cross-protected (pH 5.8) S. Typhimurium against heat (50 to 57.5°C), salt (2.5 M NaCl) and an active lactoperoxidase system (Leyer and Johnson, 1993), and provided (pH 5.5) cross-protection to sodium lactate (10 to 30%) and sodium chloride (5 to 15%) in E. coli O157:H7 (Garren et al., 1998). Adaptation to ethanol (5%, vol/vol) increased resistance of L. monocytogenes to 25% sodium chloride (Lou and Yousef, 1997). Heat shock (45°C for 1 h) increased resistance of L. monocytogenes to 1% hydrogen peroxide (Lou and Yousef, 1997), while it enhanced (48°C, 10 min) acid tolerance (pH 2.5, minimum glucose medium) of E. coli O157:H7 (Wang and Doyle, 1998). Habituation of Salmonella spp. at reduced water activity (0.95) in media with various solutes increased its heat tolerance (Mattick et al., 2000b). Listeria monocytogenes cultivated under low nutrient conditions showed increased tolerance to chlorine sanitizers (Lee and Frank, 1991). In general, research and experience have shown that foodborne bacteria display a broad spectrum of resistance responses to common food preservation techniques, and resistant bacterial pathogens, such as E. coli O157:H7 (Armstrong et al., 1996;
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Park et al., 1999), S. Typhimurium DT104 (Davis et al., 1999; Glynn et al., 1998), C. jejuni (Alterkruse et al., 1999; Humphrey, 1995), and L. monocytogenes, have emerged and are a global problem (Foster, 1997; Mead et al., 1999; Tauxe, 1997). These pathogens and other causative agents of foodborne illness outbreaks may possess unusual growth characteristics, may be multi-stress and multi-drug resistant or may be virulent at low infective doses (Archer, 1996; Gahan and Hill, 1999; Threlfall et al., 2000). The potential existence, development or establishment of such multi-stress-resistant pathogenic strains in food environments may explain why traditionally low risk foods, such as fruit juices (Besser et al., 1993), fermented meats (CDC, 1995a,b,c), fresh produce (Ackers et al., 1998), and dried products (Keene et al., 1997) have become vehicles of foodborne illnesses. This suggests that traditional preservation barriers are becoming ineffective against multi-stress resistant pathogenic bacteria developing in our food supply. Also, the intentional or inadvertent exposure of bacteria to sublethal food-related stresses may enhance their stress adaptation and survival under unfavorable conditions. Thus, development of bacterial resistances and cross-protection effects, which are well documented with pure pathogenic cultures grown under laboratory conditions, may also be a reality in foods. Since adaptive mutations of pathogens may also be associated with increased virulence (Sheridan and McDowell, 1998), stress-adapted, multi-resistant mutants that may develop in food environments may be difficult to control and may dramatically increase food safety risks. This concern is particularly important for at-risk populations, such as the elderly, infants, children, pregnant women, patients with chronic diseases and other groups of immunosuppressed individuals (Morris and Potter, 1997). In summary, any food processing, decontamination or storage method has the potential to cause shock, adaptation, or shock/adaptation, and lead to cross-protection to environmental food-related stresses. This necessitates the reconsideration of traditional food preservation barriers by including pathogen stress responses in food validation studies to avoid underestimation of potential risks and to enhance food safety (Knochel and Gould, 1995; Sofos, 1993, 2001).
RESEARCH NEEDED TO CONTROL STRESSED PATHOGENS IN FOODS The complexity of, and our developing but still limited knowledge on, the microbial responses to environmental stresses makes the control of stress-adapted pathogens in foods a difficult task. Achieving this objective, however, is a necessity that will challenge food microbiologists in the coming years. According to Lederberg (1997), “we have barely started to study the responses of microorganisms under stress, although we have examples where root mechanisms of adaptive mutability are themselves responses to stress.” Development of strategies for pathogen control in foods should aim at: (1) controlling resistant and stress-adapted pathogens; (2) minimizing resistance development; and (3) avoiding virulence changes. Thus, the overall strategic plan for pathogen control should have two major targets: (1) trying to maintain control of
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the existing situation and (2) preventing the existing situation from becoming worse. Bower and Daeschel (1999) proposed five approaches to control stress resistant bacterial pathogens in foods and, overall, in nature: (1) change antibiotic usage practices; (2) develop new antibiotics; (3) apply hurdle preservation approaches; (4) prevent bacterial adhesion; and (5) utilize competitive exclusion. These approaches are among the many useful ones that can be applied, and indicate the need to consider foods as a part of the environment, which is the main reservoir of stress-resistant microorganisms. On this basis, it is important to consider that, while antibiotic-resistant bacteria are an increasing problem for patients at home or in hospitals, current information on the potential of bacteria to overcome food preservation hurdles solely because of their drug resistance is scanty. Indeed, multidrug-resistant pathogens, such as S. Typhimurium DT104, have been isolated from animal feeding and food processing environments (Davis et al., 1999), but the impact of their antibiotic resistance on survival under stressful conditions in foods is unclear. Thus, more research is needed to address the problem by a better understanding of potential cross effects between antibiotics and other stress factors on bacteria. Research should be multidisciplinary and proactive to determine mechanisms and controls rather than only describe unpredicted bacterial responses (Buchanan, 1997; Sofos, 2001). In addition, basic research should investigate stress responses at the cellular or subcellular level. Factors involved in global stress response need to be identified, while accurate determination and correlation of the physiological reactions of bacteria responding to stress with the respective biochemical and genomic alterations occurring inside the bacterial cell are required. Continuous updating of our knowledge on microbial genetics and physiology is a prerequisite for a better understanding of bacterial responses to stress. Stress-related mutations and gene transfer, growth requirements, survival characteristics and pathogenicity of bacteria under stress and their mechanisms to cope with the stress by maintaining cell homeostasis and developing resistance or enhanced virulence need further elucidation (Buchanan, 1997; Lederberg, 1997; Sofos, 2001; Tauxe, 1997). Research should also examine whether responses similar to those triggered in vitro or in vivo in the laboratory possibly occur in situ in multifactor ecosystems, including foods. This is because, unlike pure cultures in laboratory media, bacterial pathogens in foods are usually a minor component of a complex microbial association dominated by food-specific, environmentally adapted microbial species (Aggelis et al., 1998; Dainty and Mackey, 1992; Mossel et al., 1995; Samelis et al., 1998; Sofos, 1994). While the food itself may represent an oligotrophic and stressful environment, the different microorganisms present compete strongly for uptake of limiting nutrients to initiate growth, and eventually predominate. Thus, survival and development of pathogens in foods frequently takes place under limited nutrient availability and adverse conditions of pH, osmolarity, oxidation, temperature, chemical residues and competition by other microorganisms. Therefore, to accurately determine or predict the response of a pathogen to stress, we need to consider all micro-ecological factors involved in the processing of a specific food. Intensified research is needed to clarify the behavior of foodborne pathogens in their natural environment. Despite progress in epidemiology, the origins and main reservoirs of contaminating pathogenic strains, and the sources and routes of con-
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tamination (e.g., pathways of transmission from non-food to food environments or throughout the food processing line) are largely unknown. This is because, until recently, characterization of foodborne microorganisms, including pathogens, was primarily based on physiological, biochemical or immunological criteria determined by classical methods (Farber, 1996). These methods are valuable, but they are timeconsuming and cannot follow up bacterial transmission and cross contamination involved in the chain of food processing steps. However, the recent developments in polyphasic taxonomy, based on genotypic molecular characterization and fingerprinting methods (Vandamme et al., 1996), should enable food microbiologists to compare bacterial strains originating from different natural environments, food niches or food processing steps, and thereby permit the establishment of their mode of transmission (Farber, 1996). Another limitation has been the difficulty in detecting small numbers of unpredictably distributed target pathogens among high numbers of a diverse, non-pathogenic, spoilage flora in complex and nonhomogeneous foods. As sampling and detection methods improve (Brown et al., 2000; de Boer and Beumer, 1999; Manafi, 2000; Swaminathan and Feng, 1994; Vernozy-Rozand, 1997), their application will enhance pathogen surveillance during food processing. For example, advanced molecular techniques, such as denaturating gradient gel electrophoresis (DGGE), are currently in progress and may allow rapid monitoring of microbial population dynamics directly in a food, without the need of obtaining bacterial isolates (Ben Omar and Ampe, 2000; Cocolin et al., 2001). Also, on line pathogen detection in the field and in the food industry with biosensors is receiving increasing attention. There is also a need to improve virulence testing and toxin detection methods (Pimbley and Patel, 1998) and to investigate virulence changes in relation to resistance mechanisms and the behavior and stress response of pathogenic bacteria in foods. At present, there is evidence that stress enhances virulence under laboratory conditions (Archer, 1996; Buncic and Avery, 1998; Foster, 1995; Gahan and Hill, 1999; O’Driscoll et al., 1996). However, whether this response may also be inducible to stressed pathogens present in foods, and the magnitude of increased virulence these mutants may attain compared to their non-stressed parental strains, are unknown. It is, therefore, necessary to investigate potential strain variations in stress resistance and virulence in relation to the sources and routes of food contamination and the sensitivity of at-risk populations (Takumi et al., 2000). Further areas to be investigated for more effective control of resistant pathogens in foods include the mechanisms through which bacteria examine and sense their surrounding environment (Dorman, 1994; Lazim and Rowbury, 2000; Rowbury, 1997, 2001), how they process and transmit this information to their genome, and how the mechanisms of changes in gene expression are activated (Storz and Hengge-Aronis, 2000). These are challenging research issues that require copious studies. In addition to the elucidation of the genetics and physiology of resistant pathogens and determination of mechanisms of survival and resistance development in restrictive environments, advanced food-related research approaches are required. As indicated, the ideal approach is to evaluate food systems by considering them as stressful environments. On this basis, farm to fork evaluations and measures are needed to increase the effectiveness and practical significance of the research under-
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taken. Animal and plant production practices may be decisive for induction of pathogen resistance. For example, the potential role of diet shifts on the acidresistance of E. coli in cattle has been an important, but controversial, research issue in the United States in recent years (Diez-Gonzalez et al., 1998; Russell et al., 2000). This underscores the difficulty in understanding complex responses of foodborne pathogens in complex environments. Also, pathogens resistant to antibiotics, pesticides and other chemical residues are increasingly being isolated from animal feeding (Davis et al., 1999; Tollefson and Miller, 2000) and plant cultivation (McManus, 2000) environments, which otherwise would have been exposed to normal-range magnitudes of natural environmental stresses. Exposure of multidrug-resistant isolates from these habitats to common stresses encountered in food systems may reveal potential correlation between those two types of resistances. An additional area to be examined by reasearch is bacterial stress resistance development in response to changing food preparation practices at home. As indicated, increasing numbers of consumers lack the knowledge necessary to handle and prepare foods safely, while preparation of mildly cooked or undercooked meals and application of non-validated cooking procedures in microwave ovens or other home appliances is increasing. Although these consumer-related changes in food preparation may induce bacterial resistance, their impact on food safety has been largely overlooked. During home-drying of foods, for example, multiple stresses are applied (e.g., marination, gradual heating to enhance vaporization, decrease in water activity and increase in osmolarity due to water removal and salting of cured meats, low pH and acidity due to concentration of solids, etc.). However, the magnitude of stresses and the drying times may be low and long enough, respectively, to allow stress adaptation, resistance development and eventually survival of bacterial pathogens in the final, ready-to-eat product. Following that, some bacterial pathogens may survive during storage of dried foods, temperature and atmosphere permitting, while others become inactivated. Recent research data support these concerns. For example, acidsensitive enteric pathogens were protected from killing under acidic conditions when attached onto solid foods, such as ground beef (Waterman and Small, 1998), while an increased thermotolerance may be induced by high osmolarity in Salmonella (Fletcher and Csonka, 1998; Mattick et al., 2000b). Salmonella Enteritidis and S. Typhimurium DT104 could survive at a water activity of 0.92 for long periods by increasing their biomass via filament formation, and their survival was enhanced as the temperature was lowered (Mattick et al., 2000a). As expected, increasing the osmotic pressure by addition of sodium chloride increased the lag phase and decreased the growth rate of L. monocytogenes (Vasseur et al., 1999); however, the osmotically stressed pathogenic cells survived on processed meat surfaces by accumulating osmolytes (Smith, 1996). The inactivation of E. coli exposed to osmotic stress was biphasic; the initial high death rate was followed by a slower second phase decline, or “tailing” effect (Shadbolt et al., 1999). Osmotic resistance of E. coli was greater upon starvation (Jenkins et al., 1990), while exposure of E. coli O157:H7 to acidic environments increased its heat tolerance (Buchanan and Edelson, 1999b; Ryu and Beuchat, 1999a); a major concern when drying fruits at home after acid soaking.
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Escherichia coli O157:H7 could survive in dried beef powder and other foods, but its survival was suppressed as the water activity and the pH of the product were decreased and the storage temperature was increased (Deng et al., 1998; Ryu et al., 1999b). Also, previous storage or pre-incubation of ground beef at higher temperatures resulted in increased sensitivity and decreased survival of E. coli O157:H7 following a decrease in its acid tolerance (Cheng and Kaspar, 1998). Importantly, E. coli O157:H7, as well as Salmonella and L. monocytogenes, increases its ability to recover heat damage in foods when stored under anaerobic conditions or under a low redox potential; thus, the anticipated level of safety in foods dried by heating might not be achieved (George et al., 1998; Murano and Pierson, 1993). In summary, plant cultivation, animal feeding, and food production, harvesting, transportation, decontamination, sanitation, processing, distribution and storage technologies, as well as food preparation methods in restaurants, catering or at home, need to be evaluated for contribution to bacterial resistance. This evaluation is important to perform for both the traditional and novel technologies and procedures used in food processing and handling, by considering all variables that may contribute to resistance.
RESEARCH APPROACHES FOR CONTROL OF STRESSED PATHOGENS In order to meet current research needs associated with control of stress-adapted pathogenic bacteria in foods, we also need to advance our research approaches by introducing more complex and challenging objectives and multifactorial experimental designs, and by using advanced microbiological detection and typing methods. Considering the recent salmonellosis and hemorrhagic colitis outbreaks implicating acidic, fermented, or home-dried foods, it appears necessary to reconsider critical limits at critical control points for traditional food manufacturing practices. Advanced research approaches are required to explain complex mechanisms of pathogen emergence, resistance and virulence in foods and to avoid false estimations of potential food safety risks. Significant modifications in current food production, processing and handling systems may be necessary to avoid or minimize resistance and virulence changes in bacteria and to assure food safety. Modifications should be based on results of welldesigned pathogen challenge and validation studies (e.g., finding combinations of interventions or processes that guarantee the delivery of pre-specified reductions of target pathogens) to achieve established food safety objectives (van Schothorst, 1998). These studies should be based on worst case scenarios (e.g., high levels of food contamination with very resistant pathogenic strains; pathogen protection and survival enhancement due to the food microenvironment; food storage under conditions that prevent destruction of survivors, etc.). For example, in validation studies, it is important to use multistrain and stress-resistant inocula, which originate from similar niches and, better yet, have been exposed to conditions as stressful as they are expected to encounter in real foods. Thus, inocula can be previously grown in synthetic media of minimal composition or food extracts, in the presence of chemical
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TABLE 9.5 D-Values (min) of Escherichia coli O157:H7 at Different Heating Temperatures or Concentrations of Lactic Acid as Affected by the Growth Temperature of the Pathogen Prior to Exposure to Heat or Acid and the Origin of the Strain Culture Growth Temperature (ºC)
Origin of Strain
10
Beef Cider Beef Cider
Heating Temperature (ºC)
Lactic Acid Concentration (%)
52
56
0.1
0.5
11.2 40.7 17.7 89.4
2.5 5.1 9.3 14.1
3.7 5.3 6.4 4.8
0.35 0.44 1.7 0.47
(Data from Semanchek, J.J. and Golden, D.A., J. Food Prot., 61, 395, 1998.)
or natural preservatives, or competitive flora, or harvested following prior habituation/growth in liquid foods or on solid food surfaces. Strains that have previously been isolated from outbreaks caused by related foods and are of increased resistance or virulence are the best candidates for inclusion in multistrain inocula. For example, an outbreak E. coli O157:H7 strain associated with apple cider demonstrated higher D values than a strain from beef (Table 9.5), potentially due to its origin from a more stressful, acidic food environment. For this reason, it is essential to deposit in culture collections more strains and their respective epidemiological and biochemical data, isolated from foodborne outbreaks and indicating unusual stress resistance. It is very important to understand the significance of testing resistance of stressed bacterial populations isolated directly from foods, rather than to monitor the survival of purposefully stress-adapted laboratory cultures inoculated in challenge media or foods. On this basis, research should investigate pathogen responses to sequential applications of stresses of varying intensity in situ in foods. Research with mixed microbial cultures in real foods, or in media that are simulatory to foods, should be intensified. In other words, we need to determine survival and development of resistance of surviving pathogens to initial or secondary food-related stresses, with the natural flora present, and to follow or simulate their passage from diverse environments in the field to the food processing chain and the host. Succession of stresses, as affected by their type, order of application, magnitude and duration, may allow survival and resistance development. The responses of surviving pathogen populations in the presence of food-specific natural flora should be examined. This is necessary because the metabolic activity and competition by this natural flora may induce additional stresses, may alter the intensity of existing stresses, or may affect the sensing ability and the timing of pathogen responses to these stresses. Indeed, recent data from basic microbiological studies have revealed that early sensing of environmental stresses by bacteria may be due to extracellular
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sensors to alarmones and cross-talking between cells rather than due to intracellular compounds (Lazim and Rowbury, 2000; Rowbury et al., 1998; Rowbury 2001). Thus, compounds found or added to a food or formed by natural flora may act as extracellular stress sensors to alarmones to pathogenic cells, or may suppress the formation of such alarmones to eventually sensitize pathogens. Alarmones and their precursors or inhibitors may be absent in cultivation or challenge media, which are sterile or defined in chemical composition. It appears, therefore, that the natural competitive flora may do more than simply affect growth, which is well documented in the literature (Breidt and Fleming, 1998; Buchanan and Bagi, 1999; Drosinos and Board, 1994; Duffy et al., 1999; Farrag and Marth, 1989; Janisiewicz et al., 1999b). Competitive or synergistic interactions of mixed microbial associations may alter or monitor stress resistance responses of bacterial pathogens under conditions encountered in food environments, which may have a significant impact on food safety (Samelis et al., 2001b). Advanced research approaches are needed to investigate long-term effects of food processing, decontamination and storage technologies on development of bacterial stress resistance and to examine subsequent responses of surviving bacteria exposed to a set of stressors. The benefits from the commercial application of novel technologies may be attractive enough to adopt them without much concern or skepticism as to potential negative impacts on the environment or humans and without evaluation of potential undesirable effects. For example, modified atmosphere packaging (MAP) of foods had been introduced commercially for at least 20 years before major concerns were expressed regarding the microbiological safety of this technology (Genigeorgis, 1985; Hotchkiss and Banco, 1992; Sofos, 1993). It was suggested that low-oxygen packaging methods may enhance survival and growth of facultative or anaerobic pathogens, such as L. monocytogenes and clostridia. The concerns may have been reasonable, as indicated by the early botulism outbreaks associated with consumption of vacuum packaged smoked fish in the 1960s and the recent 1998–1999 listeriosis outbreak from the consumption of ready-to-eat vacuum packaged processed meats in the United States (CDC, 1999). The latter outbreak renewed interest on hurdles for control of post-processing contamination of L. monocytogenes in processed meat products. Potential hurdles under investigation are the inclusion of chemical antimicrobials in the formulation (Bedie et al., 2001; Blom et al., 1997; Harmayani et al., 1993; Wederquist et al., 1994), immersion in post-processing antimicrobial solutions (Samelis et al., 2001e), post-packaging thermal pasteurization (Roering et al., 1998), inoculation with protective cultures (Bredholt et al., 1999; Degnan et al., 1992; Hugas, 1998; McMullen and Stiles, 1996) and potential application of emerging technologies such as high pressure and irradiation (Farber and Peterkin, 1999). Most of these hurdles have shown promise in controlling L. monocytogenes, but their potential long-term effects in inducing stress resistances to pathogen survivors in the packaged food or in the host and the environment are largely unknown. Another recent case of using a new technology without really knowing its potentially long-term negative impact on bacterial stress resistance is decontamination of fresh foods. As mentioned, the commercial use of antimicrobial interventions to reduce contamination of meat and fresh produce with pathogenic bacteria is
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steadily increasing, particularly in North America (Beuchat and Ryu, 1997; Siragusa, 1995; Smulders and Greer, 1998; Sofos and Smith, 1998). Also, following several outbreaks from fresh produce in recent years, consumers have adopted recommendations to increase washing of raw foods with disinfectants at home. Research has clearly demonstrated that decontamination technologies or treatments with disinfectants deliver significant reductions (1 to 3 logs) in populations of total and pathogenic bacteria, particularly in those of enteric pathogens such as E. coli O157:H7 and Salmonella (Cutter and Siragusa, 1994; Dorsa et al., 1998a; Hardin et al., 1995; Smulders and Greer, 1998; Sofos and Smith, 1998). However, the impact of heat, organic acids, sanitizers and other types of chemicals used as decontaminants or disinfectants in inducing resistance to pathogens surviving the process or contaminating the food post-decontamination has not been addressed adequately. Most studies are limited at determining the immediate antimicrobial effect of decontamination on the target pathogens or the total bacterial populations (Bacon et al., 2000; Beuchat, 1996a; Delmore et al., 2000; Hardin et al., 1995; Siragusa, 1995; Smulders and Greer, 1998; Sofos and Smith, 1998). In recent years, however, there is an increasing scientific interest in evaluating the consequences of decontamination on the microbial ecology of fresh meat and produce and the impact of possible implications of flora changes on product safety and shelf life (Jay, 1996, 1997; Van Netten et al., 1994). In response, the residual effect of several intervention treatments on bacterial growth during subsequent refrigeration storage of products that were intact after decontamination or originated from decontaminated primal units was evaluated (Dorsa et al., 1998b; Kenney et al., 1995; Prasai et al., 1997; Van Netten et al., 1994, 1998). Results were conflicting as to whether growth of natural flora and inoculated pathogens was affected in stored product, while experimental conditions varied among studies to allow for clear conclusions. However, an overall conclusion has been that pathogens that may survive or contaminate meat after decontamination do not seem to represent an additional health risk (Dorsa et al., 1998a,b; van Netten et al., 1998). Additional studies, however, have shown that bacterial pathogens on decontaminated meat may recover and multiply to be equal to or even higher than levels of respective pathogens surviving on or contaminating the untreated product. Specifically, high levels of natural flora inhibited growth of E. coli O157:H7 in ground beef (Vold et al., 2000), while the numbers of the pathogen on decontaminated beef (e.g., by steam vacuuming combined with spraying with 0.2 M lactic acid) increased by nearly 3 logs compared to 1 log and virtually no increase on the untreated beef stored (10°C) in air and vacuum, respectively (Nissen et al., 2001). It should be stressed though that inhibition of pathogens by the background flora on nondecontaminated meat stored at temperatures ≤10°C appears to be more due to retardation of growth because of the high population increases of the competitive flora, rather than due to an antimicrobial effect. For example, pathogens more psychrotrophic than E. coli O157:H7, such as S. Enteritidis and Y. enterocolitica, increased to similarly high levels (7 to 9 logs) on decontaminated and untreated chicken or pork skin, respectively, indicating increased competitiveness with the natural flora at 10°C (Nissen et al., 2001). It should be emphasized, however, that there is a significant percentage reduction of Salmonella or E. coli biotype 1- or
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O157:H7-positive beef carcasses in United States plants that use decontamination (Bacon et al., 2000; Elder et al., 2000; Sofos et al., 1999a, b, c). Decontamination of fresh foods packaged under vacuum or MAP may also significantly reduce the numbers of indigenous lactic acid bacteria that potentially inhibit L. monocytogenes and other pathogens during anaerobic storage (Babic et al., 1997; Carlin et al., 1996; Nissen et al., 2001; Vold et al., 2000). For this reason, the use of salts of organic acids, natural antimicrobials or protective cultures as additional microbial barriers is also under investigation for fresh vacuum packaged meats or produce (Ajjarapu and Shelef, 1999; Bennik et al., 1999; McMullen and Stiles, 1996; Smulders and Greer, 1998). However, potential effects of chemical antimicrobials, competitive natural flora or protective cultures on stress resistance of the coexisting pathogens, and on their secondary stress responses, are largely unknown. There is a need for research to examine the long-term benefits of risks of decontamination of fresh meat or other foods as they relate to pathogen reduction and potential development of long-term bacterial resistances. Such research should determine benefits and recognize risks, which may be eliminated or minimized by proper application of technologies. Numerous studies have shown that healthy E. coli O157:H7 inocula may survive for extended times in fresh fruit juices or other acidic foods, especially if previously acid-shocked or acid-adapted and the food is stored at refrigeration temperatures (Glass et al., 1992; Leyer et al., 1995; Miller and Kaspar, 1994; Tsai and Ingham, 1997; Zhao and Doyle, 1994). In contrast, few studies have evaluated whether acid-injured survivors that may be generated in such foods maintain high resistance upon subsequent exposure to acid or other stresses (Ingham and Uljas, 1998; Riordan et al., 2000; Uljas and Ingham, 1998). One study found that E. coli O157:H7 cells that had previously been exposed to acidic (pH 3.5) apple juice were of equal or lower acid tolerance in a simulated gastric fluid, compared to their parental strains habituated in nonacidic juice (pH 6.5) or broth acidified with various acids (Uljas and Ingham, 1998). Also, E. coli O157:H7 cells that had previously survived in pepperoni fermented to final pH 4.5 were of decreased thermal tolerance in heated pepperoni, compared to their parental strains that survived in pepperoni of higher (4.8) final pH (Riordan et al. 2000). Likewise, Salmonella strains previously adapted for growth in acidic conditions by sequential transfer in broth (pH 5.0; final growth 107 CFU/ml) had either equal or greater sensitivity to organic acid rinses of beef tissue and significantly lower D55 values than their parental strains (Dickson and Kunduru, 1995). It appears, therefore, that based on the results of studies with laboratory cultures, pathogen survivors from acidic foods or fresh foods decontaminated with organic acids, or survivors in the processing environments of such foods, may acquire resistance to acid or other stress to a level different from that anticipated. Recent and ongoing research in our laboratory has been undertaken to examine potential safety risks and to determine critical control points and limits associated with stress-adapted pathogens in foods (Lakkakula et al., 2001; Samelis et al., 2001a,b,c,d, 2002a,b; Stopforth et al., 2001). Based on previous data (Brackett et al., 1994; Cutter and Siragusa, 1994), it was assumed that meat decontamination may be incomplete or that poor sanitation may allow pathogenic bacteria to establish niches in the plant (Sofos and Smith, 1998). Therefore, we used decontamination
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washing residual waste fluids (washings) from fresh meat, either acidic or plain water washings, as a natural stressful and oligotrophic environment to monitor survival and growth, as well as the initial and subsequent ATR of acid-adapted, partially acid-adapted or nonadapted stationary-phase inocula of L. monocytogenes, E. coli O157:H7 and S. Typhimurium DT104 in the presence or absence of natural flora at incubation temperatures ranging from 4 to 15°C (plant conditions) to 35°C (abusive plant conditions or optimum temperature range of the pathogens in model studies). Initially, it was shown that E. coli O157:H7 has greater potential compared to L. monocytogenes or S. Typhimurium DT104 for survival (up to 7 days) in 2% organic acid meat washings, even with moderate previous adaptation to acid, and mainly when acetic rather than lactic acid is sprayed and the washings are kept at 4°C compared to 10°C (Samelis et al., 2001a). As expected, acid adaptation of E. coli O157:H7 enhanced survival in acidic washings under the same storage conditions; acid-adapted populations of the pathogen survived with minimal reductions for 14 days in 2% acetate washings or in mixtures of both 2% acetate or lactate washings with water washings at ratios of 1:1, 1:9 or 1:99 (Samelis et al., 2002b). Mixing of acidic with water washings was purposefully done to obtain decontamination waste fluids of sublethal pH and lowered acid concentration, as may be the case in meat plants. Declines of nonadapted populations followed a similar pattern, but they were much faster compared to those of acid-adapted populations (Samelis et al., 2002b). On the other hand, in water (control) washings at 4 and 10°C, E. coli O157:H7 survived but the low storage temperatures, the predominant growth (>108 CFU/ml at 2 to 4 days) of the natural meat flora and an apparently limited nutrient availability synergistically inhibited its growth (Samelis et al., 2001a,b). Importantly, nonadapted E. coli O157:H7 populations showed greater potential for survival and a tendency to initiate growth in plain water washings compared to acid-adapted populations at 10°C, suggesting that acid adaptation negatively influenced the pathogen ability to readapt upon a sudden shift to high pH conditions (e.g., pH 6.5 to 7.5) prevailing in water washings (Samelis et al., 2002b). Based on these findings, additional experiments were done at 15°C and indicated that, while the behavior of E. coli O157:H7 within treatments was unchanged, the higher incubation temperature accelerated pathogen death in acidic washings, while in nonacid (water) washings, it enhanced pathogen growth by approximately 2 logs, irrespective of type of inoculum (unpublished data). It should be also stressed that acid-containing washings diluted with water washings, at ratios up to 1:9, suppressed growth of the predominant Pseudomonas-like natural meat flora while they were simultaneously selective for growth of lactic acid bacteria and yeasts. This natural species selection was not observed in very dilute acid:water (1:99) washings where the normal Gram-negative meat flora could overcome the inhibitory effect of acid and eventually grow to high (8 logs) levels similar to those in plain water washings (Samelis et al., 2002b). These findings are in agreement with earlier studies indicating that the acid tolerance of E. coli O157:H7 may be pH-inducible, and underscored potential meat safety risks associated with this induction (Buchanan and Edelson, 1999a; Berry and Cutter, 2000). In fact, results were consistent with survival and growth of E. coli
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O157:H7 on acid-decontaminated vs. water-decontaminated or untreated meat (Berry and Cutter, 2000; Brackett et al., 1994; Dorsa et al., 1998a,b; Nissen et al., 2001; Siragusa, 1995; van Netten et al., 1998). Furthermore, it was clearly demonstrated that acid decontamination interventions do have the potential to alter the microbial ecology of meat plant environments (Samelis et al., 2002b) in addition to that of the acid-treated meat (Van Netten et al., 1994). Interesting and potentially important results were obtained by evaluating the ATR attained by E. coli O157:H7 following exposure to meat washings (unpublished data). Acid-adapted or nonadapted survivors (3.0 to 5.0 logs) of the pathogen in 1:9 or 1:99 mixtures (pH 3.1 to 4.7) of 2% lactate or 2% acetate washings with plain water (pH 6.8 to 7.1) meat washings after 2 or 7 days of storage at 10°C were reexposed to broth acidified to pH 3.5 with lactic acid. Survivors (4.2 to 5.2 logs) from plain water washings were also acid-challenged to serve as control. Day-2 survivors from acid-containing washings survived at pH 3.5 much better than survivors from water washings, especially when the mixtures contained acetic acid and the original inoculum was acid-adapted. High ATR was maintained in day-7 survivors from acetate washings, which were more numerous, as suffering less acid injury, but not in day-7 survivors from lactate washings. It may be postulated that the latter survivors were either energetically exhausted (1:9 dilution) to be able to survive the secondary acid stress, or that they shifted to acid sensitive soon after the metabolic activity of the Gram-negative natural flora eliminated the acid stress (1:99 dilution) in the washings. It should be indicated that acid-adapted populations from plain water washings continued to exhibit very low ATR at day 7, while nonadapted populations showed a clear reverse trend (Samelis and Sofos, 2002). The above findings indicate that acid stress may lead to development of acidresistant strains of E. coli O157:H7 in fresh meat environments, when its magnitude (i.e., pH and type of acid) and duration (i.e., time of exposure) are not adequate to energetically exhaust the pathogen to death, or the natural flora cannot eliminate long-term sublethal pH effects. This may lead to potential risks because, as mentioned, acid decontamination of fresh meat may be ineffective per se to completely inactivate pathogens (Brackett et al., 1994; Cutter and Siragusa, 1994; Dorsa et al., 1998a). Moreover, acid decontamination is not followed by other lethal stresses in immediate sequence, as in the case in fermenting meat products with a gradually decreasing water activity. Therefore, acid-injured pathogen survivors may have time and enough energy remaining to survive (Berry and Cutter, 2000; Samelis et al., 2001a) and grow (Nissen et al., 2001; Van Netten et al., 1998) as potentially adaptive mutants on the meat or in the plant. Similar phenomena may occur in any other type of food where the intensity of total stress is insufficient to eliminate or exhaust and irreversibly injure surviving bacterial pathogens. This postulation, which requires experimental verification, leads to a proposed novel hypothesis for development of strategies to enhance food safety (unpublished data), as discussed later. In our studies, an important case of an unexpected secondary bacterial stress response has been a dramatic acid sensitization of E. coli O157:H7 following exposure to plain water washings for 2 or 7 days at 10°C, particularly when the pathogen was previously acid-adapted (Samelis et al., 2002a). A similar trend was also noted for L. monocytogenes and S. Typhimurium DT104 in water washings under the same
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incubation conditions (Samelis et al., 2001c,d). Indeed, all three pathogens exposed to water washings at 10°C for 2 to 8 days became more acid-sensitive compared to their acid-adapted, partially acid-adapted or nonadapted inocula (day 0) previously grown at 30°C, or pure cultures previously grown in broth at 10°C. It needs to be stressed though that acid sensitization of L. monocytogenes and S. Typhimurium DT104 at 10°C was not so prominent because both pathogens were of lower acid resistance compared to E. coli O157:H7 (Samelis et al., 2001a). Also, both pathogens were acid-sensitized while they were growing exponentially (e.g., ca. 2-log increase) in water washings at 10°C (Samelis et al., 2001c,d), which was not the case for the less psychrotrophic E. coli O157:H7 (Samelis et al. 2002a). Accordingly, the logphase populations of L. monocytogenes and S. Typhimurium DT104 during the first days in water washings were of expectedly greater acid sensitivity compared to 1-week-old stationary-phase populations, which were also acid-sensitized. Thus, despite these differences between pathogens, their acid sensitization in nonacid meat decontamination waste fluids was shared, suggesting a role of the dominant natural flora, its catabolic products and potentially other compounds transferred to the washings from the meat in enhancing this response, which may be of major practical importance. As discussed, the potential effects of natural flora on pathogen responses to stresses have not been examined adequately, despite the coexistence and competition of spoilage and pathogenic bacteria in foods. To examine these effects, a logical research approach is to determine the initial and secondary stress responses of bacterial pathogens in real or model food systems under identical conditions, in the presence of natural flora or after its removal prior to processing or at the beginning of storage. Very few studies have systematically investigated this issue, mainly using laboratory media rather than foods. In culture broth incubated at 37°C, Duffy et al. (1995) reported that the addition of 108 CFU/ml viable competitors (i.e., E. coli, Citrobacter freundii and Pseudomonas fluorescens) protected an underlying, exponentially growing population (105 CFU/ml) of S. Typhimurium against thermal inactivation, as the pathogen D values at 55°C increased from 0.43 to 2.09 min. In a more recent study, Aldsworth et al. (1998) showed that the presence of 108, but not 105-107 CFU/ml, mixed viable competitors also increased the viability of S. Typhimurium (105 CFU/ml) under freezing, but addition of equal populations of heat-killed competitors did not provide this protective effect. Thus, the observed protective effect to food-related stresses, such as heating and freezing, was associated with the metabolic activity of the competitive flora, while it could not be correlated with RpoS (Aldsworth et al., 1998). It was found that by rapidly reducing levels of dissolved oxygen through active respiration, high levels of competitive flora reduced oxidative damage to exponential-phase cells of S. Typhimurium and decreased the RpoS induction time, thus arresting pathogen growth and conferring protection from stress (Aldsworth et al., 1998). Accordingly, a novel hypothesis was advanced to explain the increased sensitivity to stress of exponential compared to stationary phase bacterial cultures: the “suicide” response. Pure cultures of rapidly growing and respiring bacterial cells exposed to mild stresses will suffer growth arrest but their metabolism will continue to result in a burst of free-radical production that is lethal to the cells, rather than the stress itself (Aldsworth et al., 1999). Consistent with this
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hypothesis, E. coli O157:H7 was inhibited to a lesser extent in co-culture with a competitive flora than it was inhibited in pure culture in a simulated fermentation broth (from pH 5.8 to 4.8) at 37°C, suggesting that the increased numbers of competitive flora protected the pathogen from acid (Duffy et al., 1999). Thus, published data suggest that high numbers of natural flora may increase resistance of exponentially growing bacterial pathogens to a sudden stress by previous uptake of available oxygen to prevent oxidative damage (e.g., the burst of free radicals) and avoid “suicide” through stress. The “suicide” response of pure cultures and the protection of pathogens from sudden stress provided by a competitive flora (Aldsworth et al., 1998, 1999) seem to be in conflict with acid sensitization of E. coli O157:H7, S. Typhimurium DT104 and L. monocytogenes in nonacid meat washings (Samelis et al., 2001c,d, 2002a). No previous study, however, has investigated whether this protective effect continued to exist upon extended periods of coexistence of growth-arrested pathogenic cells lacking an active metabolism with high competitor populations under starvation, as it happened in water meat washings. Starvation is also known to increase acid tolerance of pathogens (Arnold and Kaspar, 1995; Buncic and Avery, 1998) and generally induces multiresistances in bacteria (Hengge-Aronis, 1993, 1996; Jenkins et al., 1990; Nystrom et al., 1992; Pichereau et al., 2000). Starvation of pure cultures exposed to synthetic liquid media of minimal composition, as above, or in natural but previously heat-sterilized water (Gauthier and Clement, 1994), however, may result in different pathogen responses compared to starvation in limited nutrient food environments in the presence of high numbers of natural flora. Growth-arrested and starving pathogens are most likely to occur in complex microbial associations in meat and other foods (Dainty and Mackey 1992; Labadie, 1999; Mossel et al., 1995; Samelis et al., 1998; Sofos, 1994), where stress signals, sensors and alarmones may be crucially altered by the food-specific natural flora. Considering these differences, we used original (unfiltered) and filter-sterilized water washings from fresh meat to monitor growth and ATR of inoculated (5 logs) L. monocytogenes at 35°C, an incubation temperature purposefully selected at the optimum range of the pathogen to maximize its responses (Samelis et al., 2001b). Results confirmed an acid-protective effect by the natural flora (>8 log CFU/ml) that grew rapidly in unfiltered washings, within 24 h. However, this early protective effect to acid by the natural flora on L. monocytogenes shifted to a dramatic acid sensitization of the pathogen after incubation at 35°C for 8 days. In contrast, in filter-sterilized washings the pathogen significantly increased its ATR from days 1 to 8 (Samelis et al., 2001b). As discussed previously, sensitization of E. coli O157:H7, Salmonella and L. monocytogenes to acid also occurred in unfiltered water washings at 10°C, where the protective effect of the natural flora was delayed due to the decrease in microbial growth rate (Samelis et al., 2001c,d, 2002a). However, particularly for E. coli O157:H7, additional data have shown that acid sensitization occurs only when this pathogen, at least the acid-resistant strain ATCC 43895 used, completely fails to grow in the water washings (unpublished data). This growth primarily depends on the storage temperature, rather than the competition by the natural flora present. Indeed, growth-arrested acid-adapted cultures of E. coli O157:H7 in unfiltered washings at 10°C, and any pathogen culture at 4°C, were very sensitive at pH
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3.5, while the respective stationary-phase cultures following growth (ca. 2 logs) in the same washings at 15 or 25°C were very resistant at pH 3.5, irrespective of acid adaptation of the inoculum (unpublished data). Similar behavior at each respective incubation temperature was observed in parallel cultures of acid-adapted or nonadapted E. coli O157:H7 exposed to heat-sterilized water washings, which, of course, were free of natural flora but contained organic material from meat. In contrast, pure cultures of acid-adapted and nonadapted E. coli O157:H7 in sterile water (controls), where no growth occurred, became more acid-sensitive at pH 3.5 with time when the incubation temperature increased from 4 to 10 to 15 and 25°C (unpublished data). These findings suggest that strains of E. coli O157:H7 characterized by a permanent acid resistance, such as strain ATCC 43895, may express this resistance upon their transfer from niche to niche, provided that environmental conditions are favorable for the pathogen to multiply. Additional studies are required to investigate the behavior of E. coli O157:H7 strains that are acidsensitive or their high ATR is primarily pH-inducible. From a practical point of view, these findings suggest that in order to acid-sensitize E. coli O157:H7 in meat plants, its growth potential should be minimized by either keeping nonacid conditions under environmentally low (≤10°C) temperatures or establishing acidic conditions at ambient temperatures. On this basis, E. coli O157:H7 populations that may have multiplied on slaughtered animals or the meat plant to subsequently survive on an acid-decontaminated carcass transferred to chill storage, or acid-adapted survivors that may establish niches in refrigerated plant areas may pose serious safety risks. Such survivors, if not exhausted due to prevalence of sublethal acid conditions, may maintain high ATR and potentially mutate, while if they have chances to multiply at later stages of meat processing due to abusive (>12°C) temperature conditions, risks may be maximized. In contrast, even high E. coli O157:H7 populations still attached on water-decontaminated carcasses stored under refrigeration temperatures inhibitory to their growth, would be eventually acid-sensitized. It appears that the same scenario is not the case with Salmonella and L. monocytogenes due to their lower ATR and because acid sensitization occurred following growth in a fresh meat environment, such as the washings (Samelis et al., 2001c,d). In conclusion, acid sensitization of bacterial pathogens after exposure to nonacid, but otherwise stressful, environments is a complex response which may depend on the species, temperature, microbial competition, naturally present or microbially catabolized extracellular compounds that may act as stress sensors and alarmones, limiting nutrients, etc. Overall, this response seems logical because E. coli O157:H7, Salmonella and other pathogens are neutrophiles. Alterations in ATR of starving pathogens in the presence of high numbers of competitors increases in alkali tolerance at neutral pH (Lazim and Rowbury, 2000) that may switch off genes expressing or maintaining ATR, or production of antagonistic compounds by the natural flora (Cheng et al., 1995) may contribute to acid sensitization. Intensified research is required in this field to indicate whether the natural flora as affected by food type, composition, processing and storage conditions may synergistically regulate stress responses of various bacterial pathogens or strains with varying biochemical capabilities and natural tolerances. Such a correlation may exist and may lead to different responses because food-related processing and storage factors by themselves have
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a strong impact, for example, on acid resistance of E. coli O157:H7 (Brudzinski and Harrison, 1998; Diez-Gonzalez and Russell, 1999; Ingham and Uljas, 1998; Kauppi and Tatini, 1996; Semancheck and Golden, 1998). In summary, improved understanding of the effects of decontamination and other processes on the antagonistic or symbiotic interactions among pathogenic and spoilage microorganisms in foods during subsequent processing and storage may prevent overlooked or underestimated (or indicate overestimated) risks associated with their use. In addition to physical or chemical antimicrobial factors (Cheroutre-Vialette and Lebert, 2000; Fernandez et al., 1997; George et al., 1996; McClure et al., 1994), specific parameters referring to microbial competition should be incorporated in mathematical models to enhance accuracy of their predictions regarding food safety and shelf life (Aggelis et al., 1998; Lebert et al., 2000; Vereecken et al., 2000). Based on the behavior (e.g., growth, survival, inhibition, resistance in stress sequences and death) of mixed pathogenic strains in validation studies, food processes and products can be modified to build in extra safety margins. Then, the data obtained should be used to re-validate and potentially revise or expand existing food safety models to increase accuracy of predictions (McMeekin et al., 1997), with emphasis given on the nonlinear inactivation kinetics of bacterial pathogens exposed to lethal agents (Peleg and Penchina, 2000). Quantitative predictions of pathogen responses from model experiments with real or simulated foods should be the basis for the development of reliable quantitative risk assessment schemes (Lammerding and Paoli, 1997; Walls and Scott, 1996). These schemes should enhance transfer of microbiologically validated technological developments in the food industry (Roberts, 1997), and should be the basis for developing approaches to meet food safety objectives (van Schothorst, 1998). Moreover, extension service bulletins based on realistic and product-specific quantitative risk assessment of foods should be used in public education to enhance pathogen control at the plant or kitchen level. As mentioned, a major deficiency of our modern society, with respect to food production and processing, is the lack of education of food industry personnel and consumers based on brief, but practical and straight forward, recommendations (Bruhn, 1997).
NOVEL PATHOGEN CONTROL STRATEGIES Control strategies for stressed pathogens may need to be based on novel approaches. According to Lederberg (1997), “we could imaginably adapt in a Darwinian fashion, but the odds are stacked against us. We cannot compete with microorganisms whose populations are measured in exponents of 1012, 1014, 1016, over periods of days… In the case against microbial genes, our weapon is our wits, not natural selection of our genes. New mechanisms of genetic plasticity of one microbe species or another are uncovered almost daily.” Archer (1996) postulated, “I wonder if a reduction in preservation might not in fact lead to a reduction in the immediate virulence of certain pathogens, and additionally, to a lowering of the rate of emergence of new or better host-adapted pathogens.” These statements make us pose some important questions: How can we use our wits as weapons? Should we eliminate use of common antimicrobial inhibitory © 2003 by CRC Press LLC
hurdles? How can we balance between short-term antimicrobial activity of hurdles and potential long-term negative effects? No doubt, it is difficult to decide the best way to control the evolution of resistant bacterial pathogens in our food supply and, overall, in the environment. Principally, there are two types of control strategies, which could be seen as complementary rather than as counteractive: the classical and the novel. Classical strategies suggest a continuous increase in the application of safety barriers in foods by developing new antimicrobials and incorporating more hurdle technologies or decontamination interventions, without necessarily knowing any potential secondary effects prior to application. A major concern associated with this strategy is that multihurdles with “milder” doses of stresses, although detrimental to many microorganisms in the short term, may enhance generation or evolution of pathogenic strains of increased resistance and virulence in the future (Archer, 1996). Based on recent data, however, this disadvantage may be eliminated if we assure that multihurdles are applied in the right order and to sufficient levels to cause exhaustion of pathogenic cells due to expenditure of large amounts of cellular energy to maintain viability during processing of foods. If such conditions are carefully set with either classical or novel methods, exhausted survivors from an initial stress may have insufficient energy reserves to cope successfully with secondary stresses or the final acid stress in the stomach, irrespective of their numbers (which, in any case, would expectedly be very low). In other words, the principal aim is for stationary-phase pathogen survivors in foods to become so energetically exhausted while attempting to maintain viability that their potential adaptation to the initial stress applied, or cross-protection against secondary stresses, is counteracted by exhaustion. The above hypothesis was first made following exposures to food-related stresses, such as acid, high or low temperatures and low water activity, of substratelimited pure cultures of E. coli in broth (Krist et al., 1998; Shadbolt et al., 1999, 2001). Initially it was observed that acid stress reduced the efficiency of substrate conversion to biomass by E. coli compared to osmolarity or cold and heat, potentially due to its higher demand for energy (Krist et al., 1998). In contrast, E. coli could survive low levels of water activity (e.g., 0.75 to 0.90) for long times by developing osmotic-resistant subpopulations that declined very slowly, e.g., “tailing” effect (Shadbolt et al., 1999). More recently, Shadbolt et al. (2001) demonstrated that when a non-pathogenic E. coli strain sharing high acid tolerance with E. coli O157:H7 was initially exposed to suboptimal water activity of 0.90, followed by exposure to a secondary lethal acid stress at pH 3.5 with HCl, survivors from the osmotic stress displayed a second rapid inactivation period due to acid, but eventually a resistant subpopulation emerged to maintain viability for nearly 50 h after acid addition to the broth culture. In contrast, when these experiments were performed under identical conditions, but by applying the acid stress first followed by the osmotic stress, acid survivors were inactivated very rapidly and declined below the detection limit within a few hours (Shadbolt et al. 2001). Thus, these data indicate that acid should be the initial stress in food processing, whenever used, in order to sensitize foodborne pathogens to secondary stresses due to its ability to impose large energetic burdens to bacterial cells (Krist et al., 1998; Shadbolt et al., 2001). In fact, this approach has been wisely
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applied in traditional meat fermentations since antiquity. Acid stress due to pH lowering/lactate formation by the antagonistic lactic flora is followed by a long-term lethal osmotic stress ranging from 0.92 to 0.85, during drying; conditions that ultimately exhaust pathogens to death in the ripened product (Lucke, 2000; Samelis et al., 1998). Differences in survival of E. coli O157:H7 to initial and secondary acid stress may potentially indicate different levels of pathogen exhaustion, as affected by the type (lactate or acetate) and concentration of acid (dilution factor) and the exposure times to acid-containing meat washings (Samelis et al., 2002b). These results may be directly extrapolated to meat plant conditions because pathogen survivors from the initial acid stress originated from a natural, meat-associated environment rather than from laboratory cultures. Additional studies in our laboratory (Burnham et al., 2001; Lakakkula et al., 2001; unpublished data) involving dried foods have also shown that carefully selected hurdles may enhance death of bacterial pathogens to very low or undetectable levels, due to potential depletion of cellular energy and eventual exhaustion of cells. Burnham et al. (2001) found that dipping E. coli O157:H7 inoculated apple slices in an ascorbic acid solution enhanced destruction of the pathogen during drying of the product. This synergism has also been observed in sliced dried apples of different varieties previously treated with citric acid (Lakakkula et al., 2001), and in beef jerky. Inoculated (7 logs) E. coli O157:H7, Salmonella or L. monocytogenes on beef jerky strips decreased more rapidly and extensively during drying and ambient storage of product treated before drying with marinade containing acetic acid or combinations of lactic acid with ethanol, compared to traditional or no marination (unpublished data). Notably, subsequent drying of jerky was more detrimental for previously acidadapted than nonadapted inocula, which may indicate that the amount of cellular energy expensed by E. coli O157:H7 for acid adaptation could not be replaced before and during drying to eventually reduce survival in the dried product. Another complementary rather than counteractive approach for controlling stress-adapted pathogens in foods may be to make them misinterpret the environmental signals and, thus, activate wrong genes at the right (for food safety), time (Sheridan and McDowell, 1998). This strategy was originally termed as “microbial psychosis” (Dorman, 1994). Practically, it requires avoidance of intentional or inadvertent exposure of pathogens to food-related stresses in order to drive them to become unprepared to cope with sudden stress during food processing, in the kitchen or in the host. As an inappropriate bacterial response, “microbial psychosis” may be induced effectively, provided that we have exact knowledge of the microbial regulatory processes under different sets of environmental conditions. For example, we believe that acid sensitization of E. coli O157:H7 and L. monocytogenes in nonacid food environments, such as water meat washings, indicates a “psychotic” behavior in response to absence of acid stress. However, it is difficult to elucidate the exact factors and their interactions that induce this behavior, which may differ among pathogens and be altered by different sets of environmental conditions. “Microbial psychosis” may be induced on pathogens by changing the food environment via the competitive natural flora. In other words, we can use microbial competition in foods not just to inhibit pathogen growth, which is the traditional
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approach in food preservation, but to potentially reduce stress resistance of bacterial pathogens (Samelis et al., 2001b). As more knowledge on pathogen interactions with other bacteria is being accumulated, new approaches for pathogen inhibition by a competitive flora in foods will be indicated. For example, we have recently demonstrated that Pseudomonas may completely inhibit growth of E. coli O157:H7 at 10°C, and may retard its growth at 15°C, if glucose at 1% is present in culture broth to be converted to gluconate. With no or low concentrations (0.25%) of glucose in the broth, inhibitory effects are low to zero (Samelis and Sofos, 2002). Thus, supplementation of fresh foods with higher levels of glucose than those they contain may increase control of E. coli O157:H7 via competitive exclusion (Bower and Daeschel, 1999). Also, a competitive flora, which can be purposefully added or allowed to develop in a food product or plant to exert a targeted metabolic activity, may significantly contribute to a weakening of pathogen resistance to stress factors not prevailing during the process (Samelis et al., 2001b). Intensified research is required in this field. In particular, such competitive effects should be studied on adherent pathogens with natural flora in mixed biofilms where prevention of adhesion or increased sensitization of pathogens to sanitizers may be induced by the predominant spoilage flora (Stopforth et al., 2001). Sensitization may be due to excreting of microbial metabolites, which act as “misleading” signals to pathogenic cells, or chemical compounds that inactivate alarmones excreted by the cells in order to cope with a specific type of stress. Directed subsequent exposure of the sensitized pathogen to the target stress may result in rapid cell inactivation. Another example of a novel approach may be to create a food environment that causes bacterial cell growth to cease, without application of severe stress and without adversely affecting food quality (McMeekin et al., 1997). For this purpose, compatible solutes or osmolytes widely found in foods, such as glycine betaine or carnitine, may be added to make the pathogens activate specific transport systems to equilibrate the external increase in osmotic pressure. Depending on the magnitude of this increase, osmotic conditions may either inactivate pathogenic cells, or inhibit their growth while the compatible solutes transported inside the cell allow continuation of enzymatic reactions. Thus, energy reserves of pathogen cells are once again depleted, but by other means than acid stress, and upcoming starvation may drastically extend the pathogen lag phase, or lead to death (Krist et al., 1998; McMeekin et al., 1997; Shadbolt et al., 1999).
PRACTICAL APPLICATION OF PATHOGEN CONTROL STRATEGIES The establishment of multistep barriers throughout the food chain is the recommended common approach to effectively control microbial pathogens in foods. Steps in the food chain that require barrier application include animal feeding, plant harvesting, cleaning of animals and plants, decontamination of animal carcasses and produce, plant sanitation and hygiene, food production, processing, storage and distribution, food retailing and meal preparation, food service facilities, and food handling at home by consumers. It is important not to simply establish, but to
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continuously update, antimicrobial barriers, mainly in consideration of their potential impact on bacterial stress resistance and virulence development. Advanced knowledge on bacterial responses to food-related stresses should be used to benefit the food industry and consumers. Effective application of this knowledge can only be achieved by collaborative action between all components of the food sector, including the industry, regulators, public health authorities and academic institutions (Fischhoff and Downs, 1997; Majkowski, 1997; Roberts, 1997). Thus, an integrated microbial control strategy needs to be elaborated (Sofos, 2001). Foodspecific research data should be used to increase the accuracy of predictive models (McMeekin et al., 1997; Walls and Scott, 1996), and the results from model validation studies with resistant pathogens in real food systems can lead to the development of reliable quantitative risk assessment schemes (Lammerding and Paoli, 1997). These schemes can be used to establish food safety objectives (van Schothorst, 1998) and be implemented in hazard analysis critical control point (HACCP) systems to enhance food safety (FSIS, 1996; NACMCF, 1998). Meanwhile, academic institutions need to coordinate education and provide updated, educational or extension material to food scientists, producers, handlers, processors and consumers. In fact, the entire food safety network requires continuous updating. Although research, development and application of novel food preservation approaches may help us control stress-adapted and emerging pathogens, better application of already known, common pathogen control interventions by food handlers and consumers may be adequate to greatly reduce foodborne illness episodes. Today, most outbreaks of foodborne disease are due to mishandling food in ways that we already know how to avoid; approximately 85% of all outbreaks occur as a result of food mishandling in food service establishments or homes (Hall, 1997). Lederberg (1997) stated: “It is important to prevent foodborne disease through sensible monitoring, standard of cleanliness and consumer and foodhandler education and not just care of its victims… Education, however, is a universally accepted countermeasure, especially important in foodborne disease. Food safety programs should more specifically target food handlers, examining their hands to determine, if they are carriers, to ensure they are complying with basic sanitation.” Thus, education of food handlers and consumers in basic hygiene and the consequences of temperature abuse are urgently needed, as is a greater depth of understanding of those in technical positions in the food industry or those with regulatory responsibilities (McMeekin et al., 1997). Food microbiologists must realize their responsibility to provide industry personnel and consumers with simplified knowledge on food safety aspects, while all sectors of the continuum from farm to table should share in the responsibilities (Bruhn, 1997). Governmental agencies have the responsibility to coordinate collaborative actions toward the goal of providing our society with safe foods, although the globalization of the entire food sector requires effective collaborative action and international harmonization of food safety objectives and regulations (Kaferstein et al., 1997).
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CONCLUSIONS When exposed to sublethal doses of a stress, microorganisms may adapt and progressively develop resistance to stronger doses of the stress, while microbial adaptation to one stress can confer cross-protective resistance to other stresses. Bacteria respond to stresses primarily by temporary intracellular changes, or may undergo mutations which introduce permanent genomic changes that favor their survival. Accordingly, stressed pathogens have greater potential for survival and/or proliferation in foods and under stressing conditions of temperature, acidity, water activity, chemical or biological preservatives and limited nutrients; highly resistant and virulent mutants may arise in situ. Traditional food processing safety barriers may be inadequate, and foodborne diseases may be caused even by pathogenic strains at very low infective doses. Several recent uncommon outbreaks associated with foods traditionally considered low-risk support current food safety concerns. To control the emergence and evolution of stress-resistant pathogens in foods, we need to undertake effective basic and applied research. The overall goal is to develop control strategies based on optimized, efficient, economical and integrated approaches that rely on basic knowledge to prevent resistance development and virulence enhancement, and to assure product quality, production efficiency, and control of resistant pathogens. A good approach is to work with real food systems, considering them as stressful environments, and under experimental conditions that simulate microbial associations in these environments as much as possible. Work with pure cultures under laboratory conditions may be useful in verifying mechanisms of microbial resistance development and control. Except for food processes achieving food safety by extensive microbial inactivation (e.g., canning), other approaches employed for control of pathogens in foods achieve reduction of prevalence or inhibition of proliferation. Selection of such treatments, interventions or processes is difficult and requires extensive evaluation. This is because most nondestructive food processing, decontamination or preservation technologies, although useful in reducing pathogen prevalence and controlling growth, may not deliver complete removal, inactivation or control of contamination, and may allow bacterial survivors to become stress resistant and virulent at very low infectious doses. Such doses may not affect consumers with a strong immune system, but may cause infection to immunosuppressed individuals. Thus, pathogen control strategies should be decided by considering the resistance of stressed pathogens, and by predicting the virulence of survivors in relation to the susceptibility of atrisk populations. Effective commercial application of multihurdle pathogen control strategies in foods requires multivariable research approaches and, indeed, there are many issues associated with bacterial stress resistance in complex food ecosystems that require elucidation. Recent data suggest that the energetic status of pathogenic cells, rather than their actual numbers, may be more important in deciding whether survivors from primary stresses can cope successfully with secondary stresses in foods, and the ultimate acid stress in the stomach. On this basis, food-related sequential stresses should be carefully applied to assure pathogen exhaustion or should be intelligently avoided to induce “microbial psychosis” (Dorman, 1994) to pathogen survivors, but
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should never have an intermediate effect. In practice, when during food processing and storage application or exposure to a preservation barrier is unavoidable, the barrier considered the most effective in reducing and exhausting bacterial survivors capable of recovering in the product should be selected. When it is wiser to avoid challenging of bacteria, technologies that may sensitize pathogens to types of stresses applied subsequently during food processing should be selected. Since multihurdles disrupt cell homeostasis in multiple ways, they should be selected and applied at the right magnitude and succession to minimize development of multi-stress resistant strains, and to lead to as much pathogen inactivation as possible. Monitoring and validating food processes on the basis of novel pathogen control strategies, such as “microbial psychosis,” and elucidating whether foodborne pathogens can be virulent while being “psychotic,” or still viable but energetically exhausted, are major future challenges in food microbiology. Potential discrepancies between what we believe and what may really happen in the food environment have to be hypothesized and, if possible, predicted and verified. Scientific knowledge and consumer awareness may reduce common or new routes of transmission for known or newly emerging pathogens from foods. The objective is to provide consumers with food products for preparation at home, or ready-to-eat, which are free of active pathogens, especially those of low infectious doses and high virulence for at-risk populations.
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