1005 This is to certify that the dissertation entitled ALTERATION IN SENSITIVITY OF STRESS-ADAPTED LISTERIA INNOCUA TO THE CHEMICAL SANITIZER CETRIMIDE presented by Mark A. Moorman has been accepted towards fulfillment of the requirements for the Doctoral degree in Philosophy Food Science and Human Nutrition Major Professor’s Signature 8'2 3*‘05" Date MSU is an Affirmative Action/Equal Opportunity Institution ._————-——-—-—"——"‘T LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 chlRC/DateDueindd-p. 15 ALTERATION m SENSITIVITY OF STRESS-ADAPTED LISTERIA INNOCUA TO THE CHEMICAL SANITIZER CETRIMIDE By Mark A. Moorman A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 2005 ABSTRACT ALTERATION IN SENSITIVITY OF STRESS-ADAPTED LISTERIA INNOCUA TO THE CHEMICAL SANITIZER CETRIMIDE By Mark A. Moorman Experts in food hygiene have long struggled to eliminate microorganisms established within the food-manufacturing environment. Such environments may be contaminated with pathogenic or spoilage organisms that evade sanitation and contaminate food. It was hypothesized that sensitivity of L. innocua to the quaternary ammonium compound sanitizer cetrimide is altered following adaptation to acid, starvation, cold and heat stress, stressors commonly found within the food manufacturing environment and this relates to altered cell hydrophobicity and membrane fluidity. This research demonstrated that exposure of L. innocua to acid and starvation stress diminishes sensitivity to 10 ppm cetrimide while exposure to cold and heat stress enhance sensitivity. F urthermore, acid and starvation stress increased net cell hydrophobicity and reduced cell membrane fluidity. In contrast, decreased hydrophobicity and increased membrane fluidity were observed in cold adapted L. innocua. No significant changes in hydrophobicity or indicators of membrane fluidity, aside fi'om increased C-l8 unsaturated fatty acids, were detected in heat adapted L. innocua. That certain environmental conditions within food manufacturing facilities such as acid and starvation could diminish cellular sensitivity to industrial sanitizers suggest the physiological stress response not only diminishes sensitivity to the stress, but also enables persistence upon exposure to low levels of quaternary ammonium compound sanitizers. Conversely, that other modifications of the environment, such as cold temperature, would stress-adapt and concurrently enhance sensitivity of L. innocua to quaternary ammonium compounds suggest interventions exist that enhance sanitation efficacy. The potential exists therefore, for the application of stress conditions to equipment or manufacturing sites persistently testing positive for problematic microorganisms, and thereby diminish the ability the microorganisms to survive sanitizer exposure. This dissertation is dedicated to my close friend and mentor Dr. John Silliker. While the long lunches at Olympia Fields Country Club covered innumerable topics better left undocumented, the many on the discipline of food microbiology coupled with his unending encouragement, provided me the confidence and thirst necessary to complete this dissertation. I will forever be grateful to Dr. Silliker for his friendship and interest in teaching me about the fascinating field of food microbiology. iv ACKNOWLEDGMENTS I would like to thank my dissertation adviser Dr. James J. Pestka for his guidance, support and assistance prior to, and throughout, my Ph.D. study. Your continued support and patience through the ups and downs of this project allowed me to finish this program with a sense of pride and accomplishment. I would also like to thank the remainder of my committee, Dr. John Linz, Dr, Elliot Ryser and Dr. Bradley Marks, for their valuable advice, suggestions and comments. I want to especially thank Dr. Dale Romsos who guided, encouraged and mentored me through the last year of this program. The effort you put toward helping me attain my goal taught me much about my dissertation but even more about life. Special thanks to my laboratory interns, most notably Will Nettleton and Caitlin Thelemann. Your help was invaluable to my success. Finally, I would like to thank my wife Dawn and children Eric, Sarah and Cole for their continued support, encouragement and mostly patience throughout my program. This day would not have come if Dawn had not sent me back into my office the many times I said “I Quit”. The end is finally here! I eagerly look forward to a new beginning. TABLE OF CONTENTS LIST OF TABLES viii LIST OF FIGURES ix CHAPTER I. Literature review 1 Introduction 1 Chemical Sanitation l Chlorine Sanitizers .......................................................................................... 2 Quaternary Ammonium Compounds (QAC) .................................................. 4 Bacterial Membranes 5 Bacterial Lipid Composition and Membrane Fluidity .................................... 6 Fatty acid chain length and saturation ............................................................. 7 Branched chain (anteiso/iso) fatty acids ......................................................... 8 Growth and Survival Within Acidic Environments 9 Acid Types and Emergency pH Homeostasis ................................................. 9 ATR Mechanism ........................................................................................... 10 Stress-induced Proteins ................................................................................. ll Cross-protection against multiple stresses .................................................... 12 Virulence ....................................................................................................... 13 Starvation 14 Cold Adaptation l6 Sub-lethal Heat 17 Heat Shock Proteins ...................................................................................... 19 Relevance of Heat Shock Proteins to the Food Industry .............................. 20 Alternate mechanisms for altered cetrimide sensitivity 20 Rationale and Significance 21 Aims 22 CHAPTER II. Altered Sensitivity to a Quaternary Ammonium Sanitizer in Stressed Listeria innocua 28 SUMMARY 28 Introduction 28 Materials and Methods 30 Results 32 Discussion 34 CHAPTER III. Altered Hydrophobicity and Membrane Composition in Stress- adapted Listeria innocua 42 Summary 42 Introduction 43 Materials and Methods 46 Statistical Analysis ........................................................................................ 49 vi Results Discussion Conclusions CHAPTER IV. Summary and Research Conclusions Significance Mechanism and Alternate Explanation Possible Future Studies APPENDICES APPENDIX A Acid Tolerance Induction protocol ..................................... APPENDIX B Sanitizer sensitivity protocol ................................................ Bibliography vii 49 58 59 62 63 65 LIST OF TABLES Table 1. Effect of fatty acid modification on membrane fluidity ..................................... 27 Table 2. Heat and acid tolerance of respective adapted and non-acid-adapted L. innocua. ................................................................................................................................... 39 Table 3. Log reduction of adapted and non-acid-adapted L. innocua during a 3 min exposure to 10 ppm cetrimide. ................................................................................. 40 Table 4. Net cell hydrophobrcrty of control and stress-adapted L. innocua“ .. 56 Table 5: Changes in fatty acid composition in stress-adapted L. innocua .................. 57 viii LIST OF FIGURES Figure 1. Effect of growth phase on resistance to pH 3.5. The growth curve is represented by the open circles, while the % survivors after exposure to pH 3.5 for 60 min is indicated by the closed circles (Hill, O'Driscoll et al. 1995). ................... 24 Figure 2. Phospholipid molecule (Christie 2005) ............................................................. 25 Figure 3. Lipid bilayer (Kaiser 2005) ............................................................. 26 Figure 4. Comparative effects of different stressors between stressed cetrimide-induced reduction after 1.5 minutes ................................................................... 41 ix CHAPTER 1. LITERATURE REVIEW INTRODUCTION The purpose of this introduction is to review the current literature concerning 1) the effect of the chemical sanitizers chlorine and quaternary ammonium containing compounds on microorganisms 2) the response of microorganisms to sub-lethal heat and 3) the adaptation of microorganisms to acidic cold and starvation environments. CHEMICAL SANITATION Cleaning of food manufacturing equipment is performed to remove accumulated soils (carbohydrate, proteins, fats, and minerals) and to purge the resident microorganisms. In general terms, sanitation is comprised of cleaning to remove soils, and chemical sanitizer application to destroy microorganisms remaining afier cleaning. During the chemical cleaning and sanitizing process microorganisms are exposed to a) alkali or acid conditions from cleaning compounds b) heat during equipment rinse and cleaner application and c) chemical sanitizers. The two basic classes of cleaning compounds used in the food industry are alkaline and acid cleaners (Juneja, F oglia et al. 1998). Most cleaners are alkaline while acids are used to remove highly insoluble mineral deposits on equipment (Dychdala 2001 ). The basic functions of the alkali cleaners are peptidization of proteins and emulsification and saponification of fat (Phan-Thanh and Gormon 1995; Phan-Thanh and Gonnon 1997). In chlorinated alkali cleaners, used in clean-in-place (CIP) systems, alkali or acid conditions from cleaning compounds b) heat during equipment rinse and cleaner application and c) chemical sanitizers. The two basic classes of cleaning compounds used in the food industry are alkaline and acid cleaners (J uneja, Foglia et al. 1998). Most cleaners are alkaline while acids are used to remove highly insoluble mineral deposits on equipment (Dychdala 2001). The basic functions of the alkali cleaners are peptidization of proteins and emulsification and saponification of fat (Phan-Thanh and Gormon 1995; Phan-Thanh and Gannon 1997). In chlorinated alkali cleaners, used in clean-in-place (CIP) systems, chlorine serves not as a sanitizer but to increase the peptizing efficiency of the alkaline compounds (Dychdala 2001). The soil rinse (35 - 43°C) and detergent application (46 — 48.9 °C) temperatures are sub-lethal for most microorganisms and are ideal for the induction of heat shock proteins (HSP) in Saccharomyces cerevisiae (Piper 1996), Listeria monocytogenes (F arber and Brown 1990), E. coli (Arsene, Tomoyasu et al. 2000), and Campylobacter jejuni (Konkel, Kim et al. 1998). Production of heat shock proteins permits survival of the heat-induced microorganism at higher temperatures that would otherwise be lethal. The adaptation of L. monocytogenes to heat conditions encountered in inadequately cleaned and sanitized environments may induce thermotolerance in this microbe and promote its survival in foods receiving mild heat treatment (Taormina and Beuchat 2001). Interestingly, alkali exposure (pH 12 for 45 minutes), similar to conditions encountered with alkali cleaners, induces thermotolerance in L. monocytogenes (Taormina and Beuchat 2001). It is unknown if alkali—induced thermotolerance is a result of heat shock protein expression. Since chemical sanitizers are the principal means by which the food industry controls microorganisms in wet manufacturing environments, understanding the impact of microbial adaptation on sanitizer efficacy is critical for design of interventions that may improve effectiveness of food plant sanitation. CHLORINE SANITIZERS The three most common classes of sanitizers contain chlorine, quaternary ammonium compounds and iodine as active ingredients. Among the sanitizer subclasses, the hypochlorites are the oldest and most widely used (Dychdala 2001). Hypochlorite germicidal efficacy diminishes with increasing pH and with the presence of organic matter, but is unaffected by water hardness and increases with temperature and concentration (Dychdala 2001). The bactericidal mechanism for hypochlorite (HOCl) is still incompletely understood. Early theories proposed that chlorine destroys bacteria by combining with proteins of cell membranes (Baker, 1926). Later work confirmed the irreversible oxidation of chlorine on sulfliydyl groups of vital enzymes (Knox, 1948) including ATPase within the cytoplasmic membrane which catalyzes respiration linked phosphoanhydride bond formation between ADP + Pi (Barrette, Hannum et al. 1989). Cells that are exposed to lethal concentrations of HOCl are unable to maintain the necessary levels of cytoplasmic ATP. The net loss of the cytoplasmic pool of ATP results from oxidative damage to as few as two amino acids within subunits of the Fl-ATPase complex (Hannum, Barrette et al. 1995). Increasing membrane permeability might also increase the germicidal activity of chlorine compounds. EDTA increased permeability of the cellular membrane of Salmonella Typhimurium and increased HOCl lethality (Leyer and Johnson 1997). Although the bactericidal mechanism of free chlorine is debated, the bactericidal activity of lethal concentrations of free chlorine is nevertheless established. Microorganisms have evolved systems to resist and adapt to oxidative compounds such as superoxides, peroxides and hypochlorous acid within neutrophils and macrophage. It is notable that sub-lethal HOCl concentrations (0.2 mg/liter) induce the alternative sigma factor (0”) heat shock promoter rpoH in E. coli (Dukan, Dadon et al. 1996) and heat shock proteins in E. coli OlS7:H7. 032 regulates the transcription of DnaK and other heat-shock genes (Lindquist and Craig 1988). Heat shock proteins reside within the cytoplasm and function to fold translated proteins. Thus, thermotolerance may be induced in microorganisms within the food manufacturing environment that are exposed to sub-lethal HOCl concentrations. QUATERNARY AMMONIUM COMPOUNDS (QAC) Quaternary ammonium compounds primarily act by disrupting cellular permeability of the cytoplasmic membrane (Merianos 2001). The net negative charge of the cell membrane renders the cell impermeable to polar, charged and hydrophobic molecules. This bacterial cytoplasmic membrane assumes the functions of all internal membranes of eukaryotic organisms including ATP synthesis by FlATPase, electron transport and photosynthesis, chromosome segregation, synthesis of membrane proteins, secretory proteins, lipids and the cell wall. QAC below 12 carbons in length interfere at receptor sites for many biologically active compounds (Merianos 2001). When length exceeds 12 carbons, these compounds become surface active and antimicrobial. As polycationic surface-active agents, they reduce the surface tension of electrochemically repulsed materials, including the negatively charged microorganism, thus permitting interaction of substances that are otherwise repulsed. QAC disrupt the cytoplasmic membrane after 1) adsorption of compound on the bacterial cell surface, 2) diffusion through the cell wall, 3) binding to cytoplasmic membrane, 4) disruption of cytoplasmic membrane, 5) release of potassium ions and other cytoplasmic constituents and 6) precipitation of cell contents and death of the cell (Merianos 2001). Microorganisms can modify the fatty acid composition of their membrane lipid bilayer in response to changes in their environment. Pseudomonas aeruginosa is known to be resistant to antibiotics and disinfectants and can modify its fatty acid composition when grown in the presence of two quaternary ammonium compounds. (Guerin-Mechin, Dubois-Brissonnet et al. 1999) BACTERIAL MEMBRANES The microbial response to stress involves changes in protein and membrane lipid composition that collectively enable the microorganism to persist. The bacterial membrane serves as an interface between the external environment and the cellular cytoplasm, the composition of which it helps to regulate. The membrane performs vital functions, such as maintenance of the proton-motive force and the uptake of nutrients (Russell, Evans et al. 1995). The membrane is composed of lipids that are united not by a common structural feature, but by the common physical property of being insoluble in water and soluble in organic solvents (V oet and Voet 1995). The primary lipids of a biological membrane are phospholipids, a group of molecules with a structure related to triglycerides (Fig. 2). The 3-carbon glycerol molecule is esterified to fatty acids at two carbons while the third links to a bridging phosphate group often bound to a nitrogen containing alcohol. The amphipathic phospholipid naturally orients the hydrophobic tail within the inside of the membrane exposing the hydrophilic moiety to the external aqueous environment (Fig 3). We believe it is likely the bacterial stress response induces protein and fatty acid compositional changes which collectively alter a cells sensitivity to chemical sanitizers. BACTERIAL LIPID COMPOSITION AND MEMBRANE FLUIDITY The fluidity of biological membranes is one of their important physiological attributes since it permits their embedded proteins to interact. As the cell membrane of Gram-positive bacteria is comprised of 75% protein and 25% lipid (V oet and Voet 1995), the movement or fluidity of these proteins is critical for cellular vitality. The primary way that bacteria maintain constant membrane fluidity at different growth temperatures is by adjusting their fatty acid composition (Russell, Evans et al. 1995). The ability of bacteria to change their membrane fluidities determines to some extent how well a bacterium tolerates certain environmental stresses (Li, Chikindas et al. 2002) and is based in part upon the fatty acid composition. Saturated fatty acid membranes are tightly packed and non-fluid while branched fatty acid membranes have increased fluidity. The cell will modify the fatty acid composition (chain length, branched, saturated/unsaturated) of the lipids in the membrane to sustain viability in diverse and stressful environments. Membranes are fluid when composed of unsaturated and branched fatty acids and relatively non-fluid when containing saturated or tightly packed fatty acids. High melting point fatty acids (saturated long and normal chain) decrease membrane fluidity whereas low melting point fatty acids (unsaturated and branched chain) increase membrane fluidity (Table 1) (Russell, Evans et al. 1995). Hydrophobicity of the cell is based upon the cells protein and fatty acid composition. Protein and fatty acid hydrophobicity is influenced by amino acid and fatty acid (acyl chain length, the degree of saturation and branch position) composition (Li, Chikindas et al. 2002). The interaction of the cell with the surrounding environment, and molecules that may affect the microorganism, may be influenced by the net hydrophobicity of the cell. The lipid bilayer of the microbial cell is extraordinarily impermeable to ionic and polar substances (V oet and Voet 1995). Changes in fatty acid composition likely influence the net hydrophobicity of the cell and the ability of bioactive molecules, such as quaternary ammonium compounds, to affect the cell. The complex change in proteins and fatty acid composition in response to stress may influence the cells net hydrophobicity. FATTY ACID CHAIN LENGTH AND SATURATION Lipids within a bacterial membrane have a transition temperature, below that temperature the lipids are in an orderly array or gel-like solid. This orderly array decreases membrane fluidity and cellular function. The transition temperature of the lipid increases with the chain length and the degree of saturation of its component fatty acid residues (V cot and Voet 1995). The membrane becomes more gel-like as the chain length increases or becomes more saturated. Fatty acid composition changes in three ways in response to temperature fluctuation: acyl chain length, the degree of saturation, and the branch position of the fatty acids. In L. monocytogenes, the major cell membrane response to temperature changes is alteration of the fatty acid component of the membrane’s lipids; changes in the head group composition are generally minor (Li, Chikindas et al. 2002). L. monocytogenes membrane fluidity is maintained upon temperature reduction by an increase in C15 and decrease in C17 (increased ratio) and an increase in C-18:1 (Chihib, Ribeiro da Silva et al. 2003) (Russell, Evans et al. 1995). Shortening fatty acid chain length or increasing unsaturation results in low melting point fatty acids (Annous, Becker et al. 1997). These changes in membrane composition are reversible and may occur in a few hours (Li, Chikindas et al. 2002). BRANCHED CHAIN (ANTEISO/ISO) FATTY ACIDS Branched chain fatty acids in microorganisms typically have a methyl group in the iso-methyl (branch point on the penultimate carbon 1'. e. one from the end) or anteiso- methyl (branch point on the ante-penultimate carbon atom i. e. two fi'om the end) (Christie 2004). Anteiso fatty acids have lower melting points than iso fatty acids and contribute to increased membrane fluidity. Lower melting point fatty acids are less likely to be in the gel-like or non-fluid state than higher melting point fatty acids. As the anteiso number increases, the cell is lowering its melting point. Anteiso C-15 in L. monocytogenes, coupled with unusually low levels of straight-chain saturated fatty acids plays a critical role in providing an appropriate degree of membrane fluidity for growth at low temperatures (Annous, Becker et al. 1997). The susceptibility of L. monocytogenes to the antimicrobial activity of nisin illustrates the relationship of membrane lipid composition to bioactive molecules. Nisin, a 34 amino acid-containing protein produced by a Lactococcus lactis ssp. lactis, has antimicrobial activity principally against gram-positive bacteria (Davidson and Harrison 2003). Nisin has GRAS status and is used in heat processed and low pH foods (Register 1999). The bactericidal activity of nisin is due to pore formation in the bacterial membrane, which occurs through a four-step process of binding to the anionic (net negative charge) phospholipids of the cell membrane, insertion into the membrane and pore formation (Davidson and Harrison 2003). The cell’s sensitivity to nisin is influenced by the membrane’s lipid composition, which might act on any of the fours steps. Nisin-resistant strains of L. monocytogenes had altered phospholipid compositions resulting in a decreased net negative charge that hindered binding of the cationic (net positive charge) nisin compound (Davidson and Harrison 2003). These nisin resistant strains had increased long chain fatty acids and reduced ratios of C1 5/C17 fatty acids resulting in reduced membrane fluidity (Mazzotta and Montville 1997). Mildly acidic conditions (pH 5.5) diminished nisin sensitivity of L. monocytogenes and are related to increases in C-14:0 and C-16:0 and decreases in C-18:O fatty acids (van Schaik, Gahan et al. 1999). Predicting the effect of altered hydrophobicity on the ability of the amphipathic sanitizer cetrimide to interact with the cell membrane is difficult. GROWTH AND SURVIVAL WITHIN ACIDIC ENVIRONMENTS Undoubtedly the ability of a microorganism to induce stress-response proteins and consequently adapt to a hostile environment plays a major role in the ubiquity of microorganisms in diverse environments. One such hostile environment frequently encountered by microorganisms is acidity. The acid tolerance response (ATR) describes the phenomenon whereby bacteria that have been exposed to mildly acidic conditions acquire the ability to survive at normally lethal pH values (Hill, O'Driscoll et al. 1995). Key issues are how acid adaptation increases microbe virulence and induces protection against stress encountered in food and food manufacturing environments. ACID TYPES AND EMERGENCY PH HOMEOSTASIS The outer cell membrane is intrinsically impermeable to polar, charged or hydrophobic molecules including the protons liberated by acidic compounds. Hence inorganic acids are largely incapable of penetrating the cell membrane and acidifying the cytoplasm. Undissociated lipid permeable weak acids such as acetic and citric are able to pass through the membrane with relative ease and may dissociate to liberate protons in the cytoplasm (Hill, O'Driscoll et al. 1995). Reducing cytoplasmic (intracellular) pH acidifies the menstrum that permits all vital biochemical reactions. This acidification can lead to the denaturation of acid sensitive proteins and cell death (Hill, O'Driscoll et a1. 1995) A cell can maintain intracellular pH near neutrality via passive and active mechanisms (Hill, O'Driscoll et al. 1995). Passive mechanisms include cell membrane impermeability to protons and the inherent buffering capacity of the protein-rich cytoplasm (Hill, O'Driscoll et al. 1995). Active mechanisms require energy (ATP) to transport H+ ions out of the cell while importing potassium ions. In Enterobacteriaceae, an emergency pH homeostasis system has been described that functions to keep intracellular pH (pH.) above 5 as the organism encounters severe acid outside of the cell (pHo). Within the cell are pools of positively charged amino acids such as lysine, histidine and arginine. Upon acid shock, inducible amino acid decarboxylases act to remove carbon dioxide fiom these amino acids and in turn consume a proton. One such system, lysine decarboxylase, consumes lysine and a proton generating cadaverine which is transported outside of the cell by a lysine-cadaverine antiporter (Bearson, Bearson et al. 1997). Removing protons raises the intracellular pH of the cytoplasm. 10 ATR MECHANISM The inducible ATR of microorganisms is highly dependent upon the growth phase of the organism. L. monocytogenes is tolerant to acid stress at stationary phase, but this tolerance is rapidly lost during exponential grth (Hill, O'Driscoll et al. 1995). Mid- exponential cells are most sensitive to low pH. In Enterobacteriaceae the alternate sigma factor-38 (038), encoded by RpoS is a critical regulator of stationary phase physiology and general stress resistance (Bearson, Bearson et al. 1997). This factor binds to RNA polymerase and enhances transcription of select genes during stationary phase. 038 itself is an acid shock protein and controls the expression of eight other acid shock proteins (Lee, Lin et al. 1995). The pH-inducible ATR has been described as pre-acid (log phase) acid shock response (induced at pH 5.8) or post-acid (stationary phase) acid shock response (induced at or below pH 4.0). Pre-acid phase inducible ATR in Salmonella triggers the synthesis of 43 acid shock proteins while the post-acid phase ATR induces the synthesis of 15 acid shock proteins (Lee, Lin et al. 1995). Both phases of the ATR are necessary for maximal protection against low pH. Reduction in pH during growth will induce both phases of the ATR, however, transfer of cells from pH 7 to pH 3.5 bypasses the pre-acid ATR resulting in death of the cell. STRESS-INDUCED PROTEINS Two dimensional electrophoretic gels of acid-adapted and acid stressed L. monocytogenes have revealed that the initial response to moderate stress (pH 5.5) is to increase the synthesis of predominately constitutive proteins. A second category of novel proteins, not synthesized at neutral pH, is synthesized upon acid adaptation (pH 5.5) and acid stress (pH 3.5) conditions (Phan-Thanh and Mahouin 1999). Protein bands that 11 appeared in large quantities on 2-D gels were tryptic digested to peptides and analyzed by mass spectrometry. Masses of these peptide sequences was compared against the masses of peptides for microorganisms with known genomes using NCBI database at the University of California at San Francisco. This analysis revealed that a number of these proteins including dehydrogenases, quinones, oxydoreductases and a subunit of ATP synthase function in pumping protons out of the cell (Phan-Thanh and Mahouin 1999). Interestingly, two proteins identified as chaperonins, one similar to GroEL, are induced upon sub-lethal heat shock in a number of organisms. This might explain why acid adaptation in S. Typhimurium also induces thermotolerance (Leyer and Johnson 1993). Biotinylation of proteins fiom SDS-PAGE gels demonstrated that acid adaptation alters the outer membrane structure through the synthesis of specific outer membrane proteins (Leyer and Johnson 1993). Most ATR proteins of S. Typhimurium are membrane associated (Foster and Hall 1990). While thermotolerance can be rapidly induced (approximately 1 minute) upon heat shock, full acid tolerance in L. monocytogenes can take up to 60 minutes (Davis, Coote et al. 1996). Since more time is required for full acid tolerance, major changes must occur in cellular composition rather than simple up-regulation of endogenous proteins within the cell (Davis, Coote et al. 1996). It might be predicted these acid- induced changes in the cytoplasmic membrane would affect permeability of chemical sanitizers altering their ability to destroy the cell. CROSS-PROTECTION AGAINST MULTIPLE STRESSES Many investigators have sought to determine whether adaptation to a stress induces the cell to survive other types of stress. This cross-protection permits the stress- 12 adapted cell to survive a myriad of stressful conditions (Lou and Yousef 1997). For example, acid-adapted S. Typhimurium has increased tolerance towards heat, salt, the activated lactoperoxidase system, crystal violet and polymyxin B (Leyer and Johnson 1993). The later two stressors exert their action on the cytoplasmic membrane confirming that acid adaptation and the ATR involve changes in the outer membrane structure. These acid-adapted Salmonella are more hydrophobic and more resistant to surface active agents (layer and Johnson 1993). Maldi-mass spectrophotometric analysis of peptide sequences from acid-adapted L. monocytogenes demonstrated that chaperonin GroEL was produced. This protein serves to maintain, fold or transport damaged or denatured proteins (Phan-Thanh and Mahouin 1999). Using this strain the authors demonstrated that acid-adapted L. monocytogenes had increased resistance to heat shock (52 °C), osmotic shock (25 - 30% NaCl) and alcohol stress (15%). In this study, heat-adapted Listeria (50°C for 45 minutes) also displayed increased resistance to acid shock. Chaperonins are produced in L. monocytogenes upon acid and heat adaptation and provide cross-protection against acid and heat stress. However the synthesis of heat shock proteins by heat-shocked Salmonella did not increase acid resistance (Leyer and Johnson 1993), indicating that induction pathways for acid and heat are different (Lee, Lin et al. 1995). Acid-adapted but not acid-shocked E. coli 0157:H7 cells in low pH fruit juices exhibit enhanced heat tolerance in orange juice at 52 °C (Ryu and Beuchat 1998). Interestingly, acid adaptation to pH 5.0 to 5.8 for one to two cell doublings markedly increased sensitivity of S. Typhimurium to halogen sanitizers (hypochlorite and iodine based disinfectant) (Leyer and Johnson 1997). Changes in the cytoplasmic membrane 13 that result from acid-adaptation may increase penetration or exposure of the cell to the lethal activity of hypochlorite. VIRULEN CE Since acid shock induces the cell to survive other stresses, this ATR may serve as an important signal for inducing general stress resistance. Indeed these cells may become “hardened” to innate gastric acid exposure - one of the first stresses encountered - and within macrophage. Any bacterial cell product that enhances survival in a host can be thought of as a virulence factor (Abee and Wouters 1999). Lee and Lin (Lee, Lin et al. 1995) discovered that virulence of S. Typhimurium is dependent on a sustained induction of the ATR and that this induction is dependent on the “RpoS status” of the cell. The RpoS gene product is the alternate sigma factor 38 (038) responsible for inducing transcription of stationary phase stress-response proteins. A L. monocytogenes mutant incapable of inducing an ATR was less virulent again suggesting that ATR response contributes to in-vivo survival of L. monocytogenes (Marron, Emerson et al. 1997). Four test strains of L. monocytogenes grown at 4°C were more virulent (recovery of L. monocytogenes from spleens and livers) in mice (Czuprynski, Brown et al. 1989). Therefore, the adaptive mechanisms discussed in this review (acid and temperature) may be considered virulence properties. STARVATION Most free-living heterotrophs are thought to lead a “feast and famine” existence, with famine the more habitual state (Koch 1971). The principal changes in response to nutrient deprivation in bacteria relate to nutrient scavenging systems with glucose uptake 14 being the best characterized. (F erenci 1996). The RpoS independent starvation response in E. coli regulates outer membrane proteins and transporters (porins) involved in nutrient scavenging (Ferenci 1996). These membrane proteins selectively transport carbohydrates and other select molecules across the cell membrane. Bacteria have developed a Starvation Survival Response (SSR) to permit survival during lengthy periods of nutrient limitation (Herbert and Foster, 2001). The SSR in L. monocytogenes involves both protein and cell wall biosynthesis permitting survival during starvation and cross- protection to several environmental stresses (Herbert and Foster 2001). Glucose deprived L. monocytogenes cultures decreased up to three logs over the first two days yet persisted with only one additional log decrease over the remaining 18 days (Herbert and Foster 2001). Interestingly, addition of penicillin G and chlorarnphenicol, inhibitors of cell wall and protein synthesis respectively, to starved L. monocytogenes cultures followed by enumeration demonstrated that cell wall biosynthesis stopped after 7 days while protein synthesis stopped 8 hours afier starvation. This suggests that proteins produced during the SSR response occur early upon carbon deprivation and that the cell quickly senses nutrient changes within the environment. SSR of L. monocytogenes may enable environmental and food persistence and enhance the organisms ability to establish infection. It is plausible the L. innocua starvation response results in protein and cell wall biosynthesis that alters cetrimide sensitivity. This starvation response enhances nutrient scavenging capability but also induces resistance to various environmental stressors. The D5“; value of nutrient-starved L. monocytogenes increased 13-fold during 163 hours of starvation at 30°C and significantly increased the heat resistance (56°C) of two of three strains of E. coli OlS7:H7 (Rowe and 15 Kirk 2000). Interestingly, when starved E. coli culture is supplemented with glucose, cells lose their elevated levels of DnaK, H202 resistance, and thermotolerance (Rockabrand, Arthur et al. 1995) suggesting that increased environmental resistance is related to the starvation response. Heat and freeze-thaw resistance of E. coli OlS7:H7 and nonpathogenic E. coli is enhanced after acid adaptation and starvation (Leenanon and Drake 2001). Heat, acid and freeze-thaw resistance of Vibrio parahaemolyticus adapted to starvation with or without low salinity were higher than non-adapted controls (Wong, Chang et al. 2004). As the starvation response induces cell membrane proteins to scavenge nutrients, the acid tolerance response likewise results in production of proteins that predominantly are membrane associated. The E. coli 01 57:H7 starvation response characteristically involves an increased resistance to chlorine and to deoxycholate, a membrane-active detergent (Lisle, Broadaway et al. 1998). Conversely, adaptation of L. monocytogenes to environmental stress conditions (ethanol (5%), acid (pH 4.5 to 5.0), H202 (500 ppm) or salt (7% wt/vol)) did not enhance starvation survival (Lou and Yousef 1997). Habituation of Salmonella at 0.95 Aw resulted in increased heat tolerance at 54°C (Mattick, J orgensen et al. 2000). While habituation at low AW is distinct from starvation, its relevance to starvation relates to the potential mechanism for increased heat tolerance during nutrient deprivation. During habituation at low Aw, the microorganism lacks a solvent to solubilize surrounding excess nutrients. Three solutes (glucose-fructose, NaCl and glycerol) at the same Aw induce different inactivation rates at 54°C (Mattick, J orgensen et al. 2000). This thermotolerance is independent of protein synthesis suggesting L. monocytogenes is reacting to more than A... These solutes would cause 16 substantial osmotic stress leading to the accumulation of compatible solutes. Taken together, these data suggest that cellular targets of heat activation can be protected via scavenger proteins or by accumulated solutes. COLD ADAPTATION Temperature downshift causes the production of cold shock proteins in E. coli (Abee and Wouters 1999) and L. monocytogenes (Bayles, Annous et al. 1996) concurrent with severe inhibition of general protein synthesis and cell growth arrest (Phadtare 2004). A reduction in temperature from 37°C (optimum) to 10°C in E. coli results in a 4—hour lag period followed by growth with a generation time of 24 hours (Jones and Inouye 1994). During the lag period many physiological changes occur, including modification of the fatty acid composition of the membrane bilayer, and an inhibition of DNA, RNA and protein synthesis. Membrane lipid bilayer saturated fatty acid composition is reduced with an increase in unsaturated fatty acids (Russell 1990). Such lipid change will modulate membrane fluidity and the activity of intrinsic proteins that perform functions such as electron transport, ion pumps and nutrient uptake (Russell 1990). Similar to heat shock, temperature downshifi (37°C to 10°C) induces 15 proteins in E. coli (Abee and Wouters 1999) and 12 proteins in L. monocytogenes (Bayles, Annous et al. 1996). These proteins are produced at concentrations 2 to 10 times greater than at 37°C and function at the level of transcription and translation (Jones and Inouye 1994). The cold shock protein CspA has the highest induction level reaching up to 13% of total cellular protein synthesis and may be the general activator of the cold shock regulon. Membrane bilayer modifications that affect permeability and cold shock protein 17 induction that impact metabolism, transcription, translation and protein folding might alter the effectiveness of chemical sanitizers. SUB-LETHAL HEAT The primary structure of a polypeptide dictates the final three-dimensional structure of a protein. Upon ribosomal translation of mRNA, the unfolded protein must pass through biological membranes (eukaryotes) and then properly fold to form a functional protein. Protein miss-folding will disrupt its secondary structure thereby rendering it non-fimctional. To maintain and shield unfolded newly synthesized proteins, the cell produces a set of proteins called “chaperones”. These chaperones affect newly synthesized proteins by a) preventing miss-folding or aggregation, b) allowing them to traverse biological membranes, and c) facilitating their proper folding (Ang, Liberek et al. 1991). All stresses to some extent cause protein denaturation and increase the concentration of unfolded proteins (J uneja, Foglia et al. 1998). As microorganisms encounter changes in their environmental temperatures, they sense the temperature change mainly at the level of the cell membrane, nucleic acids and ribosomes (Phadtare 2004). The cellular response to heat results in a dramatic increase in chaperone proteins called heat shock proteins (HSP) that function to prevent stress-induced accumulation of unfolded proteins. This heat stress (shock) response has been described in every organism investigated, from microorganisms to plants and animals, and represents the most highly conserved genetic system known (Lindquist and Craig 1988). This stress response system permits the cell to rapidly adapt to heat and survive under conditions that would otherwise be fatal. These heat shock proteins have been thoroughly described in 18 E. coli, Salmonella and L. monocytogenes. In both L. monocytogenes and L. innocua, heat and cold shock turns off roughly half the number of proteins synthesized at normal (25°C) temperatures (Phan-Thanh and Gormon 1995). Cells exposed to mild heat modify their cell membrane by increasing the saturation and length of the fatty acids in order to maintain optimal fluidity of the membrane and activity of intrinsic proteins (Abee and Wouters 1999). While most compounds traverse the cytoplasmic membrane through porins (Smith 1997), modifying the fluidity of the cell membrane may alter the ability of compounds to penetrate the lipid bilayer. Acid, cold, heat and starvation-induced changes in the membrane lipid bilayer may also alter permeability of chemical sanitizers which in turn affect their ability to destroy the cell. HEAT SHOCK PROTEINS Heat shock proteins function as chaperones and proteases that act together to maintain quality control of cellular proteins (Abee and Wouters 1999). The HSP 70 family of heat shock proteins is conserved across species and is named DnaK in E. coli. In response to stress, pathogenic microorganisms accumulate levels of heat shock proteins that may represent up to 20% of total cellular protein (Lindquist 1992). Salmonella also induces the DnaK protein in response to oxidative stress within macrophage, one of the most hostile environments encountered by an invading microorganism (Buchmeier and Heffron 1990). The large concentration of heat shock proteins during infection coupled, with the high sequence homology of mammalian and bacterial heat shock proteins, suggests that heat shock proteins play a role in autoimmune disease — i.e., the immune response against stress-induced pathogens may result in “self” 19 immune humoral and cell mediated response. Heat shock protein immunity is implicated in several autoimmune pathologies, including insulin-dependent diabetes mellitus (IDDM), trachoma, systemic lupus erythematosus, Graves disease and both reactive and rheumatoid arthritis (Lindquist 1992). RELEVANCE OF HEAT SHOCK PROTEINS TO THE FOOD INDUSTRY Heat-shocked pathogens are induced to produce heat shock proteins that may permit survival under lethal heat conditions. While heat-shocked pathogens are less resistant to heat than endospore forming organisms (e. g. Bacillus), they might survive in foods receiving a minimal heat process. Additionally these heat-shocked organisms might reside for longer periods of time in the manufacturing environment surrounding high-heat manufacturing processes (e. g. surrounding oven areas). A 3 to 20-fold increase in the time necessary to reduce numbers of S. Typhimurium 7 logs occurred following sub-lethal heating at 48°C (Mackey and Derrick 1986). An average 2.4-fold increase in the D64°c value occurred when L. monocytogenes inoculated fermented meat was held at 48°C followed by lethal heating at 68°C (Farber and Brown 1990). Since the authors did not state the pH of this fermented meat, it is unknown if the acidity further induced thermotolerance. ALTERNATE MECHANISMS FOR ALTERED CETRIMIDE SENSITIVITY That gram-positive cytoplasmic membranes are comprised of 70% protein and 25% lipid (V oet and Voet 1995) suggest alterations in sanitizer sensitivity in stress- adapted L. innocua may be due to fatty acid and/or protein compositional changes in the cell membrane. The response to acid stress in L. monocytogenes and Salmonella results 20 in increased expression of acid tolerance proteins of which many are membrane associated (Foster and Hall 1990; Phan-Thanh and Mahouin 1999). Interestingly, upon acid-adaptation L. monocytogenes increased expression of Fo/F 1 ATPase containing hydrophobic proteolipid Co residues (Phan-Thanh and Mahouin 1999). These hydrophobic proteolipid residues span the membrane and potentially alter net hydrophobicity. It is plausible the cells response to acid may inadvertently alter the cells sensitivity to amphipathic molecules such as quaternary ammonium compounds. RATIONALE AND SIGNIFICANCE Stress adaptation of microorganisms permits their survival under hostile conditions that would otherwise be lethal. In some instances the response to one stress leads to resistance to a myriad of stresses. Because chemical sanitizers are the principal means by which the food industry eliminates microorganisms from the food environment during wet sanitation, it is critical to understand their efficacy when the microorganisms adapt to those environments. This research will determine the effect of chemical sanitizers on stress-adapted L. innocua (Objective 1). L. innocua exposed to acid, starvation, cold and heat conditions will be exposed to the quaternary ammonium compound cetrimide followed by enumeration on tr'ypticase soy agar at 35°C for 48 hours. This work will also seek to understand the relationship of net cell hydrophobicity and fatty acid profiles of stress-adapted L. innocua (Objective 2) to sanitizer sensitivity differentials noted in objective 1. This work will determine if changes in cell membrane fatty acid composition are related to cetrimide sensitivity in stress exposed L. innocua. Microorganisms normally exist in a stressed state, yet chemical sanitizer efficacy studies 21 are rarely performed using microorganisms cultured under stress conditions. This research will evaluate chemical sanitizer efficacy using stress-adapted microorganisms. The development of interventions by the food industry to increase sanitation effectiveness and reduce cross-contamination is critical to reducing post-process contamination. Sponge sampling of beef hide, feces and carcass immediately prior and after slaughter demonstrated the presence of E. coli 01 57:H7 on carcass not detected on the beef cattle hide or within feces (Elder, Keen et al. 2000) illustrating the possible presence of a pathogenic microorganism within the manufacturing environment contaminating processed food. The December 1998 Listeriosis outbreak linked to hot dogs and sliced luncheon meats resulted in a CDC reported 101 illness, 15 adult deaths and 6 still births or miscarriages (USDA 2001). The isolate responsible for the outbreak was purported to reside in the air conditioning unit cooling air over cooked product (Perl 2000) again illustrating contamination of processed food from pathogens persisting within the manufacturing environment. Processed foods likely become contaminated with microorganisms that have adapted the environment of food processing, distribution and retail facilities. While commercially produced foods that contain pathogenic microorganisms may have been under processed, we would predict post-process contamination by pathogens in the manufacturing environment is responsible for adulteration of most foods classified by the USDA-FSIS as Ready To Eat (RTE). The draft L. monocytogenes risk assessment published by the joint effort of the USDA, FDA and CDC requests new strategies to 22 “decrease the rates of recontamination during the manufacturing and marketing of ready- to-eat foods” (Buchanan 2001). AIMS This research will determine whether sensitivity to the quaternary ammonium compound sanitizer cetrimide is altered when L. innocua is adapted to acid, starvation, cold and heat stress, conditions commonly found within food manufacturing environments. This research will investigate whether altered sanitizer sensitivity in stress exposed L. innocua is related to cell membrane changes in fatty acid composition. This research strives to further our understanding of the behavior of L. innocua within the food manufacturing environment and lead to the development of sanitation interventions providing a useful tool to the food industry and to US. Agriculture. 23 Growth phase % Survivors V CFU/ml ' Survivors Vv—r‘v‘v vrvv‘ . vjVIVYV' Time/hours Figure 1. Effect of growth phase on resistance to pH 3.5. The growth curve is represented by the open circles, while the % survivors afier exposure to pH 3.5 for 60 min is indicated by the closed circles (Hill, O'Driscoll er al. 1995). 24 R’ and R” = fatty acid cHz—ooca' R"COO —CI>H a + CH2 —0 47—0 -CH2CH2NI-h 0- FIGURE 2. PHOSPHOLIPID MOLECULE (CHRISTIE 2005) 25 / Hydrophillic ydrophobic ~forming protein pore n .l e t O I D. inner layer FIGURE 3. LIPID BILAYER (KAISER 2005). 26 TABLE 1. EFFECT OF FATTY ACID MODIFICATION ON MEMBRANE FLUIDITY Fatty Acid Effect of increased concentration of fatty acid on membrane fluidity Saturation Decrease Methyl-branched Iso Decrease Anteiso Increase acyl chain length Decrease CHAPTER II. ALTERED SENSITIVITY TO A QUATERNARY AMMONIUM SANITIZER IN STRESSED LISTERIA INNOCUA SUMMARY Chemical sanitizers are commonly used to inactivate Listeria monocytogenes and other Listeria spp. that persist in food processing environments after cleaning. In this study, sensitivity of L. innocua to the quaternary ammonium compound cetrimide was assessed following exposure to acid, heat, cold and starvation stress. Unstressed and stressed cultures were exposed to cetrimide for three minutes, neutralized and plated on Tryptic Soy Agar with yeast extract to determine the percent survivors. Relative to controls, L. innocua demonstrated diminished cetrimide sensitivity when exposed to acid and starvation conditions, whereas heat and cold stress increased cetrimide sensitivity (P<0.05). Diminished sensitivity of acid and starvation-exposed L. innocua to cetrimide suggests that these stressors might increase the persistence of this organism within food manufacturing facilities. In contrast, enhanced L. innocua sensitivity to cetrimide following heat and cold suggests that these interventions might increase sanitation efficacy. INTRODUCTION Research over the past two decades has determined that microorganisms have multiple genetic and physiological mechanisms to respond to adverse or stressful conditions (Lindquist and Craig 1988). Stressed microorganisms may exhibit changes ranging from minor metabolic alterations to more extreme modifications in cell structure (Johnson 2003). Physiological or structural changes resulting from exposure to moderate 28 or sub-lethal stress might permit the organism to survive greater amounts of stress than an organism grown under optimal conditions (Johnson 2003). In food manufacturing facilities where conditions are maintained to minimize growth of pathogens and spoilage agents, microorganisms may be sub-lethally stressed by exposure to acid, heat, cold, or nutrient depletion. These conditions retard growth of microorganisms yet might trigger stress-induced cellular changes that enable the organism to persist within these environments. “Controlling the presence and growth of Listeria species has proven to be very difficult for the food industry and this is attributed in part to the microorganisms ability to grow under refrigeration conditions. Control of L. monocytogenes in the food manufacturing environment has been a challenge despite being sensitive to commonly used chemicals such as acid anionic, quaternary ammonium compound, iodine, and chlorine based chemical sanitizers (Lopes 1986). Sanitizer efficacy is generally determined with microorganisms cultured under ideal conditions (Grab and Bennett 2001). However, there is scant information on sanitizer efficacy under conditions of stress. The known existence of stress-hardened organisms (Lou and Yousef 1997) raises questions about the applicability of lethality studies conducted when the microorganism is cultured under ideal conditions. The objective of this research was to determine efficacy of the quaternary ammonium compound cetrinride on L. innocua that was sub-lethally stressed by exposure to acid, elevated temperature, cold and starvation conditions. L. innocua was selected as the model challenge organism because this species has proven to be very difficult to eliminate in food manufacturing environments following sanitation (Tompkin 2002) and 29 it serves as a safe and scientifically appropriate laboratory surrogate for the pathogen L. monocytogenes . MATERIALS AND METHODS L. innocua ATCC strain 33090 (Microbiologics, Saint Cloud, MN), maintained at —-80 °C, was used in all experiments, and cultured in Tryptic Soy Broth (TSB) or Tryptic Soy Agar (TSA) ( BBLTM, Sparks, MD). Acid and heat stress induction was determined by exposing the culture to modest acid or heat stress, and then enumerating survivors following exposure of adapted and control cultures to the lethal stress (Buchanan and Edelson 1996; Lou and Yousef 1997). Cold and starvation stress was induced following established protocols (Rowe and Kirk 2000; Leenanon and Drake 2001). Following stress exposure, treated and control cultures were exposed to the quaternary ammonium compound cetrimide for 3 minutes. Survivors were enumerated by pour plating in TSA containing 0.6% yeast extract (TSA-YE). All plates were counted following 48 h incubation at 37°C. The acid adaptation method of Buchanan and Edelson (Buchanan and Edelson 1996) was used. Briefly, L. innocua was inoculated into TSB supplemented with 1% (w/v) glucose (EM Science, Gibbstown, NJ) (TSB+G). Following 18-20 hours of incubation, the pH decreased from pH 5.5 to pH 4.7 at which point L. innocua was classified as acid-adapted. A non-adapted control culture (pH 7.0) was generated by adding 0.15 ml of 0.25M Butterfield’s Phosphate Water (BPW) (Food and Drug Administration 1998) to 1 ml of the acid-adapted culture and incubating for l h at 37°C . The ability of acid-adapted L. innocua to survive lethal acid was evaluated by exposing 0.1 ml of the acid-adapted and control cultures to 9 ml of Brain Heart Infirsion 30 broth (adjusted to pH 2.5 with HCl) for 2 h at 37°C (Buchanan and Edelson 1996). After 2 h of exposure, L. innocua was enumerated on TSA-YE. The heat adaptation method of Lou and Yousef was used (Lou and Yousef 1997). Briefly, heat exposure was conducted using log phase (8 h) cultures grown at 37°C in TSB supplemented with 0.6% yeast extract (TSB-YE). Cultures (5 ml containing approximately 10° CFU/ml) were centrifuged (5,000 x g) twice for 10 minutes at room temperature and resuspended in 5 ml BPW. TSB-YE (0.5 ml) was added to 1 ml of washed cells, followed by l h of incubation at 45°C in a static waterbath (heat-adapted) or at room temperature (22°C) (control). Heat tolerance was evaluated by adding 0.1 ml of heat-adapted and control cultures to 55°C (lethal heat) tempered BPW (9 ml) supplemented with 0.5 ml TSB-YE. After 1 h, these cultures were rapidly cooled to 10°C and enumerated on TSA-YE. The method of Leenanon and Drake (Leenanon and Drake 2001) was used for cold exposure. Overnight (18-22 h) TSB-YE cultures of L. innocua were centrifuged (5,000 x g) twice for 10 minutes and resuspended in 1 ml of TSB-YE (1 ml containing approximately 10° CFU/ml) (Leenanon and Drake 2001). Cells were incubated in TSB- YE for 5 days at 10°C (cold culture). The control culture was similarly prepared on the day of cetrimide exposure by suspending a twice washed overnight L. innocua (10° CFU/ml) culture in 1 ml of TSB-YE. Nutrient starvation was carried out as described by Rowe and Kirk (Rowe and Kirk 2000). Overnight TSB-YE cultures (9 ml) of L. innocua were centrifuged (5,000 x g) twice for 10 minutes and resuspended in 1 ml of TSB-YE (approximately 10° CFU/ml) after which 0.2 ml was added to 20 ml of sterile distilled water. These cells were starved 31 for 24 h at 37°C (Rowe and Kirk 2000). The control culture was similarly prepared on the day of cetrimide exposure by suspending 0.2 ml of twice washed (5,000 x g for 10 min.) overnight culture (TSB-YE) in 20 ml of sterile distilled water. Sanitizer sensitivity of stress-exposed and control cultures was evaluated by exposing the cultures to 10 ppm cetrimide (J .T. Baker, Phillipsburg, NJ) prepared in distilled water. Cultures (0.1 ml) were added to 10 ml of double-distilled water containing 10-ppm cetrimide. This cetrimide concentration reduced L. innocua numbers yet was sufficiently low to discern differences in sensitivity between adapted and control groups. Initially and after 0.5, 1.5 and 3 minutes of exposure, the sanitizer-exposed culture (50 pl) was neutralized in 0.2 ml Letheen broth (Food and Drug Administration 1998) in the first column of wells in a 96 well microtiter plate (Corning, Corning, NY). Neutralized cultures (50 ul) were serially diluted in 0.2 ml BPW to permit quantification. Aliquots (0.1 ml) were plated in TSA-YE and incubated for 48 h at 37°C. Tolerance induction and sanitizer sensitivity experiments were run in duplicate or triplicate and replicated twice. Logarithms of initial and subsequent populations at the various time points were calculated. The differences between these paired sets were analyzed using analysis of variance with culture as one factor and time of analysis as a second factor. RESULTS Following exposure to sub-lethal acid (pH 4.7) and heat (45°C), L. innocua was exposed to lethal acid (pH 2.5) and heat (55°C). The lethal conditions were designed to eliminate the population at a rapid yet quantifiable rate. A 7-fold decrease in sensitivity to lethal acid was observed in acid-adapted L. innocua relative to the control (Table. 2). 32 The control culture was generated in this experiment by raising the pH of the acid- adapted culture to pH 7.0 using 0.25 M BPW. To determine if this elevated BPW ion concentration per se affected acid tolerance, acid-adapted L. innocua was exposed to BPW with pH reduced from 7.0 to pH 4.7 with l M HCL. Acid-tolerance of the raised ion concentration culture was unaffected by addition of BPW, indicating that decreased acid sensitivity in acid-adapted L. innocua is related to acid tolerance but not to elevated buffer ion concentration (data not shown). A 276-fold decrease in sensitivity to lethal heat was observed in heat-adapted L. innocua relative to the non-adapted control (Table 2). While the non-adapted control L. innocua decreased 3 logs during 1 hr min of heating at 55°C, heat-adapted L innocua was unaffected. Cetrimide at 10 ppm reduced the L. innocua control by 4 log within the first 30 sec. Acid-adapted L. innocua was less sensitive to cetrimide than the control (P<0.05) (Table 3). For example, after 1.5 min of cetrimide exposure, the acid-adapted and control cultures decreased 0.8 and 4.6 logs, respectively. Cetrimide reduction of heat-adapted L. innocua exceeded that seen in the control population at all time points. After 1.5 min of cetrimide exposure, the heat-adapted and control cultures decreased 5.5 and 2.4 log, respectively (Table 3). Cold-exposed L. innocua cells were also more sensitive to 10 ppm cetrimide than control cultures (Table 3). Cold-exposed L. innocua was nearly eliminated (3.7 log reduction) by 30 sec cetrimide exposure, while the respective control population was reduced by less than 1 log. 33 Cetrimide reduced starvation-exposed L. innocua just over 1 leg after 30 sec. (Table 3), whereas the starvation control decreased 3.4 log. The starvation-exposed and control populations were reduced to the same level (approximately 3.5 log) after 3 min exposure. DISCUSSION The general stress response induces multiple physiological changes in the cell including multiple stress resistance (Lou and Yousef 1997). These stress-induced changes have the potential to alter a microbe’s sensitivity to cetrimide. The major findings of this study were that acid and starvation stress diminished sensitivity of L. innocua when exposed to the quaternary ammonium sanitizer cetrimide, whereas heat and cold stress enhanced survival. Relative differences in cetrimide sensitivity between stress-exposed and control cultures are summarized in Fig. 4 as the differential (log reduction) between stress-exposed and control cultures. It should be noted that cetrimide sensitivity might be influenced by the cell preparation method and the suspending medium (Grab and Bennett 2001). The suspending media were identical for each stress and its respective control but varied between acid, heat, cold, and starvation experiments. These factors preclude direct statistical comparisons of cetrimide sensitivity across stress types within time periods. Further variability in sanitizer sensitivity may exist across Listeria species as this research was conducted using a single ATCC sourced strain. Quaternary ammonium compounds such as cetrimide act primarily by disrupting cellular permeability of the cytoplasmic membrane (Merianos 2001). The sequence of 34 events leading to cell death are as follows: (a) adsorption of the compound on the bacterial cell surface, (b) diffirsion through the cell wall, (c) binding to the cytoplasmic membrane, ((1) disruption of the cytoplasmic membrane, (c) release of potassium ions and other cytoplasmic constituents and (f) precipitation of cell contents and death of the cell. Microorganisms can modify the fatty acid composition of their membrane lipid bilayer in response to changes in their environment. For example, Pseudomonas aeruginosa modifies its fatty acid composition when grown in the presence of two quaternary ammonium compounds (Guerin-Mechin, Dubois-Brissonnet et al. 1999). Pseudomonas aeruginosa resistance to QAC is attributed to an increase in content of cellular fatty acids resulting in decreased penetration of sanitizer (QAC) through the cell wall. Susceptibility to QAC may be related to the appearance of phospholipids and neutral lipids in the outer layer of the outer membrane (Sakagami, Yokoyama et al. 1989). Recent research demonstrated that Pseudomonas aeruginosa QAC sensitivity is related to an outer membrane associated protein (Oer) with homology to lipoproteins of other bacterial species (Tabata, Nagarnune et a1. 2003). Oer knockout Pseudomonas aeruginosa exhibits increased sensitivity to QAC relative to the wild type strain. Heat-adapted L. innocua exhibited greater sensitivity to cetrimide than control cultures. The cellular response to heat results in a dramatic increase in chaperone proteins called heat shock proteins (HSP) that function to prevent stress-induced accumulation of unfolded proteins (Lindquist and Craig 1988). This heat stress (shock) response has been described in every organism investigated, from microorganisms to plants and animals, and represents the most highly conserved genetic system known (Kaufmann 1990). This stress response system permits the cell to rapidly adapt to heat and survive under 35 conditions that would otherwise be fatal. These heat shock proteins have been thoroughly described in E. coli, Salmonella and L. monocytogenes. In both L. monocytogenes and L. innocua, heat and cold shock turns off roughly half the number of proteins synthesized at normal (25°C) temperatures (Phan-Thanh and Gormon 1995). Down-regulation of specific proteins during heat and cold stress may sensitize the cell to cetrimide. Cold and heat stress induce divergent changes in membrane lipid composition. Organisms cultured under cold conditions respond by increasing the ratio of unsaturated to saturated fatty acids within the cellular membrane while organisms cultured under heat conditions decrease unsaturated relative to saturated fatty acids. These changes in membrane fatty acid composition presumably occur to maintain fluidity and to increase the efficiency of solute uptake at low temperatures (Rowe and Kirk 2000). For example, L. monocytogenes cells grown at 10°C were more sensitive to the bacteriocin nisin than cells grown at 30°C (Li, Chikindas et al. 2002). Cells grown at 10°C relative to 30°C had cell membranes with increased amounts of shorter, branched-chain fatty acids and increased fluidity. The short (1 h) duration of heat employed here may be insufficient time to affect a compositional change in membrane lipids. Increased resistance of L. innocua to cetrimide following exposure to acid is consistent with the diminished sensitivity of acid-adapted Salmonella Typhimurium and Vibrio parahemolyticus to surface-active agents. Acid adaptation induces cross- protection against heat, crystal violet, bile and deoxycholic acid in V. parahemolyticus (Koga, Sakamoto et al. 1999). Acid-adapted S. typhimurium (Leyer and Johnson 1993) and L. monocytogenes (Lou and Yousef1996) exhibited increased surface hydrophobicity. The diminished lethality of surface-active agents and increased cell 36 surface hydrophobicity in acid-adapted organisms suggests that the cell membrane directly or indirectly affects the ability of antimicrobials to destroy the cell. Many of the 37 known proteins induced upon acid-adaptation in L. monocytogenes are membrane- associated and firnction by pumping protons out of the cell (Phan-Thanh, Mahouin et al. 2000). Most proteins induced upon acid-adaptation in S. typhimurium are membrane- associated (Foster and Hall 1990) and likely alter outer membrane structure (Leyer and Johnson 1993). Heat shock proteins serve as chaperones within the cell protecting intracellular proteins from heat denaturation. These chaperone proteins protect the cell by protecting proteins within the cytoplasm, while the acid tolerance response proteins principally fimction at the cytoplasmic membrane. Diminished sensitivity of acid- adapted and enhanced sensitivity of heat-adapted L. innocua following cetrimide exposure might be explained by protein-induced alterations of the cell membrane in acid- adapted, but not heat-adapted L. innocua. Consistent with data reported here, L. monocytogenes starved in a low nutrient medium and PBS demonstrated decreased QAC sensitivity (Ren and Frank 1993). L. monocytogenes in a low nutrient medium was less sensitive to QAC than in PBS (Ren and Frank 1993). Starved cells have decreased membrane fluidity and permeability and increased surface hydrophobicity (Lou and Yousef 1996). The observation that microorganisms held in low nutrient and even starved conditions are less sensitive to QAC has important industrial implications. Cleaning programs aim to remove nutrients, thereby preventing unacceptable levels of microbial growth. Microorganisms within food manufacturing environments typically starved for nutrients and these studies suggest they are less likely to be destroyed upon exposure to low concentrations of QAC. The 37 levels of QAC used in this study are 20 times less than the ZOO-ppm maximum level permissible on food contact surfaces without rinsing (Federal Register 2002). While it is unlikely that acid and starvation stress encountered within the food-manufacturing environment induces survival upon exposure to 200 ppm QAC, sanitizers applied in wet environments are often diluted by water used during manufacturing to levels less than 10 ppm in which acid and starvation-induced L. innocua might survive. Taken together, this research demonstrated decreased cetrimide sensitivity in acid-adapted and starvation-exposed L. innocua, and increased sensitivity in cultures exposed to heat and cold conditions. Alteration of sanitizer sensitivity in L. innocua exposed to stress conditions suggests that the manufacturing environment may influence the sanitarians ability to purge the manufacturing environment of spoilage or pathogenic microorganisms. Evidence that acid and starvation conditions diminish cetrimide sensitivity suggests that certain conditions may enable microorganisms to persist and establish growth niches. It may be necessary to increase sanitizer concentration in environments that select for these persistent strains. Conversely, heat and cold conditions enhanced cetrimide sensitivity suggesting that sanitation efficacy might be increased by the sanitarian through application of sub-lethal stress conditions. 38 TABLE 2. HEAT AND ACID TOLERANCE OF RESPECTIVE ADAPTED AND NON-ACID-ADAPTED L.INNOCUA. Log reduction 1 Acid‘? Heat3 Adapted 0.30 (0.01) a 0.01 (0.20) a Control 2.13 (0.15) b 2.99 (0.39) b 1 Log reduction of acid or heat adapted and non-adapted (control) L. innocua following exposure to respective lethal condition. Values represent mean log reduction +/- SEM. Columns with the same letters are not significantly different (P>0.05; N=6). 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