IN VITRO SURVIVAL OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI AT LOW PH Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. _________________________ Bashar Wajeeh Shaheen Certificate of Approval: _________________________ Omar A. Oyarzabal, Chair Assistant Professor Poultry Science _________________________ Robert A. Norton Associate Professor Poultry Science _________________________ Sacit F. Bilgili Professor Poultry Science _________________________ Stephen L. McFarland Dean Graduate School IN VITRO SURVIVAL OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI AT LOW PH Bashar Wajeeh Shaheen A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Master?s Degree Auburn, Alabama May 11, 2006 iii IN VITRO SURVIVAL OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI AT LOW PH Bashar Wajeeh Shaheen Permission is granted to Auburn University to make copies of this thesis at its discretion, upon the request of individuals or institutions and at their expense. The author reserves all publication rights. _________________________ Signature of Author _________________________ Date iv VITA Bashar W. Shaheen, son of Wajeeh Raghib Shaheen and Aziza Abdulfatah Naser, was born August 29, 1975, in Kuwait, State of Kuwait. He graduated from comprehensive high school in 1993. He completed his degree of Bachelor of Veterinary Medicine and Surgery (BVMs) equivalent to DVM from Mosul University, Iraq in September, 1998. After working as a veterinarian in Greater Amman Municipality (food safety and inspector in charge) for three years, he entered Graduate School in the Department of Poultry Science at Auburn University, Alabama in January 2004 and conferred a Master of Science degree in December, 2005. v THESIS ABSTRACT IN VITRO SURVIVAL OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI AT LOW PH Bashar Wajeeh Shaheen Master?s of Science, May 11, 2006 (BVMs, Mosul University, 1998) 91 Typed Pages Directed by Omar A. Oyarzabal A series of experiments were performed to investigate the effect of pre-exposure to acidic conditions of early exponential and stationary-phase cells of Campylobacter jejuni and C. coli. The sublethal acid exposure to protect C. jejuni and C. coli against low pH treatment was also studied. Twelve C. jejuni strains, including ten fluoroquinolone- resistant C. jejuni isolated from humans, and nine C. coli strains, were grown at 42 o C under microaerophilic conditionss (85% N 2, 10% CO 2, 5% and O 2 ) in modified Campy Cefex (mCC) agar to create stationary-phase cells. The colonies were then suspended in Tryptic Soya broth (TSB, at final pH 7), and grown to the appropriate early exponential- phase under microaerophilic conditions. The cells in both phases were exposed to TSB at vi pH 5 for 4 h before the final challenge (TSB at final pH 4). The same procedures were performed utilizing Brucella broth. Survival was determined after 2-3 h of the final challenge by using viable cell counts and utilizing Campylobacter blood free selective media (mCCDA). Pre-exposure of the early exponential-phase and the late stationary- phase cells to moderate pH 5 shows no induction of resistance to the pH 4, where the control cells show better survival. Moreover, the survival time increased from 2 to 3 h when using Brucella broth instead of TSB. Campylobacter coli were very sensitive to the lethal acidic treatments. The results showed that Campylobacter jejuni and C. coli failed to produce an acid adaptation response in early-exponential or stationary-phase in Tryptic Soya broth or Brucella broth (pH 5). The images obtained from scanning electron microscopy also indicated that cells displayed different morphological changes at different pH values. The traditionally used liquid broth and chemically defined medium M 199 was also evaluated for Campylobacter jejuni in vitro. The liquid media compared were M 199, Brucella broth (BB), Brain Heart Infusion broth supplemented with yeast extract (BHIYE), and Tryptone Soya broth supplemented with yeast extract (TSBYE). An initial inoculum of 10 3 to 10 4 CFU/ml resulted in greater than or equal to 10 6 CFU/ml in BB and M 199 after incubation for up to 72 h, while lower growth rate was observed in both TSBYE and BHIYE. Among all liquid media compared, M 199 exhibited the least percent of coccoid cell formation. On M 199, C. jejuni grew over a pH range of 5 to 8. vii ACKNOWLEDGMENTS This thesis could not have been written without Dr. Omar A. Oyarzabal who not only served as my supervisor but also encouraged me and challenged me throughout my graduate program. Also I would like to thank my committee members, Dr. S.F. Bilgili, and Dr. Robert A. Norton who offered guidance and support. Special thanks are due to Dr. Michael Miller for helping me in carrying out the electron microscopy work in his laboratory. Thank you all for everything. I would like extend my thanks to the faculty members, staff, lab managers and technicians and fellow students of the Poultry Science department for their support, help, knowledge, and for making my study years joyful and memorable. I would like to thank my parents, my father and my mother, who taught me the value of hard work by their own experience. I would like to share this moment of happiness with my father and mother-in law, who rendered me enormous support during the whole study. I am grateful to my wife Suhair for the inspiration and moral support she provided throughout my research work and her patience was absolutely incredible during the long hours spent working in the laboratory. Without her love, support and understanding I would never have completed my present work. Particularly, I owe to my son Kareem and my unborn child for their silent prayer for my work to be successfully completed. viii Style manual or journal used Applied and Environmental Microbiology Computer software used Microsoft Word, Version 7, SAS, 8. ix TABLE OF CONTENTS LIST OF TABLES.................................................................................................. LIST OF FIGURES................................................................................................. I. LITERATURE REVIEW................................................................................... II. THE EFFECTS OF DIFFERENT PH VALUES ON THE SURVIVAL OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI????????????????????????????.. III. EXPOSURE TO SUBLETHAL LOW PH BY FLUOROQUINOLONE- RESISTANT C. JEJUNI AND C.COLI??????????????? IV. GROWTH AND SURVIVAL OF C. JEJUNI IN CHEMICALLY DEFINED MEDIUM M 199???????????..?????..... ?. V. REFRENCES???????????????????.?????? x xi 1 17 32 52 66 x LIST OF TABLES II. 1. SURVIVAL OF EXPONENTIAL AND STATIONARY-PHASE CELLS OF C. JEJUNI AND C. COLI AT PH 6, 5 AND 4???.?. III. 1. ANTIBIOTIC RESISTANCE PROFILE OF CAMPYLOBACTER JEJUNI STRAINS???????????....????????. 2. PERCENTAGE SURVIVAL OF C. JEJUNI AT PH 4 IN TSB AND BRUCELLA BROTH??????????????????... 3. PERCENTAGE SURVIVAL OF C. COLI AT PH 4 IN TSB AND BRUCELLA BROTH?.?????????????????.. 4. PERCENTAGE SURVIVAL OF C. JEJUNI AND C. COLI STRAINS ANALYZED FOR SURVIVAL IN TSB OR BRUCELLA BROTH??????????????????... IV. 1. EVALUATION OF THE SURVIVAL OF DIFFERENT C. JEJUNI ISOLATES IN BB, TSB, BHI, AND M 199???.?....................... 27 45 46 59 50 62 xi LIST OF FIGURES II. 1. MORPHOLOGICAL CHANGES OF C. COLI ATCC 51798 AND MCC 66 CELLS UNDER SCANNING ELECTRON MICROSCOPY?????????????????????. 2. MORPHOLOGICAL CHANGES OF C. JEJUNI CDC 2002-353 CELLS UNDER SCANNING ELECTRON MICROSCOPY??. 3. MORPHOLOGICAL CHANGES OF C. COLI MCC 66, C. JEJUNI CDC 2002-353, AND C. COLI ATCC 51798 CELLS UNDER SCANNING ELECTRON MICROSCOPY??????.. III. 1. RIBOTYPING RESULTS OF CAMPYLOBACTER STRAINS?? IV. 1. VIABILITY AFTER INCUBATION OF C. JEJUNI CDC 2002- 353 IN A DEFINED MEDIUM AT DIFFERENT PH VALUES???????????????????????... 2. VIABILITY OF ATCC 49941 IN A DEFINED MEDIUM AT DIFFERENT PH VALUES????................................................... 3. GROWTH OF C. JEJUNI STRAINS IN DIFFERENT BROTHS???????????????????????... 28 29 30 51 62 62 64 1 I. LITERATURE REVIEW The Bacterial Growth Curve In batch culture and under favorable conditions, a growing bacterial population doubles at regular intervals, and this is called exponential growth. When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over time, plotting the data will yield bacterial growth curve (Figure 1 below). Figure 1. The bacterial growth curve. When bacteria are grown in a closed system (also called a batch culture), like a test tube, a prog ression through a series of phases can observed : no net growth occurs (lag phase). This is followed by a phase of exponential growth, where the cell mass increases exponentially with time. When their growth 2 bcomes limited, the cells stop dividing (stationary phase), until eventually they show loss of viability (death phase). Note the parameters of the x and y axes. Growth is expressed as change in the number viable cells vs time. Phases of bacterial growth curve: 1. Lag Phase. When cells in the stationary phase of growth are transferred to fresh medium, a lag phase occurs. Although there is no apparent cell division occurring, the cells may be growing in volume or mass. The lag phase is due to the time required for the physiological adaptation of stationary phase cells in preparation for growth. The lag phase can be due the time required for the recovery of cells from toxic products of metabolism that may accumulate in the external medium, such as acid, bases, alcohols, or solvents. Sometimes, new enzymes or coenzymes, proteins, RNA, etc. must be synthesized before growth resumes especially if the fresh media is different from the inoculum medium. The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. 2. Exponential (log) Phase. In exponential phase, all cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the 3 composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time. 3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). It may be because of exhaustion of available nutrients, limitation of oxygen, or the accumulation of inhibitory metabolites or end products and exhaustion of space, in this case called a lack of "biological space". The stationary phase, like the lag phase, is not necessarily a period of quiescence. 4. Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. Common causes of death include the depletion of cellular energy and the activity of autolytic (self-destructive) enzymes. Some bacteria can remain viable for along time. For example, some bacteria sporulate or form cysts or form coccoid form when exponential growth ceases. Some bacteria can adapt to nutrient depletion and remain viable for along periods in stationary phase. pH homeostasis in gram-negative bacteria. Enteric pathogens try to keep the internal pH close to 7.6 to 7.8 during growth (3). The difference between internal pH and outside pH is known as delta pH (? pH). The internal pH is maintained by pumping the protons into the cell at alkaline outside pH, or by extruding the protons in an acidic environment (18). Two systems are critical to 4 maintaining the internal pH homeostasis in gram negative bacteria. These systems are potassium-proton antiporters (21) and sodium-proton antiporters (4). Briefly, shifting to acidic condition causes the potassium-proton antiporter system to alkalinize the cytoplasm, whereas shifting to alkaline media result in acidification of the cytoplasm through sodium-proton antiporter (10, 46). In addition to the above mentioned systems, adaptive acid stress response systems are ?emergency? systems which enable the enteric pathogens to induce certain regulatory pathways to cope with the damage caused by severe acidic conditions. Inducible acid stress responses in some gram negative bacteria are provided in this review. Acid resistance properties of Escherichia coli. The human stomach maintains the acidic environment around pH 2, with an approximate emptying time of 2 h (41). Escherichia coli possess remarkable acid resistance systems that enable it to cause disease at low inocula (10 ? 100 CFU/ml). These bacteria are also able to withstand a pH-2 challenge for at least 2 h (24). Four E. coli inducible acid resistance systems in stationary-phase have been reported that provide protection to severe acidic environments. The first system is acid resistance system one, AR1: which relies directly on the stationary-phase sigma factor, and protects cells at pH 2?5 in minimal media (22). There is a need for activation (not induction) by brief exposure to glutamate prior to challenge at pH 2.5. The cyclic adenosine monophosphate (cAMP) receptor protein (CRP) appears to control whether or not the sigma factor- dependent induction mechanism will operate. In rapidly growing cells in complex media, 5 the high value of CRP appears to prevent sigma factor-dependent transcription of the gad genes. In stationary-phase cells (growing on glucose), cAMP values are low, thereby allowing sigma factor -dependent gad expression (7). The second acid resistance system, AR2, requires inducible glutamate decarboxylase, an enzyme that convert intracellular glutamate to gamma amino butyric acid (GABA) and glutamate/GABA antiporter gad C (6). Two glutamate decarboxylase (GAD) genes, gadA and gadBC, encode two proteins, GadA and GadB which provide resistance at pH 2.5 as long as GadC antiporter is present (9). Inducible amino acid decarboxylase systems provide protection to low pH by consumption of intracellular protons. In this system, protons leak across the cell membrane at low pH challenge, and then are consumed by amino acid decaboxylation reactions (e.g., glutamate decarboxylase GAD to form GABA). To consume protons efficiently, the decarboxylation product should be carried out by specific substrate/product antiporters (GadC antiporter) (35). The third AR system requires inducible arginine decarboxylase (AdiA) and the AdiC antiporter, that convert intracellular arginine to agmatine with consumtion of protons in the process (15, 19). The optimum expression of AR3 occurs at low pH 2 in complex media. A fourth system was recently described as lysine dependent, and involves the inducible lysine decarboxylase (19). In these described systems, protons entering the cell are consumed by the decarboxylase reaction via exchange with the amino acid -carboxyl group. The product of decarboxylation is then returned to the exterior by antiport in exchange for more substrate (27). Consuming protons by decarboxylation would produce a less acidic internal pH and generate an inside positive potential that could help repel protons and/or prevent excessive proton motive force. This acidophilic strategy developed by E. coli 6 help to convert membrane potential from an inside negative charge to positive charge, appears to be an important system used to survive extreme acidic environments (34). In contrast, the adaptation of E. coli during the exponential-phase has been determined and called acid habituation (AH). This occurs when E. coli are adapted in nutrient broth at pH 5, with the survival measured when the cells are challenged to pH 3, and involves a short exposure time of 7 minutes (16). Many inducing-agents such as glutamate, aspartate, proline, glucose, and KCL can be added to the log-phase cells for 60 minutes that cause induction at a neutral pH (36). Phosphate and the phosphate-specific porin PhoE have been shown to play a major role in AH during the exponential-phase. Thus, high values of phosphate in the medium are proposed to block access of H + to the PhoE pore and thereby interfere with signal transduction (2, 37). Extracellular proteins made at pH 5 are thought to be important to induce acid habituation in log-phase cells grown at neutral pH (2, 38, 39). Another system that provides protection to log-phase cells is called acid tolerance response (ATR). In an ATR system, log-phase cells are protected from severe acid stress for several hours at pH 3, unlike the previously described AH which provides protection for only 7 minutes at pH 3 in complex media. Cyclopropane fatty acid (CFA) formation in the membrane is critical for ATR in E. coli (8). The potential roles of CFAs during the ATR in E. coli include interaction with membrane proteins that influence proton?s traffic. Unsaturated fatty acids are converted to their cyclopropyl derivatives by the action of a cyclopropane fatty acid (CFA) synthase encoded by the cfa gene (17). 7 Acid tolerance response of Salmonella serovar Typhimurium. Salmonella typhimurium has both a log-phase and a stationary-phase acid tolerance system called acid tolerance response (ATR). In both phases, cells grown in moderately acidic environment trigger the synthesis of certain proteins which protect the cells from severe acidic condition. This occurs when exponential cells are grown at pH 7.7 in minimal media, then, exposed to moderately acidic environment at pH 5.8. This treatment enhances the synthesis of pre-acidic proteins that allow adapted bacteria to survive severe acidic shock (13). Adaptation to pH 4.5 enhances the synthesis of other sets of proteins called acidic shock proteins (ASPs) (11). About 50 ASPs, which differ from those of pre-acidic proteins, are believed to be produced due to the lowering of the internal pH as the outer pH decreases (12). In log-phase ATR, three regulatory genes are involved in controlling the expression of subsets of ASPs. The regulatory genes include the alternate sigma factor sigma factor, which control the expression of 10 ASPs; the iron regulator Fur for 5 ASPs; and the two components signal transduction system PhoPQ for four ASPs. In addition to the ASPs, the Ada protein, which is also known as transcriptional regulator, is required to survive in low pH condition but does not appear to be induced by acid or to regulate ASPs (2). The acid tolerance of stationary-phase of Salmonella typhimurium involves both sigma factor-dependent and sigma factor-independent systems (23). The ? S -dependent stationary-phase is not induced by acid, as indicated in the log phase dependent system, but the entrance of cells into the stationary-phase was enough to induce the sigma factor system and the acid tolerance. For the acid tolerance sigma factor-independent system, 8 the only regulator found to affect this system is the OmpR response regulator. It has been shown that low pH conditions appear to induce the production of OmpR, which, in its phosphorylated state, can trigger expression of genes needed for acid-induced stationary- phase tolerance (1). There is a link between the survival at low pH and virulence. Salmonella typhimurium is able to survive and replicate in the severe acidic condition of the macrophage for 60 min (32). Other studies also indicate that mutants defective in acid tolerance attenuate the pathogenecity of the microorganism (14, 45). Helicobacter pylori acid tolerance. H. pylori colonizes the stomach and causes gastroduodenal diseases, such as gastritis, peptic ulcer disease and gastric carcinoma in humans (5). This organism has exceptional acid tolerance mechanisms that enable it to survive the severe acidic condition of the stomach. The most important mechanism involves the production of urease, which converts urea to carbon dioxide and ammonia an alkaline product (25). One model suggested for urease protection is that the external urease provides a ?cloud? of less acidic conditions around the organism, and the internal urease plays major role in supporting the internal pH homeostasis (40). Furthermore, by using urease inhibitor (fluorofamide), which poorly penetrates the cell, external urease has been shown to be unimportant for internal pH homeostasis (40). The above mentioned results are also supported by the fact that internal urease does not become active until the pH outside is below 6 and remains active through an outside pH 2.5, and that the external urease is inactivated at pH below 4.5. Considering the optimum pH for urease activity is 7.5, how 9 do the cells protect themselves from alkalinizing their internal pH when the cells grow at neutral pH outside in the presence of urea? This question has been solved by the identification of UreI which is an inner membrane proton-gated, urea-specific channel (43). The UreI pore opens as pH drops below 6.5 and the cytoplasmic urease gains access to the outside urea. The enzymatic activity neutralizes the acidic condition, and the pore closes and urea transport ceases. It has been determined that at least six periplasmic amino acid residues are important for activation of the UreI transport activity (44, 33). Protonation of these amino acid residues causes a conformation change that opens the UreI pore. Campylobacter jejuni acid tolerance. Campylobacter spp. are a common causes of food foodborn illness in humans (20). Due to the fastidious nature of the organism, Campylobacter are very sensitive to oxygen, unable to grown at temperature below 29 o C, and at pH below 4.9 or at sodium chloride values above 2.5% (42). Unlike other food borne pathogens, Campylobacter lacks many regulatory pathways in stationary-phase that enable them to cope with the stresses (31, 30). Apparently, the lack of rpoS increases the sensitivity of Campylobacter to many stress conditions. Previous studies indicated that the resistance of C. jejuni NCTC 11351 to different kind of stresses, such as heat or oxidative stress, is not induced upon the entry to the stationary-phase (20). This sensitivity is also described by acidic challenge of C. jejuni CI 120, in which acid adaptation did not provide a protection to mid exponential or the stationary-phase cells (29). More recently, by utilizing different media such as Brucella broth (BB), Tryptic Soya broth supplemented with yeast extract, 10 Mueller Hinton broth, and Brain Heart Infusion supplemented with yeast extract, no ATR was induced (28). Interestingly, upon the entry to the stationary-phase, C. jejuni displayed a change in membrane fatty acid composition, particularly a decrease of the proportion of unsaturated fatty acid and increase in the content of cyclopropane and short-chain fatty acids (26). This change in membrane composition coincided with an increase in the cellular pressure resistance, but no change in acid or heat treatment was observed. 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Acid tolerance induced by metabolites and secreted proteins, and how tolerance can be counteracted, p. 93-106. In J. C. Derek and G. Gardew (ed.), Bacterial responses to pH. John Wiley & Sons, Ltd., Chichester, England. 37. Rowbury, R. J., M. Goodson, and D. A. Wallace. 1992. The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli. J. Appl. Bacteriol. 72:233?243. 38. Rowbury, R. J., T. J. Humphrey, and M. Goodson. 1999. Properties of L- glutamate-induced acid tolerance response which involves the functioning of extracellular induction components. J. Appl. Microbiol. 86:325-330. 39. Rowbury, R. J., N. H. Hussain, and M. Goodson. 1998. Extracellular proteins and other components as obligate intermediates in the induction of a range of acid tolerance and sensitisation responses in Escherichia coli. FEMS Microbiol. Lett. 166:283-288. 40. Scott, D. R., D. L. Weeks, C. Hong, S. Postius, K, Melchers, and G. Sachs. 1998. The role of internal urease in acid resistance of Helicobacter pylori. Gastroenterology 114:58-70. 41. Smith, J. L. 2003. The role of gastric acid in preventing foodborne disease and how 16 bacteria overcome acid conditions. J. Food Prot. 66:1292-1303. 42. Solomon, E. B., and D. G. Hoover. 1999. Campylobacter jejuni: a bacterial paradox. J. Food Safety. 19:121?136. 43. Weeks, D. L., S. Eskandari, D. R. Scott, and G. Sachs. 2000. A H + -gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science. 287: 482-485. 44. Weeks, D. L., G. Sachs. 2001. Sites of pH regulation of urea channel of Helicobacter pylori. Mol. Microbiol. 40:1249-1259. 45. Wilmes-Riesenberg, M. R., B. Bearson, J. W. Foster, and R. Curtiss III. 1996. Role of the acid tolerance response in virulence of Salmonella typhimurium. Infect. Immun. 64: 6220-6228. 46. Zilberstein, D., V. Agmon, S. Schuldiner, and E. Padan. 1984. The sodium/proton antiporter is part of the pH homeostasis mechanism in Escherichia coli. J. Biol. Chem. 257:3687-3691. 17 II. THE EFFECTS OF DIFFERENT PH VALUES ON THE SURVIVAL OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI INTRODUCTION Campylobacter jejuni and C. coli are important causative agents of gastrointestinal infections in humans (15), with an estimated 2.5 million cases each year in the United States (5).Campylobacter is a normal inhabitant of the gastrointestinal tract of broiler chickens (20). Many studies linked the occurrence of Campylobacter in chickens and the increased incidence of campylobacteriosis in humans. Deming et al. (4) identified that humans could be infected through eating re-contaminated fully-cooked chicken, eating chicken that was reported to be raw or undercooked, or eating chicken meat that was in contact with cats. Another study indicated that consuming unpasteurized milk was associated with an outbreak of campylobacteriosis in Hungary (13). Some important factors involved in the epidemiology of Campylobacter in humans are: handling raw chicken, which causes sporadic Campylobacter infection (7), eating pork (19), living or working on a farm (19), barbequing (1, 19) and traveling. Traveling is proposed to be an important factor in the acquisition of bacteria resistant to antibiotics (6, 18). 18 There are many factors that influence stress tolerance of bacteria. The value of inherent stress tolerance varies among natural isolates. It has been proposed that Salmonella entritidis PT4 isolated from clinical cases had better inherent tolerance to heat, acidity, oxidative stress and surface stress than non-clinical ones (8). Those clinical isolates also showed more resistance to the host defenses systems and displayed enhanced virulence (9). Another factor playing an important role in inherent stress tolerance and survival of bacteria in foods is the growth phase of contaminating organism. In general, the stress tolerance is at the lowest in mid log-phase and increases in late log-phase and the stationary-phase. RpoS play a major role to induce the tolerance response during the stationary-phase to different kind of stresses such as heat, H 2 O 2 (10), osmotic stress (11), and irradiation (16). The purpose of this study was to investigate the survival of C. jejuni and C. coli at pH 6, 5 and 4 during the early exponential and late stationary-phase. Furthermore, scanning electron microscopy (SEM) was utilized to determine the nature of morphological changes that occur upon the exposure of C. coli and C. jejuni to different pH values. MATERIALS AND METHODS Bacterial Strains C. jejuni ATCC 33560, CDC 2002-370 and Post 5, and C. coli ATCC 43473, 43133 and mCC 66 were used in this study. All strains were maintained at -80 o C. Cultures were transferred from frozen stocks to modified Campy Cefex (mCC) and grown at 42 o C under microaerophilic conditions (BOC Gas, 5% O 2 , 10% CO 2 and 85% 19 N 2 ). The cultures were incubated for 48 h before starting each experiment. Preparation of inoculum A loop of the culture was obtained from mCC plates and suspended into 9 ml sterile phosphate buffer saline (PBS). The suspension was adjusted to MacFarland No. 0.5 turbidity to achieve a concentration of 10 6 -10 8 CFU/ml and adjusting them to an optical density at 600 of 0.1 to 0.2 (approximately 1x10 8 CFU/ml), measured with spectrophotometer (Spectronic GENESYS 20 Vis; Thermo Electron Co, Waltham, MA). From each PBS tube, one ml was transferred to 10 ml tryptic soy broth (TSB, Difco Laboratories, Detroit, MI) and incubated for 4 h under microaerophilic conditions at 42 o C to create early exponential-phase cultures. To maintain the stationary-phase cells; mCC culture plates were incubated under microaerophilic conditions for 48 h. The cells were then harvested in PBS and concentrated to MacFarland No. 0.5 turbidity (~10 6 -10 8 CFU/ml). Acid resistance of Campylobacter spp. in exponential and stationary-phase Preliminary studies were done to determine the survival of Campylobacter jejuni and C. coli at different pH values and over time. Tubes with 10 ml TSB adjusted to pH 7, 6, 5 and 4 with 1N HCL were inoculated with 0.1 to 1 ml of PBS containing exponential or stationary-phase cultures. Survival (percentage) was determined at 0.5, 1, 4 and 24 h at 42 o C under microaerophilic conditions. Initial and final counts were determined on Campylobacter blood free agar plates (mCCDA, Acumedia, Baltimore, MD) incubated at 42 o C under microaerophilic conditions for 48 h. 20 Scanning electron microscopy (SEM) To determine the value of stress induced at pH 5 and pH 4, SEM photographs were collected for two C. coli cultures (mCC 66 and ATCC 51798), and one C. jejuni (CDC 2002-353). After 2?3 h exposure to pH 4, one ml of the culture was fixed in 2% Glutaraldehyde, 1% Osmium tetroxide and 0.1 M Cacodylate buffer (pH 7.2) for 20 minutes. Cultures were mounted on aluminum stubs with ventral surface upwards and sputter?coated with gold/plalladium. Specimens were examined in a Zeiss DSM 940 SEM operated at 15 kV. Statistical analysis All experiments were performed in triplicates. Results were converted to log 10 CFU/ml and expressed as percent survival after each test. Data were analyzed using GLM procedures (SAS Institute Incorporated, Cary, NC) and means analyzed for differences by Duncan?s test (SAS Program) with significance set at P ? 0.05. RESULTS Tolerance of Campylobacter spp. to different pH value The survival percentage of Campylobacter spp. indicated that there is no significant difference after exposure to pH 6 for 1 h and 4 h and at both of exponential and stationary-phases. Under moderate acidic condition (pH 6), Campylobacter survival was slightly decreased after 24 h (P > 0.05). At pH 5, the survival of C. jejuni declined with time and at 4 h only 74 to 75% of the original population was recovered. The 21 survival of C. jejuni decreased pronouncedly at pH 4, with only 24% surviving after 4 h. C. coli strains were quickly inactivated by pH 4. After 1 h, only 55 and 42% of C. coli were recoverable at pH 4 at the exponential and stationary phases, respectively. No C. coli were recovered after 4 h, even after the enrichment of the samples in Bolton broth (Oxoid) without antibiotics. Morphological changes of Campylobacter spp. upon exposure to acid stress The morphological change and topography due to acidic stress were examined by SEM (Fig. 1, 2, 3). The control C. coli and C. jejuni cells have a normal spiral shape with smooth surface (Fig. 1 A, 2 A) and in a large magnification (Fig. 3 A, C). Cells stressed at pH 4.5 completely lost their spiral shape, resulting in roughness and amorphous collapsed bodies (Fig. 1 B, D, 2 B) and in a large magnification (Fig. 3 B, D). These results indicate the possibility that the outer membrane of the cells or the cytoplasmic membrane partially perturbed. Campylobacter cells exposed to pH 5 showed ruffling and craters on the surface (Fig. 1 C) and in a large magnification (Fig. 2 F) compared with control cells (Fig. 2 E). DISCUSSION Our results are in agreement with what has been reported previously by Kelly et al. (14) who indicated that the resistance of the cells to heat and aeration was greater during exponential-phase, and that there is a fluctuation in resistance during the early and late stationary-phases. These results correlate with the absence of RpoS in the genome of Campylobacter jejuni NCTC 1135, the strain used in their experiment. Unexpectedly, our results pointed out that all C. coli strains lost their viability 22 quickly in TSB broth at pH 4. This observation was noted in both the exponential and the stationary-phases with a better survival for the control cells compared with the exposed ones, and unlike the experiments by Kelly et al. (14) in which the C. coli strain UA585 showed a pattern of survival similar to that observed in C. jejuni following entry in the stationary-phase. Chaveerach et al. (2) found that ten strains of C. jejuni and C. coli were equally inhibited by a mixture of water and commercial broiler feed acidified at pH 4.0 with formic, acetic, propionic, or hydrochloric acid. Those ten strains also lost completely their culturability in Mueller-Hinton broth adjusted to pH 4 with formic acid and could not be recovered even when enrichment media was used (2). Variation in strains, media and methodologies used may explain the discrepancy between our results and the results obtained by previous studies. Whereas, the viable cell counts decreased upon exposure to acidic conditions, this decrease paralleled with a gradual change in Campylobacter cell morphology (Figure 1 B, D; Figure 2 B) and in a large magnification (Fig. 3 B, D). Stress-induced morphology has been also reported with E. coli, which occurs as a result of inducing gene bolA belonging to the rpoS regulon upon exposure to different stresses during the stationary- phase (17). It is apparent that Campylobacter spp. respond to the change in the environment, especially the change in pH, and these responses are reflected as altered cell morphology. These results are in agreement with previously reported morphological changes of two Gram-negative pathogens, E. coli O157:H7 and S. typhimurium, after exposure to hydrostatic pressure and bacteriocin mixture (nisin and pediocin) (3, 12). The results presented above demonstrated that acidic stress, particularly at pH 4 and 5, could be caused obviously greater morphological changes on cell surfaces of Campylobacter, 23 allowed H + to gain access to cytoplasmic components of the cell. Such changes in the cell envelope could subsequently induce lysis and an obvious reduction in viable cell counts especially in C. coli. In conclusion, these results indicate that Campylobacter, especially C. coli, are sensitive to low pH. The influence of media on acid sensitivity should be investigated. 24 REFERENCES 1. Allerberger, F., N. Al-Jazrawi, P. Kreidl, M. P. Dierich, G. Feierl, I. Hein, and M. Wagner. 2003. Barbecued chicken causing a multi-state outbreak of Campylobacter jejuni enteritis. Infection. 31:19-23. 2. Chaveerach, P., A. A. H. M. ter Huurne, L. J. A. Lipman, and F. van Knapen. 2003. Survival and Resuscitation of Ten Strains of Campylobacter jejuni and Campylobacter coli under Acid Conditions. Appl. Environ. Microbiol. 69:711-714. 3. Chaveerach, P., D. A. Keuzenkamp, H. A. Urlings, L. J. Lipman, and F. van Knapen. 2002. In vitro study on the effect of organic acids on Campylobacter jejuni/coli populations in mixtures of water and feed. Poult. Sci. 81:621-628. 4. Deming, M., R. Tauxe, P. Blake, S. Dixon, B. Fowler, S. Jones, E. Lockamy, C. Patton, and R. Sikes. 1987. Campylobacter enteritis at a university: transmission from eating chicken and from cats. Am. J. Epidemiol. 126: 526-534. 5. Friedman, C. R., J. Neimann, H. G. Wegener, and R. V. Tauxe. 2000. Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations, p. 121?39. In I. Nachamkin, M. J. Blaser, (ed.), Campylobacter. Washington: ASM Press. 6. Gaunt, P. N., and L. J. V. Piddock. 1996. Ciprofloxacin resistant Campylobacter spp. in humans: an epidemiological and laboratory study. J. Antimicrob. Chemother. 37:747-757. 7. Hopkins, R. S., and A. S. Scott. 1983. Handling raw chicken as a source for sporadic Campylobacter jejuni infections.J. Infect. Dis. 148:770. 8. Humphrey, T. J., E. Slater, K. McAlpine, R. J. Rowbury, and R. J. Gilbert. 1995. 25 Salmonella enteritidis phage type 4 isolates more tolerant of heat, acid, or hydrogen peroxide also survive longer on surfaces. Appl. Environ. Microbiol. 61:3161-4. 9. Humphrey, T. J., A. Williams, K. McAlpine, M. S. Lever, J. Guard-Petter, and J. M. Cox. 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. 10. Jenkins, D. E., J. E. Schulz, and A. Martin. 1988. Starvation-induced cross- protection against heat or H 2 O 2 ?challenge in Escherichia coli. J. Bacteriol. 170:3910-3914. 11. Jenkins, D. E., S. A. Chaisson, and A. Martin. 1990. Starvation-induced cross- protection against osmotic challenge in Escherichia coli. J. Bacteriol. 172:2779-2781. 12. Kalchayanand, N., P. Dunne, A. Sikes and B. Ray. 2004. Viability loss and morphological change of foodborne pathogens following exposure to hydrostatic pressures in the presence and absence of bacteriocins. Int. J. Food Microbiol. 91:91? 98. 13. Kalman, M., E. Szollosi, B. Czermann, M. Zimanyi, S. Szekeres, and M. Kalman. 2000. Milkborne Campylobacter infection in Hungary. J. Food Prot. 63: 1426-1429. 14. Kelly, A. F., S. F. Park, R. Bovill, and B. M. Mackey. 2001. Survival of Campylobacter jejuni during stationary-phase: evidence for the absence of a phenotypic stationary-phase response. Appl. Environ. Microbiol. 67:2248-54. 15. Nachamkin, I., J. Engberg, and F. M. Aaestrup. 2000. Diagnosis and antimicrobial susceptibility of Campylobacter species. pp. 45-66. In: I. Nachamkin and M. J. Blaser 26 eds., Campylobacter 2ed. (). ASM Press, Washington, DC. 16. Rowburry, R. J. 1972. Observation on starvation-induced resistance enhancement (SIRE) in Salmonella typhimurium. Int. J. Radiat. Biol. 21:297-302. 17. Santos, J. M., P. Freire, M. Vicente, and C. M. Arraiano. 1999. The stationary- phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Mol. Microbiol. 32:789-98. 18. Schorr, D., H. Schmid, , H. Rieder, A. Baumgartner, H. Vorkauf, and A. Burnens. 1994. Risk factors for Campylobacter enteritis in Switzerland. Zbl. Hyg. 196: 327-337. 19. Studahl, A., and Y. Andersson. 2000. Risk factors for indigenous Campylobacter infection: A Swedish case-control study. Epidemiol. Infect. 125: 269-275. 20. Zimmer, M., H. Barnhart, U. Idris, and M. D. Lee. 2003. Detection of Campylobacter jejuni strains in the water lines of a commercial broiler house and their relationship to the strains that colonized the chickens. Avian Dis. 47:101-7. 27 Table 1. Survival (% of initial inoculum) of exponential and stationary-phase cells of C. jejuni (ATCC 33560 and 43473, CDC 2002-370 and Post 5) and C. coli (ATCC 43473, 43133 and mCC 66) at pH 6, 5 and 4. All strains were unrecoverable at 24 h in pH 5 and pH 4. SEM: standard error of the mean. Means with different superscripts are statistically significant (P ? 0.05) for the same pH and time. Comparison made between exponential and stationary-phase cells. pH 6 pH 5 pH 4 Species Phase 1 h 4 h 24 h 1 h 4 h 0.5 h 1 h 4 h Exponential 88 87 79 82 A 68 76 A 55 A 0 C. coli Stationary 86 86 79 76 B 64 63 B 42 B 0 SEM 1.0 0.9 2.2 1.3 2.0 3.9 3.6 0 Exponential 88 88 75 86 75 83 77 24 C. jejuni Stationary 88 87 75 85 74 83 75 24 SEM 0.5 0.5 3.0 0.9 1.1 1.2 2.0 4.5 28 Figure 1. Morphological changes of C. coli ATCC 51798 and mCC 66 cells under SEM. mCC 66 cells with integral cytoplasmic membranes at pH 7 (A) and cells with disruption in the membranes at pH 4 (B). ATCC 51798 cells at pH 5, with arrows showed ruffling and craters on the surface (C) and pH 4 (D). Photographs were collected after 2?3 h exposure to pH 7, 5 or 4. One ml of the culture was fixed in 2% glutaraldehyde, 1% osmium tetroxide and 0.1 M cacodylate buffer (pH 7.2) for 20 minutes. Specimens were examined in a Zeiss DSM 940 scanning electron microscope operated at 15 kV. Bars= 1?m. 29 Figure 2. Morphological changes of C. jejuni CDC 2002-353 cells under scanning electron microscopy. CDC 2002-353 cells with integral cytoplasmic membranes at pH 7 (A) and cells with disruption in the membranes at pH 4 (B). A B 30 A B C D E F 31 Figure 3. Morphological changes of and C. coli mCC 66, C. jejuni CDC 2002-353, and C. coli ATCC 51798 cells under SEM. mCC 66 cells with integral cytoplasmic membranes at pH 7 (A) and cells with disruption in the membranes at pH 4 (B). Similar cell morphology has been observed for CDC 2002-353 in control cells (C) and stressed cells at pH 4 (D). ATCC 51798 cells at pH 5 showed ruffling and craters on the surface (F) compared with control cells at pH 7 (E). Photographs were collected after 2?3 h exposure to pH 7, 5 or 4. One ml of the culture was fixed in 2% glutaraldehyde, 1% osmium tetroxide and 0.1 M cacodylate buffer (pH 7.2) for 20 minutes. Specimens were examined in a Zeiss DSM 940 scanning electron microscope operated at 15 kV. Bars= 1?m. 32 III. EXPOSURE TO SUBLETHAL LOW PH BY FLUOROQUINOLONE-RESISTANT CAMPYLOBACTER JEJUNI AND C. COLI INTRODUCTION The mechanisms of acid tolerance have been discovered for many Gram positive and Gram negative bacteria (6). However, few studies have been conducted on the stress response properties of Campylobacter spp. Kelly et al. (10) indicated that the resistance of Campylobacter jejuni to heat and aeration was greater during the exponential-phase and that there are fluctuations in the resistance during the early and late stationary-phases. This study also indicated that one C. coli strain had a pattern of survival similar to that observed in C. jejuni following entry to the stationary-phase. More recently, a study was conducted to identify the adaptive tolerance response to acid and/or aerobic conditions in exponential and stationary-phases using one C. jejuni strain (13). The survival of C. jejuni CI 120 adapted cells during the early stationary- phase was better than the mid-exponential-phase cells after adaptation to mild pH, and ATR was induced under aerobic + acidic conditions in both early and late stationary- phases. Under acidic adaptation, the late stationary-phase showed inconsistent survival rates at different times and at low pH challenge. Fluoroquinolone resistance (FQ R ) in Campylobacter has increased rapidly in 33 many countries (5). Up to 80% of Campylobacter isolates are fluoroquinolone- resistant in Thailand and Spain (9). However, in many parts of the world fluoroquinolone resistance values remain low (5). In the US, data collected by the Centers for Disease Control and Prevention (CDC) through the National Antimicrobial Resistance Monitoring System (NARMS) showed that 14.2% of the C. jejuni isolates tested in 2000 were fluoroquinolone resistant (3). In vivo fitness of FQ R Campylobacter jejuni in chickens indicates that acquisition of FQ R in Campylobacter does not impair its colonization in the absence of antibiotic selective pressure (11). This finding indicates that FQ R Campylobacter is able to stay in the flock until the slaughter age, with no obvious history of using FQ antimicrobial as treatment. Furthermore, the enhanced fitness in FQ R Campylobacter was not due to compensatory mutation in the genes targeted by FQ, but was directly linked to the resistance-conferring mutation in gyr A. The previous study also proposed that other etiological factors, such as bacterial virulence, host immune system, and environmental condition and management practice play a role to increase the prevalence of FQ R in Campylobacter isolates. The purpose of this study was to determine if early-exponential or stationary- phase cells of 10 fluoroquinolone-resistant C. jejuni isolated from humans, two fluoroquinolone-sensitive C. jejuni strains and nine C. coli strains that showed an acid adaptation response when subjected to pH 5 for 4 h. Furthermore, the influence of different broth media in the response to low pH by C. jejuni and C. coli was also assessed. 34 MATERIALS AND METHODS Bacterial strains C. jejuni strains used in the studies included one chicken isolate (Post 5), ATCC 33560, and ten fluoroquinolone-resistant (FQ R ) strains (CDC 2002-341, -348, -351, -353, -370, -404, -409, -410, -420 and -439) from the Centers for Disease Control and Prevention (2). The C. coli strains included a chicken isolate (mCC 66), and ATCC numbers 33559, 43133, 43473, 43481, 43484, 49941, 51798 and BAA-371. All strains were maintained at -80 o C in glycerol (20-30%). Cultures were transferred from frozen stocks to modified Campy-Cefex (mCC) (14) and grown at 42 o C under microaerophilic conditions (BOC Gas, 5% O 2 , 10% CO 2 and 85% N 2 ). Cultures were incubated for 48 h before starting each experiment. Table 1 shows the antibiotic resistance profile of 10 FQ R strains of C. jejuni (CDC 2002). The identity of the isolates was also confirmed with an automated ribotyping system (Qualicon, Wilmington, DE) (Figure 1). Preparation of inoculum A loop of the culture was harvested from mCC plates and suspended into 9 ml sterile phosphate buffer saline (PBS). The suspension was adjusted to MacFarland No. 0.5 turbidity to achieve a concentration of 10 6 -10 8 CFU/ml and adjusting them to an optical density at 600 of 0.1 to 0.2 (approximately 1x10 8 CFU/ml), measured with spectrophotometer (Spectronic GENESYS 20 Vis; Thermo Electron Co, Waltham, MA). To create early exponential-phase cultures, one ml of the inoculated PBS was transferred to 10 ml tryptic soy broth (TSB pH 7, Difco Laboratories, Detroit, MI) and incubated for 4 h under microaerophilic conditions at 42 o C. 35 To obtain stationary-phase cells, mCC culture plates were incubated under microaerophilic conditions for 48 h. The cells were then harvested and adjusted to MacFarland No. 0.5 turbidity before each experiment. Studies to induce acid stress Forty-eight hour cultures were used as stationary-phase cells or used to induce early exponential-phase cells as described under Preparation of Inoculum. Cultures were transferred to TSB adjusted to pH 5 that was incubated at 42 o C under microaerophilic conditions for 4 h. Cultures were then transferred to TSB adjusted to pH 4. Initial counts were determined immediately after the transfer to pH 4 for both control and pH-5- exposed cells. Final counts were determined after 2 h of incubation at 42 o C under microaerophilic conditions. Similar experiments were done using Brucella broth (BB pH 7, Acumedia) but the final counts in pH 4 were determined at 3 h incubation at 42 o C under microaerophilic conditions. Automated ribotyping Automated ribotyping was performed by using RiboPrinter (Qualicon, Wilmington, DE) with the restriction enzyme PstI and according to the manufacturer? instructions. Briefly, single colonies from a 24-48h cultures on blood agar plate (mCC) were suspended in a sample buffer and heated at 80?C for 15 min. The cell suspension was lysed and the chromosomal DNA was isolated, digested with PstI, electrophoresed, and simultaneously blotted in an automated manner. Bands were detected and analyzed 36 with Bionumerics software using the band matching coefficient of Pearson?s clustering to determine profile relatedness. Statistical analysis All experiments were performed in triplicates. Results were converted to log 10 CFU/ml and expressed as percent survival after each test. Data were analyzed using GLM procedures (SAS Institute Incorporated, Cary, NC) and means analyzed for differences by Duncan?s test (SAS Program) with significance set at P ? 0.05. RESULTS The results of susceptibility testing among Campylobacter CDC 2002 isolates to fluoroquinolone are shown in Table 1. FQ R in CDC strains are noticed particularly against Nalidixic Acid (MIC>256) followed by Ciprofloxacin (MIC>32). The multi-drug resistance pattern (>2 drugs) has been observed in all Campylobacter CDC 2002 isolates, involving two drugs: Ciprofloxacin and Nalidixic Acid. Acid tolerance response of Campylobacter jejuni and C. coli During the exponential-phase, the non-exposed cells showed a pronounced survival advantage over the exposed cells in all C. jejuni strains used (Table 2). This was statistically significant in almost 50% of C. jejuni strains in TSB experiment. The survival of C. jejuni in Brucella broth was extended to 3 h; because at 2 h a higher percent of survival was ascend for both exposed and control cells in both phases (data not shown). Similar results were obtained by using Brucella broth during the exponential- 37 phase, in which the non-exposed cells showed a better survival compared with the exposed ones. However, in BB, the exposed cells showed a remarkable decrease at low pH, and this was significant in all C. jejuni strains (Table 2). CDC 2002-439 showed a very low percent survival at low pH in both exposed and control cells, with no differences for both media and growth phases (Table 2). It is clear from this data that as the time increases, survivals decrease, especially in the exposed cells during the exponential-phase. C. jejuni strains showed a fluctuation in the survival at low pH during the stationary-phase, and this was strain specific. In the TSB experiments, the non-exposed cells gave a better survival than the exposed cells. However; this was only statistically significant for CDC 2002-410, -351, post 5, and ATCC 33560. It should be noted that as time increased to 3 h in Brucella broth stationary-phase experiment, more strains displayed a larger survival rate in the non-exposed cells (Table 2). In contrast to C. jejuni, C. coli strains were very sensitive to the low pH treatment (Table 3). Statistically, the mean survival of non-exposed cells for C. coli ATCC 44941, - 43133, and -43473 was significantly larger than the exposed cells, especially during the exponential-phase. Furthermore, C. coli-stationary-phase experiments showed a remarkable sensitivity to low pH treatment compared with C. jejuni and for both exposed and non-exposed cells. In the TSB experiment, the control cells showed very low survival with almost no counts observed for the exposed cells in the stationary-phase. In Brucella broth experiments cells died quickly and almost no bacterial counts detected in the plates in the non-exposed and exposed groups (data not shown). Interestingly, a group analysis for all strains also indicated a better survival of the 38 control cells compared with the exposed cells (Table 4). The survival advantage of the control cells was noticed in both exponential and stationary-phases. DISCUSSION The increase in fluoroquinolone-resistant among C. jejuni strains in the United States is not well understood. Smith et al. (1999) suggested that foreign travel was the main contributing factor for Minnesota residents (18). Furthermore, the use of a fluoroquinolone antibiotic within a month before the collection of the stool samples appeared to be associated with increase fluoroquinolone resistance (18). Many studies focused on the great possibility acquiring antimicrobial resistance from animals (i.e. poultry) to humans (4). Other studies suggested the relationship between the use of fluoroquinolones in animals and the resistance among human isolates in the 1980s in European countries (5). Because of the complex transmission cycle, studying the transmission of antimicrobial resistance from animals to humans has become very difficult (8). In both TSB and BB experiments, the sublethal exposure to low pH during the early exponential-phase at pH 5 for 4 h caused no acid adaptation in fluoroquinolone- resistance C. jejuni cells (Table 2). Probably this is correlated with the absence of regulatory mechanisms in Campylobacter especially an alternative sigma factor, which provides protection to different environmental stresses upon the entry to the stationary- phase. On the other hand, our results demonstrated that survival advantage of fluoroquinolone-resistance C. jejuni during the stationary-phase in TSB or BB was not induced upon exposure to pH 5. The data (Table 2) showed better survival (P ? 0.05) of control cells over the exposed ones. Wu et al. (19) also failed to find any stress-induced 39 protein in Campylobacter jejuni subjected to pH 5 or 4 for 1 h, but were able to find proteins homologue of E. coli GroEL and GroES at pH 8.6. ATCC C. coli strains quickly lost their viability in TSB or BB after being challenged to low pH 4 (Table 3). This observation was noted both in the exponential or the stationary-phases with a better survival for the non-exposed cells. Our finding disagree with that reported by Kelly et al. (10) in which a C. coli strain had a pattern of survival similar to that observed in C. jejuni following entry in the stationary-phase (10). Different kinds of stresses, strains variations, different media and methodologies used may explain the discrepancy between our result and results obtained by the previous study. Our conclusion about the survival of exposed cells during early exponential-phase is consistent with what has been reported by Murphy et al. (2003), although their study investigated the mid exponential-phase rather than early exponential-phase (12). This study indicated inconsistencies with the survival of late stationary-phase cells over time and at low pH. Our findings revealed a variation in the survival among different strains studied during the late stationary-phase, with no induction observed at pH 5. Additionally, the non-exposed cells always showed better survival than the exposed cells in both TSB and Brucella Broth. The survival of non-exposed cells was statistically significant, and strain specific (Table 2 and 3). Group analysis also indicated that the survival of non-exposed was statistically significant in both experiments (Table 4). Murphy et al (2003) experiment also indicated that ATR was induced under aerobic + acidic conditions in both the early and the late stationary-phases. It can be concluded that the exposure to mild acidic treatment does not provide protection to sever 40 acidic shock in Campylobacter, and that those other stresses such as aerobic conditions as reported previously (12) could play a major role in cross protection. Campylobacter in many food systems is readily exposed to oxygen that leads to the formation of antioxidant enzymes which play a major role in protecting against oxidative stress and indirectly against low pH. Superoxide dismutase (SOD) (15, 16, 17), alkyl hydroperoxidase reductase (Ahp) (1), and catalase (KatA) (7), play a key role as an oxidative defense mechanism in Campylobacter. Those enzymes play an important role in the survival of Campylobacter in many foods such as milk, poultry, and meat, and also have a role in the survival to freezing (16, 18). SOD is believed to enhance Campylobacter colonization in animal models (16), and invasion of mammalian cells in vitro (15). Because our experiments were done completely under microaerophilic conditions, further studies are required to investigate the role of antioxidant enzyme during the exposure to low pH and their role to provide a cross protection to low pH. Ribotyping results for all Campylobacter strains indicated that two different species of C. jejuni and C. coli strains separate themselves into two distinct groups with very low similarity (25.54%) (Figure 1). If ATCC 43133 is not included, the three very closely related C. coli strains are 92.53% similar. In our attempt to investigate the role of different media to induce the survival of Campylobacter spp. against sever acidic condition, we found that when Campylobacter spp. are cultured in a rich medium such as Brucella broth, the bacterial survival in pH 4 is greater than that observed in TSB. In both phases, the data showed a close similarity in the results between TSB and BB for both exposed and non-exposed cells (Table 4). Time is a critical factor in determining the survival advantage for each exposed and non- 41 exposed cells, and time variation between TSB and BB maybe correlated with the different components of the two media. These results illustrated no difference between two media to induce acid adaptation to severe acidic environment. Murphy et al. (12) have recently described a variation in the survival of one C. jejuni strain in different broths and in three Brucella broths from different manufacturers. The strain showed similar results to ours, with exposed cells surviving less than non-exposed cells in one Brucella broth from one manufacturer. However, the same strain exhibited a tolerance response at pH 4.5 in the other two Brucella broths. This information highlights the complexity of selecting a standard strain and or broth for stress studies with Campylobacter. It is apparent that the survival of FQ R Campylobacter jejuni to low pH was not enhanced upon exposure to moderate acidic conditions. Also our findings indicated that cells grown at neutral pH showed a better survival than the exposed cells in both exponential and stationary-phase. The lack of a defined minimal medium suitable for Campylobacter growth prevented clear understanding of the nutrient requirements of the organism, and the environmental condition that play an important role in the survival of Campylobacter cells under stress conditions. Due to the growth media complexity, the effects of pH on the growth survival of Campylobacter should be assessed in a defined medium. 42 REFERENCES 1. Baillon, M. L., A. H. van Vliet, J. M. Ketley, C. Constantinidou, and C.W. Penn. 1999. An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni. J. Bacteriol. 181:4798?4804. 2. CDC. 2004. National Antimicrobial Resistance Monitoring System for Enteric Bacteria: 2002. Human isolates final. Atlanta, GA: U. S. Department of Health and Human services, Centers for Disease Control and Prevention. [Online.] http://www.cdc.gov/narms/annual/2002/2002ANNUALREPORTFINAL.pdf 3. CDC. 2000. National Antimicrobial Resistance Monitoring System for Enteric Bacteria: 2000. Annual report. NARMS. Atlanta: Center for Disease Control and Prevention. 4. Endtz, H. P., G. J. Ruijs, B. van Klingeren, W. H. Jansen, T. van der Reyden, and R. P. Mouton. 1991. Quinolone resistance in campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine. J. Antimicrobioe. Chemother. 27:199-208. 5. Engberg, J., F. M. Aarestrup, D. E. Taylor, P. Gerner-Smidt, and I. Nachamkin. 2001. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg. Infect. Dis. 7:24?34. 6. Foster, J.W. 2000. Microbial responses to acid stress, pp. 99?115. In G. Storz and R. Hengge-Aronis, Bacterial Stress Responses (ed.). ASM Press, Washington, DC. 7. Grant, K. A., and S. F. Park. 1995. Molecular characterization of katA from Campylobacter jejuni and generation of a catalase-deficient mutant of Campylobacter 43 coli by intraspecific allelic exchange. Microbiol. 141:1369?1376. 8. Guerrant, R. L., T. Van Gilder, T. S. Steiner, N. M. Thielman, L. Slutsker, R. V. Tauxe, T. Hennessy, P. M. Griffin, H. DuPont, R. B. Sack, P. Tarr, M. Neill, I. Nachamkin, L. B. Reller, M. T. Osterholm, M. L. Bennish, and L. K. Pickering. 2001. Practice guidelines for the management of infectious diarrhea. Clin. Infect. Dis. 32:331-351. 9. Hoge, C. W., J. M. Gambel, A. Srijan, C. Pitarangsi, and P. Echeverria. 1998. Trends in antibiotic resistance among diarrheal pathogens isolated in Thailand over 15 years. Clin. Infect. Dis. 26:341?5. 10. Kelly, A. F., S. F. Park, R. Bovill, and B. M. Mackey. 2001. Survival of campylobacter jejuni during stationary-phase: evidence for the absence of a phenotypic stationary-phase response. Appl. Environ. Microbiol. 67:2248-54. 11. Luo, N., S. Pereira, O. Sahin, J. Lin, S. Huang, L. Michel, and Q. Zhang. 2005. Enhanced in vivo fitness of fluoroquinolone-resistant Campylobacter jejuni in the absence of antibiotic selection pressure. Proc. Natl. Acad. Sci. USA 102:541-546. 12. Murphy, C., C. Carroll, and K. N. Jordan. 2005. The effect of different media on the survival and induction of stress responses by Campylobacter jejuni. J. Microbiol. Methods. 62:161-166. 13. Murphy, C., C. Carroll, and K. N. Jordan. 2003. Induction of an adaptive tolerance response in the foodborne pathogen, Campylobacter jejuni. FEMS Microbiol. Lett. 223:89-93. 44 14. Oyarzabal, O. A., K. S. Macklin, J. M. Barbaree, and R. S. Miller. 2005. Evaluation of agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses. Appl. Environ. Microbiol. 71:3351-3354. 15. Pesci, E. C., D. L. Cottle, and C.L. Picket. 1994. Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni. Infect. Immun. 62:2687?2695. 16. Purdy, D., S. Cawthraw, J. H. Dickinson, D. G. Newell, and S. F. Park. 1999. Generation of a superoxide dismutase-deficient mutant of Campylobacter coli: evidence for the significance of SOD in Campylobacter survival and colonization. Appl. Environ. Microbiol. 65:2540?2546. 17. Purdy, D., and S. F. Park. 1994. Cloning, nucleotide sequence, and characterisation of a gene encoding superoxide dismutase from Campylobacter jejuni and Campylobacter coli. Microbiology. 140:1203?1208. 18. Stead, D., and S. F. Park. 2000. Roles of Fe superoxide dismutase and catalase in resistance of Campylobacter coli to freeze?thaw stress. Appl. Environ. Microbiol. 66: 3110?3112. 19. Wu, Y. L., L. H. Lee, D. M. Rollins, and W. M. Ching. 1994. Heat shock- and alkaline pH-Induced proteins of Campylobacter jejuni: Characterization and immunological properties. Infect. Immun. 62:4256-4260. 45 Antimicrobial (MIC Values) Strain NA CM CL CI EM AZ GM TC CDC 2002 341 >256 0.25 1.0 >32 0.50 0.094 0.250 0.064 CDC 2002 348 >256 0.19 0.5 >32 0.75 0.125 0.190 32 CDC 2002 351 >256 0.75 3.0 >32 1.00 0.125 0.250 0.25 CDC 2002 353 >256 0.50 1.5 >32 1.00 0.094 0.125 0.19 CDC 2002 370 >256 0.75 4.0 >32 1.00 0.190 0.190 0.5 CDC 2002 404 >256 0.50 2.0 >32 0.50 0.047 0.250 0.25 CDC 2002 409 >256 0.38 6.0 >32 0.75 0.094 0.250 >256 CDC 2002 410 >256 0.38 1.5 >32 1.00 0.125 0.250 0.125 CDC 2002 420 >256 0.25 1.5 >32 0.75 0.125 0.250 0.19 CDC 2002 439 >256 0.38 6.0 >32 1.50 0.125 0.250 0.25 Table 1. Antibiotic resistance profile of Campylobacter jejuni strains. NA = nalidixic acid, CM = chloramphenicol, CL = clindamycin, CI = ciprofloxacin, EM = erythromycin, AZ = azithromycin, GM = gentamicin, TC = tetracycline. 46 Survival (%) TSB pH 4 (2 h) Brucella broth pH 4 (3 h) Strain Treatment Expo. SEM Statio. SEM Expo. SEM Statio. SEM Exposed (pH 5) 63.6 B 69.7 75.0 B 80.2 CDC 2002- 341 Control (pH 7) 88.6 A 6.26 77.4 4.65 79.8 A 0.90 85.8 2.66 Exposed (pH 5) 77.6 B 90.6 54.7 B 84.4 CDC 2002- 348 Control (pH 7) 94.3 A 3.86 94.0 1.79 82.7 A 1.29 88.8 1.57 Exposed (pH 5) 78.6 B 87.3 59.7 B 93.4 CDC 2002- 351 Control (pH 7) 96.3 A 1.39 95.6 1.85* 81.6 A 2.00 88.0 1.80 Exposed (pH 5) 85.6 87.0 83.0 B 59.8 B CDC 2002- 353 Control (pH 7) 92.3 4.05 91.8 4.87 93.6 A 2.16 74.2 A 2.85 Exposed (pH 5) 65.6 B 78.4 48.6 B 48.9 B CDC 2002- 370 Control (pH 7) 91.3 A 3.55 80.0 1.43 86.2 A 2.23 65.9 A 3.14 Exposed (pH 5) 74.0 81.3 79.6 B 76.3 B CDC 2002- 409 Control (pH 7) 93.6 6.18 84.2 1.50 89.7 A 2.70 81.1 A 1.10 47 Exposed (pH 5) 61.3 B 65.0 63.2 B 69.1 B CDC 2002- 404 Control (pH 7) 89.3 A 5.23 70.2 4.99 83.5 A 2.25 88.9 A 2.22 Exposed (pH 5) 62.3 62.9 B 68.3 B 71.9 CDC 2002- 410 Control (pH 7) 70.6 2.47 77.1 A 1.40 86.0 A 1.88 75.0 3.28 Exposed (pH 5) 72.3 B 80.4 70.7 B 75.7 CDC 2002- 420 Control (pH 7) 85.3 A 1.81 84.5 2.09 87.0 A 2.02 83.1 2.07 Exposed (pH 5) 58.3 59.6 46.2 62.0 CDC 2002- 439 Control (pH 7) 56.6 2.50 71.0 6.05 51.6 2.28 65.7 5.61 Exposed (pH 5) 85.3 69.4 B 52.6 B 79.9 Post 5 Control (pH 7) 87.6 2.18 80.6 A 1.93 82.1 A 2.54 80.2 4.26 Exposed (pH 5) 78.6 79.6 B 77.5 B 60.6 B ATCC 33560 Control (pH 7) 83.0 5.90 92.6 A 1.71 87.3 A 1.78 85.4 A 2.82 Table 2. Survival (%) of Campylobacter jejuni at pH 4 on TSB and Brucella broth. SEM: standard error of the mean. Means (for the same strain and phase) with different 48 superscripts are statistically significant (P ? 0.05). Comparison made between exposed and non-exposed (control) cells 49 Table 3. Percentage survival of Campylobacter coli at pH 4 on TSB and Brucella Broth. ATCC 51798, 33559 and 43484 were unrecoverable in TSB and Brucella. ATCC 43133 and BAA-371 gave recoverable cells (10% or less) only in Brucella broth, exponential- phase and for non-exposed cells. SEM: standard error of the mean. Means with different superscripts are statistically significant (P ? 0.05) for the same strain and phase. Comparison made between exposed and non-exposed cells. Survival (%) in TSB pH 4 (2 h) Strain Treatment Exponential SEM Stationary SEM Exposed (pH 5) 0.0 0.0 mCC 66 Control (pH 7) 20.0 14.14 4.6 3.30 Exposed (pH 5) 0.0 B 0.0 ATCC 43473 Control (pH 7) 40.2 A 5.07 0.0 0.0 Exposed (pH 5) 0.0 B 0.0 ATCC 44941 Control (pH 7) 45.6 A 1.56 5.1 3.60 Exposed (pH 5) 0.0 B 0.0 ATCC 43133 Control (pH 7) 45.7 A 2.23 0.0 0.0 Exposed (pH 5) 0.0 2.9 ATCC 43481 Control (pH 7) 0.0 0.0 3.5 3.28 Exposed (pH 5) 0.0 0.0 ATCC BAA-371 Control (pH 7) 2.6 1.8 6.4 4.52 50 Survival (%) Media Phase Treatment C. coli C. jejuni Control (pH 7) 17.1 A 85.4 A Exposed (pH 5) 0.0 B 72.3 B Exponential SEM 3.1 2.0 Control (pH 7) 2.1 81.7 Exposed (pH 5) 0.3 77.4 TSB pH 4 (2 h) Stationary SEM 0.8 1.8 Control (pH 7) 1.5 A 82.6 A Exposed (pH 5) 0.0 B 64.9 B Exponential SEM 0.5 1.9 Control (pH 7) 0.0 80.1 A Exposed (pH 5) 0.0 71.8 B Brucella broth pH 4 (3 h) Stationary SEM 0.0 1.8 Table 4. Survival (%) of C. jejuni and C. coli strains analyzed for survival in TSB or Brucella broth. SEM: standard error of the mean. Means (for the same strain and phase) with different superscripts are statistically significant (P ? 0.05). Comparison made between exposed and non-exposed (control) cells. 51 Figure 1. Dendorogram of Campylobacter strains. Restriction was done with PstI according to manufacturer?s instructions. Bands were detected and analyzed with BioNumerics (Applied-Math, Inc., Austin, TX) using the coefficient of Pearson?s clustering to determine profile relatedness. 52 IV. GROWTH AND SURVIVAL OF CAMPYLOBACTER JEJUNI IN CHEMICALLY DEFINED MEDIUM M 199 INTRODUCTION Many selective media have been designed to isolate C. jejuni from foods. These media utilize mixture of antibiotics in a rich base and are usually incubated at 42 o C under microaerophilic conditions for 24 h (7, 8). Due to the microaerophilic nature of the organism, it is important to incorporate ingredients to neutralize the toxic effect of the substances that form in the presence of oxygen and light. For most plate media, the incorporation of blood at values of 5% to 15% is common (9). Other supplements are also incorporated as a replacement of blood, primarily a combination of ferrous sulphate, sodium metabisulphite, and sodium pysulphate (FBP) at different concentrations (12, 13). Blood and FBP are believed to neutralize the toxic effect of oxygen derivatives (14, 15, 23). Haematin (16) and charcoal (3, 16) are also used to quench oxygen derivatives. Campylobacter enrichment broths have been developed to isolate the organism from samples where the numbers of Campylobacter are relatively low (5, 4). The most commonly used media for isolation of Campylobacter from a food sample are Preston and Campylobacter blood free agar plates (mCCD) media of Bolton. Both of those are 53 utilized as enrichment media (5, 9). For stress response studies, the use of complex media does not necessary give results that mimic in vivo survival of C. jejuni. Furthermore, certain nutrient requirements for Campylobacter growth can not be evaluated by using complex media, and their role are still poorly understood. A chemically defined medium has been developed previously by Dickgiesser et al. (10) for cultivation of C. jejuni strains of human origin. About 57.7% of the strains investigated required amino acid L-methionine. However, C. intestinalis were unable to grow in the previous described media. Therefore, the use chemically defined media must be investigated further to better understand the physiology of Campylobacter, and the regulatory pathway required for stress response. The purpose of the study was to evaluate the growth of C. jejuni and C. coli in a medium in which all components were chemically defined and to determine the effect of different pH values on cell viability. Furthermore, the difference in terms of cell viability, cell morphology, and cell motility of Campylobacter between the chemically defined media M 199 and the traditional media used for isolation was also evaluated. MATERIALS AND METHODS Strains used C. jejuni strains used in the studies included one human strain (CDC 2002-353), and one C. coli (ATCC 49941). Three C. jejuni strains Post 5, ATCC 700819, and CDC 2002-353 were used in the comparative study between minimal medium and the traditional media. 54 Preparation of inoculum Cultures were transferred from frozen stocks to modified Campy Cefex (mCC) plates and grown at 42 o C under microaerophilic conditions (BOC Gas, 5% O 2 , 10% CO 2 and 85% N 2 ). The cultures were incubated for 48 h before starting each experiment. The media used in this study are as follow: chemically defined medium M 199 (Gibco) (21); Brucella broth (BB; Acumedia); Brain Heart Infusion broth (Oxoid) supplemented with 0.6% (w/v) yeast extract (BHIYE) and Tryptone Soya broth (Oxoid) supplemented with 0.6% (w/v) yeast extract (TSBYE). No antibiotics or other supplements, such as blood or FBP, were added to the media. Survival of Campylobacter at different pH values utilizing M 199 C. jejuni (CDC 2002-353), and C. coli (ATCC 49941) were streaked on mCC and incubated for 48 h under micoaerophilic conditions. The pH of the defined medium was adjusted to different pH values (pH 8, pH 7, pH 6, pH 5, and pH 4) by direct addition of 1 M HCl and confirming with a pH Meter (AB 15, Fisher Scientific). The cell cultures obtained from mCC plates were harvested and suspended into 10 ml of sterile phosphate buffer saline (PBS). The suspension was adjusted to MacFarland No. 0.5 turbidity to achieve a concentration of 10 6 -10 8 CFU/ml and adjusting them to an optical density at 600 of 0.1 to 0.2 (approximately 1x10 8 CFU/ml), measured with spectrophotometer (Spectronic GENESYS 20 Vis; Thermo Electron Co, Waltham, MA). From both PBS tubes, 0.1 ml was transferred into 10 ml M 199 tubes adjusted to different pH values. The initial counts were performed immediately after transfer to different pH values. Cultures 55 tubes were tested for survival at 0, 4 and 24 h after incubation at 42 o C under microaerophilic conditions. Viable cell counts at different times were determined on Campylobacter blood free agar plates (mCCDA, Acumedia, Baltimore, MD), and incubated at 42 o C under microaerophilic conditions for 48 h. Comparison between M 199, BB, BHIYE, and TSBYE Each C. jejuni isolate was harvested from mCC plates and suspended into 9 ml sterile phosphate buffer saline (PBS). The suspension was adjusted to MacFarland No. 0.5 turbidity. Utilizing phosphate buffer saline (PBS), another serial dilution (1:10) was performed from the previous concentration to obtain a concentration of 10 5 -10 6 CFU/ml. From each diluted PBS tube, 0.1 ml was transferred to 10 ml of the four media used to achieve a further dilution of 1:1000-1:10,000. The initial count for each strain was determined on Campylobacter blood free agar plates (mCCDA, Acumedia, Baltimore, MD) incubated at 42 o C under microaerophilic conditions for 48 h. All broth cultures were incubated at 42 o C under microaerophilic conditions and the survival for each strain in different broth was determined at 24, 48, and 72 h. Bacterial counts were performed by utilizing Petroff Hausser counting chamber (Hausser Scientific). The pH for different broth cultures was also determined at different times during the experiment. RESULTS AND DISCUSSION Although the fastidious nature of the organism requires special culture conditions in vitro (22), good growth is now achieved in many complex media (9) and in a 56 chemically defined media for C. jejuni described by Dickgiesser and Czylwik (10). In this study, we describe the growth in chemically defined medium M 199 without supplements of both C. jejuni and C. coli. Good growth was achieved with overnight incubation in our available defined medium. C. jejuni grew over the range of pH 5 to pH 8, whereas C. coli became more sensitive to moderate pH 5 especially after 24 h incubation (Figure 1, 2). Both organisms, however, are very sensitive to high acidic condition of pH 4, and readily inactivated after 4 h in the defined medium. These results are in agreement with the previous study indicating that the organism is unable to growth below pH 4.9 and is killed rapidly at pH below this value (2, 16). The value of growth provided by BB and in a defined media was about the same in all C. jejuni strains examined (Figure 3). However, the difference in BHIYE and TSBYE to support growth of C. jejuni indicates that some growth factors. The growth- limiting factor could be the difference in protein content between the different broth cultures. However, supplements such as yeast extracts has been shown to support the growth of C. jejuni at moderate pH value (11). In this study, each experiment has initiated with a low inoculum (10 3 -10 4 CFU/ml), and this was satisfactory for achieving a high growth rate after 24 h. This also allowed a continual culture system that sustained growth beyond 48 h. The highest value of cells concentration (< 7 log 10 CFU/ml) was achieved by using two different isolates after 24 h (Fig. 3). Other studies have achieved peak concentrations of Helicobacter pylori resulted in ? 10 8 CFU/ml after using different initial inoculums of 10 5 , 10 3 to 10 4 , or 10 2 CFU/ml after incubation for 24 h, 48 h, or 72 h, respectively (18). Fetal calf serum supplemented to BB provided a maximum growth for H. pylori (18). No supplementation 57 was used in this study. Antibiotics utilized in most selective media and enrichment broths to inhibit the competitive microflora (7, 16) were not used in this study. The cell motility was significantly greater on BB than on any of the other three liquid media used to culture C. jejuni (Table 1). However, the conversion to coccoid form was greater on BB than on any of the other media. This was particularly noticed after incubation for 48 h and extended incubation for 72 h in C. jejuni ATCC 700819 and Post 5. In contrast, the defined medium M 199 showed the lowest rate of coccoid cell formation compared with other three media, even after 72 h in C. jejuni CDC 2002-353 and Post 5. The prolonged incubation of Campylobacter for more than 48 h stimulated the conversion to the coccoid form. This maybe easily induced following exposure to oxygen, changes in temperature, and starvation. (20). The coccoid is degenerative form that has a decreased values of nucleic acids and peptides and lacks of cellular integrity (1, 6, 17). Our previous results indicated that BB does not provide any induction to low pH in C. jejuni and C. coli. More recently, Murphy et al. (19) indicated that several media such as BHIYE, TSBYE, MH, and BB can not induce adaptation for C. jejuni to low pH treatment. The defined medium M 199 exhibited a particular advantage over all the non- selective broth cultures that have been used in this study to culture C. jejuni. Cultures of C. jejuni in defined medium showed an improvement in cell morphology and decrease in coccal cells compared with other broths. Additionally, the improvement in the growth viability using low inoculum indicated that the defined medium may be an alternative use for future studies. 58 In conclusion, our results indicate that studies of the growth of C. jejuni in a medium in which all components are chemically defined is possible. This defined medium provides enhancement in cell viability and cell morphology and could provides additional information on nutrient requirement, and new approach to study Campylobacter acid tolerance. 59 REFERENCES 1. Beumer, R. R., J. de Vries, and F.M. Rombouts. 1992. Campylobacter jejuni non- culturable coccoid cells. Int. J. Food Microbiol. 15:153?163. 2. Blaser, M. J., H. L. Hardesty, B. Powers, and W. L. L.Wang. 1980. Survival of Campylobacter fetus subsp. jejuni in biological milieus. J. Clin. Microbiol. 11:309? 313. 3. Bolton, F. J., D. N. Hutchinson, and D. Coates. 1984. A blood-free selective medium for the isolation of Campylobacter jejuni from faeces. J. Clin. Microbiol. 19:169?171. 4. Bolton, F. J., and D. Coates. 1983. Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance. J. Appl. Bacteriol. 54:115?125. 5. Bolton, F. J., and L. Robertson. 1982. A selective medium for isolating Campylobacter jejuni/coli. J. Clin. Pathol. 35:462?467. 6. Boucher, S. N., E. R. Slater, A. H. Chamberlain, and M.R. Adams. 1994. Production and viability of coccoid forms of Campylobacter jejuni. J. Appl. Bacteriol. 77:303?307. 7. Butzler, J. P., and M. B. Skirrow, 1979. Campylobacter enteritis. Clin. Gastroenterol. 8:737?765. 8. Butzler, J. P., M., De Boeck, and H. Goossens. 1983. New selective medium for isolation of Campylobacter jejuni from faecal specimens. Lancet. i:818. 9. Corry, J. E., D. E. Post, P. Colin, and M. J. Laisney. 1995. Culture media for the isolation of campylobacters. Int. J. Food Microbiol. 26:43-76. 60 10. Dickgiesser, N., and D. Czylwik. 1985. Chemically defined media for auxotyping of Campylobacter jejuni. Zentralbl. Bakteriol. Mikrobiol. 260:57-64. 11. Fletcher, R. D., A. C. Albers, A. K. Chen, and J. N. Albertson, Jr. 1983. Ascorbic acid inhibition of Campylobacter jejuni growth. Appl. Environ. Microbiol. 45:792- 795. 12. George, H. A., P. S. Hoffman, R. M. Smibert, and N.R. Krieg, 1978. Improved media for growth and aerotolerance of Campylobacter fetus. J. Clin. Microbiol. 8:36? 41. 13. Hoffman, H. A., N. R. Krieg, and R. M. Smibert. 1979. Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. I. Physiological aspects of enhanced aerotolerance. Can. J. Microbiol. 25:1?7. 14. Hoffman, P.S., H.A. George, N.R. Krieg, and R.M. Smibert. 1979. Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni II. Role of exogenous Superoxide anions and hydrogen peroxide. Can. J. Microbiol. 25:8?16. 15. Juven, B. J., and I. Rosenthal. 1985. Effect of free-radical and oxygen scavengers on photochemically generated oxygen toxicity and on the aerotolerance of Campylobacter jejuni. J. Appl. Bacteriol. 59:413?419. 16. Karmali, M. A., A. E. Simor, M. Roscoe, P. C. Fleming, S. S. Smith, and J. Lane. 1986. Evaluation of a blood-free, charcoal-based, selective medium for the isolation of Campylobacter organisms from faeces. J. Clin. Microbiol. 23:456?459. 17. Moran, A. P., and M. E. Upton. 1987. Factors affecting production of coccoid forms by Campylobacter jejuni on solid media during incubation. J. Appl. Bacteriol. 62:527?537. 61 18. Morgan, D. R., R. Freedman, C. E. Depew, and W. G. Kraft. 1987. Growth of Campylobacter pylori in liquid media. J Clin Microbiol. 25:2123?2125. 19. Murphy, C., C. Carroll, and K. N. Jordan. 2005. The effect of different media on the survival and induction of stress responses by Campylobacter jejuni. J. Microbiol. Methods. 62:161-166. 20. Park, S. F. 2002. The physiology of Campylobacter species and its relevance to their role as foodborne pathogens. . Int. J. Food Microbiol. 74:177-188. 21. Testerman., T. L., D. J. McGee, H. L. Mobley. 2001. Helicobacter pylori growth and urease detection in the chemically defined medium Ham's F-12 nutrient mixture. J. Clin. Microbiol. 39:3842-50. 22. Skirrow, M. B., J. Benjamin, M. H. H. Razi, and S. Waterman. 1982. Isolation, cultivation and identification of Campylobacter jejuni and Campylobacter coli, p. 313?328. In J.E.L. Corry, D. Roberts, and F. A. Skinner (ed.), Isolation and Identification Methods for Food Poisoning Organisms, Academic Press, London. 23. Weinrich, K., K. Winkler, and E. Heberer. 1990. Untersuchungen zur Eignung ausgew?hlter Humin-saurenchargen als Nahbodenzusatz f?r Medien sur Isolierung thermophiler Campylobacterspecies. Deusche Tierarztliche Wochenschrift. 97:511? 515. 62 Table 1. Evaluation the survival of different C. jejuni isolates in BB, TSBYE, BHIYE, and M 199 based on the motility and percent of coccoid cell formation at 24 h, 48 h, and 72 h. The coccoid cell formation was measured by taking the % of coccal cells among 100 cells counted in Triplicate. The motility test was evaluated upon microscopic examination of the sample utilizing Petroff Hausser counting champer. Motilility standard includes: (+ +) very fast, (+) fast, (-) slow, and (- -) very slow. time initial 24 h 48 h 72 h Media strain (%) coccoid Motility (%) coccoid Motility (%) coccoid Motility (%) coccoid Motility M199 0 + + 0 + 1 - 40 - - TSBYE 0 + + 0 - - 2 - - 90 - - BHIYE 0 + + 3 - - 20 - - 0 + + BB ATCC 700819 0 + + 0 50% + + 40 50% + 40 - M199 0 + + 0 - 0 - 3 - TSBYE 0 + 0 - 1 80% + + 20 80% + + BHIYE 0 + + 4 - - 1 - 9 - - BB Post 5 0 + + 0 95% + + 40 50% + + 95 - - M199 0 + 0 - 0 + 1-2 - TSBYE 0 + + 0 - 2-3 - 1-2 - - BHIYE 0 + + 3 - - 50 - - 70 - - BB CDC 353 0 + + 0 - 0 + 1 + 63 Figure. 1. Viability after incubation of C. jejuni CDC 2002-353 in a defined medium at different pH values: after 4h, and 24 h. Figure. 2. Viability after incubation of C. coli ATCC 49941 in a defined medium at different pH values: after 4h, and 24 h. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Log 10 CFU\ml initial 87654 pH Level Survival of ATCC 49941 in M 199 at different pH values 4h 24h 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Log 10 CFU/ml initial 8 7 6 5 4 pH Level Survival of CDC 353 in M 199 at different pH values 4h 24h 64 Figure. 3. Growth of C. jejuni strains in different broth cultures. Broth cultures of C. jejuni strains grown in mCC were diluted 1:1000 or 1:10,000 into fresh M 199, TSBYE, BHIYE, and BB broth and incubated at 42?C in a microaerophilic conditions containing (B) 3.0 4.0 5.0 6.0 7.0 8.0 Initial Count 24 48 72 Time (h) (A) 3.0 4.0 5.0 6.0 7.0 8.0 Initial Count 24 48 72 Time (h) M199 TSBYE BHIYE BB log10 CFU/ml 65 5% O 2 , 10% CO 2 and 85% N 2 . At different times, portions were analyzed using Petroff Hausser Counting Chamber. 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