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Survival, growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature
International Journal of Food Microbiology 87 (2003) 131 – 138 www.elsevier.com/locate/ijfoodmicro
Survival, growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature Gloria L. Tetteh, Larry R. Beuchat * Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA Received 8 July 2002; received in revised form 25 October 2002; accepted 2 December 2002
Abstract A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acidshocked Shigella flexneri 2a cells as affected by pH and temperature. The pathogen was grown at 37 jC for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5 F 0.05 with lactic acid. All three cell types were separately inoculated into tryptic soy broth (6.6 – 7.0 log10 cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5 – 5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 jC for up to 144 h. Overall, inactivation of S. flexneri cells at low pH was enhanced with an increase in incubation temperature. All three types of cells survived for 144 h at 4 jC in TSB acidified to pH 3.5, compared to < 24 h at 30 jC and 2 h at 48 jC. The population of all three cell types increased significantly (a = 0.05) within 24 h when cells were incubated at 12, 21, or 30 jC in TSB at pH 5.0, 5.5, or 7.3. Prior exposure of the S. flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions. Acid-adapted cells decreased by approximately 2.5 log10 cfu/ml when incubated at 4 jC for 144 h, compared to a 6log10 reduction in control (unadapted) cells. When cells were exposed to low pH (3.5 – 4.5) and high temperature (48 jC), significantly higher (a = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S. flexneri cells were injured. Results show that the ability of S. flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Shigella flexneri; Acid stress
1. Introduction Shigella dysenteriae, one of four Shigella species, causes classic bacillary dysentery, which is the most severe form of shigellosis (Lampel and Maurelli, * Corresponding author. Tel.: +1-770-412-4740; fax: +1-770229-3216. E-mail address: [email protected] (L.R. Beuchat).
2001). Shigella sonnei causes the mildest infection, while Shigella flexneri and Shigella boydii infections can be either mild or severe. Shigellae have no known nonhuman reservoir, and are usually transmitted from person to person through poor personal hygiene, although contaminated food and water have been associated in outbreaks of shigellosis (Smith, 1987). The infective dose of Shigella is lower than that reported for most other enteric pathogens. According
0168-1605/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(03)00052-7
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to Dupont et al. (1989), 10– 500 viable cells can cause disease in healthy adults. Small (1998) stated that, for most enteric pathogens, a higher dose is generally required to cause diarrhea. This may be due to lethality caused by low pH in the stomach, as well as inhibition of colonization in the intestine tract. The low infectious dose of Shigella suggests that, compared to some enteric pathogens, a higher proportion of cells are capable of surviving exposure to highly acidic environments. The ability of microbial cells to adapt to sublethal stresses and subsequently become more resistant to extreme environmental conditions has been documented for several foodborne pathogenic bacteria, including Salmonella typhimurium (Leyer and Johnson, 1992; Foster and Hall, 1990; Leyer and Johnson, 1993), Escherichia coli O157:H7 (Leyer et al., 1995; Buchanan and Edelson, 1996; Ryu and Beuchat, 1999), and Listeria monocytogenes (Gahan et al., 1996; Davis et al., 1996; Datta and Benjamin, 1995). Earlier work in our laboratory showed that S. flexneri cells grown in tryptic soy broth supplemented with 1% glucose (TSBG) (acid-adapted cells) were able to survive for 6 h in TSB acidified to pH 3.5 (Tetteh and Beuchat, 2001). Populations of control cells grown in TSB without glucose, i.e., cells unadapted to an acid environment, were reduced to undetectable levels within 2 to 4 h. Bacterial cells surviving exposure to acid stress may exhibit increased resistance to an unrelated stress that is subsequently applied. Cross protection against heat as a result of acid stress has been documented for E. coli O157:H7 (Wang and Doyle 1998; Ryu and Beuchat, 1998), S. typhimurium (Leyer and Johnson, 1993), and L. monocytogenes (Farber and Pagotto, 1992). Survival of S. sonnei and S. flexneri under various temperature and pH conditions has been described (Bagamboula et al., 2002; Wu et al., 1999; Zaika et al., 1989, 1991; Zaika, 2001); however, the combined effect of temperature and pH on survival and growth of acid-stressed cells has not been reported. This study was undertaken to determine the effects of temperature and pH on the survival and growth of acidstressed S. flexneri. The extent of injury resulting from exposure of these cells to an acidic environment was also determined.
2. Materials and methods 2.1. Preparation of inoculum S. flexneri strain F340-MS1, an isolate originating in Malawi and obtained from the Centers for Disease Control and Prevention, Atlanta, GA, was grown in 10 ml of sterile tryptic soy broth (TSB; contains 0.25% glucose) (BBL/Difco, Sparks, MD). Cultures were incubated at 37 jC for 24 h and transferred into TSB at 24-h intervals at least three times immediately before being used as inoculum for subsequent experiments. One milliliter of each culture was inoculated into 250-ml Erlenmeyer flasks containing 99 ml of TSB containing no glucose (TSBNG) or TSB supplemented with 1% glucose (Sigma, St. Louis, MO) (TSBG), and incubated at 37 jC for 18 h to produce stationary-phase unadapted (control) and acid-adapted cells, respectively. Unadapted and acid-adapted cells were sedimented by centrifuging at 2000 g for 15 min in a bench top Centra-CL2 centrifuge (International Equipment, Needham Heights, MA). Pellets were resuspended in TSBNG or TSBG, respectively, to give a population of 107 cfu/ml. Acid-shocked cells were prepared by centrifuging (2000 g, 15 min) 18h TSBNG cultures, resuspending the pellet in TSBNG acidified to pH 4.5 with 13 M lactic acid (Fisher Scientific, Fair Lawn, NJ), and incubating at 37 jC for 15 min. The three types of cells were used as inocula for acidified TSB, tryptic soy agar (TSA), and TSA supplemented with 4% sodium chloride (TSAS). 2.2. Survival and growth studies To prepare acidified TSB, filter-sterilized 13 M lactic acid was added to 200 ml of sterile TSB to achieve pH 3.5, 4.0, 4.5, 5.0, and 5.5; 9.9 ml was dispensed into sterile 16 150-mm screw-capped test tubes. TSB at pH 7.3 served as an unacidifed control broth. TSAS was prepared by adding 40 g of NaCl per liter of TSA. The ability of S. flexneri to survive or grow in unacidified and acidified TSB as affected by temperature was determined. TSB (9.9 ml) at pH 7.3, 5.5, 5.0, 4.5, 4.0, and 3.5 was inoculated with 0.1 ml of suspensions of unadapted, acid-adapted, or acidshocked S. flexneri cells and incubated at 4, 12, 21, 30, and 48 jC for up to 144 h. After incubation for
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0.5, 1, 2, 4, 6, 24, 48, 72, 96, and 144 h, undiluted suspensions were surface-plated (0.25 ml in quadruplicate and 0.1 ml in duplicate) on TSA (pH 7.2) and TSAS (pH 7.2) to determine if a portion of the cells were injured. Samples serially diluted in sterile 0.1% peptone water were also surface-plated (0.1 ml in duplicate) on TSA and TSAS. Plates were incubated at 37 jC for 48 h before colonies were counted. 2.3. Statistical analysis Three replicate experiments were done for each experiment. Data were analyzed using the general linear model and analysis of variance of the Statistical Analysis Systems (SAS Institute, 1987). The least significant difference test was used to determine the sources of differences between the test parameters (cell type, pH, media, temperature, and incubation time). A second-order polynomial equation was used to develop a model for the growth or inactivation of S.
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flexneri in acidified and unacidified TSB at various incubation times: Y ¼ b0 þ b1 X1 þ b2 X2 þ b11 X12 þ b22 X22 þ b12 X1 X2 þ e where bi (i = 0,1,2) are constant regression coefficients; X1 and X2 represent time and pH, respectively; Y is the population recovered at a given time, pH, and temperature; and e is the error term. Statistical software was used to generate response surface plots at constant temperature and varying pH and incubation time.
3. Results and discussion According to Leyer and Johnson (1993), the physiological state of Salmonella can influence its survival in foods and resistance to food processing conditions and chemical preservatives. Exposure to acidic environments can result in cross protection against sub-
Fig. 1. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 4 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
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sequent stress conditions. We studied the behavior of acid-adapted and acid-shocked S. flexneri exposed to acidic pH achieved using lactic acid and temperature conditions that characterize a wide range of foods during processing, preparation, and short-term storage. Lactic acid was used as an acidulant in the study. Compared to exposure of S. flexneri to acetic or propionic acids at a given pH following induction of acid shock with lactic acid, we have observed that tolerance of cells to lactic acid is greater (Tetteh and Beuchat, 2001). The use of lactic acid as a component of surface sanitizers for lettuce, which has been associated with outbreaks of shigellosis (Beuchat, 1996), has been reported (Lin et al., 2002). Information on the behavior of Shigella after exposure to lactic acid would be useful when developing treatment strategies to reduce the risk of shigellosis associated with produce that had been washed with dilute lactic
acid solution. Implications of the behavior of E. coli O157:H7 on beef carcasses washed with lactic acid solution for the purpose of killing the pathogen may also exist. Response surface plots of populations of unadapted (control), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB as affected by pH (3.5 – 7.3) and incubation time (up to 144 h) at 4, 12, 21, 30, and 48 jC are shown in Figs. 1 – 5, respectively. The general trend in population, regardless of the physiological state of the cell, indicates that death of S. flexneri was enhanced with increased temperature and decreased pH. Unadapted (control) cells (6.55 – 7.04 log10 cfu/ml) inoculated into TSB acidified to pH 3.5 survived 144, 144, 24, 6, and less than 0.5 h at 4, 12, 21, 30, and 48 jC, respectively. To determine if unadapted, acid-adapted, and acidshocked cells were injured as a result of exposure to
Fig. 2. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 12 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
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Fig. 3. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 21 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
various pH and temperature conditions, cells were surface-plated on TSA and TSAS. A lower number of colonies formed on TSAS indicates the presence of injured cells that could not be resuscitated. Significantly higher (a = 0.05) populations of all three types of cells were recovered on TSA than on TSAS at pH 3.5 –4.5, regardless of temperature at which cells were incubated before plating. More injury was observed in unadapted cells than in acid-adapted or acid-shocked cells as evidenced by the inability of larger numbers of unadapted cells to form colonies on TSAS compared to TSA. After 144 h, regardless of the incubation temperature, control cells inoculated into highly acidified TSB (pH 3.5) were not able to grow on TSAS; however, acid-adapted and acid-shocked cells held in TSB (pH 3.5) at 4 or 12 jC (Figs. 1 and 2, respectively) for 144 h formed colonies on TSAS. Similar observations on E. coli O157:H7 have been
reported by Semanchek and Golden (1998) and Ryu and Beuchat (1998). Cells inoculated into acidified media exhibited decreased tolerance to sodium chloride, indicating they were injured by exposure to the low pH. Exposure of S. flexneri to higher temperatures (21 and 30 jC; Figs. 3 and 4, respectively) and higher pH (5.0 –7.3) did not result in significant differences in numbers of colonies formed on TSA and TSAS, indicating that these conditions did not injure S. flexneri. Unadapted, acid-adapted, and acid-shocked S. flexneri cells inoculated into unacidified (pH 7.3) and acidified TSB, then held at 4 jC for up to 144 h, decreased with time (Fig. 1). The most rapid decline occurred at pH 3.5 – 4.5. Significantly higher (a = 0.05) numbers of acid-adapted and acid-shocked cells were recovered from TSB at lower pH (3.5 –4.5) compared to those recovered from TSB at pH 5.5 and
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Fig. 4. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 30 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
7.3. For example, after 144 h, 6.86 log10 cfu of acidadapted cells per ml were detected in TSB acidified to pH 4.0 and incubated at 4 jC compared to 5.82 log10 cfu/ml detected in TSB at pH 7.3. All three cell types showed significant increases in populations within 24 h when inoculated into TSB at pH 5.0, 5.5, or 7.3, and incubated at 12, 21, and 30 jC (Figs. 2 – 4, respectively). Zaika (2001) reported that S. flexneri grew at 19, 28, and 37 jC in brain heart infusion broth at pH 5.0. Populations were stable for more than 1000 h at 4 and 12 jC. This trend is similar to that observed in our experiment, although the pathogen grew in TSB (pH 5.0 – 7.3) at 12 jC. Prior exposure of S. flexneri cells to an acidic environment rendered them more resistant to extremes in acid or temperature. Acid-adapted cells decreased approximately 2.5 log10 cfu/ml when incubated at 4 jC for 144 h compared to a 6-log10 cfu/ml reduction of unadapted (control) cells (Fig. 1). A 3-log10 reduction
in acid-adapted cells inoculated into TSB acidified to pH 3.5 was observed within 0.5 h, compared to more than a 6-log reduction in unadapted cells. The effects of pH on changes in populations of S. flexneri recovered from TSB incubated at 12, 21, and 30 jC (Figs. 2 –4, respectively) were similar. These plots reveal that survival was enhanced and growth increased with an increase in pH and incubation time, with highest populations achieved at pH 5.5– 7.3 and extended incubation times (48 –144 h). The number of injured cells recovered increased with increasing temperature from 12 to 30 jC. Fig. 1 shows that the population of cells incubated at 4 jC remained constant for the first 72 h at pH 4.5– 5.5, then declined with time, regardless of pH. Survival at 48 jC decreased with a decrease in pH and increase in incubation time (Fig. 5). The highest populations were detected from TSB at pH 5.0– 7.3 during the first 6 h.
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Fig. 5. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 48 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
Acid-stressed cells of S. flexneri were cross-protected against subsequent stress at low and high temperatures (4 and 48 jC). Acid-adapted and acidshocked cells were more resistant to these temperatures than were unadapted cells. When acid-adapted cells incubated for 48 h at 48 jC in TSB (pH 7.3) were plated on TSA, 3.46 log10 cfu/ml were recovered compared to less than 1 cfu/ml and 1.76 log10 cfu/ml for unadapted and acid-shocked cells, respectively. Similar trends (i.e., higher populations of acidstressed cells than unadapted cells) were observed when cells were incubated at 48 jC, regardless of the pH of TSB. Cross protection to other environmental stresses as a result of acid or alkaline stress has been reported for other pathogenic bacteria. Leyer and Johnson (1993) reported that acid-adapted S. typhimurium cells have increased resistance to heat and osmotic stresses. They concluded that acid adaptation affects resistance to a variety of environmental stresses. Cells of E. coli O157:H7 adapted to acid by growing cells in TSBG had enhanced heat tolerance at 52 and 54
jC (Ryu and Beuchat, 1998; Ryu et al., 1999). D values of acid-adapted cells were higher than those of unadapted or acid-shocked cells, regardless of heating temperature. Other researchers have reported acidand alkaline-induced heat resistance in L. monocytogenes (Farber and Pagotto, 1992; Taormina and Beuchat, 2001). Coefficients of variables in the models generated for survival, growth, and death of S. flexneri in TSB at various pH values over a 144-h incubation period indicated that the linear effects of time and pH, the quadratic effect of pH, and the cross-product terms were significant (a = 0.05) in all the models at all incubation temperatures. This indicates that incubation time and pH are important variables in the survival or growth of S. flexneri in TSB. In summary, S. flexneri is able to grow at temperatures ranging from 12 to 30 jC at a pH range of 5.0 – 7.3. The pathogen is able to survive longer at 4 jC than at 12 – 48 jC, regardless of pH (3.5 – 7.3) or prior exposure to acidic environments. Survival and growth
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of S. flexneri at a given pH is influenced by the incubation temperature. Acid adaptation in S. flexneri induces cross protection against temperature stress (48 jC) and may be an important mechanism influencing survival in foods and in food processing environments. An understanding of the level of tolerance of S. flexneri to pH as affected by temperature is critical to predicting rates of death or growth of the pathogen in foods.
Acknowledgements This study was supported, in part, by the U.S. Agency for International Development, Bean/Cowpea Collaborative Research Support Program.
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Fundamentals and Frontiers, 2nd ed. Am. Soc. Microbiol. Press, Washington, DC, pp. 247 – 261. Chapter 12. Leyer, G.J., Johnson, E.A., 1992. Acid adaptation promotes survival of Salmonella sp. in cheese. Appl. Environ. Microbiol. 58, 2075 – 2080. Leyer, G.H., Johnson, E.A., 1993. Acid adaptation induces crossprotection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbiol. 59, 1842 – 1847. Leyer, G.J., Wang, L.L., Johnson, E.A., 1995. Acid adaptation of Escherichia coli O157:H7 increases survival in acidic foods. Appl. Environ. Microbiol. 61, 3752 – 3755. Lin, C.-M., Moon, S.S., Doyle, M.P., McWatters, K.H., 2002. Inactivation of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis, and Listeria monocytogenes on lettuce by hydrogen peroxide and lactic acid and by hydrogen peroxide with mild heat. J. Food Prot. 65, 1215 – 1220. Ryu, J.-H., Beuchat, L.R., 1998. Influence of acid tolerance responses on survival, growth, and thermal cross-protection of Escherichia coli O157:H7 in acidified media and fruit juices. Int. J. Food Microbiol. 45, 185 – 193. Ryu, J.-H., Beuchat, L.R., 1999. Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments. Food Microbiol. 16, 317 – 324. Ryu, J.-H., Deng, Y., Beuchat, L.R., 1999. Behavior of acid-adapted and unadapted Escherichia coli O157:H7 when exposed to reduced pH achieved with various organic acids. J. Food Prot. 62, 451 – 455. SAS Institute, 1987. Statistical Analysis System. SAS Institute, Cary, NC. Semanchek, J.J., Golden, D.A., 1998. Influence of growth temperature on inactivation and injury of Escherichia coli O157:H7 by heat, acid, and freezing. J. Food Prot. 61, 395 – 401. Small, P.L.C., 1998. Shigella and Escherichia coli strategies for survival at low pH. Jpn. J. Med. Sci. Biol. 51 (Suppl. 1), S81 – S89. Smith, J.L., 1987. Shigella as a foodborne pathogen. J. Food Prot. 50, 788 – 801. Taormina, P.J., Beuchat, L.R., 2001. Survival and heat resistance of Listeria monocytogenes after exposure to alkali and chlorine. Appl. Environ. Microbiol. 67, 2555 – 2563. Tetteh, G.L., Beuchat, L.R., 2001. Sensitivity of acid-adapted and acid-shocked Shigella flexneri to reduced pH achieved with acetic, lactic, and propionic acids. J. Food Prot. 64, 975 – 981. Wang, G., Doyle, M.P., 1998. Heat shock enhances acid tolerance in Escherichia coli O157:H7. Lett. Appl. Microbiol. 26, 31 – 34. Wu, F.M., Doyle, M.P., Beuchat, L.R., Wells, J.G., Mintz, E.D., Swaminathan, B., 1999. Fate of Shigella sonnei on parsley and methods of disinfection. J. Food Prot. 63, 568 – 572. Zaika, L.L., 2001. The effect of temperature and low pH on survival of Shigella flexneri in broth. J. Food Prot. 64, 1162 – 1165. Zaika, L.L., Engel, L.S., Kim, A.H., Palumbo, S.A., 1989. Effect of sodium chloride, pH, and temperature on growth of Shigella flexneri. J. Food Prot. 52, 356 – 359. Zaika, L.L., Kim, A.H., Ford, L., 1991. Effect of sodium nitrite on growth of Shigella flexneri. J. Food Prot. 54, 424 – 428.
Survival, growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature Gloria L. Tetteh, Larry R. Beuchat * Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA Received 8 July 2002; received in revised form 25 October 2002; accepted 2 December 2002
Abstract A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acidshocked Shigella flexneri 2a cells as affected by pH and temperature. The pathogen was grown at 37 jC for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5 F 0.05 with lactic acid. All three cell types were separately inoculated into tryptic soy broth (6.6 – 7.0 log10 cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5 – 5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 jC for up to 144 h. Overall, inactivation of S. flexneri cells at low pH was enhanced with an increase in incubation temperature. All three types of cells survived for 144 h at 4 jC in TSB acidified to pH 3.5, compared to < 24 h at 30 jC and 2 h at 48 jC. The population of all three cell types increased significantly (a = 0.05) within 24 h when cells were incubated at 12, 21, or 30 jC in TSB at pH 5.0, 5.5, or 7.3. Prior exposure of the S. flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions. Acid-adapted cells decreased by approximately 2.5 log10 cfu/ml when incubated at 4 jC for 144 h, compared to a 6log10 reduction in control (unadapted) cells. When cells were exposed to low pH (3.5 – 4.5) and high temperature (48 jC), significantly higher (a = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S. flexneri cells were injured. Results show that the ability of S. flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Shigella flexneri; Acid stress
1. Introduction Shigella dysenteriae, one of four Shigella species, causes classic bacillary dysentery, which is the most severe form of shigellosis (Lampel and Maurelli, * Corresponding author. Tel.: +1-770-412-4740; fax: +1-770229-3216. E-mail address: [email protected] (L.R. Beuchat).
2001). Shigella sonnei causes the mildest infection, while Shigella flexneri and Shigella boydii infections can be either mild or severe. Shigellae have no known nonhuman reservoir, and are usually transmitted from person to person through poor personal hygiene, although contaminated food and water have been associated in outbreaks of shigellosis (Smith, 1987). The infective dose of Shigella is lower than that reported for most other enteric pathogens. According
0168-1605/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-1605(03)00052-7
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to Dupont et al. (1989), 10– 500 viable cells can cause disease in healthy adults. Small (1998) stated that, for most enteric pathogens, a higher dose is generally required to cause diarrhea. This may be due to lethality caused by low pH in the stomach, as well as inhibition of colonization in the intestine tract. The low infectious dose of Shigella suggests that, compared to some enteric pathogens, a higher proportion of cells are capable of surviving exposure to highly acidic environments. The ability of microbial cells to adapt to sublethal stresses and subsequently become more resistant to extreme environmental conditions has been documented for several foodborne pathogenic bacteria, including Salmonella typhimurium (Leyer and Johnson, 1992; Foster and Hall, 1990; Leyer and Johnson, 1993), Escherichia coli O157:H7 (Leyer et al., 1995; Buchanan and Edelson, 1996; Ryu and Beuchat, 1999), and Listeria monocytogenes (Gahan et al., 1996; Davis et al., 1996; Datta and Benjamin, 1995). Earlier work in our laboratory showed that S. flexneri cells grown in tryptic soy broth supplemented with 1% glucose (TSBG) (acid-adapted cells) were able to survive for 6 h in TSB acidified to pH 3.5 (Tetteh and Beuchat, 2001). Populations of control cells grown in TSB without glucose, i.e., cells unadapted to an acid environment, were reduced to undetectable levels within 2 to 4 h. Bacterial cells surviving exposure to acid stress may exhibit increased resistance to an unrelated stress that is subsequently applied. Cross protection against heat as a result of acid stress has been documented for E. coli O157:H7 (Wang and Doyle 1998; Ryu and Beuchat, 1998), S. typhimurium (Leyer and Johnson, 1993), and L. monocytogenes (Farber and Pagotto, 1992). Survival of S. sonnei and S. flexneri under various temperature and pH conditions has been described (Bagamboula et al., 2002; Wu et al., 1999; Zaika et al., 1989, 1991; Zaika, 2001); however, the combined effect of temperature and pH on survival and growth of acid-stressed cells has not been reported. This study was undertaken to determine the effects of temperature and pH on the survival and growth of acidstressed S. flexneri. The extent of injury resulting from exposure of these cells to an acidic environment was also determined.
2. Materials and methods 2.1. Preparation of inoculum S. flexneri strain F340-MS1, an isolate originating in Malawi and obtained from the Centers for Disease Control and Prevention, Atlanta, GA, was grown in 10 ml of sterile tryptic soy broth (TSB; contains 0.25% glucose) (BBL/Difco, Sparks, MD). Cultures were incubated at 37 jC for 24 h and transferred into TSB at 24-h intervals at least three times immediately before being used as inoculum for subsequent experiments. One milliliter of each culture was inoculated into 250-ml Erlenmeyer flasks containing 99 ml of TSB containing no glucose (TSBNG) or TSB supplemented with 1% glucose (Sigma, St. Louis, MO) (TSBG), and incubated at 37 jC for 18 h to produce stationary-phase unadapted (control) and acid-adapted cells, respectively. Unadapted and acid-adapted cells were sedimented by centrifuging at 2000 g for 15 min in a bench top Centra-CL2 centrifuge (International Equipment, Needham Heights, MA). Pellets were resuspended in TSBNG or TSBG, respectively, to give a population of 107 cfu/ml. Acid-shocked cells were prepared by centrifuging (2000 g, 15 min) 18h TSBNG cultures, resuspending the pellet in TSBNG acidified to pH 4.5 with 13 M lactic acid (Fisher Scientific, Fair Lawn, NJ), and incubating at 37 jC for 15 min. The three types of cells were used as inocula for acidified TSB, tryptic soy agar (TSA), and TSA supplemented with 4% sodium chloride (TSAS). 2.2. Survival and growth studies To prepare acidified TSB, filter-sterilized 13 M lactic acid was added to 200 ml of sterile TSB to achieve pH 3.5, 4.0, 4.5, 5.0, and 5.5; 9.9 ml was dispensed into sterile 16 150-mm screw-capped test tubes. TSB at pH 7.3 served as an unacidifed control broth. TSAS was prepared by adding 40 g of NaCl per liter of TSA. The ability of S. flexneri to survive or grow in unacidified and acidified TSB as affected by temperature was determined. TSB (9.9 ml) at pH 7.3, 5.5, 5.0, 4.5, 4.0, and 3.5 was inoculated with 0.1 ml of suspensions of unadapted, acid-adapted, or acidshocked S. flexneri cells and incubated at 4, 12, 21, 30, and 48 jC for up to 144 h. After incubation for
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0.5, 1, 2, 4, 6, 24, 48, 72, 96, and 144 h, undiluted suspensions were surface-plated (0.25 ml in quadruplicate and 0.1 ml in duplicate) on TSA (pH 7.2) and TSAS (pH 7.2) to determine if a portion of the cells were injured. Samples serially diluted in sterile 0.1% peptone water were also surface-plated (0.1 ml in duplicate) on TSA and TSAS. Plates were incubated at 37 jC for 48 h before colonies were counted. 2.3. Statistical analysis Three replicate experiments were done for each experiment. Data were analyzed using the general linear model and analysis of variance of the Statistical Analysis Systems (SAS Institute, 1987). The least significant difference test was used to determine the sources of differences between the test parameters (cell type, pH, media, temperature, and incubation time). A second-order polynomial equation was used to develop a model for the growth or inactivation of S.
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flexneri in acidified and unacidified TSB at various incubation times: Y ¼ b0 þ b1 X1 þ b2 X2 þ b11 X12 þ b22 X22 þ b12 X1 X2 þ e where bi (i = 0,1,2) are constant regression coefficients; X1 and X2 represent time and pH, respectively; Y is the population recovered at a given time, pH, and temperature; and e is the error term. Statistical software was used to generate response surface plots at constant temperature and varying pH and incubation time.
3. Results and discussion According to Leyer and Johnson (1993), the physiological state of Salmonella can influence its survival in foods and resistance to food processing conditions and chemical preservatives. Exposure to acidic environments can result in cross protection against sub-
Fig. 1. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 4 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
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sequent stress conditions. We studied the behavior of acid-adapted and acid-shocked S. flexneri exposed to acidic pH achieved using lactic acid and temperature conditions that characterize a wide range of foods during processing, preparation, and short-term storage. Lactic acid was used as an acidulant in the study. Compared to exposure of S. flexneri to acetic or propionic acids at a given pH following induction of acid shock with lactic acid, we have observed that tolerance of cells to lactic acid is greater (Tetteh and Beuchat, 2001). The use of lactic acid as a component of surface sanitizers for lettuce, which has been associated with outbreaks of shigellosis (Beuchat, 1996), has been reported (Lin et al., 2002). Information on the behavior of Shigella after exposure to lactic acid would be useful when developing treatment strategies to reduce the risk of shigellosis associated with produce that had been washed with dilute lactic
acid solution. Implications of the behavior of E. coli O157:H7 on beef carcasses washed with lactic acid solution for the purpose of killing the pathogen may also exist. Response surface plots of populations of unadapted (control), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB as affected by pH (3.5 – 7.3) and incubation time (up to 144 h) at 4, 12, 21, 30, and 48 jC are shown in Figs. 1 – 5, respectively. The general trend in population, regardless of the physiological state of the cell, indicates that death of S. flexneri was enhanced with increased temperature and decreased pH. Unadapted (control) cells (6.55 – 7.04 log10 cfu/ml) inoculated into TSB acidified to pH 3.5 survived 144, 144, 24, 6, and less than 0.5 h at 4, 12, 21, 30, and 48 jC, respectively. To determine if unadapted, acid-adapted, and acidshocked cells were injured as a result of exposure to
Fig. 2. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 12 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
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Fig. 3. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 21 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
various pH and temperature conditions, cells were surface-plated on TSA and TSAS. A lower number of colonies formed on TSAS indicates the presence of injured cells that could not be resuscitated. Significantly higher (a = 0.05) populations of all three types of cells were recovered on TSA than on TSAS at pH 3.5 –4.5, regardless of temperature at which cells were incubated before plating. More injury was observed in unadapted cells than in acid-adapted or acid-shocked cells as evidenced by the inability of larger numbers of unadapted cells to form colonies on TSAS compared to TSA. After 144 h, regardless of the incubation temperature, control cells inoculated into highly acidified TSB (pH 3.5) were not able to grow on TSAS; however, acid-adapted and acid-shocked cells held in TSB (pH 3.5) at 4 or 12 jC (Figs. 1 and 2, respectively) for 144 h formed colonies on TSAS. Similar observations on E. coli O157:H7 have been
reported by Semanchek and Golden (1998) and Ryu and Beuchat (1998). Cells inoculated into acidified media exhibited decreased tolerance to sodium chloride, indicating they were injured by exposure to the low pH. Exposure of S. flexneri to higher temperatures (21 and 30 jC; Figs. 3 and 4, respectively) and higher pH (5.0 –7.3) did not result in significant differences in numbers of colonies formed on TSA and TSAS, indicating that these conditions did not injure S. flexneri. Unadapted, acid-adapted, and acid-shocked S. flexneri cells inoculated into unacidified (pH 7.3) and acidified TSB, then held at 4 jC for up to 144 h, decreased with time (Fig. 1). The most rapid decline occurred at pH 3.5 – 4.5. Significantly higher (a = 0.05) numbers of acid-adapted and acid-shocked cells were recovered from TSB at lower pH (3.5 –4.5) compared to those recovered from TSB at pH 5.5 and
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Fig. 4. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 30 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
7.3. For example, after 144 h, 6.86 log10 cfu of acidadapted cells per ml were detected in TSB acidified to pH 4.0 and incubated at 4 jC compared to 5.82 log10 cfu/ml detected in TSB at pH 7.3. All three cell types showed significant increases in populations within 24 h when inoculated into TSB at pH 5.0, 5.5, or 7.3, and incubated at 12, 21, and 30 jC (Figs. 2 – 4, respectively). Zaika (2001) reported that S. flexneri grew at 19, 28, and 37 jC in brain heart infusion broth at pH 5.0. Populations were stable for more than 1000 h at 4 and 12 jC. This trend is similar to that observed in our experiment, although the pathogen grew in TSB (pH 5.0 – 7.3) at 12 jC. Prior exposure of S. flexneri cells to an acidic environment rendered them more resistant to extremes in acid or temperature. Acid-adapted cells decreased approximately 2.5 log10 cfu/ml when incubated at 4 jC for 144 h compared to a 6-log10 cfu/ml reduction of unadapted (control) cells (Fig. 1). A 3-log10 reduction
in acid-adapted cells inoculated into TSB acidified to pH 3.5 was observed within 0.5 h, compared to more than a 6-log reduction in unadapted cells. The effects of pH on changes in populations of S. flexneri recovered from TSB incubated at 12, 21, and 30 jC (Figs. 2 –4, respectively) were similar. These plots reveal that survival was enhanced and growth increased with an increase in pH and incubation time, with highest populations achieved at pH 5.5– 7.3 and extended incubation times (48 –144 h). The number of injured cells recovered increased with increasing temperature from 12 to 30 jC. Fig. 1 shows that the population of cells incubated at 4 jC remained constant for the first 72 h at pH 4.5– 5.5, then declined with time, regardless of pH. Survival at 48 jC decreased with a decrease in pH and increase in incubation time (Fig. 5). The highest populations were detected from TSB at pH 5.0– 7.3 during the first 6 h.
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Fig. 5. Populations of control (unadapted), acid-adapted, and acid-shocked S. flexneri cells recovered from TSB (pH 3.5 – 7.3) incubated at 48 jC for up to 144 h. Cell suspensions were surface-plated on TSA and TSAS.
Acid-stressed cells of S. flexneri were cross-protected against subsequent stress at low and high temperatures (4 and 48 jC). Acid-adapted and acidshocked cells were more resistant to these temperatures than were unadapted cells. When acid-adapted cells incubated for 48 h at 48 jC in TSB (pH 7.3) were plated on TSA, 3.46 log10 cfu/ml were recovered compared to less than 1 cfu/ml and 1.76 log10 cfu/ml for unadapted and acid-shocked cells, respectively. Similar trends (i.e., higher populations of acidstressed cells than unadapted cells) were observed when cells were incubated at 48 jC, regardless of the pH of TSB. Cross protection to other environmental stresses as a result of acid or alkaline stress has been reported for other pathogenic bacteria. Leyer and Johnson (1993) reported that acid-adapted S. typhimurium cells have increased resistance to heat and osmotic stresses. They concluded that acid adaptation affects resistance to a variety of environmental stresses. Cells of E. coli O157:H7 adapted to acid by growing cells in TSBG had enhanced heat tolerance at 52 and 54
jC (Ryu and Beuchat, 1998; Ryu et al., 1999). D values of acid-adapted cells were higher than those of unadapted or acid-shocked cells, regardless of heating temperature. Other researchers have reported acidand alkaline-induced heat resistance in L. monocytogenes (Farber and Pagotto, 1992; Taormina and Beuchat, 2001). Coefficients of variables in the models generated for survival, growth, and death of S. flexneri in TSB at various pH values over a 144-h incubation period indicated that the linear effects of time and pH, the quadratic effect of pH, and the cross-product terms were significant (a = 0.05) in all the models at all incubation temperatures. This indicates that incubation time and pH are important variables in the survival or growth of S. flexneri in TSB. In summary, S. flexneri is able to grow at temperatures ranging from 12 to 30 jC at a pH range of 5.0 – 7.3. The pathogen is able to survive longer at 4 jC than at 12 – 48 jC, regardless of pH (3.5 – 7.3) or prior exposure to acidic environments. Survival and growth
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of S. flexneri at a given pH is influenced by the incubation temperature. Acid adaptation in S. flexneri induces cross protection against temperature stress (48 jC) and may be an important mechanism influencing survival in foods and in food processing environments. An understanding of the level of tolerance of S. flexneri to pH as affected by temperature is critical to predicting rates of death or growth of the pathogen in foods.
Acknowledgements This study was supported, in part, by the U.S. Agency for International Development, Bean/Cowpea Collaborative Research Support Program.
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