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Sub-lethal heat treatment affects the tolerance of Cronobacter sakazakii BCRC 13988 to various organic acids, simulated gastric juice and bile solution
International Journal of Food Microbiology 144 (2010) 280–284
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Sub-lethal heat treatment affects the tolerance of Cronobacter sakazakii BCRC 13988 to various organic acids, simulated gastric juice and bile solution Wan-Ling Hsiao a, Wei-Li Ho b, Cheng-Chun Chou a,⁎ a b
Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan Department of Food Health, Ching Kuo Institute of Management and Health, Keelung, Taiwan
a r t i c l e
i n f o
Article history: Received 8 June 2010 Received in revised form 28 September 2010 Accepted 7 October 2010 Keywords: Cronobacter sakazakii Sub-lethal heat treatment Organic acid Simulated gastric juice Bile solution
a b s t r a c t Cronobacter spp., formerly Enterobacter sakazakii, are considered emerging opportunistic pathogens and the etiological agent of life-threatening bacterial infections in infants. In the present study, C. sakazakii BCRC 13988 was first subjected to sub-lethal heat treatment at 47 °C for 15 min. Survival rates of the heat-shocked and non-shocked C. sakazakii cells in phosphate buffer solution (PBS, pH 4.0) containing organic acids (e.g. acetic, propionic, citric, lactic or tartaric acid), simulated gastric juice (pH 2.0–4.0), and bile solution (0.5 and 2.0%) were examined. Results revealed that sub-lethal heat treatment enhanced the test organism's tolerance to organic acids, although the extent of increased acid tolerance varied with the organic acid examined. Compared with the control cells, heat-shocked C. sakazakii cells after 120-min of exposure, exhibited the largest increase in tolerance in the lactic acid-containing PBS. Furthermore, although heat shock did not affect the behavior of C. sakazakii in bile solution, it increased the test organism's survival when exposed to simulated gastric juice with a pH of 3.0–4.0. © 2010 Published by Elsevier B.V.
1. Introduction Cronobacter spp., formerly Enterobacter sakazakii, are Gramnegative facultative anaerobic rods. They are considered as emerging opportunistic pathogens and the etiological agent of life-threatening bacteria infections in infants (Block et al., 2002; CDC, 2002). These organism are ubiquitous in the environment and food (Iversen and Forsythe, 2003; Friedemann, 2007). Although dried infant formula milk has been confirmed as the source of severe systemic neonatal infections caused by Cronobacter spp., these pathogens have also been detected in vegetables, cereals, legumes, minced beef, cheese products, and spices (Iversen and Forsythe, 2003; Leclercq et al., 2002; Beuchat, et al., 2009). Neonates, particularly those of low birth weight, are at the greatest risk, and have a high mortality rate. However, involvement of Cronobacter spp. in cases of infections in children, and immunocompromised adults has been reported (FAO and WHO, 2004). Due to the extremely severe consequences of infection in infants and its ubiquitous nature, C. sakazakii was ranked as “severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration.” (ICMSF, 2002) Various foodborne pathogens such as Salmonella typhimurium, Vibrio parahaemolyticus, Listeria monocytogenes, and Escherichia coli O157:H7 (Bunning et al., 1990; Wang and Doyle, 1998; Jørgensen et al., 1999; Lin ⁎ Corresponding author. Graduate Institute of Food Science & Technology, National Taiwan University 59, lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan. Tel.: +886 2 3366 4111; fax: +886 2 2362 0849. E-mail address: [email protected] (C.-C. Chou). 0168-1605/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2010.10.006
and Chou, 2004; Chang et al., 2004) have been reported as capable of enhancing their abilities to tolerate more adverse heat and other lethal stresses after being exposed to sub-lethal heat treatment. Previously, Shaker et al. (2008) reported that heat resistance of C. sakazakii reduced after exposure to heat shock at 55 °C for 5 min. While we found that heat shock at 47 °C for 15 min enhanced the survival of C. sakazakii BCRC 13988 in its subsequent exposure to thermal stresses (51 °C) and other lethal stresses such as acidity (pH 3.0), and ethanol (15%) (Chang et al., 2009, 2010). Additionally, heat shock increased the survival of C. sakazakii after freeze drying or spray drying, and during storage of lactic fermented milk products (pH 4.3) at 5 °C for 48 h (Hsiao et al., 2010). To provide more information for developing adequate control measures and risk assessments for this pathogen, the authors further investigated the effects of heat shock on the survival of C. sakazakii in the presence of various organic acids. Furthermore, this study examined the response of the heat-shocked C. sakazakii in simulated gastric juice (pH 2.0–4.0) and bile solution (0.5% and 2.0%), since C. sakazakii in food may encounter various organic acids which are frequently used to control the proliferation of microorganisms in a food system (Golden et al., 1995). Besides, the microorganisms are exposed to low pH of gastric juice and bile salt after ingestion. 2. Materials and methods 2.1. C. sakazakii and preparation of the heat-shocked cells C. sakazakii BCRC 13988 (ATCC 29544), the present study's test organism, was obtained from the Bioresources Collection and Research
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Center in Hsinchu, Taiwan. It is a clinical strain originally isolated from the throat of an infant patient (Farmer et al., 1980). Procedures described in the authors’ previous paper (Chang et al., 2009) were followed to prepare the heat-shocked cells of C. sakazakii. Briefly, the activated cultures obtained by two successive transfers of the test organism in Tryptic soy broth (TSB) (Acumedia Manufactures, Lansing, MI, USA) at 37 °C for 24 h, were grown in TSB (pH 7.2) at 37 °C for 6 h to reach late exponential growth when pH of TSB reduced to ca 5.7. The culture was centrifuged at 8000×g for 10 min. After resuspending the pellet in fresh TSB (pH 7.2), the cultures were subjected to heating at 47 °C for 15 min to obtain the heat-shocked cells of C. sakazakii. Cultures not exposed to the heat shock treatment were included as control.
2.2. Study on the survival of C. sakazakii in phosphate buffer solution (PBS) containing various organic acids To examine the survival of C. sakazakii in the presence of organic acid, 9.9 mL PBS (0.1 M, pH 4.0) containing 40 mM acetic, propionic, lactic, citric, or tartaric acid was inoculated with 0.1 mL of C. sakazaki inoculum at an initial population of approximately 106 CFU/mL. It was then incubated at room temperature (ca 23–25 °C) for 120 min. The
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viability of the test organism was determined periodically during the incubation period.
2.3. Study of the survival of C. sakazakii in simulated gastric juice and bile solutions Methods described by Charteris et al. (1998) were employed to compare the gastric and acidic resistance of heat-shocked and nonshocked C. sakazakii cells. The simulated gastric juice was prepared by suspending pepsin (3 g/L, Acumedia, Lansing, MI, USA) in saline (0.5%, v/v) and adjusting the pH to 2.0, 3.0, 3.5 or 4.0 with 1.0 N HCl. It was then sterile-filtered through a membrane (0.45 μm, Gelman Science, Ann Arbor, MI, USA). To prepare the bile solution, 10 g oxgall (Difco, Sparks, MO, USA) was dissolved in 90 mL distilled water. This solution was then used to prepare 0.5% and 2.0% of bile solution. All solutions were sterilized at 121 °C for 15 min. When the survival study was conducted, 0.1 mL of the heat-shocked or non-shocked C. sakazakii inoculum was added to 9.9 mL simulated gastric juice (pH 3.0 and 2.0) or bile solution (0.5% or 2.0%). The solution was vortexed for 20 s. Samples were taken immediately after mixing to determine the viability of C. sakazakii (0 h sample). The mixtures were
(A) Control
(B) Lactic acid
(C) Acetic acid
(D) Propionic acid
(E) Citric acid
(F) Tartaric acid
Fig. 1. Effect of heat shock treatment on the survival of C. sakazakii after exposure to phosphate buffer solution (pH 4.0) containing no organic acid (A), containing 40 mM lactic acid (B), acetic acid (C), propionic acid (D), citric acid (E), and tartaric acid (F). ○, control cells; ●, heat-shocked cells. Viability of the test organism in the 0 time sample was determined immediately after 20 s of mixing with PBS with or without organic acid. The initial population of control and heat-shocked C. sakazakii were ca 106 CFU/mL. Survival percentage was obtained by dividing the surviving population by the initial population which corresponds to 100%.
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then incubated at 37 °C with shaking (150 rpm) for 3 or 12 h. During the incubation period, viable C. sakazakii were enumerated at predetermined time intervals. 2.4. Enumeration of C. sakazakii To determine the viable population of C. sakazakii, samples were serially diluted in PBS containing 0.1% peptone. The viable counts were then made by spread plating 0.1 mL on Tryptic soy agar (Acumedia Manufactures). Colonies were counted after 18 h of incubation at 37 °C. 2.5. Statistical analysis Mean values and standard deviations were calculated from the data obtained from the three separate experiments. Data were analyzed using an unpaired two-tailed Student's t-test. Statistical significance was set at P b 0.05 (SAS, 2001). 3. Results and discussion 3.1. Susceptibility to organic acid Fig. 1 shows changes in percent of cells of control and heat-shocked C. sakazakii surviving exposure to PBS (pH 4.0) with or without organic acid for 120 min. It was found that the survival percentage of the control C. sakazakii in PBS containing no organic acid declined slightly during the exposure period (Fig. 1A). After 120 min of exposure in PBS
without organic acid, the control C. sakazakii exhibited a survival percentage of 64.1% (Fig. 1A) with a population reduction of 0.2 log CFU/mL which was obtained by subtracting the final population (log CFU/ml) from the initial population (log CFU/ml). During the exposure period, control cells in PBS containing organic acid (Fig. 1B– F) also showed various degrees of reduction in viable cells depending on the organic acid examined. However, a marked reduction in the survival percentage of control C. sakazakii was found in the PBS containing lactic (Fig. 1B), acetic (Fig. 1C) and propionic acids (Fig. 1D). At the end of the exposure period, the control C. sakazakii exhibited a population reduction of 2.0, 1.2, 0.6, 0.4 and 0.2 log CFU/mL in PBS containing lactic (Fig. 1B), acetic (Fig. 1C), propionic (Fig. 1D), citric (Fig. 1E), and tartaric acids (Fig. 1F), respectively. This indicated that at pH 4.0, C. sakazakii was most susceptible to lactic acid, followed by acetic, propionic, citric, and tartaric acids in descending order. During the exposure period, the heat-shocked C. sakazakii, similar to those observed with the control cells, also showed reductions in surviving cells as the exposure period was extended. In PBS containing no organic acid (Fig. 1A), the survival percentage of the heatshocked and control C. sakazakii determined at same specific exposure intervals showed no significant difference (P N 0.05). On the other hand, a relatively higher survival rate with a significantly smaller (P b 0.05) population of heat-shocked cells was generally noted in PBS containing lactic, acetic, propionic, and citric acids (Fig. 1B, C, D and F, respectively) when compared with that of the control C. sakazakii. For example, at the end of the 120-min exposure period, the heat-shocked C. sakazakii in PBS containing lactic acid showed a survival percentage of 13.8% with a population reduction of 0.9 log CFU/mL. Meanwhile,
(A) pH 2.0
(B) pH 3.0
(C) pH 3.5
(D) pH 4.0
Fig. 2. Effect of heat shock treatment on the survival of C. sakazakii after exposure to simulated gastric juice with various pH. ○, control cells; ●, heat-shocked cells. Viability of the test organism in the 0 time sample was determined immediately after 20 s of mixing with PBS with or without simulated gastric juice with various pH. The initial population of control and heat-shocked C. sakazakii were ca 106 CFU/mL. Survival percentage was obtained by dividing the surviving population by the initial population which corresponds to 100%.
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the control C. sakazakii showed a lower survival percentage of 1.4% with a significantly higher (P b 0.05) population reduction of 2.0 log CFU/mL. These data demonstrated that heat shock treatment might enhance the tolerance of C. sakazakii to organic acid, although the degree of enhancement varied with the organic acid tested. The phenomenon of enhanced acid tolerance exerted by C. sakazakii after heat shock treatment as observed in the present study is in agreement with those reported for Shigella flexneri (Tetteh and Beuchat, 2003), E. coli O157:H7 (Wang and Doyle, 1998) and Lactobacillus collinoides (Laplace et al., 1999), while different from that observed on V. parahaemolyticus (Chiang et al., 2005). Chiang et al. (2005) reported that V. parahaemolyticus shocked at 42 °C for 45 min resulted in a decreased tolerance to acetic, citric, tartaric and lactic acids. In addition to the heat shock conditions examined, differences in the kind of bacteria tested might lead to these discrepancies. 3.2. Susceptibility to simulated gastric acid Sondheimer et al. (1985) reported that gastric pH of infants varies with age. The gastric pH of infants six days old varies from a fasting pH of 2.9 to a value of 5.2 directly after feeding. On the other hand, the gastric pH in infants 7–15 days old is relatively higher, within a narrow range between 4.6 and 5.8. Healthy adults usually have gastric pH levels of 2.0–3.0, which is lower than in infants. Fig. 2 shows the survival of heat-shocked and control C. sakazakii cells during the exposure to simulated gastric juices with pH levels of 2.0 to 4.0. It was generally found that survival of C. sakazakii varied with the pH of the simulated gastric juices, exposure period, and heat shock treatment. Among the various pHs examined, the most pronounced population reduction occurred when the test organism was exposed to gastric juice with a pH of 2.0, regardless of heat shock treatment. The viable population of C. sakazakii, regardless of heat shock, reduced by approximately 1.0 log CFU/mL after 20 s of mixing with the pH 2.0 gastric juice. No viable cells of control or heat-shocked C. sakazakii were noted after further extending the exposure period to 1 h (Fig. 2A). However, this phenomenon was not observed with test organism exposure to simulated gastric juice having pH levels between 3.0 and 4.0 (Fig. 2B, C and D). Nevertheless, viable populations of C. sakazakii still declined when exposed to gastric juice with a pH 3.0–4.0, although not to the extent seen in pH 2.0 juice. The survival percentage usually increased with increased pH and heat shock treatment. For example, the control C. sakazakii showed a survival of 0.1% after 2 h of exposure to gastric juice having a pH of 3.0 (Fig. 2B), compared to a higher survival rate of 64.6% noted with exposure to simulated gastric juice with a pH of 4.0 (Fig. 2D). Most importantly, heat shock enabled the C. sakazakii to increase survival when exposed to the simulated gastric juice. For example, the control cells of C. sakazakii exhibited a population reduction of 3.8 log CFU/mL after 3 h of exposure to gastric juice with a pH of 3.0 (Fig. 2B). Meanwhile, a significantly less (P b 0.05) population reduction of 3.1 log CFU/mL was observed with the heat-shocked C. sakazakii (Fig. 2B). A similar phenomenon was also noted with the control and the heat-shocked C. sakazakii when exposed to pH 3.5 or 4.0 gastric juice (Fig. 2C and D). It is therefore suggested that storage of food, especially infant formula, at a temperature that induces the heat shock response of C. sakazakii should be avoided. In this way, the enhanced survival of C. sakazakii in the presence of gastric juice can be prevented.
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tolerate bile is important for their survival and subsequent colonization of the gastrointestinal tract (Begley et al., 2005). Bile concentration has been found to be approximately 2.0% 1 h after foods enter the intestine, while declining to approximately 0.5% in 2 h (Davenport, 1977). In the present study, control and heat-shocked cells of C. sakazakii were exposed to 0.5 or 2.0% bile salt for a period of 12 h and the results are shown in Fig. 3. It was found that the viable populations of control and the heat-shocked C. sakazakii increased with the extension of exposure period in the bile salt solution regardless of concentration. After 12 h of exposure to 0.5% bile salt, control and heat-shocked C. sakazakii increased from initial populations of ca 6.4 log CFU/mL to 8.3 and 8.1 log CFU/mL, respectively (Fig. 3A). The final population of the control cells and heat-shocked cells showed no significant difference (P N 0.05). Similar phenomena were also noted in the bile salt solution with a concentration of 2.0% (Fig. 3B). In this experiment, the test organism in TSB which mimics food material was inoculated in bile salt solution. It is therefore suggested that the nutrients in TSB supported the growth of the test organism, leading to the larger viable population observed during the exposure period. Gram-negative bacteria are inherently more resistant to bile than Gram-positive bacteria (Bridson, 1995). It is reported that E. coli is very bile resistant and is commonly isolated from gallbladder and bile of animals and human (Brook, 1989; Flores et al., 2003). Van Velkinburgh and Gunn (1999) also reported that S. typhimurium and S. typhi are
(A) 0.5 % bile solution
(B) 2 % bile solution
3.3. Susceptibility to bile salt Being a digestive secretion, bile plays a major role in the emulsification and solubilization of lipids. It has the ability to affect the phospholipids and proteins of cell membranes and disrupt cellular homeostasis. Therefore, the ability of pathogens and commensales to
Fig. 3. Effect of heat shock treatment on the viable population of C. sakazakii after exposure to 0.5 and 2.0% bile solution. ○, control cells; ●, heat-shocked cells. Viability of the test organism in the 0 time sample was determined immediately after 20 s of mixing with PBS with or without bile solution. The initial population of control and heat-shocked C. sakazakii were ca 106 CFU/mL.
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rather resistant to bile. They observed that minimal inhibitory concentrations of oxbile (oxgall) for S. typhimurium and S. typhi were 18% and 112%, respectively, while minimal bactericidal concentrations were N60% for S. typhimurium and 18% for S. typhi. The data shown in Fig. 3 demonstrated that bile salt at concentrations of 0.5 or 2.0% were not detrimental to C. sakazakii. Additionally, heat shock treatment did not alter the behavior of C. sakazakii BCRC 13988 in the bile salt solution, although Flahaut et al. (1996) reported that survival of Enterococcus faecalis increased in lethal levels of bile after adaptation to heat (50 °C). 4. Conclusion In summary, data obtained from the present study demonstrated that sub-lethal heat treatment resulted in enhanced tolerance to organic acids. Furthermore, although heat shock did not affect C. sakazakii BCRC 13988 survival in the bile salt solution examined, it did significantly (P b 0.05) increase the tolerance of C. sakazakii BCRC 13988 in the presence of simulated gastric acid with a pH of 3.0–4.0. It is therefore suggested that the stress-hardening phenomenon observed in the present study should be addressed in the creation of both adequate quality control measures and risk assessments on the importance of C. sakazakii in food processing operations. Acknowledgement This research was financially supported by The National Science Council, ROC (Taiwan) (NSC 98-2313-B-002-037-MY3). References Begley, M., Gahan, C.G.M., Hill, C., 2005. The interaction between bacteria and bile. FEMS Microbiology Reviews 29, 625–651. Beuchat, L.R., Kim, H., Gurtler, J.B., Lin, L.C., Ryu, J.H., Richards, G.M., 2009. Cronobacter sakazakii in foods and factors affecting its survival, growth, and inactivation. International Journal of Food Microbiology 136, 204–213. Block, C., Peleg, O., Minster, N., Bar-Oz, B., Simhon, A., Arad, I., Shapiro, M., 2002. Cluster of neonatal infections in Jerusalem due to unusual biochemical variant of Enterobacter sakazakii. European Journal of Clinical Microbiology & Infectious Diseases 21, 613–616. Bridson, E.I., 1995. The oxoid manual, 7th ed. Unipath Limited, Basingstoke, England. Brook, I., 1989. In vitro susceptibility and in vivo efficacy of antimicrobials in the treatment of Bacteroides fragilis–Escherichia coli infection in mice. The Journal of Infectious Diseases 160, 651–656. Bunning, V.K., Crawford, R.G., Tierney, J.T., Peeler, J.T., 1990. Thermotolerance of Listeria monocytogenes and Salmonella typhimurium after sublethal heat shock. Applied and Environmental Microbiology 56, 3216–3219. Centers for Disease Control and Prevention (CDC), 2002. Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. MMWR. Morbidity and Mortality Weekly Report, 51, pp. 297–300. Chang, C.M., Chiang, M.L., Chou, C.C., 2004. Response of heat-shocked Vibrio parahaemolyticus to subsequent physical and chemical stresses. Journal of Food Protection 67, 2183–2188. Chang, C.H., Chiang, M.L., Chou, C.C., 2009. The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988. International Journal of Food Microbiology 134, 184–189.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Sub-lethal heat treatment affects the tolerance of Cronobacter sakazakii BCRC 13988 to various organic acids, simulated gastric juice and bile solution Wan-Ling Hsiao a, Wei-Li Ho b, Cheng-Chun Chou a,⁎ a b
Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan Department of Food Health, Ching Kuo Institute of Management and Health, Keelung, Taiwan
a r t i c l e
i n f o
Article history: Received 8 June 2010 Received in revised form 28 September 2010 Accepted 7 October 2010 Keywords: Cronobacter sakazakii Sub-lethal heat treatment Organic acid Simulated gastric juice Bile solution
a b s t r a c t Cronobacter spp., formerly Enterobacter sakazakii, are considered emerging opportunistic pathogens and the etiological agent of life-threatening bacterial infections in infants. In the present study, C. sakazakii BCRC 13988 was first subjected to sub-lethal heat treatment at 47 °C for 15 min. Survival rates of the heat-shocked and non-shocked C. sakazakii cells in phosphate buffer solution (PBS, pH 4.0) containing organic acids (e.g. acetic, propionic, citric, lactic or tartaric acid), simulated gastric juice (pH 2.0–4.0), and bile solution (0.5 and 2.0%) were examined. Results revealed that sub-lethal heat treatment enhanced the test organism's tolerance to organic acids, although the extent of increased acid tolerance varied with the organic acid examined. Compared with the control cells, heat-shocked C. sakazakii cells after 120-min of exposure, exhibited the largest increase in tolerance in the lactic acid-containing PBS. Furthermore, although heat shock did not affect the behavior of C. sakazakii in bile solution, it increased the test organism's survival when exposed to simulated gastric juice with a pH of 3.0–4.0. © 2010 Published by Elsevier B.V.
1. Introduction Cronobacter spp., formerly Enterobacter sakazakii, are Gramnegative facultative anaerobic rods. They are considered as emerging opportunistic pathogens and the etiological agent of life-threatening bacteria infections in infants (Block et al., 2002; CDC, 2002). These organism are ubiquitous in the environment and food (Iversen and Forsythe, 2003; Friedemann, 2007). Although dried infant formula milk has been confirmed as the source of severe systemic neonatal infections caused by Cronobacter spp., these pathogens have also been detected in vegetables, cereals, legumes, minced beef, cheese products, and spices (Iversen and Forsythe, 2003; Leclercq et al., 2002; Beuchat, et al., 2009). Neonates, particularly those of low birth weight, are at the greatest risk, and have a high mortality rate. However, involvement of Cronobacter spp. in cases of infections in children, and immunocompromised adults has been reported (FAO and WHO, 2004). Due to the extremely severe consequences of infection in infants and its ubiquitous nature, C. sakazakii was ranked as “severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration.” (ICMSF, 2002) Various foodborne pathogens such as Salmonella typhimurium, Vibrio parahaemolyticus, Listeria monocytogenes, and Escherichia coli O157:H7 (Bunning et al., 1990; Wang and Doyle, 1998; Jørgensen et al., 1999; Lin ⁎ Corresponding author. Graduate Institute of Food Science & Technology, National Taiwan University 59, lane 144, Keelung Rd., Sec. 4, Taipei, Taiwan. Tel.: +886 2 3366 4111; fax: +886 2 2362 0849. E-mail address: [email protected] (C.-C. Chou). 0168-1605/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2010.10.006
and Chou, 2004; Chang et al., 2004) have been reported as capable of enhancing their abilities to tolerate more adverse heat and other lethal stresses after being exposed to sub-lethal heat treatment. Previously, Shaker et al. (2008) reported that heat resistance of C. sakazakii reduced after exposure to heat shock at 55 °C for 5 min. While we found that heat shock at 47 °C for 15 min enhanced the survival of C. sakazakii BCRC 13988 in its subsequent exposure to thermal stresses (51 °C) and other lethal stresses such as acidity (pH 3.0), and ethanol (15%) (Chang et al., 2009, 2010). Additionally, heat shock increased the survival of C. sakazakii after freeze drying or spray drying, and during storage of lactic fermented milk products (pH 4.3) at 5 °C for 48 h (Hsiao et al., 2010). To provide more information for developing adequate control measures and risk assessments for this pathogen, the authors further investigated the effects of heat shock on the survival of C. sakazakii in the presence of various organic acids. Furthermore, this study examined the response of the heat-shocked C. sakazakii in simulated gastric juice (pH 2.0–4.0) and bile solution (0.5% and 2.0%), since C. sakazakii in food may encounter various organic acids which are frequently used to control the proliferation of microorganisms in a food system (Golden et al., 1995). Besides, the microorganisms are exposed to low pH of gastric juice and bile salt after ingestion. 2. Materials and methods 2.1. C. sakazakii and preparation of the heat-shocked cells C. sakazakii BCRC 13988 (ATCC 29544), the present study's test organism, was obtained from the Bioresources Collection and Research
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Center in Hsinchu, Taiwan. It is a clinical strain originally isolated from the throat of an infant patient (Farmer et al., 1980). Procedures described in the authors’ previous paper (Chang et al., 2009) were followed to prepare the heat-shocked cells of C. sakazakii. Briefly, the activated cultures obtained by two successive transfers of the test organism in Tryptic soy broth (TSB) (Acumedia Manufactures, Lansing, MI, USA) at 37 °C for 24 h, were grown in TSB (pH 7.2) at 37 °C for 6 h to reach late exponential growth when pH of TSB reduced to ca 5.7. The culture was centrifuged at 8000×g for 10 min. After resuspending the pellet in fresh TSB (pH 7.2), the cultures were subjected to heating at 47 °C for 15 min to obtain the heat-shocked cells of C. sakazakii. Cultures not exposed to the heat shock treatment were included as control.
2.2. Study on the survival of C. sakazakii in phosphate buffer solution (PBS) containing various organic acids To examine the survival of C. sakazakii in the presence of organic acid, 9.9 mL PBS (0.1 M, pH 4.0) containing 40 mM acetic, propionic, lactic, citric, or tartaric acid was inoculated with 0.1 mL of C. sakazaki inoculum at an initial population of approximately 106 CFU/mL. It was then incubated at room temperature (ca 23–25 °C) for 120 min. The
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viability of the test organism was determined periodically during the incubation period.
2.3. Study of the survival of C. sakazakii in simulated gastric juice and bile solutions Methods described by Charteris et al. (1998) were employed to compare the gastric and acidic resistance of heat-shocked and nonshocked C. sakazakii cells. The simulated gastric juice was prepared by suspending pepsin (3 g/L, Acumedia, Lansing, MI, USA) in saline (0.5%, v/v) and adjusting the pH to 2.0, 3.0, 3.5 or 4.0 with 1.0 N HCl. It was then sterile-filtered through a membrane (0.45 μm, Gelman Science, Ann Arbor, MI, USA). To prepare the bile solution, 10 g oxgall (Difco, Sparks, MO, USA) was dissolved in 90 mL distilled water. This solution was then used to prepare 0.5% and 2.0% of bile solution. All solutions were sterilized at 121 °C for 15 min. When the survival study was conducted, 0.1 mL of the heat-shocked or non-shocked C. sakazakii inoculum was added to 9.9 mL simulated gastric juice (pH 3.0 and 2.0) or bile solution (0.5% or 2.0%). The solution was vortexed for 20 s. Samples were taken immediately after mixing to determine the viability of C. sakazakii (0 h sample). The mixtures were
(A) Control
(B) Lactic acid
(C) Acetic acid
(D) Propionic acid
(E) Citric acid
(F) Tartaric acid
Fig. 1. Effect of heat shock treatment on the survival of C. sakazakii after exposure to phosphate buffer solution (pH 4.0) containing no organic acid (A), containing 40 mM lactic acid (B), acetic acid (C), propionic acid (D), citric acid (E), and tartaric acid (F). ○, control cells; ●, heat-shocked cells. Viability of the test organism in the 0 time sample was determined immediately after 20 s of mixing with PBS with or without organic acid. The initial population of control and heat-shocked C. sakazakii were ca 106 CFU/mL. Survival percentage was obtained by dividing the surviving population by the initial population which corresponds to 100%.
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then incubated at 37 °C with shaking (150 rpm) for 3 or 12 h. During the incubation period, viable C. sakazakii were enumerated at predetermined time intervals. 2.4. Enumeration of C. sakazakii To determine the viable population of C. sakazakii, samples were serially diluted in PBS containing 0.1% peptone. The viable counts were then made by spread plating 0.1 mL on Tryptic soy agar (Acumedia Manufactures). Colonies were counted after 18 h of incubation at 37 °C. 2.5. Statistical analysis Mean values and standard deviations were calculated from the data obtained from the three separate experiments. Data were analyzed using an unpaired two-tailed Student's t-test. Statistical significance was set at P b 0.05 (SAS, 2001). 3. Results and discussion 3.1. Susceptibility to organic acid Fig. 1 shows changes in percent of cells of control and heat-shocked C. sakazakii surviving exposure to PBS (pH 4.0) with or without organic acid for 120 min. It was found that the survival percentage of the control C. sakazakii in PBS containing no organic acid declined slightly during the exposure period (Fig. 1A). After 120 min of exposure in PBS
without organic acid, the control C. sakazakii exhibited a survival percentage of 64.1% (Fig. 1A) with a population reduction of 0.2 log CFU/mL which was obtained by subtracting the final population (log CFU/ml) from the initial population (log CFU/ml). During the exposure period, control cells in PBS containing organic acid (Fig. 1B– F) also showed various degrees of reduction in viable cells depending on the organic acid examined. However, a marked reduction in the survival percentage of control C. sakazakii was found in the PBS containing lactic (Fig. 1B), acetic (Fig. 1C) and propionic acids (Fig. 1D). At the end of the exposure period, the control C. sakazakii exhibited a population reduction of 2.0, 1.2, 0.6, 0.4 and 0.2 log CFU/mL in PBS containing lactic (Fig. 1B), acetic (Fig. 1C), propionic (Fig. 1D), citric (Fig. 1E), and tartaric acids (Fig. 1F), respectively. This indicated that at pH 4.0, C. sakazakii was most susceptible to lactic acid, followed by acetic, propionic, citric, and tartaric acids in descending order. During the exposure period, the heat-shocked C. sakazakii, similar to those observed with the control cells, also showed reductions in surviving cells as the exposure period was extended. In PBS containing no organic acid (Fig. 1A), the survival percentage of the heatshocked and control C. sakazakii determined at same specific exposure intervals showed no significant difference (P N 0.05). On the other hand, a relatively higher survival rate with a significantly smaller (P b 0.05) population of heat-shocked cells was generally noted in PBS containing lactic, acetic, propionic, and citric acids (Fig. 1B, C, D and F, respectively) when compared with that of the control C. sakazakii. For example, at the end of the 120-min exposure period, the heat-shocked C. sakazakii in PBS containing lactic acid showed a survival percentage of 13.8% with a population reduction of 0.9 log CFU/mL. Meanwhile,
(A) pH 2.0
(B) pH 3.0
(C) pH 3.5
(D) pH 4.0
Fig. 2. Effect of heat shock treatment on the survival of C. sakazakii after exposure to simulated gastric juice with various pH. ○, control cells; ●, heat-shocked cells. Viability of the test organism in the 0 time sample was determined immediately after 20 s of mixing with PBS with or without simulated gastric juice with various pH. The initial population of control and heat-shocked C. sakazakii were ca 106 CFU/mL. Survival percentage was obtained by dividing the surviving population by the initial population which corresponds to 100%.
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the control C. sakazakii showed a lower survival percentage of 1.4% with a significantly higher (P b 0.05) population reduction of 2.0 log CFU/mL. These data demonstrated that heat shock treatment might enhance the tolerance of C. sakazakii to organic acid, although the degree of enhancement varied with the organic acid tested. The phenomenon of enhanced acid tolerance exerted by C. sakazakii after heat shock treatment as observed in the present study is in agreement with those reported for Shigella flexneri (Tetteh and Beuchat, 2003), E. coli O157:H7 (Wang and Doyle, 1998) and Lactobacillus collinoides (Laplace et al., 1999), while different from that observed on V. parahaemolyticus (Chiang et al., 2005). Chiang et al. (2005) reported that V. parahaemolyticus shocked at 42 °C for 45 min resulted in a decreased tolerance to acetic, citric, tartaric and lactic acids. In addition to the heat shock conditions examined, differences in the kind of bacteria tested might lead to these discrepancies. 3.2. Susceptibility to simulated gastric acid Sondheimer et al. (1985) reported that gastric pH of infants varies with age. The gastric pH of infants six days old varies from a fasting pH of 2.9 to a value of 5.2 directly after feeding. On the other hand, the gastric pH in infants 7–15 days old is relatively higher, within a narrow range between 4.6 and 5.8. Healthy adults usually have gastric pH levels of 2.0–3.0, which is lower than in infants. Fig. 2 shows the survival of heat-shocked and control C. sakazakii cells during the exposure to simulated gastric juices with pH levels of 2.0 to 4.0. It was generally found that survival of C. sakazakii varied with the pH of the simulated gastric juices, exposure period, and heat shock treatment. Among the various pHs examined, the most pronounced population reduction occurred when the test organism was exposed to gastric juice with a pH of 2.0, regardless of heat shock treatment. The viable population of C. sakazakii, regardless of heat shock, reduced by approximately 1.0 log CFU/mL after 20 s of mixing with the pH 2.0 gastric juice. No viable cells of control or heat-shocked C. sakazakii were noted after further extending the exposure period to 1 h (Fig. 2A). However, this phenomenon was not observed with test organism exposure to simulated gastric juice having pH levels between 3.0 and 4.0 (Fig. 2B, C and D). Nevertheless, viable populations of C. sakazakii still declined when exposed to gastric juice with a pH 3.0–4.0, although not to the extent seen in pH 2.0 juice. The survival percentage usually increased with increased pH and heat shock treatment. For example, the control C. sakazakii showed a survival of 0.1% after 2 h of exposure to gastric juice having a pH of 3.0 (Fig. 2B), compared to a higher survival rate of 64.6% noted with exposure to simulated gastric juice with a pH of 4.0 (Fig. 2D). Most importantly, heat shock enabled the C. sakazakii to increase survival when exposed to the simulated gastric juice. For example, the control cells of C. sakazakii exhibited a population reduction of 3.8 log CFU/mL after 3 h of exposure to gastric juice with a pH of 3.0 (Fig. 2B). Meanwhile, a significantly less (P b 0.05) population reduction of 3.1 log CFU/mL was observed with the heat-shocked C. sakazakii (Fig. 2B). A similar phenomenon was also noted with the control and the heat-shocked C. sakazakii when exposed to pH 3.5 or 4.0 gastric juice (Fig. 2C and D). It is therefore suggested that storage of food, especially infant formula, at a temperature that induces the heat shock response of C. sakazakii should be avoided. In this way, the enhanced survival of C. sakazakii in the presence of gastric juice can be prevented.
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tolerate bile is important for their survival and subsequent colonization of the gastrointestinal tract (Begley et al., 2005). Bile concentration has been found to be approximately 2.0% 1 h after foods enter the intestine, while declining to approximately 0.5% in 2 h (Davenport, 1977). In the present study, control and heat-shocked cells of C. sakazakii were exposed to 0.5 or 2.0% bile salt for a period of 12 h and the results are shown in Fig. 3. It was found that the viable populations of control and the heat-shocked C. sakazakii increased with the extension of exposure period in the bile salt solution regardless of concentration. After 12 h of exposure to 0.5% bile salt, control and heat-shocked C. sakazakii increased from initial populations of ca 6.4 log CFU/mL to 8.3 and 8.1 log CFU/mL, respectively (Fig. 3A). The final population of the control cells and heat-shocked cells showed no significant difference (P N 0.05). Similar phenomena were also noted in the bile salt solution with a concentration of 2.0% (Fig. 3B). In this experiment, the test organism in TSB which mimics food material was inoculated in bile salt solution. It is therefore suggested that the nutrients in TSB supported the growth of the test organism, leading to the larger viable population observed during the exposure period. Gram-negative bacteria are inherently more resistant to bile than Gram-positive bacteria (Bridson, 1995). It is reported that E. coli is very bile resistant and is commonly isolated from gallbladder and bile of animals and human (Brook, 1989; Flores et al., 2003). Van Velkinburgh and Gunn (1999) also reported that S. typhimurium and S. typhi are
(A) 0.5 % bile solution
(B) 2 % bile solution
3.3. Susceptibility to bile salt Being a digestive secretion, bile plays a major role in the emulsification and solubilization of lipids. It has the ability to affect the phospholipids and proteins of cell membranes and disrupt cellular homeostasis. Therefore, the ability of pathogens and commensales to
Fig. 3. Effect of heat shock treatment on the viable population of C. sakazakii after exposure to 0.5 and 2.0% bile solution. ○, control cells; ●, heat-shocked cells. Viability of the test organism in the 0 time sample was determined immediately after 20 s of mixing with PBS with or without bile solution. The initial population of control and heat-shocked C. sakazakii were ca 106 CFU/mL.
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rather resistant to bile. They observed that minimal inhibitory concentrations of oxbile (oxgall) for S. typhimurium and S. typhi were 18% and 112%, respectively, while minimal bactericidal concentrations were N60% for S. typhimurium and 18% for S. typhi. The data shown in Fig. 3 demonstrated that bile salt at concentrations of 0.5 or 2.0% were not detrimental to C. sakazakii. Additionally, heat shock treatment did not alter the behavior of C. sakazakii BCRC 13988 in the bile salt solution, although Flahaut et al. (1996) reported that survival of Enterococcus faecalis increased in lethal levels of bile after adaptation to heat (50 °C). 4. Conclusion In summary, data obtained from the present study demonstrated that sub-lethal heat treatment resulted in enhanced tolerance to organic acids. Furthermore, although heat shock did not affect C. sakazakii BCRC 13988 survival in the bile salt solution examined, it did significantly (P b 0.05) increase the tolerance of C. sakazakii BCRC 13988 in the presence of simulated gastric acid with a pH of 3.0–4.0. It is therefore suggested that the stress-hardening phenomenon observed in the present study should be addressed in the creation of both adequate quality control measures and risk assessments on the importance of C. sakazakii in food processing operations. Acknowledgement This research was financially supported by The National Science Council, ROC (Taiwan) (NSC 98-2313-B-002-037-MY3). References Begley, M., Gahan, C.G.M., Hill, C., 2005. The interaction between bacteria and bile. FEMS Microbiology Reviews 29, 625–651. Beuchat, L.R., Kim, H., Gurtler, J.B., Lin, L.C., Ryu, J.H., Richards, G.M., 2009. Cronobacter sakazakii in foods and factors affecting its survival, growth, and inactivation. International Journal of Food Microbiology 136, 204–213. Block, C., Peleg, O., Minster, N., Bar-Oz, B., Simhon, A., Arad, I., Shapiro, M., 2002. Cluster of neonatal infections in Jerusalem due to unusual biochemical variant of Enterobacter sakazakii. European Journal of Clinical Microbiology & Infectious Diseases 21, 613–616. Bridson, E.I., 1995. The oxoid manual, 7th ed. Unipath Limited, Basingstoke, England. Brook, I., 1989. In vitro susceptibility and in vivo efficacy of antimicrobials in the treatment of Bacteroides fragilis–Escherichia coli infection in mice. The Journal of Infectious Diseases 160, 651–656. Bunning, V.K., Crawford, R.G., Tierney, J.T., Peeler, J.T., 1990. Thermotolerance of Listeria monocytogenes and Salmonella typhimurium after sublethal heat shock. Applied and Environmental Microbiology 56, 3216–3219. Centers for Disease Control and Prevention (CDC), 2002. Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. MMWR. Morbidity and Mortality Weekly Report, 51, pp. 297–300. Chang, C.M., Chiang, M.L., Chou, C.C., 2004. Response of heat-shocked Vibrio parahaemolyticus to subsequent physical and chemical stresses. Journal of Food Protection 67, 2183–2188. Chang, C.H., Chiang, M.L., Chou, C.C., 2009. The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988. International Journal of Food Microbiology 134, 184–189.
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