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Effects of sublethal heat, bile and organic acid treatments on the tolerance of Vibrio parahaemolyticus to lethal low-salinity
Food Control 28 (2012) 349e353
Contents lists available at SciVerse ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Effects of sublethal heat, bile and organic acid treatments on the tolerance of Vibrio parahaemolyticus to lethal low-salinity Wei Shen Huang, Hin-chung Wong* Department of Microbiology, Soochow University, Taipei, Taiwan 111, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2012 Received in revised form 8 May 2012 Accepted 15 May 2012
As a marine foodborne pathogenic bacterium commonly found in seawater and seafood, Vibrio parahamolyticus often encounters low salinity stress when the contaminated seafood is washed with fresh water during food processing. This study investigated the influence of prior adaptation to sublethal stresses on the tolerance of Vibrio parahaemolyticus ST550 to lethal low salinity. Results revealed that tolerance to lethal low salinity (0.25% NaCl) was enhanced in the exponential phase cells by prior adaptation in sublethal bile (0.05%), heat (41 C), acetic (0.05%) or lactic acid (1.0%), but not in low acidity (pH4.48). In stationary phase cells, tolerance to lethal low salinity was enhanced by prior adaptation in bile and heat. Among the stresses examined, bile was the most efficient one to induce tolerance to lethal low salinity in this pathogen. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Vibrio parahaemolyticus Low salinity Stress Adaptation Tolerance Bile
1. Introduction Vibrio parahaemolyticus is a prevalent halophilic gram-negative enteropathogenic bacterium in many of the Asian Pacific countries. Human infections caused by this pathogen in these countries are frequently attributed to the consumption of contaminated seafood. This bacterium inhabits seawater, and has been frequently isolated from fish, clams and crustaceans (Wong, Ting, & Shieh, 1992). Bacteria share some common regulatory mechanisms, such as RpoS, in response to different kinds of environmental stressors, and thus adaptation of bacteria to a specific stress may also enhances their tolerance to some other stresses (also known as cross-protection) (Pichereau, Hartke, & Auffray, 2000). V. parahaemolyticus encounters various stresses in its natural habitat, during food processing or in a human host, while the stress adaptation and cross-protection has been characterized in this pathogen (Wong et al., 2002; Yeung & Boor, 2004). Under carbon starvation, V. parahaemolyticus exhibits cross-protection against heat, hypoosmotic (13 mM NaCl) and oxidative stresses (Koga & Takumi, 1995b), and also protects against freeze-thawing treatment (Wong, Chang, & Chen, 2004). Acid- or alkaline-adapted cells exhibit increased resistance to heat, crystal violet and deoxycholic acid (Koga, Sakamoto, Yamoto, & Takumi, 1999; Koga, Katagiri, Hori, * Corresponding author. Tel.: þ886 2 28819471x6852; fax: þ886 2 28831193. E-mail address: [email protected] (H.-c. Wong). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.037
& Takumi, 2002). Cold shock treatment increases the survival of V. parahaemolyticus against crystal violet, but reduces tolerance for heat, oxidative stress and organic acids (lactic and acetic acid) (Lin, Yu, & Chou, 2004). Fresh water washing is a common practice during food processing of seafood, and it can be regarded as a lethal stress treatment to lower the levels of V. parahaemolyticus and other marine vibrios. Prior treatment of this pathogen by other sublethal stresses may trigger the induction of cross-protection against lethal low salinity, and thus increases its risk in food. Influence of starvation and heat-shock on the increased survival of V. parahaemolyticus against hypoosmotic stress has been demonstrated (Koga & Takumi, 1995a, 1995b), while the effects of other different stresses have not been clarified. Applying different stressors in seafood processing is often practiced especially in some oriental countries, such as acid/fermentation, drying and salting (Chen, 1995), while V. parahaemolyticus is frequently isolated from these processed seafood (Yang et al., 2008). Presence of lactic acid bacteria and small amount of organic acid is demonstrated in both vacuum and modified atmosphere packaging of seafood (Francoise, 2010). Therefore, careful handling of these seafood processing methods prior to low salinity wash will be beneficial to monitor the risk of this pathogen. In this study, prior adaptation of this pathogen to sublethal heat, bile, low acidity and organic acids to enhance the tolerance of V. parahaemolyticus to lethal low salinity were investigated.
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2. Materials and methods
3. Results
2.1. Bacterial strain and culture
The V. parahaemolyticus ST550 cells were rapidly inactivated by the lethal low salinity in 10 min (Figs. 1 and 2). In the non-stress adapted groups of experiments, exponential phase cells of V. parahaemolyticus ST550 were more vulnerable than the stationary phase cells against the challenge of lethal low salinity (PBS-0.25% NaCl), and about 0.01% (Figs. 1 and 2A) or 0.1% (Figs. 1 and 2B) of the exponential phase or stationary phase cells, respectively, remained culturable after 10 min treatment. The tolerance of heat-, bile-, low acidity- or organic acid (acetic acid, lactic acid)-adapted V. parahaemolyticus ST550 cells to the challenge of lethal low salinity was examined, and the results revealed significant increase of survival in most of these stressadapted cells as compared to the control groups. The heat- and bile-adapted exponential phase and stationary phase cells were significantly protected (Figs. 1 and 2). Comparing with the nonadapted cells, the survival increased by about 90% in both of the heat-adapted cells in exponential phase or stationary phase (Fig. 1). Comparing with the non-adapted cells, the survival increment for the bile-adapted exponential phase cells was more than 99% (Fig. 2). These results suggest that bile adaptation is more efficient than heat to induce tolerance against lethal low salinity in V. parahaemolyticus.
2.2. Preparation of stress-adapted cultures A 100 ml aliquot of overnight culture of V. parahaemolyticus ST550 was inoculated into 100 ml LB-3.0% NaCl in a 250 ml Erlenmeyer flask and incubated at 37 C with shaking at 110 rpm for 4.5 h or 18 h, to reach the exponential or stationary phase, respectively. Bacterial cells in the exponential or stationary phase were harvested by centrifugation at 10,000 g for 15 min and washed using equal volumes of phosphatebuffered saline (PBS) with 3.0% NaCl at 25 C (Liu & Wong, 2008). For the preparation of acid-adapted cultures, bacterial cells were harvested and resuspended in equal volumes of PBS with 3.0% NaCl and 20 amino acids (AA-PBS-3.0% NaCl, alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, penylalanine, praline, serine, threonine, tryptophan, tyrosine and valine, all at 20 mg/l) (Wong et al., 2002) which had been acidified to pH 4.48 using 1 N HCl and incubated at 25 C for 20 min (Wong et al., 1998). In the preparation of heat-adapted cultures, bacterial cells were harvested and resuspended in AA-PBS-3.0% NaCl that had been preheated to 41 C for 20 min (Wong et al., 2002). To prepare bile-, acetic acid- or lactic acid-adapted cultures, bacterial cells were harvested and resuspended in AA-PBS-3.0% NaCl that contained 0.05% bile salts (Product number B3301, Sigma Co., St. Louis, MO, U.S.A.)(Wong & Liu, 2006), 0.05% acetic acid (pH 4.8) or 0.5% lactic acid (pH 4.8), respectively, and further incubated at 25 C for 20 min. These stress conditions were determined in preliminary experiments not to be lethal to V. parahaemolyticus ST550.
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V. parahaemolyticus ST550, a serotype O4:K13 and KPþ clinical strain isolated in Thailand, was provided by T. Arai and used in this work (Wong, Peng, Han, Chang, & Lan, 1998). This strain was frozen and stored at 85 C in beads in Microbank cryovials (PRO-LAB Diagnostics, Austin, TX, USA), and cultured in LauriaBertani broth (LB, Difco, Becton-Dickinson Diagnostic Systems, Sparks, MD, USA) that contained 3.0% NaCl (final concentration) at 25 C.
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The non-adapted control and stress-adapted cells were harvested, resuspended in PBS with 0.25% NaCl which was lethal as determined in a preliminary experiment, and incubated at 25 C with shaking at 100 rpm. The surviving bacterial cells were determined at different intervals by the standard plate count method using Lauria broth agar (LA, Difco)-3.0% NaCl following serial dilution in PBS-3.0% NaCl and were subsequently incubated at 37 C for 16 h (Wong, Liu, & Chen, 2005) and the survival percentages were calculated.
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2.4. Statistical analysis All experiments were repeated three times and at least triplicate determinations of bacterial counts were performed in each of these repeats. Data of the adapted and non-adapted control cultures after challenging by lethal low salinity for 10e30 min were analyzed by performing ANOVA using SPSS for Windows 10.0 (SPSS Inc., Chicago, Ill, U.S.A.) at a significance level of p < 0.05.
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Fig. 1. Survival of heat-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted at 41 C for 20 min in AA-PBS-3.0% NaCl, and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
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Fig. 2. Survival of bile-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that contained 0.05% bile salts, and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
The low acidity (pH 4.48)-adapted cells, regardless of their growth phase, were not protected against lethal low salinity, and were rapidly inactivated and about 0.001% or 0.1% survived after 10 min treatment for the exponential phase cells or stationary phase cells, respectively (Fig. 3). Whereas the organic acid-adapted V. parahaemolyticus cells exhibited enhanced tolerance in the exponential phase cells challenged for 20e30 min, and significant increase of tolerance was only detected in the stationary phase cells challenged for 30 min (Figs. 4 and 5). The acetic acid-adapted cells at exponential phase exhibited significant protection against lethal low salinity, with survival increment of less than 90% as compared to the control cells (Fig. 4A). Response of the lactic acid-adapted cells was similar to those of acetic acid-adapted cells, and lactic acid-adapted exponential phase cells were significantly protected against lethal low salinity (Fig. 5). 4. Discussion Responses to hyperosmotic stress in a wide range of pathogenic bacteria, including several vibrios, namely, Vibrio alginolyticus (Xu et al., 2005), Vibrio cholerae (Pflughoeft, Kierek, & Watnick, 2003), V. parahaemolyticus (Xu, Ren, Wang, & Peng, 2004) and Vibrio vulnificus (Lee, Park, Lee, & Choi, 2003), have been examined. Nevertheless, the low salinity/hypoosmotic stress responses in vibrios have not been studied in detail. We explored the low salinity
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Fig. 3. Survival of acid-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that had been acidified to pH 4.48 using 1 N HCl, and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
response of V. vulnificus and showed that cross-protected against lethal low salinity was demonstrated in the bile-adapted exponential and stationary cells and not in the acid (pH 4.4)- and heat (41 C)-adapted cells (Wong & Liu, 2006). V. parahaemolyticus is phylogenetically close to V. vulnificus (Thompson, Iida, & Swings, 2004), but it exhibited some different features in response to low salinity stress. In this study, enhanced tolerance against lethal low salinity was found in the heat- (Fig. 1), bile- (Fig. 2) and organic acid-adapted V. parahaemolyticus cells (Figs. 4 and 5). Bile was the most efficient stress to induce such response than heat and organic acids in V. parahaemolyticus (Fig. 2). Another observation in this study is that the V. parahaemolyticus cells at stationary phase were more resistant to environmental stressors and less responsive to the sublethal stress adaptation as compared to those cells at exponential phase (Figs. 1e5), and this is a common phenomenon associated with the function of RpoS. The cellular RpoS level is regulated by integrated multiple signals ivolving the transcriptional and translational control of rpoS and the RpoS proteolysis. The transcription of rpoS is known to be increased in cells which are shifting from exponential phase to stationary phase with a reduced growth rate or being challenged by environmental stressors (Dodd & Aldsworth, 2002; Hengge-Aronis, 2002; Hirsch & Elliott, 2005). Stresses that activate the transcription of rpoS in exponential phase cells are reasonably less effective in stationary phase cells in which rpoS is already at raised level.
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Time, min Fig. 4. Survival of acetic acid-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that contained 0.05% acetic acid (pH 4.8), and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
Tolerance to low salinity/hypoosmotic stress involves a combination of specific (secondary transport), non-specific and rapid (stretch-activated mechanosensitive channels, MscL and MscS) solute efflux and aquaporin-mediated water efflux (Sleator & Hill, 2001). The expression of the mechanosensitive channels in Eschereichia coli under hypoosmotic conditions is at least partially regulated by RpoS (Stokes et al., 2003), and the significant role of RpoS was also demonstrated in the low salinity response of V. vulnificus (Tan, Liu, Oliver, & Wong, 2010). While RpoS generally regulates various genes in response to a variety of environmental stressors (Bearson, Benjamin, Jr., Swords, & Foster, 1996; Rosche, Smith, Parker, & Oliver, 2005) and the function of rpoS has also been demonstrated in V. parahaemolyticus (Vasudevan & Venkitanarayanan, 2006). These results suggest that stresses which can activate RpoS of V. parahaemolyticus will consequentially affiliate the tolerance of this pathogen against low salinity. Role of RpoS in the adaptation to bile has been demonstrated in Salmonella enterica (Prouty et al., 2004) and V. vulnificus (Chen, Oliver, & Wong, 2010). Also, bile-adaptation is known to promote the production of 18 common stress proteins, including DnaK and GroEL, in Enterococcus faecalis (Flahaut et al., 1996). Bile adaptation also activates porins and other efflux pumps in Campylobacter jejuni (Lin, Sahin, Michel, & Zhang, 2003), E. coli (Rosenberg, Bertenthal, Nilles, Bertrand, & Nikaido, 2003) and V. cholerae (Chatterjee,
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Fig. 5. Survival of lactic acid-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that contained 0.5% lactic acid (pH 4.8), and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
Chaudhuri, Saha, Gupta, & Chowdhury, 2004). Accordingly, cells that have adapted to bile may be equipped with better protective machinery (porins and efflux pumps) than control cells, and this machinery participates in the immediate solute and water efflux under low-salinity stress. In conclusion, this work revealed that bile in addition to heat and organic acids were able to induce tolerance against lethal low salinity in V. parahaemolyticus. Acknowledgements The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC95-2313-B-031-001 and NSC 962313-B-031-001. Ted Knoy is appreciated for his editorial assistance. References Bearson, S. M., Benjamin, W. H., Jr., Swords, W. E., & Foster, J. W. (1996). Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium. Journal of Bacteriology, 178, 2572e2579. Chatterjee, A., Chaudhuri, S., Saha, G., Gupta, S., & Chowdhury, R. (2004). Effect of bile on the cell surface permeability barrier and efflux system of Vibrio cholerae. Journal of Bacteriology, 186, 6809e6814.
W.S. Huang, H.-c. Wong / Food Control 28 (2012) 349e353 Chen, H. C. (1995). Seafood microorganisms and seafood safety. Journal of Food and Drug Analysis, 3, 133e144. Chen, W. L., Oliver, J. D., & Wong, H. C. (2010). Adaptation of Vibrio vulnificus and an rpoS mutant to bile salts. International Journal of Food Microbiology, 140, 232e238. Dodd, C. E., & Aldsworth, T. G. (2002). The importance of RpoS in the survival of bacteria through food processing. International Journal of Food Microbiology, 74, 189e194. Flahaut, S., Hartke, A., Giard, J. C., Benachour, A., Boutibonnes, P., & Auffray, Y. (1996). Relationship between stress response toward bile salts, acid and heat treatment in Enterococcus faecalis. FEMS Microbiology Letters, 138, 49e54. Francoise, L. (2010). Occurrence and role of lactic acid bacteria in seafood products. Food Microbiology, 27, 698e709. Hengge-Aronis, R. (2002). Signal transduction and regulatory mechanisms involved in control of the sS (RpoS) subunit of RNA polymerase. Microbiology & Molecular Biology Review, 66, 373e395. Hirsch, M., & Elliott, T. (2005). Stationary-phase regulation of RpoS translation in Escherichia coli. Journal of Bacteriology, 187, 7204e7213. Koga, T., Katagiri, T., Hori, H., & Takumi, K. (2002). Alkaline adaptation induces cross-protection against some environmental stresses and morphological change in Vibrio parahaemolyticus. Microbiology Research, 157, 249e255. Koga, T., Sakamoto, F., Yamoto, A., & Takumi, K. (1999). Acid adaptation induces cross-protection against some environmental stresses in Vibrio parahaemolyticus. Journal of General & Applied Microbiology, 45, 155e161. Koga, T., & Takumi, K. (1995a). Comparison of cross-protection against some environmental stresses between cadmium-adapted an heat-adapted cells of Vibrio parahaemolyticus. Journal of General & Applied Microbiology, 41, 263e268. Koga, T., & Takumi, K. (1995b). Nutrient starvation induces cross protection against heat, osmotic, or H2O2 challenge in Vibrio parahaemolyticus. Microbiology & Immunology, 39, 213e215. Lee, J. H., Park, N. Y., Lee, M. H., & Choi, S. H. (2003). Characterization of the Vibrio vulnificus putAP operon, encoding proline dehydrogenase and proline permease, and its differential expression in response to osmotic stress. Journal of Bacteriology, 185, 3842e3852. Lin, J., Sahin, O., Michel, L. O., & Zhang, Q. (2003). Critical role of multidrug efflux pump CmeABC in bile resistance and in vivo colonization of Campylobacter jejuni. Infection and Immunity, 71, 4250e4259. Lin, C., Yu, R. C., & Chou, C. C. (2004). Susceptibility of Vibrio parahaemolyticus to various environmental stresses after cold shock treatment. International Journal of Food Microbiology, 92, 207e215. Liu, S.-H., & Wong, H. C. (2008). Characterization of the low salinity stress in Vibrio vulnificus. Journal of Food Protection, 71, 416e419. Pflughoeft, K. J., Kierek, K., & Watnick, P. I. (2003). Role of ectoine in Vibrio cholerae osmoadaptation. Applied and Environmental Microbiology, 69, 5919e5927. Pichereau, V., Hartke, A., & Auffray, Y. (2000). Starvation and osmotic stress induced multiresistances. Influence of extracellular compounds. International Journal of Food Microbiology, 55, 19e25. Prouty, A. M., Brodsky, I. E., Manos, J., Belas, R., Falkow, S., & Gunn, J. S. (2004). Transcriptional regulation of Salmonella enterica serovar Typhimurium genes by bile. FEMS Immunology & Medical Microbiology, 41, 177e185.
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Rosche, T. M., Smith, D. J., Parker, E. E., & Oliver, J. D. (2005). RpoS involvement and requirement for exogenous nutrient for osmotically induced cross protection in Vibrio vulnificus. FEMS Microbiology Ecology, 53, 455e462. Rosenberg, E. Y., Bertenthal, D., Nilles, M. L., Bertrand, K. P., & Nikaido, H. (2003). Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein. Molecular Microbiology, 48, 1609e1619. Sleator, R. D., & Hill, C. (2001). Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS Microbiology Review, 26, 49e71. Stokes, N. R., Murray, H. D., Subramaniam, C., Gourse, R. L., Louis, P., Bartlett, W., et al. (2003). A role for mechanosensitive channels in survival of stationary phase: regulation of channel expression by RpoS. Proceeding of National Academy of Science of United States of America, 100, 15959e15964. Tan, H. J., Liu, S. H., Oliver, J. D., & Wong, H. C. (2010). Role of RpoS in the susceptibility of low salinity-adapted Vibrio vulnificus to environmental stresses. International Journal of Food Microbiology, 137, 137e142. Thompson, F. L., Iida, T., & Swings, J. (2004). Biodiversity of vibrios. Microbiology & Molecular Biology Review, 68, 403e431 (table). Vasudevan, P., & Venkitanarayanan, K. (2006). Role of the rpoS gene in the survival of Vibrio parahaemolyticus in artificial seawater and fish homogenate. Journal of Food Protection, 69, 1438e1442. Wong, H. C., Chang, C. N., & Chen, M. Y. (2004). Effects of heat, acid, and freeze-thaw challenges on survival of starved Vibrio parahaemolyticus in minimal salt medium, tryptic soy broth and filtered oyster homogenate medium. Journal of Food Protection, 67, 1243e1246. Wong, H. C., & Liu, S. H. (2006). Susceptibility of the heat-, acid-, and bile-adapted Vibrio vulnificus to lethal low-salinity stress. Journal of Food Protection, 69, 2924e2928. Wong, H. C., Liu, S. H., & Chen, M. Y. (2005). Virulence and stress susceptibility of clinical and environmental strains of Vibrio vulnificus isolated from samples from Taiwan and the United States. Journal of Food Protection, 68, 2533e2540. Wong, H. C., Peng, P. Y., Han, J. M., Chang, C. Y., & Lan, S. L. (1998). Effect of mild acid treatment on the survival, enteropathogenicity, and protein production in Vibrio parahaemolyticus. Infection and Immunity, 66, 3066e3071. Wong, H. C., Peng, P. Y., Lan, S. L., Chen, Y. C., Lu, K. H., Shen, C. T., et al. (2002). Effects of heat shock on the thermotolerance, protein composition, and toxin production of Vibrio parahaemolyticus. Journal of Food Protection, 65, 499e507. Wong, H. C., Ting, S. H., & Shieh, W. R. (1992). Incidence of toxigenic vibrios in foods available in Taiwan. Journal of Applied Bacteriology, 73, 197e202. Xu, C., Ren, H., Wang, S., & Peng, X. (2004). Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus. Research in Microbiology, 155, 835e842. Xu, C., Wang, S., Ren, H., Lin, X., Wu, L., & Peng, X. (2005). Proteomic analysis on the expression of outer membrane proteins of Vibrio alginolyticus at different sodium concentrations. Proteomics, 5, 3142e3152. Yang, Z. Q., Jiao, X. A., Zhou, X. H., Cao, G. X., Fang, W. M., & Gu, R. X. (2008). Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China. International Journal of Food Microbiology, 125, 279e285. Yeung, P. S., & Boor, K. J. (2004). Effects of acid stress on Vibrio parahaemolyticus survival and cytotoxicity. Journal of Food Protection, 67, 1328e1334.
Contents lists available at SciVerse ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Effects of sublethal heat, bile and organic acid treatments on the tolerance of Vibrio parahaemolyticus to lethal low-salinity Wei Shen Huang, Hin-chung Wong* Department of Microbiology, Soochow University, Taipei, Taiwan 111, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2012 Received in revised form 8 May 2012 Accepted 15 May 2012
As a marine foodborne pathogenic bacterium commonly found in seawater and seafood, Vibrio parahamolyticus often encounters low salinity stress when the contaminated seafood is washed with fresh water during food processing. This study investigated the influence of prior adaptation to sublethal stresses on the tolerance of Vibrio parahaemolyticus ST550 to lethal low salinity. Results revealed that tolerance to lethal low salinity (0.25% NaCl) was enhanced in the exponential phase cells by prior adaptation in sublethal bile (0.05%), heat (41 C), acetic (0.05%) or lactic acid (1.0%), but not in low acidity (pH4.48). In stationary phase cells, tolerance to lethal low salinity was enhanced by prior adaptation in bile and heat. Among the stresses examined, bile was the most efficient one to induce tolerance to lethal low salinity in this pathogen. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Vibrio parahaemolyticus Low salinity Stress Adaptation Tolerance Bile
1. Introduction Vibrio parahaemolyticus is a prevalent halophilic gram-negative enteropathogenic bacterium in many of the Asian Pacific countries. Human infections caused by this pathogen in these countries are frequently attributed to the consumption of contaminated seafood. This bacterium inhabits seawater, and has been frequently isolated from fish, clams and crustaceans (Wong, Ting, & Shieh, 1992). Bacteria share some common regulatory mechanisms, such as RpoS, in response to different kinds of environmental stressors, and thus adaptation of bacteria to a specific stress may also enhances their tolerance to some other stresses (also known as cross-protection) (Pichereau, Hartke, & Auffray, 2000). V. parahaemolyticus encounters various stresses in its natural habitat, during food processing or in a human host, while the stress adaptation and cross-protection has been characterized in this pathogen (Wong et al., 2002; Yeung & Boor, 2004). Under carbon starvation, V. parahaemolyticus exhibits cross-protection against heat, hypoosmotic (13 mM NaCl) and oxidative stresses (Koga & Takumi, 1995b), and also protects against freeze-thawing treatment (Wong, Chang, & Chen, 2004). Acid- or alkaline-adapted cells exhibit increased resistance to heat, crystal violet and deoxycholic acid (Koga, Sakamoto, Yamoto, & Takumi, 1999; Koga, Katagiri, Hori, * Corresponding author. Tel.: þ886 2 28819471x6852; fax: þ886 2 28831193. E-mail address: [email protected] (H.-c. Wong). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.037
& Takumi, 2002). Cold shock treatment increases the survival of V. parahaemolyticus against crystal violet, but reduces tolerance for heat, oxidative stress and organic acids (lactic and acetic acid) (Lin, Yu, & Chou, 2004). Fresh water washing is a common practice during food processing of seafood, and it can be regarded as a lethal stress treatment to lower the levels of V. parahaemolyticus and other marine vibrios. Prior treatment of this pathogen by other sublethal stresses may trigger the induction of cross-protection against lethal low salinity, and thus increases its risk in food. Influence of starvation and heat-shock on the increased survival of V. parahaemolyticus against hypoosmotic stress has been demonstrated (Koga & Takumi, 1995a, 1995b), while the effects of other different stresses have not been clarified. Applying different stressors in seafood processing is often practiced especially in some oriental countries, such as acid/fermentation, drying and salting (Chen, 1995), while V. parahaemolyticus is frequently isolated from these processed seafood (Yang et al., 2008). Presence of lactic acid bacteria and small amount of organic acid is demonstrated in both vacuum and modified atmosphere packaging of seafood (Francoise, 2010). Therefore, careful handling of these seafood processing methods prior to low salinity wash will be beneficial to monitor the risk of this pathogen. In this study, prior adaptation of this pathogen to sublethal heat, bile, low acidity and organic acids to enhance the tolerance of V. parahaemolyticus to lethal low salinity were investigated.
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2. Materials and methods
3. Results
2.1. Bacterial strain and culture
The V. parahaemolyticus ST550 cells were rapidly inactivated by the lethal low salinity in 10 min (Figs. 1 and 2). In the non-stress adapted groups of experiments, exponential phase cells of V. parahaemolyticus ST550 were more vulnerable than the stationary phase cells against the challenge of lethal low salinity (PBS-0.25% NaCl), and about 0.01% (Figs. 1 and 2A) or 0.1% (Figs. 1 and 2B) of the exponential phase or stationary phase cells, respectively, remained culturable after 10 min treatment. The tolerance of heat-, bile-, low acidity- or organic acid (acetic acid, lactic acid)-adapted V. parahaemolyticus ST550 cells to the challenge of lethal low salinity was examined, and the results revealed significant increase of survival in most of these stressadapted cells as compared to the control groups. The heat- and bile-adapted exponential phase and stationary phase cells were significantly protected (Figs. 1 and 2). Comparing with the nonadapted cells, the survival increased by about 90% in both of the heat-adapted cells in exponential phase or stationary phase (Fig. 1). Comparing with the non-adapted cells, the survival increment for the bile-adapted exponential phase cells was more than 99% (Fig. 2). These results suggest that bile adaptation is more efficient than heat to induce tolerance against lethal low salinity in V. parahaemolyticus.
2.2. Preparation of stress-adapted cultures A 100 ml aliquot of overnight culture of V. parahaemolyticus ST550 was inoculated into 100 ml LB-3.0% NaCl in a 250 ml Erlenmeyer flask and incubated at 37 C with shaking at 110 rpm for 4.5 h or 18 h, to reach the exponential or stationary phase, respectively. Bacterial cells in the exponential or stationary phase were harvested by centrifugation at 10,000 g for 15 min and washed using equal volumes of phosphatebuffered saline (PBS) with 3.0% NaCl at 25 C (Liu & Wong, 2008). For the preparation of acid-adapted cultures, bacterial cells were harvested and resuspended in equal volumes of PBS with 3.0% NaCl and 20 amino acids (AA-PBS-3.0% NaCl, alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, penylalanine, praline, serine, threonine, tryptophan, tyrosine and valine, all at 20 mg/l) (Wong et al., 2002) which had been acidified to pH 4.48 using 1 N HCl and incubated at 25 C for 20 min (Wong et al., 1998). In the preparation of heat-adapted cultures, bacterial cells were harvested and resuspended in AA-PBS-3.0% NaCl that had been preheated to 41 C for 20 min (Wong et al., 2002). To prepare bile-, acetic acid- or lactic acid-adapted cultures, bacterial cells were harvested and resuspended in AA-PBS-3.0% NaCl that contained 0.05% bile salts (Product number B3301, Sigma Co., St. Louis, MO, U.S.A.)(Wong & Liu, 2006), 0.05% acetic acid (pH 4.8) or 0.5% lactic acid (pH 4.8), respectively, and further incubated at 25 C for 20 min. These stress conditions were determined in preliminary experiments not to be lethal to V. parahaemolyticus ST550.
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V. parahaemolyticus ST550, a serotype O4:K13 and KPþ clinical strain isolated in Thailand, was provided by T. Arai and used in this work (Wong, Peng, Han, Chang, & Lan, 1998). This strain was frozen and stored at 85 C in beads in Microbank cryovials (PRO-LAB Diagnostics, Austin, TX, USA), and cultured in LauriaBertani broth (LB, Difco, Becton-Dickinson Diagnostic Systems, Sparks, MD, USA) that contained 3.0% NaCl (final concentration) at 25 C.
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The non-adapted control and stress-adapted cells were harvested, resuspended in PBS with 0.25% NaCl which was lethal as determined in a preliminary experiment, and incubated at 25 C with shaking at 100 rpm. The surviving bacterial cells were determined at different intervals by the standard plate count method using Lauria broth agar (LA, Difco)-3.0% NaCl following serial dilution in PBS-3.0% NaCl and were subsequently incubated at 37 C for 16 h (Wong, Liu, & Chen, 2005) and the survival percentages were calculated.
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2.4. Statistical analysis All experiments were repeated three times and at least triplicate determinations of bacterial counts were performed in each of these repeats. Data of the adapted and non-adapted control cultures after challenging by lethal low salinity for 10e30 min were analyzed by performing ANOVA using SPSS for Windows 10.0 (SPSS Inc., Chicago, Ill, U.S.A.) at a significance level of p < 0.05.
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Fig. 1. Survival of heat-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted at 41 C for 20 min in AA-PBS-3.0% NaCl, and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
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Fig. 2. Survival of bile-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that contained 0.05% bile salts, and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
The low acidity (pH 4.48)-adapted cells, regardless of their growth phase, were not protected against lethal low salinity, and were rapidly inactivated and about 0.001% or 0.1% survived after 10 min treatment for the exponential phase cells or stationary phase cells, respectively (Fig. 3). Whereas the organic acid-adapted V. parahaemolyticus cells exhibited enhanced tolerance in the exponential phase cells challenged for 20e30 min, and significant increase of tolerance was only detected in the stationary phase cells challenged for 30 min (Figs. 4 and 5). The acetic acid-adapted cells at exponential phase exhibited significant protection against lethal low salinity, with survival increment of less than 90% as compared to the control cells (Fig. 4A). Response of the lactic acid-adapted cells was similar to those of acetic acid-adapted cells, and lactic acid-adapted exponential phase cells were significantly protected against lethal low salinity (Fig. 5). 4. Discussion Responses to hyperosmotic stress in a wide range of pathogenic bacteria, including several vibrios, namely, Vibrio alginolyticus (Xu et al., 2005), Vibrio cholerae (Pflughoeft, Kierek, & Watnick, 2003), V. parahaemolyticus (Xu, Ren, Wang, & Peng, 2004) and Vibrio vulnificus (Lee, Park, Lee, & Choi, 2003), have been examined. Nevertheless, the low salinity/hypoosmotic stress responses in vibrios have not been studied in detail. We explored the low salinity
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Fig. 3. Survival of acid-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that had been acidified to pH 4.48 using 1 N HCl, and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
response of V. vulnificus and showed that cross-protected against lethal low salinity was demonstrated in the bile-adapted exponential and stationary cells and not in the acid (pH 4.4)- and heat (41 C)-adapted cells (Wong & Liu, 2006). V. parahaemolyticus is phylogenetically close to V. vulnificus (Thompson, Iida, & Swings, 2004), but it exhibited some different features in response to low salinity stress. In this study, enhanced tolerance against lethal low salinity was found in the heat- (Fig. 1), bile- (Fig. 2) and organic acid-adapted V. parahaemolyticus cells (Figs. 4 and 5). Bile was the most efficient stress to induce such response than heat and organic acids in V. parahaemolyticus (Fig. 2). Another observation in this study is that the V. parahaemolyticus cells at stationary phase were more resistant to environmental stressors and less responsive to the sublethal stress adaptation as compared to those cells at exponential phase (Figs. 1e5), and this is a common phenomenon associated with the function of RpoS. The cellular RpoS level is regulated by integrated multiple signals ivolving the transcriptional and translational control of rpoS and the RpoS proteolysis. The transcription of rpoS is known to be increased in cells which are shifting from exponential phase to stationary phase with a reduced growth rate or being challenged by environmental stressors (Dodd & Aldsworth, 2002; Hengge-Aronis, 2002; Hirsch & Elliott, 2005). Stresses that activate the transcription of rpoS in exponential phase cells are reasonably less effective in stationary phase cells in which rpoS is already at raised level.
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Time, min Fig. 4. Survival of acetic acid-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that contained 0.05% acetic acid (pH 4.8), and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
Tolerance to low salinity/hypoosmotic stress involves a combination of specific (secondary transport), non-specific and rapid (stretch-activated mechanosensitive channels, MscL and MscS) solute efflux and aquaporin-mediated water efflux (Sleator & Hill, 2001). The expression of the mechanosensitive channels in Eschereichia coli under hypoosmotic conditions is at least partially regulated by RpoS (Stokes et al., 2003), and the significant role of RpoS was also demonstrated in the low salinity response of V. vulnificus (Tan, Liu, Oliver, & Wong, 2010). While RpoS generally regulates various genes in response to a variety of environmental stressors (Bearson, Benjamin, Jr., Swords, & Foster, 1996; Rosche, Smith, Parker, & Oliver, 2005) and the function of rpoS has also been demonstrated in V. parahaemolyticus (Vasudevan & Venkitanarayanan, 2006). These results suggest that stresses which can activate RpoS of V. parahaemolyticus will consequentially affiliate the tolerance of this pathogen against low salinity. Role of RpoS in the adaptation to bile has been demonstrated in Salmonella enterica (Prouty et al., 2004) and V. vulnificus (Chen, Oliver, & Wong, 2010). Also, bile-adaptation is known to promote the production of 18 common stress proteins, including DnaK and GroEL, in Enterococcus faecalis (Flahaut et al., 1996). Bile adaptation also activates porins and other efflux pumps in Campylobacter jejuni (Lin, Sahin, Michel, & Zhang, 2003), E. coli (Rosenberg, Bertenthal, Nilles, Bertrand, & Nikaido, 2003) and V. cholerae (Chatterjee,
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Fig. 5. Survival of lactic acid-adapted Vibrio parahaemolyticus ST550 against challenge of lethal low salinity. Cells in exponential phase (panel A) or stationary phase (panel B) were adapted for 20 min in AA-PBS-3.0% NaCl that contained 0.5% lactic acid (pH 4.8), and then challenged by PBS-0.25% NaCl for different intervals. B, non-adapted cells; C, adapted cells. Vertical bars represent the standard deviations of the means.
Chaudhuri, Saha, Gupta, & Chowdhury, 2004). Accordingly, cells that have adapted to bile may be equipped with better protective machinery (porins and efflux pumps) than control cells, and this machinery participates in the immediate solute and water efflux under low-salinity stress. In conclusion, this work revealed that bile in addition to heat and organic acids were able to induce tolerance against lethal low salinity in V. parahaemolyticus. Acknowledgements The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC95-2313-B-031-001 and NSC 962313-B-031-001. Ted Knoy is appreciated for his editorial assistance. References Bearson, S. M., Benjamin, W. H., Jr., Swords, W. E., & Foster, J. W. (1996). Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium. Journal of Bacteriology, 178, 2572e2579. Chatterjee, A., Chaudhuri, S., Saha, G., Gupta, S., & Chowdhury, R. (2004). Effect of bile on the cell surface permeability barrier and efflux system of Vibrio cholerae. Journal of Bacteriology, 186, 6809e6814.
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