SDRP Journal of Food Science & Technology

Effects of Stresses on the Growth and Cytotoxicity of Shiga-Toxin Producing Escherichia coli in Ground Beef and Spinach

Co-Authors

Byong Kwon Yoo, Yanhong Liu, Vijay Juneja, Lihan Huang, Cheng-An Hwang

Citation

Cheng-An Hwang, Effects of Stresses on the Growth and Cytotoxicity of Shiga-Toxin Producing Escherichia coli in Ground Beef and Spinach(2016)SDRP Journal of Food Science & Technology 1(1)

Abstract

The objectives of this study were to examine the effect of stresses on the growth and cytotoxicity of pathogenic Escherichia coli in beef and spinach. A mixture of three strains of Shiga toxin-producing E. coli (STEC) O157:H7 or 4 strains of non-O157 STEC, O26:H11, O103:H1, O104:H4, and O145:NM, was subjected to stress of 2 ppm chlorine, aw 0.97, pH 5, or 15-day starvation. Stressed or non-stressed STEC was inoculated into 5 g of irradiated ground beef or spinach. The cell populations during storage at 8, 12, or 16?C for 4 weeks were compared to evaluate the growth variation between O157 and non-O157 STEC. Supernatant from each sample after 24-h incubation at 22?C was used to determine Vero-cytotoxicity using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS] or lactate dehydrogenase (LDH) assay to evaluate the effects of stresses on the cytotoxicity exhibited by STEC. After one week at 8?C, the population of non-stressed non-O157 (3.1 log CFU/g) was significantly (p<0.05) higher than O157 (1.9 log CFU/g) in ground beef, and the difference in populations (3.9 vs. 1.7 log CFU/g, p0.05) different in beef and spinach. MTS assay showed that stressed O157 and non-O157 STEC exhibited significantly (p<0.05) higher cytotoxicity than the non-stressed controls. The numbers of surviving Vero cells were 47-52% (stressed) vs. 65% (control) in beef and 20-30% (stressed) vs. 52-53% (non-stressed) in spinach. Similarly, LDH assay also indicated an increased cytotoxicity (p<0.05) in stressed O157 and non-O157 STEC than non-stressed controls in spinach. There was no significant difference among the four stresses in inducing the levels of cytotoxicity in O157 or non-O157 STEC. Results showed that STEC cells exposed to sub-lethal stresses might have increased cytotoxicity during subsequent growth in ground beef or spinach. The findings illustrate the importance of applying suitable control measures to eliminate the presence of stressed STEC in beef and spinach processing environment or their subsequent contamination in the products.

References

  1. Allen, K.J., Lepp, D., McKellar, R.C., Griffiths, M.W., 2008. Examination of stress and virulence gene expression in Escherichia coli O157:H7 using targeted microarray analysis. Foodborne Pathogens and Disease 5, 437-447. PMid:18713062

    View Article      PubMed/NCBI     

  2. Arechabala, B., Coiffard, C., Rivalland, P., Coiffard, L.J., de Roeck-Holtzhauer, Y., 1999. Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH release. Journal of Applied Toxicololgy 19, 163-165. 1099-1263(199905/06)19:3<163::AID-JAT561>3.0.CO;2-H

    View Article           

  3. Berridge, M.V., Herst, P.M., Tan, A.S., 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review 11, 127-152. 11004-7

    View Article           

  4. Beuchat, L.R., 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection 4, 413-423. 01555-1

    View Article           

  5. Bosilevac, J.M., Koohmaraie, M., 2011. Prevalence and characterization of non-O157 Shiga toxin-producing Escherichia coli isolates from commercial ground beef in the United States. Applied and Environmental Microbiology 77, 2103-2112. PMid:21257806 PMCid:PMC3067332

    View Article      PubMed/NCBI     

  6. Buttke, T.M., McCubrey, J.A., Owen, T.C., 1993. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. Journal of Immunological Methods 157, 233-240. 90092-L

    View Article           

  7. Chagnot, C., Caccia, N., Loukiadis, E., Ganet, S., Durand, A., Bertin, Y., Talon, R., Astruc, T., Desvaux, M., 2014. Colonization of the meat extracellular matrix proteins by O157 and non-O157 enterohemorrhagic Escherichia coli. International Journal of Food Microbiology 188, 92-98. PMid:25090606

    View Article      PubMed/NCBI     

  8. Cutter, C.N., Rivera-Betancourt, M., 2000. Interventions for the reduction of Salmonella Typhimurium DT 104 and non-O157:H7 enterohemorrhagic Escherichia coli on beef surfaces. Journal of Food Protection 63, 1326-1332. PMid:11041130

    View Article      PubMed/NCBI     

  9. Fotakis, G., Timbrell, J.A., 2006. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters 160, 171-177. PMid:16111842

    View Article      PubMed/NCBI     

  10. Galluzzi, L., Aaronson, S.A., Abrams, J., Alnemri, E.S., Andrews, D.W., Baehrecke, E.H. et al., 2009. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death and Differentiation 16, 1093-1107. PMid:19373242 PMCid:PMC2757140

    View Article      PubMed/NCBI     

  11. Harris, J.N., Robinson, P., Lawrance, J., Carrington, B.M., Hopwood, P., Dougal, M., Makin, W., 2003. Symptoms of colorectal liver metastases: correlation with CT findings. Clinical Oncology 15, 78-82.

    View Article           

  12. Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Schmidt, J.W., Wang, R., Shackelford, S.D., Wheeler, T.L., 2012. Evaluation of commonly used antimicrobial interventions for fresh beef inoculated with Shiga toxin-producing Escherichia coli serotypes O26, O45, O103, O111, O121, O145, and O157:H7. Journal of Food Protection 75, 1207-1212. PMid:22980002

    View Article      PubMed/NCBI     

  13. Kolling, G.L., Matthews, K.R., 2007. Influence of enteric bacteria conditioned media on recovery of Escherichia coli O157:H7 exposed to starvation and sodium hypochlorite. Journal of Applied Microbiology 103, 1435-1441. PMid:17953554

    View Article      PubMed/NCBI     

  14. Liu, Y., Wang, C., Tyrrell, G., Li, X.F., 2010. Production of Shiga-like toxins in viable but nonculturable Escherichia coli O157:H7. Water Research 44, 711-718. PMid:19879621

    View Article      PubMed/NCBI     

  15. Niemira, B.A., Cooke, P.H., 2010. Escherichia coli O157:H7 biofilm formation on Romaine lettuce and spinach leaf surfaces reduces efficacy of irradiation and sodium hypochlorite washes. Journal of Food Science 75, M270-277. PMid:20629883

    View Article      PubMed/NCBI     

  16. Olaimat, A.N., Holley, R.A., 2012. Factors influencing the microbial safety of fresh produce: a review. Food Microbiology 32, 1-19. PMid:22850369

    View Article      PubMed/NCBI     

  17. Olmez, H., Temur, S.D., 2010. Effects of different sanitizing treatments on biofilms and attachment of Escherichia coli and Listeria monocytogenes on green leaf lettuce. LWT Food Science and Technology 43, 964-970.

    View Article           

  18. Painter, J.A., Hoekstra, R.M., Ayers, T., Tauxe, R.V., Braden, C.R., Angulo, F.J., Griffin, P.M., 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998-2008. Emerging Infectious Diseases 19, 407-415. PMid:23622497 PMCid:PMC3647642

    View Article      PubMed/NCBI     

  19. Saldana, Z., Sanchez, E., Xicohtencatl-Cortes, J., Puente, J.L., Giron, J.A., 2011. Surface structures involved in plant stomata and leaf colonization by Shiga-toxigenic Escherichia coli O157:H7. Frontier Microbiology 2, 119. PMid:21887151 PMCid:PMC3157101

    View Article      PubMed/NCBI     

  20. Smith, S.M., Wunder, M.B., Norris, D.A., Shellman, Y.G., 2011. A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time. PLoS One 6(11): e26908. doi: 10.1371/journal.pone.0026908.

    View Article           

  21. Villegas, N.A., Baronetti, J., Albesa, I., Polifroni, R., Parma, A., Etcheverria, A., Becerra, M., Padola, N., Paraje, M., 2013. Relevance of biofilms in the pathogenesis of Shiga-toxin-producing Escherichia coli infection. Article ID 607258, The Scientific World Journal.

  22. Zaborina, O., Zaborin, A., Romanowski, K., Babrowski, T., Alverdy, J., 2011. Host stress and virulence expression in intestinal pathogens: development of therapeutic strategies using mice and C. elegans. Current Pharmaceutical Design 17, 1254-1260. PMid:21470113 PMCid:PMC4862579

    View Article      PubMed/NCBI     

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