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

Abstract

†Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. 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, p<0.05) was also observed after 4 weeks. However, in spinach, the populations after 4 weeks at 8C were not significant (3.9 vs. 3.3 log CFU/g). Starvation or chlorine stress induced higher growth in non-O157 than O157 in ground beef (4.5 vs. 1.2 log CFU/g) and spinach (4.7 vs. 2.5 log CFU/g). At 12 and 16C, cell populations of stressed and nonstressed O157 or non-O157 STEC were not significantly (p>0.05) 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.