Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2007
Publication Date: 2/1/2008
Citation: Uhlich, G.A., Sinclair, J.R., Warren, N.G., Chmielecki, W.A., Fratamico, P.M. 2008. Characterization of Shiga toxin-producing Escherichia coli associated with two multi-state foodborne outbreaks in 2006. Applied and Environmental Microbiology. 74:1268-1272. Interpretive Summary: In the fall of 2006 two multi-state outbreaks of Shiga toxin-producing E. coli (STEC) serotype O157:H7 infection occurred that involved contaminated spinach and contaminated lettuce. In this study, we compare seven STEC isolates associated with those two outbreaks to a diverse but limited collection of ten food, environmental, and animal STEC isolates for genetic and/or phenotypic differences in virulence, stress tolerance, and biofilm formation. Six of the outbreak isolates were isolated from either produce or from patients that consumed the contaminated produce, and all were serotype O157:H7, the most common STEC serotype associated with foodborne outbreaks. The seventh isolate (serotype O-:H4) was cultured from the same bag of spinach that contained one of the six serotype O157:H7 isolates. When all seventeen strains were compared for the presence of genes responsible for virulence in E. coli, there were very few differences among the outbreak strains and the other STEC strains. In addition, there were very few differences among the outbreak strains and the other STEC isolates in their resistance to heat, acid stress, and biofilm formation. These results suggest that STEC strains that originated from produce do not alter their gene expression in order to persist in the environment. Sequencing of the Shiga toxin genes of the six serotype O157:H7 isolates from the two different outbreaks showed that all of the strains were 100% identical. These results together with the pulsed field gel electrophoresis fingerprinting results, which showed that the strains had very similar patterns, suggests that the strains from the two outbreaks may be closely related or have a common origin. Biofilm studies showed that only the serotype O-:H4 isolate, but none of the serotype O157:H7 strains, was able to form a biofilm on glass. However, when the serotype O-:H4 isolate and the serotype O157:H7 isolate that originated from the same spinach bag were cultured together in biofilm studies the serotype O157:H7 strain was able to form greater amounts of biofilm than the serotype O-:H4 isolate. This suggests that strains of E. coli that co-contaminate food products along with serotype O157:H7 strains, may enhance the biofilm forming abilities of the serotype O157:H7 strains and allow for their persistence on food products.
Technical Abstract: In the fall of 2006 two multi-state outbreaks of E. coli serotype O157:H7 infection occurred that involved contaminated spinach and contaminated lettuce. In this study, we compare 7 Shiga toxin-producing isolates associated with those two outbreaks to a collection of food, environmental, and animal isolates for genetic and/or phenotypic differences in virulence, stress tolerance, and biofilm formation. Sixteen of the isolates examined were serotype O157:H7, and all were positive by PCR for hly, eae, wyzO157, and either stx1, stx2 or both toxin genes. An E. coli serotype O-:H4 strain isolated from a spinach bag during the spinach-associated outbreak was negative for all tested virulence genes except for stx1. Sequencing of the Shiga toxin genes from serotype O157:H7 isolates associated with the two 2006 outbreaks revealed that all contained both a stx2 and a stx2c variant gene but not stx1. The acid tolerance and heat inactivation profiles of all 17 strains were comparable except for a serotype O157:H7 calf isolate that showed reduced acid tolerance. Biofilm assays showed that the O-:H4 strain, but none of the serotype O157:H7 strains, bound Congo red dye and formed a biofilm. However, mixed strain biofilm studies indicated that a serotype O157:H7 strain that was co-isolated from the same spinach bag as the O-:H4 strain showed increased persistence on glass when incubated together with the O-:H4 strain. These results indicate that the serotype O157:H7 isolates from produce did not show increased resistance to stress, genotypic differences in virulence, or differences in biofilm formation compared to isolates from other sources. Moreover, there were no genetic or phenotypic differences between the patient and produce isolates from paired isolates. However, this study is the first to indicate a possible cooperative mechanism for biofilm formation between a serotype O157:H7 strain and a non-O157:H7 strain of E. coli.