Distribution And Biological Characteristics Of STEC In Foods From Some Markets In Wuhan

Shiga-toxin producing Escherichia coli (STEC) are considered as important emerging food-borne bacterial pathogens of public health, which are able to cause infective epidemic cases. The infection of clinical cases caused by the bacteria have increased recently. This study aimed at investigating the epidemic distribution of STEC from commercial foods in Wuhan. At the same time, the virulence genes, Shiga toxin titers, biofilm formation and drug resistance of the isolated STEC were detected to study their biological characteristics.A total of 161 food samples were collected from 22 markets and supermarkets in Wuhan.Thirty five samples were confirmed to be contaminated with O157 STEC by PCR screening. Meanwhile,O45 STEC was detected in 4 samples.O145 STEC and O111 STEC were also detected in one sample, respectively. Among the 55 samples contaminated with STEC,1.8% samples only detected stx1 gene,74.5% samples detected stx2 gene,23.6% samples detected both stx1 and stx2 genes. None of the samples contained stx and non-0157 markers possess hly gene, while 27 samples contaminated with 0157 STEC were tested eae-positive. This study also indicated that O157 STEC were frequently present in pork products, while non-O157 STEC were prevalent in beef products; The results also showed that STEC were seldom detected from chicken samples.None of the samples from fish and vegetable were detected stx-positive.Then the 27 samples, which were eae-positive, were performed immuno-magnetic isolation, serological identification and colony PCR assay. Finally five O157 strains were isolated. Among the five O157 strains, four strains carried eae、 hly and ehxA genes. One strain EC5.11 only carried stx2 gene, while the left three isolates carried both stxl and stx2 genes. One strain EC9.102 carried none of the genes mentioned above. EC9.102 only carried rfbo157 gene. The left four isolates were both positive for rfbo157 and fliCH7 genes, indicating that they were O157:H7 serotypes.The five 0157 isolates were measured Stx production by Vero cell cytotoxicity assay. The results showed that Vero cells didn’t have significant morphological changes when treated with EC9.102 bacterial filtrate, while the left 4 isolates had detectable cytotoxicity effects. EC9.101 and EC9.12 demonstrated high toxicity to Vero cells similar to that of the positive control strain EC150 (1×104 CD50/mL).The stx genes of the 4 O157:H7 isolates were further subtyped by PCR using sequence-specific primers targeting each stx variants. The results showed that the prevalent stx genotype were stxla and stx2a. One isolate EC 5.11 carried stx2 gene but can not be subtyped by PCR. The entire stx2 gene was then amplified from EC 5.11, cloned into a vector and sequenced. Sequencing data showed that the stx2 gene of EC5.11 was 100% identical to the published sequences of stx2c. At the same time the q genes of the isolates was tested. EC5.11 was confirmed to contain q21 gene and the other three contained q933 genes.The drug resistance of the four O157:H7 isolates were tested by K-B disk diffusion method. The results showed that cefotaxime, gentamicin, chloramphenicol, ciprofloxacin, cefepime, new neomycin, ampicillin had good inhibitory effects on the four O157:H7 isolates.The O157:H7 isolates were sensitive to Tetracycline and polymyxin B except for EC 9.12.EC9.12 was presented intermediary reactions to these two antibiotics.Two selective plates were designed to isolate the non-0157 STEC in this study. The results indicated that the colonial morphology of non-0157 STEC was similar to 0157 strains.Four O157:H7 isolates were isolated in this study. EC5.11 carried stx2c gene. And stx2c genotype had been confirmed to be highly pathogenic. It proved that the virulence trendency of 0157 had changed in Wuhan and there was a high risk in commercial foods in Wuhan. Among the non-0157,045 STEC was the most common. This result was quite different from other publications. Intensive studies on non-0157 STEC should be implemented to get comprehensive data for the prevention and control of STEC in Wuhan.