| EnterohemorrhagicEscherichia coli(EHEC)is an importantZoonotic and food borne pathogen,and O157 is the predominant serotype.EHECcause human diarrhea,hemorrhagic enteritis,hemorrhagic colitis(HC),thrombotic thrombocytopenic purpura(TTP),hemolytic uremic syndrome(HUS),which could lead to over 30%fatality rate.EHEC 0157was first identified in USA in 1982,followed by Britain,Germany,Japan,Canada,Australia and China,which seriously threatened the food safety and public health.E.coli O157 can cause diarrhea disease in cattle,pigs,chickens and other livestock and poultry,leading to reduction in the productivity and quality of the products.Evidence indicated that cattle,sheep,pigs and poultry and their feces are the reservoirs of E.coli 0157,whichcould contaminate food by various ways.Humans are exposed to E.coli O157 by ingesting contaminated foods,such as unpasteurized dairy products and incompletely cooked meats,severely harming food security and public health security.Therefore this study investigated the distribution and characteristics ofE.coli O157 from animal fecal and food samplesin JiangSu province,to elucidate the extent of food contaminationby animal feces.Animalsinfected with E.coli O157:H7 by ingesting contaminated feeds,causing disease and food contamination.The conventional bacteriological method to detect EHEC O157:H7 in feeds is time-consuming,high-cost,and of low detection rate.Consequently,it is necessary to develope a rapid,sensitive and effective method for detection of E.coli O157:H7 in feeds.In this study,we have developed a duplex PCR method to detect theE.coli O157:H7 in feeds.1.Characterization of E.coli O157 from pig and poultry in JiangsuTo determine the extent of pork and chicken contamination by E.coli O157in Jiangsu Province,a total of 700 animal fecal and food samples were screened forE.coli O157 through selective culture medium and PCR for O157 lipopolysaccharide O-antigen synthesis gene rfbE.Then,phylogenetic group,virulence genes distribution,biofilm formation,and antibiotic sensitivity of each isolates were determined.E.coli O157 was detected in 2.6%(13/500)animal fecal samples and 1.5%(3/200)food samples.Phylogenetic typing showed most isolates were classified to D group,and 68.75%(11/16)isolates could produce biofilm.The stx1?stx2?hlyA?eae gene present in 25%?37.5%?68.75%?63.16%of the O157 isolates,respectively.Moreover,the isolates from animal feces and food possessed the same gene combination.Antibiotic sensitivity test indicated more than 56%E.coli O157 isolates were resistant to common antibiotics.Moreover,the multi-drug resistance strains have been very popular,and all isolates were even resistant to 7 different antibiotics.The results of antibiotic resistant gene detection showed that 50%?63%isolates harbor the antibiotic resistant genetetA?TEM?floR?cmlA?aph3?aadAl?aadA2,whereas,the prevalence of other antibiotic resistant genes is less 30%.Our results indicated that pig and poultry feces serve as reservoirs for pork amd chicken contamination by E.coli O157,which was a potential harm to human health.2.Development of duplex PCR method for detecting E.coli O157:H7 in feedsThis study developed a duplex PCR method to detect the E.coli O157:H7in feeds.Two pairs of primers were designed based on therfbE and fliCgenes,encoding O and H antigen,repespectively.The PCR results showed that rfbE and fliCgenes wasspecificly amplified in theduplex PCR.The minimum detection limit of duplex PCR was 100 CFU bacteria.The feeds were artificially-contaminated with E.coli O157:H7,selective cultured,and then detected by the duplex PCR method.The results showed that the duplex PCR could specific detect the E.coli O157:H7.The detection limit could be improved to 20 CFU/g feeds if combined with further incubation for 4 h.These results suggest that the duplex PCR developed in this study could efficiently detect theE.coli O157:H7 contaminated in feeds,which was a useful and rapid technique for epidemiological investigation. |
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