牛源性非O157大肠杆菌的分离与鉴定

Isolation and Identification of Bovine-Derived Non-O157 Escherichia coli Strains

目的:对牛粪及牛肉中的非O157大肠杆菌进行分离、鉴定和毒力基因携带情况进行检测,以了解非O157大肠杆菌的污染状况。方法:参考USDA检测方法,对样品进行选择性增菌,用多重聚合酶链式反应(polymerase chain reaction,PCR)方法进行初步筛选,检测O抗原(O157、O121、O111、O103、O26),阳性样本用选择性显色培养基rainbow agar分离纯化,可疑菌株用多重PCR方法鉴定O抗原,PCR-限制性片段长度多态性鉴定H抗原,并采用血清凝集实验进行验证,确认的阳性菌株采用多重PCR方法进行毒力基因(stx1、stx2、eae、hly)检测。结果:在153份牛粪和49份牛肉样本中,共40份样品检出1个或多个O血清型阳性,牛粪检出率高于牛肉,非O157阳性率为19.3%,O157的阳性率为0.50%;经分离纯化后,共鉴定出阳性菌株30株,其中O26最多占73.3%,O121、O103、O157分别占16.7%、6.7%、3.3%。毒力基因检测结果显示,分离自牛肉的2株O26:H11,一株stx1、hly阳性,另一株hly阳性,分离自牛粪的1株O26:H11携带hly基因,因此30株菌株中带毒菌株阳性率为10.0%,非O157产志贺毒素大肠杆菌阳性率为3.4%。结论:牛粪和牛肉中非O157大肠杆菌的污染率明显高于O157,尤其是O26:H11血清型最高,且检出含志贺毒素基因的大肠杆菌,这提示零售牛肉市场存在非O157大肠杆菌污染的安全风险,我国应该加强对非O157大肠杆菌的检测和监控。

Objectives:The aims of this study were to isolate and identify non-O157 Escherichia coli strains from bovine fecal and beef samples and to detect the virulence genes of the isolates,in order to understand the contamination status of non-O157 E.coli.Methods:After selective enrichment of beef and fecal samples by the USDA method with slight modifications,multiplex PCR was used as a prescreening method to determine the O-groups(O157,O121,O111,O103 and O26).The enriched positive sample was streaked for isolation and purification on chromogenic rainbow agar,and tested for the O-groups.The flagellar antigens were identified by PCR-restriction fragment length polymorphism(PCR-RFLP)and the positive isolates were further verified by means of serum agglutination test.The virulence genes(stx1,stx2,eae,and hly)of the positive isolates were detected by PCR.Results:A total of 153 fecal samples and 49 beef samples were collected.Overall,40 samples were tested positive for one O-group,and the detection rate in feces was higher than that in beef,19.3%of which were tested positive for non-O157,and 0.50%positive for O157.After purification,30 positive isolates were identified.O26 was detected most frequently,accounting for 73.3%of the total isolates,followed by O26,O121,O103,and O157,accounted for 73.3%,16.7%,6.7%and 3.3%,respectively.The results of virulence gene analysis showed that two strains of O26:H11 were isolated from beef,one being positive for stx1 and hly and the other being positive for hly.One strain of O26:H11 harboring the hly gene was obtained from feces.Therefore,10.0%of the 30 strains were positive for virulence genes,and 3.4%were non-O157 STEC.Conclusion:The contamination rate of non-O157 E.coli,especially O26:H11,in feces and beef was significantly higher than that of O157.Shiga toxin-producing E.coli was even detected in the beef samples,which suggested that there is a potential risk of non-O157 E.coli contamination in the retail beef market.The detection and monitoring of non-O157 E.coli should be strengthened in China.