肉类及蔬菜食品中EHEC O157污染分布及分型研究

Prevalence and ERIC−PCR Typing of Enterohemorrhagic Escherichia coli O157 in Meat and Vegetables

摘要:

为深入了解肉类及蔬菜中肠出血性大肠埃希氏菌（EHEC O157）的污染分布规律和遗传多样性，随机采集全国18个城市的食品样品，参考GB/T 4789.36-2008方法进行样品处理，并用rfbE／fliCH7双重PCR对菌株进行鉴定；分别采用单重PCR和ERIC-PCR对菌株进行毒力基因（eae、hlyA、stx1和stx2）检测和分子分型。结果表明，414份样品中检出18份EHEC O157阳性样品，总污染率4.35%，其中生鲜肉12份，速冻肉6份，蔬菜未检出；经过生化、血清鉴定和双重PCR检测，鉴定出52株EHEC O157，包括29株O157: H7，3株O157:NM（fliCH7 +，无动力），2株O157:NM（fliCH7 -，无动力）和18株O157:hund（未确定H型）；毒力基因检测结果发现，52株菌株中有50株携带毒力基因，其中40株（76.92%）携带eae，31株（59.62%）携带hlyA，20株（38.46%）携带stx1，24株（46.15%）携带stx2。ERIC-PCR分型结果表明，在相似系数为0.80时，菌株可分为12个聚类簇，F型为其主要基因簇，分离株的基因型呈现多样性。

Abstract:

The aim of this study was to investigate the prevalence and genotypic diversity of enterohemorrhagic Escherichia coli O157 (EHEC O157) in meat and vegetables. Food samples were collected randomly from 18 cites in China and processed according to the GB/T 4789.36-2008method with slight modifications. The isolates were identified by duplex PCR targeting the rfbE and fliCH7 genes. The presence of four virulence genes (eae, hlyA, stx1, and stx2) in EHEC O157 was determined by single-PCR. Additionally, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) was used for molecular typing of EHEC O157 isolates. The results showed that 18 (4.35%) of 414 samples were positive for EHEC O157, including 12 fresh meat samples and 6 frozen meat samples. None of the vegetable samples was positive. Furthermore, a total of 52 isolates were identified as EHEC O157 by serotyping, biochemical tests, and duplex-PCR, of which 29 strains were O157:H7, 3strains were O157:NM (carried fliCH7 gene, non-mobile), 2 strains were O157:NM (without fliCH7 gene, non-mobile), and 18strains were O157:hund (undetermined H type). The distribution of virulence genes indicated that 50 out of 52 EHEC O157 isolates carried at least one virulence gene,40 (76.92%) isolates possessed eae gene, 31(59.62%) isolates harbored hlyA gene, 20 (38.46%) isolates harbored stx1 gene, while 24 (46.15%) isolates harbored stx2 gene. ERIC-PCR molecular fingerprint clustering analysis showed that, when the similarity coefficient was 0.80, the strains could be divided into 12 types, and F type was the dominant gene type, indicating a high diversity of genes in these isolates.