山东省某地区奶牛源大肠埃希菌的血清型、耐药特性及分子特性

Serotyping, drug resistance and molecular characteristics of Escherichia coli from dairy cows in a region of Shandong province

摘要:

[目的] 旨在对从山东省某地区4个健康奶牛养殖场分离到的大肠埃希菌进行优势血清型、耐药特性、Ⅰ类整合子基因盒携带情况以及系统进化群分析。[方法] 采集194份来自山东省某地区4个规模化奶牛场奶牛新鲜粪便样品，进行大肠埃希菌分离和鉴定，利用常用大肠埃希菌诊断血清进行血清型鉴定；利用10%的绵羊血平板检测溶血性；利用K-B法检测对14种常规抗菌药物的敏感性；利用聚合酶链式反应（PCR）检测革兰阴性菌常见的6大类24种耐药基因、Ⅰ类整合子基因盒结构并对目的条带测序分析；利用细菌多位点序列分型（Multilocus sequence typing，MLST）技术分析大肠埃希菌的ST型并使用eBURST v3软件分析菌株之间的克隆关系。[结果] 从194份新鲜粪便样品中分离到171株大肠埃希菌，其中主要为致病性（19.9%）和侵袭性大肠埃希菌（17.0%），优势血清型分别为O128：K67（12/171）和O143：K7（12/171）。另外，具有溶血性的大肠埃希菌阳性率为9.4%（16/171）；药敏试验结果显示多重耐药菌株的比率为22.2%，其中对氨苄西林耐药率最高为33.9%，四环素次之，为24.0%；PCR检测耐药基因和整合子结果显示，59.1%的菌株携带β-内酰胺类耐药基因bla_TEM，59.1%的菌株携带氨基糖苷类耐药基因ant（2'），未检测到四环素耐药基因tetA和tetB；Ⅰ类整合子的阳性率为4.1%（7/171），dfrA12-aadA2-sul1为优势基因盒结构（4/171）；MLST将大肠埃希菌分为8种ST型，其中，ST155（10/171）和ST58（45/171）形成一个克隆复合物且没有发现新的ST型。[结论] 本研究证实，从该地区规模化健康奶牛场新鲜粪便中分离到的大肠埃希菌优势血清型为O128：K67和O143：K7；少部分大肠埃希菌具有溶血性；仅对氨苄西林、四环素等具有较高的耐药率；优势基因盒结构为dfrA12-aadA2-sul1；MLST分型显示不同奶牛场分离出亲缘关系较近的菌株，其分布具有多态性，血清型与ST型之间无相关性。本研究表明源自表观健康的奶牛的大肠埃希菌存在多重耐药现象，具有食品公共卫生安全隐患，该研究对于提升规模化奶牛场奶制品的安全生产与质量评估具有一定的理论指导意义。

Abstract:

[Objective] To analyze the dominant serotype, class I integrons gene cassette, drug resistance characteristics and phylogenetic of Escherichia coli isolated from four large-scale healthy dairy farms in Shandong Province.[Methods] We collected 194 samples of fresh feces from dairy cows in 4 large-scale dairy farms. The isolation and identification of E. coli was tested by using laboratory method, and the serotype was identified by the E. coli diagnostic serums commonly used. The hemolysis of E. coli was detected by 10% sheep blood plate while the sensitivity of 14 conventional antibiotics was detected by K-B method and the ST type of E. coli was analyzed by MLST technique and the cloning relationship among the strains was analyzed by eBURST v3 software.[Results] The results showed that 171 strains of E. coli were isolated from 194 fresh feces samples, mainly pathogenic E. coli (19.9%) and invasive E. coli (17.0%), and the dominant serotypes were O128:K67 (12/171) and O143:K7 (12/171). In addition, the positive rate of hemolytic E. coli was 9.4% (16/171) and the drug susceptibility test showed that the ratio of multi-drug resistant strains was 22.2%, among which the highest resistance rate to ampicillin was 33.9%, followed by tetracycline, which was 24.0%. The results of PCR detection of drug resistance genes and integrons showed that 59.1% of the strains carried the β-lactam resistance gene bla_TEM, 59.1% of the strains carried the aminoglycoside resistance gene ant(2'), and no tetracycline resistance gene (tetA and tetB) was detected. The positive rate of integron I was 4.1% (7/171), dfrA12-aadA2-sul1 was the dominant gene cassette structure (4/171); MLST divided E. coli into 8 ST types, of which ST155 (10/171) and ST58 (45/171) formed a clonal complex and no new ST type was found.[Conclusion] This study confirmed that the dominant serotypes of E. coli isolated from fresh feces in large-scale healthy dairy farms in the region were O128:K67 and O143:K7. Some E. coli were hemolytic and the resistance rate was only high in ampicillin and tetracycline. Moreover, the dominant gene cassette structure is dfrA12-aadA2-sul1. The MLST classification showed that the isolates from different dairy farms were closely related, indicating that the distribution of E. coli in cattle origin was polymorphic, and there was no correlation between serotype and ST type. There is a multi-drug resistance phenomenon in E. coli from dairy cows which is potential dangers of food public safety. This research has certain theoretical guiding significance for improving the safe production and quality evaluation of dairy products in large-scale dairy farms.