水产食品源大肠杆菌耐药基因传播元件Ⅰ、Ⅱ、Ⅲ型整合子多样性分析

Diversity of class I,II,and III integrons associated with transmission elements of antibiotic resistance genes of Escherichia coli from aquatic food

【背景】整合子作为一种重要的耐药基因传播元件,可以通过位点特异性重组的方式捕获和表达耐药基因,在细菌耐药传播方面有着重要作用。因此,分析水产食品源大肠杆菌整合子携带情况,阐明整合子介导的大肠杆菌多重耐药现状,对水产养殖耐药监测和用药指导具有重要意义。【目的】分析水产食品源大肠杆菌中耐药基因传播元件Ⅰ、Ⅱ、Ⅲ型整合子的多样性。【方法】采集浙江省某农贸市场凡纳滨对虾、太平洋牡蛎和太平洋鲭鱼样品各160份,利用伊红美蓝选择性培养基和PCR法分离鉴定大肠杆菌,采用Kirby-Bauer纸片扩散法分析菌株对9类19种抗生素的耐药特征,并通过PCR法分析菌株携带的整合子及其基因盒多样性。【结果】各160份凡纳滨对虾、太平洋牡蛎和太平洋鲭鱼样品中依次分离到大肠杆菌15株、59株和26株,共计100株,大肠杆菌分离株多重耐药率依次为93.3%(14/15)、76.3%(45/59)和80.8%(21/26)。大肠杆菌分离株的I型整合子携带率为71.0%(71/100),Ⅱ型整合子携带率为5.0%(5/100),未检出Ⅲ型整合子。共分离到10种不同阵列的Ⅰ型整合子-基因盒和3种不同阵列的Ⅱ型整合子-基因盒,其中Ⅱ型整合子-基因盒dfrA1-catB2-sat2-aadA1为首次在大肠杆菌中发现。Pearson相关性分析显示,水产食品源大肠杆菌的整合子携带率与菌株多重耐药性之间具有显著正相关性(相关系数r=0.99,P<0.05)。【结论】本研究对进一步揭示整合子介导的水产食品源病原微生物耐药基因传播机制、促进水产养殖业健康发展具有一定意义。

[Background]Integrons,as key mobile genetic elements associated with the transmission of antibiotic resistance genes(ARGs),can capture and express ARGs through site-specific recombination,playing a role in the transmission of bacterial antimicrobial resistance(AMR).Therefore,it is of great significance to analyze the integron-carrying status of Escherichia coli from aquatic food and clarify the status of integron-mediated multi-drug resistance(MDR)of E.coli for AMR monitoring and antibiotic use guidance in aquaculture.[Objective]To analyze the diversity of class I,II,and III integrons of E.coli in aquatic food.[Methods]One hundred and sixty samples of Pacific white shrimp(Penaeus vannamei),160 samples of Pacific oyster(Crassostrea gigas),and 160 samples of Pacific mackerel(Pneumatophorus japonicas)were collected from a farmer’s market in Zhejiang province.E.coli was isolated and identified by Eosin Methylene Bule Agar and PCR method.The AMR characteristics of E.coli to 9 categories of 19 antibiotics were analyzed by the Kirby-Bauer disc diffusion method.The integrons and diversity of gene cassettes(GCs)carried by E.coli from aquatic products were analyzed by PCR.[Results]Fifteen,59,and 26 strains of E.coli were isolated from the samples of Pacific white shrimp,Pacific oyster,and Pacific mackerel,respectively,with a total of 100 strains.The MDR rates of E.coli isolates from Pacific white shrimp,Pacific oyster,and Pacific mackerel were 93.3%(14/15),76.3%(45/59),and 80.8%(21/26),respectively.The class I,II,and III integron-carrying rates of E.coli isolates was 71.0%(71/100),5.0%(5/100),and 0.0%,respectively.A total of 10 different class I integron GC arrays and three different class II integron GC arrays were detected.The class II integron GC array dfrA1-catB2-sat2-aadA1 was identified in E.coli for the first time.The Pearson correlation analysis showed that there was a positive correlation between the integron-carrying rate and MDR of E.coli from aquatic food(r=0.99,P<0.05).[Conclusion]This study has significance for revealing the integron-mediated transmission mechanism of ARGs of aquatic food-derived pathogenic microorganisms and promoting the healthy development of aquaculture.