城市景观水体头孢噻肟耐药菌的β-内酰胺酶类抗性基因接合转移特性

Characterization of conjugative transfer of β-lactamase resistance genes in cefotaxime-resistant bacteria from urban landscape waters

抗生素抗性基因的水平转移是细菌耐药性传播的重要方式.为了解城市景观水体中细菌的抗生素抗性基因水平转移状况,利用从西安市5处景观水体分离得到的216株头孢噻肟耐药菌作为备选供体菌,以大肠杆菌NK5449作为受体菌进行接合试验,测定接合转移频率.采用PCR技术分析可发生接合转移的供体菌株质粒上3种β-内酰胺酶类抗性基因(blaCTX-M、blaTEM、blaSHV)和I型整合子整合酶基因(intI)的分布.同时,采用RT-PCR技术确定这些供体菌株中编码外膜蛋白基因(ompA、ompC、ompF)和接合转移相关基因(trbC、traA、trbBp、traF、trfAp、traJ、korA、korB、trbA)的mRNA表达情况.结果表明,共筛选出12株可发生接合转移的头孢噻肟耐药菌,分别被鉴定为大肠杆菌、蜡状芽孢杆菌和维氏气单胞菌,接合转移频率为3.95×10^-8~3.07×10^-3.在这12株供体菌株的质粒上,β-内酰胺酶类抗性基因blaCTX-M、blaTEM、blaSHV的检出率分别为58.33%、58.33%、8.33%,I型整合子的检出率为100%.外膜蛋白基因(ompA、ompC、ompF)、性菌毛编码基因(trbC)和供受体菌接合配对形成调控基因(trbBp)在这些供体菌中mRNA表达活跃.

The horizontal transfer of antibiotic resistance genes is an important way to spread antibiotic resistance. In order to understand the characteristics of horizontal transfer of antibiotic resistance genes of bacteria in urban landscape waters, the conjugation experiments with 216 cefotaxime-resistant strains isolated from five urban landscape waters in Xi′an city as candidate donor strains and Escherichia coli NK5449 as the recipient bacteria were conducted to determine the conjugative transfer frequency. PCR was used to analyze the distribution of three β-lactamase resistance genes(blaCTX-M, blaTEM, blaSHV) and class I integron integrase gene(intI) on the plasmid of the donor strains. Using RT-PCR technology, mRNA expression of outer membrane genes(ompA, ompC, ompF) and conjugative transfer-related genes(trbC, traA, trbBp, traF, trfAp, traJ, korA, korB, trbA) were detected in these donor strains. The results showed that conjugative transfer was observed in a total of 12 cefotaxime-resistant strains, which were identified as Escherichia coli, Aeromonas veronii and Bacillus cereus, respectivley. The conjugative transfer frequency ranged from 3.95×10^-8 to 3.07×10^-3. The detection rates of three β-lactamase resistance genes blaCTX-M, blaTEM and blaSHV on the plasmids of the 12 donor strains were 58.33%, 58.33% and 8.33%, as well as class I integron were 100%. Outer membrane protein genes(ompA, ompC, ompF), sex pilus-coding gene(trbC) and mating pair formation gene(trbBp) showed active mRNA expression in these donor strains.