广东省腹泻仔猪大肠杆菌blaCTX-M-65与mcr-1基因共传播特征

Co-transmission Characteristics of bla CTX-M-65 and mcr-1 Genes in Escherichia coli Isolated from Diarrhea Piglets in Guangdong Province

【目的】调查分析广东省某生猪养殖场腹泻仔猪的大肠杆菌耐药情况以及同时携带blaCTX-M-65与mcr-1两种耐药基因的阳性大肠杆菌的分子特征与其共同传播特征,为耐药性监测及风险防控提供科学依据。【方法】从广东省肇庆市某生猪养殖场采集腹泻仔猪直肠棉拭子样品90份,并进行大肠杆菌分离鉴定;采用PCR方法检测产超广谱β-内酰胺酶(ESBLs)与mcr-1基因,通过测序确定CTX-M亚型;采用琼脂二倍稀释法和微量肉汤稀释法测定分离菌对12种抗菌药物的最小抑菌浓度(MIC);使用脉冲场凝胶电泳(PFGE)及多位点序列分型(MLST)分析菌株间的遗传背景;通过接合转移方法确定质粒水平转移的能力;使用复制子分型、S1-PFGE、Southern blotting方法检测质粒类型、耐药基因的定位及定位质粒的大小;通过三代测序及序列分析确定质粒的重组过程。【结果】共分离鉴定86株大肠杆菌,检测到44株携带CTX-M型ESBLs基因,其中blaCTX-M-55基因最流行(n=16,36.4%),其次是blaCTX-M-65(n=10,22.7%)、blaCTX-M-27(n=8,18.2%)、blaCTX-M-15(n=5,11.4%),还检测出blaCTX-M-79(n=2,4.5%)、blaCTX-M-14(n=2,4.5%)和blaCTX-M-24(n=1,2.3%)。10株blaCTX-M-65基因阳性菌有8株同时携带mcr-1基因。这8株大肠杆菌均表现为多药耐药,包括氨苄西林、阿莫西林、头孢噻呋、头孢喹肟、黏菌素等多种抗生素。8株菌共分为6种ST型、7个PFGE谱型。接合转移试验发现,有6株菌的blaCTX-M-65和mcr-1基因发生了共转移,且blaCTX-M-65与mcr-1基因均位于IncHI2型(253 kb)质粒上。其中1株菌在接合转移的过程中,其所携带的一个253 kb IncHI2质粒与一个69 kb IncFⅡ质粒发生融合,形成一个大小323 kb的融合质粒。对融合质粒进行三代测序解析,结果表明,在接合转移的过程中IS26介导了两个不同大小质粒的重组。【结论】IncHI2质粒是介导blaCTX-M-65和mcr-1基因共同传播的主要媒介,IS26通过与靶位点GTTTCACT的整合导致IncHI2质粒与IncFⅡ质粒融合。质粒重组扩大了大肠杆菌的耐药谱,加速了耐药基因的传播,促使多药耐药现象更严重,对公共卫生安全构成威胁,本研究为阐明多重耐药大肠杆菌的传播机制提供了参考依据。

【Objective】This study was aimed to investigate and analyze the drug resistance of diarrheal piglets in a pig farm and the molecular characteristics and the co-transmission mechanism of bla CTX-M-65 and mcr-1 drug resistance genes positive Escherichia coli(E.coli)in Guangdong province,so as to provide scientific basis for drug resistance monitoring and risk prevention and control.【Method】E.coli strains were isolated and identified from 90 intestinal samples of diarrhea pigs in a pig farm in Zhaoqing,Guangdong province.ESBLs and mcr-1 genes were detected by PCR,and subtyping of CTX-M ESBLs was determined by sequencing.The minimum inhibitory concentration(MIC)of strains to 12 antibiotics was performed using agar double dilution method and broth microdilution method.Genetic relatedness of bla CTX-M-65 and mcr-1 genes co-carrying strains was analyzed by pulsed field gel electrophoresis(PFGE)and multiple locus sequence typing(MLST).The ability of plasmid horizontal transfer was determined by conjugation transfer,and plasmid replicon types were detected by PCR-based replicon typing.The localization of bla CTX-M-65 and mcr-1 genes and the size of plasmids were determined by S1-PFGE and Southern blotting.The recombination event of plasmid was determined by whole-genome sequencing and bioinformatics analyses.【Result】A total of 86 strains of E.coli were isolated and identified.44 strains carried CTX-M ESBLs genes,bla CTX-M-55 gene was the predominant subtype(n=16,36.4%),followed by bla CTX-M-65(n=10,22.7%),bla CTX-M-27(n=8,18.2%),bla CTX-M-15(n=5,11.4%),bla CTX-M-79(n=2,4.5%),bla CTX-M-14(n=2,4.5%)and bla CTX-M-24(n=1,2.3%).Of 10 bla CTX-M-65 positive strains,8 were confirmed co-carrying mcr-1 gene,presenting multidrug-resistance phenotypes,including resistance to gentamicin,florfenicol,aztreonam and other antibiotics.The 8 strains were divided into 6 ST types,contained 7 PFGE profiles.A total of 6 transconjugates co-carrying bla CTX-M-65 and mcr-1 genes were obtained.The bla CTX-M-65 and mcr-1 genes in all 6 conjugates were located on the IncHI2(253 kb)plasmids.The resulting 323 kb plasmid was a fusion of a 253 kb IncHI2 plasmid and a 69 kb IncFⅡplasmid,which was formed during conjugation transfer.Sequence analysis of plasmids revealed that IS26 mediated the recombination of the IncHI2 plasmid and the IncFⅡplasmid.【Conclusion】IncHI2 plasmid mediated the widely spread of bla CTX-M-65 and mcr-1 genes.IS26 led to the fusion of IncHI2 plasmid and IncFⅡplasmid through integration with the target site GTTTCACT.Such recombination events of plasmids played an important role in expanding the antibiotic resistance spectrum,accelerating the spread of resistance genes,making multidrug resistance more serious and posing a threat to public health security.This study provided a basis for elucidating the transmission mechanism of multi-drug resistant E.coli.