广东地区黄羽肉鸡肠杆菌中碳青霉烯耐药基因的流行病学调查

Epidemiological Investigation of Carbapenem-resistant Enterobacteriaceae from Yellow-feathered Broiler in Guangdong Province

为了调查广东地区黄羽肉鸡肠杆菌的耐药性以及碳青霉烯耐药基因的流行病学特征,本研究于2020年8—10月从广东省4个黄羽肉鸡养殖场累计收集的248份样品中分离肠杆菌,利用大肠杆菌基因phoA和细菌16S rRNA进行菌种鉴定,采用琼脂扩散法和微量肉汤稀释法测定肠杆菌对13种药物的敏感性,通过PCR方法检测bla_(NDM)、bla_(KPC)、bla_(OXA)和bla_(IMP)四种常见碳青霉烯类耐药基因,并对耐碳青霉烯肠杆菌进行全基因组测序分析,通过接合转移试验探究碳青霉烯耐药基因的水平传播特征。细菌分离和鉴定结果显示,共计分离肠杆菌206株,分离率为83.06%,其中包括186株大肠杆菌、18株肺炎克雷伯菌和2株阴沟肠杆菌。药物敏感性试验结果显示,肠杆菌对氨苄西林钠(94.66%)、复方新诺明(94.66%)、氟苯尼考(86.89%)、盐酸多西环素(70.87%)和硫酸新霉素(61.17%)的耐药性较高,对硫酸阿米卡星(5.83%)、美罗培南(3.40%)和硫酸黏菌素(1.46%)的敏感性较高。PCR结果显示,共有7株肠杆菌检测到碳青霉烯耐药基因,检出率为3.40%,包括5株bla_(NDM-5)基因阳性和2株bla_(OXA-181)基因阳性菌株。全基因组分析结果显示,7株耐碳青霉烯肠杆菌分别为3株大肠杆菌(携带bla_(NDM-5)基因)、2株拟肺炎克雷伯菌(携带bla_(NDM-5)基因)和2株阴沟肠杆菌(携带bla_(OXA-181)基因);7株耐碳青霉烯肠杆菌均携带有大量的耐药基因,且3株大肠杆菌可分为2个序列型(ST)。接合转移试验结果显示,7株耐碳青霉烯菌株均可通过质粒将携带的碳青霉烯耐药基因传播至工程菌C600。结果表明,广东地区黄羽肉鸡肠杆菌耐药状况严峻,耐碳青霉烯基因在肠杆菌的流行率较低,但可通过质粒接合方式发生水平传播,亟需加强监测。

To investigate antibiotic resistance of Enterobacteriaceae and epidemiological characteristics of carbapenem-resistant Enterobacteriaceae(CRE)from yellow-feathered broiler in Guangdong province,this study isolated Enterobacteriaceae from 248 samples collected cumulatively from four yellow-feathered broiler farms in Guangdong province from August to October,2020.The isolates were identified by PCR targeting Escherichia coli phoA and 16S rRNA genes.Antibiotic susceptibility to 13 drugs was determined by agar diffusion method and broth microdilution method.The carbapenem-resistant genes,including bla_(NDM),bla_(KPC),bla_(OXA)and bla_(IMP),were assessed in all isolated strains and the positive strains were examined by whole genome sequencing.The horizontal transmission characteristics of carbapenem-resistant genes was further determined by conjugation experiment.The results of bacterial isolation and identification showed that a total of 206 strains were isolated from 248 samples with the isolation rate of 83.06%,including 186 Escherichia coli,18 Klebsiella pneumonia e and 2 Enterobacter cloacae.The antibiotic susceptibility test revealed that these strains were highly resistant to ampicillin sodium(94.66%),sulfamethoxazole/trimethoprim(94.66%),florfenicol(86.89%),doxycycline hyclate(70.87%)and neomycin sulfate(61.17%),and were somewhat sensitive to amikacin sulfate(5.83%),meropenem(3.40%)and colistin sulphate(1.46%).PCR results showed that the prevalence of carbapenem-resistant genes was 3.40%(n=7),including 5 bla_(NDM-5)-positive strains and 2 bla_(OXA-181)-positive strains.Whole genome sequencing analysis showed that seven CRE strains were identified as 3 E.coli(carrying bla_(NDM-5)gene),2 K.quasipneumoniae(carrying bla_(NDM-5)gene)and 2 E.cloacae(carrying bla_(OXA-181)gene),and these seven CRE strains all carried multiple resistance genes,and the 3 strains of E.coli could be divided into 2 sequence type(ST).All CRE strains had successfully transferred carbapenem-resistant gene to the receipt E.coli C600 by conjugation experiment.Thus,antibiotic resistance was serious among Enterobacteriaceae from yellow-feather broilers in Guangdong province,the prevalence of CRE was 3.40%,and the dissemination of carbapenem-resistant genes was due to horizontal transfer by plasmid conjugation.