三类禽源碳青霉烯类耐药大肠杆菌流行病学调查及全基因组测序分析

Epidemiology Investigation and Whole Genome Sequencing Analysis of Three Types of Poultry Originated Carbapenem-resistant Escherichia coli

【目的】研究不同禽源碳青霉烯类耐药大肠杆菌(carbapenem-resistant Escherichia coli,CREC)的群间分布、耐药特征及菌株间亲缘关系,为阻断CREC的潜在危害提供技术支持。【方法】在胶东地区采集肉鸡、蛋鸡、水禽三类家禽泄殖腔拭子1131份,利用选择性分离培养、质谱鉴定、PCR、微量肉汤稀释法、多位点序列分型(MLST)及全基因组测序(WGS)等方法进行CREC菌株鉴定与分析。【结果】共分离出364株bla NDM基因阳性的CREC菌株,分离率为32.18%,阳性场总体占比为76.32%,肉鸡场和个体阳性率均最高,分别为93.33%和55.56%。三类禽源菌株均多重耐药,83.65%菌株对8类及以上药物同时耐药,对多数测试药物耐药率在80%以上。肉鸡多重耐药问题最严重,水禽次之,蛋鸡相对较轻。45株全基因组测序的CREC菌株携带12类52种耐药基因,4种bla NDM变异体,以bla NDM-5(77.78%)为主,三类家禽对同类耐药基因的检出率存在差异。共检出46种ST型,多样性比为44.23%。三类家禽CREC菌株间单核苷酸多态性(SNPs)差异位点为82~71307个,且携带的优势型不同。【结论】携带bla NDM的CREC在不同家禽养殖场尤其是肉鸡场广泛存在,以bla NDM-5亚型最为常见,对多种抗菌药普遍耐药,且携带大量耐药基因,以质粒传播为主。CREC菌株在三类家禽中呈多样化分布,且多数菌株间亲缘关系相差较远。提示应针对不同动物群体,加强监测和影响因素研究,以降低CREC的潜在风险。

【Objective】This study was aimed to investigate the inter-group distribution,drug resistance characteristics and genetic relationships of carbapem-resistant Escherichia coli(CREC)strains from different poultry,so as to provide technical support for blocking the potential hazards of CREC.【Method】A total of 1131 cloacal swabs were collected from broilers,laying hens and waterfowls in Jiaodong area,and CREC strains were identified by selective isolation and culture,mass spectrometry identification,PCR,broth microdilution,multi-locus sequence typing(MLST)and whole genome sequencing(WGS).【Result】A total of 364 bla NDM gene positive CREC strains were isolated with a separation rate of 32.18%and the overall proportion of positive farms was 76.32%.The positive rates at both farm and individual levels of broilers were the highest(93.33%and 55.56%respectively).All the strains from three types of poultry were multi-drug resistant.83.65%of the isolates were resistant to 8 or more classes of drugs simultaneously,and the resistance rate to most of the tested drugs was more than 80%.The multi-drug resistance in broilers was the most serious,followed by waterfowl and laying hens.45 whole-genome sequenced CREC strains carried 52 drug resistance genes in 12 classes within 4 kinds of bla NDM variants(mainly bla NDM-5,77.78%).The detection rates of specific drug resistant gene of CREC originated from three types of poultry were different.A total of 46 ST types were detected,with a diversity ratio of 44.23%.There were 82-71307 SNPs among the CREC strains from three types of poultry,and the dominant ST types of different poultry were quite different.【Conclusion】CREC that carrying bla NDM was widely prevalent in different poultry farms,especially in broiler farms,and the bla NDM-5 subtype was dominant.They were generally resistant to a variety of antimicrobial agents and carried a large number of drug-resistant genes,mainly plasmid transmission.CREC strains from three types of poultry showed diverse distribution,and the genetic relationship among most strains was quite far.It was suggested that surveillance and risk factors of CREC among different animals should be strengthened.