朱鹮源大肠埃希氏菌的耐药性及毒力基因与种系分型检测分析

Detection of Drug Resistance,Virulence Gene and Phylogenetic Types of Escherichia coli from Nipponia nippon

为了解陕西圈养朱鹮源大肠埃希氏菌耐药性、系统进化分群、毒力基因及耐药基因携带情况,对采集自陕西省周至县32份朱鹮源肛拭子进行肠道细菌分离鉴定,采用微量肉汤稀释法测定9种抗菌药物对分离株的最小抑菌浓度(MIC),PCR检测毒力基因、耐药基因携带情况,采用三重PCR鉴定分离菌株的系统进化分群,并进行小鼠致病性试验。结果显示,在32份朱鹮源肛拭子中分离到大肠埃希氏菌15株,分离率为46.87%;分离菌株对氨苄西林100%耐药,对多西环素、恩诺沙星、头孢噻夫耐药率较高,对美罗培南、替加环素、黏菌素表现较高敏感,耐2种以上抗菌药物的菌株达100%;从分离菌株中检测到blaTEM、blaSHV、blaCTX-M等耐药基因的检出率分别为100%、93.3%、33.3%,phop、tetx3占比较高(66.67%、53.33%),未检测到cfr、tetx4耐药基因;分离菌株携带10种不同的毒力基因,未检测到hlyA、saf、cnf1、iutB、stx1、stx2、papC、FasA等8种毒力基因,其中etrA、aer、hlyE、ompA的检出率最高,均达到93.33%,其他依次为iutA(80%)、fimC(73.33%)、rfc(60%)、ompT(53.33%)、hlyF(46.67%)、Tsh(20%),且检测到9株分离菌株携带的rfc毒力基因。该批朱鹮源大肠埃希氏菌以B2型为主(占53.33%),其次为D型(占33.34%),A型仅占比13.33%,未检测到B1型;小鼠致病性试验表明,小鼠表现较轻的临床症状。结果表明,该批朱鹮源大肠埃希氏菌耐药现象较为严重,毒力基因与耐药基因在该批朱鹮中较流行,与其他动物相比,其种系分型较特殊。研究结果可为朱鹮源大肠杆菌病的防控、治疗及圈养朱鹮的放飞提供参考依据。

The purpose of this study was to make a knowledge of drug resistance,virulence gene and phylogenetic types of Escherichia coli from captive Nipponia nippon in Shaanxi.In 2021,32 anal swab samples were collected from Zhouzhi county,Shaanxi province for Escherichia coli isolation and identification.The minimum inhibitory concentration(MIC)of 9 antimicrobial agents against the isolates was determined by broth dilution method.Virulence genes,drug resistance genes and phylogenetic types of strains were detected by PCR method,and the pathogenicity test in mice was conducted.The results showed that 15 strains Escherichia coli were isolated from 32 samples,and the isolation rate was 46.87%.The strains were 100%resistant to ampicillin,with a high resistance rate to doxycycline,enrofloxacin,and cefotaxif.They were highly sensitive to meropenem,tegacyclin,and colistin,and 100%resistant to more than two antimicrobial drugs.The detection rates of bla TEM,bla SHV,and blaCTX-M resistant genes detected from strains were 100%,93.3%,and 33.3%,respectively.Phop and tetx3 accounted for a relatively high proportion(66.67%,53.33%);Cfr and tetx4 resistance genes were not detected.The strains carried 10 different virulence genes,but eight virulence genes were not detected,including hlyA,saf,cnf1,iutB,stx1,stx2,papC,and FasA.Among them,the highest detection rates were etrA,aer,hlyE,and ompA,all of which reached 93.33%,followed by iutA(80%),fimC(73.33%),rfc(60%),ompT(53.33%),hlyF(46.67%),and Tsh(20%).The rfc virulence gene carried by 9 strains was detected.The main phylogenetic type of Escherichia coli is B2(53.33%),followed by D(33.34%),and A(13.33%),with no B1 detected.The pathogenicity test showed mild clinical symptoms in mice.The research results showed that the drug resistance of Escherichia coli from the captive Nipponia nippon was relatively serious,and their virulence genes and drug resistance genes were more prevalent.Compared with other animals,their phylogenetic types were more specific.This study can provide a reference basis for the prevention and treatment of Escherichia coli from Nipponia nippon and the release of captive Nipponia nippon.