猕猴源大肠杆菌药敏试验与高致病性毒力岛基因的检测

Drug Sensitivity Test and Detection of HPI Gene of Macaca mulatta-derived Escherichia coli

为探究腹泻猕猴粪便中分离出的大肠杆菌(E.coli)是否携带高致病性毒力岛(HPI)相关基因,并分析其耐药性和致病性,本试验采集23只腹泻猕猴的粪便,用麦康凯培养基、LB琼脂和生化鉴定管对E.coli进行分离鉴定,通过PCR试验、纸片扩散法和动物回归试验,研究分离菌株的HPI基因携带情况、耐药性、致病性,并且进行全基因组测序验证。结果显示,23份样品中均分离出E.coli,分离率为100%;分离出的E.coli中HPI标志基因irp 2携带率高达47.83%,其余基因irp 1、irp 3、irp 4、irp 5、irp 6、irp 7、irp 8、irp 9、fyuA、intB和ybtA携带率分别为65.22%、34.78%、47.83%、69.57%、56.62%、43.48%、39.13%、43.48%、34.78%、65.22%和26.09%;在分离出的23株E.coli菌株中携带HPI全岛基因的有5株,占21.74%,这5株E.coli HPI阳性菌株(HPI+)对阿米卡星、环丙沙星、头孢哌酮、头孢噻肟、苯唑西林、四环素、头孢唑林、头孢曲松的耐药性都极高,对氯霉素较敏感;接种E.coli HPI+菌株后8 h小鼠死亡率为100%,接种E.coli HPI-菌株后12 h小鼠死亡率为25%;用2株E.coli HPI+全基因检测的结果构建了2个基因组圈图,其中1株(H-7)预测到5300个编码基因,其总长4728917 bp;另1株(H-23)预测到5460个编码基因,其总长4811637 bp。结果表明,猕猴腹泻源致病性E.coli HPI基因携带率较高、耐药性普遍存在,同时致病性E.coli HPI+可提高小鼠死亡率,本试验通过COG、GO、KEGG获得大量的基因功能注释结果,从基因组水平加深了对E.coli HPI+的认识。

This study investigated whether Escherichia coli isolates from diarrheal Macaca mulatta carried the high pathogenicity island(HPI)gene,and determined their drug resistance and pathogenicity.Specifically,E.coli was isolated from the 23 feces of diarrheal Macaca mulatta and identified by MacConey agar medium,LB agar,and biochemical identification tube.The HPI gene carrier status,drug resistance and pathogenicity of the isolates were assessed by PCR,disc diffusion mothod,and animal regression test,and verified by whole-genome sequencing.The results showed that 23 E.coli strains were isolated from the 23 feces of diarrheal Macaca mulatta(isolation rate of 100%),and the carrying rate of the HPI gene irp 2 was 47.83%,the carrying rates of irp 1,irp 3,irp 4,irp 5,irp 6,irp 7,irp 8,irp 9,fyuA,intB,and ybtA were 65.22%,34.78%,47.83%,69.57%,56.62%,43.48%,39.13%,43.48%,34.78%,65.22%,26.09%,respectively.Among the 23 E.coli strains,5(21.74%)contained HPI+,were highly resistant to amikacin,ciprofloxacin,cefepime,cefotaxime,oxacillin,tetracycline,cefazolin and ceftriaxone,but sensitive to chloramphenicol.The death rate of mice after inoculation with E.coli HPI+strain for 8 h was 100%,and that of mice after inoculation with E.coli HPI-strain for 12 h was 25%.Two E.coli HPI+genome maps were constructed using whole gene test results,one of which(H-7)predicted 5300 coding genes with a total length of 4728917 bp,and the other(H-23)predicted 5460 coding genes with a total length of 4811637 bp.Thus,the carrier rate of diarrhea pathogenic E.coli HPI from Macaca mulatta is high,E.coli drug resistance is widespread,pathogenic E.coli HPI+causes increased mouse mortality.The gene function annotation results through COG,GO and KEGG reveal valuable insights on E.coli HPI+at the genomic level.