小肠结肠炎耶尔森菌的分离鉴定及毒力基因检测

TIsolation and identification of Yersinia enterocolitica and its virulence gene detection

目的了解昆明市腹泻患者粪便标本中分离出的少见致病性小肠结肠炎耶尔森菌与其他地区该菌生物特性的差异。方法采集腹泻患者新鲜粪便标本,用磷酸盐缓冲液(PSB)4℃增菌21 d后,接种CIN培养基平板,25℃培养48 h,分离获得纯培养菌,采用肠杆菌科细菌微量生化编码管(GYZ-11e系统)进行鉴定,API-20E生化鉴定编码系列进行复核,使用小肠结肠炎耶尔森菌诊断血清进行血清分型,PCR检测菌株携带的毒力基因,采用K-B法测定小肠结肠炎耶尔森菌的毒力。结果分离到7株小肠结肠炎耶尔森菌,经鉴定为O∶8血清型小肠结肠炎耶尔森菌。毒力试验结果显示,小白鼠48 h内均发病死亡。PCR检测结果表明,有6株携带的毒力基因为ail-,yst A-、yst B+、yad A-、vir F-,1株为ail-、yst A+、yst B-、yad A-、vir F-。药敏试验显示,该菌对20种常见抗生素敏感率为80%。结论本研究分离得到7株O∶8型小肠结肠炎耶尔森菌,具有潜在的致病性和较强的毒力,是国内不多见的致病性菌型。

Objective To investigate the difference in the biological characteristics between the rare pathogenic Yersinia enterocolitica isolated from the diarrhea patients＇ excrement in Kunming and that bacterium in other areas. Methods Fresh feces samples from diarrhea patients were collected. Those samples were cultured in phosphate buffer for 21 days at 4 ℃ and inoculated in CIN medium plate for 48 hours at 25 ℃. Pure bacterial colony was isolated and identified by enterobacteriaceae bacteria micro biochemical coding tube （GYZ-1 le series）. Then results were confirmed by API-20E biochemical identification coding series. The serotype was assayed by Yersinia enterocolitiea diagnostic serum. Virulence genes in the detected strains were examined by PCR. The virulence was tested by K-B method. Results Seven strains of Yersinia enterocolitiea was isolated and identified as 0：8 serotype. The virulence test showed that all mice deceased within 48 h. PCR testing results indicated that the virulence genes carried in 6 strains were ail-, ystA-, ystB＋, yadA-, and virF-; those carried in the other one strain were all-, ystA ＋, ystB-, yadA-, and virF-. Snsceptibility test indicated the sensitive rate of this bacterium against 20 common antibiotics was 80%. Conclusion Seven strains of O：8 type Yersinla enterocolitica have been isolated and obtained in this research. This bacterium has potential pathogenicity and strong virulence and is a rare pathogenetic bacterium in our country.