西藏部分地区牦牛源肺炎克雷伯菌的分离、鉴定、毒力及耐药基因分析

Isolation,identification,virulence and drug resistance genes detection of Klebsiella pneumoniae in Tibet yak

利用细菌分离纯化、生化鉴定、分子生物学及WHO推荐的K-B纸片法等研究方法,对从西藏不同地区采集的103份牦牛鼻拭子样品进行肺炎克雷伯菌的分离鉴定,对分离鉴定的疑似菌株进一步进行16S rDNA测序及系统发育分析,荚膜血清学分型、毒力基因和耐药表型及耐药基因检测。结果显示,从西藏林芝市、那曲市分离鉴定出8株牦牛源克雷伯菌,分离率为7.76%;挑取其中2株进行测序,结果显示与重庆牛源KM096433和北京土壤源JQ003560聚为一簇,亲缘关系最近;8株西藏牦牛源克雷伯菌荚膜血清型均为K20型;此外,8株菌株均检测到3种毒力基因mrkD、fimH和wabG,检出率为100%;blaTEM、sul3、tetA和tetA耐药基因检出率分别为100%,100%,75%和75%,其余耐药基因均未检出;药敏试验结果显示,8株菌株对青霉素和氨苄西林耐药率达100%,其次是克拉霉素、四环素和多西环素耐药率达75%,而对喹诺酮类药物,如:环丙沙星、氧氟沙星和诺氟沙星强敏感;致病性试验中对照组全部存活,处理组小鼠病死率为80%(4/5),病变主要表现为脾脏肿大。本试验对西藏地区牦牛源肺炎克雷伯菌的流行情况、毒力基因和耐药性进行分析,对牦牛源肺炎克雷伯菌的防制具有重要的指导意义。

A total of 103 yak nose swab samples collected from different areas of Tibet were used for isolation and identification of Klebsiella pneumoniae using the bacterial isolation and purification,biochemical identification method.They were further identified for 16 S rDNA sequencing and phylogenetic analysis,capsular serological typing,virulence gene detection and drug resistance gene detection using the K-B disk method recommended by WHO and molecular biology methods.The results showed that 8 strains of yak-derived Klebsiella were isolated from Nyingchi city and Naqu city,with an isolation rate of 7.76%.Two of them were selected for sequencing and the results showed that they have closest relationship with No.KM096433 and No.JQ003560.The capsular serotypes of 8 strains of Klebsiella derived from Tibetan yak were all K20.In addition,virulence genes mrkD,fimH,and wabG were detected in eight strains,and the detection rate was 100%.The detection rates of resistance genes blaTEM,sul3,tetA and tetA were 100%,100%,75%and 75%,respectively.No other drug resistance genes were detected.Drug susceptibility test results showed that 8 strains were 100%resistant to penicillin and ampicillin,followed by clarithromycin,tetracycline,and doxycycline with a resistance rate of 75%,however they were highly sensitive to quinolones such as:ciprofloxacin,ofloxacin,and norfloxacin.In the pathogenicity test,all the mice in the control group survived.The mortality rate of mice in the treatment group was 80%(4/5)and the lesions mainly manifested as spleen enlargement of mice.Our study analyzes the prevalence,virulence genes and drug resistance of Klebsiella pneumoniae from yak in Tibet,and has important guiding significance for the prevention and control of Klebsiella pneumoniae from yak.