1株朱鹮源致病性拟肺炎克雷伯菌的分离鉴定与分子特征分析

Isolation, identification and molecular characteristic of Klebsiella quasipneumoniae from crested ibis (Nipponia nippon)

为确定浙江省德清县下渚湖国家湿地公园1只野外朱鹮的死因及病原特性,对从肺、肝脏和腹水中分离出的病原菌进行形态特征、生化特性、16S rDNA鉴定,进而采用麻鸭腹腔接种鉴定其致病性、K-B纸片法药敏试验检测其耐药情况,并采用全基因组测序分析其耐药与毒力基因携带情况及ST型和分子血清型。结果显示,从朱鹮腹水和肺脏等样本分离到1株多重耐药致病性拟肺炎克雷伯菌,并暂定名为ZH200912。该菌株ST分型为ST3244,血清型为K165:O12,对1日龄麻鸭的LD_(50)为1×10^(6.5)CFU;携带耐药相关基因24个,其中,bla_(OKP-B-1),fosA6耐药基因与氨苄西林、哌拉西林和磷霉素耐药表型一致;对头孢噻肟耐药可能为携带多重耐药外排泵基因acrBD和耐药基因转录调节因子marAR等所致,虽然鉴定到oqxAB耐药基因但对喹诺酮类药物均不耐药;携带毒力基因67个,其中包括Ⅰ型菌毛蛋白基因fimACD、Ⅲ型菌毛蛋白基因mrkD、铁摄取相关基因肠杆菌素操纵子entABCEFS和fepABC,以及铁运输操纵子iroEN等核心毒力基因18个,缺失高黏相关基因rmpA和rpmA2。本研究分离鉴定到1株多重耐药致病性朱鹮源拟肺炎克雷伯菌,首次提出了拟肺炎克雷伯菌为朱鹮潜在致病菌,为相关疾病防治提供了数据支持,提示应重视拟肺炎克雷伯菌对动物的潜在危害。

To determine the pathogen that caused the death of one wild crested ibis(Nipponia nippon) near Xiazhu Lake National Wetland Park, Deqing city, morphological observation, biochemical characteristics and 16 S rDNA sequencing were applied to identify the isolates from lung and abdominal cavity.The pathogen and drug resistances of the isolate were validated by duckling infection test and universal Kirby-Bauer(K-B) test respectively.Whole genome sequence(WGS) of the isolate was sequenced and annotated to explore the virulence and drug resistance genes.The results showed that one Klebsiella quasipneumoniae,with ST3244 and molecular serotype K165:O12,was obtained and named as ZH200912.It had a strong pathogenicity to 1-day ducking(LD50=1×10CFU).WGS analysis showed that isolate ZH200912 carries 24 resistance genes,the antimicrobial resistance phenotype of ampicillin,piperacillin,fosmycin was consistent with the resistance genes blaOKP-B-1 and fosA6,cefotaxime phenotype may relate to multiple resistant efflux pump genes acrBDand resistant gene transcription regulators marAR.Sixty-seven virulence genes,including 18 core virulence genes were identified,such as iron uptake system genes entABCEFS,fepABCand iroEN,typeⅠandⅢfimbriae genes fimACD and mrkD,while rmpAand rmpA2 considering to hypermucoviscous phenotype were absent.In this study,one Klebsiella quasipneumoniae isolate without hypermucoviscous factor was proposed as a potential pathogen of crested ibis for the first time,which provide data for the prevention and treatment of related diseases.It suggested the potential harm of Klebsiella quasipneumoniae to animals.