1株大鲵源葡萄牙柠檬酸杆菌的全基因组测定及毒力基因和耐药基因的分析

Whole genome determination and analysis of virulence gene and drug resistance gene of a strain of Citrobacter portucalensis isolated from the Chinese giant salamander

葡萄牙柠檬酸杆菌是一种新发现的条件性致病菌,为明确重庆地区大鲵源多重耐药葡萄牙柠檬酸杆菌的基因组及其耐药特征,本试验对患病大鲵分离菌进行鉴定、药敏试验、全基因组测序及毒力、耐药基因的分析。分离菌首先通过16S rDNA进行初步鉴定,腹腔注射健康小鼠观察其致病性;利用Kirby-Bauer(K-B)纸片法进行9类共35种抗生素的敏感性试验;再利用二代测序技术进行全基因组测序,通过生物软件和数据库对分离菌的毒力基因和耐药基因进行分析。结果显示,分离菌鉴定为葡萄牙柠檬酸杆菌,将其命名为CQ-CP1,该菌对小鼠具有较强的致病性。药敏试验显示CQ-CP1对多种抗生素不敏感,为多重耐药菌株。全基因组测序该菌株共有5006960 bp,G+C含量为51.8%,共编码有4722个结构蛋白,基因组信息在GenBank中的登录号为RZIH00000000.1。病原毒力因子数据库(VFDB)比对发现CQ-CP1中共有49个与毒力相关的基因,与细菌的运动、黏附、转录等相关;耐药基因数据库比对结果显示,该菌携带6个耐药基因(ARG),分别是氨基糖苷类aadA1,喹诺酮类qnrS1、qnrB32,β内酰胺类bla_(CMY-35)、bla_(TEM-1A)和磺胺类dfrA1。本试验为葡萄牙柠檬酸杆菌的流行病学、致病性及耐药特征等研究提供参考。

Citrobacter portucalensis(C.portucalensis)is an emerging opportunistic nosocomial pathogen,in order to retrieve the characterization of a multidrug-resistant C.portucalensis strain isolated from a diseased Chinese giant salamander in Chongqing,identification,antibiotic susceptibility test,whole genome sequencing(WGS)and analysis of virulence gene and drug resistance gene of the isolate were conducted.The isolated bacteria were preliminarily identified by 16S rDNA,and were injected intraperitoneally into healthy mice to observe the pathogenicity.Kirby-Bauer agar disc diffusion method was carried out using 35 antibiotics belonging to 9 antibiotic groups.After that,next-generation sequencing technology was performed to acquire the whole genome sequence of the isolate.The virulence gene and drug resistance gene of the isolate were analyzed by biological software and database.The results showed that the isolate belonged to C.portucalensis and then it was named CQ-CP1.In the pathogenicity test,all the mice died after infection,revealing that CQ-CP1was a pathogenic agent.Antibiotic susceptibility test showed that CQ-CP1was resistant to multiple antibiotics,indicating that it was a multidrug resistant strain.Sequence analysis showed the assembled genome size was 5006960bp with 51.8% of G+C content,containing 4722 protein-coding genes.The genome sequence has been deposited in GenBank under the accession number RZIH00000000.1.The comparison results with pathogen virulence factor database(VFDB)showed that CQ-CP1harbored 49virulence related genes,they were related to bacterial movement,adhesion and transcription.Furthermore,it was found that six antibiotic resistance genes conferring resistance to four different groups of antibiotics such as aminoglycoside(aadA1),fluoroquinolone(qnrS1 and qnrB32),trimethoprim(dfrA1)andβ-lactams(bla_(CMY-35)and bla_(TEM-1A)).All these findings offered valuable resource for studies on the epidemiology,pathogenic mechanism and antibiotic resistance characterizations of C.portucalensis.