多重耐药胸膜肺炎放线杆菌GD2107株全基因组测序及生物信息学分析

Whole Genome Sequencing and Biological Characteristics Analysis of Multidrug-resistant Actinobacillus pleuropneumoniae GD2107 Strain

【目的】通过对多重耐药胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)GD2107株进行全基因组测序及生物信息学分析,丰富APP基因组数据库信息;构建基于ApxⅣ基因的系统进化树,分析该菌株的进化关系,为探索APP致病机制和临床防控猪传染性胸膜肺炎(porcine contagious pleuropneumia,PCP)提供参考。【方法】通过药敏试验测定分离菌株的耐药谱;采用全基因组测序技术(whole genome sequencing,WGS)对细菌DNA进行全基因组测序,分别利用Illumina NovaSeq、PacBio SequeI测序平台对全基因组测序结果进行基因功能注释及生物信息学分析(包括基因组基本信息、功能元件分析及亚系统分析等);基于ApxⅣ基因构建系统进化树。【结果】药敏试验结果显示,GD2107菌株对青霉素、头孢拉定(先锋Ⅵ)、卡那霉素等14种抗菌药均耐药。对GD2107株全基因组测序得到1条大小为2271987 bp的环状染色体(GC含量为41.21%)和2个大小分别为5027和3497 bp的环状质粒,共预测到2290个编码基因,包含19个rRNA(7个5S rRNA、6个16S rRNA、6个23S rRNA)、21个tRNA基因、20个ncRNA;23个基因岛、4个原噬菌体和2组CRISPR相关序列;分别有2112、1549和1866个基因在COG、KEGG和GO数据库中得到注释,且相关蛋白集中分布于APP的代谢过程;另外在毒力因子(VFDB)和耐药因子(CARD)数据库中还注释到48个毒力基因和22个耐药基因(仅floR基因位于质粒上)。绘制该菌株的全基因组圈图,将基因组信息提交至NCBI,获得染色体GenBank登录号为CP097377,质粒登录号分别为CP097378和CP097379。系统进化树分析发现,该菌株与来自中国的APP菌株(CP063424.1)进化关系最近。【结论】本研究完成了对多重耐药菌株GD2107的全基因组测序与生物信息学分析,全面认识了该菌株基因组的结构和功能并探究了耐药和致病机制中的相关基因,进化关系显示该菌株具有一定的地域流行性,为预防PCP的流行和探索APP的致病机制提供了参考。

【Objective】 Through genome sequencing and bioinformatics analysis of GD2107 strain of multi-drug resistant Actinobacillus pleuropneumoniae(APP),the genome database information of APP was enriched, the phylogenetic tree based on ApxⅣ gene was constructed, and the evolutionary relationship of the strain was analyzed, so as to provide reference for exploring the pathogenic mechanism of APP and clinical prevention and control of porcine infectious porcine contagious pleuropneumia(PCP).【Method】 The drug resistance spectrum of the isolated strains was determined by drug sensitivity test. The whole genome of bacterial DNA was sequenced by whole genome sequencing(WGS) method.Based on Illumina NovaSeq and PacBio SequeI sequencing platforms, the whole genome sequencing results were analyzed by gene function annotation and bioinformatics analysis(including basic genome information, functional element analysis and subsystem analysis, etc.). Phylogenetic tree was constructed based on ApxⅣ gene.【Result】 The results of drug sensitivity test of GD2107 strain showed that it was resistant to 14 kinds of antimicrobials including penicillin, cefradine(pioneer Ⅵ),kanamycin and so on.The whole genome of GD2107 strain was sequenced to obtain a circular chromosome of 2 271 987 bp(GC content was 41.21%) and two circular plasmids of 5 027 and 3 497 bp, respectively.A total of 2 290 coding genes were predicted, including 19 rRNA(7 5 S rRNA,6 16 S rRNA,6 23 S rRNA),21 tRNA genes, 20 ncRNA;23 gene islands, 4 prophages and 2 groups of CRISPR related sequences.2 112,1 549 and 1 866 genes were annotated in the databases of COG,KEGG and GO,respectively, and the related proteins were mainly distributed in the metabolic process of APP.In addition, 48 virulence genes and 22 drug resistance genes were annotated in VFDB and CARD databases(only floR gene was located on the plasmid).The whole genome circle map of the strain was drawn and the genome information was submitted to NCBI.The chromosome GenBank accession No. was CP097377 and the plasmids accession No. were CP097378 and CP097379, respectively. Phylogenetic tree analysis showed that the evolutionary relationship between this strain and APP strain from China(CP063424.1) was the closest.【Conclusion】 In this study, we completed the whole genome sequencing and bioinformatics analysis of the multi-drug resistant strain GD2107, fully understood the structure and function of the genome of the strain, and explored the related genes in drug resistance and pathogenic mechanism. The evolutionary relationship showed that the strain had a certain regional epidemic, which provided a reference for preventing the epidemic of PCP and exploring the pathogenic mechanism of APP.