两株驴源马链球菌兽疫亚种流行株的生物学特性及全基因组序列的比较分析

Comparison of the biological characteristics and whole genomes between two donkey-derived isolates of Streptococcus equi subsp. zooepidemicus

摘要:

目的 开展新疆2株驴源马链球菌兽疫亚种(Streptococcus equi subsp. zooepidemicus, SEZ)不同区域流行株(YLCD588和HT222)的致病特征、耐药特点、多位点序列分型(multilocus sequence typing, MLST)及其基因组比较分析，明确该菌的分子进化规律，分析该菌致病性和耐药性相关的基因和表型特征，探究SEZ的致病与耐药机制，为该菌的防控提供科学依据。方法 利用二代测序技术对分离菌株进行全基因组测序，将测序数据与数据库中已有序列构建MLST发育树；利用毒力因子数据库(https://www.mgc.ac.cn/VFs/)和基因组流行病学中心数据库(http://genomicepidemiology.org)对其毒力基因和耐药基因分别进行注释和比较；对2株菌的生长、药物敏感性和生物膜形成进行检测与比较。将菌株分别接种小鼠，观察比较实验动物的发病情况，并对发病小鼠的肺脏和脾脏组织进行病理组织学观察和组织菌体载量的检测比较。结果 测序结果显示，HT222与YLCD588基因组大小分别为2 105 005 bp和2 090 225 bp，分别有1 995个和1 905个编码区，HT222共有214个毒力基因，YLCD588共有212个毒力基因；HT222携带有235个耐药基因，YLCD588共有233个耐药基因。YLCD588为一个新的ST型ST545，MLST系统发育树显示YLCD588与山羊源SEZ具有最近的亲缘关系，而HT222则与犬源SEZ最近。YLCD588对6种抗生素耐药，HT222对4种抗生素耐药。结晶紫法(P<0.05)和共聚焦显微观察均表明YLCD588生物膜形成能力显著高于HT222，而HT222菌株对小鼠致死率(P<0.05)及组织荷菌量(P<0.01)显著高于YLCD588，且对小鼠病理损伤更严重。结论 2株流行株具有不同的基因组特征、序列分型(sequence type, ST)、致病和耐药特征，HT222比YLCD588携带毒力和耐药基因多，致病表型更强；YLCD588比HT222生物膜形成能力强，耐药表型显著；拓宽了对不同驴源SEZ分子流行、致病与耐药机制的认识，为有效控制SEZ的感染传播以及临床治疗提供了参考。

Abstract:

Objective To deeply understand the molecular evolution pattern, pathogenicity, and drug resistance mechanism and provide a scientific basis for the prevention and control of Streptococcus equi subsp. zooepidemicus (SEZ), we compared the pathogenicity, drug resistance, and genome sequences of two SEZ strains (YLCD588 and HT222) isolated from donkeys in Xinjiang.Methods Whole-genome sequences of the two strains were determined by next-generation sequencing and a phylogenetic tree was constructed based on multilocus sequence typing (MLST) of sequencing data and existing sequences in the database. The virulence factor database (VFDB) (https://www.mgc.ac.cn/VFs/) and the center for genomic epidemiology (CGE) (http://genomicepidemiology.org) were used for annotation of the virulence and drug resistance genes of the two strains. The growth curves, antimicrobial susceptibility, and biofilm formation of the two strains were examined and compared. Mice were challenged with these strains separately and the pathogenicity of the strains was observed and evaluated. Then, histopathological changes and bacterial loads in the lung and spleen tissues of the morbid mice were observed and determined.Results Sequencing data showed that the genome sizes of YLCD588 and HT222 were 2 090 225 bp (1 905 coding sequences) and 2 105 005 bp (1 995 coding sequences), respectively. HT222 and YLCD588 carried 214 and 212 virulence genes, respectively. HT222 and YLCD588 had 235 and 233 drug resistance genes, respectively. YLCD588 was assigned to a novel sequence type (ST) 545 by MLST. The MLST phylogenetic tree indicated that YLCD588 was closely related to the goat-derived SEZ strain, while HT222 was closely related to the canine SEZ strain. YLCD588 displayed resistance to six antibiotics and HT222 exhibited resistance to four. The crystal violet assay and confocal laser scanning microscopy results showed that YLCD588 exhibited stronger biofilm formation than HT222 (P<0.05), whereas HT222 caused higher mortality rate (P<0.05), higher bacterial load (P<0.01), and severer pathological damage in mice than YLCD588.Conclusion The two SEZ strains exhibit distinct genomic characteristics, sequence types, pathogenicity, and drug resistance. HT222 possesses more drug resistance genes and virulence genes and exhibits stronger pathogenicity than YLCD588, while YLCD588 showcases stronger biofilm formation and drug resistance than HT222. These findings broaden the understanding about the molecular epidemiology, pathogenicity, and drug resistance of different SEZ strains from donkey and provide references for the effective control of the infection and spread of SEZ and clinical treatment of SEZ infection.