牛源、兔源及人源无乳链球菌分子分型特征分析

摘要

为了解人源、牛源及兔源无乳链球菌(S.agalactiae)的基因型差异及分子分型的总体结构特征,本实验对30株2012年～2015年分离于人、牛及兔的S.agalactiae在cfb基因鉴定的基础上,通过荚膜多糖分子血清分型(CPS)、多位点序列分型(MLST)和脉冲场凝胶电泳(PFGE)进行基因型的确定和分子特征差异性比较.CPS结果显示,牛源和兔源S.agalactiae均为Ia血清型,而人源无乳链球菌包括Ⅰa和Ⅲ型两种血清型,以Ⅰa血清型为主要血清类型;MLST分型获得8个STs序列型(ST7、ST10、ST19、ST61、ST103、ST199、ST486、ST651)和6个克隆群(CC7、CC10、CC19、CC23、CC61、CC103),其中人源S.agalactiae拥有最多的STs和克隆群分型;PFGE聚类分析可以将其分为18个PFGE带型,且不同动物源性S.agalactiae之间基因分型差异性较大.血清分型、STs序列型、PFGE带型与宿主的来源之间无明显的相关性.不同来源菌株可以同属于血清型Ⅰa,且所有Ⅰa血清型菌株可以分布于不同的STs序列型和PFGE带型中.但分型为Ⅰa/ST651的人源S.agalactiae在MLST的克隆分群中是分型为Ⅰa/ST103牛源S.agalactiae的克隆衍生物,同属克隆群CC103,Ⅰa/ST651只是Ⅰa/ST103单一位点变化的产物,从分子水平的相似性推测二者存在交叉感染的可能性.本研究为奶牛乳房炎的有效预防和治疗提供依据.%To understand the genetic diversity and the structural characteristics of molecular typing of S.agalactiae isolated from human,bovine and rabbit.Based on the PCR dentification of cfb gene in S.agalactiae,capsular polysaccharide serotyping (CPS),multilocus sequence typing (MLST) and pulsed field gel electrophoresis(PFGE) were further used to confirmed genotypes and molecular characteristic diversity of 30 S.agalactiae strains isolated from human,bovine and rabbit between 2012 to 2015 in Sichuan province.The results of CPS revealed that only Ia serotype existed in bovine and rabbit S.agalactiae,Ⅰa and Ⅲ serotypes existed in human S.agalactiae,and Ⅰa was the preponderant serotype.Eight STs sequence types (ST7,ST10,ST19,ST61,ST103,ST199,ST486,ST651) and six clonal complexes (CC7,CC10,C19,CC23,CC61,CC103) were obtained by MLST typing,among which human S.agalactiae had the largest number genotypes of STs and CCs.These S.agalactiae could bedivided into 18 banding patterns with PFGE clustering analysis,and there were obvious genotype difference among S.agalactiae from different animal sources.Among serotyping,MLST types,PFGE types and host's sources had no obvious correlation with each other.Different sources of S.agalactiae strains could belong to serotype Ⅰa and all serotype Ⅰa strains could distribute into different STs and PFGE patterns.Ⅰa/ST651 typing of human S.agalactiae were the clone derivative of Ⅰa/ST103 typing of bovine S.agalactiae with MLST genotyping,but they all belonged to clone complexes CC103.Ⅰa/ST651 was single-locus variant (SLV) of Ⅰa/ST103,which suggested that they had the possibility of cross infection at the genetic level.This study could provide the basis for effective prevention and treatment,mastitis of dairy cows.