光唇鱼源无乳链球菌的分离鉴定及遗传特征

Identification and genetic characterization of Streptococcus agalactiae isolated from Acrossocheilus fasciatus cultured in Zhejiang

【目的】为查明浙江养殖光唇鱼大量死亡的病原,了解病原的遗传特征。【方法】本工作对患病光唇鱼进行病原分离,结合形态特征、生理生化特性和16S rRNA基因序列同源性,对分离菌株进行鉴定;采用人工回感试验确定其病原性,并对分离株的血清型、多位点序列分型(multilocus sequence typing,MLST)、毒力基因型和表面蛋白抗原基因型等遗传特征进行分析;此外,还测试了菌株的药敏特性。【结果】从患病光唇鱼体中分离得到优势菌株ACRO-0708,为革兰氏阳性球菌,不溶血,分子与生化鉴定为无乳链球菌(Streptococcus agalactiae);人工感染试验证实其对光唇鱼有较强的致病性,LD_(50)为6.47×10^(3)CFU/g,属于血清型Ⅰb和MLST型ST261,毒力基因型为sip+bibA+cfb+hylB+iagA+fbsA+fbsB+bac–bca–cylE–scpB–lmb–,不携带所检测的6种表面蛋白基因。药敏试验结果显示,对青霉素、氨苄西林等8种药物较敏感,对氯霉素、复方新诺明等7种药物耐药。【结论】引起浙江养殖光唇鱼死亡的病原菌为无乳链球菌,其分子特征与水产动物主要流行的无乳链球菌株具有显著差异,生产中可选用氨苄西林、氟苯尼考等药物进行防治。

[Objective]This study aimed to investigate pathogenesis of diseased Acrossocheilus fasciatus cultured in Zhejiang province and analyze genetic characteristics of the pathogen.[Methods]We isolated and purified pathogens from diseased A.fasciatus and identified the pathogenic bacteria by analyzing the physiology biochemistry characteristics and 16 S rRNA gene sequence.We also carried out artificial infection experiments to verify the pathogenicity of isolated strain to A.fasciatus.Then the genetic characteristics of the pathogen were analyzed by molecular serotyping,multilocus sequence typing(MLST)and PCR screening of surface protein antigen genes and virulence-related genes.Meanwhile,we tested its antimicrobial susceptibility by disc diffusion method.[Results]One predominant strain named ACRO-0708 was isolated from diseased A.fasciatus,which was gram-positive,chain-forming coccus and nonhemolytic,and was identified as Streptococcus agalactiae through the morphological characteristics,phenotypic characteristics and 16 S rRNA sequence analysis.In Challenge trials,the strain ACRO-0708 exhibited high virulence in A.fasciatus with an intra-peritoneal LD_(50) value of 6.47×10^(3) CFU/g.The strain ACRO-0708 was type of Ib-ST261,and the virulence-related genotype was sip+bibA+cfb+hylB+iagA+fbsA+fbsB+bac–bca–cylE–scpB–lmb–.However,it did not carry any of the six surface protein antigen genes which were detected.The antimicrobial susceptibility results showed strain ACRO-0708 was susceptible to 8 agents including penicillin,ampicillin and other antibiotics.Meanwhile,it showed resistance to chloroamphenicol,trimethoprim-sulfamethoxazole and other 5 antibiotics.[Conclusion]Our results demonstrated that S.agalactiae with molecular characteristics obviously different from the main epidemic strains of S.agalactiae in aquatic animals was the causative agent of the enzootic,and the disease could be prevented by using drugs such as penicillin and florfenicol in fisheries.To our knowledge,this is the first report of S.agalactiae as a pathogen of A.fasciatus.