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

Abstract:

[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 16S 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 16S rRNA sequence analysis. In Challenge trials, the strain ACRO-0708 exhibited high virulence in A. fasciatus with an intra-peritoneal LD₅₀ value of 6.47×10³ 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.

Key words:Acrossocheilus fasciatus;Streptococcus agalactiae;molecular serotyping;MLST;virulence-related genes