Phenotypic and genotypic characterization of ST103 serotype Ia Streptococcus agalactiae isolated from bovine mastitis in China

Streptococcus agalactiae is a contagious pathogen responsible for bovine mastitis, leading to significant economic losses in the global dairy industry. Our objectives were to determine the population structure, to profile the antimicrobial resistance and to investigate the pathogenicity and genes associated with pathogenicity in S. agalactiae isolated from Chinese dairy herds. A total of 266 milk samples were collected on three dairy farms in Ningxia (herd A) and Hebei provinces (herds B & C) with outbreaks of bovine mastitis from September 2020 to April 2021. There were 116 isolates identified as Streptococcus agalactiae by 16S rRNA sequencing. Twenty-seven S. agalactiae isolates were randomly selected using a stratified approach from the three farms for whole genome sequencing analysis and phenotypic analyses, including antimicrobial resistance profiling, identification of adhesion, invasion and virulence genes using in vitro bovine mammary epithelial cell models and in vivo Galleria mellonella models. Multilocus sequence typing and serotyping showed that all isolates belonged to sequence type ST103 and serotype Ia. In total, 34 genes were identified as virulence genes in Streptococcus species. Isolates from herd C had significantly higher virulence than those from herd B. Genome-wide association analysis revealed 166 virulence-related genes, 221 adhesion-related genes and 218 invasion-related genes. Furthermore, 47 genes were associated with pathogenicity in infecting G. mellonella. Resistance to tetracycline and macrolides was related to the presence of antimicrobial resistance genes tetO, tetM, and ermB. Pan-genome analyses revealed that 1,421 S. agalactiae isolates (27 from our study and 1,394 from the NCBI genome database) had 20,955 genes, including 666 and 20,289 genes in the core and accessory genomes, respectively. This study characterized the phenotypic and genotypic profiles for S. agalactiae, and identified associations between phenotypic traits and genetic determinants of virulence and antimicrobial resistance, providing new insights into controlling S. agalactiae mastitis in Chinese dairy herds.