Phenotypic and molecular differentiation of Lactococcus garvieae and Lactococcus petauri isolated from trout

Lactococcosis caused by Lactococcus garvieae leads to infective endocarditis, urinary tract infections, and peritonitis for humans and massive deaths for rainbow trout, an important zoonotic disease affecting fish, other animals and humans. However, the description of Lactococcus petauri revealed that most L. garvieae isolates belonged to L. petauri by genome analysis. In the present study, we revealed a comprehensive genomic comparison between L. garvieae and L. petauri species covering the determination of genome characteristics, overall genome-relatedness indices, genomic islands including virulence genes and antimicrobial resistance models, protein families, habitat preference and ecological distribution features. Besides, L. garvieae and L. petauri strains were also evaluated by whole-cell MALDI-TOF MS analysis. Notably, we assessed a species-specific primer design, named LG_IBS and LP_IBS, based on the genome analysis to distinguish L. garvieae and L. petauri for the first time by basic PCR. In addition to genotypic analyses, 177 L. garvieae isolates and four reference strains were tested to ferment sucrose which was determined as the main phenotypic test to differentiate these species for the first time. Our study revealed that L. garvieae and L. petauri have unique genes, and the designed primers, LG_IBS and LP_IBS, clearly differentiate both isolates. In phenotypical comparison, L. garvieae was observed as negative for sucrose fermentation, whereas L. petauri gave a positive reaction. Therefore, we confirmed that the sucrose fermentation test and designed PCR primers have 100% differentiation power for the isolates from two different laboratories for the first time. Moreover, a comparative analysis of protein families for L. garvieae and L. petauri strains revealed that the genomes of L. garvieae strains do not encode the genes required for sucrose metabolism, whereas L. petauri genomes encode genes for fructokinase, sucrose operon repressor and sucrose-6-phosphate hydrolase. Although both species exhibit similar habitat preferences, they also differ in the distribution of various genomic islands, including virulence factors and antimicrobial resistance genes. In conclusion, we propose that sucrose fermentation and newly designed primers yielding different amplicons for L. garvieae and L. petauri could be employed to differentiate the members of both species.