Phenotypic and phylogenomic characterization of Lactococcus garvieae isolates from rainbow trout (Oncorhynchus mykiss) in Türkiye.

Abstract

Lactococcosis is an important bacterial disease of farmed fish and causes substantial economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture. In this study, Lactococcus garvieae isolates recovered from rainbow trout farms in Türkiye were characterized using phenotypic, molecular, and phylogenomic methods. Among 32 presumptive Lactococcus isolates recovered from 127 dead rainbow trout, four were confirmed as L. garvieae and exhibited identical biochemical characteristics, Pulsed Field Gel Electrophoresis (PFGE) profiles, and broad growth tolerance across different pH, salinity, and temperature conditions. All isolates were presumptively classified as resistant to ciprofloxacin and florfenicol, while remaining susceptible to tetracycline and penicillin. Based on the AMR profiles, strain LG2, which exhibited the most susceptible antimicrobial profile among the isolates, was selected for whole-genome sequencing (WGS). WGS of the representative isolate LG2 generated a single 2,214,687-bp chromosomal contig with 38.5% GC content and 99.0% BUSCO completeness. In silico PCR assigned LG2 to serotype I, and the genome contained an intact capsule-associated cps/kps locus. The chromosomal lsa(D) determinant and an mdt(A)-like efflux-associated gene were detected, whereas no plasmid replicons or acquired quinolone or florfenicol resistance genes were identified, indicating discordance between the phenotypic and genomic AMR results. Taxonomic verification of 236 publicly available Lactococcus assemblies yielded 41 verified public L. garvieae genomes, which, together with LG2, formed a 42-genome within-species dataset. LG2 was most closely related to the Turkish isolate OS-37, sharing 99.96% ANI and differing by three core SNPs; both belonged to ST109, whereas the other Turkish isolates belonged to ST139. cgMLST identified a conserved genomic backbone, while pan-genome analysis identified 5,655 gene clusters and an open pan-genome characterized by a large cloud-gene fraction. These findings demonstrate the importance of species verification in Lactococcus population genomics and reveal substantial accessory-genome diversity within L. garvieae. The genomic features of LG2 provide a basis for future pathogenicity and immunogenicity studies, although experimental validation is required. Overall, these findings highlight the importance of local genomic surveillance for understanding L. garvieae population structure and provide a genomic framework for future region-specific vaccine research.