Variability at gene and genome levels revealed different genomic lineages in Lactococcus garvieae

Lactococcus garvieae is a pathogen that causes septicemia in fish and serious damage to fish aquaculture worldwide, but recently has been isolated from human, ruminant, water sources and several food matrix. In the present work we monitored the population structure of L. garvieae strains, including isolates of dairy, fish, meat, vegetable and cereal origin. This bacterial collection was explored using molecular polyphasic approach comprising PCR-ribotyping, Rep and RAPD-PCR analyses and multilocus restriction typing (MLRT) carried out on six partial genes. This approach allowed high-resolution cluster analysis in which two major groups were distinguishable: one group included dairy isolates, the other group meat isolates. Unexpectedly, some strains coming from fish grouped with dairy isolates whereas the other with meat isolates. Likewise, strains isolated from vegetables allocated between the two main groups. These findings revealed high variability within the species at both gene and genome levels. The observed genetic heterogeneity among Lactococcus garvieae strains was not entirely coherent with the ecological niche of origin of the strains, but rather supports the idea of an early separation of L. garvieae population into independent genomic lineages. To better define this result, a multilocus sequence typing (MLST) scheme was developed for L. garvieae, based on the sequence analysis of eight genes (α-acetolactate synthase als, α-subunit of ATP synthase atpA, bacterial elongation factor EF-Tu tuf, glyceraldehyde-3-phosphate dehydrogenase gapC, β-subunit DNA gyrase gyrB, β’-subunit RNA polymerase rpoC, D-alanine-D-alanyl carrier protein ligase dltA, galactose permease galP). On the 17 sequence types determined, to estimate intraspecific genetic diversity of data samples and to detect particular population structures several statistical analysis were carried out. The average nucleotide diversity, numbers of synonymous and nonsynonymous mutations, the measure of contribution of recombination and mutation in the creation of sample population were determined to delineate a phylogenetic framework of our collection of L. garvieae.