Comparative Phenotypic and Molecular Genetic Profiling of Wild Lactococcus lactis subsp.lactisStrains of the L. lactis subsp.lactisand L. lactis subsp.cremorisGenotypes, Isolated from Starter-Free Cheeses Made of Raw Milk

Abstract

ABSTRACTTwentyLactococcus lactisstrains with anL. lactissubsp.lactisphenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to theL. lactissubsp.lactisandL. lactissubsp.cremorisgenotypes (lactisandcremorisgenotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of thecremorisgenotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely,atpA,rpoA,pheS,pepN,bcaT,pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into thecremorisorlactisgenotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in thecremorisgroup showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identicalcremorisgenotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with thecremorisgenotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that thelactisandcremorisgenotypes of phenotypicLactococcus lactissubsp.lactisactually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.