title: Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.
abstract: Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, 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 the cremoris genotype, 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 the cremoris or lactis genotype 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 the cremoris group 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 identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.
authors.affiliation: Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villaviciosa, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Fernández
authors.forename: Angel
authors.initials: A
authors.lastname: Alegría
authors.forename: Susana
authors.initials: S
authors.lastname: Delgado
authors.forename: M Cruz
authors.initials: MC
authors.lastname: Martín
authors.forename: Baltasar
authors.initials: B
authors.lastname: Mayo
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D002611:Cheese / Q000382:microbiology*; D024881:Drug Resistance, Bacterial; D005516:Food Microbiology; D005838:Genotype; D013294:Lactococcus lactis / Q000235:genetics* / Q000302:isolation & purification; D008892:Milk / Q000382:microbiology*; D008969:Molecular Sequence Data; D058885:Multilocus Sequence Typing; D010641:Phenotype; D010802:Phylogeny; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D017422:Sequence Analysis, DNA
chemical_list: D012336:RNA, Ribosomal, 16S