Lactobacillus casei group effective identification

Lactobacillus casei, L. paracasei and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei-group) within the facultatively heterofermentative lactobacilli. Some strains belonging to these species have been used for a long time as probiotics in a wide range of different products, and they commonly represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavor development. The close genetic relationship among L. casei-group species, and the related starter cultures, hinders the development of an adequate selective identification method useful to understand the role played by each microbial population in the matrix and the routine biochemical tests often fail to discriminate among closely related species occurring in the same ecological environments, like inside ripened cheeses. The aim of this study was a comprehensive analysis of 75 cheese isolates previously identified as L. casei group, combining the results of multiple approaches. Alternative proposed culture media for Lb. casei- group strains selective growth were tested, giving poor discrimination among strains. Futhermore, a species-specific PCR based on existing primers designed on the 16s rDNA was adapted to laboratory conditions and tested on the isolated strains. This approach allowed the identification of the majority of the tested strains, but still leaving some strains with an uncertain identity. These isolates had to be checked with more than one primer pair in order to avoid misidentification due to possible cross-reactions. Finally, a new species-specific multiplex PCR assay was developed using primers designed on a universal protein encoding gene. The amplicon size was able to rapidly distinguish L. casei, L. paracasei and L. rhamnosus strains.