Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay

Abstract

Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level

“…Of the 29 strains belonging to L. rhamnosus, PCR products of the expected length could not be obtained in six cases (data not shown). Similar cases are also described by Bottari et al [33].…”

“…On the other hand, especially for bacteria with high genetic similarity, the design and optimization of the reaction itself can be relatively difficult [31,32]. In this study, we used two sets of primers that were designed based on the tuf and mutL gene sequences (Table S1), to identify species of the L. casei group [16,33].…”

“…The second set of primers described by Bottari et al [33] made it possible to generate single, species-specific PCR products of 666 bp (L. casei), 253 bp (L. paracasei) and 801 bp (L. rhamnosus). This multiplex PCR reaction confirmed the taxonomic relevance of 28 tested strains.…”

Comparative Genomics of the Genus Lactobacillus Reveals Robust Phylogroups That Provide the Basis for Reclassification

“…Multilocus sequence analysis based on 29 ribosomal proteins and 12 phylogenetic markers and phylogenetic tree construction. A maximum likelihood phylogeny was built from 29 ribosomal proteins and 12 housekeeping markers, which were chosen on the basis of their use in published multilocus sequence typing schemes and their presence in the genomes of the 222 strains (Table S2) (38).…”

The Lactobacillus casei Group: History and Health Related Applications

“…Another MLST study of 224 L. casei isolates from fermented foods using 10 housekeeping genes found no correlation with geographic location or food source, implying a complex evolutionary history and contradicting previous studies ( Bao et al, 2016 ). Other single target genes used for species discrimination include housekeeping genes such as the chaperone protein gene, dnaJ ( Huang et al, 2015 ), dnaK ( Huang and Lee, 2011 ), yycH ( Huang et al, 2014 ), and mutL ( Bottari et al, 2017 ). Indeed, it was found that the dnaJ gene provided better resolution than the 16S rRNA subunit gene for comparisons within and between species, and this has been suggested as an alternative marker ( Huang et al, 2015 ).…”