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

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.

Reclassification of Lactobacillus catenaformis (Eggerth 1935) Moore and Holdeman 1970 and Lactobacillus vitulinus Sharpe et al. 1973 as Eggerthia catenaformis gen. nov., comb. nov. and Kandleria vitulina gen. nov., comb. nov., respectively.

The development of molecular tools and in particular the use of 16S rRNA gene sequencing has had a profound effect on the taxonomy of many bacterial groups. Gram-positive organisms that encompass the genera Lactobacillus and Clostridium within the Firmicutes are examples of taxa that have undergone major revisions based on phylogenetic information. A consequence of these reorganizations is that a number of organisms are now recognized as being misclassified. Previous studies have demonstrated that Lactobacillus catenaformis and Lactobacillus vitulinus are phylogenetically unrelated to Lactobacillus sensu stricto, being placed within the Clostridia rRNA cluster XVII. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, it is proposed that L. catenaformis and L. vitulinus be reclassified in two new genera, named respectively Eggerthia gen. nov., with the type species Eggerthia catenaformis gen. nov., comb. nov. (type strain DSM 20559(T) = ATCC 25536(T) = CCUG 48174(T) = CIP 104817(T) = JCM 1121(T)) and Kandleria gen. nov., with the type species Kandleria vitulina gen. nov., comb. nov. (type strain LMG 18931(T) = ATCC 27783(T) = CCUG 32236(T) = DSM 20405(T) = JCM 1143(T)).

Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches.

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.

Lactobacillus durianis Leisner et al. 2002 is a later heterotypic synonym of Lactobacillus vaccinostercus Kozaki and Okada 1983.

The taxonomic status of the species Lactobacillus durianis and Lactobacillus vaccinostercus is briefly summarized and experimental evidence concerning their similarity is presented. Highly similar 16S rRNA gene sequences (99.8 % similarity over 1,523 bp), partial recA gene sequences (99.5 % similarity over 600 bp) and partial hsp60 gene sequences (99.1 % similarity over 924 bp) suggest that the two species are closely related. Moreover, a high DNA-DNA binding level (87 %) and similar genomic DNA G+C contents (41-44 mol% for both species) as well as similar biochemical characteristics support the evidence that they constitute a single species. Consequently, according to Rules 38 and 42 of the Bacteriological Code, the name Lactobacillus vaccinostercus, the oldest legitimate name, must be maintained and the name Lactobacillus durianis should be considered a later heterotypic synonym.

Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices.

Fourteen strains isolated from vegetable sources and identified as belonging to Lactobacillus plantarum presented an atypical pattern of amplification with a species-specific multiplex-PCR assay. Phylogenetic analysis of two protein-encoding genes, recA (encoding the recombinase A protein) and cpn60 (encoding the GroEL chaperonin), as well as phenotypic and genomic traits revealed a homogeneous group of very closely related strains for which subspecies status is proposed, with the name Lactobacillus plantarum subsp. argentoratensis. The type strain is DKO 22(T) (=CIP 108320(T)=DSM 16365(T)).

Lactobacillus delbrueckii subsp. indicus subsp. nov., isolated from Indian dairy products.

Four strains isolated from Indian dairy products and initially identified as Lactobacillus delbrueckii could not be assigned to a definite subspecies because molecular identification and phenotypic traits did not agree with those of recognized subspecies of L. delbrueckii. Hybridization of total DNA (78-86 % against type strains of the other three subspecies), AFLP and RAPD-PCR fingerprints, phylogenetic analysis based on 16S rRNA gene sequences and sequence analysis of two coding genes (recA and hsp60), together with phenotypic profiles, indicated that the four strains form a coherent cluster and represent a novel subspecies, for which the name Lactobacillus delbrueckii subsp. indicus subsp. nov. is proposed. The type strain is NCC725T (=LMG 22083T=DSM 15996T).