Novel approaches for the species-level identification of lactobacilli: integration of 16S rRNA gene sequencing with MALDI-TOF MS and 1H-NMR

Background: Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of Lactobacillus genus, its species identification still poses several difficulties. Since traditional genotypic methods are relatively expensive and do not relate to the physiological properties, new approaches are needed for lactobacilli identification. The aim of this study was to assess the integration of different methods, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic characterization of Lactobacillus species. Material/methods: A total of 40 Lactobacillus strains, isolated from clinical specimens, included in probiotics or belonging to collections of microorganisms were used in this study. The genotypic identification at species level was obtained by 16S rRNA gene sequencing. A phylogenetic tree based on 16S rDNA sequences was created using MEGA 6 software. MALDI-TOF MS analysis was performed after protein extraction starting from bacterial cell pellets, using a Bruker Microflex instrument. For species identification, spectra collected were compared with the ones of the reference database. A clustering analysis was performed by the generation of a score-oriented dendrogram, starting from the main spectrum profiles (MSPs) of each strain with MALDI Biotyper 3.1 software. 1H-NMR metabolomic analysis was performed using an AVANCE III spectrometer (Bruker), both on the intracellular metabolome, after appropriate bacterial lysis, and on the extracellular supernatants, after an overnight growth in culture broth. Differences in the metabolome composition were calculated by means of an unpaired Wilcoxon test Results: 16S rRNA gene sequencing led to the following identifications: 7 L. crispatus, 7 L. gasseri, 5 L. acidophilus, 5 L. delbrueckii, 2 L. vaginalis, 2 L. reuteri, 6 L. plantarum, 1 L. pentosus, 2 L. rhamnosus, 2 L. casei/paracasei and 1 L. brevis. For species level identification, an excellent agreement between MALDI-TOF and the genotypic analysis was found (97.5%), with only one discordant result. Moreover, the phylogenetic tree based on 16S rRNA gene sequences and the MALDI-TOF dendrogram were highly similar. The metabolomic approach led to identify a panel of molecules whose variations were strictly associated with the taxonomy. As an example, considering the extracellular metabolome, L. crispatus showed the highest glucose consumption (P=1 × 10-3), whereas L. acidophilus species was characterized by the highest-level production of acetone and pyruvate (P=3 × 10-4 and P=1 × 10-3). Conclusions: MALDI-TOF MS is an excellent method for lactobacilli species identification and could represent a simple and reliable tool for their taxonomic characterization. Metabolomic analysis could be useful in correlating lactobacilli taxonomy with biological properties, as their anti-microbial activity or fermentation capacity for food production.