Novel approaches for the taxonomic and metabolic characterization of lactobacilli: Integration of 16S rRNA gene sequencing with MALDI-TOF MS and 1H-NMR

Abstract

Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological 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 genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic and metabolic characterization of Lactobacillus species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 Lactobacillus strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some Lactobacillus species.

Fig 1. Phylogenetic tree based on lactobacilli 16S rRNA sequences.

The Neighbor-Joining method was used to infer evolutionary history. The evolutionary distances were computed using the Maximum Likelihood method based on Tamura-Nei model [32]. The tree is drawn to scale, with branch lengths measured in number of substitutions per site. The bootstrap values inferred from 1000 replicates is shown next to the branches. The analysis involved 40 nucleotides sequences. All positions containing gaps and missing data were eliminated. The tree was obtained by using MEGA 6 software.

Fig 2. Cluster analysis of MALDI-TOF MS spectra obtained from the Lactobacillus strains included in the study.

In the MSP dendrogram, relative distance between isolates is displayed as arbitrary units. Zero indicates complete similarity and 1,000 indicates maximum dissimilarity.

Fig 3. Variations in lactobacilli extracellular metabolome.

Box plots represent the concentration (mM) of extracellular metabolites which vary significantly among the diverse Lactobacillus species considered. Metabolites were quantified in cell free supernatants by ¹H-NMR. Lines within the boxes indicate the median values of the metabolite concentration and each box represents the interquartile range (25–75th percentile). The bottom and top bars indicate the 10th and 90th percentiles, respectively. Boxes were colored in grey to highlight the Lactobacillus species groupings that show significantly different concentration of the corresponding metabolite (P<0.05, Bonferroni-adjusted).

Fig 4. Variations in lactobacilli intracellular metabolome.

Box plots represent the concentration (mM) of intracellular metabolites which vary significantly among the diverse Lactobacillus species considered. Metabolites were quantified in cellular lysates by ¹H-NMR. Lines within the boxes indicate the median values of the metabolite concentration and each box represents the interquartile range (25–75th percentile). The bottom and top bars indicate the 10th and 90th percentiles, respectively. Boxes were colored in grey to highlight the Lactobacillus species groupings that show significantly different concentration of the corresponding metabolite (P<0.05, Bonferroni-adjusted).