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.

Identification of Species and Subspecies of Lactic Acid Bacteria Present in Spanish Cheeses Type “Torta” by MALDI-TOF MS and pheS gene Analyses

“…From the species previously identified in the "Torta del Casar" cheese [5], in the present work only L. lactis has been identified in the "Torta de Trujillo" cheese. Nevertheless, the species identified in this study have been found in some of the European artisanal cheeses elaborated with raw milk [7][8][9][10][11][12][13].…”

“…However, the 16S rRNA gene has limitations in differentiating among closely related species and subspecies of LAB needing additional techniques, such as the sequencing of protein-coding genes or MALDI-TOF MS [12]. The latter technique has been used to identify the LAB from a French artisanal cheese, showing the presence of species such as La.…”

A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production

“…13 MHz, was employed to record 1 H-NMR spectra at 298 K. The HOD residual signal was suppressed by means of presaturation. Following the procedure described in [21], the signals from broad resonances originating from large molecules were suppressed by a CPMG-filter, while the HOD residual signal was suppressed by means of presaturation. Each spectrum was acquired by summing up 256 transients using 32 K data points over a 7184 Hz (for an acquisition time of 2.28 s).…”

Identification, Classification and Screening for γ-Amino-butyric Acid Production in Lactic Acid Bacteria from Cambodian Fermented Foods

“…MALDI-TOF MS has been widely used for the rapid identification and taxonomic characterization of Lactobacillus spp. [15,30,[34][35][36][37][38][39][40][41][42], Enterococcus spp. [33], and Pediococcus spp.…”