Análise genômica e caracterização funcional de Limosilactobacillus fermentum JAC 231 como candidato a probiótico

Abstract

The genus Lactobacillus is composed of Gram-positive, catalase-negative, acid-tolerant, non- spore-forming, carbohydrate-fermenting bacteria. A recent reclassification of this genus led to

a change in nomenclature, generating 23 new genera. The species Lactobacillus fermentum was renamed Limosilactobacillus fermentum. L. fermentum strains of occupy various niches, and in humans it is considered an indicator of a normal and healthy microbiota. Many strains have been investigated for their role in maintaining the epithelial barrier function, competitive exclusion, immunomodulatory activity and synthesis of antimicrobial substances (lactic acid, H2O2 and bacteriocins) with broad activity against pathogenic microorganisms, motivating their use as probiotics. In this sense, the characterization of probiotic activity and genome sequencing technologies have been important tools to obtain information about the metabolic and safety aspects of each strain, directing research towards its biotechnological and therapeutic use. This study aimed to sequence the complete genome of the vaginal isolate, L. fermentum JAC 231, analyzing general and safety aspects of the strain, investigating the genotypic and phenotypic bases for its probiotic potential, such as tolerance to diverse gastrointestinal conditions (bile salts, adhesion to mucin, adhesion to eukaryotic cells and pH), presence of resistance and virulence genes. Here we expand the characterization of the antimicrobial and anti-adhesion activity of this strain, evaluating its effect on Klebisiella pneumoneae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonella enteritidis ATCC 13076, Enterococcus faecalis ATCC 29212 and enteropathogenic Escherichia coli O127:H6 E2348/69-EPEC. From the bioinformatics analysis, we identified that the genome contained 2.064.918 bp, with a GC content of 51.4%, presenting 1.993 protein-coding sequences related mainly to the metabolism of proteins, carbohydrates and amino acids. Comparative analysis revealed that L. fermentum JAC 231 shares 2.204 orthologous clusters with the other five probiotic strains of L. fermentum (MCC 2760, AGR 1485, ATCC 23271, CECT 5716, e IFO 3956). Genome investigation demonstrated that L. fermentum JAC 231 has genes encoding a bacteriocin (Enterolysin A), stress tolerance genes, vitamin synthesis, and survival under gastrointestinal conditions. Furthermore, we observed the absence of genes for virulence factors and the presence of genes for intrinsic resistance to vancomycin. Phenotypically, L. fermentum JAC 231, was susceptibility to most of the antibiotics tested, except vancomycin, as already visualized in the genome analysis. It was able to tolerate acid pH (with growth of 107.7% at pH 2 and 75.29% at pH 4), as well as bile salts at concentrations of 0.5% (87.34) and 1% (84.76%). It also demonstrated adhesion capacity to mucin and HeLa cells, presenting logarithmic growth of 5.9 and 5.7, respectively. Additionally, it presented antagonistic activity in the overlay test, inhibiting the growth of all bacteria used, with inhibition zones ranging from 16.6 ± 2.08 to 28.3 ± 2.88 mm, in addition to the anti-adhesion potential observed, mainly, in the displacement and exclusion tests. In this way, our study confirms the characteristics and safety profile of L. fermentum JAC 231 as a probiotic strain. Genome analysis proved to be effective in evaluating the beneficial properties of this strain, showing correlation with the data obtained in vitro. In addition, we verified the antagonistic and anti-adhesive activity displayed by L. fermentum JAC 231. Further studies are needed to elucidate the mechanisms by which this promising strain exerts its broad-spectrum antimicrobial effects.