No abstract

Despite the use of several Weissella (W.) strains for biotechnological and probiotic purposes, certain species of this genus were found to act as opportunistic pathogens, while strains of W. ceti were recognized to be pathogenic for farmed rainbow trout. Herein, we investigated the pathogenic potential of weissellas based on in silico analyses of the 13 whole genome sequences available to date in the NCBI database. Our screening allowed us to find several virulence determinants such as collagen adhesins, aggregation substances, mucus-binding proteins, and hemolysins in some species. Moreover, we detected several antibiotic resistance-encoding genes, whose presence could increase the potential pathogenicity of some strains, but should not be regarded as an excluding trait for beneficial weissellas, as long as these genes are not present on mobile genetic elements. Thus, selection of weissellas intended to be used as starters or for biotechnological or probiotic purposes should be investigated regarding their safety aspects on a strain to strain basis, preferably also by genome sequencing, since nucleotide sequence heterogeneity in virulence and antibiotic resistance genes makes PCR-based screening unreliable for safety assessments. In this sense, the application of W. confusa and W. cibaria strains as starter cultures or as probiotics should be approached with caution, by carefully selecting strains that lack pathogenic potential.

A collection of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives were screened in depth in the present study for their probiotic potential. Several strains could be considered promising probiotic candidates since they showed good growth capacity and survival under simulated gastro-intestinal conditions (acidic pH of 1.5, up to 4% of bile salts and 5 mM of nitrate), good ability to auto-aggregate which may facilitate their adhesion to host cells as multiple aggregates and the subsequent displacement of pathogens. Moreover, co-aggregation of lactobacilli with pathogenic bacteria was shown with Listeria innocua, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis as good defense strategy against gut and food pathogens. Furthermore, they exhibited adherence to intestinal and vaginal cell lines, such property could be reinforced by their capacity of biofilm formation which is also important in food matrices such as the olive surface. Their antagonistic activity against pathogenic bacteria by means of acids and plantaricins, and also their different functional properties may determine their efficacy not only in the gastro-intestinal tract but also in food matrices. Besides their ability to ferment several prebiotics, the new evidence in the present study was their capacity to ferment lactose which reinforces their use in different food matrices including dairy as a dietary adjunct to improve lactose digestibility. Lactobacillus pentosus CF2-10N was selected to have the best probiotic profile being of great interest in further studies. In conclusion, spontaneous fermented Aloreña table olives are considered a natural source of potential probiotic L. pentosus to be included as adjunct functional cultures in different fermented foods.

Lactobacillus pentosus MP-10 is a potential probiotic lactic acid bacterium originally isolated from naturally fermented Aloreña green table olives. The entire genome sequence was annotated to in silico analyze the molecular mechanisms involved in the adaptation of L. pentosus MP-10 to the human gastrointestinal tract (GIT), such as carbohydrate metabolism (related with prebiotic utilization) and the proteins involved in bacteria–host interactions. We predicted an arsenal of genes coding for carbohydrate-modifying enzymes to modify oligo- and polysaccharides, such as glycoside hydrolases, glycoside transferases, and isomerases, and other enzymes involved in complex carbohydrate metabolism especially starch, raffinose, and levan. These enzymes represent key indicators of the bacteria’s adaptation to the GIT environment, since they involve the metabolism and assimilation of complex carbohydrates not digested by human enzymes. We also detected key probiotic ligands (surface proteins, excreted or secreted proteins) involved in the adhesion to host cells such as adhesion to mucus, epithelial cells or extracellular matrix, and plasma components; also, moonlighting proteins or multifunctional proteins were found that could be involved in adhesion to epithelial cells and/or extracellular matrix proteins and also affect host immunomodulation. In silico analysis of the genome sequence of L. pentosus MP-10 is an important initial step to screen for genes encoding for proteins that may provide probiotic features, and thus provides one new routes for screening and studying this potentially probiotic bacterium.

Acidity often prevents the undesirable microbial colonization both in fermented foods and under gastric conditions. Thus, the acid resistance of Lactobacillus pentosus strains used as starter cultures and/or probiotics requires further understanding. This was investigated by means of comparative proteomic approach using three strains representing the phenotypes: resistant (AP2-15), intermediate (AP2-18) and sensitive (LP-1) to acidic conditions. Proteomic analysis of constitutive phenotypes revealed that the intrinsic resistance of L. pentosus is associated with the over-production of three principal proteins: 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 2 (PGAM-d), elongation factor G and 50S ribosomal protein L10, and additionally on ATP synthase subunit beta and chaperone protein DnaK; they are associated with metabolic pathways of proteins and carbohydrates, energy production and stress responses. Suggested protein biomarkers for acid resistance in L. pentosus include elongation factor G and PGAM-d, both being abundantly found in the constitutive proteome of the resistant phenotype under standard and acidic conditions. Furthermore, L. pentosus strains pre-exposed to acids displayed enhanced probiotic function such as auto-aggregation ability via surface proteins. We conclude that pre-exposure of probiotic L. pentosus strains to acid may strategically enhance their performance as starter cultures and probiotics.

Lactobacillus pentosus MP-10, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. The genome sequence of L. pentosus MP-10 is currently considered the largest genome among lactobacilli, highlighting the microorganism’s ecological flexibility and adaptability. Here, we analyzed the complete genome sequence for the presence of acquired antibiotic resistance and virulence determinants to understand their defense mechanisms and explore its putative safety in food. The annotated genome sequence revealed evidence of diverse mobile genetic elements, such as prophages, transposases and transposons involved in their adaptation to brine-associated niches. In-silico analysis of L. pentosus MP-10 genome sequence identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) as an immune system against foreign genetic elements, which consisted of six arrays (4–12 repeats) and eleven predicted cas genes [CRISPR1 and CRISPR2 consisted of 3 (Type II-C) and 8 (Type I) genes] with high similarity to L. pentosus KCA1. Bioinformatic analyses revealed L. pentosus MP-10 to be absent of acquired antibiotic resistance genes, and most resistance genes were related to efflux mechanisms; no virulence determinants were found in the genome. This suggests that L. pentosus MP-10 could be considered safe and with high-adaptation potential, which could facilitate its application as a starter culture and probiotic in food preparations.

We report here a 3,698,214-bp complete genome sequence of a potential probiotic Lactobacillus pentosus strain, MP-10, isolated from brines of naturally fermented Aloreña green table olives; it is considered the largest sequenced genome among lactobacilli to date. The annotated genome sequence revealed the presence of 3,558 open reading frames (ORFs) and 87 structural RNAs.

We analyzed the adhesion capacity to mucus of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives using an immobilized mucin model. On the basis of their adhesive capacity to mucin, three phenotypes were selected for cell-wall protein proteomic analysis to pinpoint proteins involved in the adhesion process: the highly adhesive L. pentosus CF1-43 N (73.49% of adhesion ability), the moderately adhesive L. pentosus CF1-37 N (49.56% of adhesion ability) and the poorly adhesive L. pentosus CF2-20P (32.79% of adhesion ability). The results revealed four moonlighting proteins over-produced in the highly adhesive L. pentosus CF1-43 N, which were under/not produced in the other two L. pentosus strains (CF1-37 N and CF2-20P). These proteins were involved in glycolytic pathway (phosphoglycerate mutase and glucosamine-6-phosphate deaminase), stress response (small heat shock protein) and transcription (transcription elongation factor GreA). Furthermore, the relative fold change in gene expression analysis showed significant up-regulation of the genes coding for these four moonlighting proteins in the highly adhesive L. pentosus CF1-43 N versus the poorly adhesive L. pentosus CF2-20P and also in response to mucin for 20 h which clearly indicate the significant role of these genes in the adhesion capacity of L. pentosus. Thus, these proteins could be used as biomarkers for mucus adhesion in L. pentosus. On the other hand, mucin exposure induced other probiotic effects in L. pentosus strains, enhancing their co-aggregation ability with pathogens and possible inactivation.