Biochemical, genetic and transcriptional characterization of multibacteriocin production by the anti-pneumococcal dairy strain Streptococcus infantarius LP90.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

Evaluation of bacteriocinogenic activity, safety traits and biotechnological potential of fecal lactic acid bacteria (LAB), isolated from Griffon Vultures (Gyps fulvus subsp. fulvus).

BACKGROUND: Lactic acid bacteria (LAB) are part of the gut microbiota and produce ribosomally synthesized antimicrobial peptides or bacteriocins with interest as natural food preservatives and therapeutic agents. Bacteriocin-producing LAB are also attractive as probiotics. Griffon vultures (Gyps fulvus subspecies fulvus) are scavenger birds that feed almost exclusively on carrion without suffering apparent ill effects. Therefore, griffon vultures might be considered a reservoir of bacteriocin-producing lactic acid bacteria (LAB) with potential biotechnological applications. RESULTS: Griffon vulture feces were screened for LAB with antimicrobial activity, genes encoding bacteriocins, potential virulence determinants, susceptibility to antibiotics, genotyping and characterization of bacteriocins. In this study, from 924 LAB evaluated 332 isolates (36 %) showed direct antimicrobial activity against Gram-positive bacteria only. The molecular identification of the most antagonistic 95 isolates showed that enterococci was the largest LAB group with antimicrobial activity (91 %) and E. faecium (40 %) the most identified antagonistic species. The evaluation of the presence of bacteriocin structural genes in 28 LAB isolates with the highest bacteriocinogenic activity in their supernatants determined that most enterococcal isolates (75 %) encoded multiple bacteriocins, being enterocin A (EntA) the largest identified (46 %) bacteriocin. Most enterococci (88 %) were resistant to multiple antibiotics. ERIC-PCR and MLST techniques permitted genotyping and recognition of the potential safety of the bacteriocinogenic enterococci. A multiple-step chromatographic procedure, determination of the N-terminal amino acid sequence of purified bacteriocins by Edman degradation and a MALDI TOF/TOF tandem MS procedure permitted characterization of bacteriocins present in supernatants of producer cells. CONCLUSIONS: Enterococci was the largest LAB group with bacteriocinogenic activity isolated from griffon vulture feces. Among the isolates, E. faecium M3K31 has been identified as producer of enterocin HF (EntHF), a bacteriocin with remarkable antimicrobial activity against most evaluated Listeria spp. and of elevated interest as a natural food preservative. E. faecium M3K31 would be also considered a safe probiotic strain for use in animal nutrition.

Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture.

BACKGROUND: The microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food. RESULTS: These LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities. CONCLUSIONS: To our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.