Aims:The objective of this study was to isolate multifunctional bacteriocin-producing strains; to characterize the expressed bacteriocin for the control of Listeria monocytogenes and vancomycin-resistant Enterococcus; to evaluate the safety of studied strains; and to explore their antifungal activity. Methods and Results: Two Pediococcus strains were isolated from silage samples obtained from an organic farm in Belogradchik, Bulgaria. The strains were identified by 16S rRNA sequencing analysis and characterized as bacteriocins producers. Strong antimicrobial activity was detected against more than 74 different strains of Listeria monocytogenes, 27 different vancomycin-resistant Enterococcus strains. In addition, studied strains were able to inhibit the growth of strains of Alternaria alternate, Aspergillus flavus, Aspergillus niger, Cladosporium sphaerospermum, Penicillium chrysogenum and Penicillium expansum. Some aspects of the antimicrobial mode of action were evaluated, including killing curves and aggregation properties. Both strains generated positive PCR results for the presence of pediocin PA-1, but not for other bacteriocins evaluated in this screening process. Metabolomic analysis of the cell-free supernatants from both strains was performed in order to explain the observed antifungal activity against different moulds. According to PCA and PLS-DA score plot, P. acidilactici ST3522BG and P. pentosaceus ST3633BG were clearly clustered from control (MRS). Increases in the production of benzoic acid, 2-hydroxyisocaproic acid, β-phenyl-lactic acid, α-hydroxybutyric acid and 1,3-butanediol were recorded, these metabolites were previously described as antifungal. Conclusions: Pediococcus acidilactici ST3522BG and P. pentosaceus ST3633BG were evaluated as producing bacteriocin strains with high specificity against Listeria and vancomycin-resistant Enterococcus species. In addition, both investigated Pediococcus strains were evaluated as producer of effective antifungal metabolites with potential for the inhibition of mycotoxin-producing moulds. Significance and Impact of the Study:To the best of our knowledge, this report is a pioneer in the evaluation of Pediococcus strains isolated from silage with highly specific bacteriocinogenic antimicrobial activity against Listeria spp. and vancomycin-resistant Enterococcus spp., and antifungal activity against mycotoxin-producing moulds.

Three out of one hundred eighty putative LAB isolates from Korean traditional fermented soybean paste were identified to be unique and bacteriocinogenic strains. Based on phenotypic and 16S rRNA sequencing analysis, selected strains were identified as Enterococcus faecium ST651ea, E. faecium ST7119ea and E. faecium ST7319ea. The bacteriocinogenic properties of the studied strains were evaluated against Listeria monocytogenes ATCC15313, Listeria innocua ATCC33090 and vancomycin-resistant E. faecium VRE19 of clinical origin. The strains E. faecium ST651ea, ST7119ea and ST7319ea expressed bacteriocins with an activity of 12,800 AU/mL, 25,600 AU/mL and 25,600 AU/mL, respectively, recorded against L. monocytogenes ATCC15131. According to the PCR-based screening of bacteriocin-related genes, which was further confirmed through amplicon sequencing, showed that strain E. faecium ST651ea carries entB and entP genes, whereas both E. faecium ST7119ea and ST7319ea strains harbor entA and entB genes. The molecular size of expressed bacteriocins was estimated by tricine-SDS-PAGE showing an approximative protein size of 4.5 kDa. The assessment of the spectrum of activity of bacteriocins ST651ea, ST7119ea and ST7319ea showed strong activity against most of clinical VRE isolates, majority of other Enterococcus spp. and Listeria spp. Bacteriocins ST651ea, ST7119ea and ST7319ea were partially purified by combination of 60% ammonium sulfate precipitation and hydrophobic chromatography on the SepPakC18 column. Challenge test with semi-purified (60% 2-propanol fraction) bacteriocins resulted in a significant reduction of viable cells for all test organisms. Thus, indicating that all the bacteriocins evaluated can be used as potential biocontrol in food and feed industries as well as an alternative treatment for VRE-related infections in both veterinary and clinical settings.

Aims: The objective of this study was to isolate a bacteriocin-producing strain and to characterize the expressed bacteriocin for the control of Listeria monocytogenes with aim of biopreservation application. Methods and Results: Soil samples from a Korean organic farm were subjected to microbiological analysis for isolation of potential bacteriocinogenic LAB, based on a three-level approach, using L. monocytogenes ATCC 15313 as an indicator test micro-organism. From a total of 17 isolates with inhibitory potential, seven were confirmed to be bacteriocin producers. The selected isolates were differentiated based on their morphology, catalase reaction, sugar fermentation profile obtained by API50CHL and by RAPD-PCR generating two unique profiles. One of the isolates, ST110LD, a specific strong producer of anti-Listeria bacteriocins (12 800 AU ml À1 ) was identified as Leuconostoc citreum. The proteinaceous nature of the inhibitory compound produced by Leuc. citreum ST110LD was confirmed through treatment with pepsin and a-chymotrypsin. Bacteriocin activity was observed to be not affected by the presence of milk, NaCl, SDS, Tween 80 or glycerol. Bacteriocin ST110LD effectively inhibited the growth of exponentially growing L. monocytogenes ATCC 15313 during a 10-h incubation period in BHI at 37°C. In addition, this bacteriocin showed specific inhibition of only Listeria spp., but did not inhibit the growth of beneficial cultures included in the microbial test panel for assessment of the spectrum of activity. Conclusions: Leuconostoc citreum ST110LD was evaluated as safe bacterium strain, producing bacteriocin with high specificity against listerial and enterococcal species. Specificity of producer strain and expressed bacteriocin can be explored in biopreservation of different fermented food products or applied in biotherapy of antibiotic resistant listerial or enterococcal infections. Significance and Impact of the Study: To the best of our knowledge, this is the first report of bacteriocin produced by Leuc. citreum strain with highly specific antimicrobial activity against Listeria sp. and Enterococcus sp.

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Significance and Impact of the Study: We evaluated the safety and beneficial features of two bacteriocinogenic strains: P. acidilactici ST3522BG and P. pentosaceus ST3633BG. The concept 'safety first' needs to be the starting point in any proposal for application of new beneficial culture, although they belong to a species with historical safety record. Two evaluated Pediococcus strains were negative for biogenic amine production, haemolytic activity and antibiotic resistance according to performed physiological tests. At the DNA level, both strains generated negative results for the majority of the tested virulence genes, biogenic amines and vancomycin resistance genes. Apart from their safety, some beneficial properties of studied strains were evaluated, including their effectiveness in control of Listeria monocytogenes on silage model system, demonstrating successful inhibition of artificially added pathogen by both the Pediococcus strains.