Characterization of Partially Purified Bacteriocins Produced by Enterococcus faecium Strains Isolated from Soybean Paste Active Against Listeria spp. and Vancomycin-Resistant Enterococci

Abstract

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.