Phenotypic and Genotypic Characteristics of Enterocin Producing Enterococci against Pathogenic Bacteria

The study investigated the antimicrobial activity of 13 enterococcal strains (E. faecalis -8, E. faecium-2, E. hirae-2, E. spp.-1) isolated from our traditional cheeses against pathogen microorganisms. Also, it includes the detection of the following enterocin structural genes: enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin Q, enterocin EJ97 and cytolysin by using PCR method. All isolates inhibited growth of L. monocytogenes and L.innocua. One isolate had a broader antimicrobial activity. None of the isolates showed inhibitory activity against S. enteritidis, E. coli and Y. enterocolitica. The genes enterocin P, cytolysin and enterocin A were the most frequently detected structural genes among the PCR positive strains. No amplification was obtained in two strains E. faecalis-25 and E. faecalis-86. Three different genes were identified in some strains. With the exclusion of strains possessing a virulence factor, such as cytolysin, producers of more than one enterocins could be of a great technological potential as protective cultures in the cheese industry.

Production, Characterization, and Antimicrobial Activity of a Bacteriocin from Newly Isolated Enterococcus faecium IJ-31

This work aimed to isolate and characterize Enterococcus spp. from indigenous dairy products in Islamabad, Pakistan. By classical microbiological techniques, one strain from a butter sample was identified to be Enterococcus faecium, and we designated it E. faecium IJ-31. The precise identity of this strain was then established by determining the sequence of its 16S and 23S rRNA genes. The sequence homology searches revealed matches with a number of previously reported strains, such as E. faecium HN-N3 and HN-N29, both isolated from swine intestines in China. The newly isolated strain was tested for hemolysis and antibiotic sensitivity; it was nonhemolytic on sheep and human blood and sensitive to vancomycin. Consistent with its vancomycin sensitivity, repeated attempts to amplify the vancomycin resistance genes vanA and vanB failed. Similar attempts to amplify the virulence genes gelE, agg, and cyl also failed, suggesting the absence of these genes. In contrast, the enterocin-P gene, entP, readily amplified with primers based on the previously reported sequences, and the deduced sequence showed near identity with a number of reported sequences from E. faecium. Further, the 71-residue enterocin-P sequence from strain IJ-31 is only the second complete sequence reported. The enterocin was partially purified and tested for antibacterial activity. It showed potent inhibitory activity against many bacteria, including Listeria monocytogenes, a routinely used test strain. Further, the enterocin showed potent activity against Bacillus subtilis and Bacillus cereus. The enterocin retained antibacterial activity even following heating to 121°C for 15 min. Further, it also retained activity after exposure to pH values ranging from 4 to 10. However, proteinase K treatment rendered the peptide nonfunctional.

Chimeras of Mature Pediocin PA-1 Fused to the Signal Peptide of Enterocin P Permits the Cloning, Production, and Expression of Pediocin PA-1 in Lactococcus lactis

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SPentP) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SPentP:pedA) and pMPP14i (SPentP:pedA+pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti–PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SPEntP is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.