Isolation and characterization of bacteriocins from enterococcus species from non-broiler chicken

Abstract

Bacteriocins are small proteins or peptides produced by varieties of microbes with antimicrobial activity against closely related species. These antimicrobial agents are gaining more attention not only as alternative therapeutics, but also as an important bio-preservative in food. The aim of this study was to isolate, identify and characterize bacteriocin and the bacteriocin producing lactic acid bacteria from Malaysian non-broiler chicken and explore on their potential biotechnological properties. Using MRS agar medium, a total number of 56 bacterial strains were isolated from several internal organs such as bile, cecum, gizzard and intestine of a local non-broiler chicken from Kuantan area. Bile was the best source for the isolation of Enterococci followed by gizzard, intestine and cecum. Standard biochemical, morphological and molecular biology analyses were carried out on 7 selected isolates. By using polymerase chain reaction (PCR), amplification of 16S rRNA gene in the presence of 16S rRNA universal primers, 1.5 kb fragments were amplified and through the nucleotide sequencing and homology searches, confirmed the identification of these isolates as strain B3L3 to be Ent. faecium, strains B4L4 and G5L5 as Ent. hirae, strains B5L6, B10L7 and I1L8 as Ent. faecalis and strain C4L10 as Ent. mundtii. These sequences were subsequently submitted to gene bank, accession number was then allocated to each strain, (KC731419 for B3L3; KC731420 for B4L4; KC731421 for B5L6; KC731422 for B10L7; KC731423 for C4L10; and KC731424 for I1L8) respectively. Antimicrobial screening revealed that strain B3L3 showed good potential as producer of bacteriocin. The bacteriocin of the 7 selected strains showed good antimicrobial activity against Methicillin resistance Staphylococcus aureus (MRSA). Extracted bacteriocins from the bacterial pellets of the selected strains gave a molecular weight of approximately 10 kDa based on SDS-PAGE analysis. The purified bacteriocin were highly thermostable, retaining their activity even after treatment at 121°C for 15min, and were stable in a pH range of 4-9 with enhanced activity in acidic pH. There was a loss of activity upon protease treatment; while catalase and lysozyme has no effect on the antimicrobial activity. PCR amplification of the genomic DNA of the selected strains using several Enterocin gene primers revealed that Enterocin_A, Enterocin_L and Enterocin_P genes were present in the genome of E. faecalis I1L8; E. hirae B4L4; while E. mundtii C4L10 has Enterocin_B and Enterocin_P. The structural genes of the bacteriocin were all chromosomally encoded. The PCR product of the enterocin genes was then sequenced, translated and subsequently checked for enterocin homology using a specialized bacteriocin database, Bactibase. Enterocin_P was found to dominate the encoding genes of the bacteriocin among these Enterococcus strains. Based on the database search, the Enterococcus sp. strains I1L8, B4L4, and C4L10 were found to have high homology to Bioticin (47% identity), Bacteriocin L-1077 (63% identity) and BacteriocinL-1077 (83% identity), respectively. In addition, novel double-glycine signals in two of these strains, B4L4 and C4L10 were detected. Furthermore, a novel YGNGP characteristic of class IIa bacteriocins was also detected in bacteriocin from E. mundtii strain C4L10. Bacteriocin sequence from strain C4L10 contained an enzyme glycotransferases conserved domain, which is implicated in anti-proliferative action. Subsequent studies to check for the possibility for an anti-proliferative effect against human cell-lines, futher tests were carried out on four human neoplastic cell lines: breast cancer (MCF7); lung cancer H1299; colon cancer HCT116; and oral cancer HSC3. Oral cancer cell lines (HSC3) was found to be the most sensitive cell line to the bacteriocin of C4L10 with cytotoxic index of IC50 of 9.009µg/ml, followed by breast cancer (IC50 of 11.51µg/ml) then lung cancer (15.25 µg/mL), colon cancer cell line (HCT116) was the least sensitive (IC50 of 20.57µg/ml). The different antimicrobial regions within the bacteriocin amino acid sequences were also predicted. Sequence analysis showed that B4L4 enterocin contained one putative bactericidal region, while strain C4L10 and I1L8 contained two bactericidal regions each. In conclusion, the isolated enterococci are promising candidate for further investigation of their biotechnological potential in relation to their use in dairy food products and antitumor potentials.