Background and Aim: Probiotics are proven beneficial to health since they enhance immunity against dangerous pathogens and increase resistance to illness. Bacteriocin produced by lactic acid bacteria (LAB), demonstrates a broad inhibitory spectrum and therapeutic potential. This study aimed to isolate LAB-producing bacteriocin and investigate the effect of crude bacteriocin on biofilm from methicillin-resistant Staphylococcus aureus (MRSA). Materials and Methods: This study used randomly collected 80 white soft local cheeses (40 each from cows and sheep) from different supermarkets in Basrah Province. The obtained samples were cultured and the bacterial suspension of S. aureus was prepared at 1.5 × 108 cells/mL. The crude bacteriocin extracted from LAB was obtained, and the tube was dried and inverted to detect the biofilm loss at the bottom. Results: There were 67 (83.75%) LAB isolates. Among 40 milk samples collected directly and indirectly, there were 36 (83.33%). Staphylococcus aureus isolates based on conventional bacteriological analysis and biochemical tests. Molecular testing was conducted to identify LAB and MRSA. Depending on genotypic results, the effect of white soft local cheese (cows and sheep) and the amplification results of the 16S rRNA gene were detected in 46 LAB isolates from white soft local cheese from cows and sheep. Based on the molecular identification of the mecA, results on Staphylococcus determined that only 2 of 36 isolates of S. aureus carried the mecA. Moreover, there were 26 (86.66%) isolates (MRSA) from samples of raw milk from local markets and subclinical mastitis in cows. The ability of LAB isolates was tested. The effects of bacteriocin production on preventing biofilm growth and formation were investigated. Results demonstrated that bacteriocin has high activity. Microtiter plates applied to investigate the ability of S. aureus to produce biofilms revealed that all isolates were either weak or moderate biofilm producers, with neither non-biofilm nor strong biofilm producers found among the tested isolates. Conclusion: Lactic acid bacteria demonstrate a high ability to produce bacteriocin. Crude bacteriocin from LAB has a restrictive effect on biofilms produced by MRSA; thus, it can be used to reduce the pathogenicity of this bacterium. Keywords: 16S rRNA gene, biofilm formation assay, mecA gene, polymerase chain reaction, subclinical mastitis.
Antimicrobial activity of certain species of Lactobacillus bacteria were known to produce a certain type of bacteriocin called Plantaricin (Pln). In this study, the PlnF (plnF) gene (666 bp) encoding plantaricin was synthetically constructed based on a sequence of predefined and valuable amino acid compositions and subcloned into pET-28 (+) expression vector. Thereafter transformation of recombinant vector into BL21 (DE3) competent E. coli was achieved and to verify the result of cloning, the PCR colony technique was conducted. The expression of the inserted gene was analyzed by the SDS-polyacrylamide gel electrophoresis method, which detected a band of approximately 30 KD molecular weight for interested protein as a natural bioactive bacteriocin.
The focus of the present study was to characterize chimeric synthetic plantaricin F which naturally produced by Lactobacillus plantarum against zoonotic pathogenic bacteria Staphylococcus aureus and Escherichia coli as antibacterial peptide. The synthetic bacteriocin by bioinformatics revealed higher stability under studied parameter, hence was taken up for further investigation. The amino acids of bacteriocin from L. plantarum were analyzed by SnapGene. Further, synthetic PLNF was characterized in silico. The translated partial amino acid sequence of the synthetic PLNF gene displayed 253 amino acids for whole and 148 without tag. The predicted properties of the peptide included theoretical isoelectric point (pI) and hydrophobicity was highly acidic. Molecular weight was 27.2KDa for whole protein and 15.8 KDa for without tag. Predication the molecular approach of using SnapGene software and the protein was having antingcity against bacteria and has B-cell epitope on the surface of protein. Prediction data base on characterization of bacteriocin is novel and predicts synthetic PLNF to be a peptide responsible for antimicrobial activity. The study provides information about a broad spectrum bacteriocin in native probiotic culture and paves a way towards its application as alternative natural antimicrobial agent against zoonotic pathogenic bacteria. Finally, the 3D peptide structure analysis in present study showed that the predicted structure of model and has more functional properties and probably the form most suitable for binding to bacterial cell walls.
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