CHARACTERIZATION OF ANTIMICROBIAL SUBSTANCE WITH ANTIBIOFILM ACTIVITY FROM Pediococcus acidilactici

Abstract

Objective: The aim of the current study was to isolate and identify the strain having broad spectrum antibacterial and antibiofilm activity and characterize its antimicrobial substance. Methods: Lactic acid bacteria isolated from the raw milk of Indian goat species Babri and Sirohi (Capra aegagrus hircus) were screened for antibacterial activity against Staphylococcus epidermidis, Micrococccus luteus, Pseudomonas fluorescens and Escherichia coli. The selected strains were also analysed for anti-biofilm activity on S. epidermidis. The potential bacterial strain was characterized at molecular level by 16S rDNA sequencing. The antimicrobial substance produced by P. acidilactici was precipitated by 90% ammonium sulphate and subsequently ultra filtered. It was analysed for size, stability under various pH, temperature, chemicals and enzymatic conditions. Its mechanism of killing was determined by ML35p assay and scanning electron microscopy (SEM). Results: The 16S rDNA sequence of the potential isolate, possessing broad spectrum antibacterial activity and antibiofilm activity against S. epidermidis, was deposited to Genbank under accession no. KP671843 and was identified as Pediococcus acidilactici. The partially purified bacteriocin ran at approximately 3500 Da on tris- tricine SDS-PAGE and showed antibacterial activity against S. epidermidis by In-gel assay. Partially purified bacteriocin was stable at wide pH and high temperature and also to the treatment of lipase, amylase, catalase and various detergents/organic solvents. However, it was sensitive to proteolytic enzymes indicating its proteinaceous nature. Results of membrane permeability assay revealed that the bacteriocin targets bacterial cell membrane as its bactericidal action. Conclusion: The bacteriocin from P. acidilactici has the potential to be used as broad spectrum antibacterial and antibiofilm agent.