Enhanced antimicrobial activity of lactic acid bacteria through genome shuffling and genetic variability among shuffled strains

In this investigation, lactic acid bacteria (LAB) isolated from milk were tested for their antibacterial properties and improved the antimicrobial activity of these isolates using genome shuffling. A total of sixty-one isolates were found in eleven samples, which were then tested using the agar diffusion method for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa. Thirty-one strains exhibited antibacterial activity against at least one of the tested pathogens, with an inhibitory zone's diameter varying between 15.0 and 24.0 mm. Two isolates that showed the highest antimicrobial activity were identified as Lactobacillus plantarum CIP 103151 and Lactobacillus plantarum JCM 1149 according to 16S rRNA analysis. In the present study, applying genome shuffling approach significantly enhanced the antibacterial activity of L. plantarum. The initial populations were obtained via ultraviolet irradiation and were treated using the protoplast fusion method. The ideal condition for the production of protoplasts was 15 mg/ml of lysozyme and 10 μg/ml of mutanolysin. After two rounds of fusion, ten recombinants exhibited a significant increase in the inhibition zones versus S. aureus, S. typhimurium, P. aeruginosa, and E. coli, reaching up to 1.34, 1.31, 1.37, and 1.37-fold increase in inhibitory zone respectively. Random Amplified Polymorphic DNA results showed clear differences in DNA banding patterns among the wild strain of L. plantarum CIP 103151 and the three selected shuffled strains using primers 1283 & OPA09. On the other hand, no change was obtained using primers OPD03 neither among the wild strain and the three recombinant strains nor among the three shuffled strains.