Abstract:
Background: Biofilm-forming pathogens are among the leading causes of hospital-acquired infections and are largely refractory to conventional antibiotic therapy. Bacteriocins — small proteinaceous antimicrobial peptides produced by lactic acid bacteria (LAB) — represent a promising alternative to traditional antibiotics. This study characterized two bacteriocins, BacF1 and BacF2, produced by Lactobacillus plantarum subsp. argentoratensis SJ33, and evaluated their antibacterial, antibiofilm, and cytotoxic properties.
Methods: BacF1 and BacF2 were purified by semi-preparative reversed-phase HPLC and characterized by Tris-tricine SDS-PAGE and Q-TOF ESI mass spectrometry. Antibacterial spectrum was assessed by agar well diffusion assay against multiple Gram-positive and Gram-negative pathogens. Minimum inhibitory concentration (MIC) and time-kill kinetics were determined against Staphylococcus aureus MTCC 96. Membrane disruption was visualized by scanning electron microscopy (SEM). Antibiofilm activity was quantified by crystal violet assay, fluorescence microscopy, and SEM against S. aureus and Pseudomonas aeruginosa. Downregulation of biofilm-associated genes (icaC, icaD in S. aureus; pelA, psl, rhlA in P. aeruginosa) was evaluated by quantitative real-time PCR. Cytotoxicity was assessed using MTT assay on HEK-293 cells and a Caenorhabditis elegans in vivo model.
Results: BacF1 (4.039 kDa) and BacF2 (1.6 kDa) demonstrated broad-spectrum antibacterial activity against both Gram-positive (Listeria monocytogenes, S. aureus) and Gram-negative (P. aeruginosa, Escherichia coli) pathogens. BacF1 achieved >90% inhibition of S. aureus at 62.5 µM (MIC), comparable to nisin. Time-kill assays confirmed rapid bactericidal action within 6–12 hours, with complete eradication at 12 h. SEM revealed pronounced membrane pore formation and cellular lysis in treated cells. At sub-MIC concentrations (31.25 µM), BacF1 and BacF2 inhibited 70% and 65% of S. aureus biofilms, and 55% and 50% of P. aeruginosa biofilms, respectively. qRT-PCR confirmed significant downregulation of ica operon genes in S. aureus and polysaccharide/rhamnolipid biosynthesis genes in P. aeruginosa. Crucially, both bacteriocins showed negligible cytotoxicity on HEK-293 cells (>80% viability at MIC) and enhanced survival of S. aureus-infected HEK-293 cells. No adverse effects were observed on C. elegans even at 2× MIC over 48 hours.
Conclusions: BacF1 and BacF2 from L. plantarum SJ33 are potent, broad-spectrum, non-cytotoxic bacteriocins with robust antibiofilm and bactericidal activity. Their ability to suppress biofilm gene expression and prevent pathogen adherence to mammalian cells, combined with an excellent safety profile, positions them as compelling candidates for development as next-generation antimicrobial agents against drug-resistant, biofilm-associated infections — a critical priority within the One Health framework.
Keywords: Bacteriocins; Lactobacillus plantarum; Antibiofilm; Staphylococcus aureus; Pseudomonas aeruginosa; Cytotoxicity; One Health; Antimicrobial resistance
Biography:
Amrita Ray Mohapatra is a researcher in the Department of Biochemistry and Molecular Biology at Pondicherry University, India. Her research focuses on the discovery, purification, and characterization of bioactive bacteriocins from lactic acid bacteria, with particular emphasis on their antibiofilm and antimicrobial properties against clinically relevant pathogens. She has contributed to studies on novel bacteriocin coatings for medical devices to combat device-associated infections, and her work explores the therapeutic potential of LAB-derived peptides as alternatives to conventional antibiotics.
