A bacteriocin-based antimicrobial formulation to effectively disrupt the cell viability of methicillin-resistant *Staphylococcus aureus* (MRSA) biofilms

Methicillin-resistant *Staphylococcus aureus* (MRSA) infections, particularly those associated with biofilms, pose a significant global health challenge. MRSA's high propensity for antibiotic resistance, coupled with the resilience of biofilms to antimicrobial treatments, makes these infections difficult to treat. Biofilms, formed by bacterial communities embedded in an extracellular matrix, exhibit significantly reduced susceptibility to antibiotics due to factors such as altered gene expression, poor antimicrobial penetration, and reduced bacterial growth rates. The emergence of community-associated MRSA (CA-MRSA) strains has further exacerbated the problem, increasing the incidence of antibiotic-resistant infections outside hospital settings. The World Health Organization (WHO) has highlighted the urgent need for novel antimicrobial strategies to combat drug-resistant infections. Bacteriocins, ribosomally synthesized antimicrobial peptides produced by bacteria, offer a promising alternative. These peptides often target closely related species, providing a selective advantage to the producer. Many bacteriocins produced by lactic acid bacteria (LAB) are considered generally recognized as safe (GRAS) and have shown efficacy against various pathogens, including MRSA. This study focuses on garvicin KS, a novel multi-peptide bacteriocin with broad-spectrum activity, and micrococcin P1, another promising bacteriocin, to investigate their potential as therapeutic agents against MRSA biofilms. The researchers hypothesized that these bacteriocins, potentially in combination with traditional antibiotics, could offer a new strategy to combat MRSA biofilm infections.

The introduction extensively reviews the literature on MRSA infections, antibiotic resistance, biofilm formation, and the potential of bacteriocins as antimicrobial agents. It highlights the challenges posed by MRSA's resistance mechanisms, including the acquisition of the *mecA* gene, and the protective nature of biofilms, which significantly reduces the effectiveness of antibiotics. The review emphasizes the need for alternative therapeutic strategies to address the growing problem of drug-resistant bacterial infections. Specific bacteriocins, like nisin and pediocin PA-1/AcH, are mentioned as examples of LAB-produced bacteriocins that have been approved for use as food preservatives and are being explored for medicinal applications. The review sets the stage for the study by introducing garvicin KS and micrococcin P1 as promising candidate bacteriocins with potential against MRSA biofilms.

The study employed several methods to assess the antimicrobial activity of garvicin KS and micrococcin P1, both individually and in combination, against various *S. aureus* strains, including MRSA strains. Minimum inhibitory concentrations (MIC50) were determined for planktonic cells and biofilms using modified biofilm-oriented antimicrobial tests (BOAT). The BOAT assay measured metabolic activity using triphenyl-tetrazolium chloride (TTC) and colony-forming units (CFU) to assess cell viability. Antibiotic susceptibility testing (AST) was performed using the Kirby-Bauer disc diffusion method and the VITEK-2 system. Biofilm formation ability was quantified using crystal violet staining. Confocal laser scanning microscopy (CLSM) with LIVE/DEAD staining (SYTO 9 and propidium iodide) visualized biofilm architecture and cell viability after antimicrobial treatments. Scanning electron microscopy (SEM) examined the morphological changes in biofilms after treatment with the tricomponent formulation (TCF). Fractional inhibitory concentration (FIC) values determined synergistic interactions between antimicrobials. The study included both laboratory strains of *S. aureus* and clinical isolates obtained from skin ulcers of leprosy patients in India. Statistical analysis using R Studio was performed to determine the significance of the results.

Garvicin KS effectively eradicated biofilms from most *S. aureus* strains, including a MRSA strain (USA300), but not ATCC 33591, showing over 100-fold MIC increase compared to planktonic state. Micrococcin P1 alone showed limited efficacy against biofilms. The combination of garvicin KS and micrococcin P1 synergistically reduced metabolic activity in all strains, with significant reductions in CFU observed for most strains except ATCC 33591. Significantly, the combination of garvicin KS and micrococcin P1 sensitized MRSA strains to penicillin G. A tricomponent formulation (TCF) comprising garvicin KS, micrococcin P1, and penicillin G demonstrated the strongest antimicrobial activity against all strains, including a significant reduction in the viability of the highly resistant ATCC 33591 strain. Confocal microscopy revealed that the TCF induced a substantial shift from live (SYTO 9-positive) to dead (propidium iodide-positive) cells in both MRSA and methicillin-sensitive strains. SEM showed severe cell damage and debris in TCF-treated biofilms. The TCF also effectively inhibited biofilm growth in clinical isolates from leprosy patients, including MRSA strains. Overall, the MIC50 values for the planktonic and biofilm states were significantly different for the tested antimicrobials.

The study's findings demonstrate the potential of a bacteriocin-based formulation for treating MRSA biofilm infections. The synergistic effect of garvicin KS and micrococcin P1, especially in combination with penicillin G, highlights a novel approach to overcome antibiotic resistance. The ability of the TCF to effectively inhibit the growth of both laboratory and clinical MRSA strains, including the highly resilient ATCC 33591 strain, is noteworthy. The observation that garvicin KS and micrococcin P1 can restore sensitivity to penicillin G suggests the potential to revive the use of β-lactam antibiotics against MRSA. The discrepancy between metabolic activity (BOAT) and cell viability (CFU) could be attributed to biofilm-associated cell dormancy, a phenomenon where metabolically inactive cells persist and contribute to the long-term persistence of infections. The high efficacy of the TCF against clinical isolates from leprosy patients confirms its potential clinical relevance.

This research demonstrates the potential of a tricomponent formulation consisting of garvicin KS, micrococcin P1, and penicillin G as a potent antimicrobial agent against *Staphylococcus aureus* biofilms, including MRSA strains. The synergistic action of the bacteriocins in restoring sensitivity to penicillin G offers a valuable strategy to combat antibiotic resistance. Future research should focus on in vivo studies to evaluate the efficacy and safety of the TCF in animal models and eventually clinical trials in humans. Investigating the mechanisms of action of the bacteriocins in greater detail and exploring potential modifications to enhance their properties are also important areas for future investigation.

The study's limitations primarily involve the in vitro nature of the experiments. While the results demonstrate strong antimicrobial activity in laboratory settings, further in vivo studies are necessary to confirm the efficacy and safety of the TCF in animal models before clinical trials can be conducted. The use of a limited number of clinical isolates might not fully capture the diversity of MRSA strains found in various clinical settings. Additional research is needed to investigate the mechanism through which the combined treatment induces cell death in the biofilm and the potential effect of the drug combination on human cells or the microbiome.