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

Staphylococcus aureus is a prominent opportunistic human pathogen and major biofilm former, with methicillin-resistant S. aureus (MRSA) posing substantial clinical challenges due to antibiotic resistance. Biofilm growth confers 10–1000-fold decreased susceptibility to antimicrobials through mechanisms including altered gene expression, reduced penetration, and slowed growth. The World Health Organization highlights a lack of innovation in antibiotic development, motivating complementary strategies that combine antibiotics with alternative antimicrobials and anti-biofilm agents. Bacteriocins, ribosomally synthesized peptides produced broadly among bacteria (including lactic acid bacteria), are promising due to established safety in food applications and activity against pathogens such as MRSA. The authors previously identified garvicin KS, a leaderless, three-peptide bacteriocin from Lactococcus garvieae with broad-spectrum activity and synergy with nisin. This study investigates whether garvicin KS alone or with micrococcin P1 (a thiopeptide protein synthesis inhibitor) can inhibit S. aureus biofilms, and whether these bacteriocins can re-sensitize MRSA to β-lactam antibiotic penicillin G.

Study design: In vitro evaluation of bacteriocins garvicin KS (GAK; three synthesized peptides mixed equimolar) and micrococcin P1 (MP1), alone and in combinations with penicillin G (PenG), against biofilms and planktonic cultures of S. aureus reference strains (ATCC 10832, Newman, USA300, ATCC 29213, ATCC 33591, and S. aureus 3255) and clinical S. aureus isolates (14 total; 8 MSSA and 6 MRSA) from plantar ulcers of leprosy patients. Antimicrobials and preparation: GAK peptides (90–99% purity) solubilized in 0.1% TFA; MP1 (≥95% purity) in 50% isopropanol with 0.1% TFA; PenG in sterile water (100 mg/ml stock). Vehicles matched in all controls. Storage at −20 °C. Planktonic MIC50 and synergy: 96-well microtiter broth microdilution in TSB with twofold serial dilutions; inoculated with overnight cultures to 150 µl total volume; incubated 24 h at 37 °C. MIC50 defined as concentration reducing OD600 by ≥50% vs untreated. Fractional inhibition concentration (FIC) used to assess synergy: for two drugs, synergy if FIC ≤ 0.5; for three drugs, synergy if FIC ≤ 0.75. FIC calculated as sum of MIC in combination/MIC alone for each component. Biofilm formation: Static biofilms grown 24 h at 37 °C in TSB supplemented with 1% glucose and 1% NaCl (TSB-GN) in 96-well plates. Biofilm biomass quantified by crystal violet staining (OD600) in preliminary assays to confirm good biofilm formation. Biofilm-oriented antimicrobial test (BOAT): After 24 h biofilm growth, wells were washed and challenged 24 h with serial twofold dilutions of antimicrobials: typically starting at 5 mg/ml (GAK), 0.1 mg/ml (MP1), 10 mg/ml (PenG), alone or in combinations including GAK+MP1 and tricomponent formulation (TCF: GAK+MP1+PenG). Matched vehicle controls: water (PenG), 0.02% TFA (GAK), 0.013% TFA/6.25% 2-propanol (MP1), 0.033% TFA/6.25% 2-propanol (GAK/MP1 and TCF). Post-challenge, metabolic activity was measured via TTC reduction to formazan (OD492). Biofilm MIC50 defined as the concentration reducing metabolic activity by ≥50% vs control. Viability (CFU) after BOAT: Following antimicrobial challenge and washing (as above), biofilm cells were resuspended, serially diluted, plated on BHI agar, incubated 24 h at 37 °C, and CFU enumerated. Results reported as Log10 CFU. Microscopy: Confocal laser scanning microscopy on biofilms formed in chambered coverglass, stained with LIVE/DEAD kit (SYTO-9 for live; propidium iodide for dead). Z-stacks acquired with 488 nm (SYTO-9) and 561 nm (PI) lasers. Scanning electron microscopy (SEM) performed on biofilms grown on glass coverslips, treated with TCF or vehicle, fixed in 3% glutaraldehyde, ethanol-dehydrated, critical-point dried, sputter-coated (Pd/Au), imaged at 15 kV at multiple magnifications. Antibiotic susceptibility testing: Reference strains assessed by Kirby–Bauer disc diffusion per CLSI; clinical isolates by VITEK-2, with identification confirmed by MALDI-TOF and 16S rRNA genotyping. Statistics: Three independent experiments for all quantifications. Welch’s t test for group comparisons; analyses in R Studio.

- Planktonic susceptibility: Garvicin KS inhibited S. aureus strains with MIC50 0.025–0.05 mg/ml; micrococcin P1 with MIC50 0.00063–0.01 mg/ml (Table 1). The GAK+MP1 combination showed synergy against MRSA USA300 (FIC 0.31) and MRSA ATCC 33591 (FIC 0.39) in planktonic culture (synergy threshold ≤0.5). - Biofilm susceptibility to bacteriocins: Using BOAT, garvicin KS alone eradicated metabolic activity in 5/6 strains (including USA300) with MICs ~1.3–2.5 mg/ml; ATCC 33591 remained insensitive within the tested range (>5 mg/ml). MP1 alone up to 0.1 mg/ml did not abolish metabolic activity, causing only modest reductions. GAK+MP1 reduced metabolic activity across all strains; synergy by FIC was observed for S. aureus 3255 (FIC 0.5), with reduced MICs in several strains (Table 2). - Biofilm viability by CFU: Significant reductions in Log10 CFU after GAK (p=0.022) and GAK+MP1 (p=0.0013) vs vehicle. ATCC 33591 retained the highest viability among tested strains despite treatment. - Penicillin G in biofilms: PenG at 10 mg/ml inhibited metabolic activity for non-MRSA strains but not MRSA (USA300, ATCC 33591). GAK+PenG further delayed metabolic recovery and lowered MICs in non-MRSA but did not affect MRSA. - Tricomponent formulation (TCF: GAK+MP1+PenG): Produced stronger inhibition of metabolic activity across all strains and significant CFU reductions. Median Log10 CFU decreased significantly overall in non-MRSA (p=0.0027) and in MRSA (p=0.0013). Notably, ATCC 33591 showed a strong, significant viability reduction with TCF (p=0.00067). In planktonic tests, tri-component FIC indicated synergy for ATCC 29213 (FIC 0.49) and ATCC 33591 (FIC 0.52) (synergy threshold ≤0.75 for three components) (Table 3). In biofilms, synergy by FIC with TCF was evident for several non-MRSA strains (e.g., ATCC 10832 FIC 0.12; ATCC 29213 FIC 0.41; S. aureus 3255 FIC 0.41) (Table 4); MRSA FIC in biofilms was not determined due to exceeding maximum testable concentrations, despite clear efficacy by metabolic and CFU measures. - Microscopy: LIVE/DEAD confocal imaging showed a shift from live (SYTO-9) to dead (PI) staining after treatment, with TCF having the most pronounced effect. MRSA ATCC 33591 displayed resistance to single agents but marked loss of viability with GAK+MP1 and especially TCF. SEM revealed reduced biofilm density and extensive cell damage (deformed morphology, surface debris) after TCF treatment vs control. - Clinical isolates: Of 31 wound swabs from plantar ulcers, 14 were S. aureus (∼50% MRSA), all biofilm producers. Garvicin KS alone reduced metabolic activity across isolates; addition of MP1 and PenG increased efficacy, with TCF showing the strongest and broadest activity across MSSA and MRSA clinical strains.

This study demonstrates that bacteriocin-based approaches can overcome biofilm-associated tolerance and antibiotic resistance in S. aureus. Garvicin KS, likely acting via membrane disruption typical of leaderless bacteriocins, and micrococcin P1, a thiopeptide inhibitor of protein synthesis, together potentiate β-lactam activity. While penicillin G alone was ineffective against MRSA biofilms, combining it with both bacteriocins (TCF) significantly reduced MRSA viability, including the highly resilient ATCC 33591 strain, indicating bacteriocin-mediated sensitization to β-lactams. Confocal microscopy showed uniform penetration/effects within biofilms, arguing against poor penetration as the sole barrier and suggesting biofilm-specific gene expression and physiology contribute to tolerance. The discrepancy between metabolic inactivity (TTC) and residual CFU likely reflects dormant persister subpopulations that regain growth upon plating. The findings support bacteriocin-antibiotic combinations as a strategy to restore efficacy of legacy antibiotics and to target biofilm-associated infections, with efficacy validated against diverse reference strains and clinically derived MSSA/MRSA isolates.

A tricomponent formulation comprising garvicin KS, micrococcin P1, and penicillin G effectively inhibits S. aureus biofilm metabolic activity and viability in vitro and sensitizes MRSA strains, including ATCC 33591, to β-lactam treatment. Garvicin KS alone and in combination with micrococcin P1 also showed broad anti-biofilm effects, and all approaches were effective against clinical MSSA and MRSA isolates. These results highlight bacteriocins as promising therapeutic tools, especially in combination with antibiotics, to manage biofilm-associated and drug-resistant staphylococcal infections. Future work should elucidate detailed mechanisms of sensitization, optimize dosing and formulations, expand strain coverage, assess resistance development, and validate efficacy and safety in relevant in vivo infection models.

Findings are based on in vitro biofilm models; in vivo efficacy and safety were not assessed. Effects were strain-dependent, with MRSA ATCC 33591 more tolerant to individual agents. Maximum test concentrations limited FIC determinations for some MRSA biofilm combinations. Discrepancies between metabolic activity assays (TTC) and CFU suggest dormant persister cells not detected by metabolic readouts. Mechanistic details of garvicin KS action and the precise basis of β-lactam re-sensitization were not fully elucidated. Micrococcin P1 historical solubility constraints may affect formulation; the study did not address pharmacokinetics or toxicity.