EXPLORING THE INTERACTION BETWEEN ANTIMICROBIALS AND CUTIBACTERIUM ACNES IN VITRO AND IN THE GALLERIA MELLONELLA IN VIVO MODEL

Aim Cutibacterium acnes remains susceptible to a broad range of antibiotics, but resistant strains have emerged. This study evaluated the therapeutic activity of ceftriaxone (CTX), vancomycin (VA), and gentamicin (GEN) on C. acnes using the Galleria mellonella infection model, and the interactions of these compounds with bioactive glass S53P4 (BAG) supernatant on biofilm formation. We also assessed the in vitro elution efficacy of CTX, VA and GEN released from antibioticloaded polymethylmethacrylate (PMMA) and calcium sulfate (CaSO4) on C. acnes strains. Method Sequenced C. acnes strains 30.3 (phylotype IB, ST42, CC5) and 64.1 (phylotype IB, ST5, CC5) were selected. Antimicrobial interactions were evaluated using a checkerboard assay, and combinations exhibiting synergism or partial synergism were analyzed for their effects on biofilm formation, alone and in combination with BAG supernatant. Biofilm formation was quantified. Therapeutic potential was assessed in the G. mellonella infection model by infecting larvae with C. acnes (OD600 = 3.0) and administering 10 µL of antimicrobials, their combinations, or BAG supernatant under prophylactic and therapeutic regimens. Zone of inhibition (ZOI) testing assessed in vitro elution efficacy. Results Partial synergism we identified between VA (0.25 µL/mL) and CTX (0.01 µL/mL) for C. acnes ATCC 6919. Strain 30.3 exhibited partial synergism between GEN (0.06 µL/mL) and CTX (1 µL/mL), while strain 64.1 showed synergism with GEN (0.06 µL/mL) and CTX (0.5 µL/mL). Biofilm reduction was observed across all tested strains, with CTX and GEN combinations achieving a threefold reduction. The addition of BAG supernatant reduced the efficacy of certain antimicrobial combinations, though notable biofilm reductions were still observed in specific strain-treatment pairs (e.g., ATCC 6919 + CTX; 30.3 + CTX; 64.1 + CTX; 64.1 + BG supernatant; 30.3 + BG supernatant + CTX + GEN; and ATCC + BG supernatant). The untreated G. mellonella larvae exhibited 100% mortality within 24 hours, while prophylactic and immediate post-infection treatments significantly improved survival, with some larvae surviving the full five-day experimental period. ZOI of VA and VA+CTX persisted for over 40 days, with PMMA exhibiting longer elution. GEN elution was completed within 25 days for both biomaterials. Conclusions We identified potential antimicrobial combinations, particularly CTX and GEN in reducing C. acnes biofilm formation, enabling the use of lower therapeutic concentrations. While BAG supernatant exhibited limited synergistic effects, the larvae model emphasized the importance of early antimicrobial interventions for improved survival. VA and VA+CTX beads demonstrated prolonged elution in both CaSO4 and PMMA.