Development and comparative evaluation of in vitro, ex vivo, and in vivo models for the assessment of broad-spectrum topical antimicrobial efficacy

Abstract

The escalating prevalence of multidrug-resistant skin and soft tissue infections (SSTIs) undermines current decolonization strategies. While mupirocin remains the gold standard for MRSA, its inactivity against Pseudomonas aeruginosa creates a critical therapeutic gap in polymicrobial wounds. The development of effective alternatives is currently impeded by in vitro assays that fail to mimic the physical barriers of the wound microenvironment. To address these challenges, this study aimed to validate a comprehensive multi-tiered screening platform and evaluate the efficacy of a novel 5% ceftriaxone gel as a broad-spectrum alternative. We established a comprehensive, multi-tiered validation platform to assess a novel 5% ceftriaxone gel formulation. The pipeline included: (1) Agar Well Diffusion to quantify vehicle release kinetics; (2) a Gauze-Disk Biofilm Prevention assay to evaluate penetration through standard dressings; (3) an Ex Vivo Porcine Ear Skin model to challenge efficacy against the stratum corneum and wound lipids; and (4) an In Vivo Galleria mellonella model to assess the prevention of systemic lethality. The formulation was benchmarked against mupirocin, and triple antibiotic ointment. In diffusion assays, the 5% ceftriaxone gel exhibited broad-spectrum release, producing inhibition zones > 44 mm against staphylococci and a mean zone of 50.1 mm against P. aeruginosa . In the Gauze model, the formulation successfully permeated standard cotton dressings, achieving complete bacterial eradication (No Growth) across all tested strains, including P. aeruginosa , significantly outperforming positive controls ( p < 0.001). Crucially, in the ex vivo porcine model, the gel maintained potent bioactivity despite the lipid barrier, yielding significant > log reductions at 24 h across all strains: MRSA (> 2.5-log), MSSA (> 3-log), S. pyogenes (> 4-log), and P. aeruginosa (> 4-log). Furthermore, topical application in the in vivo G. mellonella model significantly reduced the systemic bacterial burden across all pathogens ( p < 0.05), validating trans-cuticular protective efficacy. This study evaluates a robust, ethical preclinical screening platform for topical antimicrobials. The findings suggest that a high-concentration ceftriaxone gel has the potential to address the “mupirocin gap” by providing broad-spectrum coverage against both resistant staphylococci and Pseudomonas in experimental models.