Model-dependent efficacy of sequential phage-antibiotic-antifungal therapy against dual-species biofilms.

The rising rate of antimicrobial resistance presents a major global health risk. Staphylococcus aureus and Candida albicans often co-infect and create dual-species biofilms, which increase virulence and reduce the effectiveness of treatment of human infections. This study explores the potential of sequentially and simultaneously administering ciprofloxacin (CIP), caspofungin (CASP), and bacteriophages (AD) as an alternative method to control dual-species biofilms. The therapeutic approach was tested using biofilms formed in ex vivo fluids (human urine [HU] and heat-inactivated human plasma blood [HIP-B]) and an in vivo model (Galleria mellonella larvae). In the HIP-B model, the microbial loads of both S. aureus and C. albicans tended to decrease across treatment combinations. Notably, the AD→CIP + CASP and CIP + CASP→AD regimens produced the largest reduction in biofilm biomass, reaching up to 40%. Conversely, the therapy was ineffective in the HU model: although microbial counts showed slight reductions, these were not statistically significant, and no decrease in biofilm biomass was observed. Using Galleria mellonella larvae as a model, the strongest protective effect was seen with the CIP + CASP→AD sequence. This combination achieved the highest median survival and the greatest reduction in mortality risk. The simultaneous triple therapy (AD + CIP + CASP) was nearly as effective. In summary, the three-factor therapy shows variable effectiveness depending on the infection environment. These results highlight the importance of testing phage-based combination therapies in the specific biological environments in which they are intended to act, as treatment efficacy proved to be strongly model-dependent.