Problems connected with biofilm-related infections and antibiotic resistance necessitate the investigation and development of novel treatment strategies. Given their unique characteristics, one of the most promising alternatives to conventional antibiotics are bacteriophages. In the in vitro and in vivo larva model study, we demonstrate that phages vB_SauM-A, vB_SauM-C, and vB_SauM-D are effective antibiofilm agents. The exposure of biofilm to phages vB_SauM-A and vB_SauM-D led to 2–3 log reductions in the colony-forming unit number in most of the multidrug-resistant S. aureus strains. It was found that phage application reduced the formed biofilms independently of the used titer. Moreover, the study demonstrated that bacteriophages are more efficient in biofilm biomass removal and reduction in staphylococci count when compared to the antibiotics used. The scanning electron microscopy analysis results are in line with colony forming unit (CFU) counting but not entirely consistent with crystal violet (CV) staining. Additionally, phages vB_SauM-A, vB_SauM-C, and vB_SauM-D can significantly increase the survival rate and extend the survival time of Galleria mellonella larvae.

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