In vitro concentration-dependent inhibition of early biofilm formation by Staphylococcus aureus isolated from dairy cattle using a bacteriophage cocktail

Bovine mastitis remains one of the most economically important diseases in dairy cattle, with Staphylococcus aureus being a major etiological agent, particularly in chronic and subclinical infections. The ability of S. aureus to form biofilms significantly contributes to antimicrobial tolerance and treatment failure, highlighting the need for alternative or adjunctive therapeutic approaches. Bacteriophage therapy has re-emerged as a promising strategy; however, data on its anti-biofilm efficacy against mastitis-associated S. aureus isolates are limited. In this study, we evaluated the anti-biofilm activity of a newly developed anti-Staphylococcus aureus bacteriophage cocktail against clinical isolates obtained from subclinical bovine mastitis. Twenty-eight non-duplicate field isolates of Staphylococcus aureus obtained from dairy cattle with subclinical mastitis on different farms in north-eastern Poland were examined using a MIC-like assay, followed by biofilm inhibition experiments conducted under high bacterial inoculum conditions. Biofilm formation was assessed after 24 and 48 h using crystal violet staining and confocal laser scanning microscopy with LIVE/DEAD™ fluorescence staining. Ultrastructural changes were analyzed by scanning electron microscopy. MIC-like values did not correspond to concentrations effective against biofilm formation. Biofilm biomass and viability were reduced in a concentration- and time-dependent manner, with the most pronounced effects observed at higher bacteriophage concentrations. Microscopic analyses confirmed biofilm disruption and bacteriophage-induced cellular damage. These findings demonstrate the potential of bacteriophage cocktails as anti-biofilm agents and support further investigation of bacteriophage-based strategies targeting early biofilm development in veterinary staphylococcal infections. This in vitro study was designed to evaluate the concentration-dependent effects of bacteriophages on early biofilm development rather than clinical treatment efficacy.