Targeted elimination of Staphylococcus aureus mastitis infections with synthetic phage-based CRISPR-Cas delivery systems

Treatment options for Staphylococcus aureus infections are increasingly limited, particularly in livestock, where S. aureus causes mastitis requiring prolonged antibiotic therapy. This study engineered Phage Inducible Chromosomal Islands (ePICIs) to deliver CRISPR-Cas9 modules targeting small RNA genes. ePICIs exhibit bactericidal activity without chromosomal integration, an expanded host range compared to their parental phages, and biofilm-dependent efficacy influenced by the extracellular matrix composition. Biofilms mediated by the Bap protein strongly protect bacteria from ePICIs, whereas PIA/PNAG-based biofilms do not. Despite Bap-mediated protection in vitro, ePICIs achieved bactericidal effects comparable to vancomycin in a mouse mastitis model caused by Bap-producing strains. These findings reveal key factors affecting phage-delivered CRISPR-Cas efficacy and highlight that antibiofilm therapies should not be dismissed based solely on in vitro performance. Non-replicative ePICIs thus represent a promising alternative for treating localized infections such as mastitis.