Isolation and phenotypic characterization of a lytic bacteriophage with quantitative host range and antibiofilm...

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major public health concern due to multidrug resistance and robust biofilm formation, limiting the effectiveness of conventional antibiotics. Bacteriophages have re-emerged as alternative biological tools for targeted antibacterial and antibiofilm strategies.

This study aimed to isolate and phenotypically characterize a bacteriophage active against clinical MRSA isolates and evaluate its replication kinetics, environmental stability, host range, and antibiofilm activity.

Bacteriophages were isolated from hospital sewage by enrichment against all 51 confirmed MRSA isolates obtained from 207 clinical specimens, and a single isolate was selected based on highest plaque-forming titer, clear plaque morphology, and broadest lysis spectrum. Phage morphology was examined using transmission electron microscopy. Replication kinetics were assessed by one-step growth analysis, and stability was evaluated under varying pH and temperature conditions. Host range was determined using efficiency of plating (EOP) across four MRSA isolates representing the spectrum of qualitative susceptibility responses. Antibiofilm activity was assessed using crystal violet biomass assay and CFU-based viability analysis.

Among the 51 confirmed MRSA isolates, 98% (50/51) carried the mecA gene. The phage produced clear plaques with a titer of 1.38 × 10¹⁰ PFU/mL. EOP analysis showed strain-dependent infectivity, indicating a moderate-to-broad host range. The phage exhibited optimal stability at pH 7.0 and 37 °C, with reduced activity at extreme conditions. A latent period of ∼30 min and a burst size of 16 phages per cell were observed. Biofilm assays demonstrated up to 96.9% biomass reduction and >4 log₁₀ reduction in viable cells.

The study provides comprehensive phenotypic characterization of an MRSA-targeting bacteriophage. Although whole-genome sequencing was not performed, the findings establish a functional baseline supporting future genomic and translational investigations.