Withatanung P.Apiratwarrasakul S.Sresuwadjarey P.Phumthanakorn N.Lerdsittikul V.Mahidol University2026-01-072026-01-072025-12-01Scientific Reports Vol.15 No.1 (2025)https://repository.li.mahidol.ac.th/handle/123456789/113802The rise of methicillin-resistant Staphylococcus pseudintermedius (MRSP), a multidrug-resistant zoonotic pathogen with strong biofilm-forming ability, highlights the urgent need for alternative therapies. Here, we report the isolation and comprehensive characterization of vB_SpsS_VL4 (VL4), a novel temperate phage obtained from sewage using a clinical MRSP isolate. VL4 genome (39,788 bp) contains 68 open reading frames (ORFs) with no virulence, resistance, or toxin genes and encodes lysis proteins with Cysteine, Histidine-dependent Amidohydrolase/Peptidase (CHAP) domains. Genomic and phylogenetic analyses classified VL4 as a new species within the Coventryvirus genus. Despite its temperate nature, VL4 demonstrated efficient replication (latent period 50 min; burst size 56 PFU per cell), remained viable at neutral pH and temperatures up to 37 °C, and significantly suppressed the host bacterial growth. Among 31 S. pseudintermedius clinical isolates, 27 (87%) strains were susceptible to VL4, while no activity was detected against Staphylococcus aureus. Moreover, VL4 inhibited biofilm formation and partially degraded mature MRSP biofilms. Collectively, these findings identify VL4 as a promising Coventryvirus phage with potential for future engineering and therapeutic development against difficult-to-treat MRSP infections.MultidisciplinaryArticleSCOPUS10.1038/s41598-025-32942-72-s2.0-10502621867320452322