Comparative genomics and cefepime synergy of a lytic Vectrevirus phage Eco-MTCMU-01, targeting extended spectrum β-lactamase-producing Escherichia coli

The increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has significantly reduced the effectiveness of β-lactam antibiotics, necessitating alternative or adjunct antimicrobial strategies. Here, we report the isolation and comprehensive characterization of a novel lytic bacteriophage, Eco-MTCMU-01, targeting ESBL-producing E. coli. The phage was isolated from wastewater in Thailand and morphologically classified by transmission electron microscopy as a member of the Autographiviridae, exhibiting an icosahedral capsid and short tail. A complete genome was resolved using hybrid assembly of short- and long-read sequencing data, enabling robust phylogenomic assignment to the genus Vectrevirus. Comparative genomic analysis indicated that Eco-MTCMU-01 shares core genomic architecture with Vectrevirus members but lacks identifiable genes associated with lysogeny, antimicrobial resistance, virulence factors, or anti-CRISPR systems, supporting a strictly lytic lifestyle and favorable safety profile. Host range analysis demonstrated that Eco-MTCMU-01 exhibits a highly specific lytic spectrum, infecting E. coli ATCC 25,922 and a subset of clinical isolates, including ESBL-producing strains, while showing no activity against other tested Gram-negative or Gram-positive species. Functionally, although phage treatment alone did not achieve complete bacterial eradication after 24 h, combination therapy with cefepime resulted in marked synergistic effects. Checkerboard assays yielded a fractional inhibitory concentration index (FICI) of 0.25, indicating strong synergy, and reduced the minimum inhibitory and bactericidal concentrations of cefepime from 32 to 64 µg/mL to < 8 µg/mL against a representative ESBL-producing isolate. Collectively, Eco-MTCMU-01 represents a genomically well-characterized lytic phage with selective activity against clinically relevant E. coli and demonstrated capacity to enhance cefepime efficacy. These findings support further investigation of phage-antibiotic combination strategies and highlight the importance of integrating genomic safety assessment with quantitative functional validation in the development of phage therapeutics against antimicrobial-resistant pathogens.