[Isolation and biological characteristics of a strain of broad spectrum phage infecting Enterobacter xiangfangensis].

To isolate and investigate the biological properties and antibacterial efficacy of phages targeting carbapenem-resistant Enterobacter cloacae complex (CR-ECC), thereby to contribute to the expansion of the phage library against ECC. This study constitutes a comprehensive experimental investigation encompassing bacteriophage isolation and purification, along with both in vitro and in vivo assessments. First, using a CR-ECC clinical isolate preserved in the clinical laboratory as the host, bacteriophages were isolated from untreated hospital sewage at the Second Affiliated Hospital of Soochow University by the double-layer agar plate method in March 2025. Key biological characteristics-including morphology, host range, environmental adaptability, optimal multiplicity of infection (MOI), and one-step growth kinetics-were analyzed. Furthermore, the antibacterial efficacy of the phage was assessed through in vitro growth inhibition assays, time-kill assay and Galleria mellonella infection models. The results showed that a lytic phage, designated Pxf1, was isolated and identified as a member of the Myoviridae family. Host range analysis demonstrated that phage Pxf1 could lyse ECC strains of sequence types (STs), including CR-ECC strains. Phage Pxf1 exhibited tolerance to a pH range of 2 to 11 and withstood temperatures up to 60 ℃. The optimal MOI was determined to be 10-³, and one-step growth curve analysis revealed a latent period of 10 minutes and a burst size of (40±3) PFU/cell. Phage Pxf1 rapidly adsorbed to host cells within 5 minutes of co-incubation, it demonstrated inhibitory effects on host bacteria within 6 hours under various MOI conditions. Furthermore, time-kill curve assays demonstrated that phage Pxf1 effectively suppressed the viable bacterial count to a low level for up to 8 hours. In a Galleria mellonella infection model, the phage-treated group exhibited a significantly higher survival rate compared to the control group (χ2=9.78, P<0.01). Genomic analysis revealed that phage Pxf1 possesses a double-stranded DNA genome of 174 207 bp with a (G+C) content of 39.8%. The genome was predicted to encode 299 open reading frames (ORFs) and 8 tRNAs. Notably, no genes associated with antibiotic resistance, virulence factors, or lysogeny were identified, indicating its potential suitability for therapeutic applications. In conclusion, a novel broad-host-range lytic phage, designated Pxf1, was isolated from hospital sewage samples using a clinically high-risk ST177 E. xiangfangensis strain as the host. Pxf1 exhibits a broad host range, capable of lysing CR-ECC strains across multiple sequence types (STs), coupled with robust environmental stability, making it a promising candidate for phage therapy against CR-ECC infections.