Isolation, Genomic Analysis, and Evaluation of the Novel Lytic Phage vB_Cf_HW01: Potent Antibiofilm Activity and Therapeutic Efficacy Against a Clinical Isolate of <i>Citrobacter freundii</i>

Abstract Aims Multidrug-resistant Citrobacter freundii, particularly isolates carrying extended-spectrum β-lactamases and carbapenemases are increasingly difficult to treat in clinical settings. To explore alternatives to antibiotics, we isolated a novel lytic bacteriophage from hospital wastewater in Zhengzhou and designated it vB_Cf_HW01. Methods and Results TEM revealed a siphovirus with a 50 nm capsid and 160 nm tail. Genomic analysis identified vB_Cf_HW01 as a novel Drexlerviridae species. Our results indicated an optimal MOI of 0.001, defined as the initial infection ratio that yielded the highest progeny titer. The lysis curve showed that vB_Cf_HW01 effectively inhibited host cell proliferation at this low MOI. Adsorption kinetics and one-step growth assays showed 97.17% adsorption within 14 min, a 10 min latent period, and a burst size of 256 PFU cell−1. The phage remained stable from -20 to 60°C and pH 4 to 10, resisted chloroform exposure, and retained residual infectivity after 90 min of UV irradiation. Sequencing revealed a 49563 bp linear dsDNA genome (Accession: PX694430.1) containing 94 open reading frames. Genomic analysis identified no known virulence or antibiotic resistance genes. CLSM with live/dead staining showed severe disruption of mature C. freundii biofilms after a 12 h of vB_Cf_HW01 treatment. Live cell coverage decreased from 9.17 ± 0.70% to 3.03 ± 0.92%, whereas dead cells increased from 0.14 ± 0.01% to 7.87 ± 0.18%. In an in vivo Galleria mellonella infection model, a single phage dose (MOI 10) rescued 36.67% of infected larvae. Conclusions Thus, vB_Cf_HW01 is a promising candidate against drug-resistant C. freundii infections.