**Background:** Antibiotic resistance, particularly among Gram-negative Enterobacteriaceae like Klebsiella pneumoniae, is a growing global health threat. Carbapenem-resistant Enterobacteriaceae (CRE) are listed as priority pathogens by the WHO. Bacteriophage therapy is being explored as a promising alternative to conventional antibiotics, especially for multidrug-resistant (MDR) infections. This study aimed to isolate and characterize a novel lytic phage, vB_Kpn_ZC2 (ZCKP2), targeting MDR K. pneumoniae, and assess its safety and efficacy for potential therapeutic use.
**Methods:** Phage ZCKP2 was isolated from sewage water in Giza, Egypt, using the clinical K. pneumoniae isolate KP/08 as the host. The phage was purified and amplified using standard enrichment and double-layer agar techniques. Morphology was examined by transmission electron microscopy (TEM). Genome size was estimated by pulsed-field gel electrophoresis (PFGE) and confirmed by whole genome sequencing (Illumina MiSeq platform, 150 bp paired-end reads). Host range was tested against 30 MDR K. pneumoniae clinical isolates using spot assays and relative efficiency of plating (EOP). One-step growth curves were performed at an MOI of 0.1 to determine eclipse period, latent period, and burst size. Time-killing curves were conducted over 325 minutes at MOIs of 0.1, 1, 10, and 100, measuring optical density at 600 nm. Thermal stability was assessed after 4-hour incubation at temperatures from -20°C to 80°C, and pH stability after 4-hour incubation at pH values from 2 to 13. Whole genome annotation was performed using NCBI ORF finder, BLASTp, PHASTER, RASTtk, and BV-BRC. Safety was assessed using PhageLeads to screen for temperate markers, antibiotic resistance genes, and virulence genes. Transmembrane topology was predicted using DeepTMHMM. Phylogenetic analysis included VIRIDIC intergenomic similarity calculations, ViPTree proteomic trees, and maximum likelihood phylogenetic trees of signature proteins (major capsid protein, terminase large subunit, single-strand DNA-binding protein, DNA polymerase III).
**Key Results:** TEM revealed ZCKP2 has an icosahedral head (~65 nm diameter) and a non-contractile tail (~160 nm), characteristic of siphoviruses. The genome was assembled as a single 48,258 bp contig with 47.5% G+C content, containing 69 open reading frames (ORFs). Twenty-eight putative proteins were assigned functions including DNA replication/transcription/repair, DNA packaging, structural proteins, and cell lysis proteins. One tRNA-Arg gene (anticodon TCT) was identified. No lysogeny-related genes, antibiotic resistance genes, or virulence genes were detected. Phage ZCKP2 infected 7 of 30 K. pneumoniae isolates. The one-step growth curve showed an eclipse period of ~22 min, a latent period of 25±3 min, and a burst size of ~650±50 PFU per infected cell. Time-killing curves showed MOI-dependent bacterial growth inhibition: after 325 min, untreated bacteria had OD600 of 10.8±0.96, while at MOI 0.1 OD600 was 0.275±0.05, at MOI 1 it was 0.125±0.01, at MOI 10 it was 0.085±0.01, and at MOI 100 it was 0.095±0.01. Higher MOIs (10 and 100) showed faster reduction but some regrowth suggestive of phage resistance. The phage was stable from -20°C to 60°C, with titers dramatically reducing above 70°C and becoming undetectable at 80°C. Optimal pH was 7.0, with acceptable stability at pH 4-9, but no survival at pH ≤3 or ≥11. VIRIDIC analysis showed ZCKP2 shares 93% intergenomic similarity with Klebsiella phage ZCKP8 (same genus, different species) and is distantly related to the family Drexlerviridae. Proteomic and pan-genome analyses suggest ZCKP2 represents a new, unclassified family. DeepTMHMM predicted two transmembrane domains in the putative holin (ORF 15, 81 amino acids), consistent with class II holin topology.
**Clinical Implications:** Phage ZCKP2 demonstrates several characteristics desirable for therapeutic application: strictly lytic lifecycle, absence of harmful genetic elements (no lysogeny, virulence, or antibiotic resistance genes), high burst size (~650 PFU/cell), rapid lysis kinetics, and stability across a wide range of environmental conditions (pH 4-9, temperatures up to 60°C). The relatively narrow host range (7/30 isolates) suggests that ZCKP2 may be best suited for use in phage cocktails or combined with antibiotics to broaden coverage. The predicted lytic enzymes (holin and lysozyme) and the presence of a putative novel antimicrobial protein (ORF 28) warrant further investigation. The authors conclude that ZCKP2 is a safe and effective candidate for further in vivo studies and phage therapy applications against MDR K. pneumoniae.