Isolation and characterization of bacteriophage against gram-negative bacterial species in diabetic foot ulcers

This study focused on the isolation and characterization of 18 bacteriophages targeting Gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella aerogenes, and Escherichia coli, isolated from diabetic foot ulcer patients admitted to Çukurova University Balcalı Hospital's Endocrinology Department. Among these, three phages with the broadest host ranges PAP5 (targeting P. aeruginosa), KPP3 (targeting K. pneumoniae), and ECP1 (targeting E. coli) were selected for further study. The selected phages formed plaques approximately 1 mm in diameter. Transmission electron microscopy (TEM) analysis classified PAP5 (head: 68 nm, tail: 35 nm) as a member of the Podoviridae family, while KPP3 (head: 80 nm, tail: 20 nm) and ECP1 (head: ~75 nm, tail: 165 nm) were identified as members of the Myoviridae family. Phage growth dynamics revealed single-step growth curves, with latent periods of 35-45 minutes for PAP5, 10-15 minutes for KPP3, and 20-25 minutes for ECP1. Burst sizes were measured at 57 pfu/cell for PAP5, 80 pfu/cell for KPP3, and 73.3 pfu/cell for ECP1. The multiplicity of infection (MOI) for all three phages was optimized at 0.01. Stability tests showed that the phages remained active across a broad pH range (3–12) and temperature range (4°C-65°C), with strong lytic activity observed at 37°C. Whole-genome sequencing using Illumina NovaSeq technology provided detailed genomic insights. PAP5 was found to have a linear dsDNA genome of 302,823 bp, with a GC content of 46.5%, 660 coding sequences (CDS), 13 tRNAs, and 2 tmRNAs. KPP3 had a linear dsDNA genome of 167,858 bp, a GC content of 39.5%, 287 CDS, and 16 tRNA-coding sequences. ECP1's linear dsDNA genome was 88,275 bp, with a GC content of 39.2% and 155 CDS. Functional gene regions were identified, and comparative genomic analyses were conducted to construct phylogenetic trees, providing valuable insights into the evolutionary relationships of these phages.