Whole-Genome Characterization and Comparative Genomic Analysis of Three Bacteriophages Active against Multidrug-Resistant Escherichia coli

Abstract

Background: Multidrug-resistant (MDR) Escherichia coli is an important clinical challenge, and bacteriophages are being investigated as targeted antibacterial agents. Whole-genome sequencing is essential for defining phage genome organization, taxonomic relationships and genomic features relevant to phage biology and safety.
Methods: Three purified bacteriophages active against MDR E. coli, isolated from environmental water samples and designated Phage-1 (P1; sequencing sample 1A), Phage-2 (P2; sample 2A) and Phage-3 (P3; sample 3A), were subjected to Illumina paired-end whole-genome sequencing. Raw-read quality was assessed using FastQC and MultiQC, and reads were trimmed using Trim Galore. Genomes were assembled using Unicycler and annotated using Pharokka. MASH/INPHARED was used for comparative genomic identification. Predicted lysis-associated genes and CARD/VFDB hits were assessed, and terminase large-subunit (terL)-based phylogenetic analysis was performed.
Results: Single-contig assemblies of 50,343 bp, 31,564 bp and 39,699 bp were obtained for P1, P2 and P3, respectively, with GC contents of 44.395%, 51.384% and 49.777%. MASH identified Escherichia phage vB_Eco_mar001J1, Escherichia phage 11W and Escherichia phage K1F as the closest database references for P1, P2 and P3, respectively, each with a MASH distance of 0.0 and 1000/1000 matching hashes. In the terL-based phylogeny, P1 clustered with vB_Eco_mar001J1, P2 with phage 11W and other P2-like (Peduovirus) phages, and P3 with Escherichia phage MirsaeidiM_Esher2. Lysis-associated functions were detected in all three genomes. No CARD antimicrobial-resistance-gene hits or VFDB virulence-factor hits were detected. An integrase annotation was identified in P2.
Conclusion: The three bacteriophages showed distinct genome sizes and functional organizations and were closely related to previously described E. coli phages. The absence of detected CARD/VFDB hits and integration-associated genes supports further evaluation of P1 and P3, whereas the integrase annotation and P2-like affiliation of P2 suggest a potentially temperate lifestyle that limits its therapeutic suitability. The genomic data provide a foundation for further biological and taxonomic characterization.