A Novel Broad-Spectrum Lytic Phage vB_EcoM_P3322: Isolation, Characterization, and Therapeutic Potential Against Avian Pathogenic Escherichia coli

Panpan Tong^1*Ying Huang¹Mengfei Zhang¹Yuan Tian¹Xueqin Lan¹Wanjing Jin¹Yixin Bai¹Qirui Zang¹Mingshuai Chen¹Zhanqiang Su¹Wei Zhang²Gulina • Aishan³Mingyang Geng³Jinxin Xie¹

• ¹Xinjiang Agricultural University, Ürümqi, China
• ²Nanjing Agricultural University, Nanjing, China
• ³Ili Kazakh Autonomous Prefecture General Animal Husbandry Station, Xinjiang Uighur Autonomous Region, China

The widespread misuse of antibiotics has accelerated the emergence of multidrug-resistant bacterial strains, presenting a major threat to global public health. Bacteriophages (phages), owing to their host-specific lytic activity and self-replicating nature, have emerged as promising alternatives or adjuncts to conventional antibiotic therapies. In this study, we isolated and characterized a lytic phage, designated vB_EcoM_P3322, from farm wastewater targeting avian pathogenic Escherichia coli (APEC). Transmission electron microscopy classified vB_EcoM_P3322 within the Myoviridae family. The phage exhibited broad lytic activity against five Escherichia coli serotypes (O8:H10, O15:H18, O51:H20, O149:H20, and O166:H6). Optimal biological parameters included a multiplicity of infection (MOI) of 1, a latent period of 10 minutes, an 80-minute burst period, and a burst size of 252 PFUs/cell. vB_EcoM_P3322 maintained stable lytic activity across a pH range of 5–9 and temperatures from 4°C to 50°C, although it was sensitive to chloroform. In vitro, the phage effectively suppressed bacterial growth within 6 hours at MOIs of 0.1, 1, and 10. Whole-genome sequencing revealed a 151,674 bp double-stranded DNA genome encoding 279 predicted open reading frames. No virulence factors, toxin genes, antibiotic resistance genes, or lysogeny-related elements were identified, affirming its safety for therapeutic application. Phylogenetic analysis indicated 98.44% nucleotide identity (97% coverage) with phage vB_EcoM_Ro121c4YLVW (GenBank: NC_052654), suggesting a close evolutionary relationship. In a pigeon infection model, vB_EcoM_P3322 treatment significantly improved survival and reduced histopathological damage in the liver and spleen. Metagenomic analysis of duodenal contents revealed a marked reduction (P < 0.01) in E. coli abundance in the treatment group, indicating selective pathogen clearance and modulation of gut microbiota. In summary, vB_EcoM_P3322 displays broad-spectrum lytic activity, robust environmental stability, potent antibacterial efficacy both in vitro and in vivo, and a safe genomic profile. These attributes support its potential as a novel biocontrol agent for managing APEC infections in poultry farming.