Spontaneous wbbL and waaG mutations selected by phage vB_EcoP_P61425 confer resistance and attenuate virulence in ExPEC
- 1Department of Clinical Laboratory, The First People's Hospital of Zhangjiagang City, Zhangjiagang Hospital Affiliated to Soochow University, Zhangjiagang, China
- 2MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing, China
- 3College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, China
Abstract
Bacteriophage therapy is a promising strategy against multidrug-resistant (MDR) extraintestinal pathogenic Escherichia coli (ExPEC), yet bacterial resistance evolution remains a major clinical challenge. In this study, we isolated and comprehensively characterized a novel lytic phage, vB_EcoP_P61425, belonging to the genus Suseptimavirus. The phage has a latent period of 15 min and a burst size of approximately 68 PFU/cell. Phage vB_EcoP_P61425 exhibits stability across temperatures of 4 °C to 50 °C and a pH range of 3 to 10, displaying potent lytic activity against common ExPEC serotypes including O25 and O75. Genomic analysis revealed a 77933 bp double-stranded DNA genome with a GC content of 42.60% and 118 predicted coding sequences (CDSs), devoid of known lysogeny-associated, toxin, or antimicrobial resistance genes. In vitro assays confirmed its strong bactericidal efficacy, and it significantly reduced bacterial colonization in a medical catheter model. To elucidate the genetic mechanisms underlying bacterial resistance, spontaneous phage-resistant mutants were isolated and analyzed using whole-genome resequencing, targeted gene deletion, and genetic complementation assays. We demonstrated that mutational inactivation or directed deletion of the lipopolysaccharide (LPS) biosynthesis genes wbbL and waaG significantly impaired phage adsorption, thereby conferring resistance to vB_EcoP_P61425 in EC1649. Crucially, these resistant mutants exhibited phenotypic and virulence-associated trade-offs, manifested by altered cell morphology, markedly impaired adherence to host epithelial cells, and significantly attenuated virulence in a Galleria mellonella larval infection model. Collectively, this study demonstrates that mutations in the LPS biosynthesis genes wbbL and waaG in E. coli EC1649 confer phage resistance while imposing an evolutionary trade-off that attenuates bacterial pathogenicity, providing new insights for optimizing phage therapy regimens against ExPEC.
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BibTeX
@article{chen2026spontaneous,
title = {{Spontaneous wbbL and waaG mutations selected by phage vB_EcoP_P61425 confer resistance and attenuate virulence in ExPEC}},
author = {Long Chen and Sixiang Xu and Xiaoyue Li and Pei Li and Mengmeng Su and Peifan Liu and Yilin Fang and Wei Zhang},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1953743},
url = {https://doi.org/10.3389/fmicb.2026.1953743},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Spontaneous wbbL and waaG mutations selected by phage vB_EcoP_P61425 confer resistance and attenuate virulence in ExPEC
AU - Long Chen
AU - Sixiang Xu
AU - Xiaoyue Li
AU - Pei Li
AU - Mengmeng Su
AU - Peifan Liu
AU - Yilin Fang
AU - Wei Zhang
PY - 2026
DA - 2026/10/12
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1953743
UR - https://doi.org/10.3389/fmicb.2026.1953743
AB - Bacteriophage therapy is a promising strategy against multidrug-resistant (MDR) extraintestinal pathogenic Escherichia coli (ExPEC), yet bacterial resistance evolution remains a major clinical challenge. In this study, we isolated and comprehensively characterized a novel lytic phage, vB_EcoP_P61425, belonging to the genus Suseptimavirus. The phage has a latent period of 15 min and a burst size of approximately 68 PFU/cell. Phage vB_EcoP_P61425 exhibits stability across temperatures of 4 °C to 50 °C and a pH range of 3 to 10, displaying potent lytic activity against common ExPEC serotypes including O25 and O75. Genomic analysis revealed a 77933 bp double-stranded DNA genome with a GC content of 42.60% and 118 predicted coding sequences (CDSs), devoid of known lysogeny-associated, toxin, or antimicrobial resistance genes. In vitro assays confirmed its strong bactericidal efficacy, and it significantly reduced bacterial colonization in a medical catheter model. To elucidate the genetic mechanisms underlying bacterial resistance, spontaneous phage-resistant mutants were isolated and analyzed using whole-genome resequencing, targeted gene deletion, and genetic complementation assays. We demonstrated that mutational inactivation or directed deletion of the lipopolysaccharide (LPS) biosynthesis genes wbbL and waaG significantly impaired phage adsorption, thereby conferring resistance to vB_EcoP_P61425 in EC1649. Crucially, these resistant mutants exhibited phenotypic and virulence-associated trade-offs, manifested by altered cell morphology, markedly impaired adherence to host epithelial cells, and significantly attenuated virulence in a Galleria mellonella larval infection model. Collectively, this study demonstrates that mutations in the LPS biosynthesis genes wbbL and waaG in E. coli EC1649 confer phage resistance while imposing an evolutionary trade-off that attenuates bacterial pathogenicity, providing new insights for optimizing phage therapy regimens against ExPEC.
SN - 1664-302X
ER -