Genome-wide transposon screening uncovers phage resistance mechanisms and bidirectional effects on antibiotic susceptibility in carbapenem-resistant Klebsiella pneumoniae
6 affiliations
- 1Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
- 2Center for Discovery and Innovation, Hackensack-Meridian Health, Nutley, New Jersey, USA
- 3Department of Pharmacy Practice, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, New York, USA
- 4Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China
- 5Department of Clinical Laboratory, The Second People’s Hospital of Zhangjiagang, Suzhou, Jiangsu, China
- 6MOE Key Laboratory of Geriatric Diseases and Immunology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
Abstract
Phage therapy and phage-antibiotic combinations are promising strategies against multidrug-resistant pathogens like carbapenem-resistant Klebsiella pneumoniae (CRKP). However, the mechanisms underlying phage resistance and the molecular basis of the rational design of phage-antibiotic combinations require further investigation. Using genome-wide transposon screening, CRISPR-Cas9 knockout, and plasmid complementation in a clinical ST11-KL64 CRKP strain, we investigated susceptibility determinants for phage P545 and their impact on antibiotic susceptibility. We identified multiple phage resistance mechanisms, including disruption of phage receptor lipopolysaccharide (LPS) biosynthesis genes (involving deletions of waaQ, wabH, wabG, ugd, galU, and wcaG), inhibition of phage burst through an undefined pathway (involving deletion of sirB1), and increased mutation frequency (involving deletions of mutS and mutL). Furthermore, we found that deletions of LPS-related genes reduced resistance to multiple antibiotics, while mutS and mutL deletions increased resistance to several antibiotics. Further synergy assays showed that P545 in combination with meropenem, colistin, or ceftazidime had synergistic effects in the in vitro killing of host bacteria. By integrating genetic screening and functional validation, our approach offers a versatile platform for dissecting phage targets, understanding resistance mechanisms, and evaluating phage-antibiotic interactions. These findings provide valuable tools and insights for optimizing phage-phage and phage-antibiotic combination therapies against clinically significant multidrug-resistant pathogens.
Related
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BibTeX
@article{zhu2026genomewide,
title = {{Genome-wide transposon screening uncovers phage resistance mechanisms and bidirectional effects on antibiotic susceptibility in carbapenem-resistant Klebsiella pneumoniae}},
author = {Zhichen Zhu and Tengfei Long and Bingjie Wang and Weijia Ding and Yuxin Yan and Yicheng Wen and Yapin Zhao and Jie Zhu and Liang Wang and Jinnan Lv and Barry N. Kreiswirth and Hong Du and Liang Chen},
journal = {Antimicrobial Agents and Chemotherapy},
year = {2026},
month = aug,
doi = {10.1128/aac.00506-26},
url = {https://doi.org/10.1128/aac.00506-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Genome-wide transposon screening uncovers phage resistance mechanisms and bidirectional effects on antibiotic susceptibility in carbapenem-resistant Klebsiella pneumoniae
AU - Zhichen Zhu
AU - Tengfei Long
AU - Bingjie Wang
AU - Weijia Ding
AU - Yuxin Yan
AU - Yicheng Wen
AU - Yapin Zhao
AU - Jie Zhu
AU - Liang Wang
AU - Jinnan Lv
AU - Barry N. Kreiswirth
AU - Hong Du
AU - Liang Chen
PY - 2026
DA - 2026/08/05
JO - Antimicrobial Agents and Chemotherapy
DO - 10.1128/aac.00506-26
UR - https://doi.org/10.1128/aac.00506-26
AB - Phage therapy and phage-antibiotic combinations are promising strategies against multidrug-resistant pathogens like carbapenem-resistant Klebsiella pneumoniae (CRKP). However, the mechanisms underlying phage resistance and the molecular basis of the rational design of phage-antibiotic combinations require further investigation. Using genome-wide transposon screening, CRISPR-Cas9 knockout, and plasmid complementation in a clinical ST11-KL64 CRKP strain, we investigated susceptibility determinants for phage P545 and their impact on antibiotic susceptibility. We identified multiple phage resistance mechanisms, including disruption of phage receptor lipopolysaccharide (LPS) biosynthesis genes (involving deletions of waaQ, wabH, wabG, ugd, galU, and wcaG), inhibition of phage burst through an undefined pathway (involving deletion of sirB1), and increased mutation frequency (involving deletions of mutS and mutL). Furthermore, we found that deletions of LPS-related genes reduced resistance to multiple antibiotics, while mutS and mutL deletions increased resistance to several antibiotics. Further synergy assays showed that P545 in combination with meropenem, colistin, or ceftazidime had synergistic effects in the in vitro killing of host bacteria. By integrating genetic screening and functional validation, our approach offers a versatile platform for dissecting phage targets, understanding resistance mechanisms, and evaluating phage-antibiotic interactions. These findings provide valuable tools and insights for optimizing phage-phage and phage-antibiotic combination therapies against clinically significant multidrug-resistant pathogens.
SN - 0066-4804
SN - 1098-6596
ER -