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Life sciences · Journal article
Antimicrobial Agents and Chemotherapy · August 5, 2026
Early or partial results. Treat as a signal, not a conclusion.
This is a mechanistic screening study using transposon mutagenesis and genetic knockout to identify phage resistance determinants in a single clinical CRKP strain and to assess their bidirectional effects on antibiotic susceptibility. Multiple phage resistance pathways were identified (LPS biosynthesis, burst inhibition, mutation frequency) with conflicting impacts on antibiotic resistance, and in vitro synergy was demonstrated between phage P545 and three antibiotics, but the work remains preclinical and does not establish clinical utility.
Genome-wide transposon screening with CRISPR-Cas9 knockout and plasmid complementation. A single clinical ST11-KL64 carbapenem-resistant Klebsiella pneumoniae strain. Intervention: Genome-wide transposon mutagenesis, CRISPR-Cas9 knockout of identified genes, and phage P545 alone and in combination with meropenem, colistin, or ceftazidime. Compared with: Wild-type strain and complemented mutants.
Deletions of LPS biosynthesis genes (waaQ, wabH, wabG, ugd, galU, wcaG) conferred phage resistance but reduced resistance to multiple antibiotics Deletions of sirB1 inhibited phage burst through an undefined pathway Deletions of mutS and mutL increased mutation frequency and increased resistance to several antibiotics despite conferring phage resistance
In vitro synergy assays only; no in vivo pharmacodynamics, animal infection model, or clinical outcome data
These findings identify specific genetic targets for phage resistance and reveal conflicting effects of phage resistance mutations on antibiotic susceptibility, suggesting rational design of phage-antibiotic combinations may be possible. However, the single-strain, in vitro design means this work is exploratory and requires validation in multiple strains and animal models before clinical application.
Mechanistic screening study in a single clinical strain identifying phage resistance genes and antibiotic interactions, without clinical outcomes or in vivo validation to support practice change.
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These findings identify specific genetic targets for phage resistance and reveal conflicting effects of phage resistance mutations on antibiotic susceptibility, suggesting rational design of phage-antibiotic combinations may be possible. However, the single-strain, in vitro design means this work is exploratory and requires validation in multiple strains and animal models before clinical application.
Graded across the dimensions that decide whether you should act, each from what the source actually supports. There is no single score, and where a dimension was not assessed it says so.
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.
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