Genome-wide transposon screening uncovers phage resistance mechanisms and bidirectional effects on antibiotic susceptibility in carbapenem-resistant Klebsiella pneumoniae

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.