Phage resistance bidirectionally altered antibiotic susceptibility in Klebsiella pneumoniae via galE mutation

OBJECTIVE: Multidrug-resistant (MDR) Gram-negative bacteria, particularly Klebsiella pneumoniae, represent a significant threat to global health. Given the shortage of new antibiotics, bacteriophages (phages) offer a promising alternative. Understanding bacteria-phage interactions and resistance mechanisms is vital for optimising phage therapy. We aimed to investigate the resistance mechanism of an MDR clinical isolate, K. pneumoniae UCSD1 (designated as KpUCSD1) against a newly isolated phage ΦKpUCSD1. METHODS: A phage-resistant mutant KpUCSD1R was collected at 24 h following treatment of KpUCSD1 with ΦKpUCSD1. Comparative genomic analysis identified mutations in KpUCSD1R, and complementation was conducted to evaluate the impact of these genes towards phage resistance. Spot tests and time-kill kinetics were performed to evaluate the bacterial susceptibility to ΦKpUCSD1. The role of galE in phage infection was evaluated with adsorption and lipopolysaccharide (LPS) assays. RESULTS: Mutations were identified in ubiH and galE of KpUCSD1R and galE was crucial for phage infection, as complementing KpUCSD1R with wild-type galE restored its susceptibility to ΦKpUCSD1. The clean knockout strain, KpUCSD1ΔgalE, exhibited complete loss of phage susceptibility owing to defective phage adsorption. Further analyses revealed that mutated and loss of galE resulted in truncated LPS. Interestingly, phage resistance imposed a fitness trade-off associated with galE disruption, resulting in collateral antibiotic effects, namely increased susceptibility to aminoglycosides, chloramphenicol, and polymyxins, alongside reduced susceptibility to tetracycline and meropenem. CONCLUSION: This study demonstrates, for the first time, that galE mutation in K. pneumoniae diminishes phage adsorption by causing truncation of the LPS. The trade-off leads to increased susceptibility to several antibiotics, offering a potential therapeutic advantage in treating MDR infections.