A conserved KL2-capsule-related phage-resistant mechanism in carbapenem-resistantis surmountable by a rationally selected dual-phage cocktail.

Abstract

INTRODUCTION: As the use of bacteriophages to combat drug-resistant bacteria increases, phage resistance, which may lead to infection recurrence, has gradually gained attention. The intricate mechanisms underlying phage resistance, which are crucial for improving the efficacy of phage therapy, have not yet been fully delineated. This research focuses on uncovering the mechanisms of phage resistance and formulating robust strategy to address infections caused by carbapenem-resistant(CRAB). METHODS: A specific bacterium-phage pair (CRAB strain MRAB11 and lytic phage MRABP9) was employed. Phage-resistant strains were isolated and subjected to whole-genome sequencing, followed by comparative genomics analysis. A novel phage targeting phage-resistant strains, was screened and combined with MRABP9. The efficacy of the phage combination was validated in bacterial liquid growth and in mouse wound CRAB infection model. The antimicrobial spectra of phage MRABP9 and the phage combination were evaluated using multi-center clinicalisolates, and the fitness of phage-resistant strains was determined. RESULTS: All sequenced phage-MRABP9-resistant strains harbored mutations on KL2 locus under bothandconditions. A novel phage MRABphi22 targeting phage-resistant strains was introduced and its combination with phage MRABP9 effectively inhibited the growth and phage resistance development of CRAB, demonstrating promising efficacy in mouse wound infection treatment. The fitness of phage-resistant isolates in terms of antibiotic resistance, virulence and growth rate, was diminished, with dual-phage resistant strains bearing higher fitness costs. CONCLUSIONS: The mechanism underlying the response of MRAB11 to phage MRABP9 invasion exhibits high consistency on KL2 locus and is surmountable by a well-designed dual-phage cocktail. This study offers valuable guidance for future experimental design of clinical phage therapy, and points out potential therapeutic phage candidates for treating CRAB infections.