Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility

Objectives Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilisation of phage therapy has been severely compromised by the potential emergence of phage resistance. While certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. Methods Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated via time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. Results We observed a repertoire of phage resistance mechanisms in K. pneumoniae, including disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitises antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin. Conclusions Our findings demonstrate that K. pneumoniae employs multiple strategies to overcome phage infection which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.