Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility

Sue C Nang; Jing Lu; Heidi H Yu; Hasini Wickremasinghe; Mohammad A K Azad; Meiling Han; Jinxin Zhao; Gauri Rao; Phillip J Bergen; Tony Velkov; Norelle Sherry; David T McCarthy; Saima Aslam; Robert T Schooley; Benjamin P Howden; Jeremy J Barr; Yan Zhu; Jian Li

doi:10.1016/j.cmi.2024.03.015

Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility

Clin Microbiol Infect. 2024 Mar 22:S1198-743X(24)00145-9. doi: 10.1016/j.cmi.2024.03.015. Online ahead of print.

Authors

Sue C Nang¹, Jing Lu¹, Heidi H Yu¹, Hasini Wickremasinghe¹, Mohammad A K Azad¹, Meiling Han¹, Jinxin Zhao¹, Gauri Rao², Phillip J Bergen¹, Tony Velkov³, Norelle Sherry⁴, David T McCarthy⁵, Saima Aslam⁶, Robert T Schooley⁶, Benjamin P Howden⁴, Jeremy J Barr⁷, Yan Zhu⁸, Jian Li⁹

Affiliations

¹ Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.
² Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
³ Biomedicine Discovery Institute, Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
⁴ Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
⁵ Department of Civil Engineering, Monash University, Clayton, Victoria, Australia.
⁶ Division of Infectious Diseases and Global Public Health, Department of Medicine, University of California San Diego, La Jolla, CA, USA.
⁷ School of Biological Sciences, Monash University, Clayton, Victoria, Australia.
⁸ Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
⁹ Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia. Electronic address: jian.li@monash.edu.

PMID: 38522841
DOI: 10.1016/j.cmi.2024.03.015

Abstract

Objectives: Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although 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 by 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, such as 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-sensitizes 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.

Discussion: 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.

Keywords: Antibiotic; Antimicrobial resistance; Bacteriophage therapy; Klebsiella pneumoniae; Phage resistance; Phage steering.