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J Antimicrob Chemother. 2019 Jul 31. pii: dkz314. doi: 10.1093/jac/dkz314. [Epub ahead of print]

Polymyxin resistance in Klebsiella pneumoniae: multifaceted mechanisms utilized in the presence and absence of the plasmid-encoded phosphoethanolamine transferase gene mcr-1.

Author information

1
Department of Microbiology, Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.
2
College of Veterinary Medicine, China Agricultural University, Beijing, China.
3
Department of Pharmacology and Therapeutics, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria, Australia.

Abstract

OBJECTIVES:

Until plasmid-mediated mcr-1 was discovered, it was believed that polymyxin resistance in Gram-negative bacteria was mainly mediated by the chromosomally-encoded EptA and ArnT, which modify lipid A with phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), respectively. This study aimed to construct a markerless mcr-1 deletion mutant in Klebsiella pneumoniae, validate a reliable reference gene for reverse transcription quantitative PCR (RT-qPCR) and investigate the interactions among mcr-1, arnT and eptA, in response to polymyxin treatments using pharmacokinetics/pharmacodynamics (PK/PD).

METHODS:

An isogenic markerless mcr-1 deletion mutant (II-503Δmcr-1) was generated from a clinical K. pneumoniae II-503 isolate. The efficacy of different polymyxin B dosage regimens was examined using an in vitro one-compartment PK/PD model and polymyxin resistance was assessed using population analysis profiles. The expression of mcr-1, eptA and arnT was examined using RT-qPCR with a reference gene pepQ, and lipid A was profiled using LC-MS. In vivo polymyxin B efficacy was investigated in a mouse thigh infection model.

RESULTS:

In K. pneumoniae II-503, mcr-1 was constitutively expressed, irrespective of polymyxin exposure. Against II-503Δmcr-1, an initial bactericidal effect was observed within 4 h with polymyxin B at average steady-state concentrations of 1 and 3 mg/L, mimicking patient PK. However, substantial regrowth and concomitantly increased expression of eptA and arnT were detected. Predominant l-Ara4N-modified lipid A species were detected in II-503Δmcr-1 following polymyxin B treatment.

CONCLUSIONS:

This is the first study demonstrating a unique markerless deletion of mcr-1 in a clinical polymyxin-resistant K. pneumoniae. The current polymyxin B dosage regimens are suboptimal against K. pneumoniae, regardless of mcr, and can lead to the emergence of resistance.

PMID:
31365098
DOI:
10.1093/jac/dkz314

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