Fitness cost of mcr-1-mediated polymyxin resistance in Klebsiella pneumoniae

Abstract

Objectives: The discovery of mobile colistin resistance mcr-1, a plasmid-borne polymyxin resistance gene, highlights the potential for widespread resistance to the last-line polymyxins. In the present study, we investigated the impact of mcr-1 acquisition on polymyxin resistance and biological fitness in Klebsiella pneumoniae.

Methods: K. pneumoniae B5055 was used as the parental strain for the construction of strains carrying vector only (pBBR1MCS-5) and mcr-1 recombinant plasmids (pmcr-1). Plasmid stability was determined by serial passaging for 10 consecutive days in antibiotic-free LB broth, followed by patching on gentamicin-containing and antibiotic-free LB agar plates. Lipid A was analysed using LC-MS. The biological fitness was examined using an in vitro competition assay analysed with flow cytometry. The in vivo fitness cost of mcr-1 was evaluated in a neutropenic mouse thigh infection model.

Results: Increased polymyxin resistance was observed following acquisition of mcr-1 in K. pneumoniae B5055. The modification of lipid A with phosphoethanolamine following mcr-1 addition was demonstrated by lipid A profiling. The plasmid stability assay revealed the instability of the plasmid after acquiring mcr-1. Reduced in vitro biological fitness and in vivo growth were observed with the mcr-1-carrying K. pneumoniae strain.

Conclusions: Although mcr-1 confers a moderate level of polymyxin resistance, it is associated with a significant biological fitness cost in K. pneumoniae. This indicates that mcr-1-mediated resistance in K. pneumoniae could be attenuated by limiting the usage of polymyxins.

Figure 1.

Stability of the empty vector (pBBR1MCS-5) and mcr-1 recombinant plasmid (pmcr-1) in K. pneumoniae B5055 strains over 10 days (∼10 generations per day) in the absence of antibiotic. Error bars represent the standard deviation of the mean (n = 3).

Figure 2.

Mass spectra of lipid A from the (a) K. pneumoniae B5055 parental strain, (b) vector-only K. pneumoniae B5055_pBBR1MCS-5 strain and (c) mcr-1-expressing K. pneumoniae B5055_pmcr-1 strain. The pEtN-modified lipid A species are shown by the arrows. (d) Structures of predominant hexa-acylated lipid A species from each strain, with the corresponding pEtN-modified species on the right with pEtN shown in the boxes.

Figure 3.

Selection coefficient for K. pneumoniae B5055 parental strain, vector-only K. pneumoniae B5055_pBBR1MCS-5 strain and mcr-1-expressing K. pneumoniae B5055_pmcr-1strain against the reference strain E. coli JW1. Error bars represent the standard deviation of the mean (n = 4). *P ≤ 0.05.

Figure 4.

In vivo effect of mcr-1 in a neutropenic mouse thigh infection model. Recovered bacterial burdens at 24 h (a) without any treatment and (b) treated with polymyxin B (30 mg/kg/day). Error bars represent the standard deviation of the mean (n = 4). *P ≤ 0.05.