Multifaceted mechanisms of colistin resistance revealed by genomic analysis of multidrug-resistant Klebsiella pneumoniae isolates from individual patients before and after colistin treatment

Abstract

Objectives: Polymyxins (i.e., polymyxin B and colistin) are used as a last-line therapy to combat multidrug-resistant (MDR) Klebsiella pneumoniae. Worryingly, polymyxin resistance in K. pneumoniae is increasingly reported worldwide. This study identified the genetic variations responsible for high-level colistin resistance in MDR K. pneumoniae clinical isolates.

Methods: Sixteen MDR K. pneumoniae isolates were obtained from stool samples of 8 patients before and after colistin treatment. Their genomes were sequenced on Illumina MiSeq to determine genetic variations.

Results: Fifteen of 16 isolates harboured ISKpn26-like element insertion at nucleotide position 75 of mgrB, abolishing its negative regulation on phoPQ; while colistin-susceptible ATH7 contained intact mgrB and phoQ. Interestingly, each of the 7 mgrB-disrupted, colistin-susceptible isolates contained a nonsynonymous substitution in PhoQ (G39S, L239P, N253T or V446G), potentially impairing its function and intergenically suppressing the effect caused by mgrB inactivation. Additionally, three of the 7 corresponding mgrB-disrupted, colistin-resistant isolates harboured a secondary nonsynonymous substitution in PhoQ (N253P, D438H or T439P).

Conclusions: This is the first report of phoQ mutations in mgrB-disrupted, colistin-susceptible K. pneumoniae clinical isolates. We also discovered multiple phoQ mutations in mgrB-disrupted, colistin-resistant strains. Our findings highlight the multifaceted molecular mechanisms of colistin resistance in K. pneumoniae.

Keywords: Colistin; Genomics; Klebsiella pneumoniae; mgrB; phoQ.

Figure 1.

ML-based phylogeny of K. pneumoniae, containing the 16 isolates and 143 isolates with complete genomes (A). Bootstrap values (percentage) are indicated by a colour scale from red (1%) to green (100%). Specifically, the closely related K. pneumoniae genomes are shown in (B). The ST147 isolates ATH9 and ATH10 are highlighted in orange; and the 14 ST258 isolates are in blue. Reference strains for genetic variation analysis are in bold. The sequence type is shown after the isolate name for each genome. Scale bars in (A) and (B) indicate the number of substitutions per nucleotide site.

Figure 2.

Predicted insertion sequences in the 16 K. pneumoniae isolates.

Figure 3.

The absence (blank) and presence (colour) of the predicted antibiotic resistance genes in the 16 K. pneumoniae isolates.

Figure 4.

Unique nonsynonymous SNPs of the 16 K. pneumoniae isolates. The circular tracks, from outermost to innermost, represent the genes on positive and negative strands of the reference genomes, SNPs in colistin-susceptible (green) and -resistant isolates (red), amino acid substitutions, and the affected genes. The genes are colour-coded according to the predicted Clusters of Orthologous Groups (COG) functional categories of reference genomes using eggNOG tool. For SNP tracks, from outermost to innermost they are ATH9 and ATH10 in (A), and ATH7, ATH8, ATH15, ATH16, ATH17, ATH18, ATH21, ATH22, ATH23, ATH24, ATH25, ATH26, ATH 30 and ATH29 in (B); in each track, the SNPs are denoted by solid circles according to their quality score, with a range of 20–100 (from the lightest to darkest shading). The phoQ SNPs are highlighted by red arrows. Within 1.35–1.40 Mb (B), 73 SNPs were identified mostly in CI103_06880 (integrase) and CI103_07080–7095 (hypothetical proteins). No SNP track is depicted in (B) if there is no SNPs on reference plasmids.

Figure 5.

Mutations in PhoQ and MgrB. The mgrB inactivation (via ISKpn26-like integration) is denoted by red ‘×’. The nonsynonymous SNPs in phoQ are indicated by blue (identified exclusively in colistin-resistant strains) and red (the rest) dots. The function domains of PhoQ were assigned based on the literature.