Colistin is one of the last resort antibiotics used to treat infections by carbapenemase-producing Klebsiella pneumoniae (CPKP). Insertional inactivation of mgrB, a gene encoding a negative regulator of the PhoPQ two-component system (TCS), and crrAB (a sensory TCS) have recently gained attention as mediators of colistin resistance. In this study, broth microdilution colistin susceptibility testing and whole-genome sequencing were used to resolve phenotypic and genotypic resistance profiles in 11 clinical carbapenem- and colistin- resistant K. pneumoniae (KP). Whole-genome sequencing (WGS) was performed using short-paired end reads technology on an Illumina Miseq. Core genome single nucleotide polymorphisms (cg-SNP) were called by the Snippy pipeline, and recombination events were highlighted using Gubbins. The pan-genome was generated using Roary. Chromosomally encoded genes were also screened for synonymous and non-synonymous mutations, in particular, pmrAB, pmrD, pmrC, and phoPQ. The genetic environment of mgrB was manually validated by Sanger sequencing. Lipid A was extracted using mild acetic acid hydrolysis and profiled using MALDI-TOF MS to examine noteworthy modifications linked to decreased susceptibility to colistin. The lipid A major mass ion was observed at (m/z 1840) in all KP isolates. PCR amplification of mgrB revealed insertional inactivation ∆mgrB in three of the studied isolates (designated as KP5, KP6, and KP16) showing MICs ≥16 mg/L. ISKpn14 was associated with KP5 and KP6, while IS903 was detected in KP16. Wildtype mgrB gene in the remaining 8 isolates might suggest the involvement of other mechanisms underlying their nonsusceptibility to colistin. Recombination analysis highlighted genomic loci involved in both toxin-antitoxin and MFS efflux systems as favored hotspots for recombination. All 11 isolates were negative for the crrAB genes. Further biochemical and molecular analysis is in progress to characterize genetic determinants that play key roles in colistin resistance. Along with the escalating prevalence of CRKP and the lack of novel antibiotics, colistin resistance has imposed a worldwide concern. With the power of WGS and lipidomic approaches, genetic alterations in pathways responsible for lipid A modification can be detected with high precision, enabling us to better understand the molecular mechanisms involved in resistance.
