Multifactorial Chromosomal Variants Regulate Polymyxin Resistance in Extensively Drug-ResistantKlebsiella pneumoniae
Authors: Pitt Miranda E , Elliott Alysha G , Cao Minh Duc , Ganesamoorthy Devika , Karaiskos Ilias , Giamarellou Helen , Abboud Cely S , Blaskovich Mark AT , Cooper Matthew A , Coin Lachlan 
Published: May 2017

ABSTRACTExtensively drug-resistantKlebsiella pneumoniae(XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including nineteen polymyxin-resistant and five polymyxin-susceptible strains, underwent whole genome sequencing. Approximately 90% of polymyxin resistance was enabled by alterations upstream or withinmgrB. The most common mutation identified was an insertion at nucleotide position 75 inmgrBvia an ISKpn26-like element in the ST258 lineage and ISKpn13in one ST11 isolate. Three strains acquired an IS1 element upstream ofmgrBand another strain had an ISKpn25insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D31E) affectingmgrB. Complementation assays revealed allmgrBperturbations contributed to resistance. Missense mutations inphoQ(T281M, G385C) were also found to facilitate resistance. Several variants inphoPQco-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance conferring mutations, including the ISKpn26-like insertion colonising with a novel mutation inpmrB(P158R), both confirmed via complementation assays. We also characterized a new multi-drug resistantKlebsiella quasipneumoniaestrain ST2401 which was susceptible to polymyxins. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins inK. pneumoniae.DATA SUMMARYWhole genome sequencing of the 24 clinical isolates has been deposited under BioProject PRJNA307517 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA307517).IMPACT STATEMENTKlebsiella pneumoniaecontributes to a high abundance of nosocomial infections and the rapid emergence of antimicrobial resistance hinders treatment. Polymyxins are predominantly utilized to treat multidrug-resistant infections, however, resistance to the polymyxins is arising. This increasing prevalence in polymyxin resistance is evident especially in Greece and Brazil. Identifying the genomic variations conferring resistance in clinical isolates from these regions assists with potentially detecting novel alterations and tracing the spread of particular strains. This study commonly found mutations in the genemgrB, the negative regulator of PhoPQ, known to cause resistance in KP. In the remaining isolates, missense mutations inphoQwere accountable for resistance. Multiple novel mutations were detected to be segregating withmgrBperturbations. This was either due to a mixed heterogeneous sample of two polymyxin-resistant strains, or because of multiple mutations within the same strain. Of interest was the validation of novel mutations inphoPQsegregating with a previously known ISKpn26-like element in disruptedmgrBisolates. Complementation of thesephoPQmutations revealed a reduction in minimum inhibitory concentrations and suggests the first evidence of partial suppressor mutations in KP. This research builds upon our current understanding of heteroresistance, lineage specific mutations and regulatory variations relating to polymyxin resistance.