Genomic and Transcriptomic Analyses of Colistin-Resistant Clinical Isolates of Klebsiella pneumoniae Reveal Multiple Pathways of Resistance

Abstract

fThe emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col r ) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col r in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col r clinical isolates. Col r was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N) modification of lipid A was found in all Col r strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Col r mechanisms may be dependent on the genetic background.T he increasing occurrence of multidrug-resistant (MDR) Klebsiella pneumoniae has expanded reliance on last-line therapies like colistin, a cationic antimicrobial peptide, for effective treatment (1). Of concern is the growing recovery of colistin-resistant (Col r ) strains from clinical settings (2-4). Colistin disrupts membrane integrity through displacement of cations like Mg 2ϩ and Ca 2ϩ in the outer membrane, leading to cell lysis (5). Resistance mechanisms described to date involve lipopolysaccharide (LPS) modification, particularly through derivatization of lipid A phosphate moieties with a sugar or ethanolamine. These modifications reduce the electrostatic affinity between the cationic colistin and anionic LPS. Mutations in the transcriptional regulatory systems controlling these LPS modifications are a common genetic mechanism leading to colistin resistance. For example, the PhoPQ and PmrAB two-component regulatory systems (TCRS) regulate expression of the gene (pmrC) that codes for the addition of phosphoethanolamine (pETN) and genes encoding biosynthesis and lipid A transfer of 4-amino-4-deoxy-L-arabinose (Ara4N) (pmrHFIJKLM) (Fig. 1A). Other regulatory components in this pathway include PmrD and MgrB, two connector proteins that convey feedback between the PmrAB and PhoPQ TCRS ...

Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes

“…However, inactivation of the crrB gene leads to overexpression of the pmrAB operon, thus causing activation of the pmrHFIJKLM operon and of the pmrC and pmrE genes, consequently leading to the production of L-Ara4N and pEtN, both of which are responsible for the acquisition of colistin resistance (Fig. 3) (128). CrrB inactivation may also modify lipid A through activation of a glycosyltransferase-like protein (128).…”

“…CrrB inactivation may also modify lipid A through activation of a glycosyltransferase-like protein (128). Six amino acid substitutions in the CrrB protein have been identified as being responsible for acquired resistance to polymyxins in K. pneumoniae (Table 3) (128,137).…”

“…Several mutations in the phoP and phoQ genes are responsible for acquired resistance to polymyxins in K. pneumoniae (81,105,117,118,120,128) (Table 3). One mutation potentially involved in colistin resistance in E. coli has also been described (65).…”

Large Nosocomial Outbreak of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Traced to Clonal Expansion of an mgrB Deletion Mutant

“…Inactivation of the mgrB gene, which encodes a negative feedback regulator of the PhoQ/PhoP signal transduction system, was found to be one of the most common mutational mechanisms responsible for polymyxin resistance among clinical isolates of CRKP (12)(13)(14). Alterations of the PmrA/PmrB and other twocomponent signal transduction systems have also been identified as causes of polymyxin resistance in K. pneumoniae (15)(16)(17)(18)(19).…”

From Pigs to Patients: Transmissible, Single Gene-mediated Resistance to Colistin

“…These additions reduce the negative charge and limit antimicrobial peptide binding and membrane disruption [4,5]. The emergence of clinical resistance to colistin is due to point mutations in regulatory proteins that control the genes responsible for these additions, PhoPQ and/or PmrAB (and MgrB in K. pneumoniae) and were transmitted only vertically [6][7][8][9]. These data suggest that contrary to a common argument put forward by those developing novel antimicrobial peptides, resistance to this antibiotic class is common and now transmissable as single gene determinants.…”