ABSTRACT The 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.
