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

Abstract

ABSTRACTThe emergence of multidrug-resistant (MDR)Klebsiella pneumoniaehas resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Colr) is increasingly reported from clinical settings. The genetic mechanisms that lead to ColrinK. pneumoniaeare 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 Colrclinical isolates. Colrwas related to mutations in three different genes inK. pneumoniaestrains, with distinct impacts on gene expression. Upregulation of thepmrHoperon encoding 4-amino-4-deoxy-l-arabinose (Ara4N) modification of lipid A was found in all Colrstrains. Alteration of themgrBgene was observed in six strains. One strain had a mutation inphoQ. Common among these seven strains was elevated expression ofphoPQand unaltered expression ofpmrCAB, 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 designatedcrrAB. In these strains, expression ofpmrCAB,crrAB, and an adjacent glycosyltransferase gene, but not that ofphoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. ThecrrABgenes are present in mostK. pneumoniaegenomes, but not inEscherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because thecrrABgenes are present in only some strains, Colrmechanisms may be dependent on the genetic background.