Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing

Abstract

The present study was undertaken to find novel genes associated with colistin resistance in Klebsiella pneumoniae. Five colistin-resistant mutants were derived from four colistin-susceptible parental K. pneumoniae strains belonging to different clones. Whole-genome sequencing was performed for the nine K. pneumoniae strains to screen altered candidate genes. Expression levels of genes with amino acid alterations in derivative strains were determined using quantitative real-time Polymerase chain reaction (PCR). Colistin susceptibility was examined in a parental strain complemented with altered candidate genes. Overall, 13 genetic alterations were identified in five pairs of isogenic K. pneumoniae strains. Genetic alterations related to KP1_3468, including the insertion of an IS5-like element in an intergenic or coding region and amino acid substitutions, were identified in three separate derivative strains. Amino acid substitutions and deletion of PhoQ were determined in one derivative strain. With inactivation of CrrA and substituted CrrB, amino acid substitutions and deletion were identified in a repressor of galETK operon (KP1_0061) and hypothetical protein (KP1_3620), respectively. Decreased colistin susceptibility was observed in a parental strain complemented with KP1-0061, but not a KP1-3620 gene. This study demonstrated diverse genetic paths to colistin resistance in K. pneumoniae. Our results suggest that a repressor of galETK operon may play an important role in colistin resistance in K. pneumoniae.