Diverse genetic alterations responsible for post-exposure colistin resistance in populations of the same strain of Klebsiella pneumoniae

The intra- and interstrain diversity of genetic alterations in Klebsiella pneumoniae after exposure to colistin was investigated. Four colistin-susceptible K. pneumoniae strains with different sequence types were exposed to 2 mg/L colistin, the average steady-state plasma concentration. After exposure, 10 single-step colistin-resistant mutants were selected in vitro from each K. pneumoniae strain. Minimum inhibitory concentrations (MICs) were evaluated by broth microdilution. Nucleotide sequences of genes associated with colistin resistance (phoPQ, pmrAB, mgrB and crrAB) were determined, and the mRNA expression levels of pbgP, phoP, phoQ, pmrD, pmrA and pmrB were measured by quantitative real-time polymerase chain reaction. Colistin MICs for single-step mutants ranged from 4 to >64 mg/L. Diverse amino acid alterations in pmrB, phoP, phoQ and mgrB, including insertion sequences, were identified in the colistin-resistant mutants. Different mutations were found even among mutants originating from the same parental strain, and several mutants displayed no mutations in the tested genes. While most colistin-resistant mutants showed elevated expression of pbgP, seven showed no increase compared with their susceptible parental strain, and some mutants exhibited no overexpression of pmrAB, phoPQ or pbgP. These results demonstrate the diversity of genetic variations in colistin-resistant populations derived from the same parental strain, and suggest that there are still unknown mechanisms of colistin resistance in K. pneumoniae.