In VivoEvolution to Colistin Resistance by PmrB Sensor Kinase Mutation in KPC-Producing Klebsiella pneumoniaeIs Associated with Low-Dosage Colistin Treatment

ABSTRACTColistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceaeproducing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniaestrains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrAand of pmrK, which is part of the pmrHFIJKLMoperon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrBin KPB-2 restored colistin susceptibility and reduced the transcription of pmrAand pmrKto basal levels, while expression of PmrBL82Rin KPB-1 did not alter colistin susceptibility or upregulate pmrAand pmrKexpression, confirming the dominance of wild-type PmrB versus the PmrBL82Rmutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.