In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment

Abstract

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains 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 pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB(L82R) in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB(L82R) mutant. 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.

FIG 1

Organization of the pmrCAB operon and flanking regions in K. pneumoniae KPB-2 and location of the T245G mutation in the pmrB gene, leading to the L82R amino acid substitution in the transmembrane domain of the PmrB protein. The locations of primers used for PCR cloning of the pmrB gene for complementation experiments are indicated by thin black arrows. A, AvaI_pmrAB_F; E, EcoRI_pmrAB_R. In Salmonella enterica, the pmrC gene product catalyzes the addition of phosphoethanolamine to lipid A, which also could contribute to decreasing the negative charge of lipid A (14). The role of pmrC in K. pneumoniae has not been specifically reported, but given the conservation of pmrC and of the whole Pmr system, it likely retains the same function.

FIG 2

Growth curves for the KPB-1 (colistin-susceptible) and KPB-2 (colistin-resistant) K. pneumoniae isolates obtained in the competition experiment. The competition index (CI) values were determined at various time points, as previously reported (21).