In VivoEmergence of Colistin Resistance in Klebsiella pneumoniae Producing KPC-Type Carbapenemases Mediated by Insertional Inactivation of the PhoQ/PhoPmgrBRegulator

Abstract

ABSTRACTColistin is one of the few agents that retain activity against extensively drug-resistant strains ofKlebsiella pneumoniaeproducing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of themgrBgene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role ofmgrBinactivation in acquired colistin resistance was confirmed by complementation experiments with wild-typemgrB, which restored colistin susceptibility in KKBO-4, and by construction of anmgrBdeletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. InsertionalmgrBinactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of themgrBgene, upregulated transcription ofphoP,phoQ, andpmrK(which is part of thepmrHFIJKLMoperon) was detected. These findings confirmed the MgrB regulatory role inK. pneumoniaeand were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of thepmrHFIJKLMoperon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.