ABSTRACTKlebsiella pneumoniaeis emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniaeisolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producing K. pneumoniaeisolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in these K. pneumoniaeisolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in the lpxM, mgrB, phoQ, and yciMgenes in colistin-resistant K. pneumoniaeisolates. In two strains, mgrBwas inactivated by an IS3-like or ISKpn14insertion sequence element. Complementation in transwith the wild-type mgrBgene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2- to 4-fold in the presence of the mutated phoQ, lpxM, and yciMalleles. In conclusion, the present study indicates that heteroresistant K. pneumoniaesubpopulations may be selected for upon exposure to colistin. Mutations in mgrBand phoQhave previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in the yciMand lpxMgenes in the emergence of colistin resistance in K. pneumoniae.