Rapid induction of high-level carbapenem resistance in heteroresistant KPC-producing Klebsiella pneumoniae

Abstract

Enterobacteriaceae strains producing the Klebsiella pneumoniae carbapenemase (KPC) have disseminated worldwide, causing an urgent threat to public health. KPC-producing strains often exhibit low-level carbapenem resistance, which may be missed by automated clinical detection systems. In this study, eight Klebsiella pneumoniae strains with heterogeneous resistance to imipenem were used to elucidate the factors leading from imipenem susceptibility to high-level resistance as defined by clinical laboratory testing standards. Time-kill analysis with an inoculum as low as 3 × 10(6) CFU/ml and concentrations of imipenem 8- and 16-fold higher than the MIC resulted in the initial killing of 99.9% of the population. However, full recovery of the population occurred by 20 h of incubation in the same drug concentrations. Population profiles showed that recovery was mediated by a heteroresistant subpopulation at a frequency of 2 × 10(-7) to 3 × 10(-6). Samples selected 2 h after exposure to imipenem were as susceptible as the unexposed parental strain and produced the major outer membrane porin OmpK36. However, between 4 to 8 h after exposure, OmpK36 became absent, and the imipenem MIC increased at least 32-fold. Individual colonies isolated from cultures after 20 h of exposure revealed both susceptible and resistant subpopulations. Once induced, however, the high-level imipenem resistance was maintained, and OmpK36 remained unexpressed even without continued carbapenem exposure. This study demonstrates the essential coordination between blaKPC and ompK36 expression mediating high-level imipenem resistance from a population of bacteria that initially exhibits a carbapenem-susceptibility phenotype.

FIG 1

Outer membrane fractions of 8-h imipenem-exposed OmpK36-deficient heteroresistant K. pneumoniae strain BR7 samples analyzed by SDS-PAGE. The identities of OmpA and OmpK36 were confirmed by ESI-MS with locations marked on the image; a 170-kDa molecular mass ladder is shown on the left. Lanes 1 to 4, samples from 2-μg/ml imipenem wells; lanes 5 to 7, samples from 2-μg/ml imipenem and 100 μM KPC enzyme inhibitor (PBA) wells (maximum concentration of sample growth); lane 8, BR7 wild-type control, initially expressing OmpK36, exposed to 16 μg/ml imipenem. Note that strain BR7 loses expression of OmpK36 with lethal imipenem exposure, but regains expression of OmpK36 upon drug-free passage (refer to text).