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 these studies, eight Klebsiella pneumoniae strains, one Enterobacter cloacae strain and one Escherichia coli strain with heterogeneous resistance to imipenem were used to elucidate the factors leading from imipenem susceptibility (1-2ug/ml) to high-level resistance (>32ug/ml) as determined by clinical laboratory testing standards. These strains had highly similar heteroresistance phenotypes, yet were genetically diverse in their plasmid content other than carriage of the blaKPC-containing Tn4401.
We showed that the heteroresistant strains had effective KPC-mediated inactivation of low levels of imipenem, combined with reduced or absent expression of ompK35, but as the imipenem concentration increased to 8- and 16-fold higher than the MIC, a majority (>99%) of the population was killed. Time-kill analysis with an inoculum as low as 3x106 cfu/ml showed that full recovery of the population occurred by 20 hours of incubation in the same drug concentrations. We showed that it was not the density of the cultures per se, but cells with a distinct physiology, present at a frequency of 2x10-7 to 3x10-6 in starting cultures, and selected by lethal concentrations of imipenem, that coordinate population recovery subsequent to the killing of the majority of the initial cells. Samples selected 2 hours 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 hours after exposure, OmpK36 became absent and the imipenem MIC increased at least 32-fold. Individual colonies isolated from cultures after 20 hours of exposure revealed both susceptible and resistant subpopulations. We hypothesize that these heterogeneous populations arose from the small population of cells that initially survived imipenem-mediated killing.
There were two types of OmpK36 production among the K. pneumoniae heteroresistant strains. The majority (6 of 8) of the strains permanently abolished OmpK36 upon lethal imipenem exposure due to mobile insertion element interruptions in the coding region of ompK36. High-level imipenem resistance was maintained and OmpK36 remained absent even without continued carbapenem exposure. Two strains reverted to the heteroresistance phenotype and resumed production of OmpK36 once imipenem exposure was removed.
Through transposon mutagenesis of a reverter-type heteroresistant K. pneumoniae strain, we showed that acquisition of specific nutrients was essential to abolish OmpK36 production and for population recovery. Addition of the KPC enzyme inhibitor phenylboronic acid (PBA) at any point during imipenem exposure prior to loss of OmpK36 inhibited subsequent OmpK36 loss and prevented population recovery, showing the essentiality of blaKPC in the expression of heteroresistance.
These studies demonstrated the coordination between bacterial physiology, blaKPC and ompK36 expression that led to the rapid induction of high-level imipenem resistance from a population of bacteria that initially exhibited a carbapenem-susceptibility phenotype. The finding of specific nutritional requirements for full expression of heteroresistance highlights potential therapeutic avenues of exploration for these strains that pose an urgent and increasing threat to public health.