Heteroresistance of KPC-producing Klebsiella pneumoniae results of three subpopulations associated with a broad landscape of mutations

Carbapenemase-producing Klebsiella pneumoniae (CPKp) have disseminated globally and represent a major threat in hospitals with few therapeutic options and high mortality rates. K. pneumoniae isolates producing the carbapenemase KPC (KPC-Kp) might be misclassified as susceptible by antibiotic susceptibility testing (AST), allowing the use of imipenem or meropenem for treatment of infections, but with a high chance of failure. KPC-Kp frequently show heteroresistance (HR), with colonies growing in the inhibition halo of agar-based AST. By analysing a set of 82 CPKp, we showed that HR is indeed frequent among KPC-Kp and specific to these isolates compared to those producing other carbapenemases (VIM, NDM or OXA-48). By monitoring the single colony appearance at 8-fold the imipenem minimal inhibitory concentration using the ScanLag setup, we discriminated two surviving subpopulations: a Gaussian-like distribution of early-appearing colonies, with a delayed emergence compared to colonies arising in the absence of antibiotics, and a long tail of late-appearing colonies. A subset of colonies showed a reduced growth rate, likely resulting from costly mutations. For in-depth characterization of survival dynamics, 333 colonies were subjected to whole-genome sequencing. We identified a broad landscape of genetic alterations in 28% of these colonies, including gene loss, DNA amplification and point mutations. hypothesis of an heterogeneous population encompassing tolerant and persistent cells, but also resistant bacteria showing genetic alterations. Altogether, these data revealed the complexity of imipenem HR among KPC-Kp isolates.