Purpose: Antibiotic resistance is a growing health crisis that is further complicated by treatment failures caused by bacteria that exhibit heterogeneous susceptibility to antibiotics. The aim of this study was to describe imipenem (IPM)-heteroresistant strains among multidrug-resistant (MDR) ESBL/AmpC-producing Klebsiella pneumoniae  clinical isolates, investigate their molecular phenotypic characteristics, and elucidate the outcome of antibiotic treatment in mice infected with the heteroresistant isolates.
Materials and Methods: Antimicrobial susceptibility of K. pneumoniae  isolates was determined by the disk diffusion and E-test methods. Heteroresistance to IPM was confirmed by population analysis profile (PAP) assays. PCR and sequencing were employed to detect MDR determinants. Molecular differences between the susceptible and resistant subpopulations were evaluated by sequencing and quantitative real-time reverse transcription PCR (qRT-PCR) analysis. The effect of the carbapenem-heteroresistant strains on antibiotic treatment was assessed using a mouse model of peritonitis with heteroresistant K. pneumoniae  and subsequent treatment with IPM.
Results: In total, 37 MDR ESBL/AmpC-producing clinical isolates of K. pneumoniae  were identified between September 2018 and December 2019. These strains were notably resistant to conventional antimicrobials other than carbapenems. Among the isolates, three strains exhibited heteroresistance to IPM and carried several ESBL and/or AmpC genes. Mice infected with a lethal dose of any of the three heteroresistant isolates were unable to survive in the presence of IPM treatment, as the percentage of the IPM-resistant subpopulation of each strain was increased in the peritoneum of these mice at 24 h after infection. The resistant subpopulation of the strains presented pulsed-field gel electrophoresis (PFGE) profiles that were identical to those of the susceptible subpopulation, but ompK36 porin showed a reduction in gene expression (0.09- to 0.50-fold) in the resistant subpopulation.
Conclusion: Carbapenem-heteroresistant strains were present among the MDR K. pneumoniae  isolates producing ESBL/AmpC β-lactamases, and these heteroresistant strains failed IPM therapy in experimentally infected mice.
Keywords: Enterobacteriaceae , imipenem, heterogeneous susceptibility, treatment failure, OmpK porin, in vivo