The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a new threat to healthcare. In this study, we analyzed nine CR-hvKp isolates of different sequence-types (ST) recovered from patients with nosocomial infections in two hospitals in Saint Petersburg. Whole-genome sequencing showed that eight of them harbored large mosaic plasmids carrying resistance to carbapenems and hypervirulence simultaneously, and four different types of hybrid plasmids were identified. BLAST analysis showed a high identity with two hybrid plasmids originating in the UK and Czech Republic. We demonstrated that hybrid plasmids emerged due to the acquisition of resistance genes by virulent plasmids. Moreover, one of the hybrid plasmids carried a novel New Delhi metallo-beta-lactamase (NDM) variant, differing from NDM-1 by one amino acid substitution (D130N), which did not provide significant evolutionary advantages compared to NDM-1. The discovery of structurally similar plasmids in geographically distant regions suggests that the actual distribution of hybrid plasmids carrying virulence and resistance genes is much wider than expected.

The minimal inhibitory concentration (MIC) is conventionally used to define in vitro levels of susceptibility or resistance of a specific bacterial strain to an antibiotic and to predict its clinical efficacy. Along with MIC, other measures of bacteria resistance exist: the MIC determined at high bacterial inocula (MIC_HI) that allow the estimation of the occurrence of inoculum effect (IE) and the mutant prevention concentration, MPC. Together, MIC, MIC_HI and MPC represent the bacterial “resistance profile”. In this paper, we provide a comprehensive analysis of such profiles of K. pneumoniae strains that differ by meropenem susceptibility, ability to produce carbapenemases and specific carbapenemase types. In addition, we have analyzed inter-relations between the MIC, MIC_HI and MPC for each tested K. pneumoniae strain. Low IE probability was detected with carbapenemase-non-producing K. pneumoniae, and high IE probability was detected with those that were carbapenemase-producing. MICs did not correlate with the MPCs; significant correlation was observed between the MIC_HIs and the MPCs, indicating that these bacteria/antibiotic characteristics display similar resistance properties of a given bacterial strain. To determine the possible resistance-related risk due to a given K. pneumoniae strain, we propose determining the MIC_HI. This can more or less predict the MPC value of the particular strain.

The inoculum effect (IE) is a well-known phenomenon with beta-lactams. At the same time, the IE has not been extensively studied with carbapenem/carbapenemase inhibitor combinations. The antibiotic-to-inhibitor concentration ratio used in susceptibility testing can influence the in vitro activity of the combination. To explore the role of these factors, imipenem/relebactam and doripenem/relebactam MICs were estimated against six Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae strains at standard inocula (SI) and high inocula (HI) by two methods: with a fixed relebactam concentration and with a fixed, pharmacokinetic-based carbapenem-to-relebactam concentration ratio. The combination MICs at HI, compared to SI, increased with most of the tested strains. However, the IE occurred with only two K. pneumoniae strains regardless of the MIC testing method. The relationship between the MICs at SI and the respective inoculum-induced MIC changes was observed when the MICs were estimated at pharmacokinetic-based carbapenem-to-relebactam concentration ratios. Thus, (1) IE was observed with both carbapenem/relebactam combinations regardless of the MIC testing method; however, IE was not observed frequently among tested K. pneumoniae strains. (2) At HI, carbapenem/relebactam combination MICs increased to levels associated with carbapenem resistance. (3) Combination MICs determined at pharmacokinetic-based carbapenem-to-inhibitor concentration ratios predict susceptibility elevations at HI in KPC-producing K. pneumoniae.

Background/Objectives: Assessing antibiotic MICs at high bacterial counts is likely to disclose hidden bacterial resistance and the inoculum effect if present and therefore also reveal potential decreased antibiotic effectiveness. In the current study, we evaluated the predictive potential of MICs determined at high bacterial inocula to evaluate meropenem effectiveness and emergence of resistance in Klebsiella pneumoniae. Methods: Nine carbapenemase-free or carbapenemase-producing K. pneumoniae strains were exposed to meropenem in an in vitro hollow-fiber infection model (HFIM). The treatment effects were correlated with simulated antibiotic ratios of the area under the concentration–time curve (AUC) to the MIC (AUC/MIC) and to MICs determined at high inocula (AUC/MIC_HI). Results: Based on MICs determined at standard inocula, meropenem effects at different AUC/MIC ratios for both carbapenemase-free and carbapenemase-producing K. pneumoniae strains were stratified and could not be described by a single relationship. In contrast, when AUC/MIC_HI ratios were used, a single relationship with the antibiotic effect was obtained for all tested strains. Similarly, the emergence of meropenem resistance in HFIM was concordant with AUC/MIC_HI, but not with AUC/MIC ratios. Conclusions: MICs determined at high bacterial inocula enable the prediction of meropenem effects both for carbapenemase-free and for carbapenemase-producing K. pneumoniae strains. Also, MICs at standard and high inocula can identify carbapenemase-producing strains by revealing the inoculum effect.