Mutations of the ompK36 Porin Gene and Promoter Impact Responses of Sequence Type 258, KPC-2-Producing Klebsiella pneumoniae Strains to Doripenem and Doripenem-Colistin

Abstract

d Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 g/ml) and colistin (2 g/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 g/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n ‫؍‬ 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n ‫؍‬ 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values < 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS5, and wild-type/other mutants, respectively (P ‫؍‬ 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was <8 g/ml (P ‫؍‬ 0.0007 and 0.09, respectively), but not if the MIC was >8 g/ml (P ‫؍‬ 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5 mutants (P ‫؍‬ 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P ‫؍‬ 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin. Carbapenem-resistant (CR) Klebsiella pneumoniae has emerged as a major pathogen (1-9), causing infections that are associated with crude mortality rates as high as 50% (1, 10-13). Observational studies indicate that outcomes may be improved with combination antimicrobial therapy compared to monotherapy (12,14,15), particularly if the regimens include a carbapenem (16). Nevertheless, optimal combinations are not defined, and treatment failure rates remain unacceptably high. Our group and others have reported bactericidal activity and synergy for doripenem-colistin against some, but not all, CR K. pneumoniae strains in vitro (17-19). Indeed, "one-size-fits-all" approaches to identifying optimal antimicrobial regimens against CR K. pneumoniae and other carbapenem-resistant Enterobacteriaceae are not likely to be effective. Carbapenem resistance is mediated through multiple mechanisms, including production of metallo-␤-lactamases and nonmetallocarbapenemases (such as Klebsiella pneumoniae carbapenemase [KPC] and OXA-type carbapenemase). Alternatively, strains with defects in outer membrane proteins (OMPs) may express AmpC ␤-lactamase or extended-spectrum ␤-lactamases (ESBLs). Levels of carbapenem resistance in individual strains can be impacted by the presence of several mechanisms. We hypothesize that the specific resistance mechanisms mani...

Carbapenem-Resistant Klebsiella pneumoniae Strains Exhibit Diversity in Aminoglycoside-Modifying Enzymes, Which Exert Differing Effects on Plazomicin and Other Agents

“…Our data add to a growing body of evidence that ST258 K. pneumoniae strains are highly heterogenous, despite being considered clonal by conventional molecular epidemiologic criteria (5,(26)(27)(28)(29)(30). We previously showed that differences in ompK36 genotypes and gene expression among ST258 strains at our center predict susceptibility or resistance to carbapenemcolistin combinations during time-kill assays (27,29). Recent studies from our group and others have demonstrated striking diversity in the core genome of ST258 K. pneumoniae strains, including strains recovered from patients at single centers or longitudinally from the bloodstreams of individual patients (26,28).…”

“…Indeed, ST258 strains were distinct by genotypes (presence of particular molecular mechanisms of antimicrobial resistance) and/or phenotypes (antimicrobial susceptibility profiles). Our data add to a growing body of evidence that ST258 K. pneumoniae strains are highly heterogenous, despite being considered clonal by conventional molecular epidemiologic criteria (5,(26)(27)(28)(29)(30). We previously showed that differences in ompK36 genotypes and gene expression among ST258 strains at our center predict susceptibility or resistance to carbapenemcolistin combinations during time-kill assays (27,29).…”

“…Taken together, the data indicate that "one size fits all" approaches to identifying effective antimicrobial regimens against carbapenem- resistant K. pneumoniae strains are not likely to be useful. Rather, increased effort should be given to establishing correlations between strain genetics and responsiveness to particular antimicrobial agents and combinations (27,29) and then using these data to define how best to utilize new drugs.…”

“…Susceptible, intermediate, and resistant MICs were defined according to Clinical and Laboratory Standards Institute (CLSI) criteria (19); the Food and Drug Administration (FDA) breakpoint for susceptibility was used for tigecycline (Յ2 g/ml). Strains were typed by multilocus sequence typing (MLST), and mechanisms of carbapenem resistance determined using standard methods, as previously reported by our groups (20,21). Heterogeneity among strains was demonstrated by a combination of geographic origin, MLST typing, ompK36 genotypes, AME patterns, and MICs of various antimicrobials.…”

Predictability of Phenotype in Relation to Common β-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae

“…Nutrients and other hydrophilic molecules, such as ␤-lactam antibiotics, diffuse through these channels, and changes in these pores are linked to decreases in ␤-lactam susceptibility (7-9, 37-39). Previously described changes include disruption of the porin gene or promoter by insertion sequences (IS), frameshifts, nonsense mutations, and insertions or duplications that increase electronegativity of the inner channel-exposed loop 3 region of OmpK porins (40)(41)(42)(43)(44)(45).…”