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

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 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/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 μg/ml (P = 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/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.

Fig 1

Nucleotide sequences of the upstream regions of the ompK36 gene in strains with wild-type sequence, a full-length IS5 insertion, and an IS5 insertion along with a promoter deletion. Nucleotide positions shared by the three strains are marked with asterisks; missing bases are indicated by dashes. The ompK36 ribosome binding site (rbs), start codon, and −10 and −35 promoter sequences are shown in boldface, while the presumed IS5 element integration sites (target site duplications [TSD]) are shown in boldface italics. Open reading frames (ORFs) are portrayed by arrows, with the IS5 transposase ORF indicated in black and the upstream ORF (KPHS_37020) and ompK36 ORF in white. The inserted IS5 element, including the IS5 transposase ORF (black arrow) and its right and left inverted repeats (IR-R and IR-L), are shown in gray italic letters. Single-line arrows beneath the inverted repeats indicate the direction of the repeats. A 1,200-bp IS5 element is inserted 33 bp upstream of the ompK36 start code from the TSD site (TTAA), which qRT-PCR data indicate is disrupting the transcription of ompK36. The gray shading denotes a 395-bp deletion region, encompassing a partial sequence of an unknown ORF (KPHS_37020) and the −30 and −10 promoters. The IS5 insertion and promoter deletion also disrupt ompK36 expression.

Fig 2

Killing activity of doripenem (Dori) against KPC-K. pneumoniae strains, stratified by doripenem MICs (A) and the presence of ompK36 mutations (B). The data represent the median log₁₀ kills by doripenem. (A) P values indicate the differences in killing by doripenem against 4 strains with doripenem MICs of ≤8 μg/ml and 19 strains with MICs of >8 μg/ml. (B) P values indicate the differences in killing by doripenem against 8 ins aa134-135 GD mutants, 7 IS5 mutants, and 8 ompK36 other mutant/wild-type strains (ANOVA).

Fig 3

Killing activity of colistin (COL) against KPC-K. pneumoniae strains, stratified by colistin MICs. The data represent median log₁₀ kills by colistin; the P values indicate the differences between KPC-K. pneumoniae strains for which colistin MICs were ≤2 μg/ml and >2 μg/ml.

Fig 4

Killing activities of doripenem, colistin, and colistin-doripenem against KPC-K. pneumoniae strains, stratified by doripenem MICs (A) and the presence of ompK36 mutations (B). The P values indicate the differences in median time kills by colistin versus doripenem-colistin in combination. (A) Median log₁₀ kills by doripenem, colistin, and doripenem-colistin against KPC-K. pneumoniae strains with MICs of ≤8 μg/ml (upper graph) and >8 μg/ml (lower graph). Note the lower level of kills for strains with MICs of >8 μg/ml at each time point. (B) Median log₁₀ kills by doripenem, colistin, and doripenem-colistin against other mutant/wild-type ompK36 strains (upper graph) and strains with ins aa134-135 GD or IS5 mutations (lower graph). Note the lower level of kills for ins aa134-135 GD or IS5 mutants.

Fig 5

Model for predicting responses of KPC-K. pneumoniae strains to doripenem-colistin. The model is based on data demonstrating that lower doripenem MICs were associated with the extent of synergy at 24 h and that the absence of ins aa134-135 GD and IS5 mutations was independently associated with levels of killing and extent of synergy at 24 h.