In Vitro Synergism of Azithromycin Combination with Antibiotics against OXA-48-Producing Klebsiella pneumoniae Clinical Isolates

Abstract

Carbapenem-resistant Klebsiella pneumoniae has globally emerged as an urgent threat leading to the limitation for treatment. K. pneumoniae carrying bla_OXA-48, which plays a broad magnitude of carbapenem susceptibility, is widely concerned. This study aimed to characterize related carbapenem resistance mechanisms and forage for new antibiotic combinations to combat bla_OXA-48-carrying K. pneumoniae. Among nine isolates, there were two major clones and a singleton identified by ERIC-PCR. Most isolates were resistant to ertapenem (MIC range: 2->256 mg/L), but two isolates were susceptible to imipenem and meropenem (MIC range: 0.5-1 mg/L). All bla_OXA-48-carrying plasmids conferred carbapenem resistance in Escherichia coli transformants. Two ertapenem-susceptible isolates carried both outer membrane proteins (OMPs), OmpK35 and OmpK36. Lack of at least an OMP was present in imipenem-resistant isolates. We evaluated the in vitro activity of an overlooked antibiotic, azithromycin, in combination with other antibiotics. Remarkably, azithromycin exhibited synergism with colistin and fosfomycin by 88.89% and 77.78%, respectively. Bacterial regrowth occurred after exposure to colistin or azithromycin alone. Interestingly, most isolates were killed, reaching synergism by this combination. In conclusion, the combination of azithromycin and colistin may be an alternative strategy in dealing with bla_OXA-48-carrying K. pneumoniae infection during a recent shortage of newly effective antibiotic development.

Keywords: Klebsiella pneumoniae; OXA-48; OmpK35; OmpK36; azithromycin; carbapenem; carbapenem resistance; colistin.

Figure 1

Genetic similarity of OXA-48-producing K. pneumoniae isolates. Dendrogram of genetic similarity based on ERIC-PCR generated by BioNumerics using UPGMA. The genetic similarity between the isolates was 90% (dashed line).

Figure 2

Relative bla_OXA-48 expression levels of K. pneumoniae. RT-qPCR assay of bla_OXA-48 expression was performed in K. pneumoniae cluster A (a), a singleton isolate KP162 (a), and cluster B (b). The relative number of bla_OXA-48 transcripts of K. pneumoniae isolates was normalized to 16S rRNA expression and calculated using the 2^−ΔΔct method compared to the expression of imipenem-susceptible K. pneumoniae isolates KP203 and KP221. p-values were calculated using unpaired t-test (**, p-value < 0.01; ***, p-value < 0.001 and ns, non-significant).

Figure 3

OMP profiles of nine OXA-48-producing K. pneumoniae isolates. OMP profiles were performed by SDS-PAGE. The arrow lines indicate OmpK35, OmpK36, and OmpA, respectively.

Figure 4

Relative ompK35 and ompK36 expression levels in K. pneumoniae. RT-qPCR assay of ompK35 and ompK36 expression was performed in K. pneumoniae cluster A (a,c), a singleton isolate KP162 (a,c), cluster B (b,d). The relative number of ompK35 and ompK36 transcripts of K. pneumoniae isolates was normalized to 16S rRNA expression and calculated using the 2^−ΔΔct method compared to the expression of imipenem-susceptible K. pneumoniae isolates KP203 and KP221. p-values were calculated using unpaired t-test (*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001 and ns, non-significant). Killing effect of imipenem against K. pneumoniae cluster A and a singleton (e), and cluster B (f). Mean of bacterial viable cells at the treatment with 1 × MIC of imipenem was plotted at 0, 2, 4, 6, and 24 h of incubation. All experiments were performed in triplicate and the detection limit of the viable cells was 10² CFU/mL (dashed lines).

Figure 5

Time–kill curves of azithromycin and colistin combination against OXA-48-producing K. pneumoniae isolates. Synergism of azithromycin and colistin was confirmed by time–killing assays against KP162 (a), KP221 (b), KP241 (c), KP197 (d), KP203 (e), KP166 (f), KP260 (g), KP262 (h), and KP1184 (i). Mean of bacterial viable cells at each condition: growth control or without antibiotic (circles), colistin = 1 × MIC (squares), azithromycin = 1 × MIC (triangles), and colistin = 1 × MIC combination with azithromycin = 1 × MIC (upside down triangles) was plotted at 0, 2, 4, 6, and 24 h of incubation. All experiments were performed in triplicate and the detection limit of the viable cells was 102 CFU/mL (dashed lines).