In vitro activity of celastrol in combination with thymol against carbapenem-resistant Klebsiella pneumoniae isolates

Abstract

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial and community-acquired infections. Klebsiella has developed resistance against antimicrobials including the last resort class; carbapenem. Currently, treatment options for carbapenem-resistant-Klebsiella (CRK) are very limited. This study aims to restore carbapenem effectiveness against CRK using celastrol and thymol. Clinical Klebsiella isolates were identified using biochemical and molecular methods. Antimicrobial susceptibility was determined using disk-diffusion method. Carbapenemase-production was tested phenotypically and genotypically. Celastrol and thymol-MICs were determined and the carbapenemase-inhibitory effect of sub-MICs was investigated. Among 85 clinical Klebsiella isolates, 72 were multi-drug-resistant and 43 were meropenem-resistant. Phenotypically, 39 isolates were carbapenemase-producer. Genotypically, bla_NDM1 was detected in 35 isolates, bla_VIM in 17 isolates, bla_OXA in 18 isolates, and bla_KPC was detected only in 6 isolates. Celastrol showed significant inhibitory effect against carbapenemase-hydrolytic activity. Meropenem-MIC did not decrease in presence of celastrol, only 2-fold decrease was observed with thymol, while 4-64 fold decrease was observed when meropenem was combined with both celastrol and thymol. Furthermore, thymol increased CRK cell wall-permeability. Molecular docking revealed that celastrol is superior to thymol for binding to KPC and VIM-carbapenemase. Our study showed that celastrol is a promising inhibitor of multiple carbapenemases. While meropenem-MIC were not affected by celastrol alone and decreased by only 2-folds with thymol, it decreased by 4-64 folds in presence of both celastrol and thymol. Thymol increases the permeability of CRK-envelope to celastrol. The triple combination (meropenem/celastrol/thymol) could be useful for developing more safe and effective analogues to restore the activity of meropenem and other β-lactams.

Fig. 1

Molecular identification and phylogenetic analysis of bacterial isolates. a Gel electrophoresis of PCR products (440 bp) of genes encoding 16S rRNA of suspected K. pneumoniae, b representative phylogenetic tree of the partial 16S rRNA gene sequence from isolate 11 K compared to sequences of the most related K. pneumoniae strains recognized by BLASTN

Fig. 2

Phenotypic and genotypic detection of carbapenemase activity in Klebsiella isolates. a The Carba NP test was used for phenotypic analysis, positive results were indicated by the change of color of inoculated tube (t) to yellow: b Genotypic detection of carbapenemase genes by PCR in CRK isolates: lane 1: bla _NDM-1 gene (209 bp), lane 2: bla _VIM gene (382 bp), lane 3: bla _OXA gene (315 bp), lane 4: bla _KPC gene (209 bp), lane 5: negative control, M: 50 bp DNA-ladder

Fig. 3

Cell viability measurement with Alamar Blue. a Color change of resazurin dye 1: blank, 2: Celastrol, 3: Thymol, 4: Control. b fluorescence intensity of the treated culture was not significantly decreased from that of the control culture

Fig. 4

Celastrol inhibitory effect on the hydrolytic activity of the carbapenemases. a significant inhibitory effect of different celastrol concentrations on the activities carbapenemases detected by enzyme inhibition assays following co-incubation, b Percentage of carbapenemase activities inhibited by celastrol in a concentration-dependent manner. **indicates P < 0.01; ***indicates P < 0.001

Fig. 5

The putative binding modes (2D & 3D) of celastrol and thymol and their free binding energies expressed in Kcal/mol in the active site of the predicted 3D structure of K. pneumoniae carbapenemases a Class A KPC (KPC-2-3DW0), b Class D OXA (OXA-181 5OE0) c Class-B NDM (NDM-1 3SPU), d Class-B Verona Integron-encoded MBL (VIM-2 5YD7). The blue and cyan shadow of the ligand and active site amino acids respectively indicated strong hydrophobic/hydrophilic interactions