Bacterial isolates and chemicals

A total of 85 clinical isolates of Klebsiella were recovered from specimens sent to clinical laboratory of Zagazig University hospital, Zagazig, Egypt. Clinical specimens were from different sources including blood, urine, sputum, pus, and endotracheal tube aspirate. Celastrol, Dimethyl sulphoxide (DMSO), Ethylene-diamine tetra-acetic acid (EDTA), Resazurin, and thymol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bacterial media and antibiotics disks were purchased from Oxoid (UK).

Phenotypic and molecular identification of Klebsiella species by 16S-rRNA gene sequencing

Klebsiella isolates were identified as being Gram-negative rods with lactose fermenting activity on MacConkey agar. Confirmatory tests include: IMViC tests, growth on triple sugar iron and motility tests [18].

Molecular identification was based on sequencing of 16S rRNA gene, gDNA was extracted as described previously [19]. Briefly, a colony of isolate was suspended in 50 µl of nuclease-free water, heated to 100 °C for 10 min using Biometra T-GRADIENT thermocycler (Rudolf-Wissell-Str. Göttingen, Germany). The bacterial debris were removed by centrifugation at 21,000 × g for 10 min, the supernatant was used as template in a polymerase chain reaction (PCR). The universal primers 341 F and R806 were used for amplification of bacterial 16S rRNA [20]. These primers were supplied by Sigma Aldrich (Petaluma, USA).

The COSMO PCR RED 2x Master Mix (Willowfort UK) was used. The PCR cycling conditions were as follows: an initial denaturation for 5 min at 94 °C, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min, and then a final extension for 5 min at 72 °C. The amplified PCR products were electrophoresed on 2% agarose gel, and photographed with gel documentation system (Cleaver Scientific Ltd, UK). The PCR products were purified using Thermo scientific GeneJET PCR purification kits (Vilnius, Lithuania), according to the manufacturer’s instructions. Purified PCR samples were used for sequencing according to service requirements, where 5 μl of template DNA (20–80 ng) were mixed with 5 μl of 341 F primer (5 pmol μl−1). PCR samples were sequenced using the Illumina HiSeq platform using 300 PE chemistry (GATC-Biotech, Konstanz, Germany). The obtained sequences were used to draw the phylogenetic tree and to perform multiple sequence alignment. Phylogenetic analyses were conducted in MEGA11 [21].

Antimicrobial susceptibility testing

Antimicrobial susceptibility of Klebsiella isolates was performed by disk diffusion method according to the clinical and laboratory standard institute (CLSI) guidelines [22]. Briefly, a sterile cotton swab was dipped in Mueller Hinton broth (MHB) with a turbidity equivalent to 0.5 McFarland standards, and used to inoculate the surface of dried Mueller Hinton agar (MHA) plate. The inoculated plates were left to dry for 3–5 min, and the antibiotic disks were placed on them. Plates were incubated at 37 °C for 18 h. The diameter of inhibition zone was measured, recorded, and interpreted according to CLSI (2). The used antibiotic disks include meropenem (MEM, 10 μg), piperacillin-tazobactam (TZP, 100/10 μg), ceftriaxone (CRO, 30 μg), cefepime (FEP, 30 μg), cefoperazone (CFP, 75 μg), aztreonam (ATM, 30 μg), gentamicin (GN, 10 μg), amikacin (AK, 30 μg), azithromycin (AZM, 15 μg), tetracycline (TE, 30 μg), tigecycline (TGC, 15 μg), levofloxacin (LEV, 5 μg), ofloxacin (OFX, 5 μg), trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 μg), and chloramphenicol (C, 30 μg).

Phenotypic detection of carbapenemase-producing Klebsiella by Carba NP test

The Carba NP colorimetric assay for detection of carbapenemase production is based on detection of acidic products due to hydrolysis of imipenem using phenol red indicator [22]. A modified protocol that uses colonies (instead of bacterial extracts) and using 0.1% Triton X-100 as cells lytic agent was used [23]. Briefly, a colony of pure bacterial culture was suspended in tube containing 100 µl of solution A (phenol red 0.05% + ZnSO410 mM + 0.1% (vol/vol) Triton X-100 solution, adjusted to pH= 7.8), or solution B (solution A + 12 mg ml−1 imipenem) then vortexed for 10 s and incubated at 35 °C for up to 2 h. The tube containing solution A was used as control and tube containing solution B as test. Carbapenemase-production was indicated by the appearance of orange or yellow color in the test tube while the control tube remained red [23].

Detection of carbapenemase encoding genes by PCR

Conventional PCR was conducted for the detection of presence of Metallo-β-lactamase genes (blaNDM and blaVIM) or carbapenemases genes (blaOXA-9 -and blaKPC-1) in Klebsiella isolates. The primers were supplied by IDT (Integrated DNA Technologies, Coralville, Iowa, USA). The sequences of the primers: blaNDM, blaOXA, blaKPC-1 [24], and blaVIM [25] are listed in Supplementary Table 1. PCR mixture contained 25 μl of MasterMix, 2 μl of each primer, 2 μl of DNA template, and nuclease free water to 50 μl. The ampilification conditions were: initial denaturation at 95 °C for 3 min followed by 30 cycles of denaturation at 95 °C for 5 s, annealing for 30 s at temperature indicated in supplementary Table 1, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 5 min.

Determination of minimum inhibitory concentration (MIC) of meropenem, celastrol, and thymol against CRK isolates

Meropenem-MIC was determined by broth microdilution method for CRK isolates that were positive for carbapenemase production. Briefly, 3 colonies were used of each isolate (from overnight culture) to inoculate 5 ml of MHB. Broth cultures were incubated for 18 h at 37 °C. Cultures were then diluted in sterile saline and the turbidity was adjusted to 0.5 McFarland’s standard. Then 1/100 dilution of this suspension was made in sterile MHB. In sterile 96 wells-microplate, serial dilutions of tested chemicals were prepared in sterile MHB including: meropenem (0.5–1024 µg ml−1), celastrol (0.5–1024 µg ml−1) and thymol (5–2400 µg ml−1). 50 µl of the bacterial suspension was added to each well containing the serially diluted chemicals. The plates were incubated for 18 h at 37 °C and examined for bacterial growth. The lowest concentration that inhibited visible bacterial growth was reported as MIC [22]. After determination of meropenem-MIC, isolates with the highest MIC were used in the next analysis, where the MICs of celastrol and thymol were determined by the broth microdilution method.

Cell viability measurement with Alamar Blue assay

Alamar Blue (Resazurin) assay was performed to assess the effect of sub-MIC of celastrol and thymol on the metabolic activity of CRK as described previously [26]. Briefly, bacterial isolates were incubated alone, with 128 µg ml−1 of celastrol, with 300 µg ml−1 of thymol, or with a combination of celastrol (128 µg ml−1) and thymol (300 µg ml−1) for 24 h at 37 °C. Cells were collected by centrifugation at 8000 rpm for 10 min and resuspended in freshly prepared Phosphate-buffered saline (PBS). 0.1 ml of 6.5 mg ml−1 resazurin stock solution (prepared in PBS) was added to 0.9 ml of cell suspension. The reaction solutions were incubated in dark for 4 h at 37 °C. Sterile PBS with resazurin was used as blank. The fluorescent intensity of the reduced resazurin (resorufin) was observed at 590 nm emission and 560 nm excitation wavelengths.

Carbapenemase inhibition assay of crude periplasmic extract

This technique measured the hydrolytic activity of meropenem at a final concentration of (100 μM) using ultraviolet-visible (UV-Vis) spectrophotometry in the presence and absence of enzyme inhibitor. EDTA (reported inhibitor of MBLs) was used to validate the test [27]. Briefly, bacteria were cultured overnight on MHA supplemented with meropenem (4 μg ml−1). A standard number of bacterial cells [~5 × 108 cells ml−1; optical density (OD) at 600 nm (OD 600) = 0.65] were suspended in 1 ml of 20 mM Tris–HCl buffer containing 2% v/v Triton X. After vigorous mixing, the suspension was left at room temperature for 10 min and then centrifuged at 4000 rpm for 15 min. The supernatant was collected, and 180 µl of the supernatant was added to 96 sterile microtiter-plate. The first well was used to estimate the background absorbance (blank). The second well contained 100 μM of meropenem, the third well contained 100 μM of meropenem and 64 µg ml−1 of celastrol, the fourth well contained 100 μM meropenem and 128 µg ml−1 of celastrol and the fifth well contained 100 μM meropenem and 300 µg ml−1 of thymol. Finally, ddH2O was added to each well to make the final volume 200 μl. Hydrolysis of meropenem was measured using a microplate reader (synergy HT BioTek) at 297 nm. Meropenem initial absorbance was measured immediately after the inclusion of the antibiotic. and the reaction plate was incubated at 37 °C for 60 min [28]. The hydrolysis of meropenem was measured after the incubation period. And The hydrolysis index was calculated as follows:

hydrolysis index  =  [Initial Absorbance – Absorbance after 60 min]/Initial Absorbance.

The half maximal inhibitory concentration (IC50) was calculated using Microsoft Excel. IC50 is defined as the minimum concentration of celastrol required for inhibiting the activity of the extracted carbapenemases by 50% Four concentrations of celastrol (0, 32, 64, and 128 µg ml−1) were used to determine the percentage of carbapenemases inhibition, then the IC50 was calculated.

Determination of meropenem MIC in presence of sub-MIC of celastrol and/or thymol

The MIC of meropenem was determined in presence of sub-MIC (64 µg ml−1 and 128 µg ml−1) of celastrol. Furthermore, meropenem MIC was determined in presence of ¼ (300 µg ml−1) and 1/8 (150 µg ml−1) MIC of thymol. Finally, a triple combination of meropenem, celastrol (64, 128 µg ml−1), and thymol (150, 300 µg ml−1) was evaluated. The change of meropenem MIC in presence of tested inhibitors was determined using the broth microdilution method as described previously [22].

Effect of sub-MIC concentrations of thymol on the lytic activities of SDS and Triton X 100 on K. pneumoniae outer membrane

This experiment was used to investigate thymol ability to increase the permeability of Klebsiella outer membrane to boost the activity of less permeable celastrol [29]. This technique is based on the sensitization of cells to lytic action of the detergents sodium dodecyl sulfate (SDS) and Triton X-100 by thymol. Briefly, a standard inoculum of bacteria (OD630 of 0.5) was treated with 300 µg ml−1 of thymol for 10 min at room temperature and added to microplate wells that already contained either SDS (0.1 and 1%), Triton X-100 (0.1 and 1%) or buffer solution. Turbidity of the cell suspensions was then monitored with Synergy microplate reader (Agilent, Santa Clara, USA) as described previously [30]. Cell death caused by the sudden influx of these lytic agents was determined by measuring the decrease in OD (Relative turbidity %).

In silico analysis by molecular docking

The crystal structures of the proteins were retrieved from the Protein Data Bank (https://www.rcsb.org/). These proteins include: the class A carbapenemase KPC-2 (PDB-ID: 3DW0) [31], NDM-1 (PDB-ID: 3SPU) [32], β-lactamase OXA-181 (PDB-ID: 5OE0) [33] and VIM-2 MBL (PDB-ID: 5YD7) [34]. Both celastrol and thymol were drawn into Marvin Sketch of Marvin suite (http://www.chemaxon.com) and lowest energy three-dimensional conformer for each was generated. Dock module of MOE (Molecular Operating Environment) version MOE 2019.0102,2 [35] on a computer having Pentium 1.6 GHz workstation, 512 MB memory using windows operating system, was utilized in docking studies. Tested compounds were docked into the rigid binding pocket of the protein using flexible ligand mode. The free energy of binding of the ligand is estimated using the GBVI/WSA ΔG as a force field-based scoring function [36]. Molecular docking was performed to investigate how ligand binds to protein target [37].

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 5.0.1 for Windows, GraphPad Software (San Diego, California USA). Carbapenemase inhibition assay and cell viability measurement with Alamar Blue data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. The Paired t-test was used for analysis of the permeability assay of the outer membrane data. The probability value (P < 0.05) was considered as the level of significance.