Bacterial zincophore [S,S]-ethylenediamine-N,N′-disuccinic acid is an effective inhibitor of MBLs

Bacterial zincophore [S,S]-ethylenediamine-N,N′-disuccinic acid is an effective inhibitor of MBLs Abstract Objectives Carbapenemases such as MBLs are spreading among Gram-negative bacterial pathogens. Infections due to these MDR bacteria constitute a major global health challenge. Therapeutic strategies against carbapenemase-producing bacteria include β-lactamase inhibitor combinations. [S,S]-ethylenediamine-N,N′-disuccinic acid (EDDS) is a chelator and potential inhibitor of MBLs. We investigated the activity of EDDS in combination with imipenem against MBL-producing bacteria in vitro as well as in vivo. Methods The inhibitory activity of EDDS was analysed by means of a fluorescence-based assay using purified recombinant MBLs, i.e. NDM-1, VIM-1, SIM-1 and IMP-1. The in vitro activity of imipenem ± EDDS against mutants as well as clinical isolates expressing MBLs was evaluated by broth microdilution assay. The in vivo activity of imipenem ± EDDS was analysed in a Galleria mellonella infection model. Results EDDS revealed potent MBL inhibitory activity against purified NDM-1, weaker activity against VIM-1 and SIM-1, and marginal activity against IMP-1. EDDS did not exhibit any intrinsic antibacterial activity, but enabled a concentration-dependent potentiation of imipenem against mutants as well as clinical isolates expressing various MBLs. The in vivo model showed a significantly better survival rate for imipenem + EDDS-treated G. mellonella larvae infected with NDM-1-producing Klebsiella pneumoniae compared with monotherapy with imipenem. Conclusions The bacterial natural zincophore EDDS is a potent MBL inhibitor and in combination with imipenem overcomes MBL-mediated carbapenem resistance in vitro and in vivo. Introduction Antibiotic resistance in bacterial pathogens constitutes one of the major threats regarding human health.1–3 An alarming trend is the spread of carbapenemases among Gram-negative pathogens that can confer resistance to almost all β-lactams, including carbapenems.4 Production of β-lactamases is the most common mechanism of resistance to β-lactam antibiotics in Gram-negative pathogens.5 According to the Ambler classification, β-lactamases can be divided into two major groups, i.e. serine-β-lactamases (SBLs) and MBLs. MBLs have been further divided into subclasses B1–B3, the B1 subclass being relevant for Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Whereas there are efficient inhibitors of SBLs available, i.e. clavulanic acid, tazobactam, sulbactam and avibactam,6,7 no clinically useful antagonist of MBL activity has yet been reported. Hence, to overcome MBL-mediated resistance, a combination of a β-lactam and a β-lactamase inhibitor, which protects the β-lactam antibiotic from the activity of the MBL, is urgently needed.6,8 Zinc chelators are able to remove zinc ions from the active site of MBLs; hence, these compounds are discussed as therapeutic options concerning development of new MBL inhibitors. Potent zinc chelators, e.g. EDTA, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, di-(2-picolyl)amine and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid, have been proved to restore carbapenem activity against MBL-producing bacteria.9 Besides synthetic chelators, many microorganisms secrete metallo-chelating natural compounds such as siderophores and zincophores, providing an unexplored pool of potential MBL inhibitors. Recently, the fungal natural product aspergillomarasmine A, isolated from Aspergillus versicolor, showed good inhibitory activity against NDM-1 and VIM-2, in vitro and in vivo.10 Consequently, we assumed that naturally produced zincophores with similar structures may show similar properties regarding MBL inhibition. [S,S]-ethylenediamine-N,N′-disuccinic acid (EDDS) is produced by a wide range of bacteria and is described as a zincophore, contributing to zinc uptake (Figure 1).11 Figure 1. View largeDownload slide Chemical structures of EDTA (a), EDDS (b) and aspergillomarasmine A (c). Figure 1. View largeDownload slide Chemical structures of EDTA (a), EDDS (b) and aspergillomarasmine A (c). This study was undertaken to evaluate the potential of EDDS to function as an MBL inhibitor and, in combination with imipenem, to confer activity against MBL-producing bacteria in vitro and in vivo. Materials and methods Bacterial strains Clinical isolates as well as bacterial mutants expressing various MBLs were used for antimicrobial susceptibility testing. For the generation of bacterial mutants, genes encoding NDM-1, IMP-1, VIM-1 and SIM-1 were amplified by conventional PCR using the primers shown in Table S1 (available as Supplementary data at JAC Online). Purified PCR amplicons were cloned into pCR-Blunt II-TOPO vectors and transformed into Escherichia coli TOP10 (Invitrogen, Darmstadt, Germany). Plasmids were isolated from selected clones and the inserted sequences were subjected to nucleotide sequencing to verify that no mutations had been introduced during the PCR and cloning procedures. E. coli ATCC 25922 was used as a control for the broth microdilution assay. Bacterial strains used for MBL expression and purification were generated as described previously.12 Briefly, genes encoding NDM-1, VIM-1, IMP-1 and SIM-1 MBLs were amplified by conventional PCR using the primers shown in Table S2. The amplified DNA fragments were cleaved with respective restriction enzymes as given in Table S2 and then cloned into a correspondingly digested pET-24a expression vector. Competent E. coli BL21(DE3) cells were transformed with respective plasmids and transformants were selected on LB agar plates containing kanamycin (100 mg/L). Plasmids were isolated from selected clones and the inserted sequences were subjected to nucleotide sequencing to verify that no mutations had been introduced during PCR and cloning procedures. MBL purification E. coli clones harbouring the MBL plasmid were grown in 1 L of LB medium containing kanamycin (100 mg/L) at 37 °C to an OD600 of 0.8–1 before induction with 400 μM IPTG. Cells were allowed to grow for another 18–20 h at 21°C and harvested (10866 g, 20 min, 4 °C). Cell pellets were resuspended in 50 mL of buffer A (50 mM Tris/HCl pH 8, 500 mM NaCl, 5% glycerol, 20 mM imidazole/HCl pH 7) and supplemented with one EDTA-free protease inhibitor complete tablet (Roche, Basel, Switzerland) and a trace amount of DNase I (Applichem, Darmstadt, Germany) before passage through a cell disruptor (Constant Systems, Peterhead, UK) three times at 14500 psi. Broken cells were centrifuged for 60 min at 51632 g at 4°C to remove unbroken cells and cell debris. The supernatant was applied to a 5 mL HisTrap HP (GE Healthcare, Frankfurt, Germany) column, with buffer A as a running buffer and buffer B (50 mM Tris/HCl pH 8, 500 mM NaCl, 5% glycerol, 400 mM imidazole/HCl pH 7) as an elution buffer. The hexa-his-tagged MBL was purified using a step gradient protocol in which the protein of interest was eluted at an imidazole concentration of 115 mM. To remove imidazole, the fractions containing the MBL were pooled and concentrated by ultrafiltration through a CentriPrep concentrator (Merck Millipore, Billerica, MA, USA) with a 3 kDa membrane cut-off. The concentrated protein sample was loaded onto a Superdex 200 HiLoad 16/600 column (GE Healthcare, Frankfurt, Germany) equilibrated and run at 1 mL/min with buffer C (50 mM Tris/HCl pH 8, 500 mM NaCl, 5% glycerol). Protein concentrations were determined by NanoDrop (Thermo Fisher, Waltham, MA, USA), and the purity and identity of the MBLs (>95%) were determined by SDS–PAGE. Purified proteins for the assays were aliquoted, flash frozen in liquid nitrogen and stored at −80 °C. Fluorescence-based assay Activity assays for MBLs were performed at room temperature in black polystyrol 96-well plates (Corning, Corning, NY, USA) using dicefalotinodifluorofluorescein (Fluorocillin, Invitrogen, Darmstadt, Germany) as a substrate. Proteins were diluted in assay buffer (HEPES 50 mM, pH 7.5 containing 0.01% Triton X-100), with final protein concentrations of (NDM-1) 3 nM, (VIM-1) 4 nM, (IMP-1) 1 nM and (SIM-1) 0.35 nM. Samples were supplemented with an equimolar amount of ZnCl2. An amount of 1 μL of EDDS (Sigma–Aldrich, Steinheim, Germany) at different concentrations was incubated with 89 μL of enzyme in assay buffer. After an incubation period of 30 min at room temperature, 10 μL of Fluorocillin substrate was added to yield the final assay volume of 100 μL. The fluorescence emitted by the fluorescent product difluorofluorescein was monitored every 45 s for 30 cycles using a Tecan fluorescent plate reader (Infinite 200; excitation at 495 nm and emission at 525 nm) and was compared with a standard curve. The rate of the enzymatic reaction was obtained by dividing the quantity of the fluorescent product (RFU) by time (min). Negative controls were measured in the absence of enzyme, whereas the positive controls were measured in the presence of enzyme and in the absence of inhibitors. The inhibitory effect of each substance was measured in triplicate in three independent experiments. IC50 values were calculated using data obtained from measurements with at least six different inhibitor concentrations, applying a sigmoidal dose–response (variable slope with four parameters) equation using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) software. In order to investigate the inhibitory activity of EDDS in the presence of a high Zn2+ concentration, assay buffer containing an additional 100 μM ZnCl2 was used. The assay was performed as described above with NDM-1 and either 30 or 100 μM EDDS. Differential scanning fluorimetry (DSF) DSF, also referred to as a thermal shift assay,13 was performed in transparent 96-well PCR plates (MicroAmp, Applied Biosystems, Germany). The final assay volume was 40 μL. Test compounds EDDS and ZnCl2 solution were dissolved in Milli-Q water and diluted in wells to a final concentration of 100 μM. Thirty-two microlitres of enzyme master mix containing assay buffer (50 mM HEPES, pH 7.8) and recombinant NDM-1 at a final concentration of 5 μM was mixed with 4 μL of compound solution and 4 μL of SYPRO orange (Sigma–Aldrich) in assay buffer (2.5× final concentration). Melting points of untreated NDM-1 were measured without inhibitor. Temperature-dependent increase in fluorescence was measured from 25.0°C to 94.8 °C, in steps of 0.2°C every 24 s, using an iCycler iQ single-colour real-time PCR (Bio-Rad, Munich, Germany). Excitation was set to 490 nm and emission wavelength to 570 nm. All measurements were performed in triplicate in three independent experiments. The first derivative of the melting curve was calculated using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). The respective maxima of the resulting curve, which were considered to be the melting point, were determined in Microsoft Excel. Broth microdilution assay MICs of piperacillin, cefuroxime, ceftazidime, imipenem monohydrate (all purchased from Sigma–Aldrich) and meropenem (Hikma, Graefeling, Germany) as well as gentamicin (Merck, Darmstadt, Germany) ± EDDS or ± EDTA for transformed E. coli strains producing different recombinant MBLs as well as MICs of imipenem/EDDS for MBL-positive and MBL-negative clinical isolates were determined according to the microdilution method established by the CLSI.14 Galleria mellonella infection model G. mellonella caterpillars infected with a lethal dosage of Klebsiella pneumoniae expressing NDM-1 were treated with imipenem ± EDDS. Therefore, prior to inoculation into G. mellonella caterpillars, bacterial cells were washed with PBS and then diluted to an appropriate cell density, as determined by the optical density at 600 nm. A 10 μL Hamilton syringe was used to inject 10 μL aliquots of the inoculum into the haemocoel of each caterpillar via the last left proleg. Bacterial colony counts on blood agar were used to confirm the presence of inocula. Ten microlitres of imipenem ± EDDS was then administered by injection into a different proleg within 30 min to yield a final concentration in the larvae of 0.4 mg/kg imipenem ± 128 mg/kg EDDS. Sixteen randomly chosen caterpillars were used for each group of an experiment. For all experiments, two control groups were used: the first group included caterpillars that were inoculated with PBS to monitor for killing due to physical trauma; the second group included caterpillars that received no injection. Survival kinetics in terms of Kaplan–Meier plots were established to demonstrate differences between treated and untreated caterpillars over a timeframe of 4 days. Experiments were performed independently in triplicate and data were pooled. Statistics The log-rank (Mantel–Cox) test was used to compare survival curves by means of the software GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). P values <0.05 were considered statistically significant. Results Purified recombinant NDM-1, VIM-1, SIM-1 and IMP-1 were used to analyse the inhibitory activity of EDDS in vitro (Figure 2a). EDDS showed potent concentration-dependent inhibition of NDM-1 with an IC50 of 0.18 μM. The related MBL VIM-1 and SIM-1 were inhibited less potently, with an IC50 of 8 and 9 μM, respectively. IMP-1 was only marginally affected by EDDS at higher concentrations. Owing to the high Hill slope values obtained in the activity assay (NDM-1, 2.8; VIM-1, 6.2; SIM-1, 16.6), it could be assumed that the inhibition mode of EDDS is not competitive. Thus, thermal shift experiments were performed to investigate the inhibitory mode of action of EDDS. While NDM-1 is stabilized by ZnCl2, the addition of EDDS destabilized the protein. Furthermore, the addition of ZnCl2 to NDM-1 destabilized by EDDS could restore the melting point, indicating that EDDS chelates Zn2+ and thus the enzyme loses its activity (Figure 2b). This assumption could be confirmed in the fluorescence-based activity assay, in which the addition of ZnCl2 restored the activity of NDM-1 in the presence of EDDS. Figure 2. View largeDownload slide (a) Inhibition of recombinant NDM-1, VIM-1, IMP-1 and SIM-1 by EDDS, determined by a fluorescence-based activity assay. (b) Melting point (Tm) of recombinant NDM-1 measured by DSF. (c) Influence of ZnCl2 on the enzymatic activity of recombinant NDM-1, determined by a fluorescence-based activity assay. The columns show mean values of at least three independent experiments; error bars indicate SEM. c, concentration of EDDS. Figure 2. View largeDownload slide (a) Inhibition of recombinant NDM-1, VIM-1, IMP-1 and SIM-1 by EDDS, determined by a fluorescence-based activity assay. (b) Melting point (Tm) of recombinant NDM-1 measured by DSF. (c) Influence of ZnCl2 on the enzymatic activity of recombinant NDM-1, determined by a fluorescence-based activity assay. The columns show mean values of at least three independent experiments; error bars indicate SEM. c, concentration of EDDS. The considerable inhibition of purified NDM-1, VIM-1 and SIM-1 in vitro suggested that EDDS can potentially restore the activity of β-lactam antibiotics against bacterial isolates producing MBL. To prove this, imipenem combined with different concentrations of EDDS ranging from 1 to 128 mg/L was tested against an E. coli mutant expressing NDM-1 (Table 1). EDDS at a concentration of 8 mg/L already significantly reduced the MIC of imipenem for the MBL-expressing E. coli. Moreover, the addition of EDDS at a constant concentration of 32 mg/L reduced the MICs of all tested β-lactams for the E. coli mutant expressing NDM-1, whereas no synergistic effect of EDDS was observed for the MIC of gentamicin (Table 2). The potentiating effect of EDDS against MBL-positive E. coli mutants is comparable to another chelator, i.e. EDTA, as shown in Table 3. Finally, imipenem ± EDDS at a constant concentration of 32 mg/L was tested against clinical isolates expressing various MBLs, i.e. NDM-1, NDM-5, NDM-7, IMP-1, IMP-7, VIM-1, VIM-2, VIM-4, SIM-1, SPM-1 and GIM-1, as well as carbapenem-resistant MBL-negative Enterobacteriaceae and A. baumannii. The activity (MIC) of imipenem alone and in combination with EDDS is shown in Table 4. EDDS did not exhibit any intrinsic antimicrobial activity at the tested constant concentration of 32 mg/L. However, combination of the MBL inhibitor and imipenem revealed that EDDS restored antibiotic activity, as demonstrated by a significant reduction in imipenem MICs for all tested MBL-positive but not MBL-negative isolates. Table 1. Effects of various EDDS concentrations on MICs of imipenem for E. coli TOP10 (NDM-1)   Imipenem MIC (mg/L)  +EDDS (128 mg/L)  0.25  +EDDS (64 mg/L)  0.25  +EDDS (32 mg/L)  0.25  +EDDS (16 mg/L)  0.25  +EDDS (8 mg/L)  0.25  +EDDS (4 mg/L)  8  +EDDS (2 mg/L)  64  +EDDS (1 mg/L)  64  Without EDDS  128    Imipenem MIC (mg/L)  +EDDS (128 mg/L)  0.25  +EDDS (64 mg/L)  0.25  +EDDS (32 mg/L)  0.25  +EDDS (16 mg/L)  0.25  +EDDS (8 mg/L)  0.25  +EDDS (4 mg/L)  8  +EDDS (2 mg/L)  64  +EDDS (1 mg/L)  64  Without EDDS  128  MBL inhibitor (EDDS) in combination with imipenem was tested at various concentrations ranging from 1 to 128 mg/L. Table 2. Effects of EDDS on MICs of piperacillin, cefuroxime, ceftazidime, imipenem, meropenem and gentamicin for E. coli TOP10 (NDM-1) Antibiotic  EDDS (32 mg/L)  MIC (mg/L)a  Piperacillin  −  >128  +  2  Cefuroxime  −  >128  +  4  Ceftazidime  −  >128  +  2  Imipenem  −  128  +  0.25  Meropenem  −  >128  +  0.015  Gentamicin  −  0.5  +  0.5  Antibiotic  EDDS (32 mg/L)  MIC (mg/L)a  Piperacillin  −  >128  +  2  Cefuroxime  −  >128  +  4  Ceftazidime  −  >128  +  2  Imipenem  −  128  +  0.25  Meropenem  −  >128  +  0.015  Gentamicin  −  0.5  +  0.5  a Median of three experiments. Table 3. Effects of EDDS and EDTA on MIC of imipenem Transformant  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  +EDTAc,d  E. coli TOP10 T2359 (NDM-1)  128  0.25 (512)  0.25 (512)  E. coli TOP10 T2360 (IMP-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2361 (VIM-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2362 (SIM-1)  2  0.125 (16)  0.125 (16)  Transformant  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  +EDTAc,d  E. coli TOP10 T2359 (NDM-1)  128  0.25 (512)  0.25 (512)  E. coli TOP10 T2360 (IMP-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2361 (VIM-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2362 (SIM-1)  2  0.125 (16)  0.125 (16)  a Median of three experiments. b Fold change was calculated as MIC of imipenem/MIC of imipenem + inhibitor. Significant fold changes (≥4) are indicated in bold. c EDDS and EDTA in combination with imipenem were tested at a constant concentration of 32 mg/L. d EDDS and EDTA did not exhibit any antibacterial activity at the given concentration. Table 4. Effects of EDDS on MIC of imipenem for carbapenemase (MBL and non-MBL)-producing clinical isolates Clinical isolate  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  Serratia marcescens T2352 (NDM-1)  256  0.5 (512)  K. pneumoniae T2301 (NDM-1)  16  0.125 (128)  Enterobacter cloacae T2311 (NDM-1)  16  0.5 (32)  A. baumannii T2304 (NDM-1)  128  16 (8)  E. coli T2351 (NDM-5)  32  0.5 (64)  E. coli T2239 (NDM-7)  32  0.5 (64)  P. aeruginosa T2325 (IMP-1)  16  1 (16)  P. aeruginosa T1098 (IMP-7)  128  32 (4)  Acinetobacter genospecies 3 T2236 (SIM-1)  32  0.125 (256)  Citrobacter freundii T2354 (VIM-4)  4  1 (4)  E. cloacae T2353 (VIM-1)  8  1 (8)  P. aeruginosa T2357 (VIM-1)  256  8 (32)  E. coli T2228 (VIM-1)  8  0.5 (16)  K. pneumoniae T2216 (VIM-1)  8  0.125 (64)  P. aeruginosa T2217 (VIM-2)  128  16 (8)  P. aeruginosa T2282 (VIM-2)  512  8 (64)  P. aeruginosa T2283 (VIM-2)  16  0.5 (32)  P. aeruginosa T2229 (SPM-1)  512  16 (32)  E. cloacae T2218 (GIM-1)  2  0.5 (4)  E. coli T3261 (KPC-2)  8  8 (1)  C. freundii T2482 (KPC-3)  8  8 (1)  K. pneumoniae T2743 (KPC-9)  64  64 (1)  A. baumannii T3161 (OXA-23)  64  64 (1)  E. coli T3124 (OXA-48)  2  2 (1)  Clinical isolate  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  Serratia marcescens T2352 (NDM-1)  256  0.5 (512)  K. pneumoniae T2301 (NDM-1)  16  0.125 (128)  Enterobacter cloacae T2311 (NDM-1)  16  0.5 (32)  A. baumannii T2304 (NDM-1)  128  16 (8)  E. coli T2351 (NDM-5)  32  0.5 (64)  E. coli T2239 (NDM-7)  32  0.5 (64)  P. aeruginosa T2325 (IMP-1)  16  1 (16)  P. aeruginosa T1098 (IMP-7)  128  32 (4)  Acinetobacter genospecies 3 T2236 (SIM-1)  32  0.125 (256)  Citrobacter freundii T2354 (VIM-4)  4  1 (4)  E. cloacae T2353 (VIM-1)  8  1 (8)  P. aeruginosa T2357 (VIM-1)  256  8 (32)  E. coli T2228 (VIM-1)  8  0.5 (16)  K. pneumoniae T2216 (VIM-1)  8  0.125 (64)  P. aeruginosa T2217 (VIM-2)  128  16 (8)  P. aeruginosa T2282 (VIM-2)  512  8 (64)  P. aeruginosa T2283 (VIM-2)  16  0.5 (32)  P. aeruginosa T2229 (SPM-1)  512  16 (32)  E. cloacae T2218 (GIM-1)  2  0.5 (4)  E. coli T3261 (KPC-2)  8  8 (1)  C. freundii T2482 (KPC-3)  8  8 (1)  K. pneumoniae T2743 (KPC-9)  64  64 (1)  A. baumannii T3161 (OXA-23)  64  64 (1)  E. coli T3124 (OXA-48)  2  2 (1)  a Median of three experiments. b Fold change was calculated as MIC of imipenem/MIC of imipenem + EDDS. Significant fold changes (≥4) are indicated in bold. c EDDS in combination with imipenem was tested at a constant concentration of 32 mg/L. d EDDS did not exhibit any antibacterial activity at the given concentration. The G. mellonella infection model demonstrated 100% killing of larvae infected with NDM-1-producing K. pneumoniae (Figure 3). Treatment of infected G. mellonella with either EDDS or imipenem did not result in a significantly higher survival rate. However, treatment with EDDS + imipenem resulted in a significantly higher survival rate of larvae inoculated with NDM-1-producing K. pneumoniae (Figure 3). Figure 3. View largeDownload slide Survival curves of G. mellonella larvae infected with NDM-1-producing K. pneumoniae and treated with imipenem, EDDS or imipenem + EDDS. Curves represent pooled data from three independent experiments using 16 G. mellonella in each experiment. *P < 0.0001. IPM, imipenem; K.p., K. pneumoniae. Figure 3. View largeDownload slide Survival curves of G. mellonella larvae infected with NDM-1-producing K. pneumoniae and treated with imipenem, EDDS or imipenem + EDDS. Curves represent pooled data from three independent experiments using 16 G. mellonella in each experiment. *P < 0.0001. IPM, imipenem; K.p., K. pneumoniae. Discussion Gram-negative bacteria such as Enterobacteriaceae, P. aeruginosa and A. baumannii are considered to be major nosocomial pathogens causing acute infections such as sepsis, pneumonia, urinary tract infections and skin and soft tissue infections.15–17 Antibiotic therapy plays a crucial role in the management and cure of these infections in humans. The broad spectrum of antibiotic resistance associated with these versatile pathogens is worrying because antibiotic resistance in general increases the morbidity and mortality.18–21 The spread of carbapenemases among these pathogens, which can confer resistance to almost all β-lactams, including carbapenems, is especially alarming.2 This study aimed to provide a new anti-infective β-lactam/β-lactamase inhibitor combination, i.e. imipenem/EDDS, to combat infections in humans due to MBL-producing pathogens. EDDS showed potent concentration-dependent inhibition of purified NDM-1, VIM-1 and SIM-1. These results are in agreement with the broth microdilution assays. EDDS affected the imipenem MIC for NDM-1-expressing transformants (512-fold) more strongly than the imipenem MIC for VIM-1- and SIM-1-expressing transformants (8- and 16-fold, respectively) at concentrations slightly higher than 100 μM. These results correspond to the findings by King et al.,10 showing that aspergillomarasmine A potently inhibited NDM-1 whereas the inhibition of related MBLs VIM-2 and IMP-7 was less pronounced. IMP-1 was almost unaffected by EDDS in concentrations up to 300 μM. The experiments performed by Laraki et al.22 also show that IMP-1 is only weakly affected by EDTA. Different susceptibility of the investigated MBLs to EDDS might be explained by structural differences between these enzymes. There is a large difference in the flexibility of loops surrounding the active site in NDM-1 compared with MBLs of the VIM and IMP family.23 Furthermore, the binding cavity is wider,24,25 which enables more efficient catalysis and a broader substrate spectrum,26 and it might also have an impact on the binding affinity of zinc ions to the catalytic site. One might speculate that MBLs with low binding affinity to zinc ions are more susceptible to inhibition by zinc chelators like EDTA, EDDS and aspergillomarasmine A; however, this hypothesis has to be verified experimentally. With regard to clinical isolates, EDDS significantly reduced the imipenem MIC for all MBL-expressing bacteria under investigation, with the most pronounced effect, i.e. up to 512-fold imipenem MIC reduction, for bacteria expressing the clinically relevant NDM. The activity of the synthetic chelator Ca-EDTA in combination with ceftazidime or imipenem against MBL-producing bacteria has been analysed in vitro as well as in vivo and favourable results could be achieved.27,28 Ca-EDTA is widely used in clinics for the treatment of lead intoxication and is applied intravenously in doses between 25 and 75 mg/kg/day.27 EDDS might be applicable intravenously as well and in vivo pharmacokinetic studies need to be performed.29 EDDS is produced by bacteria as a natural compound that is biodegradable. As such, it offers an advantage over the poorly biodegradable EDTA, which cannot be removed by conventional water treatments, thereby exerting a constant environmental stress.22 In conclusion, we demonstrated that the bacterial zincophore EDDS is a highly efficient inhibitor of MBL. In combination with imipenem, EDDS demonstrated excellent activity against various MBL-producing Gram-negative pathogens in vitro as well as in vivo. Further investigation of EDDS as a chelating agent against infections due to MBL-producing pathogens, including animal studies, is warranted. Furthermore, to the best of our knowledge, EDDS is the first MBL inhibitor of bacterial origin. Although EDDS is described as a zincophore, its role in interspecies competition is unclear and should be further investigated. Acknowledgements We thank Denia Frank for excellent technical support; the German National Reference Laboratory for MDR Gram-negative bacteria for kindly providing us with isolates T1098, T2216 and T2217; the Robert Koch Institute for kindly providing us with isolates T2226 and T2228; the Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, for kindly providing us with isolate T2236; and the Universitätsspital Basel for kindly providing us with isolate T2229. Funding This work was supported by internal funding. E. P. thanks the German Research Foundation (DFG, Heisenberg-Professur PR1405/4–1) for financial support. Transparency declarations None to declare. Supplementary data Tables S1 and S2 are available as Supplementary data at JAC Online. References 1 Walker B, Barrett S, Polasky S et al.   Looming global-scale failures and missing institutions. Science  2009; 325: 1345– 6. Google Scholar CrossRef Search ADS PubMed  2 Fischbach MA, Walsh CT. Antibiotics for emerging pathogens. Science  2009; 325: 1089– 93. Google Scholar CrossRef Search ADS PubMed  3 Spellberg B, Blaser M, Guidos RJ et al.   Combating antimicrobial resistance: policy recommendations to save lives. Clin Infect Dis  2011; 52 Suppl 5: 397– 428. Google Scholar CrossRef Search ADS   4 Cornaglia G, Giamarellou H, Rossolini GM. Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect Dis  2011; 11: 381– 93. Google Scholar CrossRef Search ADS PubMed  5 Pfeifer Y, Cullik A, Witte W. 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Nature  2014; 510: 503– 6. Google Scholar CrossRef Search ADS PubMed  11 Spohn M, Wohlleben W, Stegmann E. Elucidation of the zinc-dependent regulation in Amycolatopsis japonicum enabled the identification of the ethylenediamine-disuccinate ([S,S]-EDDS) genes. Environ Microbiol  2016; 18: 1249– 63. Google Scholar CrossRef Search ADS PubMed  12 Klingler FM, Wichelhaus TA, Frank D et al.   Approved drugs containing thiols as inhibitors of metallo-β-lactamases: strategy to combat multidrug-resistant bacteria. J Med Chem  2015; 58: 3626– 30. Google Scholar CrossRef Search ADS PubMed  13 Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc  2007; 2: 2212– 21. Google Scholar CrossRef Search ADS PubMed  14 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Ninth Edition: Approved Standard M07-A9 . CLSI, Wayne, PA, USA, 2012. 15 Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev  2008; 21: 538– 82. Google Scholar CrossRef Search ADS PubMed  16 Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med  2012; 18: 263– 72. Google Scholar CrossRef Search ADS PubMed  17 Lister PD, Wolter DJ, Hanson ND. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev  2009; 22: 582– 610. Google Scholar CrossRef Search ADS PubMed  18 Hirsch EB, HT V. Impact of multidrug-resistant Pseudomonas aeruginosa infection on patient outcomes. Expert Rev Pharmacoecon Outcomes Res  2010; 10: 441– 51. Google Scholar CrossRef Search ADS PubMed  19 Lemos EV, de la Hoz FP, Einarson TR et al.   Carbapenem resistance and mortality in patients with Acinetobacter baumannii infection: systematic review and meta-analysis. Clin Microbiol Infect  2014; 20: 416– 23. Google Scholar CrossRef Search ADS PubMed  20 Tzouvelekis LS, Markogiannakis A, Psichogiou M et al.   Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev  2012; 25: 682– 707. Google Scholar CrossRef Search ADS PubMed  21 Potron A, Poirel L, Nordmann P. Emerging broad-spectrum resistance in Pseudomonas aeruginosa and Acinetobacter baumannii: mechanisms and epidemiology. Int J Antimicrob Agents  2015; 45: 568– 85. Google Scholar CrossRef Search ADS PubMed  22 Laraki N, Franceschini N, Rossolini GM et al.   Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-β-lactamase IMP-1 produced by Escherichia coli. Antimicrob Agents Chemother  1999; 43: 902– 6. Google Scholar PubMed  23 Brown MC, Verma D, Russell C et al.   A case study comparing quantitative stability-flexibility relationships across five metallo-β-lactamases highlighting differences within NDM-1. Methods Mol Biol  2014; 1084: 227– 38. Google Scholar CrossRef Search ADS PubMed  24 Chiou J, Leung TY, Chen S. Molecular mechanisms of substrate recognition and specificity of New Delhi metallo-β-lactamase. Antimicrob Agents Chemother  2014; 58: 5372– 8. Google Scholar CrossRef Search ADS PubMed  25 Guo Y, Wang J, Niu G et al.   A structural view of the antibiotic degradation enzyme NDM-1 from a superbug. Protein Cell  2011; 2: 384– 94. Google Scholar CrossRef Search ADS PubMed  26 Kim Y, Tesar C, Mire J et al.   Structure of apo- and monometalated forms of NDM-1—a highly potent carbapenem-hydrolyzing metallo-β-lactamase. PLoS One  2011; 6: e24621. Google Scholar CrossRef Search ADS PubMed  27 Aoki N, Ishii Y, Tateda K et al.   Efficacy of calcium-EDTA as an inhibitor for metallo-β-lactamase in a mouse model of Pseudomonas aeruginosa pneumonia. Antimicrob Agents Chemother  2010; 54: 4582– 8. Google Scholar CrossRef Search ADS PubMed  28 Yoshizumi A, Ishii Y, Livermore DM et al.   Efficacies of calcium-EDTA in combination with imipenem in a murine model of sepsis caused by Escherichia coli with NDM-1 β-lactamase. J Infect Chemother  2013; 19: 992– 5. Google Scholar CrossRef Search ADS PubMed  29 Lin-Tan DT, Lin JL, Yen TH et al.   Long-term outcome of repeated lead chelation therapy in progressive non-diabetic chronic kidney diseases. Nephrol Dial Transplant  2007; 22: 2924– 31. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Bacterial zincophore [S,S]-ethylenediamine-N,N′-disuccinic acid is an effective inhibitor of MBLs

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Oxford University Press
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© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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0305-7453
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1460-2091
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10.1093/jac/dkx403
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Abstract

Abstract Objectives Carbapenemases such as MBLs are spreading among Gram-negative bacterial pathogens. Infections due to these MDR bacteria constitute a major global health challenge. Therapeutic strategies against carbapenemase-producing bacteria include β-lactamase inhibitor combinations. [S,S]-ethylenediamine-N,N′-disuccinic acid (EDDS) is a chelator and potential inhibitor of MBLs. We investigated the activity of EDDS in combination with imipenem against MBL-producing bacteria in vitro as well as in vivo. Methods The inhibitory activity of EDDS was analysed by means of a fluorescence-based assay using purified recombinant MBLs, i.e. NDM-1, VIM-1, SIM-1 and IMP-1. The in vitro activity of imipenem ± EDDS against mutants as well as clinical isolates expressing MBLs was evaluated by broth microdilution assay. The in vivo activity of imipenem ± EDDS was analysed in a Galleria mellonella infection model. Results EDDS revealed potent MBL inhibitory activity against purified NDM-1, weaker activity against VIM-1 and SIM-1, and marginal activity against IMP-1. EDDS did not exhibit any intrinsic antibacterial activity, but enabled a concentration-dependent potentiation of imipenem against mutants as well as clinical isolates expressing various MBLs. The in vivo model showed a significantly better survival rate for imipenem + EDDS-treated G. mellonella larvae infected with NDM-1-producing Klebsiella pneumoniae compared with monotherapy with imipenem. Conclusions The bacterial natural zincophore EDDS is a potent MBL inhibitor and in combination with imipenem overcomes MBL-mediated carbapenem resistance in vitro and in vivo. Introduction Antibiotic resistance in bacterial pathogens constitutes one of the major threats regarding human health.1–3 An alarming trend is the spread of carbapenemases among Gram-negative pathogens that can confer resistance to almost all β-lactams, including carbapenems.4 Production of β-lactamases is the most common mechanism of resistance to β-lactam antibiotics in Gram-negative pathogens.5 According to the Ambler classification, β-lactamases can be divided into two major groups, i.e. serine-β-lactamases (SBLs) and MBLs. MBLs have been further divided into subclasses B1–B3, the B1 subclass being relevant for Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Whereas there are efficient inhibitors of SBLs available, i.e. clavulanic acid, tazobactam, sulbactam and avibactam,6,7 no clinically useful antagonist of MBL activity has yet been reported. Hence, to overcome MBL-mediated resistance, a combination of a β-lactam and a β-lactamase inhibitor, which protects the β-lactam antibiotic from the activity of the MBL, is urgently needed.6,8 Zinc chelators are able to remove zinc ions from the active site of MBLs; hence, these compounds are discussed as therapeutic options concerning development of new MBL inhibitors. Potent zinc chelators, e.g. EDTA, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, di-(2-picolyl)amine and 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid, have been proved to restore carbapenem activity against MBL-producing bacteria.9 Besides synthetic chelators, many microorganisms secrete metallo-chelating natural compounds such as siderophores and zincophores, providing an unexplored pool of potential MBL inhibitors. Recently, the fungal natural product aspergillomarasmine A, isolated from Aspergillus versicolor, showed good inhibitory activity against NDM-1 and VIM-2, in vitro and in vivo.10 Consequently, we assumed that naturally produced zincophores with similar structures may show similar properties regarding MBL inhibition. [S,S]-ethylenediamine-N,N′-disuccinic acid (EDDS) is produced by a wide range of bacteria and is described as a zincophore, contributing to zinc uptake (Figure 1).11 Figure 1. View largeDownload slide Chemical structures of EDTA (a), EDDS (b) and aspergillomarasmine A (c). Figure 1. View largeDownload slide Chemical structures of EDTA (a), EDDS (b) and aspergillomarasmine A (c). This study was undertaken to evaluate the potential of EDDS to function as an MBL inhibitor and, in combination with imipenem, to confer activity against MBL-producing bacteria in vitro and in vivo. Materials and methods Bacterial strains Clinical isolates as well as bacterial mutants expressing various MBLs were used for antimicrobial susceptibility testing. For the generation of bacterial mutants, genes encoding NDM-1, IMP-1, VIM-1 and SIM-1 were amplified by conventional PCR using the primers shown in Table S1 (available as Supplementary data at JAC Online). Purified PCR amplicons were cloned into pCR-Blunt II-TOPO vectors and transformed into Escherichia coli TOP10 (Invitrogen, Darmstadt, Germany). Plasmids were isolated from selected clones and the inserted sequences were subjected to nucleotide sequencing to verify that no mutations had been introduced during the PCR and cloning procedures. E. coli ATCC 25922 was used as a control for the broth microdilution assay. Bacterial strains used for MBL expression and purification were generated as described previously.12 Briefly, genes encoding NDM-1, VIM-1, IMP-1 and SIM-1 MBLs were amplified by conventional PCR using the primers shown in Table S2. The amplified DNA fragments were cleaved with respective restriction enzymes as given in Table S2 and then cloned into a correspondingly digested pET-24a expression vector. Competent E. coli BL21(DE3) cells were transformed with respective plasmids and transformants were selected on LB agar plates containing kanamycin (100 mg/L). Plasmids were isolated from selected clones and the inserted sequences were subjected to nucleotide sequencing to verify that no mutations had been introduced during PCR and cloning procedures. MBL purification E. coli clones harbouring the MBL plasmid were grown in 1 L of LB medium containing kanamycin (100 mg/L) at 37 °C to an OD600 of 0.8–1 before induction with 400 μM IPTG. Cells were allowed to grow for another 18–20 h at 21°C and harvested (10866 g, 20 min, 4 °C). Cell pellets were resuspended in 50 mL of buffer A (50 mM Tris/HCl pH 8, 500 mM NaCl, 5% glycerol, 20 mM imidazole/HCl pH 7) and supplemented with one EDTA-free protease inhibitor complete tablet (Roche, Basel, Switzerland) and a trace amount of DNase I (Applichem, Darmstadt, Germany) before passage through a cell disruptor (Constant Systems, Peterhead, UK) three times at 14500 psi. Broken cells were centrifuged for 60 min at 51632 g at 4°C to remove unbroken cells and cell debris. The supernatant was applied to a 5 mL HisTrap HP (GE Healthcare, Frankfurt, Germany) column, with buffer A as a running buffer and buffer B (50 mM Tris/HCl pH 8, 500 mM NaCl, 5% glycerol, 400 mM imidazole/HCl pH 7) as an elution buffer. The hexa-his-tagged MBL was purified using a step gradient protocol in which the protein of interest was eluted at an imidazole concentration of 115 mM. To remove imidazole, the fractions containing the MBL were pooled and concentrated by ultrafiltration through a CentriPrep concentrator (Merck Millipore, Billerica, MA, USA) with a 3 kDa membrane cut-off. The concentrated protein sample was loaded onto a Superdex 200 HiLoad 16/600 column (GE Healthcare, Frankfurt, Germany) equilibrated and run at 1 mL/min with buffer C (50 mM Tris/HCl pH 8, 500 mM NaCl, 5% glycerol). Protein concentrations were determined by NanoDrop (Thermo Fisher, Waltham, MA, USA), and the purity and identity of the MBLs (>95%) were determined by SDS–PAGE. Purified proteins for the assays were aliquoted, flash frozen in liquid nitrogen and stored at −80 °C. Fluorescence-based assay Activity assays for MBLs were performed at room temperature in black polystyrol 96-well plates (Corning, Corning, NY, USA) using dicefalotinodifluorofluorescein (Fluorocillin, Invitrogen, Darmstadt, Germany) as a substrate. Proteins were diluted in assay buffer (HEPES 50 mM, pH 7.5 containing 0.01% Triton X-100), with final protein concentrations of (NDM-1) 3 nM, (VIM-1) 4 nM, (IMP-1) 1 nM and (SIM-1) 0.35 nM. Samples were supplemented with an equimolar amount of ZnCl2. An amount of 1 μL of EDDS (Sigma–Aldrich, Steinheim, Germany) at different concentrations was incubated with 89 μL of enzyme in assay buffer. After an incubation period of 30 min at room temperature, 10 μL of Fluorocillin substrate was added to yield the final assay volume of 100 μL. The fluorescence emitted by the fluorescent product difluorofluorescein was monitored every 45 s for 30 cycles using a Tecan fluorescent plate reader (Infinite 200; excitation at 495 nm and emission at 525 nm) and was compared with a standard curve. The rate of the enzymatic reaction was obtained by dividing the quantity of the fluorescent product (RFU) by time (min). Negative controls were measured in the absence of enzyme, whereas the positive controls were measured in the presence of enzyme and in the absence of inhibitors. The inhibitory effect of each substance was measured in triplicate in three independent experiments. IC50 values were calculated using data obtained from measurements with at least six different inhibitor concentrations, applying a sigmoidal dose–response (variable slope with four parameters) equation using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) software. In order to investigate the inhibitory activity of EDDS in the presence of a high Zn2+ concentration, assay buffer containing an additional 100 μM ZnCl2 was used. The assay was performed as described above with NDM-1 and either 30 or 100 μM EDDS. Differential scanning fluorimetry (DSF) DSF, also referred to as a thermal shift assay,13 was performed in transparent 96-well PCR plates (MicroAmp, Applied Biosystems, Germany). The final assay volume was 40 μL. Test compounds EDDS and ZnCl2 solution were dissolved in Milli-Q water and diluted in wells to a final concentration of 100 μM. Thirty-two microlitres of enzyme master mix containing assay buffer (50 mM HEPES, pH 7.8) and recombinant NDM-1 at a final concentration of 5 μM was mixed with 4 μL of compound solution and 4 μL of SYPRO orange (Sigma–Aldrich) in assay buffer (2.5× final concentration). Melting points of untreated NDM-1 were measured without inhibitor. Temperature-dependent increase in fluorescence was measured from 25.0°C to 94.8 °C, in steps of 0.2°C every 24 s, using an iCycler iQ single-colour real-time PCR (Bio-Rad, Munich, Germany). Excitation was set to 490 nm and emission wavelength to 570 nm. All measurements were performed in triplicate in three independent experiments. The first derivative of the melting curve was calculated using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). The respective maxima of the resulting curve, which were considered to be the melting point, were determined in Microsoft Excel. Broth microdilution assay MICs of piperacillin, cefuroxime, ceftazidime, imipenem monohydrate (all purchased from Sigma–Aldrich) and meropenem (Hikma, Graefeling, Germany) as well as gentamicin (Merck, Darmstadt, Germany) ± EDDS or ± EDTA for transformed E. coli strains producing different recombinant MBLs as well as MICs of imipenem/EDDS for MBL-positive and MBL-negative clinical isolates were determined according to the microdilution method established by the CLSI.14 Galleria mellonella infection model G. mellonella caterpillars infected with a lethal dosage of Klebsiella pneumoniae expressing NDM-1 were treated with imipenem ± EDDS. Therefore, prior to inoculation into G. mellonella caterpillars, bacterial cells were washed with PBS and then diluted to an appropriate cell density, as determined by the optical density at 600 nm. A 10 μL Hamilton syringe was used to inject 10 μL aliquots of the inoculum into the haemocoel of each caterpillar via the last left proleg. Bacterial colony counts on blood agar were used to confirm the presence of inocula. Ten microlitres of imipenem ± EDDS was then administered by injection into a different proleg within 30 min to yield a final concentration in the larvae of 0.4 mg/kg imipenem ± 128 mg/kg EDDS. Sixteen randomly chosen caterpillars were used for each group of an experiment. For all experiments, two control groups were used: the first group included caterpillars that were inoculated with PBS to monitor for killing due to physical trauma; the second group included caterpillars that received no injection. Survival kinetics in terms of Kaplan–Meier plots were established to demonstrate differences between treated and untreated caterpillars over a timeframe of 4 days. Experiments were performed independently in triplicate and data were pooled. Statistics The log-rank (Mantel–Cox) test was used to compare survival curves by means of the software GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). P values <0.05 were considered statistically significant. Results Purified recombinant NDM-1, VIM-1, SIM-1 and IMP-1 were used to analyse the inhibitory activity of EDDS in vitro (Figure 2a). EDDS showed potent concentration-dependent inhibition of NDM-1 with an IC50 of 0.18 μM. The related MBL VIM-1 and SIM-1 were inhibited less potently, with an IC50 of 8 and 9 μM, respectively. IMP-1 was only marginally affected by EDDS at higher concentrations. Owing to the high Hill slope values obtained in the activity assay (NDM-1, 2.8; VIM-1, 6.2; SIM-1, 16.6), it could be assumed that the inhibition mode of EDDS is not competitive. Thus, thermal shift experiments were performed to investigate the inhibitory mode of action of EDDS. While NDM-1 is stabilized by ZnCl2, the addition of EDDS destabilized the protein. Furthermore, the addition of ZnCl2 to NDM-1 destabilized by EDDS could restore the melting point, indicating that EDDS chelates Zn2+ and thus the enzyme loses its activity (Figure 2b). This assumption could be confirmed in the fluorescence-based activity assay, in which the addition of ZnCl2 restored the activity of NDM-1 in the presence of EDDS. Figure 2. View largeDownload slide (a) Inhibition of recombinant NDM-1, VIM-1, IMP-1 and SIM-1 by EDDS, determined by a fluorescence-based activity assay. (b) Melting point (Tm) of recombinant NDM-1 measured by DSF. (c) Influence of ZnCl2 on the enzymatic activity of recombinant NDM-1, determined by a fluorescence-based activity assay. The columns show mean values of at least three independent experiments; error bars indicate SEM. c, concentration of EDDS. Figure 2. View largeDownload slide (a) Inhibition of recombinant NDM-1, VIM-1, IMP-1 and SIM-1 by EDDS, determined by a fluorescence-based activity assay. (b) Melting point (Tm) of recombinant NDM-1 measured by DSF. (c) Influence of ZnCl2 on the enzymatic activity of recombinant NDM-1, determined by a fluorescence-based activity assay. The columns show mean values of at least three independent experiments; error bars indicate SEM. c, concentration of EDDS. The considerable inhibition of purified NDM-1, VIM-1 and SIM-1 in vitro suggested that EDDS can potentially restore the activity of β-lactam antibiotics against bacterial isolates producing MBL. To prove this, imipenem combined with different concentrations of EDDS ranging from 1 to 128 mg/L was tested against an E. coli mutant expressing NDM-1 (Table 1). EDDS at a concentration of 8 mg/L already significantly reduced the MIC of imipenem for the MBL-expressing E. coli. Moreover, the addition of EDDS at a constant concentration of 32 mg/L reduced the MICs of all tested β-lactams for the E. coli mutant expressing NDM-1, whereas no synergistic effect of EDDS was observed for the MIC of gentamicin (Table 2). The potentiating effect of EDDS against MBL-positive E. coli mutants is comparable to another chelator, i.e. EDTA, as shown in Table 3. Finally, imipenem ± EDDS at a constant concentration of 32 mg/L was tested against clinical isolates expressing various MBLs, i.e. NDM-1, NDM-5, NDM-7, IMP-1, IMP-7, VIM-1, VIM-2, VIM-4, SIM-1, SPM-1 and GIM-1, as well as carbapenem-resistant MBL-negative Enterobacteriaceae and A. baumannii. The activity (MIC) of imipenem alone and in combination with EDDS is shown in Table 4. EDDS did not exhibit any intrinsic antimicrobial activity at the tested constant concentration of 32 mg/L. However, combination of the MBL inhibitor and imipenem revealed that EDDS restored antibiotic activity, as demonstrated by a significant reduction in imipenem MICs for all tested MBL-positive but not MBL-negative isolates. Table 1. Effects of various EDDS concentrations on MICs of imipenem for E. coli TOP10 (NDM-1)   Imipenem MIC (mg/L)  +EDDS (128 mg/L)  0.25  +EDDS (64 mg/L)  0.25  +EDDS (32 mg/L)  0.25  +EDDS (16 mg/L)  0.25  +EDDS (8 mg/L)  0.25  +EDDS (4 mg/L)  8  +EDDS (2 mg/L)  64  +EDDS (1 mg/L)  64  Without EDDS  128    Imipenem MIC (mg/L)  +EDDS (128 mg/L)  0.25  +EDDS (64 mg/L)  0.25  +EDDS (32 mg/L)  0.25  +EDDS (16 mg/L)  0.25  +EDDS (8 mg/L)  0.25  +EDDS (4 mg/L)  8  +EDDS (2 mg/L)  64  +EDDS (1 mg/L)  64  Without EDDS  128  MBL inhibitor (EDDS) in combination with imipenem was tested at various concentrations ranging from 1 to 128 mg/L. Table 2. Effects of EDDS on MICs of piperacillin, cefuroxime, ceftazidime, imipenem, meropenem and gentamicin for E. coli TOP10 (NDM-1) Antibiotic  EDDS (32 mg/L)  MIC (mg/L)a  Piperacillin  −  >128  +  2  Cefuroxime  −  >128  +  4  Ceftazidime  −  >128  +  2  Imipenem  −  128  +  0.25  Meropenem  −  >128  +  0.015  Gentamicin  −  0.5  +  0.5  Antibiotic  EDDS (32 mg/L)  MIC (mg/L)a  Piperacillin  −  >128  +  2  Cefuroxime  −  >128  +  4  Ceftazidime  −  >128  +  2  Imipenem  −  128  +  0.25  Meropenem  −  >128  +  0.015  Gentamicin  −  0.5  +  0.5  a Median of three experiments. Table 3. Effects of EDDS and EDTA on MIC of imipenem Transformant  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  +EDTAc,d  E. coli TOP10 T2359 (NDM-1)  128  0.25 (512)  0.25 (512)  E. coli TOP10 T2360 (IMP-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2361 (VIM-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2362 (SIM-1)  2  0.125 (16)  0.125 (16)  Transformant  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  +EDTAc,d  E. coli TOP10 T2359 (NDM-1)  128  0.25 (512)  0.25 (512)  E. coli TOP10 T2360 (IMP-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2361 (VIM-1)  1  0.125 (8)  0.125 (8)  E. coli TOP10 T2362 (SIM-1)  2  0.125 (16)  0.125 (16)  a Median of three experiments. b Fold change was calculated as MIC of imipenem/MIC of imipenem + inhibitor. Significant fold changes (≥4) are indicated in bold. c EDDS and EDTA in combination with imipenem were tested at a constant concentration of 32 mg/L. d EDDS and EDTA did not exhibit any antibacterial activity at the given concentration. Table 4. Effects of EDDS on MIC of imipenem for carbapenemase (MBL and non-MBL)-producing clinical isolates Clinical isolate  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  Serratia marcescens T2352 (NDM-1)  256  0.5 (512)  K. pneumoniae T2301 (NDM-1)  16  0.125 (128)  Enterobacter cloacae T2311 (NDM-1)  16  0.5 (32)  A. baumannii T2304 (NDM-1)  128  16 (8)  E. coli T2351 (NDM-5)  32  0.5 (64)  E. coli T2239 (NDM-7)  32  0.5 (64)  P. aeruginosa T2325 (IMP-1)  16  1 (16)  P. aeruginosa T1098 (IMP-7)  128  32 (4)  Acinetobacter genospecies 3 T2236 (SIM-1)  32  0.125 (256)  Citrobacter freundii T2354 (VIM-4)  4  1 (4)  E. cloacae T2353 (VIM-1)  8  1 (8)  P. aeruginosa T2357 (VIM-1)  256  8 (32)  E. coli T2228 (VIM-1)  8  0.5 (16)  K. pneumoniae T2216 (VIM-1)  8  0.125 (64)  P. aeruginosa T2217 (VIM-2)  128  16 (8)  P. aeruginosa T2282 (VIM-2)  512  8 (64)  P. aeruginosa T2283 (VIM-2)  16  0.5 (32)  P. aeruginosa T2229 (SPM-1)  512  16 (32)  E. cloacae T2218 (GIM-1)  2  0.5 (4)  E. coli T3261 (KPC-2)  8  8 (1)  C. freundii T2482 (KPC-3)  8  8 (1)  K. pneumoniae T2743 (KPC-9)  64  64 (1)  A. baumannii T3161 (OXA-23)  64  64 (1)  E. coli T3124 (OXA-48)  2  2 (1)  Clinical isolate  Imipenem MIC (mg/L)a (fold changeb in MIC)     +EDDSc,d  Serratia marcescens T2352 (NDM-1)  256  0.5 (512)  K. pneumoniae T2301 (NDM-1)  16  0.125 (128)  Enterobacter cloacae T2311 (NDM-1)  16  0.5 (32)  A. baumannii T2304 (NDM-1)  128  16 (8)  E. coli T2351 (NDM-5)  32  0.5 (64)  E. coli T2239 (NDM-7)  32  0.5 (64)  P. aeruginosa T2325 (IMP-1)  16  1 (16)  P. aeruginosa T1098 (IMP-7)  128  32 (4)  Acinetobacter genospecies 3 T2236 (SIM-1)  32  0.125 (256)  Citrobacter freundii T2354 (VIM-4)  4  1 (4)  E. cloacae T2353 (VIM-1)  8  1 (8)  P. aeruginosa T2357 (VIM-1)  256  8 (32)  E. coli T2228 (VIM-1)  8  0.5 (16)  K. pneumoniae T2216 (VIM-1)  8  0.125 (64)  P. aeruginosa T2217 (VIM-2)  128  16 (8)  P. aeruginosa T2282 (VIM-2)  512  8 (64)  P. aeruginosa T2283 (VIM-2)  16  0.5 (32)  P. aeruginosa T2229 (SPM-1)  512  16 (32)  E. cloacae T2218 (GIM-1)  2  0.5 (4)  E. coli T3261 (KPC-2)  8  8 (1)  C. freundii T2482 (KPC-3)  8  8 (1)  K. pneumoniae T2743 (KPC-9)  64  64 (1)  A. baumannii T3161 (OXA-23)  64  64 (1)  E. coli T3124 (OXA-48)  2  2 (1)  a Median of three experiments. b Fold change was calculated as MIC of imipenem/MIC of imipenem + EDDS. Significant fold changes (≥4) are indicated in bold. c EDDS in combination with imipenem was tested at a constant concentration of 32 mg/L. d EDDS did not exhibit any antibacterial activity at the given concentration. The G. mellonella infection model demonstrated 100% killing of larvae infected with NDM-1-producing K. pneumoniae (Figure 3). Treatment of infected G. mellonella with either EDDS or imipenem did not result in a significantly higher survival rate. However, treatment with EDDS + imipenem resulted in a significantly higher survival rate of larvae inoculated with NDM-1-producing K. pneumoniae (Figure 3). Figure 3. View largeDownload slide Survival curves of G. mellonella larvae infected with NDM-1-producing K. pneumoniae and treated with imipenem, EDDS or imipenem + EDDS. Curves represent pooled data from three independent experiments using 16 G. mellonella in each experiment. *P < 0.0001. IPM, imipenem; K.p., K. pneumoniae. Figure 3. View largeDownload slide Survival curves of G. mellonella larvae infected with NDM-1-producing K. pneumoniae and treated with imipenem, EDDS or imipenem + EDDS. Curves represent pooled data from three independent experiments using 16 G. mellonella in each experiment. *P < 0.0001. IPM, imipenem; K.p., K. pneumoniae. Discussion Gram-negative bacteria such as Enterobacteriaceae, P. aeruginosa and A. baumannii are considered to be major nosocomial pathogens causing acute infections such as sepsis, pneumonia, urinary tract infections and skin and soft tissue infections.15–17 Antibiotic therapy plays a crucial role in the management and cure of these infections in humans. The broad spectrum of antibiotic resistance associated with these versatile pathogens is worrying because antibiotic resistance in general increases the morbidity and mortality.18–21 The spread of carbapenemases among these pathogens, which can confer resistance to almost all β-lactams, including carbapenems, is especially alarming.2 This study aimed to provide a new anti-infective β-lactam/β-lactamase inhibitor combination, i.e. imipenem/EDDS, to combat infections in humans due to MBL-producing pathogens. EDDS showed potent concentration-dependent inhibition of purified NDM-1, VIM-1 and SIM-1. These results are in agreement with the broth microdilution assays. EDDS affected the imipenem MIC for NDM-1-expressing transformants (512-fold) more strongly than the imipenem MIC for VIM-1- and SIM-1-expressing transformants (8- and 16-fold, respectively) at concentrations slightly higher than 100 μM. These results correspond to the findings by King et al.,10 showing that aspergillomarasmine A potently inhibited NDM-1 whereas the inhibition of related MBLs VIM-2 and IMP-7 was less pronounced. IMP-1 was almost unaffected by EDDS in concentrations up to 300 μM. The experiments performed by Laraki et al.22 also show that IMP-1 is only weakly affected by EDTA. Different susceptibility of the investigated MBLs to EDDS might be explained by structural differences between these enzymes. There is a large difference in the flexibility of loops surrounding the active site in NDM-1 compared with MBLs of the VIM and IMP family.23 Furthermore, the binding cavity is wider,24,25 which enables more efficient catalysis and a broader substrate spectrum,26 and it might also have an impact on the binding affinity of zinc ions to the catalytic site. One might speculate that MBLs with low binding affinity to zinc ions are more susceptible to inhibition by zinc chelators like EDTA, EDDS and aspergillomarasmine A; however, this hypothesis has to be verified experimentally. With regard to clinical isolates, EDDS significantly reduced the imipenem MIC for all MBL-expressing bacteria under investigation, with the most pronounced effect, i.e. up to 512-fold imipenem MIC reduction, for bacteria expressing the clinically relevant NDM. The activity of the synthetic chelator Ca-EDTA in combination with ceftazidime or imipenem against MBL-producing bacteria has been analysed in vitro as well as in vivo and favourable results could be achieved.27,28 Ca-EDTA is widely used in clinics for the treatment of lead intoxication and is applied intravenously in doses between 25 and 75 mg/kg/day.27 EDDS might be applicable intravenously as well and in vivo pharmacokinetic studies need to be performed.29 EDDS is produced by bacteria as a natural compound that is biodegradable. As such, it offers an advantage over the poorly biodegradable EDTA, which cannot be removed by conventional water treatments, thereby exerting a constant environmental stress.22 In conclusion, we demonstrated that the bacterial zincophore EDDS is a highly efficient inhibitor of MBL. In combination with imipenem, EDDS demonstrated excellent activity against various MBL-producing Gram-negative pathogens in vitro as well as in vivo. Further investigation of EDDS as a chelating agent against infections due to MBL-producing pathogens, including animal studies, is warranted. Furthermore, to the best of our knowledge, EDDS is the first MBL inhibitor of bacterial origin. Although EDDS is described as a zincophore, its role in interspecies competition is unclear and should be further investigated. Acknowledgements We thank Denia Frank for excellent technical support; the German National Reference Laboratory for MDR Gram-negative bacteria for kindly providing us with isolates T1098, T2216 and T2217; the Robert Koch Institute for kindly providing us with isolates T2226 and T2228; the Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, for kindly providing us with isolate T2236; and the Universitätsspital Basel for kindly providing us with isolate T2229. Funding This work was supported by internal funding. E. P. thanks the German Research Foundation (DFG, Heisenberg-Professur PR1405/4–1) for financial support. Transparency declarations None to declare. Supplementary data Tables S1 and S2 are available as Supplementary data at JAC Online. References 1 Walker B, Barrett S, Polasky S et al.   Looming global-scale failures and missing institutions. Science  2009; 325: 1345– 6. Google Scholar CrossRef Search ADS PubMed  2 Fischbach MA, Walsh CT. Antibiotics for emerging pathogens. Science  2009; 325: 1089– 93. Google Scholar CrossRef Search ADS PubMed  3 Spellberg B, Blaser M, Guidos RJ et al.   Combating antimicrobial resistance: policy recommendations to save lives. Clin Infect Dis  2011; 52 Suppl 5: 397– 428. Google Scholar CrossRef Search ADS   4 Cornaglia G, Giamarellou H, Rossolini GM. Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect Dis  2011; 11: 381– 93. Google Scholar CrossRef Search ADS PubMed  5 Pfeifer Y, Cullik A, Witte W. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 1, 2018

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