ABSTRACT
Multidrug-resistant Acinetobacter baumannii has rapidly spread worldwide, resulting in a serious threat to hospitalized patients. Zidebactam and WCK 5153 are novel non-β-lactam bicyclo-acyl hydrazide β-lactam enhancer antibiotics being developed to target multidrug-resistant A. baumannii. The objectives of this work were to determine the 50% inhibitory concentrations (IC50s) for penicillin-binding proteins (PBP), the OXA-23 inhibition profiles, and the antimicrobial activities of zidebactam and WCK 5153, alone and in combination with β-lactams, against multidrug-resistant A. baumannii. MICs and time-kill kinetics were determined for an A. baumannii clinical strain producing the carbapenemase OXA-23 and belonging to the widespread European clone II of sequence type 2 (ST2). Inhibition of the purified OXA-23 enzyme by zidebactam, WCK 5153, and comparators was assessed. All of the compounds tested displayed apparent Ki values of >100 μM, indicating poor OXA-23 β-lactamase inhibition. The IC50s of zidebactam, WCK 5153, cefepime, ceftazidime, meropenem, and sulbactam (range of concentrations tested, 0.02 to 2 μg/ml) for PBP were also determined. Zidebactam and WCK 5153 demonstrated specific high-affinity binding to PBP2 of A. baumannii (0.01 μg/ml for both of the compounds). The MICs of zidebactam and WCK 5153 were >1,024 μg/ml for wild-type and multidrug-resistant Acinetobacter strains. Importantly, combinations of cefepime with 8 μg/ml of zidebactam or WCK 5153 and sulbactam with 8 μg/ml of zidebactam or WCK 5153 led to 4- and 8-fold reductions of the MICs, respectively, and showed enhanced killing. Notably, several of the combinations resulted in full bacterial eradication at 24 h. We conclude that zidebactam and WCK 5153 are PBP2 inhibitors that show a potent β-lactam enhancer effect against A. baumannii, including a multidrug-resistant OXA-23-producing ST2 international clone.
INTRODUCTION
Multidrug-resistant (MDR) Acinetobacter baumannii is one of the most challenging Gram-negative pathogens and is difficult to treat due to its extraordinary ability to acquire resistance determinants affecting all the antibacterial classes (1, 2). The percentage of multidrug-resistant A. baumannii clinical isolates is over 70% worldwide (3–6). Moreover, MDR A. baumannii is associated with 30-day mortality rates well above 60% when an inappropriate empirical therapy and/or dosing regimen is provided (7). As a result of the worldwide spread of MDR A. baumannii and the increased prevalence of carbapenem-resistant A. baumannii (CRAB), no appropriate therapy is available to successfully treat infections caused by this pathogen (8–10). A. baumannii is intrinsically resistant to various antibiotics owing to the low permeability of its outer membrane (11, 12). Moreover, the diminished or truncated expression of outer membrane porins further decreases carbapenem permeation (13, 14).
The Acinetobacter baumannii carbapenem resistance repertoire can be further extended by the acquisition of an oxacillinase (10, 15, 16) and/or a class B carbapenemase enzyme (17, 18). blaOXA-23 is the most widely prevalent A. baumannii-associated carbapenem-hydrolyzing β-lactamase.
Sulbactam remained a treatment option until the widespread emergence of OXA-23 in A. baumannii took place (19, 20). Tigecycline showed initial promise for the treatment of A. baumannii infections; however, its limited tolerability in critically ill patients and pharmacokinetic/pharmacodynamic (PK/PD) limitations often lead to suboptimal clinical outcomes (21, 22). Certain old revived drugs, like fosfomycin, temocillin, rifampin, minocycline, or colistin, have been deployed against MDR A. baumannii with variable success rates, besides the limitations of narrow indications and ill-defined PK/PD (9, 23–29), and β-lactamase inhibitors such as clavulanic acid (CLA), tazobactam (TAZ), avibactam (AVI), relebactam (REL), and RG6080 have not been able to fully overcome the class D OXA carbapenemase-associated resistance in A. baumannii (30–35).
In view of the excellent safety profiles of cephalosporins, a cephalosporin-based combination active against MDR A. baumannii would provide an attractive therapeutic option. The new bicyclo-acyl hydrazides (BCH) zidebactam (ZID; previously WCK 5107) and WCK 5153 are enhanced β-lactam antibiotics displaying a high-affinity for penicillin binding protein 2 (PBP2) of Gram-negative bacteria and overcoming diverse β-lactam resistance mechanisms, such as metallo-β-lactamases (MBL) in Pseudomonas aeruginosa. Zidebactam in combination with cefepime (WCK 5222) is being developed as a treatment for infections caused by MDR and extensively drug-resistant bacteria and is slated to enter phase 3 clinical trials (36–38).
The objectives of this work were to determine the PBP-binding affinity, the OXA-23 inhibition profiles, and the antimicrobial activities of zidebactam and WCK 5153 in combination with cefepime (WCK 5222) and sulbactam against MDR A. baumannii.
RESULTS AND DISCUSSION
Table 1 summarizes the MICs of zidebactam, WCK 5153, the comparators, and their combinations with cefepime and sulbactam against the wild-type strain A. baumannii ATCC 19606 and an A. baumannii OXA-23-producing sequence type 2 (ST2) strain. The MICs of imipenem, cefepime, and sulbactam for the OXA-23-producing isolate were 32, 64, and 16 μg/ml, respectively. The addition of zidebactam or WCK 5153 (8 μg/ml) lowered the MICs of cefepime and sulbactam (Table 1), suggesting a notable enhancer effect.
Susceptibility to β-lactams and WCK bicyclo-acyl hydrazides in the studied strainsa
As previously shown in P. aeruginosa (38), zidebactam and WCK 5153 demonstrated a potent affinity for A. baumannii PBP2 with a 50% inhibitory concentration (IC50) of 0.01 μg/ml, which was 7 to 8 times lower than that of imipenem and comparable to that of meropenem, both of which are well-documented potent PBP2-binding agents (Table 2; Fig. 1). Such high-affinity binding to A. baumannii PBP2 has not been previously reported for other diazabicyclooctanes (DBOs), such as relebactam, avibactam, and RG6080 (nacubactam). Similar to a recent report (39), our study also shows that cefepime and sulbactam bind to multiple A. baumannii PBPs, showing the highest affinities for PBP3 (IC50s, 0.08 and 0.64 μg/ml, respectively), as well as inhibit PBP1a and PBP1b, respectively (Table 2).
IC50s of cefepime, ceftazidime, imipenem, meropenem, sulbactam, zidebactam, and WCK 5153 for A. baumannii ATCC 19606 PBPs
Illustrative example of SDS-polyacrylamide gels. Twenty micrograms of an A. baumannii PBP-containing membrane preparation was incubated in the presence of increasing concentrations of cefepime (FEP), imipenem (IMP), ceftazidime (CAZ), meropenem (MEM), sulbactam (SUL), and zidebactam (ZID) and afterwards was labeled with 25 μM Bocillin FL. The range of concentrations tested was 0.0156 to 2 μg/ml.
PBP-binding determinations reported in the literature suggest that most of the drugs tested have a higher affinity for A. baumannii PBP2 than for the orthologues in other species (40, 41), possibly because the number/ratio of PBP2 molecules per cell is lower in A. baumannii than other Gram-negative bacteria. Such a limited availability of PBP2 molecules lends a high functional criticality, thus making PBP2 an even more valuable target (42). Microscopy-based observations confirmed that significantly sub-MICs of zidebactam or WCK 5153 (<1/128× MIC) induced spheroplast formation; furthermore, the combination of either drug with cefepime resulted in the formation of larger spheroplasts (38, 39).
OXA-23 inhibition studies with zidebactam, WCK 5153, and the comparator compounds revealed apparent Ki (Ki app) values of >100 μM for all compounds tested, consistent with previously reported data for avibactam and relebactam (1, 30).
The killing kinetics of the A. baumannii OXA-23 ST2 isolate showed that zidebactam and WCK 5153 alone did not elicit a bactericidal effect owing to their sole PBP2-binding feature. Nevertheless, combinations of 8 μg/ml zidebactam or WCK 5153 with 8 μg/ml of cefepime (0.125× MIC) displayed an initial 1.1- to 1.5-log10 kill for the first 8 h, followed by subsequent regrowth at 24 h (Fig. 2. On the other hand, higher concentration of cefepime (16 μg/ml, 0.25× MIC) in combination with 8 μg/ml of zidebactam or WCK 5153 provided sustained bactericidal activity of >3 log10, reaching complete eradication at 24 h. This enhanced bactericidal effect mediated through spheroplast formation was also confirmed through microscopy-based live-dead staining, in which a higher proportion of dead cells was observed (Fig. 3).
Results of the killing curves measured in terms of the reduction in the number of viable CFU per milliliter over time of the A. baumannii ST2 OXA-23-producing isolate (an extensively drug-resistant OXA-23-producing A. baumannii ST2 international clone) by combinations of cefepime and sulbactam with zidebactam and WCK 5153. The concentrations of the drugs tested alone were 16 μg/ml for cefepime (FEP; 0.25× MIC), 0.5 μg/ml for tigecycline (TGC; 0.5× MIC), 16 μg/ml for imipenem (IPM; 0.5× MIC), 16 μg/ml for sulbactam (SUL; 1× MIC), and 8 μg/ml for zidebactam (ZID) and WCK 5153 (<0.003× MIC). For the combinations, the concentrations used were 8 μg/ml for zidebactam and WCK 5153 and 4, 8, and 16 μg/ml for the partner β-lactams cefepime and sulbactam. The mean values for three experiments ± standard deviations are shown. Dashed lines represent the limits of detection.
LIVE/DEAD staining performed on A. baumannii MDR ST2 expressing OXA-23 (initial inoculum, 107 CFU/ml). The morphological changes observed after 3 h of exposure after inoculation in cation-adjusted-MHB (A), 8 μg/ml cefepime (B), 16 μg/ml cefepime (C), 8 μg/ml zidebactam (D), 8 μg/ml cefepime plus 8 μg/ml zidebactam (E), 16 μg/ml cefepime plus 8 μg/ml zidebactam (F), 8 μg/ml WCK 5153 (G), 8 μg/ml cefepime plus 8 μg/ml WCK 5153 (H), or 16 μg/ml cefepime plus 8 μg/ml WCK 5153 (I) are shown. The cefepime MIC was 64 μg/ml, the zidebactam MIC was ≥1,024 μg/ml, and the WCK 5153 MIC was ≥1,024 μg/ml. Bars, 10 μm.
Moreover, sulbactam at concentrations as low as 1/4 MIC (4 μg/ml) in combination with 8 μg/ml of either zidebactam or WCK 5153 elicited a faster response, resulting in a ≥3-log10 kill within 4 h which persisted until 24 h.
Concluding remarks.Combinations of zidebactam and WCK 5153 with PBP3-binding β-lactams exhibited synergistic killing of MDR A. baumannii, signifying the pivotal role of complementary PBP binding (43). Such combinations achieved pronounced bactericidal activity, even in the absence of OXA-23 carbapenemase inhibition, thus indicating the vulnerability of the spheroplasts induced by zidebactam to PBP3-binding agents, which otherwise are labile to the β-lactamases expressed by A. baumannii isolates.
Unlike the restoration of antibacterial activity reported for β-lactam and β-lactamase inhibitor combinations, zidebactam and WCK 5153 impart to PBP3-binding β-lactams rapid and consistent bactericidal effects, resulting in a pharmacodynamic action mimicking that of carbapenems, the most effective β-lactam class. Moreover, because zidebactam and WCK 5153 are non-β-lactams, they are not affected by classical β-lactam resistance mechanisms (34, 37, 38). The outcomes of this study demonstrate for the first time the principle of the activity of β-lactam enhancers against A. baumannii (37), which trigger bactericidal activity through complementary PBP binding, besides eluding the plethora of resistance mechanisms encountered in multidrug-resistant A. baumannii.
In vivo studies involving a neutropenic mouse lung infection undertaken with the cefepime-zidebactam combination have further demonstrated the in vivo relevance of the enhancer mechanism against MDR A. baumannii (44). Therefore, our results encourage further evaluations, including in vivo and clinical studies, of the efficacy of the β-lactam–zidebactam or β-lactam–WCK 5153 combination for the treatment of infections caused by MDR A. baumannii. Our study provides insights into the β-lactam enhancer-based approach as an alternative to β-lactamase inhibition in overcoming multiple mechanisms of resistance in A. baumannii.
MATERIALS AND METHODS
Susceptibility testing.The MICs of zidebactam, WCK 5153, cefepime, sulbactam, imipenem, and tigecycline were determined by the standard Clinical and Laboratory Standards Institute (CLSI) broth microdilution method for the wild-type A. baumannii ATCC 19606 strain and for the class D oxacillinase (OXA-23)-producing A. baumannii MDR ST2 strain belonging to European clone II (45). For determination of the MICs of drug combinations, zidebactam and WCK 5153 were used at a fixed concentration of 8 μg/ml.
Cloning of blaOXA-23.The blaOXA-23 gene and its upstream ISAba1 were amplified from A. baumannii strain AB0057 and cloned into a modified pWH1266 vector (a vector that replicates in A. baumannii) in which ampicillin resistance was eliminated (ΔblaTEM-1) by inverse PCR (46). blaOXA-23 without the leader peptide sequence was amplified from pWH1266-blaOXA-23 and cloned into the pET28a(+) vector downstream of the 6× His tag and electroporated into Escherichia coli DH10B. The sequences of the resulting constructs were verified. E. coli BL21(DE3) cells were used for protein expression and purification.
Purification and inhibition of OXA-23. E. coli BL21(DE3) containing pET28(+)blaOXA-23 was grown in Super Optimal broth containing 50 μg/ml kanamycin at 37°C with shaking to achieve an optical density at 600 nm (OD600) of 0.8 and induced with 0.2 mM isopropyl-β-d-1-thiogalactopyranoside for 3 h. The cells were centrifuged and frozen at −20°C. Cells were lysed using a QIAexpress nickel-nitrilotriacetic acid (Ni-NTA) fast-start kit per the manufacturer's protocol (Qiagen Inc.). To remove the His tag, the eluted protein was incubated with thrombin overnight at 4°C. The cleaved protein was separated from the His-tagged peptides by size exclusion chromatography (SEC) using a HiLoad 16/60 Superdex 75 column. The apparent Ki (Kiapp) values of zidebactam, WCK 5153, avibactam, and relebactam for OXA-23 were determined as previously described (38).
PBP-binding assays.The PBP-binding affinities for zidebactam, WCK 5153, and the comparator β-lactams (cefepime, sulbactam, ceftazidime, and meropenem) were determined by using membrane preparations from the reference strain A. baumannii ATCC 19606 following previously described protocols (47).
Briefly, 400-ml late-log-phase (OD600 = 1) cultures of A. baumannii ATCC 19606 were collected by centrifugation, washed, and resuspended in 20 mM KH2PO4 with 140 mM NaCl, pH 7.5 (buffer A). Cells were sonicated and centrifuged at 4,000 × g for 20 min. Bacterial membranes were collected by ultracentrifugation. Twenty micrograms of the A. baumannii PBP-containing membrane preparation was incubated for 30 min at 35°C in the presence of increasing concentrations of zidebactam, WCK 5153, and the comparator β-lactams (range of concentrations tested, 0.0156 to 2 μg/ml). Membrane preparations were incubated for an additional 30 min with 25 μM Bocillin FL (48). PBPs were separated by 10% SDS-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA) and visualized using a Typhoon FLA 9500 biomolecular imager (GE Healthcare Bio-Sciences AB, Björkgatan, Uppsala, Sweden) (excitation at 488 nm and emission at 530 nm). The IC50s for the different PBPs were determined from three independent experiments.
In addition, the morphological changes induced by cefepime and/or zidebactam or WCK5153 on the A. baumannii MDR ST2 isolate (expressing OXA-23) were visualized via live-dead staining using a LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and an AxioScope A1 fluorescence microscope (Carl Zeiss MicroImaging, GmbH).
Time-kill assays.Overnight Mueller-Hinton broth (MHB) cultures of the OXA-23-producing A. baumannii MDR ST2 strain were diluted (1/100) in fresh medium and incubated (37°C, 180 rpm) to an OD600 of 0.2 (mid-log-phase growth). Kill kinetics were initiated by inoculating 100 μl of mid-log-phase culture onto plates with 100 μl of MHB (initial inoculum, ∼106 CFU/ml) containing zidebactam or WCK 5153 at a final concentration of 8 μg/ of (MIC > 1,024 μg/ml) alone and in combination with 8 and 16 μg/ml of cefepime (0.125× and 0.25× cefepime MIC, respectively) or 4 μg/ml of sulbactam (0.25× MIC). Cefepime and sulbactam (both at 16 μg/ml), imipenem at 16 μg/ml, and tigecycline at 0.5 μg/ml were used alone as comparators. Cultures were then enumerated by plating serial dilutions on Mueller-Hinton agar at 1, 2, 4, 6, 8, and 24 h. All experiments were performed in triplicate.
ACKNOWLEDGMENTS
We thank Javier Piérola for his technical assistance with the fluorescence microscopy.
We thank Susan D. Rudin and Christopher R. Bethel for the cloning of blaOXA-23 and for the OXA-23 expression, purification, and kinetic experiments, respectively.
This work was supported by Wockhardt Bio AG, Switzerland, and by the Ministerio de Economía y Competitividad of Spain, Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF; A Way To Achieve Europe), through the Spanish Network for Research in Infectious Diseases (RD12/0015 and RD16/0016). The research reported in this publication was also supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs to K.M.P.-W. and R.A.B., Veterans Affairs Merit Review Program award 1I01BX002872 to K.M.P.-W., and Veterans Affairs Merit Review Program award 1I01BX001974 to R.A.B. from the U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service and by the Geriatric Research Education and Clinical Center VISN 10 to R.A.B. R.A.B. is also supported by the Harrington Foundation and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R21AI114508, R01AI100560, R01AI063517, and R01AI072219.
The contents do not represent the views of the U.S. Department of Veterans Affairs or the U.S. Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
B.M. and A.O. have received funds for research from Wockhardt Bio AG, Switzerland. M.P. is an employee and director of drug discovery research of Wockhardt Ltd., Wockhardt Research Centre. S.B. is an employee and shareholder of Wockhardt Ltd., Wockhardt Research Centre. All other authors have no conflict of interest to declare.
FOOTNOTES
- Received 15 June 2017.
- Returned for modification 9 July 2017.
- Accepted 22 August 2017.
- Accepted manuscript posted online 28 August 2017.
- Copyright © 2017 American Society for Microbiology.
