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Overcoming an extreme drug resistant (XDR) pathogen: Avibactam restores susceptibility to ceftazidime for Burkholderia cepacia complex isolates from Cystic Fibrosis patients Krisztina M. Papp-Wallace, Scott A. Becka, Elise T. Zeiser, Nozumi Ohuchi, Maria F. Mojica, Julian A. Gatta, Monica Falleni, Delfina Tosi, Elisa Borghi, Marisa L. Winkler, Brigid M. Wilson, John J. LiPuma, Michiyoshi Nukaga, and Robert A. Bonomo ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00020 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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ACS Infectious Diseases

Overcoming an extreme drug resistant (XDR) pathogen: Avibactam restores susceptibility to ceftazidime for Burkholderia cepacia complex isolates from Cystic Fibrosis patients

Krisztina M. Papp-Wallace1-2,8,*, Scott A. Becka1, Elise T. Zeiser1, Nozomi Ohuchi3, Maria F. Mojica1,8, Julian A. Gatta1, Monica Falleni4, Delfina Tosi4, Elisa Borghi4, Marisa L. Winkler1,5, Brigid M. Wilson1, John J. LiPuma6, Michiyoshi Nukaga3, and Robert A. Bonomo1-2,5,7-8,* 1

Research Service, Louis Stokes Cleveland Department of Veterans Affairs, 10701 East Blvd,

Cleveland, Ohio 44106 United States of America 2

Department of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, Ohio

44106 United States of America 3

Department of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane City,

Chiba, 283-8555 Japan 4

Department of Health Sciences, University of Milan, Via di Rudinì, 8-20142 Milan, Italy

5

Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900

Euclid Ave, Cleveland, Ohio 44106 United States of America 6

Department of Pediatrics and Communicable Disease, University of Michigan Medical School,

4810 Jackson Rd, Ann Arbor, Michigan, 48103 United States of America 7

Department of Pharmacology, Case Western Reserve University, 10900 Euclid Ave, Cleveland,

Ohio 44106 United States of America 8

Department of Biochemistry, Case Western Reserve University, 10900 Euclid Ave, Cleveland,

Ohio 44106 United States of America

*Corresponding author Email: [email protected] and [email protected]

ACS Paragon Plus Environment


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Abstract

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF).

Infections are difficult to treat as this pathogen is inherently resistant to multiple

antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-βlactam β-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤ 8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major β-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of 2 ± 1 X 106 µM-1s-1 and a slow koff of 2 ± 1 x 10-3 s-1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 hours and that avibactam recyclized off of PenA reforming active compound. Crystallographic analysis of PenA-avibactam revealed several interactions that stabilized the acyl-enzyme complex. The deacylation water molecule possessed decreased nucleophilicity preventing decarbamylation. In addition, the hydrogen bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime-avibactam was shown to significantly (p 1 doublingdilution difference between experiments) in triplicate results was resolved by the disk diffusion (Becton-Dickinson) method according to the CLSI guidelines. Avibactam was tested at 4 mg/L in combination with increasing antibiotic concentrations of ceftazidime or aztreonam.

Steady-state kinetic analysis. Steady-state kinetic parameters were determined using an Agilent 8453 Diode Array spectrophotometer. Briefly, each assay was performed in 10 mM phosphatebuffered saline at pH 7.4 (PBS) at room temperature. For determination of Vmax and Km, PenA was maintained at 14 nM with nitrocefin in excess molar concentration to establish pseudo-first order kinetics, as previously described16.


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The proposed interactions between PenA and avibactam are depicted in Figure 2B based on previous studies with class A β-lactamases and avibactam6b,

17

. Determination of kinetic

values Ki apparent (app), k2/K, kcat/kinact and koff were previously described and full methodology and equations used were defined in Papp-Wallace et al. 16. Briefly, for the determination of the Ki app value, PenA was maintained at 14 nM and using a direct competition assay with the reporter substrate, nitrocefin at 100 µM mixed with varying concentrations of avibactam (100 nM to 500 nM). For the k2/K assessment, progress curves were obtained by incubating 14 nM of PenA with increasing concentrations of avibactam (50 nM to 800 nM) using nitrocefin (50 µM) as a reporter substrate. The partition ratio (kcat/kinact) at 5 min for PenA with avibactam was determined by incubating 1 µM of PenA with 1 µM avibactam at which point >90% inhibition of 100 µM nitrocefin hydrolysis was obtained. The koff value was determined by incubating 10 µM of PenA with an avibactam concentration of 50 µM for 30 min. Samples were serially diluted to a final enzyme concentration of 2.0 nM and hydrolysis of 100 µM nitrocefin was measured. The progress curves were fit to a single exponential decay equation.

Electrospray ionization (ESI) mass spectrometry (MS).

To

discern

the

nature

of

the

intermediates of inactivation by avibactam in the reaction pathway with the PenA β-lactamase, ESI-MS was performed on a Waters SynaptG2-Si quadrupole-time-of-flight mass spectrometer equipped with a LockSpray dual electrospray ion source, as previously described18. For the experiments, 10 µM PenA was incubated with 10 µM avibactam for set times (i.e., 5 min, 5 h, and 24 h) at room temperature in 10 mM PBS, pH 7.4. Reactions were terminated by the addition of 0.1% formic acid and 1% acetonitrile.

Acyl-transfer experiment.

To assess if avibactam recyclizes to reform active compound an

acyl-tranfer ESI-MS experiment was conducting using PenA as the donor and TEM-1 as the recipient6b.

10 µM PenA was fully acylated with < 10 µM avibactam such that no free

avibactam was present. 10 µM TEM-1 β-lactamase was added and at time points of 15 sec, 2, 5, 10, 20, and 30 min, samples were terminated and prepared for ESI-MS as described above.

Crystallization and structure refinement. The PenA β-lactamase was crystallized by the vapor diffusion method using a 250 µl reservoir with a 4 µl hanging drop (2 µl reservoir solution + 2 µl



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protein solution). For PenA, the well solution contained 25% polyethylene glycol of 8 kilodaltons (PEG8K), 0.2 M sodium chloride, 0.1 M HEPES at pH 7.0 and 15 mg/ml of PenA in 2mM HEPES, pH 7.5. Crystals appeared in 1-2 weeks reaching sizes of 0.4-0.6 mm. Before data collection, a pre-grown crystal was soaked at room temperature for 30 min in the 30% PEG holding solution (pH 7.5) containing 1 mM avibactam. The crystals were cryoprotected by dipping them into a reservoir solution containing 20% glycerol, flush-cooled, and kept at 100 K with a nitrogen gas stream. The 0.5 deg. oscillation images were collected on a ADSC quantum Q210r CCD detector with synchrotron radiation (λ = 1.00 Å at beamline NW12E of the Advanced Ring of the Photon Factory, Tsukuba, Japan). The HKL2000 program was used to index and scale X-ray intensities (Table 2). The 27.5 kDa PenA β-lactamase crystallized in space group C2 with three molecules in the asymmetric unit and the following cell dimensions: a = 120.1, b = 69.4, and c = 84.6 Å, α = 90.000, β = 90.049 and γ = 90.000 (deg.) (100 K). Using the apo-PenA structure (PDB# 3W4Q) as a model, initial rigid body refinement was done with PHENIX. Model building and further refinement were performed with the programs COOT and PHENIX, respectively. Table 2 lists the final refinement statistics. The coordinates were deposited in the Protein Data Bank (PDB# 3WZR).

G. mellonella survival assays.

To assess the activity of ceftazidime-avibactam against B.

multivorans in vivo, the G. mellonella insect model of infection was used19. Larvae weighing between 200 mg and 400 mg were maintained on wood chips in the dark at (4ºC). Briefly, B. multivorans AU14786 was harvested from an 18 h culture by re-suspending 1x109 cells in 500 µl of cold PBS. Ceftazidime and avibactam were also suspended in cold PBS. A Hamilton syringe was used to inject 5 µl of the diluted bacterial suspension via the left proleg of each larva. After 30 min, the infected larvae were then treated with either ceftazidime (4 µg) or ceftazidime-avibactam (4 µg/4 µg) by injecting 5 µl into the right proleg. Two control groups of larva were used. The first group was untreated. The second group, to test the impact of trauma, was injected with 5 µl PBS and after 30 min, 5 µl of ceftazidime/avibactam (4 µg/4 µg). One hundred G. mellonella larvae were used in each condition and incubated at (37ºC) in a sterile petri dish for 24 h intervals for 48 h total. After 24h of incubation, infected and control


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larvae were subsequently treated with ceftazidime (4 µg), ceftazidime/avibactam (4 µg/4 µg) or PBS+ceftazidime/avibactam (4 µg/4 µg) respectively, by injecting 5 µl in the right proleg. Larvae were considered dead when they displayed no movement in response to gentle prodding with a glass rod. Three replicates with 100 larvae per petri dish were performed for each condition. The proportion that survived under each of the five treatments was compared using a test of proportions. Pairwise treatment comparisons were then performed, using a Bonferroni pvalue adjustment for the multiple tests. All data analysis was performed using R version 3.2.2.

Larval histology. The Histology Core facility at Case Western Reserve University embedded a larva from each treatment condition in paraffin, sectioned the larva, and stained slides using hematoxylin-eosin staining. Scientists at the Division of Human Pathology, University of Milan analyzed the stained slides.



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Supporting information Supplemental Table 1. MICs in mg/L of selected antibiotics against clinical isolates of B. multivorans. Supplemental Table 2. MICs in mg/L against clinical isolates of Burkholderia spp.


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Author Information Krisztina M. Papp-Wallace and Robert A. Bonomo, Louis Stokes Cleveland Veterans Affairs Medical Center, Research Service 151W, Rooms K102 and K104, Cleveland, Ohio 44106



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Author Contributions Conceived and designed the experiments: KMPW and RAB. Performed the experiments: KMPW, MN, NO, MLW, ETZ, SAB, JAG, and MFM. Analyzed the data: KMPW, MN, MF, DT EB, BMW, and RAB. Contributed reagents/materials/analysis tools: KMPW, MN, JJL, and RAB. Wrote the paper: KMPW, SAB, MN, and RAB


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Conflict of Interest The authors do not have any conflicts of interests to declare.



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Acknowledgements These studies were partly supported by the Grant-in-Aid for Scientific Research (C), No. 26460534, Japan Society for the Promotion of Science. Research reported in this publication was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program BX002872 (KMP-W) and BX001974 (RAB) from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory Research and Development Service, and the Geriatric Research Education and Clinical Center VISN 10 to RAB.

The contents do not represent the views of the U.S.

Department of Veterans Affairs or the United States Government. The National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers R21AI114508, R01AI100560, R01AI063517, and R01AI072219 to RAB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by funding (to JJL) from the Cystic Fibrosis Foundation.

We thank David Kuo and Dr. Menachem Shoham in the Department of Biochemistry at Case Western Reserve University for providing guidance on working with G. mellonella. We thank AstraZeneca for providing the avibactam powder used in this study.


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Figure Legends

Figure 1. Characteristics of the 50 clinical B. multivorans isolates. A. Summary pie charts of the susceptibility testing results (susceptible (blue) and resistant (red)) conducted with tobramycin,

imipenem,

ciprofloxacin,

minocycline,

trimethroprim-sulfamethoxazole,

ceftazidime, and ceftazidime-avibactam. B. Bar graph representing the number of isolates that are MDR and XDR. C. Dendrogram of the rep-PCR results.

Figure 2. Structure of avibactam and reaction scheme. A. Chemical structure of avibactam. B. Scheme representing the interactions of PenA with avibactam6a. In this model, formation of the non-covalent complex, E:I is represented by the dissociation constant, Kd, which is equivalent to k-1/k1. k2 is the first order rate constant for the acylation step, or formation of E-I. k-2 is the first order rate constant for the recyclization step or re-formation of E:I. C. Inhibition of nitrocefin hydrolysis by PenA using increasing concentrations of avibactam measured in absorbance at λ482nm (absorbance units (a.u.)). D. The data from Panel C were fit to obtain kobs values and here the kobs values was plotted vs [avibactam]. E. Recovery of nitrocefin hydrolysis activity by PenA after inhibition by avibactam (green line); PenA alone (black line) without inhibition. F. Acyl-transfer of avibactam from PenA to TEM-1 during a time course started at 15 sec and up to 30 min. Molecular weights of apo-PenA, acyl-PenA, apo-TEM-1 and acylTEM-1 are 29419 ± 3 Da, 29685 ± 3 Da, 28907 ± 3 Da, and 29172 ± 3 Da, respectively. By 20 min, most of the avibactam transferred to TEM-1 and by 30 min, some of the avibactam transferred back to PenA.

Figure 3. Active site structure of carbamylated PenA β-lactamase. A. Electron density map of acylated Ser-70 and avibactam with omit map. B. Snapshot of the active site of PenA crystal structure (PDB# 3WZR) with avibactam (gray); potential hydrogen bonding interactions are indicated by dashed green lines. C. Overlay of the crystal structures of apo-PenA (PDB# 3W4Q) (green) and PenA-avibactam (purple-gray). D. Overlay of the crystal structures of PenAavibactam (purple-gray) and KPC-2-avibactam (PDB# 4ZBE) (cyan-green). E.

Proposed

mechanistic schemes of avibactam carbamylation, decarbamylation, and recyclization with PenA based on crystallographic and biochemical analyses.


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Figure 4. G. mellonella survival assays. A. Percent survival of G. mellonella after infection by B. multivorans AU14786 (bacteria) treated with ceftazidime (CAZ) or ceftazidime-avibactam (CAZ-AVI) or mock-infected (PBS+CAZ-AVI); *** = P value 0σ(F)] 89812 a Rwork/Rfree (%) 0.164/0.199 Rtotal (%) 0.165 Residues in Ramachandran zones (%) 99.0/1.0/0 Favored/allowed/disallowed r.m.s.d. values from ideality Bond lengths (Å) 0.0006 Bond angles (deg.) 1.10 Mean B-factors (number of atoms) Protein (no.) 15.1(5852) avibactam (no.) 17.4 (51) Water molecules (no.) 29.8 (746) All atoms (no.) 16.0 (6649) aRfree

was calculated from 4512 reflections (5 %).



Figure 1.

A.

Imipenem

Tobramycin

0%

8%

36%

92%

96%

TrimethroprimSulfamethoxazole

Minocycline

Ciprofloxacin

4%

100%

Ceftazidime

64% CeftazidimeAvibactam

0% 32%

38%

68%

62% Resistant

100% Susceptible

B. 40 # of isolates

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30

70%

20 10

22%

0 MDR

XDR


C.

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Figure 2.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


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Figure 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Figure 4.


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Page 33 of 33

Figure 5.

A.

Tobramycin

Ciprofloxacin

Imipenem

7%

17%

Minocycline

24% 46%

83%

93% TrimethoprimSulfamethoxazole

54%

76% Ce azidime

Ce azidimeAvibactam

10% 36%

37%

63%

64%

Resistant

B. # of isolates

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


60 50 40 30 20 10 0

90% Susceptible

57%

19% MDR


XDR

Avibactam Restores Susceptibility to Ceftazidime ... - ACS Publications

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