Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients

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-β-lactam 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) × 10⁶ μM^-1 s^-1 and a slow k_off of (2 ± 1) × 10^-3 s^-1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the 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 < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime-avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime-avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.

Keywords: Burkholderia; avibactam; ceftazidime; cystic fibrosis; β-lactamase; β-lactamase inhibitor.

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

The avibactam inhibition mechanism of PenA. (A) Chemical structure of avibactam. (B) Scheme representing the interactions of PenA with avibactam. In this model, formation of the noncovalent complex, E:I is represented by the dissociation constant, K_d, 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 λ_{482 nm} (absorbance units (a.u.)). (D) Data from panel C were fit to obtain k_obs values, and here the k_obs values were plotted versus [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 s and up to 30 min. Molecular weights of apo-PenA, acyl-PenA, apo-TEM-1, and acyl-TEM-1 are 29419 ± 3, 29685 ± 3, 28907 ± 3, and 29172 ± 3 Da, respectively. By 20 min, most of the avibactam had transferred to TEM-1, and by 30 min, some of the avibactam had 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 PenA–avibactam (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.

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 <0.001. Histological sections of G. mellonella. Tissue labels: trachea (T), gastrointestinal tract (GI), fat body (FB), subcuticular region (Ct). (B) Uninfected. (C) Mock-infected. (D) Bacteria alone. (E) Bacteria + CAZ. (F) Bacteria + CAZ–AVI.

Figure 5

Susceptibility testing of the 96 clinical non-B. multivorans Burkholderia spp. isolates from CF patients. (A) Summary pie charts of the susceptibility testing results (susceptible (blue) and resistant (purple)) 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.