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 koff 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.
Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to β-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-β-lactamases.
is a member of the complex, a group of>20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due to , which possesses two β-lactamase genes, and PenA is a carbapenemase with a substrate profile similar to that of the carbapenemase (KPC); in addition, expression of PenA is inducible by β-lactams in Here, we characterize AmpC from ATCC 17616. AmpC possesses only 38 to 46% protein identity with non- AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of , we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward β-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 ( > 600 μM), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible in ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 μg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in compared to PenA, which is the dominant β-lactamase in ATCC 17616.
PER -lactamases are an emerging family of extended-spectrum -lactamases (ESBL) found in Gram-negative bacteria. PER -lactamases are unique among class A enzymes as they possess an inverted omega (⍀) loop and extended B3 -strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non--lactam -lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 /K ϭ 2 ϫ 10 3 Ϯ 0.1 ϫ 10 3 M Ϫ1 s Ϫ1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D -lactamases (i.e., k 2 /K range of Ϸ10 3 M Ϫ1 s Ϫ1 ) and not class A -lactamases (i.e., k 2 /K range, 10 4 to 10 5 M Ϫ1 s Ϫ1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 Ϯ 3 atomic mass units [amu] ¡ 31,604 Ϯ 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the -lactamase and that the sulfate of AVI formed interactions with the -lactam carboxylate binding site of the PER-2 -lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 -strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2 /K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 -lactamase, we tested different -lactam-AVI combinations. By lowering MICs to Յ2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2 . Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.
Carbapenem-resistant Enterobacteriaceae are a significant threat to public health, and a major resistance determinant that promotes this phenotype is the production of the OXA-48 carbapenemase. The activity of OXA-48 towards carbapenems is a puzzling phenotype as its hydrolytic activity against doripenem is non-detectable. To probe the mechanistic basis for this observation, we determined the 1.5 Å resolution crystal structure of the deacylation deficient K73A variant of OXA-48 in complex with doripenem. Doripenem is observed in the Δ1R and Δ1S tautomeric states covalently attached to the catalytic S70 residue. Likely due to positioning of residue Y211, the carboxylate moiety of doripenem is making fewer hydrogen bonding/salt-bridge interactions with R250 compared to previously determined carbapenem OXA structures. Moreover, the hydroxyethyl side chain of doripenem is making van der Waals interactions with a key V120 residue, which likely affects the deacylation rate of doripenem. We hypothesize that positions V120 and Y211 play important roles in the carbapenemase profile of OXA-48. Herein, we provide insights for the further development of the carbapenem class of antibiotics that could render them less effective to hydrolysis by or even inhibit OXA carbapenemases.
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