Inhibition of β-lactamases of mycobacteria by avibactam and clavulanate

Abstract

Objectives: Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A β-lactamases, BlaC and BlaMab, which are inhibited by clavulanate and avibactam, respectively. BlaC differs from BlaMab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of β-lactamases from slowly and rapidly growing mycobacteria. Methods: Enzyme kinetics were determined to assess the impact of the substitutions G132N in BlaC and N132G in BlaMab on β-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the β-lactamases in Escherichia coli. Results: The substitution G132N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. BlaMab efficiently hydrolysed clavulanate, but the substitution N132G led to a 5600-fold reduction in the hydrolysis rate constant kcat due to stabilization of BlaMab-clavulanate covalent adducts. The N132G substitution also led to a 610-fold reduction in the efficacy of BlaMab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the β-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa. Conclusions: G132N and N132G had opposite effects on the inhibition of BlaC and BlaMab, indicating that these substitutions might lead to acquisition of resistance to either of the β-lactamase inhibitors, but not to both of them.