Functional and Structural Characterization of OXA-935, a Novel OXA-10-Family β-Lactamase from Pseudomonas aeruginosa

Abstract

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic β-lactamase AmpC. However, OXA-10-family β-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA β-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of bla_OXA-10, named bla_OXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of bla_OXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing bla_OXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family β-lactamases are concerning, given the rising reliance on novel β-lactam/β-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

Keywords: OXA-β-lactamase; Pseudomonas aeruginosa; antimicrobial resistance; ceftazidime; crystal structure.

FIG 1

Genomic organization and structures of OXA-935 and OXA-14. (A) Sequence alignment of the Ω-loop of OXA-10, OXA-14, and OXA-935, indicating in green the change in the residue 153 (F→S) and in cyan the change in residue 157 (G→D). Cartoon representations of the asymmetric dimeric structures of (B) OXA-14 and (C) OXA-935, showing the Ω-loop in orange for OXA-14 and in green for OXA-935.

FIG 2

The F153S substitution disrupts the interactions of W154 in the Ω-loop of OXA-935 with the catalytic residue K70. (A) Structural alignment of OXA-14 (blue) and OXA-935 (pink), highlighting the Ω-loops in orange and green, respectively. Zoomed in view of the Ω-loops of (B) OXA-14 and (C) OXA-935. Positions of the Ω-loops and interactions of W154 and the catalytic residue K70 in (D) OXA-14 and (E) OXA-935. Dashed lines represent hydrogen bond interactions. The continuous black line shows the distance between K70 and W154 in OXA-935. Surface charge representation of the Ω-loops and the active sites of (F) OXA-14 and (G) OXA-935.

FIG 3

Hydrolysis of various β-lactam compounds by OXA-14 and OXA-935. Using purified OXA-14 (blue, circles) or OXA-935 (orange, squares), we measured the cleavage of β-lactam compounds in a saturated sodium bicarbonate solution, including nitrocefin (A), penicillin-G (B), cephalothin (C), cefotaxime (D), cefepime (E), and ceftazidime (F). Hydrolysis of each compound (1/s) was plotted versus the concentration of substrate and analyzed via Michaelis-Menten nonlinear analysis. Each enzyme-drug concentration was assayed in at least triplicate, and the data are presented as the mean (points) and standard deviation (bars). (G) The catalytic efficiency (k_cat/K_m) of every enzyme-drug pair is plotted as a percentage of the catalytic efficiency of pencillin-G cleavage by OXA-14 when normalized to 100%.