Molecular Basis of AmpC β-Lactamase Induction by Avibactam in Pseudomonas aeruginosa: PBP Occupancy, Live Cell Binding Dynamics and Impact on Resistant Clinical Isolates Harboring PDC-X Variants.

Silvia López-Argüello,María Montaner,Antonio Oliver,Bartolome Moya

doi:10.3390/ijms22063051

Silvia López-Argüello, María Montaner + Show 2 more

Open Access

https://doi.org/10.3390/ijms22063051

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Abstract

Avibactam belongs to the new class of diazabicyclooctane β-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including P. aeruginosa AmpC. Nonetheless, recent reports have revealed strain-dependent avibactam AmpC induction. In the present work, we wanted to assess the mechanistic basis underlying AmpC induction and determine if derepressed PDC-X mutated enzymes from ceftazidime/avibactam-resistant clinical isolates were further inducible. We determined avibactam concentrations that half-maximally inhibited (IC50) bocillin FL binding. Inducer β-lactams were also studied as comparators. Live cells’ time-course penicillin-binding proteins (PBPs) occupancy of avibactam was studied. To assess the ampC induction capacity of avibactam and comparators, qRT-PCR was performed in wild-type PAO1, PBP4, triple PBP4, 5/6 and 7 knockout derivatives and two ceftazidime/avibactam-susceptible/resistant XDR clinical isolates belonging to the epidemic high-risk clone ST175. PBP4 inhibition was observed for avibactam and β-lactam comparators. Induction capacity was consistently correlated with PBP4 binding affinity. Outer membrane permeability-limited PBP4 binding was observed in the live cells’ assay. As expected, imipenem and cefoxitin showed strong induction in PAO1, especially for carbapenem; avibactam induction was conversely weaker. Overall, the inducer effect was less remarkable in ampC-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.

Highlights

The worldwide emergence of multidrug-resistant Gram-negative Pseudomonas aeruginosa isolates has caused a substantial public health problem, which is exacerbated by few therapeutic options remaining [1]
We examined the basis for the underlying AmpC induction in the presence of clinically relevant avibactam concentrations (≈50% of Cmax after 500 mg standard dosage) [21] and the behavior of mutated AmpC enzymes (PDC-221 and -223) [15] upon induction in CAZ/AVI-resistant P. aeruginosa clinical isolates
The results presented in this work are in accordance with previous studies suggesting a link between avibactam ampC induction and PBP4 inhibition

Summary

Introduction

The worldwide emergence of multidrug-resistant Gram-negative Pseudomonas aeruginosa isolates has caused a substantial public health problem, which is exacerbated by few therapeutic options remaining [1]. Clinical isolates of P. aeruginosa resistant to ceftolozane/tazobactam (TOL/TAZ) and ceftazidime/avibactam (CAZ/AVI), expressing mutant variants of AmpC (i.e., PDC-221, -222, -223 and -322), have already been reported [14,15,16]. The studied AmpC mutants showed structural mutations in the omega (Ω) loop or adjacent residues Such mutations appear to exert a concurrent effect on the catalytic properties of AmpC, reducing its susceptibility towards avibactam inhibition and enhancing its catalytic efficiencies towards ceftolozane and ceftazidime hydrolysis [17]. According to their work, such induction capacity would become clinically significant only if two conditions were met for the AmpC enzyme: loss of inhibition by avibactam while retaining inducibility [20] Under these circumstances, knowing whether a novel BLI would be able to induce AmpC expression in avibactam-insensitive fifth-generation cephalosporin-resistant strains would be of great interest. Enzymes (PDC-221 and -223) [15] upon induction in CAZ/AVI-resistant P. aeruginosa clinical isolates

Molecular Basis of Avibactam AmpC Induction

Bacterial Strains and Antibiotics

Intact Cells’ Time-Course of PBP-Binding Assay