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

• Main Authors: Silvia López-Argüello, María Montaner, Antonio Oliver, Bartolome Moya
• Format: Article
• Language: English
• Published: MDPI AG 2021-03-01
• Series: International Journal of Molecular Sciences
• Subjects: penicillin-binding proteins; PBP; avibactam; AmpC induction; PDC; β-lactam resistance
• Online Access: https://www.mdpi.com/1422-0067/22/6/3051

Avibactam belongs to the new class of diazabicyclooctane β-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including <i>P. aeruginosa</i> 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 <i>ampC</i> 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 <i>ampC</i>-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.