title: AmpG inactivation restores susceptibility of pan-beta-lactam-resistant Pseudomonas aeruginosa clinical strains.
abstract: Constitutive AmpC hyperproduction is the most frequent mechanism of resistance to the weak AmpC inducers antipseudomonal penicillins and cephalosporins. Previously, we demonstrated that inhibition of the β-N-acetylglucosaminidase NagZ prevents and reverts this mechanism of resistance, which is caused by ampD and/or dacB (PBP4) mutations in Pseudomonas aeruginosa. In this work, we compared NagZ with a second candidate target, the AmpG permease for GlcNAc-1,6-anhydromuropeptides, for their ability to block AmpC expression pathways. Inactivation of nagZ or ampG fully restored the susceptibility and basal ampC expression of ampD or dacB laboratory mutants and impaired the emergence of one-step ceftazidime-resistant mutants in population analysis experiments. Nevertheless, only ampG inactivation fully blocked ampC induction, reducing the MICs of the potent AmpC inducer imipenem from 2 to 0.38 μg/ml. Moreover, through population analysis and characterization of laboratory mutants, we showed that ampG inactivation minimized the impact on resistance of the carbapenem porin OprD, reducing the MIC of imipenem for a PAO1 OprD mutant from >32 to 0.5 μg/ml. AmpG and NagZ targets were additionally evaluated in three clinical isolates that are pan-β-lactam resistant due to AmpC hyperproduction, OprD inactivation, and overexpression of several efflux pumps. A marked increase in susceptibility to ceftazidime and piperacillin-tazobactam was observed in both cases, while only ampG inactivation fully restored wild-type imipenem susceptibility. Susceptibility to meropenem, cefepime, and aztreonam was also enhanced, although to a lower extent due to the high impact of efflux pumps on the activity of these antibiotics. Thus, our results suggest that development of small-molecule inhibitors of AmpG could provide an excellent strategy to overcome the relevant mechanisms of resistance (OprD inactivation plus AmpC induction) to imipenem, the only currently available β-lactam not significantly affected by P. aeruginosa major efflux pumps.
authors.affiliation: Servicio de Microbiología, Unidad de Investigación, Hospital Son Espases, IUNICS, Ctra. Valldemossa 79, 07010 Palma de Mallorca, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Zamorano
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Reeve
authors.forename: Carlos
authors.initials: C
authors.lastname: Juan
authors.forename: Bartolomé
authors.initials: B
authors.lastname: Moyá
authors.forename: Gabriel
authors.initials: G
authors.lastname: Cabot
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D001398:Aztreonam / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000077723:Cefepime; D002442:Ceftazidime / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology; D000077725:Piperacillin, Tazobactam Drug Combination; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics; D013845:Thienamycins / Q000494:pharmacology; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: C084284:AmpG protein, Bacteria; D001426:Bacterial Proteins; D002511:Cephalosporins; D026901:Membrane Transport Proteins; D013845:Thienamycins; D047090:beta-Lactams; D000077725:Piperacillin, Tazobactam Drug Combination; D000077723:Cefepime; D010397:Penicillanic Acid; D002442:Ceftazidime; D000077731:Meropenem; D001398:Aztreonam; D010878:Piperacillin