AmpG Inactivation Restores Susceptibility of Pan-β-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 ␤-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 g/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 g/ml. AmpG and NagZ targets were additionally evaluated in three clinical isolates that are pan-␤-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 ␤-lactam not significantly affected by P. aeruginosa major efflux pumps.

The Pseudomonas aeruginosa CreBC Two-Component System Plays a Major Role in the Response to β-Lactams, Fitness, Biofilm Growth, and Global Regulation

“…The complete list and description of the laboratory strains and plasmids used in this study are shown in Table 1. P. aeruginosa single or multiple knockout mutants involving ampD, ampR, ampC, ampG, creBC, creD, dacB, or nagZ were constructed according to well-established procedures (15,17,25) based on the Cre-lox system for gene deletion and antibiotic resistance marker recycling in P. aeruginosa (26). The previously constructed plasmids were used as the donor for the generation of knockout mutants by conjugational transfer to the corresponding P. aeruginosa strains (15,25).…”

“…P. aeruginosa single or multiple knockout mutants involving ampD, ampR, ampC, ampG, creBC, creD, dacB, or nagZ were constructed according to well-established procedures (15,17,25) based on the Cre-lox system for gene deletion and antibiotic resistance marker recycling in P. aeruginosa (26). The previously constructed plasmids were used as the donor for the generation of knockout mutants by conjugational transfer to the corresponding P. aeruginosa strains (15,25). For competition experiments, the P. aeruginosa PAO1, PA⌬CreBC, and PA⌬CreD strains were tagged at the att intergenic neutral chromosomal locus with two mini-Tn7 constructs harboring fluorescent and antibiotic resistance markers (Table 1), as previously described (27,28).…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…In Enterobacteriaceae members, an ampG mutant is unable to recycle PG and loses the ability to induce ampC expression [7,198] (Table 3). Inactivation of ampG can restore susceptibility to b-lactams even in pan-resistant P. aeruginosa clinical isolates [199]. P. aeruginosa also has an additional AmpG homologue known as AmpP (PA4218), both of which are presumed to be involved in ampC induction [151,153].…”

Structural and Functional Characterization of Pseudomonas aeruginosa Global Regulator AmpR

“…All amp gene homologs (ampC, ampR, ampD, and ampG) have been identified and studied in P. aeruginosa (30)(31)(32)(33)(34)(35)(36)(37). Whether a similar induction mechanism is employed by P. aeruginosa is not yet known; however, recent work illustrates significant departures from the classical enterobacterial induction system.…”