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Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1
Diansy Zincke1, Deepak Balasubramanian1, Lynn L Silver2
1Department of Biological Sciences, College of Arts and Sciences, Florida International University, Miami, Florida, USA.
Abstract:
Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC β-lactamase. A second, chromosomally encoded β-lactamase, PoxB, has previously been reported in P. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frame ampC, poxB, and oprD deletions, as well as complementation by expression under the control of an inducible promoter. While poxB deletions failed to alter β-lactam sensitivities, expression of poxB in ampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in an ampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding gene oprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing β-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-β-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity. In vitro susceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition, poxB was found to be cotranscribed with an upstream open reading frame, poxA, which itself was shown to encode a 32-kDa protein of yet unknown function.
Insights
Pseudomonas aeruginosa harbors the PoxB enzyme, a class D beta-lactamase. PoxB contributes to carbapenem resistance, particularly when outer membrane porin OprD is absent, impacting treatment options for severe infections.
Area of Science:
- Microbiology
- Antimicrobial Resistance
- Enzymology
Background:
- Pseudomonas aeruginosa is a problematic opportunistic pathogen known for severe, difficult-to-treat infections.
- Antimicrobial resistance is a significant challenge, with P. aeruginosa exhibiting widespread resistance to various drugs.
- AmpC beta-lactamase is a common cause of penicillin-like compound resistance in P. aeruginosa.
Purpose of the Study:
- To investigate the role of the chromosomally encoded PoxB beta-lactamase in P. aeruginosa antimicrobial resistance.
- To determine the contribution of PoxB to resistance against different classes of beta-lactam antibiotics, including carbapenems.
- To assess the efficacy of inhibitors like avibactam against PoxB activity.
Main Methods:
- Utilized clean in-frame deletions of ampC, poxB, and oprD genes.
- Employed inducible promoter-driven complementation to study gene expression and function.
- Performed in vitro susceptibility testing (MICs) and enzyme inhibition assays.
Main Results:
- Deletion of poxB alone did not affect beta-lactam sensitivity.
- Expression of PoxB in ampC-deficient strains reduced susceptibility to meropenem and doripenem.
- PoxB significantly increased resistance to imipenem, meropenem, and doripenem in strains lacking both ampC and oprD.
- Avibactam showed limited inhibition of PoxB in vitro and failed to restore susceptibility to carbapenems at clinical concentrations.
- PoxB was found to be cotranscribed with poxA, encoding a protein of unknown function.
Conclusions:
- The class D beta-lactamase PoxB contributes to carbapenem resistance in P. aeruginosa, especially in the absence of the OprD porin.
- PoxB's activity is poorly inhibited by standard beta-lactamase inhibitors but shows slight susceptibility to avibactam, though not sufficient to overcome resistance in vivo.
- Understanding PoxB's role is crucial for developing effective therapeutic strategies against multidrug-resistant P. aeruginosa infections.
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