title: NagZ inactivation prevents and reverts beta-lactam resistance, driven by AmpD and PBP 4 mutations, in Pseudomonas aeruginosa.
abstract: AmpC hyperproduction is the most frequent mechanism of resistance to penicillins and cephalosporins in Pseudomonas aeruginosa and is driven by ampD mutations or the recently described inactivation of dacB, which encodes the nonessential penicillin-binding protein (PBP) PBP 4. Recent work showed that nagZ inactivation attenuates beta-lactam resistance in ampD mutants. Here we explored whether the same could be true for the dacB mutants with dacB mutations alone or in combination with ampD mutations. The inactivation of nagZ restored the wild-type beta-lactam MICs and ampC expression of PAO1 dacB and ampD mutants and dramatically reduced the MICs (for example, the MIC for ceftazidime dropped from 96 to 4 microg/ml) and the level of ampC expression (from ca. 1,000-fold to ca. 50-fold higher than that for PAO1) in the dacB-ampD double mutant. On the other hand, nagZ inactivation had little effect on the inducibility of AmpC. The NagZ inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate attenuated the beta-lactam resistance of the AmpC-hyperproducing strains, showing a greater effect on the dacB mutant (reducing the ceftazidime MICs from 24 to 6 microg/ml) than the ampD mutant (reducing the MICs from 8 to 4 microg/ml). Additionally, nagZ inactivation in the dacB mutant blocked the overexpression of creD (blrD), which is a marker of the activation of the CreBC (BlrAB) regulator involved in the resistance phenotype. Finally, through population analysis, we show that the inactivation of nagZ dramatically reduces the capacity of P. aeruginosa to develop ceftazidime resistance, since spontaneous mutants were not obtained at concentrations > or = 8 microg/ml (the susceptibility breakpoint) for the nagZ mutant but were obtained with wild-type PAO1. Therefore, NagZ is envisaged to be a candidate target for preventing and reverting beta-lactam resistance in P. aeruginosa.
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Instituto Universitario de Investigación en Ciencias de la Salud, 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: Lehua
authors.initials: L
authors.lastname: Deng
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: D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D002442:Ceftazidime / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018440:beta-Lactam Resistance / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D047090:beta-Lactams; D002442:Ceftazidime; C062460:AmpD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase