title: Molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains.
abstract: The molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in natural strains of Pseudomonas aeruginosa were investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with beta-lactams. All 10 ceftazidime-resistant mutants hyper-produced AmpC (beta-lactamase activities were 12- to 657-fold higher than those of the parent strains), but none of them harbored mutations in ampR or the ampC-ampR intergenic region. On the other hand, six of them harbored inactivating mutations in ampD: four contained frameshift mutations, one had a C-->T mutation, creating a premature stop codon, and finally, one had a large deletion, including the complete ampDE region. Complementation studies revealed that only three of the six ampD mutants could be fully trans-complemented with either ampD- or ampDE-harboring plasmids, whereas one of them could be trans-complemented only with ampDE and two of them (including the mutant with the deletion of the ampDE region and one with an ampD frameshift mutation leading to an ampDE-fused open reading frame) could not be fully trans-complemented with any of the plasmids. Finally, one of the four mutants with no mutations in ampD could be trans-complemented, but only with ampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to the ampDE operon.
authors.affiliation: Servicio de Microbiología, Hospital Son Dureta, C. Andrea Doria No. 55, 07014 Palma de Mallorca, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Juan
authors.forename: María D
authors.initials: MD
authors.lastname: Maciá
authors.forename: Olivia
authors.initials: O
authors.lastname: Gutiérrez
authors.forename: Carmen
authors.initials: C
authors.lastname: Vidal
authors.forename: José L
authors.initials: JL
authors.lastname: Pérez
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002442:Ceftazidime / Q000494:pharmacology; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009876:Operon; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases