title: Inactivation of the ampD gene in Pseudomonas aeruginosa leads to moderate-basal-level and hyperinducible AmpC beta-lactamase expression.
abstract: It has been shown in enterobacteria that mutations in ampD provoke hyperproduction of chromosomal beta-lactamase, which confers to these organisms high levels of resistance to beta-lactam antibiotics. In this study, we investigated whether this genetic locus was implicated in the altered AmpC beta-lactamase expression of selected clinical isolates and laboratory mutants of Pseudomonas aeruginosa. The sequences of the ampD genes and promoter regions from these strains were determined and compared to that of wild-type ampD from P. aeruginosa PAO1. Although we identified numerous nucleotide substitutions, they resulted in few amino acid changes. The phenotypes produced by these mutations were ascertained by complementation analysis. The data revealed that the ampD genes of the P. aeruginosa mutants transcomplemented Escherichia coli ampD mutants to the same levels of beta-lactam resistance and beta-lactamase expression as wild-type ampD. Furthermore, complementation of the P. aeruginosa mutants with wild-type ampD did not restore the inducibility of beta-lactamase to wild-type levels. This shows that the amino acid substitutions identified in AmpD do not cause the altered phenotype of AmpC beta-lactamase expression in the P. aeruginosa mutants. The effects of AmpD inactivation in P. aeruginosa PAO1 were further investigated by gene replacement. This resulted in moderate-basal-level and hyperinducible expression of beta-lactamase accompanied by high levels of beta-lactam resistance. This differs from the stably derepressed phenotype reported in AmpD-defective enterobacteria and suggests that further change at another unknown genetic locus may be causing total derepressed AmpC production. This genetic locus could also be altered in the P. aeruginosa mutants studied in this work.
authors.affiliation: Centre de Recherche en Infectiologie, Université Laval, Québec, Canada G1V 4G2.
authors.forename: T Y
authors.initials: TY
authors.lastname: Langaee
authors.forename: L
authors.initials: L
authors.lastname: Gagnon
authors.forename: A
authors.initials: A
authors.lastname: Huletsky
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002439:Cefotaxime / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D004790:Enzyme Induction; D005816:Genetic Complementation Test; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000378:metabolism; D010406:Penicillins / Q000494:pharmacology; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D010406:Penicillins; D000667:Ampicillin; C062460:AmpD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D002439:Cefotaxime