title: Molecular characterization of the gene encoding a new AmpC beta-lactamase in Acinetobacter baylyi.
abstract: OBJECTIVE
  The main objective of the present study was to demonstrate the presence of a beta-lactamase ampC gene in the chromosome of the non-pathogenic bacterium Acinetobacter baylyi ADP1.
  
  
  METHODS
  beta-Lactam MICs were determined by Etest. The ampC gene was amplified by PCR, with specific oligonucleotides, then cloned into pBGS18 and pAT-RA plasmids and transformed into Escherichia coli TG1 and parental A. baylyi as hosts. The gene was sequenced and analysed. The AmpC protein was expressed, purified by affinity chromatography and the kinetic parameters determined.
  
  
  RESULTS
  An ampC gene was amplified from the ADP1 genome. Sequencing of the gene showed typical SVSK and KTG domains and the typical YXN Class C motif. The amplified gene showed significant identity (48.5% to 49.3%) with the AmpC enzymes of Acinetobacter baumannii and AG3 strains, which have recently been renamed ADC-1 to ADC-7. MIC analysis revealed a cephalosporinase profile for the E. coli TG1 clone as well as for the parental A. baylyi strain that overexpressed the ampC gene cloned under the control of an external promoter. Analysis of kinetic parameters of the purified enzyme showed higher catalytic efficiency for cefalotin than for ampicillin.
  
  
  CONCLUSIONS
  This study represents the first report of an AmpC beta-lactamase in A. baylyi, which was shown by biochemical and microbiological experiments to have a typical cephalosporinase profile. The presence of the respective gene in the chromosome of A. baylyi ADP1 suggested that this ampC gene is the naturally occurring cephalosporinase in this species, as previously reported for other Acinetobacter spp. We tentatively named the enzyme ADC-8.
authors.affiliation: Servicio de Microbiología-Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, Xubias de Arriba 84, 15006 La Coruña, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Beceiro
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Pérez-Llarena
authors.forename: Astrid
authors.initials: A
authors.lastname: Pérez
authors.forename: Ma Del Mar
authors.initials: Mdel M
authors.lastname: Tomás
authors.forename: Ana
authors.initials: A
authors.lastname: Fernández
authors.forename: Susana
authors.initials: S
authors.lastname: Mallo
authors.forename: Rosa
authors.initials: R
authors.lastname: Villanueva
authors.forename: Germán
authors.initials: G
authors.lastname: Bou
mesh_terms: D000150:Acinetobacter / Q000145:classification / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D047090:beta-Lactams; C048795:AmpC beta-lactamases; D001618:beta-Lactamases