title: Diversity of β-lactam resistance mechanisms in cystic fibrosis isolates of Pseudomonas aeruginosa: a French multicentre study.
abstract: OBJECTIVE
  To investigate the resistance mechanisms of β-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France.
  
  
  METHODS
  Two-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to β-lactams were investigated. Their β-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic β-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations.
  
  
  RESULTS
  MLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to β-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to β-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary β-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene.
  
  
  CONCLUSIONS
  β-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of β-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.
authors: CHRU Jean Minjoz, Centre National de Référence de la résistance aux antibiotiques, Besançon, France.
authors: Catherine
authors: C
authors: Llanes
authors: Christine
authors: C
authors: Pourcel
authors: Charlotte
authors: C
authors: Richardot
authors: Patrick
authors: P
authors: Plésiat
authors: Gwennaele
authors: G
authors: Fichant
authors: Jean-Didier
authors: JD
authors: Cavallo
authors: Audrey
authors: A
authors: Mérens
mesh_terms: D000293:Adolescent; D000328:Adult; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002648:Child; D002675:Child, Preschool; D003550:Cystic Fibrosis / Q000150:complications*; D005260:Female; D005602:France; D020869:Gene Expression Profiling; D005798:Genes, Bacterial; D014644:Genetic Variation; D005838:Genotype; D006785:Hospitals, University; D006801:Humans; D007223:Infant; D007525:Isoelectric Focusing; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D018598:Minisatellite Repeats; D058889:Molecular Typing; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D055815:Young Adult; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000032:analysis / Q000235:genetics; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D001618:beta-Lactamases
keywords: AmpC; OprD; antibiotic resistance surveillance; efflux systems; molecular typing