title: False extended-spectrum {beta}-lactamase phenotype in clinical isolates of Escherichia coli associated with increased expression of OXA-1 or TEM-1 penicillinases and loss of porins.
abstract: OBJECTIVE
  Two clinical isolates of Escherichia coli, EC18 and EC21, were non-susceptible (MICs 4-16 mg/L) to cefpirome and cefepime, with marked synergy with clavulanate, yet were susceptible to cefotaxime and ceftazidime (MICs ≤ 1 mg/L). EC19, from the same patient as EC21, was susceptible to all four cephalosporins. We sought to characterize the molecular basis of resistance in isolates EC18 and EC21.
  
  
  METHODS
  PFGE was used to study the genetic relationships of the isolates, and MICs were determined. β-Lactamases were characterized by PCR, isoelectric focusing (IEF), construction of genomic libraries and sequencing. A double mutant of E. coli J53 was constructed, lacking OmpC and OmpF porins. Plasmids from clinical isolates were transformed into E. coli J53 and J53ΔompCF. Outer membrane proteins (OMPs) were analysed by SDS-PAGE and OmpA by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. Expression of omp and bla genes was analysed by RT-PCR.
  
  
  RESULTS
  Isolates EC19 and EC21 had identical PFGE profiles, whereas EC18 was distinct. PCR and IEF confirmed β-lactamases with pIs of 5.4 (TEM-1) in EC18 and 7.4 (OXA-1) in both EC19 and EC21. EC18 had bla(TEM-1b) with the strong promoter P5 and lacked OmpC and OmpF. RT-PCR showed stronger expression of bla(OXA-1) in EC21 versus EC19, along with diminished expression of OmpC, though with increased OmpF. Plasmids extracted from EC18 and EC21 conferred increased MICs of cefpirome and cefepime, although susceptibility to cefotaxime and ceftazidime was retained.
  
  
  CONCLUSIONS
  The 'cefpiromase' or 'cefepimase' ESBL phenotype of the clinical isolates non-susceptible to cefpirome and cefepime resulted from high expression of TEM-1 or OXA-1 β-lactamases combined with loss of porins.
authors: Health Protection Agency, Microbiology Services-Colindale, London, UK. alejandro.beceiro.casas@sergas.es
authors: Alejandro
authors: A
authors: Beceiro
authors: Sunil
authors: S
authors: Maharjan
authors: Tom
authors: T
authors: Gaulton
authors: Michel
authors: M
authors: Doumith
authors: Nelson C
authors: NC
authors: Soares
authors: Hiran
authors: H
authors: Dhanji
authors: Marina
authors: M
authors: Warner
authors: Maeve
authors: M
authors: Doyle
authors: Mary
authors: M
authors: Hickey
authors: Gordon
authors: G
authors: Downie
authors: Germán
authors: G
authors: Bou
authors: David M
authors: DM
authors: Livermore
authors: Neil
authors: N
authors: Woodford
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D000077723:Cefepime; D002511:Cephalosporins / Q000494:pharmacology; D019818:Clavulanic Acid / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004274:DNA, Recombinant / Q000096:biosynthesis / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D004927:Escherichia coli Infections / Q000382:microbiology*; D007525:Isoelectric Focusing; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics; D018272:Porins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*; D000097572:Cefpirome
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D000077723:Cefepime; D002511:Cephalosporins; D019818:Clavulanic Acid; D004247:DNA; D004274:DNA, Recombinant; D004791:Enzyme Inhibitors; D018272:Porins; D001618:beta-Lactamases; D000097572:Cefpirome; C113724:OmpC protein; C109237:OmpF protein; C039404:beta-lactamase OXA-2; C034413:beta-lactamase TEM-1