title: Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK.
abstract: OBJECTIVE
  The aim of this study was to investigate relatedness and molecular mechanism(s) of ertapenem resistance in clinical isolates of Klebsiella spp. (n = 28) and Enterobacter spp. (n = 27) referred from multiple hospitals to the UK national reference laboratory.
  
  
  METHODS
  Investigations included genotyping by PFGE, resistance gene analysis by PCR and antimicrobial susceptibility testing with and without inhibitors of efflux and beta-lactamase activity. Outer membrane proteins (OMPs) were profiled by SDS-PAGE; porin genes were sequenced and their expression was examined by RT-PCR. The contribution of porin deficiency to resistance was investigated by restoring functional porin genes on plasmids.
  
  
  RESULTS
  PFGE showed significant clonal diversity among ertapenem-resistant isolates, with only small clusters identified. SHV- and CTX-M-type extended-spectrum beta-lactamases were identified in the Klebsiella spp. isolates, whereas AmpC overexpression or KPC carbapenemase was detected in the Enterobacter cloacae isolates. SDS-PAGE showed that Klebsiella pneumoniae and Enterobacter aerogenes with high-level ertapenem resistance (MICs > or = 16 mg/L) consistently lacked both of the two major non-specific porins, whereas variable patterns of OmpC and OmpF were seen in E. cloacae with lower-level ertapenem resistance. Various point mutations or insertion sequences were identified as disrupting the porin-coding sequences, as well as mutations in the promoter region. Functional restoration of OmpK35 or OmpK36 in Klebsiella and OmpC or OmpF in Enterobacter spp. isolates significantly decreased the MICs of all carbapenems, but particularly of ertapenem. We found no evidence of efflux contributing to resistance.
  
  
  CONCLUSIONS
  Ertapenem resistance was exclusively due to combinations of beta-lactamases with impermeability caused by loss of OMPs. Efflux was not implicated and there was no national spread of resistant clones.
authors.affiliation: Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency, Centre for Infections, 61 Colindale Avenue, London NW95EQ, UK. michel.doumith@hpa.org.uk
authors.forename: Michel
authors.initials: M
authors.lastname: Doumith
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellington
authors.forename: David M
authors.initials: DM
authors.lastname: Livermore
authors.forename: Neil
authors.initials: N
authors.lastname: Woodford
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D000077727:Ertapenem; D020869:Gene Expression Profiling; D006801:Humans; D007709:Klebsiella / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D018272:Porins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D006113:United Kingdom; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D018272:Porins; D047090:beta-Lactams; D001618:beta-Lactamases; D000077727:Ertapenem