JAC Advance Access originally published online on February 20, 2009
Journal of Antimicrobial Chemotherapy 2009 63(4):659-667; doi:10.1093/jac/dkp029
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Original research |
Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK
Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency, Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK
Received 23 October 2008; returned 11 December 2008; revised 19 January 2009; accepted 21 January 2009
* Corresponding author. Tel: +44-20-8327-7236; Fax: +44-20-8327-6264; E-mail: michel.doumith{at}hpa.org.uk
Objectives: 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 β-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 β-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 
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 β-lactamases with impermeability caused by loss of OMPs. Efflux was not implicated and there was no national spread of resistant clones.
Keywords: impermeability , carbapenems , Enterobacteriaceae
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