False extended-spectrum β-lactamase phenotype in clinical isolates of Escherichia coli associated with increased expression of OXA-1 or TEM-1 penicillinases and loss of porins

Beceiro, Alejandro, Maharjan, Sunil, Gaulton, Tom, Doumith, Michel, Soares, Nelson C., Dhanji, Hiran, Warner, Marina, Doyle, Maeve, Hickey, Mary, Downie, Gordon, Bou, Germán, Livermore, David M. ORCID: https://orcid.org/0000-0002-9856-3703 and Woodford, Neil (2011) False extended-spectrum β-lactamase phenotype in clinical isolates of Escherichia coli associated with increased expression of OXA-1 or TEM-1 penicillinases and loss of porins. Journal of Antimicrobial Chemotherapy, 66 (9). pp. 2006-2010. ISSN 0305-7453

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Abstract

Objectives 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. ß-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 J53?ompCF. 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 ß-lactamases with pIs of 5.4 (TEM-1) in EC18 and 7.4 (OXA-1) in both EC19 and EC21. EC18 had blaTEM-1b with the strong promoter P5 and lacked OmpC and OmpF. RT–PCR showed stronger expression of blaOXA-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 ß-lactamases combined with loss of porins.

Item Type:	Article
Uncontrolled Keywords:	anti-bacterial agents,bacterial outer membrane proteins,cephalosporins,clavulanic acid,dna,dna, recombinant,drug resistance, bacterial,electrophoresis, gel, pulsed-field,enzyme inhibitors,escherichia coli,escherichia coli infections,isoelectric focusing,microbial sensitivity tests,plasmids,porins,reverse transcriptase polymerase chain reaction,spectrometry, mass, matrix-assisted laser desorption-ionization,transformation, bacterial,beta-lactamases
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups:	Faculty of Medicine and Health Sciences > Research Groups > Public Health and Health Services Research (former - to 2023) Faculty of Medicine and Health Sciences > Research Groups > Epidemiology and Public Health
Depositing User:	Users 2731 not found.
Date Deposited:	24 Oct 2011 10:58
Last Modified:	11 Jan 2024 01:20
URI:	https://ueaeprints.uea.ac.uk/id/eprint/35139
DOI:	10.1093/jac/dkr265

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