Phenotypic detection of AmpC in E. coli: comparison of cloxacillin, boronic acid and EDTA disk synergy assays

Abstract number: 1733_369

Giske C., Haldorsen B., Lundblad E., Aasnæs B., Simonsen G., Sundsfjord A.

Objectives: Disk synergy tests using cephalosporins ± boronic acid or cloxacillin have been found to be both sensitive and specific for phenotypic detection of chromosomal hyperproduction of AmpC and plasmid-mediated AmpC in E. coli. Also, an assay based on EDTA permeabilisation followed by analysis of distortion of cefoxitin inhibiton zone has been described. The purpose of this study was to compare the three methods against a previously characterised strain collection of E. coli either hyperproducing chromosomal AmpC or featuring plasmid-mediated AmpC.

Materials: A collection of 23 isolates from 12 different Norwegian laboratories were characterised with respect to hyperproduction of chromosomal AmpC (mediated by promotor mutations) and the presence of plasmid-mediated AmpC b-lactamases. Control strains of Enterobacteriaceae with defined plasmid-mediated AmpC or hyperproducing chromosomal AmpC as well as negative controls (ESBL-producers and wild-type E. coli) were included. Thirteen isolates were AmpC hyperproducers, whereas CMY b-lactamases were detected in 10 isolates. All isolates were subjected to disk synergy testing using cefoxitin, cefotaxime and ceftazidime ± boronic acid (400 ug) or cloxacillin (750 ug). A zone diameter difference of ≥5 mm was considered indicative of AmpC production.

Results: Synergy between cefoxitin and cloxacillin was found in all isolates, while synergy between cefoxitin and boronic acid could be demonstrated in all but two isolates. However, the latter two isolates were positive when results obtained with cefotaxime and ceftazidime ± boronic acid were taken into consideration. The EDTA test was negative in two isolates hyperproducing chromosomal AmpC.

Conclusions: The cefoxitin ± cloxacillin assay correctly identified all AmpC hyperproducers and CMY-producers. The cefoxitin ± boronic acid assay failed to identify two isolates, but both of these were positive with cefotaxime and ceftazidime ± boronic acid. This is in agreement with earlier observations that synergy between cefoxitin and boronic acid can sometimes be masked in strains featuring porin defects. Although the strain collection is relatively small the results indicate that cefoxitin ± cloxacillin may be promising as a stand alone test for phenotypic detection of AmpC production in E. coli. Further studies are warranted to determine whether cefotaxime and ceftazidime should be included in the cloxacillin assay.