JAC Advance Access originally published online on August 8, 2005
Journal of Antimicrobial Chemotherapy 2005 56(3):600-601; doi:10.1093/jac/dki278
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Correspondence |
Disc methods for detecting AmpC ß-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae
1 Department of Microbiology and 2 Hospital Infection Research Laboratory, City Hospital, Dudley Road, Birmingham, UK
* Corresponding author. Tel: +44-121-507-4228; Fax: +44-121-551-7763; E-mail: nigel.brenwald{at}swbh.nhs.uk
Keywords: susceptibility testing , ß-lactamase inhibitor , disc diffusion
Sir,
The increasing prevalence of AmpC ß-lactamase-mediated resistance among Escherichia coli and Klebsiella pneumoniae is of clinical concern.1 Both of these organisms can acquire AmpC ß-lactamases on plasmids and additionally, E. coli can hyper-produce its chromosomal AmpC ß-lactamase, which is normally only produced at very low levels.2 The recognition of AmpC ß-lactamase producers can be difficult, although resistance to cefoxitin can help in identifying them. Unfortunately, cefoxitin resistance is not only due to AmpC ß-lactamase production, but may also be due to decreased permeability. Several methods, based on the ability of cell-free extracts of organisms to hydrolyse cefoxitin, have been proposed as confirmatory tests for the production of AmpC ß-lactamases.2,3 Most are too time-consuming for use in routine diagnostic laboratories and may not detect all AmpC ß-lactamases.4 There is a need, therefore, for alternative methods that can be integrated into diagnostic laboratories and ideally do not rely on cefoxitin as the indicator antibiotic. We report our preliminary findings with a combination disc susceptibility method that uses cefpodoxime as an indicator and the AmpC ß-lactamase inhibitor benzo(b)thiophene-2-boronic acid (BZBTH2B).1,5
Sixty non-replicate AmpC ß-lactamase-producing clinical isolates of E. coli (n = 57) and K. pneumoniae (n = 3), collected from City Hospital, Birmingham (August 2001–March 2004) were tested. All of the isolates hydrolysed cefoxitin, as determined by the method of Nasim et al.,3 and their cefoxitin MICs, as determined by the BSAC standardized agar dilution method,6 were reduced 
4-fold in the presence of a fixed 100 mg/L concentration of cloxacillin.7 Fourteen of the isolates were positive by PCR for plasmid-borne blaampC using primers and conditions described by Pérez-Pérez and Hanson.8 Seventy cefoxitin-resistant, AmpC ß-lactamase-negative isolates of E. coli (n = 50) and K. pneumoniae (n = 20) were used as negative controls. All isolates were identified by API 20E (bioMérieux, Basingstoke, UK). Six laboratory strains of E. coli producing known AmpC ß-lactamases (BIL-1, ACC-1, ACT-1, MIR-1, FOX-4, CMY-2) were also tested.
Combination discs were produced in-house using commercially available susceptibility discs (Oxoid, Basingstoke, UK and Mast, Bootle, UK) to which the inhibitor BZBTH2B was added. Stock solutions of BZBTH2B (VWR International Ltd, Lutterworth, UK) were made in DMSO and further diluted in 0.1 M NaOH and water. BZBTH2B was added to discs containing cefpodoxime 10 µg or cefpodoxime 10 µg + clavulanic acid 1 µg to give 64 µg BZBTH2B per disc. Blank discs containing inhibitor alone were used to check for possible intrinsic antimicrobial activity. IsoSensitest agar plates were inoculated with the test organisms to give semi-confluent growth using the BSAC standardized disc susceptibility method. Cefpodoxime and cefpodoxime + clavulanic acid discs with and without BZBTH2B inhibitor, and discs with the inhibitor alone, were spaced over the agar surface. After overnight incubation in air at 35–37°C, the zones of inhibition were measured. For comparison, the method was repeated using cefoxitin 30 µg discs with and without BZBTH2B (64 µg) or cloxacillin (100 µg).
A 5 mm increase in the zone diameter around the combined disc compared with that for cefoxitin, cefpodoxime or cefpodoxime + clavulanic acid alone, was considered significant. Using this cut-off, none of the combined discs gave a positive result with the cefoxitin-resistant, non-AmpC ß-lactamase-producing isolates. BZBTH2B alone gave no zones of inhibition with any of the organisms tested. The combined disc methods cefoxitin + cloxacillin, cefoxitin + BZBTH2B, cefpodoxime + BZBTH2B and cefpodoxime + clavulanic acid + BZBTH2B correctly identified 57 (86.4%), 59 (89.4%), 64 (97%) and 66 (100%) of the AmpC ß-lactamase producers, respectively. Significantly, the cefoxitin-based disc methods failed to detect the ACC-1 ß-lactamase-producing strain, which is cefoxitin susceptible. ACC-1 ß-lactamase-producing clinical isolates of both E. coli and K. pneumoniae have been described.4 Cefpodoxime + BZBTH2B did not detect two isolates, which concomitantly produced an AmpC ß-lactamase and an extended spectrum ß-lactamase (ESBL). The activity of the ESBL masked the effects of BZBTH2B. Cefpodoxime + clavulanic acid + BZBTH2B was the only method to detect all the AmpC ß-lactamase producers, as the addition of clavulanic acid inhibited the activity of the ESBLs. This test is simple enough to be easily integrated into routine diagnostic laboratories and has the potential to greatly simplify the detection of AmpC ß-lactamases in E. coli and K. pneumoniae.
Acknowledgements
We would like to thank Dr G. Jacoby, Lahey Clinic, Burlington, USA, for supplying control strains and Professor P. Hawkey and Dr J. Xiong, University of Birmingham, Birmingham, UK for their help.
References
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