False-positive extended-spectrum ß-lactamase tests for Klebsiella oxytoca strains hyperproducing K1 ß-lactamase

Nicola A. C. Potz1,*, Melissa Colman1, Marina Warner1, Rosy Reynolds2 and David M. Livermore1

1 Antibiotic Resistance Monitoring & Reference Laboratory, Specialist & Reference Microbiology Division, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT; 2 British Society for Antimicrobial Chemotherapy, 11 The Wharf, 16 Bridge Street, Birmingham B1 2JS, UK

Keywords: interpretation of ESBL test results, ceftazidime, cefotaxime, cefepime, cefpodoxime

Sir,

The growing complexity of extended-spectrum ß-lactamases (ESBLs) presents new detection challenges. Until recently, the predominant ESBLs in the UK were TEM- and SHV-derived ceftazidimases, occurring mostly in Klebsiella spp. On this basis it seemed adequate to screen with ceftazidime and to perform ceftazidime–clavulanate synergy tests on Enterobacteriaceae found to be resistant. Now, however, CTX-M ESBLs are spreading and ESBLs are reaching genera, principally Enterobacter, where inducible chromosomal AmpC ß-lactamases can interfere with clavulanate synergy tests. To detect CTX-M as well as TEM and SHV ESBLs, laboratories are advised to screen with cefotaxime as well as ceftazidime, or to use cefpodoxime,1 which is a good substrate for all ESBLs. Resistant isolates should then have a clavulanate synergy test performed, using whatever cephalosporin(s) they were resistant to. Alternatively, cefepime–clavulanate synergy is proposed as a general indicator of ESBL production, applicable even for Enterobacter spp.2

During the 2002 BSAC Bacteraemia Resistance Surveillance Programme (http://www.bsacsurv.org) and recent reference work, we have screened Enterobacteriaceae with cefotaxime and ceftazidime, and performed ceftazidime–clavulanate, cefotaxime–clavulanate and cefepime–clavulanate synergy tests on those found to be resistant. We observed ostensibly positive results (eight-fold potentiation or higher) with cefotaxime and cefepime, rarely ceftazidime, for Klebsiella oxytoca isolates with only borderline resistance to these cephalosporins (MICs 2–8 mg/L) but with high-level resistance to cefuroxime and piperacillin–tazobactam. This antibiogram suggests hyperproduction of K1 (KOXY) chromosomal ß-lactamase rather than ESBL production, and we therefore assessed the behaviour of known K1 hyperproducers in multiple tests now advocated for ESBL production.

Twenty-five K1 ß-lactamase-producing K. oxytoca strains were tested; in addition, five ESBL-positive Klebsiella pneumoniae were used as controls to confirm ESBL test performance. Agar dilution MIC tests were carried out using BSAC methodology,3 with ESBL production defined by a cephalosporin:cephalosporin + clavulanate (4 mg/L) MIC ratio >=8. Antibiotic powders were from suppliers as follows: cefotaxime (Aventis, West Malling, UK), cefepime (Bristol-Myers Squibb, Hounslow, UK), and ceftazidime and clavulanate (GlaxoSmithKline, Welwyn Garden City, UK). Cephalosporin–clavulanate ESBL Etests (AB Biodisk, Solna, Sweden) were used on Mueller–Hinton agar and with inocula as per the manufacturer’s instructions. Once again, an MIC ratio of >=8 was taken as a general indicator of ESBL production, but isolates were also scored as positive if ‘phantom’ or deformed ellipses were seen, as advised by the manufacturer. Disc tests with cefpodoxime ± clavulanate (10 µg ± 1 µg) combination discs (Oxoid, Basingstoke, UK) were carried out using BSAC methodology,4 and organisms were considered positive for ESBL production if the clavulanate enlarged the zone diameter by >=5 mm.

Among the 25 K. oxytoca isolates hyperproducing K1 ß-lactamase but lacking ESBLs, 20 gave MIC ratios >=8 with cefotaxime–clavulanate by agar dilution and 17 gave ratios >=8 by Etest. Similar behaviour was seen with cefepime: 21/25 isolates gave ratios >=8 by agar dilution and 18/25 by Etest. Only one isolate gave a ratio >=8 by agar dilution with ceftazidime–clavulanate, and none did so by Etest. Using the combination discs, the zone diameter differences between the cefpodoxime discs with and without clavulanate were consistently <=3.5 mm for the K1 hyperproducers. In comparison, the K. pneumoniae ESBL-positive controls consistently gave ratios of >=8 for all cephalosporin–clavulanate synergy-based ESBL tests, whether by Etest or agar dilution, and zone diameter differences of >15 mm in the cefpodoxime combination disc tests.

This analysis confirmed the suspicion, initially formed during testing for the BSAC Bacteraemia Programme, that hyperproducers of K1 ß-lactamase often give apparently positive ESBL tests, by both agar dilution and Etest, with cefotaxime–clavulanate and cefepime–clavulanate combinations. The ceftazidime–clavulanate combination escaped this problem, but is unreliable for the detection of CTX-M ESBLs. False-positive ESBL results with K1 ß-lactamase hyperproducing K. oxytoca isolates (Table 1) have been shown previously with the VITEK2 system and (as here) with the cefotaxime ESBL Etest.5,6


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Table 1. ESBL test results for the Klebsiella isolates studied 
 
Cefpodoxime–clavulanate combination disc tests were reliable in distinguishing ESBL producers from hyperproducers of K1 enzyme, but are unlikely to be ideal for AmpC-inducible organisms, e.g. Enterobacter spp. Herein is the key point: as ESBLs become more complex, diverse and widespread, the likelihood of any single test being universally appropriate for their detection must diminish. Rather than depending solely on such tests, the best chance of accurate ESBL detection lies in considering the overall MIC profile together with the results of any ‘specific’ ESBL test. Hyperproducers of K1 ß-lactamase are not difficult to distinguish from ESBL producers, being consistently resistant to cefuroxime, piperacillin–tazobactam and aztreonam, having borderline resistance to cefotaxime and cefepime, but remaining fully susceptible to ceftazidime. This is not the typical pattern for any ESBL. Hyperproducers of K1 enzyme are only likely to be mistaken for ESBL producers if ‘ESBL tests’ are naively taken as the sole criterion for defining ESBL production, or if inadequately narrow batteries of antibiotics are tested.

Footnotes

* Corresponding author. Tel: +44-20-8200-4400 ext. 8493; Fax: +44-20-8358-3292; E-mail: Nicola.Potz{at}hpa.org.uk Back

References

1 . Andrews, J. (2003). Detection of extended-spectrum ß-lactamases (ESBLs) in E. coli and Klebsiella species. [Online.] http://www.bsac.org.uk/uploads/Ecoliklebsiella.pdf (12 December 2003, date last accessed).

2 . Thomson, K. S. (2001). Controversies about extended-spectrum and AmpC ß-lactamases. Emerging Infectious Diseases 7, 333–6.[ISI][Medline]

3 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract/Free Full Text]

4 . Livermore, D. M. & Brown, D. F. J. (2001). Detection of ß-lactamase-mediated resistance. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 59–64.[Abstract/Free Full Text]

5 . Midolo, P. D., Matthews, D., Fernandez, C. D. et al. (2002). Detection of extended spectrum ß-lactamases in the routine clinical microbiology laboratory. Pathology 34, 362–4.[CrossRef][ISI][Medline]

6 . Sturenburg, E., Sobottka, I., Feucht, H.-H. et al. (2003). Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum ß-lactamase detection in Escherichia coli and Klebsiella species clinical isolates. Diagnostic Microbiology and Infectious Disease 45, 29–34.[CrossRef][ISI][Medline]