Institut für Infektionsmedizin, Zentrum für Klinisch-Theoretische Medizin I, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany
Received 20 February 2004; returned 31 March 2004; revised 13 April 2004; accepted 14 April 2004
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Abstract |
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Methods: Overall, a total of 54 clinical isolates of ESBL-producing Enterobacteriaceae species were evaluated: Enterobacter aerogenes (n=3), Enterobacter cloacae (n=10), Escherichia coli (n=10), Klebsiella oxytoca (n=3), Klebsiella pneumoniae (n=25) and Proteus mirabilis (n=3). To check Etest behaviour with resistance phenotypes similar to ESBL, our panel was expanded by six clinical isolates of K. oxytoca that were identified as putative producers of their chromosomal K1 ß-lactamase.
Results: With this panel, ESBL Etest was 98% sensitive with cefepimeclavulanate, 83% with cefotaximeclavulanate, and 74% with ceftazidimeclavulanate strips. Concentrating on Enterobacter spp., reliable ESBL detection could only be achieved by the new cefepimeclavulanate strip since it confirmed ESBL production in all strains (100% sensitivity) whereas only 4/13 (31%) of Enterobacter strains were positive using cefotaximeclavulanate or ceftazidimeclavulanate strips. A limitation of using the new cefepime strip was less than optimal specificity with K1 phenotypes of K. oxytoca: among six strains, four isolates were scored false-positive by Etest strips containing cefepimeclavulanate.
Conclusion: The new Etest ESBL strip containing cefepimeclavulanate is a valuable supplement to current methods for detection of ESBLs. In our study collection, the cefepimeclavulanate strip was the best configuration for detection of ESBLs, particularly in Enterobacter spp.
Keywords: ESBL detection , Etest methodology
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Introduction |
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In this study, we evaluated the performance of the new cefepimeclavulanate Etest configuration with the other two existing strips in a collection of 54 clinical ESBL isolates, with special focus on Enterobacter spp. Furthermore, in order to gain an impression of how well the newly available Etest strip would perform with a resistant phenotype very similar to ESBL, we expanded our panel of test strains with six clinical K. oxytoca isolates that were identified as putative hyperproducers of their chromosomal K1 enzymes. Like TEM-, SHV- or CTX-M ESBLs, K1 ß-lactamase is an Ambler class A enzyme too and is naturally susceptible to inhibition by clavulanate.
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Materials and methods |
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The bacterial isolates selected for this study included 54 clinical non-duplicate ESBL isolates, the majority of which were collected and characterized in a previous evaluation study.17 A few other ESBL strains were sent from the laboratories acknowledged. The organisms were considered ESBL-positive when TEM-, SHV-, or CTX-M related ESBL genes had been identified by PCR and DNA sequencing and when a reduction of at least eight-fold in the MICs of either ceftazidime or cefotaxime in the presence of clavulanic acid had been detected, using broth microdilution according to the NCCLS.18,19 The clinical isolates consisted of E. aerogenes (n=3), E. cloacae (n=10), E. coli (n=10), K. oxytoca (n=3), K. pneumoniae (n=25) and P. mirabilis (n=3). The E. aerogenes strains analysed harboured CTX-M-1 (n=2) and SHV-5 (n=1) ESBLs. The 10 non-repetitive isolates of E. cloacae, which were isolated over a period of 2 years, included TEM type (ABL: A184V) (n=7) and SHV-12 (n=3) ESBLs. The E. coli strains included SHV-2 (n=1), SHV-5 (n=1), SHV-12 (n=3), TEM-26 (n=1), TEM-52 (n=1), TEM-111 (n=1), CTX-M-1 (n=1) and CTX-M-23 (n=1). The K. oxytoca isolates harboured CTX-M-1 (n=2) and SHV-12 (n=1). The P. mirabilis strains included CTX-M-1 (n=1), CTX-M-22 (n=1) and TEM-92 (n=1). The K. pneumoniae strains harboured SHV-2 (n=5), SHV-2a (n=1), SHV-5 (n=4), SHV-12 (n=11), SHV-19 (n=1), LEN type (ABL: N53S, A201P, P218A) (n=1), TEM-47 (n=1) and TEM-110 (n=1). The two NCCLS recommended ESBL test quality control strains ATCC 25922 E. coli (negative control) and ATCC 700603 K. pneumoniae (ESBL positive, SHV-18) were also included in all experiments. To check Etest behaviour with resistance phenotypes very similar to ESBL, our panel was expanded by clinical K. oxytoca isolates (six strains) that were identified as putative hyperproducers of their K1 chromosomal ß-lactamase, based on susceptibility to ceftazidime (MIC8 mg/L) but resistance to cefuroxime (MIC
32 mg/L) and negative results with the blaSHV, blaTEM and blaCTX-M PCR assays.
Etest procedure, reading and interpretation
The Etest ESBL strip (AB Biodisk, Solna, Sweden) is a plastic drug-impregnated strip, one end of which generates a stable concentration gradient of ceftazidime (MIC test range, 0.532 mg/L) and the remaining end of which generates a gradient of ceftazidime (MIC test range, 0.0644 mg/L) plus 4 mg/L clavulanic acid. An Etest ESBL strip containing cefotaxime (MIC test range, 0.2516 mg/L) and cefotaxime (MIC test range, 0.0161 mg/L) plus 4 mg/L clavulanic acid is also available. Very recently, a third strip for ESBL detection containing cefepime (MIC test range, 0.2516 mg/L) and cefepime (MIC test range, 0.0644 mg/L) plus 4 mg/L clavulanic acid has been produced. The Etest procedure, reading and interpretation were carried out according to the manufacturer's instructions. Isolated colonies from an overnight agar plate were suspended in saline (0.85% NaCl) to achieve an inoculum equivalent to 0.5 McFarland Standard. This suspension was swabbed on a MuellerHinton agar plate (Oxoid, UK) and allowed to dry completely. An ESBL Etest strip was then applied to the agar surface with sterile forceps and the plate was incubated at 36°C for 18 h. ESBL results were read either as MIC values or observation of phantom zones or deformation of inhibition ellipses. Reduction of MIC by 3 two-fold dilutions in the presence of clavulanic acid is indicative of ESBL production. Deformation of ellipses or the presence of a phantom zone is also indicative of ESBL production even if the MIC ratio is <8 or cannot be read.
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Results |
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Discussion |
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In line with previous reports,20 our study confirmed that neither ceftazidimeclavulanate nor cefotaximeclavulanate strips were suited for detecting ESBL in Enterobacter species. The cefepimeclavulanate strip was the only configuration that enabled accurate detection (100%) within this group of organisms, where inducible chromosomal AmpC ß-lactamase can interfere with clavulanate synergy. Detection of ESBL production among such strains is known to be challenging,14,21 and false-negative results using cefotaxime or ceftazidime have also been described in other studies.22,23 A limitation using the new cefepimeclavulanate strip was less than optimal specificity with resistant phenotypes of K. oxytoca. As demonstrated, cefepimeclavulanate strips as well as cefotaximeclavulanate strips were considerably susceptible to false-positive results with strains hyperproducing their chromosomal K1 ß-lactamase but lacking ESBL. Careless use of the new strip may thus result in an unacceptably high number of false-positive results. In order to assess the ESBL status in K. oxytoca, it might be more prudent to consider the overall susceptibility profile rather than depending solely on singular Etest results. K1 hyperproducer organisms have a typical pattern of resistance, being consistently resistant to cefuroxime and aztreonam, having borderline resistance to cefotaxime and cefepime, but remaining fully susceptible to ceftazidime (as shown here with the ceftazidime ESBL Etest).21
In conclusion, today's most commonly used practice to confirm ESBL enzymes by carrying out clavulanate synergy tests with ceftazidime, cefotaxime or cefpodoxime may no longer be sufficient in populations with a high prevalence of ESBL-producing Enterobacter species. For such situations, where inducible chromosomal AmpC ß-lactamase can interfere with clavulanate synergy from a co-existing ESBL, the new cefepimeclavulanate strip could be a more sensitive alternative. Finally, facing the growing complexity of ESBLs, it becomes more and more apparent that regular species identification forms the base on which accurate ESBL detection should be built.
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Acknowledgements |
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Footnotes |
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References |
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