Journal of Antimicrobial Chemotherapy (2000) 45, 881-885
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Detection of extended-spectrum ß-lactamases in members of the family Enterobacteriaceae: comparison of the MAST DD test, the double disc and the Etest ESBL
Division of Microbiology, University of Leeds, Leeds LS2 9JT, UK
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Abstract |
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 Top Abstract IntroductionMaterial and methodsResultsDiscussionReferences |
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A technically simple method—the MAST double disc (MDD) test—for the detection of extended-spectrum ß-lactamase (ESBL) production by bacteria is described. A wide range of ESBL, non-ESBL and Class 1 ß-lactamase-producing isolates was examined. The MDD test, which uses discs containing ceftazidime and a complementary disc containing ceftazidime and clavulanate and a second pair containing cefotaxime and cefotaxime and clavulanate was compared with the standard double disc diffusion test and an Etest method. Both the Etest and the MDD correctly identified 93% of ESBL producers. The MDD is an inexpensive alternative to current methods for the detection of ESBL production.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Extended spectrum ß-lactams are commonly included in empirical antibiotic regimens for the treatment of Gram-negative sepsis. The emergence of extended-spectrum ß-lactamase (ESBL)-producing bacteria poses a serious threat to the continued use of this family of antibiotics.1,2 Current disc diffusion and automated susceptibility test methods do not reliably detect ESBL production. Other techniques such as polymerase chain reaction (PCR), PCR–restriction fragment length polymorphism and direct nucleotide sequencing, which demonstrate ESBL production, are of variable sensitivity and may be time consuming, expensive or technically difficult to perform. There is a need for an easy, rapid and reproducible method for the detection of ESBLs, suitable for use in the routine diagnostic laboratory. Additionally, if the method could utilize the same methodology as antimicrobial sensitivity testing, the use of an extra one or two discs only would enable all clinical isolates to be screened during routine sensitivity testing. A novel method of ESBL detection, which satisfies these criteria, is described and compared with two other diffusion methods in current use.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Antimicrobial agents
Antibiotic discs (cefotaxime 30 µg, cefotaxime 30 µg plus clavulanic acid 10 µg, ceftazidime30 µg, ceftazidime 30 µg plus clavulanic acid 10 µg, amoxycillin 30 µg plus clavulanic acid 10 µg) were supplied by MAST Laboratories Ltd (Bootle, Merseyside, UK). The Etest strips were from Biodisk (Solna, Sweden).
Bacterial strains
A total of 100 epidemiologically distinct strains including ESBL and non-ESBL derepressed Class 1 ß-lactamase-producing strains and reference strains, were used in this study. The ß-lactamases were characterized using a combination of PCR to detect blaTEM, blaSHV, blaBIL and blaPER, iso-electric focusing, substrate profiles and in some cases DNA sequencing. The blaSHV amplimer was further characterized by polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP).3
Methods
Isolates were grown overnight on Iso-Sensitest agar (Unipath, Basingstoke, UK) plates at 37°C in air and then resuspended in 0.9% saline to give an inoculum density equivalent to 0.5 McFarland. Zone diameters of each combination were compared with those of each antibiotic alone. A ratio was calculated using the formula:
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A ratio of 1.5 or greater was taken to signify the presence of ESBL activity. This MAST double disc diffusion (MDD) test was then compared with the double disc diffusion (DDD) test described previously1 and the commercially available Etest.4 All tests were prepared by one individual and read independently by another who was blinded to the identity of each strain.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The sensitivity of the MDD test for detecting ESBLs using discs containing ceftazidime is 86%. When using cefotaxime, the sensitivity is 65.5%. The sensitivity is increased to 93% if the results obtained using both agents are taken into consideration (Table I
). From the data, it appears that the use of both cefotaxime and ceftazidime MAST discs is important as ESBLs can be missed if just a single disc is used (Tables I and II). The clinical strains producing SHV-6 ESBL were not detected by either the DDD or the MDD test. The strains appeared to be both cefotaxime and ceftazidime sensitive and therefore there was no detectable increase in zone diameter. The SHV-6 ESBL is unusual, in that the substrate profile is fully susceptible to cefotaxime and only slightly resistant to ceftazidime.5 As expected, the AmpC-type ß-lactamase producers gave negative results with all three tests. All three tests failed to detect the plasmid-mediated BIL-1 enzyme when present either on its own, or with an ESBL in the same host. This was expected, as BIL-1 producers are resistant to inhibition by ß-lactamase inhibitors such as clavulanic acid.
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The work described here attempts to address some of the problems associated with the detection of ESBL-producing strains in the clinical laboratory. Although molecular methods appear sensitive6 they are expensive, time consuming, and require specialized equipment and expertise. The standard DDD test requires careful spacing of discs for accurate results. The limitations of this test have been described.7,8 More recently, the commercially available Etest has been proposed as a simple technique for the detection of ESBL production.4
In this study, all tests showed high sensitivity for detection of ESBLs. However, the Etest requires an extra agar plate to be inoculated to enable the augmentation phenomenon to be seen and therefore cannot easily be incorporated into a routine disc testing system. It is also comparatively expensive. The DDD test uses just three discs but these have to be placed on a separate plate. Assuming that a laboratory is currently testing ceftazidime and cefotaxime with discs, the MDD only requires two discs to be added to the sensitivity plate, thus enabling all Gram-negative bacteria to be screened in the diagnostic laboratory.
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Acknowledgments |
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We are grateful to Professor P. Bradford, Professor G. Jacoby, Professor M. H. Nicolas-Chanoine, and Professor D. Collatz and Dr Haluk Vahaboglu for providing the strains that produce the standard SHV, TEM and PER-1 ß-lactamases and to Dr A. Ait-Ameur (Nedir Hospital, Algeria), Dr Jian-Hui Xiong (First Municipal People's Hospital of Guangzhou, The People's Republic of China) and Aroonlug Luilitanond (Srinagarind Hospital, Khon Kaen, Thailand) for providing the clinical strains.
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Notes |
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* Corresponding author. Tel: +44-113-233-5597; Fax: +44-113-233-5649; E-mail: michfm{at}leeds.ac.uk
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References |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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1 . Jarlier, V., Nicolas, M. H., Fournier, G. & Philippon, A. (1988). Extended broad-spectrum ß-lactamases conferring transferable resistance to newer ß-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility pattern. Reviews of Infectious Diseases 10, 867–8.[Web of Science][Medline]
2 . Livermore, D. M. (1995). ß-lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]
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M'Zali, F. H., Gascoyne-Binzi, D. M., Heritage, J. & Hawkey, P. M. (1996). Detection of mutations conferring extended-spectrum activity on SHV ß-lactamases using polymerase chain reaction single strand conformational polymorphism (PCR–SSCP). Journal of Antimicrobial Chemotherapy 37, 797–802.
4 . Bolmström, A., Karlsson, A. & Mills, K. (1995). Detection of ESBLs using a new test strip. Proceedings of the Seventh European Congress of Clinical Microbiology and Infectious Diseases, Vienna, Austria.
5 . Arlet, G., Rouveau, M., Bengoufa, D., Nicolas, M. H. & Philippon, A. (1991). Novel transferable extended-spectrum ß-lactamase (SHV-6) from Klebsiella pneumoniae conferring selective resistance to ceftazidime. FEMS Microbiology Letters 65, 57–62.[Medline]
6 . Nuesch-Inderbinen, M. T., Hachler, H. & Kayser, F. H. (1996). Detection of genes coding for extended-spectrum SHV betalactamases in clinical isolates by a molecular genetic method and comparison with the Etest. European Journal of Clinical Microbiology and Infectious Diseases 15, 398–402.[Web of Science][Medline]
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Thomson, K. S. & Sanders, C. C. (1992). Detection of extended spectrum ß-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three dimensional tests. Antimicrobial Agents and Chemotherapy 36, 1877–82.
8 . Bush, K. (1996). Is it important to identify extended-spectrum beta-lactamase-producing isolates? European Journal of Clinical Microbiology and Infectious Diseases 15, 361–4.[Web of Science][Medline]
Received 13 July 1999; returned 8 November 1999; revised 24 December 1999; accepted 24 January 2000
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