Calcium-supplemented daptomycin Etest strips for susceptibility testing on Iso-Sensitest agar

Alan P. Johnson*, Shazad Mushtaq, Marina Warner and David M. Livermore

Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency, Specialist and Reference Microbiology Division, Colindale, London NW9 5HT, UK

Received 27 January 2004; returned 12 February 2004; revised 16 February 2004; accepted 17 February 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: Iso-Sensitest agar (ISA), which is recommended by the BSAC for routine susceptibility testing of staphylococci and enterococci, contains insufficient calcium for testing daptomycin. Isotonic agar supplemented with 50 mg/L calcium has been advocated, but is not routinely available in many laboratories. We evaluated a daptomycin Etest that incorporates a constant level of calcium throughout the daptomycin gradient, designed to give an appropriate concentration around the strip during testing, as an alternative for susceptibility testing on ISA.

Methods: Ninety-one isolates of Staphylococcus aureus (45 methicillin-susceptible, 46 methicillin-resistant) and 90 enterococci (47 Enterococcus faecalis, 43 Enterococcus faecium) were tested. Daptomycin Etest MICs were determined on ISA, whereas agar dilution MICs were determined in parallel on Isotonic agar supplemented with calcium to 50 mg/L as a control.

Results: The agar dilution and Etest MIC ranges of daptomycin for S. aureus were 0.25–1 mg/L (mode 0.5 mg/L), and 0.125–2 mg/L (mode 0.25 mg/L), respectively. The corresponding MIC values for enterococci were 0.25–4 mg/L (mode, 1 mg/L) and 0.125–4 mg/L (mode, 2 mg/L). For staphylococci, 86% of the Etest MIC results were within one dilution of the agar dilution values, and for enterococci, 90% of the Etest MIC results met these criteria. When results from the two methods were not identical, there was a tendency for the Etest MIC values to be lower than the agar dilution values.

Conclusions: This study shows that calcium-supplemented daptomycin Etests on ISA are an accurate and convenient alternative to calcium-supplemented Isotonic agar.

Keywords: lipopeptides, enterococci, Staphylococcus aureus, MRSA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, has potent activity against most Gram-positive bacteria,1 and has recently received approval by the US Food and Drug Administration for the treatment of complicated skin and skin structure infections.2 Should daptomycin subsequently be licensed for clinical use in the UK, microbiology laboratories will need to include it among the antibiotics they test for activity against Gram-positive pathogens. However, testing is problematic, as daptomycin requires the use of medium containing calcium at 50 mg/L for optimal activity, and levels of calcium vary between different media and batches.35

A particular concern is that Iso-Sensitest agar (ISA), which is recommended for routine susceptibility testing by the British Society for Antimicrobial Chemotherapy (BSAC), is unsuitable for testing daptomycin, as the calcium content is insufficient.4 Although Isotonic agar supplemented with calcium to 50 mg/L has been evaluated and found suitable for use with daptomycin,4 the requirement to test daptomycin on a different medium from that used with other antibiotics is inconvenient at best. As an alternative, AB Biodisk (Solna, Sweden) has developed a daptomycin Etest strip that incorporates calcium at a constant level throughout the daptomycin gradient, designed to give an appropriate concentration around the strip during testing. The strip was developed by determining the amount of calcium that provided good correlation between the NCCLS reference method (broth microdilution with 50 mg/L calcium) and the strip result. We evaluated this Etest product for determining MICs for Staphylococcus aureus and enterococci on ISA. For comparison, daptomycin MICs were determined in parallel by incorporation in Isotonic agar with Ca2+ at 50 mg/L.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

Ninety-one isolates of S. aureus (45 methicillin-susceptible, 46 methicillin-resistant) and 90 enterococci (47 Enterococcus faecalis, 43 Enterococcus faecium) from geographically diverse UK hospitals were tested.

Susceptibility testing

Daptomycin powder and calcium-supplemented daptomycin Etest strips were provided by Cubist Pharmaceuticals (Lexington, MA, USA). Agar dilution MICs of daptomycin were determined on Isotonic agar supplemented with Ca2+ to 50 mg/L (Mast Laboratories, Bootle, UK). Etest MICs were determined on Iso-Sensitest agar (Oxoid, Basingstoke, UK). Intermediate MIC values were rounded up to the nearest doubling dilution concentration.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As shown in Figure 1, the agar dilution MICs of daptomycin for S. aureus were in the range 0.25–1 mg/L (mode 0.5 mg/L), whereas the Etest MICs were in the range 0.125–2 mg/L (mode, 0.25 mg/L). For enterococci, the corresponding values were 0.25–4 mg/L (mode, 1 mg/L) and 0.125–4 mg/L (mode, 2 mg/L).



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Figure 1. Distribution of daptomycin MICs for S. aureus and enterococci obtained by agar dilution (filled bars) and by Etest (open bars).

 
The concordance between the MICs of daptomycin obtained by agar dilution and Etest for individual isolates is shown in Figure 2. With staphylococci, 86% of the Etest MIC results were either the same as (36%) or within one two-fold dilution (50%) of the agar dilution values, with the remainder of the isolates (14%) showing a four-fold difference in MIC between the two methods. For enterococci, 90% of the Etest MIC results were either identical to the agar dilution values (53.3%) or within one two-fold dilution (36.7%). Among seven enterococcal isolates (8%) showing a greater level of discrepancy, six showed a four-fold difference in MIC between the two methods, whereas one showed an eight-fold difference. For those results that were not concordant, there was a tendency, seen with both staphylococci and enterococci, for the Etest values to be lower than the agar dilution MIC values.



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Figure 2. Correlation between the MICs of daptomycin obtained by agar dilution and Etest for individual isolates of S. aureus and enterococci. The numbers of isolates with indicated agar dilution and Etest MIC values are shown. Isolates for which the Etest MIC results were the same as the agar dilution values are indicated by dark shading, whereas isolates for which the Etest MIC results were within a two-fold dilution of the agar dilution values are indicated by light shading.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Studies by several groups have shown that testing for susceptibility to daptomycin requires the use of medium containing calcium at 50 mg/L for optimal activity.35 ISA, which is recommended for routine susceptibility testing by the BSAC, is unsuitable for testing daptomycin due to the low concentration of calcium.4 Although Isotonic agar in a commercially available formulation supplemented with calcium is suitable, it is inconvenient to have to routinely stock or source two different media for susceptibility testing, when daptomycin is to be tested alongside other agents.

This pilot study indicated that calcium-supplemented daptomycin Etest strips could be used on ISA to test staphylococci and enterococci for susceptibility to daptomycin. For 86% of the S. aureus isolates and 90% of the enterococci tested, the MIC values obtained by agar dilution on Isotonic agar supplemented with calcium, and calcium-supplemented Etests on ISA, were within one doubling dilution of each other. With only one of 181 isolates tested was the difference in MIC greater than four-fold.


    Acknowledgements
 
We thank Cubist Pharmaceuticals for financial support. These data were presented at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA.


    Footnotes
 
* Corresponding author. Present address: Division of Healthcare-Associated Infection and Antimicrobial Resistance, Communicable Disease Surveillance Centre, Colindale, London NW9 5EQ, UK. Tel: +44-20-8200-6868, ext. 3043; Fax: +44-20-8205-9185; E-mail: Alan.Johnson{at}hpa.org.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Thorne, G. M. & Alder, J. (2002). Daptomycin: a novel lipopeptide antibiotic. Clinical Microbiology Newsletter 24, 33–40.[CrossRef]

2 . US Food and Drug Administration. (2003). FDA approves first in new class of antibiotics. [Online.] http://www.fda.gov/bbs/topics/ANSWERS/2003/ANS01252.html (13 January 2004, date last accessed).

3 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (2000). Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests. Diagnostic Microbiology and Infectious Disease 38, 51–8.[CrossRef][ISI][Medline]

4 . King, A. & Phillips, I. (2001). The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates. Journal of Antimicrobial Chemotherapy 48, 219–23.[Abstract/Free Full Text]

5 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (2001). Evaluation of daptomycin susceptibility testing by Etest and the effect of different batches of media. Journal of Antimicrobial Chemotherapy 48, 557–61.[Abstract/Free Full Text]





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