The Clinical Microbiology Institute, 9725 SW Commerce Circle, Suite A-1, Wilsonville, OR 97070, USA
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Abstract |
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Introduction |
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The in vitro activity of daptomycin has been shown to be significantly affected by the concentration of calcium in the test medium.2,7,8 For broth dilution susceptibility testing of daptomycin it has been recommended to supplement the MuellerHinton broth to a physiological concentration of 50 mg/L of calcium.7 Batches of MuellerHinton agar (MHA) currently available may vary considerably in calcium content and daptomycin disc diffusion tests on MHA with low calcium levels produce significantly smaller inhibitory zone diameters than tests on MHA with c. 25 mg/L of calcium.7 The daptomycin disc diffusion interpretive criteria and quality control (QC) limits proposed recently were based on tests performed on MHA with c. 25 mg/L of calcium.7
The present study was designed to: (i) test the performance of daptomycin Etest strips on MHA with c. 25 mg/L of calcium in comparison with broth microdilution tests as the standard, and (ii) evaluate the effect of calcium concentration in the medium on Etest results by performing tests on 12 different batches of agar media that were available for susceptibility tests at that time. Calcium and magnesium concentrations in all 12 batches were measured and found to be variable.
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Materials and methods |
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The 195 Gram-positive bacterial isolates represented 17 species, which are listed in Table 1. From these a subset of 20 isolates representing eight species was selected for Etests on 12 different batches of media10 organisms each on media with and without 5% sheep blood.
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Daptomycin standardized powder and daptomycin Etest strips (AB Biodisk, Solna, Sweden) were provided by Cubist Pharmaceuticals, Inc. (Cambridge, MA, USA).
Media
For broth microdilution tests, cation-adjusted Mueller Hinton broth (Difco Laboratories, Detroit, MI, USA) was further supplemented to contain 50 mg/L of calcium. For testing fastidious species such as streptococci, 3% lysed horse blood was also added. Etests were performed on a batch of MHA with 24 mg/L of calcium, which was supplemented with 5% defibrinated sheep blood when testing fastidious organisms. For the multiple batch study, 11 batches of agar media with blood and 11 batches of agar without blood were compared. These included three batches of MHA produced by Becton Dickinson (Cockeysville, MD, USA), two batches each of MHA produced by Difco Laboratories, Accumedia (now Neogen, Baltimore, MD, USA) and Remel (Lenexa, KS, USA), one batch of MHA produced by Oxoid (Basingstoke, UK) and Criterion (Santa Monica, CA, USA) and one batch of IsoSensitest agar produced by Oxoid.
Susceptibility tests
Broth microdilution tests were performed by the method outlined by the NCCLS.9 The concentrations of daptomycin tested represented 0.5 two-fold dilution intervals ranging from 16 to 0.015 mg/L. For Etest procedures, the manufacturer's instructions were followed.
Quality control
The following QC strains were tested at the same time as the test strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Streptococcus pneumoniae ATCC 49619. Calcium and magnesium levels were assayed (Beckman/Coulter Syncron CX7 with Arsenazo III as dye indicator) for each batch of media at the Chemistry Laboratory (Peter Anderson, Director) of Providence St Vincent Medical Center (Portland, OR, USA).
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Results |
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For individual isolates the Etest MICs varied from 1.5 two-fold concentrations below to three two-fold concentrations above the broth microdilution MIC. Despite the clear tendency toward higher daptomycin MICs by Etest, 84% of Etest results were within one two-fold dilution of the broth microdilution MIC.
The concentrations of calcium and magnesium in the test media are listed in Table 2. Etest MICs correlated inversely (R = 0.76) with the calcium concentration of the agar medium. Calcium concentrations ranged from 4 to 36 mg/L and the corresponding geometric mean daptomycin Etest MICs ranged from 11.7 to 0.44 mg/L for the 10 non-fastidious strains and 3.63 to 0.28 mg/L for the 10 strains tested on blood-containing media. Because of the inverse correlation (R = 0.70) of calcium and magnesium concentrations in the different agars, a modest positive correlation (R = +0.60) between magnesium concentration and daptomycin MICs was not unexpected. In general, MHA (with or without added sheep blood) containing >20 mg/L of calcium yielded daptomycin Etest MICs within one two-fold concentration of the broth microdilution MICs. Etest MICs on media with
20 mg/L of calcium were more than double broth microdilution MICs. The batch of IsoSensitest agar (a medium more commonly used in Europe) that we evaluated was also deficient in calcium and gave Etest daptomycin MICs more than eight-fold higher than broth microdilution MICs.
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Discussion |
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Although the calcium concentration in MuellerHinton broth can be standardized and/or controlled with relative ease, incorporation into agar is more difficult to adjust. The calcium and magnesium content of current batches of MHA vary considerably (Table 2) and the concentration of calcium has been shown to have a significant effect on daptomycin disc diffusion zone diameters.7 Most currently available batches of MHA in the USA conform to the manufacturer's performance test limits as published by the NCCLS.10 However, that performance test does not include daptomycin discs, since it is still an investigational drug. To further judge cation content of the 12 batches of agar, 10 µg gentamicin discs were tested in duplicate against Pseudomonas aeruginosa ATCC 27853. For all but one batch of MHA the zone diameters were within the control range. Only batch 2 (total cation content 22 mg/L) and the only batch of IsoSensitest agar yielded results outside the control range. If and when daptomycin is approved, it will be important that media manufacturers resolve this problem as quickly as possible.
In the current study we have demonstrated that the calcium effect is also very pronounced with daptomycin Etests. We are unaware of other instances in which media variations have had such a significant effect on Etest MICs. Until MHA becomes standardized with respect to calcium concentration, this will be a challenge for the Etest manufacturer as well as Etest users. Although the number of QC tests in this study is too small to be conclusive, they do indicate that the QC strains S. aureus ATCC 29213 and E. faecalis ATCC 29212 may be used to identify media with insufficient calcium. The daptomycin MIC QC ranges proposed for broth microdilution tests appear to work satisfactorily for Etests on MHA with >20 mg/L of calcium. Routine testing of these QC strains will be important for Etest users who test daptomycin susceptibility of clinical isolates.
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Acknowledgements |
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Notes |
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References |
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6 . Tally, F. P., Zeckel, M., Wasilewski, M. M., Carini, C., Berman, C. L., Drusano, G. L. et al. (1999). Daptomycin: a novel agent for Gram-positive infections. Expert Opinion in Investigational Drugs 8, 122338.
7 . 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, 518.[ISI][Medline]
8 . Jones, R. N. (1989). Effects of reduced cation supplement recommendations (National Committee for Clinical Laboratory Standards) on daptomycin antistaphylococcal activity. Journal of Antimicrobial Chemotherapy 33, 16523.
9 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Wayne, PA.
10 . National Committee for Clinical Laboratory Standards. (1996). Protocols for Evaluating Dehydrated MuellerHinton Agar: Approved Standard M6-A. NCCLS, Wayne, PA.
Received 1 February 2001; returned 21 May 2001; revised 29 June 2001; accepted 19 July 2001