Department of Infection, KCL, St Thomas' Hospital Campus, London SE1 7EH, UK
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Abstract |
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Introduction |
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Daptomycin was discovered in the early 1980s and was shown to be effective in patients with skin and soft tissue infections; however, because of mild skeletal muscle toxicity at high doses and treatment failures in patients with S. aureus endocarditis,1,3 clinical trials were suspended. Since this time the marked increase in isolation of resistant Gram-positive organisms prompted Cubist Pharmaceuticals to conduct further clinical trials with dosing regimens unlikely to cause toxicity. The precise mechanism of action of daptomycin is not completely understood, but it is know that it kills bacteria by disrupting membrane function; possible mechanisms include inhibition of peptidoglycan synthesis, inhibition of lipotechoic acid synthesis and alterations of cytoplasmic membrane potential.46 The bactericidal activity of daptomycin is concentration-dependent and is influenced by pH and ionized calcium concentration.7
In this study we compared the in vitro activity of daptomycin with that of penicillin and vancomycin against a wide variety of Gram-positive aerobic bacteria, including those known to be resistant to other antibiotics. We also compared results obtained by NCCLS/EUCAST810 and BSAC11,12 methods.
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Materials and methods |
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The following antimicrobial agents were tested as powders of known potency: daptomycin (Cubist Pharmaceuticals, Cambridge, MA, USA), penicillin (SmithKline Beecham, Harlow, UK) and vancomycin (SigmaAldrich Co. Ltd, Poole, UK). Breakpoints for penicillin and vancomycin were as stated either in the NCCLS8,9 or BSAC11,12 methods. Tentative MIC breakpoints for daptomycin are: susceptible 2 mg/L, intermediate 4 mg/L and resistant
8 mg/L; tentative NCCLS zone diameter breakpoints are:
12 mm for resistant and
16 mm for susceptible.13
Organisms
The organisms (n = 514) included in the study were all clinical isolates from St Thomas' Hospital and were selected to represent the different Gram-positive aerobic species isolated from infections. The isolates were further selected to include, where possible, those known to have specific resistance mechanisms including vancomycin, methicillin and erythromycin resistance. For commonly isolated species the isolates were recent but for less common species the isolates were from collections made over the past 10 years.
Susceptibility testing
MICs were determined by an agar dilution method on MuellerHinton agar (Oxoid Ltd, Basingstoke, UK) and Isotonic agar adjusted to contain 50 mg/L Ca2+ supplemented Isotonic agar (Mast Laboratories, Bootle, UK). Both media were enriched with 5% horse blood (E & O Laboratories, Bonnybridge, UK) for fastidious organisms. Organisms were grown overnight in brainheart infusion broth (Oxoid) and diluted in sterile distilled water, or suspended in water directly from a fresh culture to match a 0.5 McFarland turbidity standard. These suspensions were further diluted 1/10 in IsoSensitest broth (Oxoid) and inoculated on the agar with a multipoint inoculator (Denley, Burgess Hill, UK) to give a final inoculum size of c. 104 cfu/spot. The plates were incubated for 2024 h at 37°C in air (with 5% added CO2 for S. pneumoniae).
Disc diffusion susceptibility was determined on either MuellerHinton agar (NCCLS) or supplemented Isotonic agar (BSAC). Both media were enriched with 5% horse blood for fastidious organisms. The inoculum for the NCCLS method was prepared by making a suspension in sterile distilled water to match a 0.5 McFarland turbidity standard. For the BSAC method the inoculum was prepared by diluting the suspension equivalent to 0.5 McFarland standard either 1/10 or 1/100, depending on the species, to produce semi-confluent growth. The disc contents tested were 30 µg daptomycin (BBL, Becton Dickinson, Cockysville, MD, USA) for both methods, 10 µg (NCCLS) or 1 µg (BSAC) penicillin (Oxoid), and 30 µg (NCCLS) or 5 µg (BSAC) vancomycin (Oxoid). Plates were incubated for 2024 h at 37°C in air (with 5% added CO2 for S. pneumoniae). Methicillin susceptibility was determined for all staphylococci by disc diffusion11 with 5 µg discs (Oxoid). The presence of mec(A) in all S. aureus and Staphylococcus epidermidis shown to be resistant by disc testing was confirmed by PCR.14
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Results |
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Correlation between NCCLS and BSAC methods for daptomycin disc diffusion was also good (r = 0.815) but daptomycin zone diameters were 23 mm larger on supplemented Isotonic agar than on MuellerHinton agar because of the smaller inoculum. However, the correlation between MIC and zone diameter was poor for both methods (r = 0.472 NCCLS and 0.437 BSAC), as shown in the Figure. A tentative NCCLS breakpoint of 16 mm is appropriate for the susceptible isolates. However, the two isolates with decreased susceptibility (daptomycin MICs 4 mg/L) had zone diameters >16 mm. Similar discrimination by the BSAC method would be a zone breakpoint of 18 mm, but again the four isolates with decreased susceptibility would not be detected. Correlations between the two methods of MIC determination were very good for penicillin (r = 0.975) and vancomycin (r = 0.988), as were the correlations between the methods for zone diameters of penicillin (r = 0.921) and vancomycin (r = 0.937). Correlations between MICs and zone diameters for both penicillin (r = 0.933 for NCCLS and 0.903 for BSAC) and vancomycin (r = 0.888 for NCCLS and 0.881 for BSAC) were also very good. Species-specific breakpoints for penicillin discriminated well in both methods and there were no errors of interpretation. The breakpoints for vancomycin were likewise species-specific for the NCCLS method but discriminated well, with the exception of one enterococcus, with a vancomycin MIC of 8 mg/L and a zone diameter of 17 mm. The lower disc content of vancomycin for the BSAC method and smaller zone breakpoint discriminated well between susceptible and resistant isolates.
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Discussion |
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The potency of daptomycin is known to be dependent on free calcium content.13 Preliminary results on both Oxoid IsoSensitest agar and Mast Isotonic agar showed that the Ca2+ content was insufficient, resulting in high MICs and small zone diameters. The recommended BSAC media for susceptibility testing were therefore initially considered unsuitable for testing daptomycin. However, Mast Laboratories supplemented Isotonic agar with 50 mg/L Ca2+ and this agar was used for this study. MIC results on supplemented Isotonic agar were similar to those on Mueller Hinton agar and for the majority of isolates were either the same or differed by only one doubling dilution. Where there were differences, MICs were more often higher on supplemented Isotonic agar (mean MIC 0.306 mg/L) than on MuellerHinton (mean MIC 0.201 mg/L). This appears to reflect either the difference in Ca2+ content, another chemical difference in the media or the fact that most isolates grew slightly better on supplemented Isotonic agar than on MuellerHinton agar. However, since results for penicillin and vancomycin did not differ in this way, the discrepancy is probably not associated with the quality of growth. This small difference in MICs had no implications for designating isolates as resistant. However, two isolates were susceptible on MuellerHinton but intermediate on supplemented Isotonic agar. No isolate had a daptomycin MIC in the resistant range on either agar. Penicillin MICs for methicillin-susceptible, penicillin-resistant S. aureus were lower on supplemented Isotonic agar than on MuellerHinton agar, possibly because of sodium chloride content, but this had no implications in designating the isolates as resistant. MIC determinations were repeated for some of the isolates and the results were reproducible. This phenomenon clearly needs to be investigated further.
Zone diameters determined by the NCCLS method were smaller than those determined by the BSAC method. This is not surprising since the inoculum size is different. The tentative breakpoints suggested by NCCLS seem relevant and a tentative breakpoint of 18 mm for susceptible organisms would be suitable for the BSAC method. However, the two isolates of intermediate susceptibility included in this study both had zone diameters larger than the breakpoint and would not have been detected by either method. This is consistent with an earlier study13 in which 12 enterococci with daptomycin MICs of 4 mg/L all had zone diameters >18 mm with a 30 µg daptomycin disc. There were no resistant isolates to test the proposed resistance breakpoint. In view of the two isolates of intermediate susceptibility and the poor correlation between MIC and zone diameter when all isolates are analysed together (NCCLS r = 0.472, BSAC r = 0.437), it may be necessary to introduce species-specific breakpoints, as with penicillin and vancomycin. Furthermore, the tentative breakpoints suggested may well be altered when the results of Phase III clinical trials have been analysed.
The distribution of daptomycin MICs seen in this study, which agree with those of other reports,1,3 show daptomycin to be active in vitro against a wide range of Gram-positive aerobic species, including those resistant to ß-lactams and glycopeptides. Daptomycin is potentially a useful antimicrobial agent, particularly against isolates resistant to other classes of antibiotic, but its clinical role will also depend on pharmacokinetic and safety profiles.
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Acknowledgments |
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Notes |
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References |
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2 . Jacobus, N. V., McDermott, L., Lonks, J. R., Boyce, J. M. & Snydman, D. R. (1998). In-vitro activity of daptomycin against resistant gram-positive pathogens. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract F112, p. 260. American Society for Microbiology, Washington, DC.
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8 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
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10 . European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). (2000). Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. Clinical Microbiology and Infection 6, 50915.[ISI][Medline]
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Andrews, J. M. for the BSAC Working Party on Susceptibility Testing. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 4357.
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Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 516.
13 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (1999). Daptomycin susceptibility tests: Provisional criteria, Quality Control, and importance of Ca++ concentrations in test media. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA. Abstract 350, p. 199. American Society for Microbiology, Washington, DC.
14 . Kobayashi, N., Wu, H., Kojima, K., Taniguchi, K., Urasawa, S., Uehara, N. et al. (1994). Detection of mecA, femA and femB genes in clinical strains of staphylococci using polymerase chain reaction. Epidemiology and Infection 113, 25966.[ISI][Medline]
Received 30 January 2001; returned 9 April 2001; revised 9 May 2001; accepted 22 May 2001