Journal of Antimicrobial Chemotherapy (2001) 48, 563-567
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Activity of daptomycin against Gram-positive pathogens: a comparison with other agents and the determination of a tentative breakpoint
Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK
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Abstract |
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The in vitro activity of daptomycin was studied in comparison with other agents active against 328 recent clinical isolates of Gram-positive pathogens. MIC data showed that the addition of calcium ions to a final concentration of 50 mg/L enhanced the activity of daptomycin generally by eight- to 16-fold. In the presence of calcium ions daptomycin was uniformly active against the strains of Staphylococcus spp. and Streptococcus spp. studied with a MIC90 of
1 mg/L. Enterococcus faecalis and Enterococcus faecium were slightly less susceptible (MIC90 2 mg/L). Vancomycin-, fluoroquinolone- and quinupristin/dalfopristin-resistant strains were all susceptible to daptomycin. The presence of serum reduced the apparent activity of daptomycin to only a moderate extent. Employing the BSAC methodologies, a tentative breakpoint of 2 mg/L for daptomycin is proposed. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Daptomycin is a novel cyclic lipopeptide agent that is currently being reassessed for the treatment of infections caused by Gram-positive pathogens. It is bactericidal against susceptible and resistant Gram-positive bacteria, including MRSA, VRE, MRSE, PRSP and GISA strains. One feature of daptomycin not found to such a marked degree in other antimicrobials is the dependence of its activity upon physiological concentrations of calcium ions in the medium.1,2 The mechanism is unknown but possibly related to the divalent ion being required for drug attachment or penetration into the bacterial cell. It has been recommended that a concentration of 50 mg/L of Ca2+ be present to accurately test the activity.2 This accords with the physiological free Ca2+ concentration of 1.15–1.31 mM found in plasma.3
This study compared the in vitro activity of daptomycin (with and without a 50 mg/L Ca2+ supplement) with that of other agents commonly used to treat Gram-positive infections. In addition, a study to establish a tentative breakpoint between resistant and susceptible pathogens, which should be of use in the clinical laboratory, was undertaken.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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A total of 328 Gram-positive organisms were studied, the majority of which were recent clinical isolates. The remainder were strains with well-characterized resistance mechanisms. The following agents were employed: daptomycin (Cubist Pharmaceutical Inc., Boston, MA, USA), vancomycin (Lilly Products, Basingstoke, UK), quinupristin/ dalfopristin and teicoplanin (Aventis Pharmaceutical, Denham, UK), gemifloxacin and co-amoxiclav (GlaxoSmithKline, Worthing, UK) and linezolid (Pharmacia, Milton Keynes, UK).
Susceptibilities were determined by a standard agar plate dilution method following the recommendations of Oliver et al.4 Briefly, isotonic sensitivity test agar (pH 7.2) (Mast, Bootle, UK) was employed, both alone and supplemented to 50 mg/L Ca2+. For streptococci, 5% defibrinated horse blood was also added. All strains were tested at a final inoculation of 104 cfu using a multipoint inoculator (Denley Instruments, Billinghurst, UK). Plates were incubated at 37°C for 18–24 h in air (except for Streptococcus pneumoniae and Streptococcus milleri, which were incubated in 4–6% CO2 in air). Co-amoxiclav estimations employed amoxicillin and clavulanate in a 2:1 ratio with the result recorded in terms of the amoxicillin concentration. The effect of serum on the MIC and minimum bactericidal concentration (MBC) of daptomycin was studied for two strains each of Streptococcus pyogenes, S. pneumoniae, Enterococcus faecium (including one vancomycin resistant strain) and two methicillin susceptible Staphylococcus aureus strains. An agar dilution (as described above) and a broth microdilution method were compared. The microdilution method employed isotonic broth [supplemented with 50 mg/L Ca2+ both alone and also containing 20% or 70% human serum (Bradsure Biologicals, Market Harborough, UK)] to which was added calcium to a final Ca2+ concentration of 50 mg/L. The final inoculum was 105 cfu/mL. The MIC was defined as the lowest concentration at which there was no visible growth. Following incubation, 100 µL of broth was subcultured on to appropriate antibiotic-free media for MBC determination. The MBC was defined as the lowest antibiotic concentration to show no growth (99.9% kill).
Disc susceptibility testing was undertaken on the same media utilizing the same organisms as noted above. The inoculum was adjusted to yield semi-confluent growth. Plates were incubated under the same conditions as above following the application of 30 µg daptomycin discs. The zone sizes were recorded following overnight incubation, as above.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The activity of daptomycin against the Gram-positive pathogens tested is shown in the Table
. The addition of Ca2+ to the media to physiological concentrations of 50 mg/L had a marked effect on all species tested. The MIC90 was eight- to 16-fold lower in the presence of Ca2+, the only exception being Staphylococcus saprophyticus where the decrease in MIC90 was four-fold. Further discussion of the results will refer to those obtained with the calcium supplement.
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The addition of calcium did not affect the susceptibilities of the other agents studied by more than one dilutional step.
All S. aureus, both methicillin susceptible and resistant, were inhibited by 0.5 mg/L or less of daptomycin. Similar results were obtained for quinupristin/dalfopristin. All strains of S. epidermidis and S. saprophyticus were inhibited by 1 mg/L or less of daptomycin and quinupristin/ dalfopristin was more active (all strains inhibited by 0.5 mg/L). All strains of Staphylococcus spp. tested were susceptible to 2 mg/L of linezolid.
E. faecalis and E. faecium were equally susceptible to the daptomycin plus Ca2+, with all the MICs being 4 mg/L and the MIC90 2 mg/L, whereas the strains of E. faecalis were 16-fold less susceptible to quinupristin/dalfopristin than the strains of E. faecium and four strains of E. faecalis were resistant to vancomycin (MIC > 8 mg/L). All these strains were as susceptible to daptomycin and linezolid as the vancomycin susceptible population. Co-amoxiclav and gemifloxacin had activity against E. faecalis but E. faecium strains were markedly less susceptible. All the agents studied displayed good activity against S. pneumoniae. Twenty-four strains were less susceptible to co-amoxiclav (MIC 
1 mg/L) but these were as susceptible to daptomycin, gemifloxacin, linezolid and the glycopeptides as the co-amoxiclav susceptible strains. Two strains of S. pneumoniae were resistant to quinupristin/dalfopristin (MICs 16 mg/L) but no decrease in susceptibility was noted against the other agents. S. milleri, 20 Lancefield group A and 20 group B strains were all susceptible to 1 mg/L of daptomycin.
Human serum had little effect on the activity of daptomycin. In broth there was very little difference between the MIC and the MBC and results in broth mirrored those in agar. The MIC in the presence of serum tended to show a single dilution step increase when compared with that in broth. There was a more marked effect on the MBC.
In order to establish a zone diameter breakpoint, the MIC determined for each strain was plotted against the zone diameter obtained for the 30 µg daptomycin disc (Figure). Using a zone diameter breakpoint of 20 mm to denote susceptibility, no false resistance was observed and a false susceptibility rate of 0.61% was found (which consisted of two strains of E. faecium).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The mode of action of daptomycin is poorly understood. The target is thought to be the cytoplasmic membrane and a disruption of the electrical potential across this membrane has been described, as have putative daptomycin binding proteins at this site.4 These authors also report very low frequencies (<10–10) of spontaneous resistance in S. aureus. The in vitro activity of this compound is highly dependent upon the calcium ion content of the culture medium5,6 yet the mechanism underlying this observation is poorly understood and it has been thought that Ca2+ may aid penetration of the bacterial membrane.2 We confirm the observation of enhanced activity in the presence of the cation;2,5,6 the earlier reports employed Mueller–Hinton agar, whereas we used an isotonic medium of the type more commonly employed in Europe. An eight- to 16-fold reduction in the MIC was generally observed when Ca2+ was added compared with the unsupplemented medium. As the physiological concentration of calcium in the body is 45–55 mg/L,3 this calcium dependence is an important testing issue.
The protein binding of daptomycin has been reported as 92% (B. Dvorchik, personal communication). As would be expected, there is a moderate effect upon the antimicrobial in the presence of human serum, the MIC increasing two- to four-fold and the MBC four- to eight-fold.
The tentative breakpoint of 2 mg/L of daptomycin was derived from the knowledge of the pharmacokinetics of the agent. Following a 4 mg/kg dose, a mean Cmax of c. 70 mg/L is attained.7 Application of the BSAC breakpoint formula8 would suggest a breakpoint of c. 4–8 mg/L. As so few strains had an MIC > 2 mg/L it was considered reasonable to choose a lower figure, which still yielded satisfactory results. The results we obtained are, naturally, somewhat different from those described by Fuchs et al.,6 who employed the NCCLS methodology, but equally reproducible. We did not believe that an ‘intermediate’ designation of susceptibility was appropriate.
These studies suggest that daptomycin should be a valuable addition to the agents available to treat infections caused by Gram-positive pathogens. The results of clinical trials are awaited to confirm this view and to assess the suitability of the proposed tentative breakpoint.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Thanks to G. Thorne of Cubist Pharmaceuticals for her advice and Cubist Pharmaceuticals for financial support.
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Notes |
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* Corresponding author. Tel: +44-121-507-4255; Fax: +44-121-551-7763; E-mail: r.wise{at}bham.ac.uk
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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1 . Benson, C. A., Beaudette, F. & Trenholm, G. (1987). Comparative in vitro activity of LY 146032, a new peptolide, with vancomycin and eight other agents against Gram-positive organisms. Antimicrobial Agents and Chemotherapy 20, 191–6.
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Hanberger, H., Nilsson, L. E., Maller, R. & Isakson, B. (1991). Pharmacodynamic of daptomycin and vancomycin or Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs. Antimicrobial Agents and Chemotherapy 35, 1710–6.
3 . Larsson, L., Finnstrom, O., Nilsson, B. & Ohman, S. (1983). Evaluation of radiometer ICAI as a routine instrument for serum caused calcium and its application for whole blood capillary samples from newborn infants. Scandinavian Journal of Clinical Laboratory Investigations 43, Suppl. 165, 21–6.
4 . Oliver, N., Andrew, T., Li, T. & Silverman, J. (1998). In vitro studies on resistance to the lipopeptide antibiotic daptomycin. In Program and Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, 1998. Abstract F117, p. 262. American Society for Microbiology, Washington, DC.
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Eliopoulos, G. M., Willey, S., Reiszner, E., Spitzer, P. G., Caputo, G. & Moellering, R. C., Jr (1986). In vitro and in vivo activity of LY 146032, a new cyclic lipopeptide antibiotic. Antimicrobial Agents and Chemotherapy 30, 532–5.
6 . Fuchs, P. C., Barry, A. L. & Brown, S. D. (2000). Daptomycin susceptibility tests: interpretative criteria, quality control and effect of calcium on in vitro tests. Diagnostic Microbiology and Infectious Diseases 38, 51–8.[Web of Science][Medline]
7 . Wise, R., Gee, T., Andrews, J. M. & Marshall G. (2001) The pharmacokinetics and inflammatory fluid penetration of intravenous daptomycin in volunteers. Antimicrobial Agents and Chemotherapy 45, in press.
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Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.
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