Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK
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Abstract |
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Introduction |
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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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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 1824 h in air (except for Streptococcus pneumoniae and Streptococcus milleri, which were incubated in 46% 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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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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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. 48 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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Notes |
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References |
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2 . 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, 17106.[ISI][Medline]
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, 216.
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.
5 . 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, 5325.[ISI][Medline]
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, 518.[ISI][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.
8 . 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, 150.[ISI][Medline]