The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA
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Abstract |
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Introduction |
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For the treatment of some infections such as bacterial endocarditis, and the treatment of serious infections in immunocompromised patients, bactericidal activity is desirable.4 Although not universally accepted, there are data that indicate that the timekill curve method is the in vitro method that correlates best with clinical cure in cases of bacterial endocarditis.5 The present study was designed to assess the in vitro bactericidal activity of daptomycin against staphylococci, in comparison with vancomycin, linezolid and Q-D by two methods, namely determination of minimum bactericidal concentrations (MBCs) and time kill studies.
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Materials and methods |
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From our collection of recent (2 years) North American clinical isolates, 108 staphylococcal strains were selected to give a broad range of daptomycin and vancomycin MICs. These included 53 strains of S. aureus, 28 of which were MRSA, and three of which were GISA. The remaining 55 strains included 44 Staphylococcus epidermidis and 11 Staphylococcus haemolyticus. MBCs were determined for the entire set, and timekill studies were performed on 25 strains from this set.
Antibacterial agents
Daptomycin standardized powder was provided by Cubist Pharmaceuticals, Inc. (Lexington, MA, USA). Linezolid was obtained from Pharmacia Corporation (Kalamazoo, MI, USA), Q-D was obtained from Aventis Pharmaceuticals (Bridgewater, NJ, USA) and vancomycin was obtained from Sigma Chemical Company (St Louis, MO, USA).
Susceptibility testing
MICs were determined by the broth microdilution method as described by the NCCLS.6 Cation-adjusted Mueller Hinton broth (CAMHB) was used for all antibiotics except daptomycin, for which the CAMHB was supplemented with additional calcium to a physiological concentration of 50 mg/L.3 Serial two-fold dilutions of each antibiotic were prepared in microdilution trays with concentrations ranging from 32 to 0.004 mg/L for daptomycin, 64 to 0.5 mg/L for linezolid, 4.0 to 0.03 mg/L for Q-D and 32 to 0.25 mg/L for vancomycin. For each test, the control strain S. aureus ATCC 29213 was included, and all results with this strain were within the published NCCLS quality control ranges.7
MBCs were determined by subculturing on to sheep blood agar plates 10 µL of broth from each well with no visible growth after 24 h incubation and from the highest concentration well with visible growth. The MBC was defined as the lowest concentration of drug yielding colony counts <0.1% of the initial inoculum (determined by colony counts from the growth control well immediately after inoculation).
Timekill studies were performed as recommended by the NCCLS.8 CAMHB was used for all drugs except daptomycin, for which the CAMHB was supplemented with calcium to a final concentration of 50 mg/L. Antibiotics were tested at two concentrations: (i) the MIC susceptibility breakpoint concentration, and (ii) twice the MBC (2 x MBC). When 2 x MBC was the same as the susceptible breakpoint, only one test was performed. Also, when the MBC was greater than the highest concentration tested, 2 x MBC timekill tests were not performed. Colony counts were performed on broth from the growth control flasks at the time of inoculation (0 h), and from the growth control flasks as well as all antibiotic-containing flasks after 1, 2, 4, 8, 12 and 24 h of incubation. Bactericidal activity was defined as a decrease in colony count to <0.1% of the initial inoculum count (>99.9% kill).
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Results and discussion |
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When vancomycin was tested in timekill studies at 2 x MBC, only 16 of 23 (70%) were killed. In the case of Q-D, nine of 15 (60%) strains tested at 2 x MBC were killed. The bactericidal activity of linezolid was difficult to assess. Linezolid had MBCs of 4.0 mg/L for five (20%) strains, but this was not confirmed by the timekill studies. The only strain for which linezolid was bactericidal in the timekill test system was a GISA strain with a linezolid MBC of 8.0 mg/L, and this strain was killed at both 4.0 and 16 mg/L at 24 h. The latter tests were repeated with the same results. The significance of these discrepancies remains unclear at this time, but linezolid is regarded as bacteriostatic against staphylococci.14
When tested at 2 x MBC in timekill tests, the mean time to reach >99.9% reduction in colony counts (for those that were killed) was 5.9 h for daptomycin, 6.0 h for Q-D and 15.5 h for vancomycin.
We conclude from these data that daptomycin has good in vitro bactericidal activity against staphylococci, which in this study surpassed that of vancomycin, Q-D and linezolid.
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Acknowledgements |
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Notes |
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References |
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2 . Paradisi, F., Corti, G. & Messeri, D. (2001). Antistaphylococcal (MSSA, MRSA, MSSE, MRSE) antibiotics. Medical Clinics of North America 85, 117.[ISI][Medline]
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5 . Drake, T. A. & Sande, M. A. (1985). Studies of the chemotherapy of endocarditis: correlation of in vitro, animal model, and clinical studies. Review of Infectious Diseases 5, Suppl. 2, S34553.
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7 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Susceptibility TestingTenth Informational Supplement, M100-S10. NCCLS, Wayne, PA.
8 . National Committee for Clinical Laboratory Standards. (1998). Methods for Determining Bactericidal Activity of Antimicrobial AgentsApproved Guideline, M26-A. NCCLS, Wayne, PA.
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Received 31 August 2001; returned 7 November 2001; revised 12 December 2001; accepted 14 December 2001