Department of Pathology, C606 GH, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 522421009, USA
Received 13 January 2003; returned 7 March 2003; revised 14 April 2003; accepted 15 April 2003
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Abstract |
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Methods: The activity of daptomycin was compared with the activities of seven other agents against 1483 enterococcal and S. aureus clinical isolates, including 303 GRE and 193 methicillin-resistant S. aureus (MRSA) strains. Susceptibility testing was performed by the NCCLS broth microdilution method, with one exception: MuellerHinton (MH) broth was supplemented to a physiological level of 50 mg/L Ca2+ when testing daptomycin. Daptomycin zone diameters were determined by disc diffusion with MH agar plates containing Ca2+ 50 mg/L.
Results: All staphylococcal isolates tested, and the majority of enterococcal isolates (96.5%), would be considered susceptible to daptomycin if the breakpoint previously proposed of ≤2 mg/L was applied. The activity of daptomycin against MRSA and methicillin-susceptible S. aureus was essentially equal. Daptomycin also had similar activity against GRE and glycopeptide-susceptible enterococci. Every S. aureus isolate had a daptomycin zone diameter ≥20 mm, and all of the enterococcal isolates had daptomycin zone diameters ≥17 mm.
Conclusions: Overall, daptomycin showed potent activity against S. aureus and enterococcal isolates, comparable to quinupristindalfopristin and linezolid.
Keywords: lipopeptides, staphylococci, enterococci
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Introduction |
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When performing broth microdilution susceptibility testing, the activity of daptomycin is enhanced two- to four-fold by increasing the calcium (Ca2+) concentration of MuellerHinton (MH) broth from the 2025 mg/L concentration currently recommended by the NCCLS2 to 50 mg/La level that more closely approximates the physiological Ca2+ level of human serum.36 This higher 50 mg/L Ca2+ level was recommended for MH broth by the NCCLS in 1985 standards, but subsequently lowered to achieve the optimal Ca2+ level determined for accurate testing of aminoglycosides against Pseudomonas aeruginosa and tetracycline against all bacteria.2,7 Multiple investigators have recommended that physiological levels of Ca2+ (4555 mg/L) be used for broth dilution susceptibility testing of daptomycin.36 A recent study, examining the Ca2+ dependence of daptomycin activity, suggests Ca2+ binding by the lipopeptide antibiotic increases its hydrophobicity, enhancing interaction with cytoplasmic membrane bilayers.8
The purpose of this study was to examine the in vitro activity of daptomycin, using broth microdilution with an optimal Ca2+ concentration (50 mg/L), and compare it with the activities of other agents against a large, diverse collection of recent enterococcal and Staphylococcus aureus clinical isolates, including GRE and MRSA. A daptomycin zone diameter was also determined for each isolate using media enhanced with 50 mg/L Ca2+, and the result compared with the microdilution MIC.
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Materials and methods |
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MICs were determined according to NCCLS broth microdilution guidelines,2 with one exception: cation-adjusted MH broth (Difco Laboratories, Detroit, MI, USA) was supplemented to a physiological level of 50 mg/L Ca2+ when testing daptomycin. Two-fold serial dilutions of eight antimicrobials were tested: daptomycin (0.01532 mg/L), quinupristindalfopristin (0.0364 mg/L), linezolid (0.01532 mg/L), vancomycin (0.06128 mg/L), teicoplanin (0.06128 mg/L), oxacillin (0.01532 mg/L), erythromycin (0.12128 mg/L) and clindamycin (0.0332 mg/L). Resistance was defined according to NCCLS MIC interpretive standards.11 A zone diameter was determined for each isolate with a 30 µg daptomycin disc (Cubist Pharmaceuticals, Inc., Lexington, MA, USA) according to NCCLS disc diffusion guidelines,12 except for a higher Ca2+ concentration (50 mg/L) in the MH agar (Remel, Lenexa, KS, USA). Daily quality control was performed using S. aureus ATCC 29213 (broth microdilution), S. aureus ATCC 25923 (disc diffusion) and E. faecalis ATCC 29212 (both methods).
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Results |
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There was an absence of correlation between the daptomycin zone diameters and MICs because of the unimodal MIC distribution for each species of organism tested (data not shown). Every S. aureus isolate (MICs ≤ 1 mg/L) had a daptomycin zone diameter ≥20 mm. The enterococcal isolates (MICs ≤ 4 mg/L) had daptomycin zone diameters ≥19 mm (E. faecalis) and ≥17 mm (E. faecium).
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Discussion |
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Wise et al.6 noted that British Society for Antimicrobial Chemotherapy (BSAC) methodology would support a daptomycin MIC breakpoint of 48 mg/L for Gram-positive pathogens, but endorsed a breakpoint of ≤2 mg/L because few isolates had MICs above 2 mg/L. The same report proposed a zone diameter breakpoint of ≥20 mm and found only two isolates (E. faecium: MICs, 4 mg/L; zone diameters, 21 mm) of 328 Gram-positive organisms (0.6%) falsely susceptible.
King & Phillips13 concluded that the ≥16 mm susceptible breakpoint was appropriate when using NCCLS methods, but proposed ≥18 mm for BSAC methods that employ an extra 1/10 or 1/100 inoculum dilution step (to yield semi-confluent growth) resulting in larger zones. King & Phillips13 identified two enterococcal isolates with non-susceptible daptomycin MICs of 4 mg/L, but zone diameters larger than proposed zone breakpoints.
Table 2 summarizes daptomycin susceptibility data obtained using 50 mg/L Ca2+ for S. aureus and enterococci from five different studies, including the current report. Among the studies, no S. aureus isolate with a daptomycin MIC > 2 mg/L was described, and the activity of daptomycin against MRSA and MSSA was essentially the same (MIC50s and MIC90s no more than one dilution higher). For enterococci, GRE and GSE isolates were also inhibited by similar concentrations of daptomycin, but isolates with MICs as high as 8 mg/L were reported by one study.5
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Overall, these in vitro studies document potent activity by daptomycin against isolates of enterococci and S. aureus, including GRE and MRSA, comparable with quinupristindalfopristin and linezolid. Definite interpretive criteria have not yet been established. The results of ongoing Phase III clinical trials are needed to establish firmly whether serious infections caused by staphylococcal isolates with daptomycin MICs ≤ 2 mg/L, and enterococcal isolates with daptomycin MICs ≤ 4 mg/L, can be treated effectively with this investigational lipopeptide antibiotic.
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Acknowledgements |
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Presented in part at the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, 29 September 2002, San Diego, CA, USA (Abstract E-1450).
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Footnotes |
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References |
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2 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
3 . 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.[CrossRef][ISI][Medline]
4 . Jones, R. N. & Barry, A. L. (1987). Antimicrobial activity and spectrum of LY146032, a lipopeptide antibiotic, including susceptibility test recommendations. Antimicrobial Agents and Chemotherapy 31, 6259.[ISI][Medline]
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8 . Oliver, N. S., Laganas, V., Bouchard, M. et al. (2001). Structural basis for calcium dependence of the lipopeptide daptomycin. In Programs and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract C1-1801, p. 98. American Society for Microbiology, Washington, DC, USA.
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