The in vitro activity of daptomycin against Staphylococcus aureus and Enterococcus species

S. S. Richter*, D. E. Kealey, C. T. Murray, K. P. Heilmann, S. L. Coffman and G. V. Doern

Department of Pathology, C606 GH, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242–1009, USA

Received 13 January 2003; returned 7 March 2003; revised 14 April 2003; accepted 15 April 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: The purpose of this study was to examine the in vitro activity of daptomycin using an optimal calcium (Ca2+) concentration (50 mg/L) against a diverse collection of enterococcal and Staphylococcus aureus clinical isolates, including glycopeptide-resistant enterococci (GRE) and methicillin-resistant S. aureus (MRSA).

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: Mueller–Hinton (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 quinupristin–dalfopristin and linezolid.

Keywords: lipopeptides, staphylococci, enterococci


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Daptomycin is an investigational cyclic lipopeptide antibiotic with a novel mechanism of action against Gram-positive organisms, such as methicillin-resistant Staphylococcus aureus (MRSA) and glycopeptide-resistant enterococci (GRE), for which a limited number of effective therapeutic agents are available.1 Although the bactericidal activity of daptomycin is not yet completely understood, interference with plasma membrane function appears to be an important effect.1

When performing broth microdilution susceptibility testing, the activity of daptomycin is enhanced two- to four-fold by increasing the calcium (Ca2+) concentration of Mueller–Hinton (MH) broth from the 20–25 mg/L concentration currently recommended by the NCCLS2 to 50 mg/L—a 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+ (45–55 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility testing was performed on 499 isolates of S. aureus (39% MRSA), 504 isolates of Enterococcus faecalis (8% GRE) and 480 isolates of Enterococcus faecium (55% GRE) obtained from North American medical centres during 1996–2000. All of the S. aureus and E. faecalis isolates and 72% of the E. faecium isolates were collected during 1997–2000 from 34 North American SENTRY Antimicrobial Surveillance Program9 centres in 25 states or provinces. The majority of SENTRY isolates were from blood cultures (85% of S. aureus, 91% of E. faecalis, and 71% of E. faecium). The remaining S. aureus SENTRY isolates were from the lower respiratory tract, and the non-invasive enterococcal SENTRY isolates were from skin and soft tissue. Twenty-eight percent of the E. faecium isolates were collected during 1996–1997 as part of the Synercid Microbiological Assessment of Resistance Trends (SMART) surveillance study10 from 21 hospital laboratories in the USA and Canada.

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 (0.015–32 mg/L), quinupristin–dalfopristin (0.03–64 mg/L), linezolid (0.015–32 mg/L), vancomycin (0.06–128 mg/L), teicoplanin (0.06–128 mg/L), oxacillin (0.015–32 mg/L), erythromycin (0.12–128 mg/L) and clindamycin (0.03–32 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The daptomycin MICs for S. aureus ranged from 0.12–1 mg/L (Table 1). The daptomycin MICs, at which 90% of strains were inhibited (MIC90s) for methicillin-susceptible S. aureus (MSSA) and MRSA for daptomycin, were similar (0.25 mg/L and 0.5 mg/L, respectively), as were the MSSA and MRSA MIC90s of quinupristin–dalfopristin (0.5 mg/L, 1 mg/L), linezolid (2 mg/L, 2 mg/L) and vancomycin (1 mg/L, 2 mg/L).


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Table 1. Activities of daptomycin and seven other agents against 499 staplylococcal and 984 enterococcal isolates  
 
For enterococci, the daptomycin MICs ranged from ≤0.015–4 mg/L. The activity of daptomycin against GRE was essentially the same as glycopeptide-susceptible enterococci (GSE). For E. faecium, the MIC90s of daptomycin and linezolid against GSE and GRE isolates were identical (2 mg/L), whereas the MIC90s of quinupristin–dalfopristin were 4 mg/L (GSE) and 2 mg/L (GRE). For E. faecalis, the MIC90s of GSE and GRE isolates of daptomycin (2 mg/L, 4 mg/L) and linezolid (2 mg/L, 2 mg/L) were similar.

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).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 1987, Jones & Barry4 proposed daptomycin MIC and zone diameter breakpoints for susceptible Gram-positive isolates of ≤2 mg/L and ≥16 mm (resistant isolates: MIC, ≥8 mg/L; zone diameter, ≤12 mm) using media containing 50 mg/L Ca2+. Every staphylococcal, and 96.5% of the enterococcal isolates from the current study, had daptomycin MICs that would be classified as susceptible based on this tentative MIC breakpoint of ≤2 mg/L. If the previously proposed zone diameter breakpoint of ≥16 mm was applied,4 all of the 34 enterococcal isolates with MICs of 4 mg/L (zone diameters ranging from 19–27 mm) would be considered falsely susceptible by disc diffusion. Application of the intermediate category of susceptibility (MIC, 4 mg/L) proposed by Jones & Barry,4 would make these minor rather than very major errors.

Wise et al.6 noted that British Society for Antimicrobial Chemotherapy (BSAC) methodology would support a daptomycin MIC breakpoint of 4–8 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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Table 2. In vitro activities of daptomycin against Staphylococcus aureus and Enterococcus faecium isolates reported by various studies using media containing calcium 50 mg/L
 
An MIC breakpoint for the susceptible category other than ≤2 mg/L may be warranted for enterococci. Louie et al.14 utilized a neutropenic mouse model of S. aureus thigh infection in order to study daptomycin activity, and concluded that the AUC/MIC ratio is the optimal pharmacodynamic parameter with which to predict outcome. A mouse model of enterococcal renal infection was used to demonstrate efficacy of daptomycin at doses producing AUCs of 400–600 mg·h/L against isolates with daptomycin MICs of ≤4 mg/L.15 Based on these results, the following MIC interpretive criteria for enterococci have been proposed: susceptible, ≤4 mg/L; intermediate, 8 mg/L; resistant, ≥16 mg/L.15

Overall, these in vitro studies document potent activity by daptomycin against isolates of enterococci and S. aureus, including GRE and MRSA, comparable with quinupristin–dalfopristin 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.


    Acknowledgements
 
We thank R. N. Jones (The JONES Group, North Liberty, IA, USA) for providing the isolates characterized in this study. Financial support for this project was provided by Cubist Pharmaceuticals, Inc.

Presented in part at the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, 29 September 2002, San Diego, CA, USA (Abstract E-1450).


    Footnotes
 
* Corresponding author. Tel: +1-319-356-2990; Fax: +1-319-356-4916; E-mail: sandra-richter@uiowa.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Tally, F. P. & DeBruin, M. F. (2000). Development of daptomycin for Gram-positive infections. Journal of Antimicrobial Chemotherapy 46, 523–6.[Free Full Text]

2 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth 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, 51–8.[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, 625–9.[ISI][Medline]

5 . Barry, A. L., Fuchs, P. C. & Brown, S. D. (2001). In vitro activities of daptomycin against 2,789 clinical isolates from 11 North American medical centers. Antimicrobial Agents and Chemotherapy 45, 1919–22.[Abstract/Free Full Text]

6 . Wise, R., Andrews, J. M. & Ashby, J. P. (2001). Activity of daptomycin against Gram-positive pathogens: a comparison with other agents and the determination of a tentative breakpoint. Journal of Antimicrobial Chemotherapy 48, 563–7.[Abstract/Free Full Text]

7 . Jones, R. N. (1989). Effects of reduced cation supplement recommendations (National Committee for Clinical Laboratory Standards) on daptomycin antistaphylococcal activity. Antimicrobial Agents and Chemotherapy 33, 1652–3.[ISI][Medline]

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.

9 . Pfaller, M. A., Jones, R. N., Doern, G. V. et al. (1998). Bacterial pathogens isolated from patients with bloodstream infection: frequencies of occurrence and antimicrobial susceptibility patterns from the SENTRY antimicrobial surveillance program (United States and Canada, 1997). Antimicrobial Agents and Chemotherapy 42, 1762–70.[Abstract/Free Full Text]

10 . Jones, R. N., Ballow, C. H., Biedenbach, D. J. et al. (1998). Antimicrobial activity of quinupristin-dalfopristin (RP 59500, Synercid®) tested against over 28,000 recent clinical isolates from 200 medical centers in the United States and Canada. Diagnostic Microbiology and Infectious Disease 30, 437–51.[CrossRef]

11 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing. Tenth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

12 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Seventh Edition. Approved Standard M2-A7. NCCLS, Wayne, PA, USA.

13 . King, A. & Phillips, I. (2001). The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates. Journal of Antimicrobial Chemotherapy 48, 219–23.[Abstract/Free Full Text]

14 . Louie, A., Kaw, P., Liu, W. et al. (2001). Pharmacodynamics of daptomycin in a murine thigh model of Staphylococcus aureus infection. Antimicrobial Agents and Chemotherapy 45, 845–51.[Abstract/Free Full Text]

15 . Tongchaun, L., Thorne, G., Silverman, J. et al. (2001). Efficacy and breakpoint analysis of daptomycin versus Enterococcus faecalis and Enterococcus faecium renal infection in mice. In Abstracts of the Thirty-ninth Meeting of the Infectious Diseases Society of America, San Francisco, CA, 2001. Abstract 446. Infectious Diseases Society of America, Alexandra, VA, USA.