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Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests
Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
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Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests Peter C. Fuchs*, Arthur L. Barry, Steven D. Brown The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA Received 21 December 1999; received in revised form 16 June 2000; accepted 16 June 2000
Abstract Daptomycin MICs were determined for 844 Gram-positive bacteria in three concentrations of Ca⫹⫹ and compared with the MICs of vancomycin and teicoplanin. Daptomycin was twofold to fourfold more active against most species when tested in 50 g/ml of Ca⫹⫹ than in 25 g/ml. In 50 g/ml of Ca⫹⫹ daptomycin was more active against methicillin-resistant staphylococci and vancomycin-resistant enterococci than teicoplanin or vancomycin; 100% of these isolates were susceptible to ⱕ2.0 g/ml of daptomycin. Different lots of Mueller-Hinton agar were variable in Ca⫹⫹ content, and daptomycin disk diffusion zone diameters were affected, i.e., zones were 1 to 15 mm smaller on one lot of agar with only 6 g/ml of Ca⫹⫹ compared to another lot with 28 g/ml. The previously proposed daptomycin interpretive breakpoints performed satisfactorily when MICs were determined in Mueller-Hinton broth with 50 g/ml of Ca⫹⫹ and when the agar gave appropriate zones with quality control strains. To define those control limits, replicate tests with four quality control strains were performed in ten laboratories using broth microdilution tests (with Ca⫹⫹ supplemented broth) and disk diffusion tests on MuellerHinton agar without cation adjustments. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Daptomycin is a cyclic lipopeptide antibiotic that has been reported to have good antimicrobial activity against most Gram-positive bacteria (Appelbaum et al. 1989, Eliopoulos et al. 1986, Greenwood & Palfreyman 1987, Low et al. 1989, Pohlod et al. 1987, Swenson et al. 1990). Despite encouraging results with daptomycin in experimental animals (Bryant et al. 1987, Cantoni et al. 1990, Ramos et al. 1992), further development of the drug was suspended after early clinical trials showed that relatively low doses of daptomycin (total of 2 mg/kg/day) were less effective than conventional therapy (Chambers, 1991) and high doses of 4 mg/kg q12h (8 mg/kg/day) produced muscle toxicity in a few patients. In animal studies this muscle toxicity has been shown to be significantly reduced by once-daily dosing (Oleson et al., 1999). Furthermore, since the Cmax has been shown to be the major pharmacokinetic parameter associated with infection eradication in animal efficacy models * Corresponding author. Tel.: ⫹1-503-682-3232; fax: ⫹1-503-6822065. E-mail address: [email protected] (P.C.Fuchs). A portion of this material was previously presented at the 39th ICAAC in San Francisco, California, September 26 –29, 1999. Abstract No. 350.
(Leggett et al., 1987), once-daily dosing could be more effective. With the increasing prevalence of vancomycinresistant enterococci (VRE), methicillin-resistant staphylococci (MRS) and penicillin-resistant pneumococci (PRP), there has been renewed interest in developing daptomycin for clinical use. Daptomycin Phase II trials using once-daily dosing are currently in progress with encouraging preliminary safety results (Tally et al., 1999). The in vitro activity of daptomycin is enhanced by increased concentrations of Ca⫹⫹ when tested in broth (Eliopoulos et al. 1986, Greenwood & Palfreyman 1987, Louie et al. 1992). The interpretive criteria for daptomycin that were proposed in 1987 (Jones & Barry 1987) were based on MIC tests performed in cation-supplemented Mueller-Hinton broth (CSMHB) containing 50 g/ml of Ca⫹⫹, as recommended at that time by the National Committee for Clinical Laboratory Standards (NCCLS) (1985). Since then the NCCLS has revised its recommendations, and currently recommends the use of cation-adjusted Mueller-Hinton broth (CAMHB) containing 20 –25 g/ml of Ca⫹⫹ for routine susceptibility testing (NCCLS 1997b). Jones (1989) suggested that when testing staphylococci, daptomycin MICs were higher when CAMHB was used, but the interpretive criteria developed in CSMHB could still be applied as all isolates were susceptible in both concentrations of Ca⫹⫹.
0732-8893/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 0 ) 0 0 1 6 4 - 4
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Enterococci were not a concern when that study was undertaken and thus only staphylococci were evaluated. The present studies were designed to: 1) Confirm the effects of Ca⫹⫹ on in vitro activity of daptomycin against Gram-positive bacteria including enterococci, 2) Compare the activity of daptomycin to that of vancomycin and teicoplanin, 3) Assess the daptomycin interpretive criteria in light of the current recommendation to use CAMHB (ca. 25 g/ml Ca⫹⫹) instead of CSMHB (ca. 50 g/ml Ca⫹⫹) for MIC determinations, and 4) Determine the daptomycin quality control (QC) ranges that can be used to evaluate the suitability of Mueller-Hinton broth or agar lots for testing daptomycin.
2. Materials and methods 2.1. Microorganisms A total of 844 Gram-positive bacterial isolates, representing 14 species were tested (Table 1). A large proportion of antibiotic-resistant strains was selected, including 50 VRE, 147 MRS, 52 PRP, and 111 penicillin-intermediate pneumococci. On each day of testing appropriate QC strains were tested and all results were within the published NCCLS QC ranges (NCCLS, 1999).
g/ml of Ca⫹⫹ at two separate times; MICs were determined simultaneously both times. 2.4. Quality Control Study This study was conducted in the ten different laboratories, listed in the Acknowledgments. Daptomycin MICs were determined by the broth microdilution method with S. aureus ATCC 29213, E. faecalis ATCC 29212, and S. pneumoniae ATCC 49619 on each of ten different days, following the NCCLS procedure (1997b). Each tray contained daptomycin diluted in 6 different lots of CAMHB, each with additional Ca⫹⫹ (50 g/ml) and vancomycin in two lots of CAMHB. Thus, each laboratory produced a total of 60 MIC results for daptomycin and 20 for vancomycin. Daptomycin zone diameter measurements were recorded for S. aureus ATCC 25923 and S. pneumoniae ATCC 49619 on 3 lots of MHA (Ca⫹⫹ content of 28 to 32 g/ml) using 2 lots of commercially prepared daptomycin 30 g disks on each of ten different days. Each laboratory followed the NCCLS procedure for disk diffusion tests (NCCLS 1997a) to generate 60 zone diameter measurements for daptomycin and 30 for vancomycin. 3. Results 3.1. Effect of Calcium
2.2. Antimicrobial Agents Daptomycin was provided by Cubist Pharmaceuticals, Inc., Cambridge, MA. The comparison drugs were procured from their U.S. manufacturer or other commercial sources. For disk diffusion tests commercially prepared 30 g daptomycin and vancomycin disks were used. 2.3. Test Procedures MICs were determined by broth microdilution, following the procedure outlined by the NCCLS (1997b). The CAMHB was supplemented with 3% lysed horse blood when testing streptococci, Listeria monocytogenes or Corynebacterium jeikeium. In addition to tests in CAMHB, daptomycin was also tested in MHB supplemented to contain 50 and 75 g/ml of Ca⫹⫹. Drug concentrations tested were serial twofold dilutions ranging from 32 to 0.002 g/ml for daptomycin and 64 to 0.004 g/ml for teicoplanin and vancomycin. Disk diffusion tests were performed by the method outlined by the NCCLS (1997a). When testing streptococci, L. monocytogenes, and C. jeikeium, the Mueller-Hinton agar (MHA) was supplemented with 5% defibrinated sheep blood. Six lots of MHA meeting the M-6 standard of the NCCLS (1996) were assayed for Ca⫹⫹ concentration; five of these had levels ranging from 28 to 36 g/ml, but the sixth contained only 6 g/ml. Disk diffusion tests were performed on the latter lot as well as a lot containing 28
The effect of three concentrations of Ca⫹⫹ on daptomyin MICs is summarized in Table 1. Against all species daptomycin was significantly more active in 50 g/ml than in 25 g/ml of Ca⫹⫹. When tested in 75 g/ml of Ca⫹⫹ there was a further increase in daptomycin activity for some species, but this was generally much less than the differences between 25 and 50 g/ml of Ca⫹⫹. For other species there was no increase in activity when tested in 75 g/ml of Ca⫹⫹ (e.g., streptococci). If one accepts the previously proposed susceptible MIC breakpoint for daptomycin of ⱕ 2.0 g/ml (Jones and Barry 1987), tests in 25 and 50 g/ml of Ca⫹⫹ generally yielded results with the same interpretive categories for all but two species –Enterococcus faecium and L. monocytogenes. In the case of E. faecium, 24% of 62 strains were not susceptible to daptomycin when tested in 25 g/ml of Ca⫹⫹, but all were susceptible when tested in 50 g/ml of Ca⫹⫹ . Fig. 1 is a scattergram showing the daptomycin 30 g disk diffusion zone diameter distribution for 289 isolates (189 staphylococci and 100 enterococci) tested on two lots of MHA, one containing 6 g/ml and the other 28 g/ml of Ca⫹⫹. The zone diameters on the medium with 28 g/ml of Ca⫹⫹ were 1 to 15 mm (mean, 6 mm) larger than those on the medium with 6 g/ml of Ca⫹⫹. 3.2. Comparative Activity Table 1 summarizes the activity of teicoplanin and vancomycin compared to that of daptomycin. In 50 g/ml of
P.C.Fuchs et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
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Fig. 1. Daptomycin Zone Diameters on MHA with 6 vs. 28 g/ml of CA⫹⫹
Ca⫹⫹, daptomycin had the lowest MICs of the three drugs against staphylococci; 100% of these 227 strains were susceptible to daptomycin, and 96% were susceptible to teicoplanin. While teicoplanin was more active than daptomycin against vancomycin-susceptible enterococci, daptomycin was the most active against VRE. Streptococci were very susceptible to all three drugs. Daptomycin had poor activity against L. monocytogenes (12% susceptible), but was the most active agent against C. jeikeium with a geometric mean MIC of 0.25 g/ml.
g/ml or zones ⱖ16 mm for susceptible, and MIC ⱖ8.0 g/ml or zones ⱕ12 mm for resistant) (Jones & Barry, 1987) are shown. There were no major or very major discrepancies, and 13 (2.8%) minor discrepancies. Twelve of the 13 minor discrepancies included enterococci with intermediate MICs and susceptible zone diameters. The overall low discrepancy rate is due in large part to the absence of resistant organisms.
3.3. Interpretive Criteria
With the MIC tests one of the 6 lots of broth gave very poor growth of S. aureus ATCC 29213 and low to off-scale endpoints. Consequently, that lot was deleted from subsequent calculations for this strain. One of the ten laboratories was “out of control” with vancomycin vs. E. faecalis ATCC 29212 on all ten test days, and the daptomycin data from
A scattergram of daptomycin MICs (in 50 g/ml of Ca⫹⫹) and disk diffusion zone diameters (on media with 28 g/ml of Ca⫹⫹) for enterococci and staphylococci is shown in Fig. 2. Previously proposed breakpoints (MIC ⱕ2.0
3.4. Quality Control Ranges
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Table 1 In vitro activity of daptomycin in 3 Ca⫹⫹ concentrations compared to vancomycin and teicoplanin MICs expressed in g/ml Antimicrobial agenta
Range
50%
90%
Geometric mean
% Susceptibleb
Microorganism
No.
Staphylococcus aureus Methicillin-Resistant
51
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.5–1.0 0.25–2.0 0.06–2.0 0.5–2.0 0.12–2.0
1.0 0.5 0.25 1.0 0.5
1.0 1.0 0.5 2.0 1.0
0.96 0.47 0.29 0.90 0.48
100 100 100 100 100
Staphylococcus aureus Methicillin-Susceptible
57
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.5–1.0 0.12–2.0 0.06–2.0 0.5–2.0 0.25–2.0
1.0 0.5 0.25 1.0 0.5
1.0 1.0 1.0 1.0 1.0
0.94 0.45 0.32 0.68 0.53
100 100 100 100 100
S. haemolyticus
20
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.03–2.0 0.03–1.0 0.03–1.0 0.5–4.0 0.25–⬎64
0.5 0.25 0.25 1.0 1.0
1.0 0.5 0.5 2.0 16
0.57 0.34 0.29 1.19 3.86
100 100 100 100 80.0
Coag-neg Staphylococci other species Methicillin-Resistant
61
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.004–4.0 0.004–1.0 0.03–1.0 0.25–4.0 0.12–64
1.0 0.5 0.25 1.0 2.0
1.0 1.0 0.5 2.0 8.0
0.99 0.35 0.23 1.46 2.52
98.4 100 100 100 91.2
Coag-neg Staphylococci other species Methicillin-Susceptible
38
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.03–2.0 0.03–0.5 0.03–0.5 0.5–4.0 0.12–16
1.0 0.25 0.25 2.0 1.0
2.0 0.5 0.5 2.0 4.0
0.97 0.33 0.17 1.43 1.61
100 100 100 100 94.7
Enterococcus faecalis Vancomycin-Resistant
14
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
1.0–1.0 0.5–1.0 0.5–1.0 8.0–⬎64 0.12–⬎64
1.0 1.0 0.5 ⬎64 0.25
1.0 1.0 1.0 ⬎64 64
1.00 0.78 0.64 60.9 0.82
100 100 100 0 78.6
Enterococcus faecalis Vancomycin-Susceptible
36
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–1.0 0.06–1.0 0.06–1.0 0.5–4.0 0.06–0.25
1.0 1.0 0.5 1.0 0.25
Enterococcus faecium Vancomycin-Resistant
34
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
1.0–16 1.0–2.0 1.0–2.0 32–⬎64 0.25–⬎64
Enterococcus faecium Vancomycin-Susceptible
28
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
Listeria monocytogenes
25
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
1.0 1.0 1.0 2.0 0.25
0.92 0.63 0.41 1.36 0.17
100 100 100 100 100
4.0 1.0 1.0 ⬎64 64
8.0 2.0 1.0 ⬎64 ⬎64
4.08 1.15 1.02 118 8.68
14.7 100 100 0 44.1
1.0–16 0.5–2.0 0.5–1.0 0.5–4.0 0.12–1.0
4.0 1.0 1.0 1.0 0.5
8.0 2.0 1.0 2.0 1.0
3.05 1.08 0.95 1.05 0.51
35.7 100 100 100 100
4.0–16 2.0–8.0 2.0–4.0 1.0–1.0 0.25–0.5
8.0 4.0 4.0 1.0 0.5
16 4.0 4.0 1.0 0.5
8.69 3.89 3.12 1.00 0.47
0 12.0 40.0 100 100
(continued on next page)
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Table 1 (continued) MICs expressed in g/ml Microorganism
No.
Antimicrobial agenta
Range
50%
90%
Geometric mean
% Susceptibleb
Corynebacterium jeikeium
21
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.06–2.0 0.12–0.5 0.06–0.5 0.12–1.0 0.25–4.0
0.5 0.25 0.25 0.5 0.5
2.0 0.5 0.5 0.5 2.0
0.67 0.26 0.27 0.44 0.65
100 100 100 100 100
Streptococcus pyogenes
51
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.06–1.0 0.015–0.5 0.03–0.5 0.25–0.5 0.03–0.25
0.06 0.03 0.03 0.25 0.06
0.12 0.06 0.06 0.5 0.12
0.08 0.03 0.04 0.27 0.07
100 100 100 100 100
Streptococcus agalactiae
31
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–1.0 0.12–0.25 0.06–0.5 0.25–0.5 0.015–0.25
0.5 0.25 0.25 0.5 0.12
1.0 0.25 0.25 0.5 0.25
0.57 0.22 0.22 0.46 0.13
100 100 100 100 100
Group C Streptococci
9
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.06–1.0 0.03–0.25 0.06–0.5 0.25–0.5 0.015–0.25
0.5 0.12 0.25 0.5 0.06
0.34 0.11 0.15 0.43 0.07
100 100 100 100 100
Group G Streptococci
10
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.03–0.25 0.015–0.06 0.015–0.25 0.25–0.5 0.06–0.5
0.06 0.03 0.03 0.25 0.06
0.25 0.06 0.06 0.25 0.25
0.09 0.03 0.04 0.27 0.11
100 100 100 100 100
Streptococcus pneumoniae Penicillin-Susceptible
158
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.25–1.0 0.06–0.25 0.06–0.25 0.03–0.5 0.015–0.12
0.25 0.12 0.12 0.25 0.06
0.5 0.12 0.25 0.5 0.06
0.31 0.10 0.12 0.25 0.06
100 100 100 100 100
Streptococcus pneumoniae Penicillin-Intermediate
111
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–2.0 0.06–0.5 0.06–0.5 0.06–0.5 0.03–1.0
0.25 0.12 0.12 0.25 0.06
0.5 0.25 0.25 0.5 0.12
0.37 0.12 0.14 0.28 0.07
100 100 100 100 100
Streptococcus pneumoniae Penicillin-Resistant
52
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.25–1.0 0.06–0.25 0.12–0.25 0.25–0.5 0.06–0.12
0.5 0.12 0.12 0.25 0.06
0.5 0.25 0.25 0.5 0.12
0.42 0.14 0.15 0.31 0.08
100 100 100 100 100
Viridans Streptococci
37
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–2.0 0.12–2.0 0.06–1.0 0.5–1.0 0.03–1.0
1.0 0.25 0.25 0.5 0.12
2.0 1.0 1.0 1.0 0.25
0.72 0.29 0.30 0.58 0.11
100 100 100 100 100
a b
Numbers following Daptomycin indicate Ca⫹⫹ concentration in g/ml: vancomycin and teicoplanin were tested in CAMHB. NCCLS susceptible breakpoints used for vancomycin and teicoplanin; ⱕ2.0 g/ml for daptomycin susceptibility.
this laboratory were omitted from subsequent calculations for this strain. Fig. 3 displays the distribution of daptomycin MICs and
zone diameters along with the proposed QC ranges for each control strain. These proposed ranges have been approved by the NCCLS.
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Fig. 2. Daptomycin Disk Test vs. MICs with 50 g/ml of CA⫹⫹
4. Discussion The effect of Ca⫹⫹ on daptomycin broth dilution MICs has been well-documented previously (Eliopoulos et al. 1986, Greenwood & Palfreyman 1987, Louie et al. 1992), and our data confirm that daptomycin MICs are generally twofold to fourfold lower when tested in broth containing 50 g/ml of Ca⫹⫹ than when tested in 25 g/ml of Ca⫹⫹. To our knowledge, it has not been previously demonstrated that Ca⫹⫹ levels in MHA can significantly affect the daptomycin disk diffusion zone diameters. In this study, daptomycin zone diameters on MHA containing 6 g/ml of Ca⫹⫹ averaged 6 mm smaller than those on MHA with 28 g/ml of Ca⫹⫹. It cannot be assumed that MHA lots that meet the per-
formance standards of the NCCLS (M6-A, 1996) have the same chemical composition. We assayed the calcium levels of 6 lots of NCCLS compliant MHA from three different manufacturers and obtained concentrations of 28, 32, 32, 28, 36, and 6 g/ml. The daptomycin zone diameters with S. aureus ATCC 29213 were 20 to 21 mm on the first five lots (middle of QC range) and 14 mm for the lot with 6 g/ml of Ca⫹⫹ (well below the QC range). Since daptomycin is not currently included among the drugs tested for the M-6A protocol, it becomes even more important for laboratories that wish to test daptomycin by disk diffusion to test each lot of MHA with S. aureus ATCC 29213. It is noteworthy that the gentamicin zone diameters for Pseudomonas aeruginosa ATCC 27853 and tetracycline zones with S. aureus ATCC 25923 (both known to be cation depen-
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57
Fig. 3. Ten laboratory distribution of Daptomycin MICs and zone diameters for QC organisms.a
dent) were within 1 mm of each other on all six of the aforementioned MHA lots. We have tentatively adopted the daptomycin MIC breakpoints previously proposed by Jones and Barry (1987) and recommend that for broth dilution tests of daptomycin the medium should contain physiological concentrations of Ca⫹⫹ (45–55 g/ml) (Eliopoulos et al., 1986, Jones & Barry, 1987). We feel this is reasonable until clinical data become available. Although daptomycin is highly (90 –95%) protein-bound (Lee et al. 1991, Ryback et al. 1992, Woodworth et al. 1992), serum levels were linearly related to dose, and a Cmax of 82 g/ml was achieved with a single dose of 6 mg/kg in healthy volunteers (Woodworth et al. 1992). Thus, from a pharmacokinetic viewpoint, a daptomycin susceptible MIC breakpoint of ⱕ2.0 g/ml does not seem to be unreasonable at this time. This breakpoint is also supported by the MIC population distribution; a breakpoint lower than this (e.g., 1.0 g/ml) would cut through the middle of the enterococcal MIC distribution. In any event, the final decision on daptomycin breakpoints must await the results of clinical trials, particularly with enterococcal infections. As for the zone diameter breakpoints, those proposed by Jones & Barry (1987) performed satisfactorily with our data. However, the absence of daptomycin-resistant isolates in this data set forces us to continue to consider them tentative. The in vitro activity of daptomycin against multi-resistant Gram-positive bacteria such as VRE and MRS is noteworthy, and because of the therapeutic dilemmas caused by infections with such strains, further development of this agent would appear justified.
Acknowledgments We gratefully acknowledge the participation of the following in the ten-laboratory quality control study: Mary Bauman, St. Vincent’s Hospital, Portland, OR; Mary Jane Ferraro, Massachusetts General Hospital, Boston, MA; Dwight Hardy, University of Rochester Medical Center, Rochester, NY; Janet Hindler, UCLA Medical Center, Los Angeles, CA; Stephen Jenkins, Carolinas Medical Center, Charlotte, NC; Cindy Knapp, AccuMed International, Westlake, OH; Gary Overturf, University of New Mexico Medical Center, Albuquerque, NM; Robert Rennie, University of Alberta Hospital, Edmonton, Alberta; Michael Saubolle, Good Samaritan Hospital, Phoenix, AZ. We also wish to thank Cubist Pharmaceuticals Inc. for financial subsidy of these studies.
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National Committee for Clinical Laboratory Standards (NCCLS). (1997a). Performance Standards for Antimicrobial Disk Susceptibility Tests, 6th Ed, Approved Standard M2–A6. Wayne, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS). (1997b). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 4th Ed. Approved Standard M7–A4. Wayne, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS), (1999). Performance Standards for Antimicrobial Susceptibility Testing, Ninth Informational Supplement M100 –S9. Wayne, PA: NCCLS. Oleson, F. B. Jr, Berman, C. L., Kirkpatrick, J. B., Regan, K. S., Lai J-J., & Tally, F. P. (1999). Once-daily dosing decreases toxicity of daptomycin. Toxicol Sci, 48, 322. Pohlod, D. J., Saravolatz, L. D., & Somerville, M. M. (1987). In-vitro susceptibility of gram-positive cocci to LY146032, teicoplanin, sodium fusidate, vancomycin and rifampicin. J Antimicrob Chemother, 20, 197–202. Ramos, M. C., Grayson, M. L., Elioupoulos, G. M., & Bayer, A. S. (1992). Comparison of daptomycin, vancomycin and ampicillin-gentamicin for treatment of experimental endocarditis caused by penicillin-resistant enterococci. Antimicrob Agents Chemother, 36, 1864 –2353. Ryback, M. J., Bailey, E. M., Lamp, K. C., & Kaatz, G. W. (1992). Pharmacokinetics and bactericidal rates of daptomycin and vancomycin in intravenous drug abusers being treated for gram-positive endocarditis and bacteremia. Antimicrob Agents Chemother, 36, 1109 –1114. Swenson, J. M., Facklam, R. R., & Thornsberry, C. (1990). Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus and Lactobacillus species. Antimicrob Agents Chemother, 34, 543–549. Tally, F. P., Zeckel, M., Wasilewski, M. M., Carini, C., Berman, C. L., Drusano, G. L., & Oleson, F. B. Jr. (1999). Daptomycin: a novel agent for Gram-positive infections. Exp Opin Invest Drugs, 8, 1223–1238. Woodworth, J. R., Nyhart, E. H. Jr, Brier, G. L., Wolny, J. D., & Black, H. R. (1992). Single-dose pharmacokinetics and antibacterial activity of daptomycin, a new lipopeptide antibiotic, in healthy volunteers. Antimicrob Agents Chemother, 36, 318 –325.
www.elsevier.com/locate/diagmicrobio
Daptomycin susceptibility tests: interpretive criteria, quality control, and effect of calcium on in vitro tests Peter C. Fuchs*, Arthur L. Barry, Steven D. Brown The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA Received 21 December 1999; received in revised form 16 June 2000; accepted 16 June 2000
Abstract Daptomycin MICs were determined for 844 Gram-positive bacteria in three concentrations of Ca⫹⫹ and compared with the MICs of vancomycin and teicoplanin. Daptomycin was twofold to fourfold more active against most species when tested in 50 g/ml of Ca⫹⫹ than in 25 g/ml. In 50 g/ml of Ca⫹⫹ daptomycin was more active against methicillin-resistant staphylococci and vancomycin-resistant enterococci than teicoplanin or vancomycin; 100% of these isolates were susceptible to ⱕ2.0 g/ml of daptomycin. Different lots of Mueller-Hinton agar were variable in Ca⫹⫹ content, and daptomycin disk diffusion zone diameters were affected, i.e., zones were 1 to 15 mm smaller on one lot of agar with only 6 g/ml of Ca⫹⫹ compared to another lot with 28 g/ml. The previously proposed daptomycin interpretive breakpoints performed satisfactorily when MICs were determined in Mueller-Hinton broth with 50 g/ml of Ca⫹⫹ and when the agar gave appropriate zones with quality control strains. To define those control limits, replicate tests with four quality control strains were performed in ten laboratories using broth microdilution tests (with Ca⫹⫹ supplemented broth) and disk diffusion tests on MuellerHinton agar without cation adjustments. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Daptomycin is a cyclic lipopeptide antibiotic that has been reported to have good antimicrobial activity against most Gram-positive bacteria (Appelbaum et al. 1989, Eliopoulos et al. 1986, Greenwood & Palfreyman 1987, Low et al. 1989, Pohlod et al. 1987, Swenson et al. 1990). Despite encouraging results with daptomycin in experimental animals (Bryant et al. 1987, Cantoni et al. 1990, Ramos et al. 1992), further development of the drug was suspended after early clinical trials showed that relatively low doses of daptomycin (total of 2 mg/kg/day) were less effective than conventional therapy (Chambers, 1991) and high doses of 4 mg/kg q12h (8 mg/kg/day) produced muscle toxicity in a few patients. In animal studies this muscle toxicity has been shown to be significantly reduced by once-daily dosing (Oleson et al., 1999). Furthermore, since the Cmax has been shown to be the major pharmacokinetic parameter associated with infection eradication in animal efficacy models * Corresponding author. Tel.: ⫹1-503-682-3232; fax: ⫹1-503-6822065. E-mail address: [email protected] (P.C.Fuchs). A portion of this material was previously presented at the 39th ICAAC in San Francisco, California, September 26 –29, 1999. Abstract No. 350.
(Leggett et al., 1987), once-daily dosing could be more effective. With the increasing prevalence of vancomycinresistant enterococci (VRE), methicillin-resistant staphylococci (MRS) and penicillin-resistant pneumococci (PRP), there has been renewed interest in developing daptomycin for clinical use. Daptomycin Phase II trials using once-daily dosing are currently in progress with encouraging preliminary safety results (Tally et al., 1999). The in vitro activity of daptomycin is enhanced by increased concentrations of Ca⫹⫹ when tested in broth (Eliopoulos et al. 1986, Greenwood & Palfreyman 1987, Louie et al. 1992). The interpretive criteria for daptomycin that were proposed in 1987 (Jones & Barry 1987) were based on MIC tests performed in cation-supplemented Mueller-Hinton broth (CSMHB) containing 50 g/ml of Ca⫹⫹, as recommended at that time by the National Committee for Clinical Laboratory Standards (NCCLS) (1985). Since then the NCCLS has revised its recommendations, and currently recommends the use of cation-adjusted Mueller-Hinton broth (CAMHB) containing 20 –25 g/ml of Ca⫹⫹ for routine susceptibility testing (NCCLS 1997b). Jones (1989) suggested that when testing staphylococci, daptomycin MICs were higher when CAMHB was used, but the interpretive criteria developed in CSMHB could still be applied as all isolates were susceptible in both concentrations of Ca⫹⫹.
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Enterococci were not a concern when that study was undertaken and thus only staphylococci were evaluated. The present studies were designed to: 1) Confirm the effects of Ca⫹⫹ on in vitro activity of daptomycin against Gram-positive bacteria including enterococci, 2) Compare the activity of daptomycin to that of vancomycin and teicoplanin, 3) Assess the daptomycin interpretive criteria in light of the current recommendation to use CAMHB (ca. 25 g/ml Ca⫹⫹) instead of CSMHB (ca. 50 g/ml Ca⫹⫹) for MIC determinations, and 4) Determine the daptomycin quality control (QC) ranges that can be used to evaluate the suitability of Mueller-Hinton broth or agar lots for testing daptomycin.
2. Materials and methods 2.1. Microorganisms A total of 844 Gram-positive bacterial isolates, representing 14 species were tested (Table 1). A large proportion of antibiotic-resistant strains was selected, including 50 VRE, 147 MRS, 52 PRP, and 111 penicillin-intermediate pneumococci. On each day of testing appropriate QC strains were tested and all results were within the published NCCLS QC ranges (NCCLS, 1999).
g/ml of Ca⫹⫹ at two separate times; MICs were determined simultaneously both times. 2.4. Quality Control Study This study was conducted in the ten different laboratories, listed in the Acknowledgments. Daptomycin MICs were determined by the broth microdilution method with S. aureus ATCC 29213, E. faecalis ATCC 29212, and S. pneumoniae ATCC 49619 on each of ten different days, following the NCCLS procedure (1997b). Each tray contained daptomycin diluted in 6 different lots of CAMHB, each with additional Ca⫹⫹ (50 g/ml) and vancomycin in two lots of CAMHB. Thus, each laboratory produced a total of 60 MIC results for daptomycin and 20 for vancomycin. Daptomycin zone diameter measurements were recorded for S. aureus ATCC 25923 and S. pneumoniae ATCC 49619 on 3 lots of MHA (Ca⫹⫹ content of 28 to 32 g/ml) using 2 lots of commercially prepared daptomycin 30 g disks on each of ten different days. Each laboratory followed the NCCLS procedure for disk diffusion tests (NCCLS 1997a) to generate 60 zone diameter measurements for daptomycin and 30 for vancomycin. 3. Results 3.1. Effect of Calcium
2.2. Antimicrobial Agents Daptomycin was provided by Cubist Pharmaceuticals, Inc., Cambridge, MA. The comparison drugs were procured from their U.S. manufacturer or other commercial sources. For disk diffusion tests commercially prepared 30 g daptomycin and vancomycin disks were used. 2.3. Test Procedures MICs were determined by broth microdilution, following the procedure outlined by the NCCLS (1997b). The CAMHB was supplemented with 3% lysed horse blood when testing streptococci, Listeria monocytogenes or Corynebacterium jeikeium. In addition to tests in CAMHB, daptomycin was also tested in MHB supplemented to contain 50 and 75 g/ml of Ca⫹⫹. Drug concentrations tested were serial twofold dilutions ranging from 32 to 0.002 g/ml for daptomycin and 64 to 0.004 g/ml for teicoplanin and vancomycin. Disk diffusion tests were performed by the method outlined by the NCCLS (1997a). When testing streptococci, L. monocytogenes, and C. jeikeium, the Mueller-Hinton agar (MHA) was supplemented with 5% defibrinated sheep blood. Six lots of MHA meeting the M-6 standard of the NCCLS (1996) were assayed for Ca⫹⫹ concentration; five of these had levels ranging from 28 to 36 g/ml, but the sixth contained only 6 g/ml. Disk diffusion tests were performed on the latter lot as well as a lot containing 28
The effect of three concentrations of Ca⫹⫹ on daptomyin MICs is summarized in Table 1. Against all species daptomycin was significantly more active in 50 g/ml than in 25 g/ml of Ca⫹⫹. When tested in 75 g/ml of Ca⫹⫹ there was a further increase in daptomycin activity for some species, but this was generally much less than the differences between 25 and 50 g/ml of Ca⫹⫹. For other species there was no increase in activity when tested in 75 g/ml of Ca⫹⫹ (e.g., streptococci). If one accepts the previously proposed susceptible MIC breakpoint for daptomycin of ⱕ 2.0 g/ml (Jones and Barry 1987), tests in 25 and 50 g/ml of Ca⫹⫹ generally yielded results with the same interpretive categories for all but two species –Enterococcus faecium and L. monocytogenes. In the case of E. faecium, 24% of 62 strains were not susceptible to daptomycin when tested in 25 g/ml of Ca⫹⫹, but all were susceptible when tested in 50 g/ml of Ca⫹⫹ . Fig. 1 is a scattergram showing the daptomycin 30 g disk diffusion zone diameter distribution for 289 isolates (189 staphylococci and 100 enterococci) tested on two lots of MHA, one containing 6 g/ml and the other 28 g/ml of Ca⫹⫹. The zone diameters on the medium with 28 g/ml of Ca⫹⫹ were 1 to 15 mm (mean, 6 mm) larger than those on the medium with 6 g/ml of Ca⫹⫹. 3.2. Comparative Activity Table 1 summarizes the activity of teicoplanin and vancomycin compared to that of daptomycin. In 50 g/ml of
P.C.Fuchs et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
53
Fig. 1. Daptomycin Zone Diameters on MHA with 6 vs. 28 g/ml of CA⫹⫹
Ca⫹⫹, daptomycin had the lowest MICs of the three drugs against staphylococci; 100% of these 227 strains were susceptible to daptomycin, and 96% were susceptible to teicoplanin. While teicoplanin was more active than daptomycin against vancomycin-susceptible enterococci, daptomycin was the most active against VRE. Streptococci were very susceptible to all three drugs. Daptomycin had poor activity against L. monocytogenes (12% susceptible), but was the most active agent against C. jeikeium with a geometric mean MIC of 0.25 g/ml.
g/ml or zones ⱖ16 mm for susceptible, and MIC ⱖ8.0 g/ml or zones ⱕ12 mm for resistant) (Jones & Barry, 1987) are shown. There were no major or very major discrepancies, and 13 (2.8%) minor discrepancies. Twelve of the 13 minor discrepancies included enterococci with intermediate MICs and susceptible zone diameters. The overall low discrepancy rate is due in large part to the absence of resistant organisms.
3.3. Interpretive Criteria
With the MIC tests one of the 6 lots of broth gave very poor growth of S. aureus ATCC 29213 and low to off-scale endpoints. Consequently, that lot was deleted from subsequent calculations for this strain. One of the ten laboratories was “out of control” with vancomycin vs. E. faecalis ATCC 29212 on all ten test days, and the daptomycin data from
A scattergram of daptomycin MICs (in 50 g/ml of Ca⫹⫹) and disk diffusion zone diameters (on media with 28 g/ml of Ca⫹⫹) for enterococci and staphylococci is shown in Fig. 2. Previously proposed breakpoints (MIC ⱕ2.0
3.4. Quality Control Ranges
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P.C.Fuchs et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
Table 1 In vitro activity of daptomycin in 3 Ca⫹⫹ concentrations compared to vancomycin and teicoplanin MICs expressed in g/ml Antimicrobial agenta
Range
50%
90%
Geometric mean
% Susceptibleb
Microorganism
No.
Staphylococcus aureus Methicillin-Resistant
51
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.5–1.0 0.25–2.0 0.06–2.0 0.5–2.0 0.12–2.0
1.0 0.5 0.25 1.0 0.5
1.0 1.0 0.5 2.0 1.0
0.96 0.47 0.29 0.90 0.48
100 100 100 100 100
Staphylococcus aureus Methicillin-Susceptible
57
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.5–1.0 0.12–2.0 0.06–2.0 0.5–2.0 0.25–2.0
1.0 0.5 0.25 1.0 0.5
1.0 1.0 1.0 1.0 1.0
0.94 0.45 0.32 0.68 0.53
100 100 100 100 100
S. haemolyticus
20
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.03–2.0 0.03–1.0 0.03–1.0 0.5–4.0 0.25–⬎64
0.5 0.25 0.25 1.0 1.0
1.0 0.5 0.5 2.0 16
0.57 0.34 0.29 1.19 3.86
100 100 100 100 80.0
Coag-neg Staphylococci other species Methicillin-Resistant
61
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.004–4.0 0.004–1.0 0.03–1.0 0.25–4.0 0.12–64
1.0 0.5 0.25 1.0 2.0
1.0 1.0 0.5 2.0 8.0
0.99 0.35 0.23 1.46 2.52
98.4 100 100 100 91.2
Coag-neg Staphylococci other species Methicillin-Susceptible
38
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.03–2.0 0.03–0.5 0.03–0.5 0.5–4.0 0.12–16
1.0 0.25 0.25 2.0 1.0
2.0 0.5 0.5 2.0 4.0
0.97 0.33 0.17 1.43 1.61
100 100 100 100 94.7
Enterococcus faecalis Vancomycin-Resistant
14
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
1.0–1.0 0.5–1.0 0.5–1.0 8.0–⬎64 0.12–⬎64
1.0 1.0 0.5 ⬎64 0.25
1.0 1.0 1.0 ⬎64 64
1.00 0.78 0.64 60.9 0.82
100 100 100 0 78.6
Enterococcus faecalis Vancomycin-Susceptible
36
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–1.0 0.06–1.0 0.06–1.0 0.5–4.0 0.06–0.25
1.0 1.0 0.5 1.0 0.25
Enterococcus faecium Vancomycin-Resistant
34
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
1.0–16 1.0–2.0 1.0–2.0 32–⬎64 0.25–⬎64
Enterococcus faecium Vancomycin-Susceptible
28
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
Listeria monocytogenes
25
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
1.0 1.0 1.0 2.0 0.25
0.92 0.63 0.41 1.36 0.17
100 100 100 100 100
4.0 1.0 1.0 ⬎64 64
8.0 2.0 1.0 ⬎64 ⬎64
4.08 1.15 1.02 118 8.68
14.7 100 100 0 44.1
1.0–16 0.5–2.0 0.5–1.0 0.5–4.0 0.12–1.0
4.0 1.0 1.0 1.0 0.5
8.0 2.0 1.0 2.0 1.0
3.05 1.08 0.95 1.05 0.51
35.7 100 100 100 100
4.0–16 2.0–8.0 2.0–4.0 1.0–1.0 0.25–0.5
8.0 4.0 4.0 1.0 0.5
16 4.0 4.0 1.0 0.5
8.69 3.89 3.12 1.00 0.47
0 12.0 40.0 100 100
(continued on next page)
P.C.Fuchs et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
55
Table 1 (continued) MICs expressed in g/ml Microorganism
No.
Antimicrobial agenta
Range
50%
90%
Geometric mean
% Susceptibleb
Corynebacterium jeikeium
21
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.06–2.0 0.12–0.5 0.06–0.5 0.12–1.0 0.25–4.0
0.5 0.25 0.25 0.5 0.5
2.0 0.5 0.5 0.5 2.0
0.67 0.26 0.27 0.44 0.65
100 100 100 100 100
Streptococcus pyogenes
51
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.06–1.0 0.015–0.5 0.03–0.5 0.25–0.5 0.03–0.25
0.06 0.03 0.03 0.25 0.06
0.12 0.06 0.06 0.5 0.12
0.08 0.03 0.04 0.27 0.07
100 100 100 100 100
Streptococcus agalactiae
31
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–1.0 0.12–0.25 0.06–0.5 0.25–0.5 0.015–0.25
0.5 0.25 0.25 0.5 0.12
1.0 0.25 0.25 0.5 0.25
0.57 0.22 0.22 0.46 0.13
100 100 100 100 100
Group C Streptococci
9
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.06–1.0 0.03–0.25 0.06–0.5 0.25–0.5 0.015–0.25
0.5 0.12 0.25 0.5 0.06
0.34 0.11 0.15 0.43 0.07
100 100 100 100 100
Group G Streptococci
10
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.03–0.25 0.015–0.06 0.015–0.25 0.25–0.5 0.06–0.5
0.06 0.03 0.03 0.25 0.06
0.25 0.06 0.06 0.25 0.25
0.09 0.03 0.04 0.27 0.11
100 100 100 100 100
Streptococcus pneumoniae Penicillin-Susceptible
158
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.25–1.0 0.06–0.25 0.06–0.25 0.03–0.5 0.015–0.12
0.25 0.12 0.12 0.25 0.06
0.5 0.12 0.25 0.5 0.06
0.31 0.10 0.12 0.25 0.06
100 100 100 100 100
Streptococcus pneumoniae Penicillin-Intermediate
111
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–2.0 0.06–0.5 0.06–0.5 0.06–0.5 0.03–1.0
0.25 0.12 0.12 0.25 0.06
0.5 0.25 0.25 0.5 0.12
0.37 0.12 0.14 0.28 0.07
100 100 100 100 100
Streptococcus pneumoniae Penicillin-Resistant
52
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.25–1.0 0.06–0.25 0.12–0.25 0.25–0.5 0.06–0.12
0.5 0.12 0.12 0.25 0.06
0.5 0.25 0.25 0.5 0.12
0.42 0.14 0.15 0.31 0.08
100 100 100 100 100
Viridans Streptococci
37
Daptomycin-25 Daptomycin-50 Daptomycin-75 Vancomycin Teicoplanin
0.12–2.0 0.12–2.0 0.06–1.0 0.5–1.0 0.03–1.0
1.0 0.25 0.25 0.5 0.12
2.0 1.0 1.0 1.0 0.25
0.72 0.29 0.30 0.58 0.11
100 100 100 100 100
a b
Numbers following Daptomycin indicate Ca⫹⫹ concentration in g/ml: vancomycin and teicoplanin were tested in CAMHB. NCCLS susceptible breakpoints used for vancomycin and teicoplanin; ⱕ2.0 g/ml for daptomycin susceptibility.
this laboratory were omitted from subsequent calculations for this strain. Fig. 3 displays the distribution of daptomycin MICs and
zone diameters along with the proposed QC ranges for each control strain. These proposed ranges have been approved by the NCCLS.
56
P.C.Fuchs et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
Fig. 2. Daptomycin Disk Test vs. MICs with 50 g/ml of CA⫹⫹
4. Discussion The effect of Ca⫹⫹ on daptomycin broth dilution MICs has been well-documented previously (Eliopoulos et al. 1986, Greenwood & Palfreyman 1987, Louie et al. 1992), and our data confirm that daptomycin MICs are generally twofold to fourfold lower when tested in broth containing 50 g/ml of Ca⫹⫹ than when tested in 25 g/ml of Ca⫹⫹. To our knowledge, it has not been previously demonstrated that Ca⫹⫹ levels in MHA can significantly affect the daptomycin disk diffusion zone diameters. In this study, daptomycin zone diameters on MHA containing 6 g/ml of Ca⫹⫹ averaged 6 mm smaller than those on MHA with 28 g/ml of Ca⫹⫹. It cannot be assumed that MHA lots that meet the per-
formance standards of the NCCLS (M6-A, 1996) have the same chemical composition. We assayed the calcium levels of 6 lots of NCCLS compliant MHA from three different manufacturers and obtained concentrations of 28, 32, 32, 28, 36, and 6 g/ml. The daptomycin zone diameters with S. aureus ATCC 29213 were 20 to 21 mm on the first five lots (middle of QC range) and 14 mm for the lot with 6 g/ml of Ca⫹⫹ (well below the QC range). Since daptomycin is not currently included among the drugs tested for the M-6A protocol, it becomes even more important for laboratories that wish to test daptomycin by disk diffusion to test each lot of MHA with S. aureus ATCC 29213. It is noteworthy that the gentamicin zone diameters for Pseudomonas aeruginosa ATCC 27853 and tetracycline zones with S. aureus ATCC 25923 (both known to be cation depen-
P.C.Fuchs et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 51–58
57
Fig. 3. Ten laboratory distribution of Daptomycin MICs and zone diameters for QC organisms.a
dent) were within 1 mm of each other on all six of the aforementioned MHA lots. We have tentatively adopted the daptomycin MIC breakpoints previously proposed by Jones and Barry (1987) and recommend that for broth dilution tests of daptomycin the medium should contain physiological concentrations of Ca⫹⫹ (45–55 g/ml) (Eliopoulos et al., 1986, Jones & Barry, 1987). We feel this is reasonable until clinical data become available. Although daptomycin is highly (90 –95%) protein-bound (Lee et al. 1991, Ryback et al. 1992, Woodworth et al. 1992), serum levels were linearly related to dose, and a Cmax of 82 g/ml was achieved with a single dose of 6 mg/kg in healthy volunteers (Woodworth et al. 1992). Thus, from a pharmacokinetic viewpoint, a daptomycin susceptible MIC breakpoint of ⱕ2.0 g/ml does not seem to be unreasonable at this time. This breakpoint is also supported by the MIC population distribution; a breakpoint lower than this (e.g., 1.0 g/ml) would cut through the middle of the enterococcal MIC distribution. In any event, the final decision on daptomycin breakpoints must await the results of clinical trials, particularly with enterococcal infections. As for the zone diameter breakpoints, those proposed by Jones & Barry (1987) performed satisfactorily with our data. However, the absence of daptomycin-resistant isolates in this data set forces us to continue to consider them tentative. The in vitro activity of daptomycin against multi-resistant Gram-positive bacteria such as VRE and MRS is noteworthy, and because of the therapeutic dilemmas caused by infections with such strains, further development of this agent would appear justified.
Acknowledgments We gratefully acknowledge the participation of the following in the ten-laboratory quality control study: Mary Bauman, St. Vincent’s Hospital, Portland, OR; Mary Jane Ferraro, Massachusetts General Hospital, Boston, MA; Dwight Hardy, University of Rochester Medical Center, Rochester, NY; Janet Hindler, UCLA Medical Center, Los Angeles, CA; Stephen Jenkins, Carolinas Medical Center, Charlotte, NC; Cindy Knapp, AccuMed International, Westlake, OH; Gary Overturf, University of New Mexico Medical Center, Albuquerque, NM; Robert Rennie, University of Alberta Hospital, Edmonton, Alberta; Michael Saubolle, Good Samaritan Hospital, Phoenix, AZ. We also wish to thank Cubist Pharmaceuticals Inc. for financial subsidy of these studies.
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