Journal of Antimicrobial Chemotherapy (2002) 49, 467–470

JAC
In vitro bactericidal activity of daptomycin against staphylococci

Peter C. Fuchs*, Arthur L. Barry and Steven D. Brown

The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA

MICs and minimum bactericidal concentrations (MBCs) of daptomycin, vancomycin, linezolid

and quinupristin–dalfopristin (Q-D) were determined for 108 staphylococcal isolates. All
strains were susceptible (MICs) to daptomycin ( < 2.0 mg/L) and Q-D ( < 1.0 mg/L). All but three
isolates were susceptible to vancomycin ( < 4.0 mg/L) and all but one methicillin-resistant
Staphylococcus aureus strain were susceptible to linezolid ( < 4.0 mg/L). Q-D had the lowest
geometric mean MIC (0.29 mg/L) and daptomycin had the lowest geometric mean MBC
(0.57 mg/L). Time–kill tests were performed on 25 isolates. Bactericidal activity (>99.9% kill)
was observed with daptomycin at 2 mg/L and at 2  MBC for 92% of strains tested. In
comparison, the bactericidal rates for the other drugs at breakpoint concentrations and at
2  MBC were 72% and 70% for vancomycin, 46% and 60% for Q-D, and 7% and 14% for
linezolid. Of the four drugs tested, daptomycin was bactericidal against the most strains
and had the most rapid cidal activity.

Introduction Materials and methods

Staphylococci are important Gram-positive pathogens Bacterial strains
in the hospital setting and they have demonstrated their
From our collection of recent (2 years) North American
capacity to develop resistance to standard antibiotic regi-
clinical isolates, 108 staphylococcal strains were selected to
mens. Methicillin-resistant Staphylococcus aureus (MRSA)
give a broad range of daptomycin and vancomycin MICs.
strains have become increasingly prevalent worldwide, and
These included 53 strains of S. aureus, 28 of which were
the isolated reports of glycopeptide-intermediate S. aureus
MRSA, and three of which were GISA. The remaining
(GISA) suggest that vancomycin resistance is only a step
55 strains included 44 Staphylococcus epidermidis and
away.1 Quinupristin–dalfopristin (Q-D) and linezolid, both
11 Staphylococcus haemolyticus. MBCs were determined
recently FDA approved, have been reported to have good
for the entire set, and time–kill studies were performed on
activity against MRSA.2 Daptomycin is a novel parenteral
25 strains from this set.
lipopeptide antibiotic with potent activity against Gram-
positive pathogens, including MRSA.3
For the treatment of some infections such as bacterial Antibacterial agents
endocarditis, and the treatment of serious infections in Daptomycin standardized powder was provided by Cubist
immunocompromised patients, bactericidal activity is desir- Pharmaceuticals, Inc. (Lexington, MA, USA). Linezolid
able.4 Although not universally accepted, there are data was obtained from Pharmacia Corporation (Kalamazoo,
that indicate that the time–kill curve method is the in vitro MI, USA), Q-D was obtained from Aventis Pharmaceut-
method that correlates best with clinical cure in cases of icals (Bridgewater, NJ, USA) and vancomycin was obtained
bacterial endocarditis.5 The present study was designed to from Sigma Chemical Company (St Louis, MO, USA).
assess the in vitro bactericidal activity of daptomycin
against staphylococci, in comparison with vancomycin,
Susceptibility testing
linezolid and Q-D by two methods, namely determination
of minimum bactericidal concentrations (MBCs) and time– MICs were determined by the broth microdilution method
kill studies. as described by the NCCLS.6 Cation-adjusted Mueller–

*Corresponding author. Tel: 1-503-682-3232; Fax: 1-503-682-2065; E-mail: cmi@hevanet.com

467
© 2002 The British Society for Antimicrobial Chemotherapy
 P. C. Fuchs et al.

Hinton broth (CAMHB) was used for all antibiotics except with calcium to a final concentration of 50 mg/L. Anti-
daptomycin, for which the CAMHB was supplemented biotics were tested at two concentrations: (i) the MIC sus-
with additional calcium to a physiological concentration of ceptibility breakpoint concentration, and (ii) twice the
50 mg/L.3 Serial two-fold dilutions of each antibiotic were MBC (2  MBC). When 2  MBC was the same as the
prepared in microdilution trays with concentrations rang- susceptible breakpoint, only one test was performed. Also,
ing from 32 to 0.004 mg/L for daptomycin, 64 to 0.5 mg/L when the MBC was greater than the highest concentration
for linezolid, 4.0 to 0.03 mg/L for Q-D and 32 to 0.25 mg/L tested, 2  MBC time–kill tests were not performed.
for vancomycin. For each test, the control strain S. aureus Colony counts were performed on broth from the growth
ATCC 29213 was included, and all results with this strain control flasks at the time of inoculation (0 h), and from the
were within the published NCCLS quality control ranges.7 growth control flasks as well as all antibiotic-containing
MBCs were determined by subculturing on to sheep flasks after 1, 2, 4, 8, 12 and 24 h of incubation. Bactericidal
blood agar plates 10 L of broth from each well with no activity was defined as a decrease in colony count to 0.1%
visible growth after 24 h incubation and from the highest of the initial inoculum count (99.9% kill).
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 Results and discussion
colony counts from the growth control well immediately
after inoculation). The MICs and MBCs of daptomycin and the three com-
Time–kill studies were performed as recommended parison drugs against 108 staphylococcal isolates are sum-
by the NCCLS.8 CAMHB was used for all drugs except marized in Table 1. Aside from the three GISA strains not
daptomycin, for which the CAMHB was supplemented susceptible to vancomycin and one MRSA strain not sus-

Table 1. MICs and MBCs of daptomycin and three comparison agents against 108 staphylococci

MIC (mg/L) MBC (mg/L)

Staphylococcus Antimicrobial
group No. agent range 50% 90%a range 50% 90%a

GISA 3 daptomycin 1.0–2.0 2.0 2.0–8.0 2.0

vancomycin 8.0–8.0 8.0 8.0–>32 16
linezolid 0.5–2.0 1.0 8.0–>64 16
Q-D 0.5–0.5 0.5 0.5–>4.0 0.5
MRSA 25 daptomycin 0.25–1.0 0.5 1.0 0.25–2.0 0.5 1.0
vancomycin 0.5–2.0 1.0 2.0 0.2–2.0 1.0 2.0
linezolid 2.0–8.0 2.0 4.0 >64 >64 >64
Q-D 0.25–1.0 1.0 1.0 >4.0 >4.0 >4.0
MSSA 25 daptomycin 0.25–1.0 0.5 0.5 0.25–1.0 0.5 0.5
vancomycin 0.5–2.0 1.0 1.0 0.5–2.0 1.0 1.0
linezolid 1.0–4.0 2.0 4.0 >64 >64 >64
Q-D 0.12–0.5 0.25 0.5 0.25–>4.0 >4.0 >4.0
MRSEb 40 daptomycin 0.12–1.0 0.5 0.5 0.25–2.0 0.5 1.0
vancomycin 0.5–2.0 2.0 2.0 0.5–4.0 2.0 4.0
linezolid 0.5–2.0 1.0 2.0 2.0–>64 >64 >64
Q-D 0.12–0.5 0.25 0.5 0.12–>4.0 >4.0 >4.0
MSSEb 4 daptomycin 0.25–1.0 0.5 0.5–2.0 1.0
vancomycin 0.5–2.0 1.0 0.5–2.0 1.0
linezolid 0.5–2.0 2.0 >64 >64
Q-D 0.12–0.5 0.5 0.5–2.0 2.0
S. haemolyticus 11 daptomycin 0.25–0.5 0.5 0.5 0.25–0.5 0.5 0.5
vancomycin 0.5–2.0 1.0 2.0 1.0–4.0 2.0 2.0
linezolid 1.0–2.0 2.0 2.0 2.0–>64 8.0 >64
Q-D 0.25–0.5 0.25 0.25 0.25–>4.0 0.5 >4.0
a
MIC90 and MBC90 values were not calculated for groups with 10 isolates.
b
MRSE, methicillin-resistant S. epidermidis; MSSE, methicillin-susceptible S. epidermidis.

468
 Daptomycin MBCs

ceptible to linezolid, all isolates were susceptible (by MIC)

C/Tb timec
to the four drugs tested. The lowest MICs were generated

2  MBC

24

24
–
–
–
–
–
by Q-D (geometric mean MIC of 0.29 mg/L), followed

bactericidal at:
by daptomycin (0.47 mg/L), vancomycin (1.34 mg/L) and

1/2

1/7
0/1
0/4
linezolid (1.67 mg/L). The most active bactericidal drug

0
0
0
Table 2. Time–kill test results for daptomycin and three comparator drugs at susceptible breakpoint concentrations and 2  MBC

Linezolid
was daptomycin (geometric mean MBC of 0.57 mg/L),

C/Tb timec
followed by vancomycin (1.50 mg/L), Q-D (3.24 mg/L) and

breakpointa

24

24
linezolid (64 mg/L). Although Q-D is considered a bac-

–
–
–
–
–
tericidal drug, it has been shown to be bacteriostatic rather

1/14
than bactericidal against clindamycin-resistant staphylo-

1/3
0/2
0/1
0/1
0/2
0/5
cocci,9 which include the majority of methicillin-resistant
strains. Since the majority of strains in this study were

C/Tb timec
methicillin resistant, it is not surprising that Q-D was not

Quinupristin–dalfopristin

18.0
4.0

6.0
1.5
1.5

4.0
2  MBC
bactericidal for as many strains as one would normally

–
expect. Daptomycin MBCs were 2.0 mg/L for all of the

9/15
challenge strains except one GISA strain for which the

2/2
0/1
2/2
2/2
2/4
1/4
MBC was 8.0 mg/L.

bactericidal at:
Table 2 summarizes the time–kill studies with 25 staphylo-

C/Tb timec
breakpointa
coccal strains. When tested at 2  MBC daptomycin

14.7
4.0

7.2
1.5
9.0

4.0
was bactericidal for 23 strains (92%). The two strains not

Susceptible breakpoints: 2.0 mg/L for daptomycin (tentative), 4.0 mg/L for vancomycin and linezolid, and 1.0 mg/L for Q-D.
killed by that concentration were both S. haemolyticus with

12/25
2/3
0/4
2/3
3/4
3/5
1/5
daptomycin MBCs of 0.25 mg/L. However, both were
killed when tested at 2.0 mg/L, the tentative daptomycin
susceptible breakpoint. At the breakpoint concentration,

C/Tb timec
all but two strains were killed by daptomycin. The two

12.0

16.0
12.0
12.0
15.2
2  MBC

24

24
exceptions were one GISA strain with a daptomycin MBC
of 8.0 mg/L and one isolate of S. epidermidis with a dapto-

16/23
bactericidal at:

Mean time (hours) to achieve 3 log10 reduction in colony counts for those strains killed within 24 h.
1/1
3/5
2/3
2/4
5/5
3/5
mycin MBC of 2.0 mg/L.
Vancomycin

In human volunteers, daptomycin is 89–94% bound to

plasma protein.10 Earlier in vitro studies had demonstrated
C/Tb timec
breakpointa

15.2

15.3
14.7
10.0
15.2
20.0
an increase in daptomycin MIC values in the presence of
–

C/T number cidal (3 log10 reduction in colony counts within 24 h)/number tested.
human serum; these studies also showed that there was a
decrease in free Ca2 in the media.11 However, there is only

18/25
0/3
5/5
3/3
2/4
5/5
a two- to four-fold increase in daptomycin MICs in media 3/5
containing 4% albumin or human serum if a physiological
concentration of 50 mg/L Ca2 is maintained (J. Alder,
C/Tb timec

Cubist Pharmaceuticals, personal communication). Current

8.0
3.0

5.9
2.3
7.5
7.2
8.0
2  MBC

clinical trials for treatment of complicated skin and soft

bactericidal at:

23/25

tissue infections utilize an od iv dose of 4 mg/kg dapto-

Daptomycin

3/3
5/5
3/3
4/4
5/5
3/5

mycin.12 Single dose (4 mg/kg) pharmacokinetic studies in

healthy volunteers exhibited plasma Cmax values of 52 mg/L
with a half-life of nearly 9 h.13 Assuming 10% of the drug
C/Tb timec
breakpointa

10.0
1.8

4.4
1.3
6.5
5.0
4.5

level represents unbound daptomycin, the unbound drug

concentration would be 2.0 mg/L for 6–8 h, 1.0 mg/L
23/25

for more than half the 24 h dosing interval and 0.5 mg/L
2/3
5/5
3/3
4/4
4/5
5/5

for the entire 24 h dosing interval. Daptomycin MIC90s for

staphylococcal species ranged from 0.5 to 1.0 mg/L.3 In our
tests, free daptomycin at a concentration of 2.0 mg/L was
No.

25

bactericidal for 23 (95.6%) of the 24 staphylococcal strains

3
5
3
4
5
5

tested with MBCs of 2.0 mg/L. Of 17 strains with dapto-

mycin MBCs of 0.5 mg/L, 15 (88.3%) were killed by free
S. haemolyticus

daptomycin at 2  MBC (0.5 and 1.0 mg/L). The two strains

that were not killed (both S. haemolyticus) at 2  MBC
Organism

were both killed at 2.0 mg/L of daptomycin.

MRSA

MRSE
MSSA
MSSE
GISA

Total

When vancomycin was tested in time–kill studies at

2  MBC, only 16 of 23 (70%) were killed. In the case of
b
a

469
 P. C. Fuchs et al.

Q-D, nine of 15 (60%) strains tested at 2  MBC were 5. Drake, T. A. & Sande, M. A. (1985). Studies of the chemotherapy
killed. The bactericidal activity of linezolid was difficult to of endocarditis: correlation of in vitro, animal model, and clinical
studies. Review of Infectious Diseases 5, Suppl. 2, S345–53.
assess. Linezolid had MBCs of 4.0 mg/L for five (20%)
strains, but this was not confirmed by the time–kill studies. 6. National Committee for Clinical Laboratory Standards. (2000).
The only strain for which linezolid was bactericidal in the Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically—Fifth Edition: Approved Standard M7-A5.
time–kill test system was a GISA strain with a linezolid
NCCLS, Wayne, PA.
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 7. National Committee for Clinical Laboratory Standards. (2000).
Performance Standards for Antimicrobial Susceptibility Testing—
same results. The significance of these discrepancies
Tenth Informational Supplement, M100-S10. NCCLS, Wayne, PA.
remains unclear at this time, but linezolid is regarded as
bacteriostatic against staphylococci.14 8. National Committee for Clinical Laboratory Standards. (1998).
Methods for Determining Bactericidal Activity of Antimicrobial
When tested at 2  MBC in time–kill tests, the mean
Agents—Approved Guideline, M26-A. NCCLS, Wayne, PA.
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 9. Fuchs, P. C., Barry, A. L. & Brown, S. D. (2000). Bactericidal
activity of quinupristin–dalfopristin against Staphylococcus aureus:
and 15.5 h for vancomycin.
clindamycin susceptibility as a surrogate indicator. Antimicrobial
We conclude from these data that daptomycin has good Agents and Chemotherapy 44, 2880–2.
in vitro bactericidal activity against staphylococci, which in
10. Lee, B. L., Sachdeva, M. & Chambers, H. F. (1991). Effect of
this study surpassed that of vancomycin, Q-D and linezolid.
protein binding of daptomycin on MIC and antibacterial activity.
Antimicrobial Agents and Chemotherapy 35, 2505–8.
Acknowledgement 11. Hanberger, H., Nilsson, L. E., Maller, R. & Isaksson, B. (1991).
Pharmacodynamics of daptomycin and vancomycin on E. faecalis
This study was supported by a grant from Cubist Pharma- and S. aureus demonstrated by studies of initial killing and post-
ceuticals, Inc., Lexington, MA, USA. antibiotic effect and influence of Ca2 and albumin on these drugs.
Antimicrobial Agents and Chemotherapy 35, 1710–6.
12. Tally, F. P., Zeckel, M., Wasilewski, M. M., Carini, C., Berman,
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