ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2009, p. 2719–2724
0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00047-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 7
Efficacy of Daptomycin in Implant-Associated Infection Due to
Methicillin-Resistant Staphylococcus aureus: Importance of
Combination with Rifampin䌤
Anne-Kathrin John,1 Daniela Baldoni,1 Manuel Haschke,2 Katharina Rentsch,3
Patrick Schaerli,4 Werner Zimmerli,5 and Andrej Trampuz1,6*
Infectious Diseases, Department of Biomedicine, University Hospital Basel, Basel, Switzerland1; Division of Clinical Pharmacology and
Toxicology, University Hospital Basel, Basel, Switzerland2; Institute of Clinical Chemistry, University Hospital Zurich, Zurich,
Switzerland3; Infectious Diseases, Transplantation and Immunology, Novartis Pharma Schweiz AG, Bern, Switzerland4;
Basel University Medical Clinic, Kantonsspital, Liestal, Switzerland5; and Division of Infectious Diseases and
Hospital Epidemiology, University Hospital Basel, Basel, Switzerland6
Received 13 January 2009/Returned for modification 26 March 2009/Accepted 8 April 2009
Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin
[rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig
foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 g/ml, respectively. In time-kill
studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase.
At concentrations above 20 g/ml, daptomycin reduced the counts by >3 log10 CFU/ml in 2 to 4 h. In sterile
cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 g/ml were reached 4 to 6 h after the
administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment
studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log10 CFU/ml, whereas in combination
with rifampin, a reduction in the counts of >6 log10 CFU/ml was observed. Vancomycin and daptomycin (at
both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas
rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure
rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a
cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg)
completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest
activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for
implant-associated MRSA infections.
Implants are increasingly used in modern medicine to replace a compromised biological function or missing anatomical
structure. Periprosthetic infections represent a devastating
complication, causing high rates of morbidity and consuming
considerable health care resources. Implant-associated infections are caused by microorganisms growing adherent to the
device surface and embedded in an extracellular polymeric
matrix, a complex three-dimensional structure called a microbial biofilm (8). Bacterial communities in biofilms cause persistent infection due to increased resistance to antibiotics and
the immune system and the difficulty with eradicating them
from the implant (6).
Staphylococcus aureus is one of the leading pathogens causing implant-associated infections. Successful treatment requires the use of bactericidal drugs acting on surface-adhering
microorganisms, which predominantly exist in the stationary
growth phase. Previous in vitro, experimental, and clinical
studies demonstrated that rifampin (rifampicin)-containing antimicrobial regimens were able to eradicate staphylococcal biofilms and cure implant-associated infections (23, 25). Quinolones are often used in combination with rifampin in order to
prevent the emergence of rifampin resistance (4, 19, 21). However, methicillin (meticillin)-resistant S. aureus (MRSA)
strains are often resistant to quinolones. In addition, MRSA
strains were recently shown to have decreased susceptibility to
vancomycin, reducing the efficacy of this drug. Therefore, alternative drugs for use in combination with rifampin against
implant-associated infections are needed (12, 20).
Daptomycin is a negatively charged cyclic lipopeptide with
bactericidal activity against gram-positive organisms, including
MRSA (17). The drug inserts into the bacterial cytoplasmic
membrane in a calcium-dependent fashion, leading to rapid
cell death without lysis, and causing only minimal inflammation (15). Daptomycin has been well tolerated in healthy volunteers dosed with up to 12 mg/kg of body weight intravenously for 14 days (2). Only limited data on the use of
daptomycin in combination with rifampin against staphylococcal implant-associated infections are available.
In this study, we investigated the activity of daptomycin
against MRSA ATCC 43300 in vitro. In addition, we evaluated
* Corresponding author. Present address: Infectious Diseases Service, Department of Internal Medicine, University Hospital and University of Lausanne (CHUV), Rue du Bugnon 46, Lausanne CH-1011,
Switzerland. Phone: 41 21 314 3992. Fax: 41 21 314 28 76. E-mail:
[email protected].
䌤
Published ahead of print on 13 April 2009.
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JOHN ET AL.
the activity of daptomycin in combination with rifampin in a
cage-associated infection model in guinea pigs and compared
the efficacy of the treatment with the efficacies of three other
antibiotics commonly used against MRSA, vancomycin, linezolid, and levofloxacin (alone and in combination with rifampin).
(Part of the results of the present study were presented at
the 48th Interscience Conference on Antimicrobial Agents and
Chemotherapy, Washington, DC, 24 to 29 October 2008 [abstr.
B-1000].)
MATERIALS AND METHODS
Study microorganisms. S. aureus strain ATCC 43300, which is resistant to
methicillin and which is susceptible to rifampin, vancomycin, linezolid, and levofloxacin, was studied. For the testing of rifampin resistance, rifampin-resistant
clinical S. aureus strain T4050 and rifampin-susceptible laboratory S. aureus
strain ATCC 29213 were used. The strains were stored at ⫺70°C by using the
cryovial bead preservation system (Microbank; Pro-Lab Diagnostics, Richmond
Hill, Ontario, Canada). For preparation of the inoculum, single beads were
transferred to 1 ml of sterile trypticase soy broth (TSB; Becton Dickinson and
Company, Le Pont de Claix, France) and incubated for 7 h at 37°C. This
preculture was then diluted 1:100 in fresh TSB and incubated overnight at 37°C
without shaking. The bacteria were washed twice and resuspended in sterile and
pyrogen-free 0.9% saline to the desired concentration. Bacterial concentrations
were determined by plating of aliquots from appropriate dilutions on agar,
followed by colony counting after 24 h of incubation at 37°C.
Antimicrobial agents. Daptomycin for injection was supplied by Novartis
Pharma Schweiz AG (Bern, Switzerland). A stock solution of 50 mg/ml was
prepared in sterile and pyrogen-free 0.9% saline. All other solutions were prepared in sterile water. Rifampin (Sandoz AG, Steinhausen, Switzerland) was
prepared as a 60-mg/ml stock solution. Levofloxacin hemihydrate injectable
solution (5 mg/ml) was purchased from Aventis Pharma AG (Zurich, Switzerland). Vancomycin was supplied by Teva Pharma AG (Aesch, Switzerland), and
a stock solution of 50 mg/ml was prepared. Linezolid was provided as a purified
powder from the manufacturer (Pfizer AG, Zurich, Switzerland), and a stock
solution of 2.5 mg/ml was prepared.
In vitro antimicrobial susceptibility. A standard inoculum of 1 ⫻ 105 to 5 ⫻
105 CFU/ml of MRSA strain ATCC 43300 was used. The MIC and the minimal
bactericidal concentration (MBC) in the logarithmic growth phase (MBClog)
were determined by using twofold dilutions of antimicrobial agents in MuellerHinton broth supplemented with 50 mg/liter calcium ions (CaCl2), according to
the CLSI (formerly the NCCLS) guidelines (3). This concentration of calcium is
necessary for the antimicrobial activity of daptomycin to be exhibited (1). The
MIC was the lowest drug concentration that inhibited visible bacterial growth.
The MBClog was defined as the lowest antimicrobial concentration which killed
ⱖ99.9% of the initial bacterial count (i.e., ⱖ3 log10 CFU/ml) in 24 h (10). In
addition, the MBC was determined also in the stationary (nongrowing) growth
phase (MBCstat), reflecting the characteristics of microorganisms causing implant-associated infections. MBCstat was determined by using overnight cultures
of S. aureus in nutrient-limited medium (0.01 M phosphate buffered saline [PBS],
pH 7.4) containing 0.1% glucose and 50 mg/liter calcium ions. In this medium,
the bacterial counts remained stable for up to 48 h. MBCstat was defined as the
lowest concentration which reduced the inoculum by ⱖ99.9% in 24 h. The
experiments were performed in triplicate.
Time-kill study in stationary growth phase. Glass tubes containing 10 ml PBS
supplemented with 50 mg/liter calcium ions and 0.1% glucose were incubated
with daptomycin at concentrations representing 4⫻, 8⫻, 16⫻, 32⫻, 64⫻, and
128⫻ the MIC of the test strain at 37°C without shaking. Bacterial survival in the
antimicrobial-free culture served as a control. To determine whether the inoculum size affects the killing activity of daptomycin, a low initial inoculum (3 ⫻ 105
CFU/ml) and a high initial inoculum (5 ⫻ 106 CFU/ml) were tested. For the
high-inoculum assays, PBS with 50 mg/liter calcium ions was supplemented with
0.001% TSB to keep the bacterial counts in the antimicrobial-free culture stable
for at least 24 h. Colony counts were determined immediately before addition of
daptomycin (0 h) and after 2, 4, 6, 8, and 24 h of incubation with daptomycin at
the appropriate concentrations. Before sampling of the probes, the tubes were
gently vortexed and colony counts were determined by plating aliquots of appropriate dilutions on Mueller-Hinton agar. A bactericidal effect was defined as
a ⱖ3-log10 (ⱖ99.9%) reduction of the initial bacterial count (11). The experiments were performed in triplicate.
ANTIMICROB. AGENTS CHEMOTHER.
Animal model. We used a guinea pig model of foreign-body infection which
was established by Zimmerli et al. (24). Guinea pigs (Charles River, Sulzfeld,
Germany) were kept in the Animal House of the Department of Biomedicine,
University Hospital Basel. The animal experiments were performed according to
the regulations of Swiss veterinary law. In brief, four sterile polytetrafluoroethylene (Teflon) tubes (10 by 30 mm) perforated with 130 holes (Angst ⫹ Pfister
AG, Zurich, Switzerland) were aseptically implanted into the flanks of male
guinea pigs weighing at least 500 g. The animals were anesthetized with an
intramuscular injection of ketamine (20 mg/kg; Parke-Davis, Zurich, Switzerland) and xylazine (4 mg/kg; Gräub, Bern, Switzerland). The experiments were
started after complete wound healing (i.e., approximately 2 weeks after surgery).
Before each experiment, the cages were checked for sterility by culturing the
aspirated cage fluid. The guinea pigs were weighed daily to monitor their wellbeing during the experiment and to adjust the antibiotic doses.
Pharmacokinetic study. Pharmacokinetic studies were performed with sterile
tissue cages. A single dose of 20, 30, and 40 mg/kg daptomycin was injected
intraperitoneally (three animals and 12 cages per dose group). Cage fluid was
aspirated by percutaneous cage puncture at 1, 2, 4, 6, 8, 10, 12, and 24 h after
drug administration. For each drug dose, fluid from six cages per time point (two
cages per time point and animal) was collected. Aliquots of 150 l of cage fluid
were transferred to tubes containing 15 l of filter-sterilized 5% polyanetholsulfonic acid sodium salt (Sigma-Aldrich, Buchs, Switzerland), mixed by hand, and
centrifuged at 2,100 ⫻ g for 7 min. The supernatant was stored at ⫺20°C until
further analysis.
(i) High-performance liquid chromatography assay, followed by mass spectrometry. Daptomycin standards were prepared in cage fluid by spiking cage fluid
from untreated animals with daptomycin solution in water-methanol (1/1) to give
concentrations in the range of 0.2 to 150 g/ml. Two hundred microliters of
precipitation solution (methanol, acetonitrile, 1 mM zinc sulfate) containing 2 g
of CB183253 (internal standard) was added to 50 l of each of the standards,
samples, and controls. After vortexing of the samples and centrifugation, 100 l
of the supernatant was diluted with water-methanol (1/1) and 10 l was injected
into the liquid chromatography-mass spectrometry apparatus (TSQ; Thermo
Fisher Scientific). Separation of the components was performed on a C18 column
(Uptisphere; particle size, 5 m; 125 by 2 mm) by using acetonitrile and 0.1%
formic acid as the mobile phase. Daptomycin was quantified by analyzing m/z 811 3
341, and the internal standard was quantified by analyzing m/z 837 3 393. The
daptomycin concentrations were calculated by linear regression of the peak
ratios between daptomycin and the internal standard.
(ii) Pharmacokinetic parameters. Individual concentration-time data were
analyzed by using the WinNonlin software package (Pharsight Corp., Mountain
View, CA). For each time point, the mean fluid concentration of the six cages
was used. Mean ⫾ standard deviation (SD) values of the peak (maximum)
concentration (Cmax), the time required to reach Cmax (Tmax), the trough (minimum) concentration at 24 h after dosing (Cmin), the half-life (t1/2), and the area
under the concentration-time curve (AUC) from time zero to 24 h (AUC0–24)
were calculated.
Antimicrobial treatment study. Cages were infected with the MRSA test strain
by percutaneous injection of 200 l bacterial suspension containing 4 ⫻ 106 CFU
(day 0). The establishment of an infection was confirmed by quantitative culture
of cage fluid 3 days later, immediately before the start of treatment. Three
animals were randomized into each of the following 10 treatment groups: saline
(control), rifampin at 12.5 mg/kg alone, linezolid at 50 mg/kg plus rifampin at
12.5 mg/kg, levofloxacin at 10 mg/kg plus rifampin at 12.5 mg/kg, vancomycin at
15 mg/kg alone and in combination with rifampin at 12.5 mg/kg, and daptomycin
at 20 mg/kg and 30 mg/kg alone and in combination with rifampin at 12.5 mg/kg.
The antimicrobial agents were given intraperitoneally for 4 days. The dosing
interval was 12 h for all drugs except daptomycin, which was given every 24 h.
(i) Efficacy of treatment against planktonic bacteria. Bacterial counts (median
and interquartile range) were determined before the start of treatment (i.e., day
3), during treatment (i.e., day 5), and 5 days after the completion of treatment
(i.e., day 12). The efficacy of each treatment against planktonic bacteria in cage
fluid was expressed as the difference in the bacterial counts (⌬log10 CFU/ml)
before and 5 days after the completion of treatment and the clearance rate (in
percent), defined as the number of cage fluid samples without growth of MRSA
divided by the total number of cages in the individual treatment group.
(ii) Efficacy of treatment against adherent bacteria. Five days after the end of
treatment (i.e., day 12), the animals were sacrificed and the tissue cages were
removed under aseptic conditions and incubated at 37°C in 5 ml TSB. After 48 h
of incubation, 100 l of the cage culture was spread on Columbia sheep blood
agar plates (Becton Dickinson) and analyzed for bacterial growth. A positive
culture of MRSA was defined as a treatment failure. The efficacy of treatment
against adherent bacteria was expressed as the cure rate (in percent), defined as
VOL. 53, 2009
DAPTOMYCIN AGAINST MRSA FOREIGN-BODY INFECTION
2721
TABLE 1. In vitro susceptibility of MRSA ATCC 43300
Antibiotica
MIC
(g/ml)
MBClog
(g/ml)
MBCstat
(g/ml)
MBCstat/MBClog
ratio
DAP
RIF
VAN
LZD
LVX
0.625
0.01
1
2.5
0.16
0.625
0.08
2
⬎20
0.63
20
2.5
32
⬎20
⬎20
32
31
16
NAb
⬎32
a
DAP, daptomycin; RIF, rifampin; VAN, vancomycin; LZD, linezolid; LVX,
levofloxacin.
b
NA, not applicable.
the number of cages without growth divided by the total number of cages in the
individual treatment group.
Emergence of antimicrobial resistance in vivo. Positive cultures of samples
from explanted cages were screened for the in vivo emergence of resistance to
rifampin, vancomycin, and daptomycin. In addition, all positive cultures of samples from cage fluid were screened for rifampin resistance. Colonies were collected from subcultures on agar; suspended in saline to the turbidity of a Mc-
FIG. 2. Pharmacokinetics of daptomycin in sterile cage fluids after
administration of single intraperitoneal doses of daptomycin at 20
mg/kg (circles), 30 mg/kg (squares), and 40 mg/kg (diamonds). Values
are means ⫾ SDs. The horizontal dotted line indicates the MBCstat of
MRSA for daptomycin.
Farland 0.5 standard; and spread on Mueller-Hinton agar plates containing 2
g/ml of daptomycin, 1 g/ml of rifampin, or 16 g/ml of vancomycin. The plates
were incubated at 37°C and screened for growth after 24 h.
Evaluation of antimicrobial toxicity. To evaluate the potential toxicity of
daptomycin (20 mg/kg) administered with or without rifampin (three animals per
group), histopathologic analysis of liver, kidney, and skeletal muscle tissues was
performed. The corresponding organs of the saline-treated animals served as
controls. The organs were fixed overnight in 4% buffered formalin, rinsed with
PBS, and embedded into paraffin immediately after the animals were killed.
Sections of 3 to 4 m were mounted on slides and dried overnight at 37°C. The
specimen sections were stained with hematoxylin-eosin and inspected by light
microscopy.
Statistical calculations. Comparisons were performed by the Mann-Whitney
U test for continuous variables and the two-sided 2 test or Fisher’s exact test for
categorical variables, as appropriate. For all tests, differences were considered
significant when P values were ⬍0.05. The graphs in the figures were plotted with
Prism (version 5.0a) software (GraphPad Software, La Jolla, CA).
RESULTS
FIG. 1. Time-kill curve of a low inoculum (3 ⫻ 105 CFU/ml)
(A) and a high inoculum (5 ⫻ 106 CFU/ml) (B) of MRSA in stationary
growth phase exposed to increasing daptomycin concentrations (2.5
g/ml to 80 g/ml) corresponding to 4⫻ to 128⫻ MIC. Values are
means ⫾ SDs. The experiments were performed in triplicate. The
horizontal dotted lines indicates a 3-log10 reduction of the numbers of
CFU/ml from the initial inoculum.
In vitro antimicrobial susceptibility. Table 1 summarizes the
in vitro susceptibility of MRSA ATCC 43300. Of the antibiotics tested, rifampin showed the lowest MBCstat (2.5 g/ml),
followed by daptomycin (20 g/ml) and vancomycin (32 g/
ml), whereas linezolid and levofloxacin did not kill MRSA in
the stationary growth phase. The MBCstat was at least 16-fold
higher than the MBClog for all agents (except linezolid, which
had only a bacteriostatic effect).
In vitro time-kill study in stationary growth phase. In the
low-inoculum and the high-inoculum studies, the bacterial
counts remained within ⫾5% of the initial inoculum in the
antimicrobial-free culture for 24 h. The time-kill curves in Fig.
1 demonstrate that daptomycin had rapid and concentrationdependent bactericidal activity against stationary-phase
MRSA with a low inoculum (Fig. 1A) as well as a high inoculum (Fig. 1B). At 20 g/ml (32⫻ MIC), which corresponded
to the MBCstat, daptomycin reduced the counts by ⱖ3 log10
2722
JOHN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Pharmacokinetic parameters of daptomycin in cage fluid after a single intraperitoneal dose and linked to the in vitro susceptibility
parameters of the MRSA strain testeda
Dose
(mg/kg)
Cmax
(g/ml)
Cmin
(g/ml)
Tmax (h)
t1/2 (h)
AUC0–24
(g · h/ml)
Cmax/MIC
Cmax/MBClog
Cmax/MBCstat
AUC ⬎
MBCstat/
AUC0–24
20
30
40
23.1 ⫾ 7.0
46.3 ⫾ 8.8
53.7 ⫾ 1.3
1.5 ⫾ 1.1
9.8 ⫾ 2.9
4.1 ⫾ 2.3
6.0 ⫾ 2.0
4.7 ⫾ 1.2
6.0 ⫾ 0.0
4.8 ⫾ 1.7
8.7 ⫾ 0.5
4.5 ⫾ 1.2
247 ⫾ 52
548 ⫾ 65
662 ⫾ 10
36.9 ⫾ 11.2
74.0 ⫾ 14.1
85.8 ⫾ 2.1
36.9 ⫾ 11.2
74.0 ⫾ 14.1
85.8 ⫾ 2.1
1.2 ⫾ 0.3
2.3 ⫾ 0.4
2.7 ⫾ 0.1
0.05 ⫾ 0.04
0.26 ⫾ 0.04
0.39 ⫾ 0.01
a
Values represent means ⫾ SDs.
CFU after 4 to 6 h. At concentrations above 20 g/ml, daptomycin reduced the counts by ⬎3 log10 CFU/ml in 2 to 4 h.
Pharmacokinetic study. Figure 2 shows the concentrationtime curves in sterile cage fluid after the administration of a
single intraperitoneal dose of 20, 30, or 40 mg/kg daptomycin.
Table 2 summarizes the values of the pharmacokinetic parameters calculated. For all three doses administered, the peak
Cmaxs were above the MBCstats, whereas the concentrations of
daptomycin after 24 h (Cmin) remained above the MIC and
MBClog but not above the MBCstat. The AUC0–24 increased
with the dose from 247 to 662 g · h/ml. The ratio of the AUC ⬎
MBCstat to AUC0–24 increased in a dose-dependent manner
from 5% (at 20 mg/kg) to 26% (at 30 mg/kg) and 39% (at 40
mg/kg).
FIG. 3. Killing of planktonic MRSA in cage fluid 5 days after the
completion of therapy. Positive values on the y axis denote the net
growth and negative values denote the net killing. Values are medians ⫾ interquartile ranges. The numbers in parentheses indicate the
dose (in mg/kg) administered twice daily for all drugs except daptomycin, which was administered once daily. DAP, daptomycin; RIF,
rifampin; VAN, vancomycin; LZD, linezolid; LVX, levofloxacin; ⴱ,
P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.
Antimicrobial treatment study. Three days after inoculation,
the bacterial counts surpassed the initial inoculum two- to
threefold in all infected animals (data not shown). The planktonic bacterial counts (median ⫾ interquartile range) in the
cage fluid of the control group (treated with saline) increased
by 1.4 ⫾ 0.1 log10 CFU/ml (Fig. 3); no bacterial clearance (Fig.
4A) or spontaneous cure (Fig. 4B) was observed in the untreated group.
(i) Efficacy of treatment against planktonic bacteria. Figure
3 shows the killing of planktonic bacteria in cage fluid 5 days
after the completion of therapy (compared to the bacterial
counts before treatment start). By the use of monotherapy, the
planktonic bacterial counts increased by ⬍1 log10 CFU/ml with
vancomycin or daptomycin at 20 mg/kg and decreased by 0.3
log10 CFU/ml with daptomycin at 30 mg/kg. In combination
FIG. 4. Clearance rate of planktonic MRSA (A) and cure rate of
adherent MRSA in explanted cages (B). The numbers in parentheses
indicate the dose (in mg/kg) administered twice daily for all drugs
except daptomycin, which was administered once daily. DAP, daptomycin; RIF, rifampin; VAN, vancomycin; LZD, linezolid; LVX, levofloxacin; *, P ⬍ 0.05; **, P ⬍ 0.01.
VOL. 53, 2009
DAPTOMYCIN AGAINST MRSA FOREIGN-BODY INFECTION
TABLE 3. Rates of emergence of rifampin resistance in cage fluid
during and after treatment (planktonic bacteria) and in
culture from explanted cages (adherent bacteria)
Planktonic bacteriab
Treatment (dose)
a
RIF (12.5)
VAN (15) ⫹ RIF (12.5)
LZD (50) ⫹ RIF (12.5)
LVX (10) ⫹ RIF (12.5)
DAP (20) ⫹ RIF (12.5)
DAP (30) ⫹ RIF (12.5)
During
treatment
(day 6)
After
treatment
(day 12)
Adherent
bacteriac
after
treatment
(day 12)
2/12 (17)
4/12 (33)
0/12 (0)
0/12 (0)
0/12 (0)
0/12 (0)
2/12 (17)
5/12 (42)
0/12 (0)
0/12 (0)
0/12 (0)
0/12 (0)
3/12 (25)
7/12 (58)
1/12 (8)
0/12 (0)
2/12 (17)
0/12 (0)
a
The doses are in mg/kg and were administered every 12 h for all drugs except
daptomycin, which was administered every 24 h. RIF, rifampin; VAN, vancomycin; LZD, linezolid; LVX, levofloxacin; DAP, daptomycin.
b
The data represent the number of cage fluid specimens with rifampin-resistant colonies/total number of all cage fluids (percent).
c
The data represent the number of cage cultures with rifampin-resistant
colonies/total number of cage cultures (percent).
with rifampin, levofloxacin and daptomycin at 20 and 30 mg/kg
killed planktonic MRSA more efficiently (7.4 log10, 6.6 log10,
and 6.5 log10 CFU/ml, respectively) than linezolid or vancomycin (3.3 log10 and 2.8 log10 CFU/ml, respectively) (P ⬍ 0.01
for all groups). In comparison to monotherapy, vancomycin
plus rifampin was significantly more active against planktonic
bacteria (P ⫽ 0.019). Similarly, daptomycin performed significantly better in combination with rifampin (P ⬍ 0.0001) in a
manner that was independent of the dose administered.
Figure 4A shows the rate of clearance of planktonic bacteria
in cage fluid. Vancomycin and daptomycin monotherapy were
unable to clear planktonic MRSA. In combination with rifampin, levofloxacin and daptomycin showed higher clearance
rates (all 75%) than linezolid (17%), vancomycin (33%), and
rifampin (50%) alone.
(ii) Efficacy of treatment against adherent bacteria. Figure
4B shows the efficacy of treatment against adherent bacteria.
Vancomycin and daptomycin (at both doses) were unable to
cure any cage-associated infection when they were given as
monotherapy, whereas rifampin alone cured the infections in
33% of the cages. In combination with rifampin, levofloxacin
(58%) and daptomycin at 30 mg/kg (67%) cured significantly
more infected cages than vancomycin (8%) and linezolid (0%).
Emergence of antimicrobial resistance in vivo. Table 3
shows the rates of emergence of rifampin resistance in planktonic MRSA during and after rifampin monotherapy (both
17%) as well as in adherent MRSA after treatment (25%).
Rifampin resistance emerged more often during therapy with
vancomycin plus rifampin (58%) than during therapy with linezolid plus rifampin (8%) or daptomycin at 20 mg/kg plus
rifampin (17%). Levofloxacin plus rifampin and daptomycin at
30 mg/kg plus rifampin completely prevented the emergence of
rifampin resistance in planktonic as well as adherent bacteria.
No MRSA strain in cage fluid cultures from animals treated
with daptomycin or vancomycin alone or in combination with
rifampin developed resistance to daptomycin or vancomycin
(data not shown).
Evaluation of antimicrobial toxicity. In animals treated with
daptomycin (20 mg/kg), no acute lesions in the kidneys, liver,
or skeletal muscles, such as acute muscle fiber necrosis (rhab-
2723
domyolysis), were observed. In animals treated with daptomycin and rifampin, liver histology showed mild inflammation.
DISCUSSION
Daptomycin was highly bactericidal in the logarithmic
growth phase as well as in the stationary growth phase of
MRSA ATCC 43300. These in vitro studies suggested that
daptomycin may be efficacious in eradicating MRSA implantassociated infections. We used the cage-associated infection
model in guinea pigs, which has been validated for use for the
evaluation of drug activity against implant-associated infections (7, 9, 25). In contrast to the cage model in mice and rats
(14), no spontaneous cure of infected cages occurs in guinea
pigs, which resembles the situation in humans. Assuming an
approximately 50% penetration into cage fluid, daptomycin
doses of 20, 30, and 40 mg/kg in guinea pig correspond to
human doses of 4, 6, and 8 mg/kg, respectively (2, 5, 22).
Therefore, daptomycin was used at 20 and 30 mg/kg in subsequent treatment studies with guinea pigs.
In the treatment studies, none of the monotherapy regimens
tested (except rifampin monotherapy) cleared planktonic
MRSA or eradicated adherent MRSA from the cages. It might
be possible that the concentrations of daptomycin administered were not sufficiently high to eradicate biofilm-associated
MRSA. In a recent study, daptomycin at a concentration of 64
g/ml had improved activity against staphylococci embedded
in a biofilm (16). Therefore, a higher concentration of daptomycin corresponding to human doses above 6 mg/kg should be
examined in future studies with animals.
In contrast, when levofloxacin or daptomycin at a high dose
(30 mg/kg) were combined with rifampin, they showed high
degrees of efficacy against the adherent bacteria. These data
suggest that addition of rifampin to quinolones or lipopetides
is important for the eradication of staphylococcal implantassociated infections. Interestingly, in combination with rifampin, vancomycin and linezolid, both first-line drugs used
against MRSA, had lower cure rates. Furthermore, a higher
daptomycin dose (30 mg/kg versus 20 mg/kg) in combination
with rifampin was associated with a higher cure rate. The
importance of rifampin-containing regimens was also demonstrated in vitro, when rifampin in combination with daptomycin
was significantly more effective in eliminating MRSA from the
biofilm than daptomycin alone (13).
In a previous study (18), levofloxacin alone was unable to
eradicate methicillin-susceptible S. aureus, even though quinolone monotherapy cured about half of the staphylococcal
implant-associated infections in the clinical setting (25). This
reflects the stringent experimental conditions which were applied in the present experiments, in which a high infecting
inoculum, a lack of debridement of the infected cages, and a
short duration of antibiotic treatment (4 days) were used.
These conditions were chosen in order to better discriminate
the differences in efficacies of the antibiotics tested and to
determine the risk of emergence of rifampin resistance. Antimicrobial regimens effective in the present animal model will
probably also be effective in the clinical setting.
Rifampin resistance emerged in adherent MRSA from cage
cultures with rifampin monotherapy; the rate of resistance was
higher with addition of vancomycin and lower with addition of
2724
JOHN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
daptomycin at 20 mg/kg or linezolid. Addition of levofloxacin
and daptomycin at a high dose completely prevented the emergence of rifampin resistance. These data show the importance
of combining rifampin with an effective antibiofilm drug administered at a sufficient dose.
In conclusion, daptomycin at a high once-daily dose, corresponding to 6 mg/kg in humans, in combination with rifampin
showed the highest activity against planktonic and adherent
MRSA and prevented the emergence of rifampin resistance.
The cure rate achieved with this combination was comparable
to that achieved with levofloxacin plus rifampin but higher than
the one with vancomycin plus rifampin, which could not prevent emergence of rifampin resistance. This raises concern
about vancomycin combination therapy. Since health care-associated MRSA strains are increasingly resistant to quinolones, daptomycin in combination with rifampin presents a
promising treatment option for implant-associated staphylococcal infections.
ACKNOWLEDGMENTS
We thank Gerhard R. F. Krueger from the Pathology & Laboratory
Medicine at the University of Texas Health Science Center in Houston
for interpretation of the findings for the histopathological specimens,
Andrea Steinhuber for critical review of the manuscript, and Brigitte
Schneider and Zarko Rajacic for laboratory assistance.
This study was supported by the Swiss National Science Foundation
(grant 3200B0-112547/1) and by an educational grant from Novartis
Pharma Schweiz AG, Bern, Switzerland.
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18.
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