Comparative evaluation of tigecycline and vancomycin, with and without rifampicin, in the treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis in a rabbit model

Li-Yan Yin1, Luca Lazzarini2, Fan Li3, C. Melinda Stevens3 and Jason H. Calhoun1,*

1 Department of Orthopaedic Surgery, University of Missouri, Columbia, MO, USA; 2 Infectious Diseases Unit, Department of Internal Medicine, San Bortolo Hospital, Vicenza, Italy; 3 Department of Orthopedics and Rehabilitation, The University of Texas Medical Branch, Galveston, TX, USA


* Corresponding author. Tel: +1-573-882-7189; Fax: +1-573-882-1760; Email: calhounj{at}health.missouri.edu

Received 6 October 2004; returned 15 November 2004; revised 13 January 2005; accepted 1 March 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Staphylococcus aureus is the most common organism isolated in osteomyelitis. Methicillin-resistant S. aureus (MRSA) infections are particularly difficult to treat. We evaluated the efficacy of tigecycline and vancomycin with and without rifampicin in a rabbit model of MRSA osteomyelitis.

Methods: A 28 day antibiotic therapy with a subcutaneous injection of tigecycline (14 mg/kg twice daily), with and without oral rifampicin (40 mg/kg twice daily); or subcutaneous administration of vancomycin (30 mg/kg twice daily), with and without oral rifampicin (40 mg/kg twice daily) were compared. Osteomyelitis was induced with an intramedullary injection of 106 colony-forming units of MRSA. Infected rabbits were randomly divided into six groups: tigecycline, tigecycline with oral rifampicin, vancomycin, vancomycin with oral rifampicin, and no treatment control and tigecycline bone penetration groups. Treatment began 2 weeks after infection. After 4 weeks of therapy, the rabbits were left untreated for 2 weeks. Rabbits were then euthanized, and the tibias were harvested. The bones were cultured, and bacterial counts of MRSA were performed.

Results: Rabbits that received tigecycline and oral rifampicin therapy (n=14) showed a 100% infection clearance. Rabbits treated with tigecycline (n=10) showed a 90% clearance. Rabbits treated with vancomycin and oral rifampicin (n=10) also showed a 90% clearance. Rabbits treated with vancomycin (n=11) showed an 81.8% clearance. Untreated controls (n=15) demonstrated only a 26% clearance. For the tigecycline bone penetration group, the bone concentrations of tigecycline in the infected tibia were significantly higher than the non-infected ones.

Conclusions: Tigecycline may be an effective alternative to vancomycin in the treatment of MRSA osteomyelitis.

Keywords: MRSA , antibiotics , infections


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The treatment of acute and chronic orthopaedic infections is difficult and often requires prolonged antibiotic therapy and surgical treatment.1 Antibiotic treatment of osteomyelitis is traditionally administered intravenously. However, oral regimens for osteomyelitis have been successfully tested in human trials.24 Unfortunately, the choice of oral antimicrobials is restricted when dealing with multidrug-resistant organisms, and this may require the use of parenteral drugs.5 Methicillin-resistant staphylococci are the most common organisms in orthopaedic infections.6 the options for treatment of infections caused by these microorganisms are limited: the sensitivity of clinical strains to quinolones, clindamycin, co-trimoxazole and rifampicin is variable, and the sensitivity is often limited to glycopeptides, which must be administered by the parenteral route. Resistance of staphylococci to glycopeptides has already been described and represents a major concern, since those drugs are considered the gold standard for the treatment of serious infections due to methicillin-resistant staphylococci.7 Novel drugs for the treatment of methicillin-resistant staphylococcal infections, such as quinupristin–dalfopristin and linezolid, have recently been introduced in clinical practice.8,9 However, none has been fully investigated in clinical studies on the treatment of osteomyelitis. Tigecycline (formerly GAR-936), a novel injectable glycylcycline antibiotic, has demonstrated excellent in vitro and in vivo activity against a large number of Gram-positive, Gram-negative, aerobic and anaerobic organisms, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (including Enterococcus faecalis), penicillin-resistant/macrolide-resistant pneumococci, Prevotella spp., peptostreptococci, mycobacteria, and minocycline-resistant organisms.1013 The drug has generally been well tolerated, and the experimental animal and clinical studies for safety and efficacy are ongoing. We tested the major pharmacokinetic parameters, efficacy, and safety of tigecycline, alone and in conjunction with oral rifampicin, in the treatment of experimental MRSA osteomyelitis in a rabbit model.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All procedures involving animals were approved by the Animal Care and Use Committee at the University of Texas Medical Branch, Galveston. All microbiological procedures were performed with proper aseptic techniques in accordance with National Committee for Clinical Laboratory Standards (NCCLS, Wayne, PA, USA) guidelines.

Generation of standard curves for diffusion bioassays

Normal New Zealand White rabbit serum (Fisher Scientific) and normal uninfected rabbit tibia bone were used to generate standard curves for tigecycline (Wyeth-Ayerst Research, Pearl River, NY, USA), vancomycin (Abbott Laboratories, Chicago, IL, USA), and rifampicin (Merrell Pharmaceuticals Inc., Kansas City, MO, USA). Bioassays were performed with each drug to generate the standard curves for antibiotic concentration in serum and/or tibial bone.

The organism used for the bioassay was Bacillus cereus ATCC 11778. Serum standards were prepared using 2-fold serial dilutions with either antibiotic to yield concentrations of 25 to 0.20 mg/L drug in normal New Zealand White rabbit serum. Bone eluate standards were prepared for tigecycline by thoroughly cleaning non-infected rabbit tibias with 70% ethanol in a sterilized fume hood. Each tibia was broken into small chips of ~0.5 cm2 using a grinder. The chips were placed into a sterile, 50 mL conical centrifuge tube and weighed. Sterile, 0.9% normal saline (1 mL) was added for each gram of bone chips. The solution was thoroughly vortexed for 2 min. The resulting bone eluate was allowed to shake at 180 rpm in a cold room at 4°C, for 12 h. The samples were centrifuged at 4000 rpm for 3 min prior to assay to pellet the chips.

The diameter of the zone of growth inhibition (mm) around each well was measured. A standard curve was generated for tigecycline concentration in both serum and bone eluate and for vancomycin in serum by plotting the known antibiotic concentration against its resulting zone of inhibition measurement.

Pharmacokinetics of tigecycline

A baseline group of six uninfected rabbits was subcutaneously administered 14 mg/kg tigecycline, reconstituted in sterile water, every 12 h for a period of 8 days. Blood samples were drawn at the following approximate intervals, post-initial antibiotic treatment: 1, 3, 6, 12, 171 and 180 h (time of sacrifice). Blood (0.5 mL) was collected with standard techniques. Samples were immediately placed into sterile, 1.5 mL centrifuge tubes. Following euthanasia, both tibias were thoroughly cleansed with 70% ethanol and then harvested after removal of all soft tissue. Tibias were placed into separate, sterile 50 mL centrifuge tubes and stored at –70°C.

Serum samples were stored at –70°C until bioassay was performed. Bone samples were prepared as previously described. Seeded agar plates were prepared, and samples were loaded in triplicate to the seeded plates and incubated at 30°C for 18 h. The diameter of the zone of growth inhibition around each well was measured, and concentrations of tigecycline were extrapolated from the standard curve.

Induction of tibial osteomyelitis

The strain of MRSA was obtained from a patient with osteomyelitis undergoing treatment. The MIC/MBC of tigecycline, vancomycin and rifampicin were determined using an antibiotic 2-fold tube-dilution method in a cation-supplemented Mueller–Hinton broth (CSMHB) (Difco Laboratories, Detroit, MI, USA). The limits of sensitivity of this method were 25–0.20 mg/L. S. aureus was incubated overnight in CSMHB medium spiked with 40 mg/L oxacillin, at 37°C.

New Zealand White rabbits (Ray Nicholl's Rabbitry, Lumberton, TX, USA), 8–12 weeks old and weighing 2.0–3.5 kg, were used for the study. After anaesthesia was given, an 18-gauge needle was inserted percutaneously through the lateral aspect of the left tibial metaphysis into the intramedullary cavity. Next, 0.15 mL of 5% sodium morrhuate (American Regent Laboratories, Inc., Shirley, NY, USA), 0.1 mL of S. aureus (106 cfu/mL), and 0.2 mL of sterile, normal saline, 0.9%, were injected sequentially. The infection was allowed to progress for 2 weeks, at which time the severity of osteomyelitis was determined radiographically (Table 1). Rabbits with stage 2 to 4 osteomyelitis were considered infected animals and were divided into six groups.


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Table 1. Criteria for radiographic osteomyelitis severity grading in rabbits

 
Treatment groups

Group 1 (control group, n=15) was infected but left untreated for the duration of the study. Group 2 (n=11) rabbits were treated for 4 weeks with subcutaneous vancomycin at 30 mg/kg twice daily. Group 3 (n=10) rabbits were treated for 4 weeks with subcutaneous vancomycin at 30 mg/kg twice daily plus oral rifampicin at 40 mg/kg twice daily in 0.5% methylcellulose. Group 4 (n=16) rabbits were treated for 4 weeks with subcutaneous tigecycline at 14 mg/kg twice daily. Group 5 (n=14) rabbits were treated for 4 weeks with subcutaneous tigecycline at the same dose as in the rabbits in Group 4, plus oral rifampicin at 40 mg/kg twice daily in 0.5% methylcellulose. Rabbits receiving oral rifampicin (Groups 3 and 5) were given an oral nutritional supplement (Ensure Plus®, Abbott Laboratories, Columbus, OH, USA) and a Lactobacillus spp. preparation (Kvvet Supply, David City, NE, USA) daily. Group 6 (n=10) rabbits were treated for 1 week with subcutaneous tigecycline at the same dose as in Group 4, but were sacrificed 3 h after administration of the last dose. At that time, blood and infected bone samples were collected, and tigecycline concentration was determined. Groups 1 to 5 were left untreated for 2 weeks after the treatment phase of the experiment and sacrificed at 8 weeks after infection. Radiographs of bilateral tibias were taken at initiation of therapy (2 weeks after infection), at the end of antibiotic therapy (6 weeks after infection), and at sacrifice (8 weeks after infection). Radiographs were scored according to a visual scale (Table 1) by three investigators, and the grades were averaged. Peak and trough levels of antibiotic were determined for Groups 2 and 4 at 1 h (peak) and 12 h (trough) after the initial antibiotic administration.

Determination of bacterial concentration per gram of bone and bone marrow

After sacrifice, gross cultures were performed for right and left tibias. Quantitative counts (cfu/g) of S. aureus in left tibial bone and marrow were determined for all study groups. The bone marrow and the intramedullary canal of bilateral tibias were swabbed with sterile cotton tip applicators for gross culture analysis of left tibias and quality assurance checks of right tibias. The inoculated applicator was streaked onto blood plates and then placed into 5 mL of sterile TSB. The plates and tubes were then incubated at 37°C for 24 h and growth and/or turbidity was recorded.

The bone marrow was placed into a sterile, 50 mL centrifuge tube and weighed. The bone fragments were broken into 0.5 cm2 chips, placed into a sterile, 50 mL centrifuge tube, and the final product weighed. Normal sterile saline, 0.9%, was added in a 3:1 ratio (3 mL saline/g of bone or marrow), and the suspensions were vortexed for 2 min. Six 10-fold dilutions of each suspension were prepared with sterile, normal saline, 0.9%. Samples (20 µL) of each dilution, including the initial suspension, were plated, in triplicate, onto blood agar plates and incubated at 37°C for 24 h; colony forming units were counted at the greatest dilution for each tibia sample. The S. aureus concentration was calculated in cfu/g of bone or bone marrow. The calculated resultant was multiplied by three for bone samples and by four for bone marrow in order to account for their initial dilutions in saline and for the adsorption of marrow into the saline. The mean log of the S. aureus concentration for each was calculated.

Statistical analysis of experimental data

The standard deviation and standard error of the mean were calculated for all raw data, including disc diffusion measurements, weight variances, radiograph grades, and bacterial counts for all rabbits that completed the entire study period. Linear regression analysis, least squares method, was performed for the antibiotic diffusion standard curves using the base 10 log of the antibiotic concentrations to plot the concentration (in mg/L) versus the zone of inhibition measured (mm). All subsequent diffusion measurements were extrapolated to mg/L of antibiotic concentration from the standard curve utilizing the slope and y-intercept values derived from the least squares calculations. To determine whether there was a significant difference in bacterial concentrations in the bone matrix and bone marrow of treated but still infected animals at the end of the study compared with untreated, infected controls, a Student's t-test was used. This test was also used to compare radiographic scores between the first (14 days post-infection) and third (56 days post-infection) sets of radiographs. Differences between groups was deemed statistically significant if P ≤ 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MIC and MBC

For the strain of MRSA used in the study, the MIC and MBC levels for tigecycline were less than 0.2 and 0.2 mg/L, respectively. The MIC and MBC levels for vancomycin were 0.39 and 0.78 mg/L, respectively, yielding MIC/MBC ratio of 0.5. The MIC and MBC levels for rifampicin were 0.78 and 1.56 mg/L, respectively, yielding a ratio of 0.5.

Drug kinetic levels in bone and serum

All concentrations of antibiotic were derived from the respective standard curves. The logarithmic trends of the concentrations of tigecycline (14 mg/kg twice daily) in the sera of the uninfected animals group are shown in Figure 1. The tigecycline, as depicted in Figure 1, was eliminated slowly, maintaining a steady level higher than MIC (0.2 mg/L) by 12 h (trough). Peaks and troughs of tigecycline in Group 4 (14 mg/kg twice daily) in the serum of infected rabbits were 1.62 ± 0.00 mg/L and 0.48 ± 0.00 mg/L. Peaks and troughs for vancomycin in Group 2 (30 mg/kg twice daily) in the serum of infected rabbits after administration of the first dosage were 3.26 ± 0.03 mg/L and 0.44 ± 0.03 mg/L separately. Therapeutic levels (mean peak level obtained/MIC level) in serum concentrations obtained in the vancomycin group (8.36 ± 0.08 mg/L) were similar to the levels (ratio of vancomycin to tigecycline) found in the tigecycline group (8.1 ± 0 mg/L). The bone concentrations of tigecycline (14 mg/kg, twice daily) in the infected rabbits group were measured separately in the infected tibia at the end of treatment, in which they averaged 0.78 ± 0.01 mg/L, and in the uninfected tibia, in which they averaged 0.49 ± 0.01 mg/L. The difference was statistically significant (P=0.05). The bone concentrations of tigecycline (14 mg/kg, twice daily) for the infected bone penetration group were found to be greater than the MIC (0.2 mg/L). Previous studies have shown that vancomycin bone penetration yields concentrations less than serum levels, but greater than the MIC for most strains of MRSA.14



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Figure 1. Pharmacokinetics in normal New Zealand White rabbits.

 
Radiographic findings

A stage 2 to 4 osteomyelitis, according to Table 1, was induced in all the infected animals. The initial radiographic grades were similar between the groups. The average grades for tigecycline, tigecycline + rifampicin and vancomycin + rifampicin groups at t=14 days were significantly greater than the average grades at t=56 days (P=0.05). The control group showed the least amount of improvement radiographically (0.2 ± 0.2 or 9.1%), when compared with vancomycin (0.5 ± 0.2 or 25%), with tigecycline (0.9 ± 0.1 or 40.9%), with vancomycin + rifampicin (0.9 ± 0.1 or 40.9%) or with tigecycline + rifampicin (0.8 ± 0.1 or 40.0%) groups. Figure 2 depicts the average radiographic severity for each group at t=14 and t=56 days. At the end of the study (t=56 days), average radiographic grades were compared between different groups. The average grades for tigecycline group, tigecycline + rifampicin group and vancomycin + rifampicin group at t=56 days were significantly lower than the average grades for the control group at t=56 days (P=0.05).



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Figure 2. Mean radiographic grades at day 14 and day 56 in each group.

 
Bone cultures

A high percentage of tibias from untreated infected controls (n=15) revealed positive cultures (74%) for MRSA at a mean concentration of 9.21 x 104 cfu/g of bone. When compared to untreated controls of positive MRSA recovery rate in bone, all other groups demonstrated a significantly lower percentage of positive MRSA recovery than untreated control group (P=0.02, Fisher's exact test by Wyeth-Ayerst). In marrow, the proportion of positive cultures in each of the antibiotic treated groups was not statistically significantly lower than the proportion in the control group. Figure 3(a) shows the positive recovery of MRSA from rabbits post 8 weeks of infection.



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Figure 3. (a) Positive recovery of MRSA from rabbits post 8 weeks of infection. The data in this figure were analysed by Wyeth-Ayerst company with Fisher's exact test; (b) cfu/g of marrow and bone.

 
In the vancomycin group, two out of 11 samples (18.2%) were positive for MRSA, and the average bacterial concentration of the group was 1.4 x 102 cfu/g of bone, which is lower than the control group (P=0.05). In the tigecycline group, one out of 10 samples was positive for MRSA, and the average bacterial concentration in the group was 20 cfu/g of bone, which is lower than either the controls or the vancomycin group (P=0.05). One rabbit in the vancomycin + rifampicin group showed higher bacteria concentration than the control. The rabbits receiving tigecycline + rifampicin treatment demonstrated complete eradication of bacteria from the tibia (0.0 cfu/g of bone in all the samples). Figure 3(b) compares the cfu/g of marrow and bone between all groups.

Adverse events

Of the 76 infected rabbits (including Group 6, tigecycline bone penetration study), a total of eight (10.5%) died before completion of treatment (Table 2). One rabbit in Group 6 died before the treatment with tigecycline due to intolerance to anaesthesia. Of the six rabbits that died in the tigecycline treatment group, one was euthanized at day 19 because of severe impairment of nutritional status. Another two rabbits died at day 17 and 25 of tigecycline treatment due to gastroenterocolitis. Three of the rabbits in this group died at day 28 due to gastroenterocolitis and intolerance to anaesthesia. One rabbit in the tigecycline + rifampicin group died during treatment at day 15 due to gastroenterocolitis. All rabbits were monitored weekly for weight variance. The control group showed the greatest mean gain (0.58 ± 0.27 kg), vancomycin the second greatest (0.39 ± 0.26 kg), and the vancomycin + rifampicin group the third (0.21 ± 0.32 kg). The tigecycline group (–0.05 ± 0.32 kg) and the tigecycline + rifampicin group (–0.39 ± 0.31 kg) both lost weight after the antibiotic treatment. Nearly all rabbits in the tigecycline group and tigecycline + rifampicin group presented with mild to severe symptoms of gastric dysfunction ~1.0–1.5 weeks post-antibiotic initiation, including decreased appetite, dehydration, diarrhoea, and/or weight loss. Figure 4 shows the weight variances between all the groups.


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Table 2. Adverse events

 


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Figure 4. Average initial and end weight in each group.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic treatment of osteomyelitis is still a challenge for the physician. Many orthopaedic infections are acquired in the nosocomial environment.15 The causative agents of such infections are often multidrug-resistant. Staphylococci are the most common organisms, but Gram-negative pathogens may be involved as well.16 Infections due to MRSA, compared with those due to MSSA, are more difficult to treat and may have a poorer prognosis.17 Therapeutic options for these infections are limited. The only drugs with a constant efficacy against all the staphylococcal strains, and which have been extensively studied in the treatment of bone infections, are glycopeptides. Unfortunately, resistance to these antibiotics has already been recognized as a major problem in the treatment of Gram-positive pathogens. Enterococci resistant to vancomycin are diffused worldwide, and such a resistance has been demonstrated as potentially transmittable to other Gram-positive organisms initially in vitro and more recently in patients.18,19 Moreover, sporadic strains of vancomycin-resistant S. aureus have been isolated in several countries.20,21 Therefore, the availability of alternative antimicrobial agents for the treatment of multidrug-resistant pathogens is of paramount importance.

Tigecycline (formerly GAR-936) is a 9-t-butylglycylamido synthetic derivative of minocycline, and a member of a new class of antibiotics called glycylcyclines. This new class of tetracycline derivatives has demonstrated excellent in vitro activity against a large number of Gram-positive and Gram-negative, aerobic and anaerobic organisms, including MRSA, vancomycin-resistant enterococci (including Enterococcus faecalis), penicillin-resistant/macrolide-resistant pneumococci, Prevotella spp., peptostreptococci, and Mycobacterium spp.1013 Tetracyclines are bacteriostatic agents, acting to inhibit bacterial protein synthesis. The glycylcyclines have been developed to overcome the bacterial mechanisms of resistance to tetracyclines, even though their exact mechanism of action has not yet been determined.22 In an animal model of MRSA endocarditis, tigecycline given at 14 mg/kg twice daily was shown to be more effective than 40 mg/kg vancomycin.23 In such a rat model, dosages as high as 80 mg/kg per day were administered. However, in our rabbit model, the administration of dosages higher than 14 mg/kg per day caused relevant morbidity and mortality in the animals (data not shown). Therefore, we decided to use the above cited dosage in our study. Even though our goal was not to study the pharmacokinetics of tigecycline in rabbits, we performed some drug-level measurements in order to ensure that we were using an adequate dosage in our animal model. Our data confirm that drug levels in serum were still above the MIC for the staphylococcal strain used 12 h after the last administration. Moreover, the drug has displayed a relevant bone penetration, and therapeutic levels of tigecycline have been found in the infected and uninfected bone. The higher concentration of drug found in the infected bone is another relevant finding, which requires further study.

Eradication of bacteria from the bone with a systemic antibiotic is a difficult task. Several studies have been performed using various animal models of osteomylitis.24 An important lesson learned from these studies is that, despite a prolonged antibiotic treatment, viable bacteria may be found in the bone. Eradication of more bacteria from the bone has been associated with a prolonged duration of antibiotic treatment.25 After 4 weeks of antibiotic treatment, the majority of antibiotic regimens were unable to eradicate staphylococci from the bone. This fact suggests the possibility of comparing the efficacy of various antimicrobial agents in achieving the goal of bacterial eradication. In Norden's rabbit model, only a few regimens were able to achieve bacterial eradication from the bone.26 In that model, the association of rifampicin and nafcillin gave complete eradication of bacteria from the bone, whereas a percentage of eradication ranging from 50% to 90% was documented with the use of other antibiotics.2629 In general, the association of rifampicin with another antimicrobial agent permitted a better outcome than the second antimicrobial alone.26,27 It should be noted that all of these experimental data are from studies on staphylococcal osteomyelitis and, to our knowledge, studies on osteomyelitis due to other bacteria are still very limited.

In our trial, the group treated with tigecycline showed lower colony forming units in bone and marrow than the infected, untreated control group or the group treated with vancomycin at the end of the treatment period. The association of tigecycline and rifampicin allowed the complete eradication of bacteria from the bone and marrow, whereas in the vancomycin + rifampicin group, a sample was still positive. The differences in the outcome of our treatment groups seem even more remarkable than those of the cited studies. Despite the low number of treated animals in each group, our findings are statistically significant.

As for the safety, a higher number of deaths and side effects were seen in the groups of rabbits treated with tigecycline. Enterocolitis due to tigecycline may be caused by an extensive destruction of the normal microbial flora of the bowel. The symptoms were attenuated by the administration of oral probiotics. The broad antimicrobial spectrum of tigecycline, in contrast with the narrower spectrum of vancomycin, may help explain the difference that we observed between the treatment groups.

Tigecycline appears to be a potential drug in the treatment of severe orthopaedic infections. The antimicrobial spectrum is broad, including all the pathogens found in nosocomial orthopaedic infections. In our experimental model, the pharmacokinetic properties were favourable, since the drug may be administered twice daily. Moreover, the bone penetration is adequate, and drug levels above the MIC were found in almost every sample collected. As to the safety profile in humans, recent studies have shown that tigecycline was efficacious with an acceptable safety profile in the treatment of complicated intra-abdominal infections and complicated skin and skin-structure infections.3033 Our current research further suggests that tigecycline may be suitable for clinical studies on orthopaedic infections with patients infected with MRSA.


    Acknowledgements
 
We thank the late Dr Jon T. Mader for his contributions. We also thank Maurice M. Manring, Mark E. Shirtliff and Kristi Overgarrd for their assistance with manuscript preparation; and Jay Rain, Zachary P. Hileman, Rebecca Lynn Skains, Shazia Amina and Jue Wang for technical assistance. This research was sponsored by a grant from Wyeth-Ayerst Research.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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