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INTRODUCTION
The emergence of glycopeptide intermediate Staphylococcus aureus (GISA) has caused serious concerns about the antimicrobial treatment of S. aureus endocarditis. Moore et al. (1) reported vancomycin treatment failure associated with GISA in a patient with endocarditis and in the rabbit model of endocarditis. Pavie et al.(2) reported teicoplanin was approximately 100-fold less active than the standard dose of vancomycin against the GISA strain and selected for the emergence of more resistant subpopulations in a rabbit model of endocarditis.
The benefit of combination therapy with a cell wall active agents and aminoglycosides has not been definitively established by human clinical trials of S. aureus infective endocardits. However, Perry et al. (3) recommended that if a glycopeptide is indicated for the treatment of endocarditis, combination therapy with a suitable aminoglycoside should be considered.
Hanaki et al. (4) reported the synergistic interactions between arbekacin and teicoplanin against GISA strains. And, Lee et al. (5) reported that the combinations of arbekacin with vancomycin, teicoplanin, or ampicillinsulbactam showed the synergistic interaction against hetero-GISA strains. But, no reported study has evaluated the usefulness of glycopeptide-arbekacin combination therapy in endocarditis due to GISA using an in vivo model.
In this study, we investigate the efficacy of teicoplanin and arbekacin on GISA in a rabbit model of endocarditis.
1. MATERIALS AND METHODS
Strains and animals
Mu50 strain (a gift from Dr. Hiramatsu) was used for the experiment. MICs were determined by broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS) (6). MIC determinations were performed using cation-adjusted Mueller -Hinton broth with an inoculums of 5×105 CFU/mL. Two antimicrobial agents were used in the experiment; teicoplanin (Aventis, Seoul) and arbekacin (Meiji Seika, Ltd, Tokyo, Japan). The experimental animals were 2.5 kg, white New Zealand rabbits.
2. Experimental staphylococcal endocarditis
To evaluate the efficacy of teicoplanin and arbekacin on GISA, we used a rabbit model of aortic valve endocarditis, which has been described previously (7). The animals received 50-60 mg of sodium pentobarbital intravenously and were anesthetized by ethyl ether inhalation. The right carotid artery was exposed through a 5 cm longitudinal incision in the neck, ligated and then incised. A polyethylene catheter was inserted a distance of about 9 cm until resistance was met. It was then withdrawn slightly and tied in place. The tip thus remained in a position just above the semilunar cusps of the aortic valve. The catheter was then filled with a sterile saline, and the skin incision was closed with silk over the free end of the catheter. Twenty-four hours after catheter placement, the rabbits were infected with 107 CFU of S. aureus.
3. Treatment with antibiotics and quantitative culture
The pretreatment burden of the organisms in the vegetations was determined using untreated control rabbits, which were killed 20 hours after inoculation. Treatment was started 20 hours after inoculation with teicoplanin alone (at 20 mg/kg of body weight intramuscularly every 12 hours for 4 days after loading dose of 40 mg/kg of body weight intramuscularly), arbekacin alone (at 5 mg/kg of body weight intramuscularly every 12 hours for 4 days), or teicoplanin plus arbekacin. The teicoplanin dose of 20 mg/kg every 12 hours was based on the previous report that showed a peak and trough concentration in serum of 44±5 µg/mL and 21±5 µg/mL, respectively (2). The arbekacin dose of 5 mg/kg every 12 hours was based on the previous reports that showed a 1-h postdose concentration in serum of 18 ± 0.46 µg/ml at 5 mg/kg intramuscularly (8). Treated rabbits were euthanized 20 hours after the administration of the last antibiotic dose. Aortic valve vegetations were harvested, weighed, and homogenized in 0.5 mL of 0.9% saline with a tissue homogenizer (Wheaton Science, Milleville, NJ, USA). Serial ten-fold dilutions of the homogenate were made in 0.9% saline and quantitatively cultured on Mueller-Hinton agar containing 8 µg of teicoplanin per ml. These dilutions avoided any significant in vivo carryover (9). After incubation at 37℃ for 24 hours, colonies were counted and the results obtained were expressed in log10CFU per gram of vegetation. The lower detection limit for this method is 1 CFU per 50 µL of undiluted vegetation homogenate
4. Statistical analysis
Comparisons of mean bacterial counts (log10CFU per gram of vegetation) between groups were determined by analysis of variance (one-way ANOVA). Turkey's post hoc comparison was used. A P value≤0.05 was considered significant. Statistical analysis was performed using SPSS 11.0 (SPSS, Chicago, IL, USA).
RESULTS
The MICs of arbekacin and teicoplanin for M50 strain were 8 µg/mL and 16 µg/mL.
The results obtained using the 4-days antibiotic treatment regimen, for experimental endocarditis due to GISA M50, are presented in Table 1. A total of 12 rabbits infected with GISA M50 were assigned to the control group (n=3) and to the various treatment regimens groups (3 for teicoplain alone, 3 for arbekacin alone and 3 for arbekacin/teicoplanin). Control rabbits had a mean±standard deviation aortic valve vegetation bacterial count of 8.51±0.67 log10 CFU per gram. Treatment with arbekacin alone or with teicoplanin alone did not reduce the aortic valve vegetation bacterial count significantly. However, treatment with arbekacin/teicoplanin reduced the aortic valve vegetation bacterial count significantly (P<0.05).
DISCUSSION
Methicillin-resistant Staphylococcus aureus (MRSA) has become a major nosocomial pathogen, and community-acquired infections caused by MRSA are increasing. Glycopeptide has been the main antimicrobial agent for treating severe infections caused by gram-positive bacteria, and is used for the treatment of infections caused by antibiotics-resistant gram-positive bacteria such as MRSA, and β-lactam resistant enterococci. However, vancomycin-resistant enterococci and S. aureus strains with reduced susceptibilities to glycopeptides have emerged (10-12), and the emergence of GISA has raised the question of the clinical relevance of this low level of resistance and of the efficacies of glycopeptides for the treatment of infections due to GISA. However, vancomycin treatment failures and slow clinical responses in cases of S. aureus endocarditis have been reported (13).
Arbekacin, a derivative of the aminoglycoside dibekacin has been used in Japan to treat infections caused by MRSA. S. aureus resistant to aminoglycoside has been reported to produce the enzymes 4'-aminoglcoside adenyltransferase, 3'-aminoglycoside phosphotransferase, or 2'-aminoglycoside phosphotransferase. However, arbekacin has a low modification rate for the several aminoglycoside-modifying enzymes, so this antibiotic is expected to be used to treat MRSA infection (14, 15).
Hanaki et al. (4) reported that. there were synergistic interaction between arbekacin and teicoplanin against GISA Mu50 strain by checkerboard synergy testing with fractional inhibitory concentration (FIC) index of 0.375. Antibiotic combinations for the treatment of endocarditis should produce a rapid bactericidal effect. In experimental animals, it has been shown that the rate of bactericidal action expressed by combination of drugs in broth is predictive of the relative rate at which the organisms would be eradicated from the cardiac vegetations in vivo (16). So, the combination of glycopeptides and arbekacin could be an alternative option in the treatment of GISA endocarditis.
This study showed that the treatment with teicoplanin and arbekacin in combination may be more effective than treating with either teicoplanin or arbekacin alone in a rabbit model of endocarditis. This finding is the first report about the usefulness of glycopeptide-arbekacin combination therapy in endocarditis due to GISA using an in vivo model.
Because we used in vivo model for evaluating the usefulness of combination treatment and the definitions of synergism in this model have not been defined, we could not evaluate the synergistic or additive effects of the combination treatment. However, we could verify that the treatment with teicoplanin and arbekacin in combination could significantly decrease the mean bacterial counts of vegetation by about 3.0 log10 CFU/g.
Although we did not measure the serum levels of the antimicrobial agents, the peak and trough concentrations of those agents would be higher than MICs of the agents for Mu50 strain (2, 8). However, the treatment with teicoplanin or arbekacin could not decrease the mean bacterial counts, compared with the control group. The suboptimal efficacy of those agents may be related to the maldistribution of the agents within vegetations (17). And, the bacteria in the vegetations could reach the large population.
However, there are several limitations in this study. First, the small number of animals per group were used. Although there were statistical significance between the combination therapy group and the single drug group, this is not a sample size big enough for a study from which fundamental conclusions are being drawn. Second, the serum levels of the agents were not measured and the doses of teicoplanin and arbekacin may have been lower than the therapeutic doses.
In spite of the limitations, the results of our study suggests that the combination of glycopeptide and arbekacin may be useful in the treatment of endocarditis caused by GISA. Further study should be performed about the effectiveness of glycopeptide and arbekacin combination.
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