ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2006, p. 654–657 0066-4804/06/$08.00⫹0 doi:10.1128/AAC.50.2.654–657.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 2
Linezolid in Prophylaxis against Experimental Aortic Valve Endocarditis Due to Streptococcus oralis or Enterococcus faecalis George Athanassopoulos,1 Angelos Pefanis,2* Vissaria Sakka,1 Dimitrios Iliopoulos,3,4 Despina Perrea,3,4 and Helen Giamarellou1 Fourth Department of Internal Medicine, General Hospital “Attikon,”1 Third Department of Internal Medicine, General Hospital “Sotiria,”2 Laboratory of Experimental Surgery and Surgical Research,3 and Second Department of Propedeutic Surgery, General Hospital “Laikon,” 4 Athens University Medical School, Athens, Greece Received 27 July 2005/Returned for modification 31 August 2005/Accepted 15 November 2005
There are no experimental studies regarding the prophylactic efficacy of linezolid against infective endocarditis. Nonbacterial thrombotic endocarditis of the aortic valve was induced in rabbits by the insertion of a polyethylene catheter. Twenty-four hours later, animals were randomly assigned to a control group, and groups receiving either ampicillin (two doses of 40 mg/kg of body weight each, given intravenously, 2 h apart) or linezolid (a single per os dose of 75 mg/kg). The first dose of ampicillin and the single dose of linezolid were administered 0.5 and 1 h, respectively, prior to the intravenous inoculation of ⬃107 CFU of Streptococcus oralis or Enterococcus faecalis. Linezolid peak levels in rabbit serum were similar to the peak serum levels in humans following a 600-mg oral dose of linezolid. Linezolid prevented endocarditis in 87% of S. oralis-challenged rabbits (P < 0.001 versus controls; P ⴝ 0.026 versus ampicillin). In rabbits challenged with E. faecalis, linezolid prevented endocarditis in 73% (P ⴝ 0.003 versus controls; P ⴝ 0.049 versus ampicillin). Ampicillin prevented endocarditis due to S. oralis or due to E. faecalis in 47% (P ⴝ 0.005 versus controls) and in 30% (P ⴝ not significant versus controls) of the challenged animals, respectively. In conclusion, linezolid was effective as prophylaxis against endocarditis caused by a strain of S. oralis and to a lesser degree against that caused by a strain of E. faecalis. Its prophylactic efficacy was superior to that of ampicillin. subunit, thus preventing formation of a functional initiation complex in bacterial translation systems. Linezolid is highly active in vitro against a wide range of gram-positive bacteria (25). It also exhibits quite a long half-life (4.5 to 5.5 h), and it can be given per os with excellent bioavailability (100%) (22). Thus, it could be eligible as a prophylactic agent against I⌭, particularly in patients allergic to -lactams. Since there are n experimental studies of the prophylactic activity of linezolid against I⌭, this study was designed to evaluate its prophylactic efficacy against the most common preventable etiologic agents responsible for the development of I⌭, namely, viridans group streptococci and enterococci, by applying the rabbit model (21).
Gram-positive cocci are the most frequent etiologic agents of infective endocarditis (IE), responsible for up to 80 to 90% of cases (23). Amoxicillin and newer macrolides are the primary prophylactic regimens for most patients (7). However, the frequency of resistance or reduced susceptibility to amoxicillin (11%) (12) and the high frequency of high-level resistance to penicillin (up to 45%) (11, 16, 24) and resistance to macrolides (up to 55%) (24) among some species of viridans group streptococci raise concerns regarding their use as therapeutic or prophylactic agents for infections caused by these organisms. Moreover, the need for parenteral administration of glycopeptides (which in the case of vancomycin should last at least 60 min), which are the recommended antibiotics for prophylaxis in patients allergic to -lactam antibiotics who undergo genitourinary or gastrointestinal procedures, especially in those at high risk (i.e., with previous endocarditis or a prosthetic valve) (7, 26), is the main disadvantage of their use as prophylactic agents. Thus, new compounds that combine the advantages of (i) exhibiting no cross-allergy to -lactam antibiotics, (ii) having a low level of toxicity, (iii) being effective against streptococci and other gram-positive cocci as well, and (iv) having an oral route of administration and a prolonged half-life in the serum are warranted. Linezolid belongs to the oxazolidinones, a new class of antimicrobials with a unique mechanism of action. They inhibit bacterial protein synthesis by binding to the 50S ribosomal
MATERIALS AND METHODS Microorganisms. The two strains (Streptococcus oralis and Enterococcus faecalis) used in this study were isolated from the blood of patients with endocarditis and were identified by API 20 STREP and VITEK (bioMe´rieux, Marcy l’ Etoile, France). The bacteria were stored at ⫺80°C in skim milk and were subcultured on blood agar plates (BAPs) 3 days before each experiment. Susceptibility testing. The MICs of linezolid and ampicillin were determined by a microdilution technique in volumes of 0.1 ml by using logarithmic-growthphase inocula of S. oralis in Todd-Hewitt broth and of E. faecalis in cationsupplemented Mueller-Hinton II broth (BBL Microbiology Systems, Cockeysville, Md.) adjusted to a final inoculum of ⬃5 ⫻ 105 CFU per ml (15). The MIC was defined as the lowest concentration causing no visible turbidity after incubation for 18 h at 37°C (S. oralis) or 35°C (E. faecalis). The minimum bactericidal concentration (MBC) of moxifloxacin was determined by subculture of 0.1 ml from each clear well onto BAPs and was defined as the lowest concentration that reduced the number of organisms of the initial inoculum by ⱖ99.9%. Induction of and prophylaxis against endocarditis. Nonbacterial thrombotic endocarditis of the aortic valve was induced in female white rabbits weighing ⬃3.0 kg by using the model described by Perlman and Freedman, with the polyethylene catheter left in place throughout the experiment (21). Twenty-four hours after catheterization, the rabbits were randomly assigned to a control
* Corresponding author. Mailing address: Third Department of Medicine, Athens University School of Medicine, “Sotiria” General Hospital, 152 Mesogion Avenue, 11527 Athens, Greece. Phone: 30-2109582565. Fax: 30-2107778838. E-mail:
[email protected]. 654
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TABLE 1. Results of prophylaxis in animals with aortic valve vegetations challenged with S. oralis or E. faecalis S. oralis Drug
Control Ampicillin Linezolid
E. faecalis
MIC/MBC (mg/liter)
Sterile/nonsterile vegetation (%)
Amt of vegetation (log10 CFU/g) (mean ⫾ SD)
1/1 1/2
1/19 (5) 8/9 (47)a 14/2 (87)b,c
8.80 ⫾ 2.06 4.73 ⫾ 3.07b 2.37 ⫾ 1.04b
MIC/MBC (mg/liter)
Sterile/nonsterile vegetation (%)
Amt of vegetation (log10 CFU/g) (mean ⫾ SD)
1/1 2/16
2/11 (15) 3/7 (30) 11/4 (73)d
7.91 ⫾ 3.16 5.20 ⫾ 2.34e 3.25 ⫾ 2.25b, f
Significantly different from the value obtained for the control group (P ⫽ 0.005). Significantly different from the value obtained for the control group (P ⬍ 0.001). Significantly different from the value obtained for the group treated with ampicillin (P ⫽ 0.026). d Significantly different from the values obtained for the control group (P ⫽ 0.003). e Significantly different from the values obtained for the control group (P ⫽ 0.023). f Significantly different from the values obtained for the group treated with ampicillin (P ⫽ 0.046). a b c
group, a group receiving linezolid at a single dose of 75 mg per kg of body weight per os, and a group receiving ampicillin (two doses of 40 mg per kg of body weight, intravenously, 2 hours apart). The dose of linezolid was chosen because in previous studies (3) the achieved peak (at 1 h) serum levels in rabbits (9.8 ⫾ 7.8 mg/liter) were quite similar to the maximum concentration of drug in serum in humans after a single per os dose of 600 mg (12.7 mg/liter) (22). However, in other studies, peak serum levels (in rabbits treated with the same dose of linezolid) of 18.5 ⫾ 15 mg/liter to 25.2 ⫾ 4.7 mg/liter have been reported (5, 17). The dose of ampicillin was chosen because of its use in previous studies of endocarditis prophylaxis (1, 20). The first dose of ampicilin and the single dose of linezolid were administered 0.5 and 1 h, respectively, prior to the intravenous inoculation of ⬃107 CFU of S. oralis or E. faecalis. This inoculum was suspended in 1 ml of saline and injected via the marginal ear vein. A second dose of ampicillin was administered 1.5 h after the bacterial challenge (2 h after the first dose) because of the sort half-life of the antibiotic in rabbits. The rabbits were sacrificed 3 days (72 h) after bacterial challenge by a rapid intravenous injection of 30 mg of sodium phenobarbital per kg, in order to avoid the carryover effect, since no detectable levels of antibiotics were expected to exist 72 h after their administration. Use of this time interval could also allow the detection of any possible relapses due to the regrowth of persistent, viable bacteria in vegetations after the complete elimination of antibiotics from the body. At the time of sacrifice, aortic valve vegetations were excised, weighed, homogenized in 1 ml of saline, and quantitatively cultured in duplicate onto BAPs after eight dilutions with a 1-log-unit inoculum difference between dilutions. The colonies were counted after incubation for 48 h at 37°C in ambient air with 5% CO2 or for 24 h at 35°C for S. oralis or E. faecalis, respectively. The results were expressed as the log10 numbers of CFU per gram of vegetation. The limit of detection for CFU counts was 2 log10 CFU per gram of vegetation. The macroscopic and/or bacteriologic data obtained at the time of sacrifice provided confirmation of the
successful induction of vegetative endocarditis. Rabbits with sterile vegetations were considered uninfected. The study received a permit from the veterinary directorate of the prefecture of Athens according to the Greek legislation in conformance with the European Union council directive. Antibiotic concentrations in serum. Linezolid levels were determined in serum samples at 40 and 75 min and at 2, 4, 8, and 24 h postdosing. The concentrations of linezolid were measured by high-performance liquid chromatography (HPLC) (19). The lower limit of detection of the HPLC assay applied was 0.4 g/ml. Ampicillin levels were determined in serum samples at 0.5, 1, and 2 h (trough levels) after the first dose and 2 h after the second dose. 〈n agar well bioassay technique was applied for ampicillin, with Micrococcus luteus as the test organism. Normal rabbit serum was used as the diluent. The lower limit of detection by the bioassay applied was 0.1 g/ml. Statistical analysis. ⌻ compare the differences between sterile (successful prophylaxis) and nonsterile vegetations, the Fisher exact test for probabilities was used. ⌻ compare the differences between the mean log10 CFU per gram of vegetations, the ⌲ruskal-Wallis test was applied. 〈 P value of ⬍ 0.05 was considered significant.
RESULTS MICs. The MICs and the MBCs of linezolid and ampicillin for the two strains tested are presented in Table 1. Antibiotic pharmacokinetics in serum. The mean concentrations of linezolid, after the administration of a single per os dose of 75 mg/kg, in rabbit serum over time are shown in Fig. 1. The mean ⫾ standard deviation concentrations of ampicillin in
FIG. 1. Mean concentrations of linezolid in rabbit serum over time.
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serum 0.5, 1, and 2 h after the first dose and 2 h after the second dose were 11.0 ⫾ 2.9 mg/liter (n ⫽ 13), 3.2 ⫾ 1.6 mg/liter (n ⫽ 18), 1.0 ⫾ 0.7 mg/liter (n ⫽ 14), and 0.8 ⫾ 0.4 mg/liter (n ⫽ 14), respectively. The 24-h area under the curve of linezolid was 62.4 mg · h/liter. Prophylaxis against endocarditis. The results of prophylaxis against the two strains are presented in Table 1. Ninety-five percent of the control animals challenged with 107 CFU of S. oralis and 85% of the control animals challenged with 107 CFU of E. faecalis developed infected vegetations. In rabbits challenged with this very high inoculum of S. oralis or E. faecalis, linezolid prevented endocarditis in 87% (P ⬍ 0.001 versus controls) and in 73% (P ⫽ 0.003 versus controls) of them, respectively. Ampicillin prevented endocarditis due to S. oralis or E. faecalis in 47% (P ⫽ 0.005 versus controls) and in 30% (P ⫽ 0.62 versus controls) of the challenged animals, respectively. Linezolid was superior to ampicillin in preventing streptococcal (P ⫽ 0.026) or enterococcal (P ⫽ 0.049) endocarditis. The mean log10 CFU of S. oralis per gram of vegetation in those vegetations that were not sterilized by ampicillin or linezolid were 4.73 (n ⫽ 9) and 2.37 (n ⫽ 2), respectively. The absolute log reductions were 4.07 and 6.43, respectively. The mean log10 CFU of E. faecalis per gram of vegetation in those vegetations that were not sterilized by ampicillin or linezolid were 5.20 (n ⫽ 7) and 3.25 (n ⫽ 4), respectively. The absolute log reductions were 2.71 and 4.66, respectively. DISCUSSION In the present study, we evaluated the efficacy of a single dose of linezolid for the prophylaxis of experimental endocarditis due to S. oralis or E. faecalis. Despite the fact that linezolid is bacteriostatic, it prevented endocarditis in 87% of the rabbits challenged with S. oralis and in 73% of the rabbits challenged with E. faecalis. This linezolid “paradox” has also been observed in recent studies of experimental endocarditis in which, despite bacteriostatic effects in vitro, linezolid has been proven to exhibit significant antibacterial effects (3). Conversely, despite in vitro susceptibility, ampicillin prevented endocarditis in only 47% of the rabbits challenged with S. oralis and in 30% of the rabbits challenged with E. faecalis. Glauser and Francioli, in a summary of their experimental observations of antibiotic prophylaxis of streptococcal endocarditis (9), concluded that single doses of antibiotics, such as amoxicillin, penicillin G, clindamycin, and vancomycin, are successful in reliably preventing endocarditis induced by bacterial inocula corresponding to the 90% infective dose (ID90), while multiple doses are needed to achieve prolonged inhibitory concentrations when bacterial challenges higher than the ID90 are used. In the present study, endocarditis was induced by bacterial inocula of S. oralis or E. faecalis corresponding to the ID95 and to the ID85, respectively. Buchanan et al. (3) provided evidence that linezolid exhibits time-dependent antibacterial effects in experimental rabbit endocarditis. As stated by Moreillon and the Swiss Working Group for Endocarditis Prophylaxis (13) the duration of antibiotic presence in the serum is critical. Under experimental conditions, the drugs must remain above their MIC for the organisms for ⱖ10 h to allow time for bacterial clearance from the valves. This is in agreement with the results of the present study, in which the
ANTIMICROB. AGENTS CHEMOTHER.
prolonged growth inhibition, achieved by the sustained supraMIC levels of linezolid for more than 8 h (Fig. 1) after per os administration of the drug, seems to be the most plausible explanation for the prophylactic efficacy of linezolid. However, the possible antiadherence effect of linezolid was not evaluated in our study. There are a few trials reported in the literature of the therapeutic efficacy of linezolid against staphylococcal (4, 5, 6, 10, 17) or enterococcal (18) experimental endocarditis. However, to our knowledge, this is the first study of the prophylactic efficacy of linezolid against experimental endocarditis due to viridans group streptococci or E. faecalis. In the only study of the prophylactic efficacy of linezolid yet published, Giacometti et al. (8) demonstrated, in a rat subcutaneous-pouch model of graft infection caused by Staphylococcus epidermidis with intermediate resistance to glycopeptides, that linezolid (as a single intraperitoneal injection) alone or combined with levofloxacin and vancomycin resulted in significant inhibition of bacterial growth on the Dacron prostheses, even when high concentrations of resistant organisms were inoculated topically. In conclusion, in the present experimental study, linezolid was effective as prophylaxis against endocarditis caused by a strain of S. oralis and to a lesser degree against that caused by a strain of E. faecalis. Its prophylactic efficacy was superior to that of ampicillin. These results and the facts that (i) the incidence of infections due to linezolid-resistant enterococci remains quite low (2, 14) and (ii) only a few strains of linezolidresistant viridans group streptococci have been reported (14) suggest that linezolid given as a single dose of 600 mg per os should be considered an alternative to standard regimens for the prophylaxis of endocarditis, especially in the outpatient setting. However, since its use as a first-line agent might have implications for the selection or emergence of resistance, linezolid must be reserved only for patients at risk for enterococcal endocarditis and with a history of allergy to -lactams. For allergic patients at risk for streptococcal endocarditis, current recommended prophylactic regimens, such as clindamycin or azithromycin, should be preferred to linezolid. ACKNOWLEDGMENTS We thank Thomas Tsaganos for his help in the measurement of linezolid plasma levels by HPLC and Zoi Chrissouli for technical assistance. REFERENCES 1. Bayer, A. S., and J. Tu. 1990. Chemoprophylactic efficacy against experimental endocarditis caused by -lactamase-producing, aminoglycoside-resistant enterococci is associated with prolonged serum inhibitory activity. Antimicrob. Agents Chemother. 34:1068–1074. 2. Brauers, J., M. Kresken, D. Hafner, P. M. Shah, and the German Linezolid Resistance Study Group. 2005. Surveillance of linezolid resistance in Germany, 2001–2002. Clin. Microbiol. Infect. 11:39–46. 3. Buchanan, L. V., C. F. Dailey, R. J. LeMay, R. J. Zielinski, M. S. ⌻. Kuo, and J. K. Gibson. 2002. Time-dependent antibacterial effects of linezolid in experimental rabbit endocarditis. J. Antimicrob. Chemother. 50:440–442. 4. Chiang, F. Y., and M. Climo. 2003. Efficacy of linezolid alone or in combination with vancomycin for treatment of experimental endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 47:3002–3004. 5. Dailey, C. F., C. L. Dileto-Fang, L. V. Buchanan, ⌴. ⌹. Oramas-Shirey, D. H. Batts, C. W. Ford, and J. K. Gibson. 2001. Efficacy of linezolid in treatment of experimental endocarditis caused by methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 45:2304–2308. 6. Dailey, C. F., P. J. Pagano, L. V. Buchanan, J. A. Paquette, J. V. Haas, and J. K. Gibson. 2003. Efficacy of linezolid plus rifampin in an experimental
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