Successful moxifloxacin prophylaxis against experimental streptococcal aortic valve endocarditis

Vissaria Sakka1, Lambrini Galani1, Angelos Pefanis2,*, Dimitrios Iliopoulos3,4, George Athanasopoulos1, Ismini Donta3,4 and Helen Giamarellou1

1 4th Department of Internal Medicine, General Hospital ‘Attikon’, University of Athens, Medical School, Athens, Greece; 2 3rd Department of Internal Medicine, General Hospital ‘Sotiria’, University of Athens, Medical School, 152 Mesogion Avenue 11527, Athens, Greece; 3 Laboratory of Experimental Surgery and Surgical Research, General Hospital ‘Laikon’, University of Athens, Medical School, Athens, Greece; 4 2nd Department of Propedeutic Surgery, General Hospital ‘Laikon’, University of Athens, Medical School, Athens, Greece


* Corresponding author. Tel: +30-1-9582565; Fax: +30-1-7778838; E-mail: apefan{at}med.uoa.gr

Received 10 July 2005; returned 13 August 2005; revised 1 September 2005; accepted 13 September 2005


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Objectives: Studies related to the prophylactic efficacy of fluoroquinolones against infective endocarditis are scarce. The aim of this study was to evaluate the efficacy of moxifloxacin, a quinolone active in vitro against Gram-positive cocci, in preventing streptococcal aortic valve endocarditis.

Methods: Non-bacterial thrombotic endocarditis of the aortic valve was induced by the insertion of a polyethylene catheter. Twenty-four hours later, rabbits were randomly assigned to a control group, and groups receiving either two doses of ampicillin (40 mg/kg, intravenously), 2 h apart, or a single dose of moxifloxacin (15 mg/kg, intravenously). Ampicillin and moxifloxacin were administered 0.5 and 1 h, respectively, prior to the intravenous inoculation of 107 cfu of Streptococcus oralis.

Results: Eighty-nine percent of the control animals developed infected vegetations. In rabbits challenged with this very high inoculum, moxifloxacin and ampicillin prevented endocarditis in 80% (P < 0.001 versus controls) and in 50% (P = 0.022 versus controls) of animals, respectively. The difference between ampicillin and moxifloxacin was not statistically significant (P = 0.128).

Conclusions: Moxifloxacin was at least as effective as ampicillin in preventing streptococcal endocarditis.

Keywords: Streptococcus oralis , fluoroquinolones , rabbits , ampicillin


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Gram-positive cocci are the most frequent aetiological agents, responsible for up to 80–90% of the cases, of infective endocarditis (IE). Amoxicillin and newer macrolides are the primary prophylactic regimens for most high risk patients.1 However, the frequency of resistance or reduced susceptibility to amoxicillin (11%),2 and resistance to macrolides (up to 55%) among some species of viridans group streptococci,3 raise concerns regarding their use as prophylactic agents for infections caused by these organisms. Moreover, the need for parenteral administration of glycopeptides is the main disadvantage of their use as prophylactic agents. Thus, new compounds (i) exhibiting no cross-allergy to ß-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.

Moxifloxacin is active in vitro against Gram-positive bacteria. It is also exhibits a long half-life (9–15 h), and good oral bioavailability while it is well tolerated when administered orally as a single dose in adults.4 Thus, it could be eligible as a prophylactic agent against IE.

This study was designed to evaluate the prophylactic efficacy of moxifloxacin against a strain of Streptococcus oralis, by applying the rabbit model.


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Microorganism

The strain of S. oralis used in this study was isolated from the blood of a patient with endocarditis and was identified by standard methods. The bacteria were stored at –80°C in skimmed milk and were subcultured on blood agar plates (BAPs) 3 days before each experiment.

Susceptibility testing

The MICs of penicillin, ampicillin and moxifloxacin were determined by a microdilution technique in volumes of 0.1 mL, by using logarithmic-growth-phase inocula of S. oralis in Todd–Hewitt broth (BBL Microbiology Systems, Cockeysville, MD, USA) adjusted to a final inoculum of ~5 x 105 cfu/mL. The MIC was defined as the lowest concentration causing no visible turbidity after incubation for 18 h at 37°C. The 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 ≥99.9%.

Induction and prophylaxis of endocarditis

For the production of non-bacterial thrombotic endocarditis of the aortic valve, the model described by Perlman and Freedman5 was applied. Female white rabbits weighing 3.0 to 3.9 kg were anaesthetized by intramuscular injection of ketamine hydrochloride (15 mg/kg of body weight). The left carotid artery was exposed in the neck and was cannulated with a polyethylene catheter (22Fr). The tip of the catheter was placed across the aortic valve into the left ventricle, and the proximal end was then secured in place in the neck for the duration of the experiment. Twenty-four hours after catheterization, the rabbits were randomly assigned to a control group, a group receiving moxifloxacin (kindly provided by Bayer Hellas) at a single dose of 15 mg per kg of body weight intravenously, and a group receiving two doses of 40 mg per kg of body weight of ampicillin (supplied by Bristol-Myers Squibb), intravenously, 2 h apart. The dose of moxifloxacin was chosen because in pilot studies the achieved peak serum levels in rabbits were similar to Cmax in humans after a single oral dose of 400 mg.4 The dose of ampicillin was chosen because it has been used in previous studies in endocarditis prophylaxis.6 Animals treated with ampicillin or moxifloxacin were challenged 0.5 h or 1 h later, respectively, with an inoculum of ~107 cfu of S. oralis. This inoculum was suspended in 1 mL of saline and injected via the marginal ear vein. The rabbits were sacrificed 3 days 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 moxifloxacin were expected to exist 72 h after their administration. Use of this time interval could also allow for the detection of any possible relapses due to the regrowth of persistent, viable bacteria in vegetation, 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 inoculum difference between each dilution. The colonies were counted after incubation for 24 h at 37°C in room air with 5% CO2. The results were expressed as the log10 numbers of cfu per gram of vegetation. The macroscopic and/or bacteriological 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 Greek legislation in conformance with the council directive of the EU.

Antibiotic concentrations in serum

Moxifloxacin levels were determined in serum samples obtained at 1, 2, 4, 8 and 24 h post-dosing. Ampicillin levels were determined in serum samples at 0.5, 1 and 2 h after the first dose and 2 h after the second dose. An agar well bioassay technique was applied. Bacillus subtilis ATCC 6633 was used as the test organism for moxifloxacin and Micrococcus luteus for ampicillin, and normal rabbit serum was used as the diluent. The lower limit of detection of this assay was 0.15 mg/L.

Statistical analysis

To compare the differences between sterile and non-sterile vegetations, the Fisher exact test for probabilities was used. To compare the differences between the mean log10 cfu per gram of non-sterile vegetations, the Kruskal–Wallis test was used. A P value of <0.05 was considered significant.


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The MICs of penicillin, ampicillin and moxifloxacin for the strain of S. oralis were <0.03, 0.025 and <0.03 mg/L, respectively. The MBC of moxifloxacin was 0.03 mg/L.

The mean ± standard deviation (mean ± SD) serum concentrations of moxifloxacin at 1, 2, 4, 8 and 24 h post-dosing were: 2.8 ± 0.7 mg/L (n = 7), 2.1 ± 0.7 mg/L (n = 8), 1.1 ± 0.5 mg/L (n = 7), 0.3 ± 0.1 mg/L (n = 8) and undetectable, respectively. The mean ± SD (n = 15) concentrations of ampicillin in serum 0.5, 1 and 2 h after the first dose and 2 h after the second dose were: 9.56 ± 3.72 mg/L, 3.24 ± 1.19 mg/L, 0.58 ± 0.48 mg/L (in four animals the concentrations were below the level of detection) and 0.73 ± 0.47 mg/L, respectively.

The results of prophylaxis against the strain of S. oralis are presented in Table 1. Sixteen out of 18 (89%) of the untreated animals developed infected vegetations. Moxifloxacin prevented endocarditis in 12 out of 15 (80%) animals (P < 0.001 versus controls). Ampicillin prevented endocarditis in 7 out of 14 (50%) animals (P = 0.022 versus controls, P = 0.128 versus moxifloxacin). One animal in the ampicillin group was excluded because of incorrect placement of the catheter. The mean ± SD bacterial densities in vegetations from rabbits infected despite prophylaxis with moxifloxacin (8.38 ± 0.77 log10 cfu/g) or ampicillin (7.78 ± 2.07 log10 cfu/g) were not statistically different from those found in control animals (9.21 ± 0.94 log10 cfu/g).


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Table 1. Results of prophylaxis with moxifloxacin in rabbits challenged with S. oralis

 

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The in vivo efficacy of the newer fluoroquinolones has been assessed in various experimental models. However, there are few experimental studies in which a fluoroquinolone was used successfully as prophylaxis against IE. Using a rat model, Voorn et al.7 studied ciprofloxacin against a cloxacillin-tolerant strain of Staphylococcus aureus and its non-tolerant variant at two different doses (6 and 30 mg/kg). The higher dose afforded almost full protection against both strains, whereas the lower dose had a significantly lower protective effect. Katsarolis et al.8 reported successful prophylaxis (in 100% of the animals) with trovafloxacin, against experimental aortic valve endocarditis due to an ampicillin-tolerant but trovafloxacin-susceptible strain of S. oralis. However, the necessity for a second dose of trovafloxacin, despite the fact that supra-MIC levels persisted in serum for close to 18 h after the administration of a single dose, points out substantial weaknesses of trovafloxacin as a potential prophylactic agent. Sparfloxacin has also been used successfully as a prophylactic agent against experimental staphylococcal and enterococcal endocarditis, but the results against streptococcal endocarditis were disappointing.9 In this study, a single dose of moxifloxacin was effective as prophylaxis against experimental aortic valve endocarditis, in rabbits challenged with an inoculum of S. oralis corresponding to the ID90. As stated by Moreillon and the Swiss Working Group for Endocarditis Prophylaxis,10 the duration of antibiotic presence in the serum is critical. Under experimental conditions, the drugs must remain above their MIC for the organisms for ≥10 h, to allow time for bacterial clearance from the valves. The concentrations of moxifloxacin in the serum were sustained at supra-MIC levels for more than 24 h after the administration of the drug, and this fact seems to be the most plausible explanation for the prophylactic efficacy of moxifloxacin.

In conclusion, in this study, moxifloxacin was effective as prophylaxis against endocarditis caused by a strain of S. oralis. Its prophylactic efficacy was at least equivalent to that of ampicillin, and thus could be considered as an alternative to standard regimens for the prophylaxis of endocarditis, especially in patients with a history of allergy to ß-lactams.


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No declarations were made by the authors of this paper.


    Acknowledgements
 
We thank Z. Chrissouli for technical assistance.


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1. Dajani AS, Taubert KA, Wilson W et al. Prevention of bacterial endocarditis: recommendations by the American Heart Association. Clin Infect Dis 1997; 25: 1448–58.[ISI][Medline]

2. McWhinney PH, Patel S, Whiley RA et al. Activities of potential therapeutic and prophylactic antibiotics against blood culture isolates of viridans group streptococci from neutropenic patients receiving ciprofloxacin. Antimicrob Agents Chemother 1993; 37: 2943–5.

3. Teng LJ, Hsueh PR, Chen YC et al. Antimicrobial susceptibility of viridans group streptococci in Taiwan with an emphasis on the high rates of resistance to penicillin and macrolides in Streptococcus oralis. J Antimicrob Chemother 1998; 41: 621–7.[Abstract]

4. Keating GM, Scott LG. Moxifloxacin: a review of its use in the management of bacterial infections. Drugs 2004; 64: 2347–77.[ISI][Medline]

5. Perlman BB, Freedman LR. Experimental endocarditis II: staphylococcal infection of the aortic valve following placement of a polyethylene catheter in the left side of the heart. Yale J Biol Med 1971; 44: 206–21.[ISI][Medline]

6. Bayer AS, Tu J. Chemoprophylactic efficacy against experimental endocarditis caused by ß-lactamase-producing, aminoglycoside-resistant enterococci is associated with prolonged serum inhibitory activity. Antimicrob Agents Chemother 1990; 34: 1068–74.[ISI][Medline]

7. Voorn GP, Thompson J, Goessens WHF et al. Efficacy of ciprofloxacin in treatment and prophylaxis of experimental Staphylococcus aureus endocarditis caused by a cloxacillin-tolerant strain and its non-tolerant variant. J Antimicrob Chemother 1994; 33: 785–94.[Abstract]

8. Katsarolis I, Pefanis A, Iliopoulos D et al. Successful trovafloxacin prophylaxis against experimental streptococcal aortic valve endocarditis. Antimicrob Agents Chemother 2000; 44: 2564–6.[Abstract/Free Full Text]

9. Pefanis A, Perdicaris G, Delis D et al. Sparfloxacin in the prophylaxis of experimental endocarditis due to Gram-positive cocci. In: Program and Abstracts of the Thirty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 1996. Abstract B65, p. 33. American Society for Microbiology, Washington, DC, USA.

10. Moreillon P. Endocarditis prophylaxis revisited: experimental evidence of efficacy and new Swiss recommendations. Swiss Working Group for Endocarditis Prophylaxis. Schweiz Med Wochenschr 2000; 130: 1013–26.[ISI][Medline]





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