1 Biomedical Sciences, Dstl Porton Down, Salisbury, Wiltshire SP4 OJQ; 2 Department of Medical Microbiology, Royal Free and University College Medical School, University College London, London; 3 Department of Medical Microbiology, Barts and the London, Queen Mary's School of Medicine & Dentistry, London, UK
Received 30 June 2003; returned 29 July 2003; revised 2 April 2004; accepted 14 April 2004
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Abstract |
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Methods: Treated mice and controls were inoculated subcutaneously with 5x104 spores/mouse of Bacillus anthracis Ames strain and observed for 37 days after challenge. Treated mice were given 100 mg/kg of antibiotic orally twice daily for 14 days, starting at various times post-challenge.
Results: Treatment starting 6 h post-challenge resulted in survival rates of 90%, 15% and 40% for gatifloxacin, moxifloxacin and ciprofloxacin, respectively. Treatment commencing 24 h post-challenge resulted in survival rates of 65%, 10% and 5%, respectively. Treatment starting more than 24 h after exposure had little effect on survival.
Conclusions: Gatifloxacin appeared to be more effective than moxifloxacin or ciprofloxacin, at similar doses, for early post-exposure treatment of murine systemic anthrax. However, these results might be due to differences in potency or pharmacokinetic properties.
Keywords: Bacillus anthracis , quinolones , murine model , therapy
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Introduction |
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The Health Protection Agency, UK, recommends ciprofloxacin, a fluoroquinolone with a broad spectrum of activity, as treatment for respiratory and gastrointestinal anthrax.3 It is also currently recommended for prophylaxis of anthrax infection following a deliberate release, in the USA.4
Newer fluoroquinolones, such as moxifloxacin and gatifloxacin, are now available. Gatifloxacin has been shown to be effective in vitro against a number of strains of B. anthracis,5,6 and the susceptibility of B. anthracis to gatifloxacin and moxifloxacin is comparable to that with ciprofloxacin (0.060.125 mg/L).5,7
Gatifloxacin and moxifloxacin, however, have improved intracellular pharmacokinetic properties and good activity against both Gram-positive and Gram-negative bacteria.8,9 In particular, moxifloxacin has improved activity against Gram-positive species.10 These newer agents therefore may be more appropriate agents than ciprofloxacin for prophylaxis and treatment of B. anthracis infection.
This study examined the efficacy of the two newer fluoroquinolone antibiotics, gatifloxacin and moxifloxacin, in comparison with ciprofloxacin against experimental infection with B. anthracis Ames strain in the BALB/c mouse.
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Materials and methods |
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All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986 and the Code of Practice for the Housing and Care of Animals used in Scientific Procedures 1989.
Female BALB/c mice aged between 6 and 8 weeks (i.e. sexually mature) were obtained from Charles River Laboratories (Maidstone, Kent, UK) and randomized into cages of five animals each. The mice were allowed free access to water and rodent diet (Harlan Teklad, UK). We have demonstrated previously that BALB/c mice are susceptible to challenge with injected anthrax spores (unpublished data), and BALB/c mice are used as the standard animal model in most of our experiments.
For the challenge study, the animals were housed in a custom built ACDP Animal Containment Level 3 rigid wall isolation unit (B & B Environmentals Ltd, Manchester, UK) and allowed to acclimatize to their new environment for 7 days before any procedures were undertaken. All procedures were carried out using nitrile gloves worn with a half-suit to provide protection against accidental bites.
Preparation of challenge material
The Ames strain of B. anthracis was obtained from the Defence Science and Technology Laboratory (Dstl Porton Down, Salisbury, Wiltshire, UK) culture collection. All bacteriological procedures were carried out in a Class III microbiological safety cabinet within a Containment Level 3 laboratory. Briefly, a fresh preparation of spores was prepared by streaking out the stock culture onto nutrient agar plates (Oxoid, Basingstoke, UK) and incubating at 37°C overnight. A single colony from an overnight culture was then inoculated into 100 mL of L-broth (Oxoid) and incubated with shaking at 37°C for 6 h. Then 2.5 mL of this culture was inoculated into a Roux container containing L-agar (Oxoid) and incubated at 37°C for 710 days. When spores were observed by phase contrast microscopy, the culture was washed and serially diluted with sterile distilled water. The suspension was then heated at 60°C for 1 h to destroy any vegetative cells. Spores appear highly refractive, regular in shape, and are easily distinguishable from smaller and less refractive vegetative cells. The final material had a titre of 5 x 105 spore/mL, as determined by serial dilution and plate counts on nutrient agar (Oxoid). The challenge dose of 5 x 104 spores/mouse (100 µL) was administered by subcutaneous injection in the scruff of the neck. The MLD of B. anthracis strain Ames in BALB/c mice by the subcutaneous route is 50 spores per mouse (T.J.G. Brooks, unpublished data).
Antibiotics and treatment regimens
Commercially available tablets of gatifloxacin (Bristol-Myers Squibb, USA), moxifloxacin (Bayer, Germany) and ciprofloxacin (Bayer, UK) were purchased through IDIS World Medicines (Surrey, UK). Pure antibiotic substance was not available in sufficient quantities to carry out these experiments and liquid formulations of these antibiotics were not available to us. Other workers have used antibiotics prepared from tablet formulations for similar studies.1113 Tablets were dissolved in sterile deionized water to give a stock solution of 100 mg/mL and filtered through a 0.2 µm syringe filter (Sartorius, UK). Antibiotic concentrations from tablets were checked against stock solutions made up from antibiotic pure substance (kindly provided by the manufacturers) by agar well diffusion assay. Each test animal was given 100 mg/kg of antibiotic per dose orally by dropping 20 µL of antibiotic onto the back of the oropharynx using a displacement pipette with a 20P aerosol-resistant tip (ART, Molecular BioProducts, CA, USA). This procedure triggers the swallowing reflex, and as mice cannot vomit, gives an atraumatic delivery of drug by the oral route.
Each group contained 20 animals. All treatments were given twice daily at 12 h intervals continuing for 14 days, with treatment commencing either at 6, 24, or 48 h post-exposure. Treatment was started at these various time intervals after exposure to mimic delays in provision of treatment. The mock-treated control group were given diluent only, twice daily at 12 hourly intervals continuing for 14 days. Mice were then observed for up to 37 days post-challenge (the experiment had to be terminated at this point, due to time constraints).
Statistical design and analysis
The group sizes were chosen to demonstrate a difference of less than 50% in survival between regimens or controls, with 80% power and 95% confidence limits. The significance of the results was tested using the 2test.14
Bacteriological analysis
Samples of blood were taken from the thoracic cavity of dead animals post-mortem to confirm the presence of anthrax. A single drop of blood was smeared on a glass microscope slide and stained with Loeffler's polychrome Methylene Blue (VWR International, Dorset, UK aged for 3 years). B. anthracis was identified by the characteristic pink capsule (McFadyean's reaction).15,16 At the end of the experiment, surviving mice were culled and blood samples were taken for staining in the same way. Samples with a negative smear were cultured on Columbia blood agar with 5% horse blood (bioMérieux, Hampshire, UK) after diluting 1:10 in nutrient broth (bioMérieux). Plates were incubated in air for 2 days at 37°C.
Minimum inhibitory concentrations (MICs)
All tests were conducted using standard NCCLS methodology.17 MICs were determined as the lowest concentration of antibiotic that prevented visible growth.
Pharmacokinetic analysis
A total of 135 BALB/c mice were divided into three groups containing 45 animals per group. Each group of mice received five doses (100 mg/kg) orally of gatifloxacin, moxifloxacin or ciprofloxacin, every 12 h. Different groups were culled at 0.5,1.0,1.5,2.0,4,6,8 or 12 h after the fifth dose. Antibiotic concentrations in the spleen, liver, lungs and serum were determined using a well diffusion assay, with Klebsiella edwardsii NCTC 10896 as the indicator organism. K. edwardsii was selected as the indicator organism as preliminary experiments demonstrated that this organism gave the best zone definition (data not shown).18 Zones of inhibition were measured and compared with antibiotic controls diluted in tissue homogenate from untreated animals. Antibiotic controls were prepared both from pure antibiotic powders and for comparison, from the tablet formulations used to dose animals.
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Results |
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Survival curves for each of the antibiotic treatment groups are shown in Figures 1, 2 and 3Figures 1, 2 and 3Figures 1, 2 and 3. Antibiotic treatment starting at 6 h post-challenge proved to be the most effective regimen for all three antibiotics. At the end of the experiment (day 37) survival rates of 90%, 40% and 15% were observed in mice treated with gatifloxacin, ciprofloxacin or moxifloxacin, respectively. Survival rates were significantly higher in mice treated with gatifloxacin (P < 0.001) and ciprofloxacin (P < 0.01) compared with mock-treated controls.
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Survival rates were poor when treatment was initiated 48 h after challenge. Only 10% of mice treated with gatifloxacin survived to day 37 post-infection, and this was not significantly better than the mock-treated control mice. There were no survivors in those mice given ciprofloxacin or moxifloxacin starting at 48 h post-infection.
B. anthracis vegetative cells were observed in blood smears taken from all mice that died and from sick animals that were culled during antibiotic treatment. There was no evidence of B. anthracis in blood smears from any surviving mice culled at the end of the experiment.
Pharmacokinetic data
Peak serum concentrations were reached at 0.5 h for moxifloxacin and 1 h for ciprofloxacin and gatifloxacin (Table 1). Peak serum concentrations (Cmax) were 3.66, 2.37 and 1.29 mg/L for ciprofloxacin, gatifloxacin and moxifloxacin, respectively. Tissue levels peaked within 1 h and were highest in the liver, where antibiotic could still be detected at 8 and 12 h, for moxifloxacin and gatifloxacin, respectively. These data indicate that multiple doses of 100 mg/kg can achieve serum levels in mice which are comparable to those found in man, although the half-life is reduced (as expected due to differences in the metabolic rate of humans and mice).19
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Minimum inhibitory concentration tests
The MICs measured against B. anthracis Ames in this study were 0.25, 0.015 and 0.03 mg/L for ciprofloxacin, gatifloxacin and moxifloxacin, respectively. Antibiotic controls prepared from pure substance and from the oral formulations gave similar results.
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Discussion |
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BALB/c mice were chosen for this study as they proved to be susceptible to B. anthracis strain Ames (a virulent toxin-producing and encapsulated strain) with a MLD of 50 spores per mouse. Other strains of mice tested, such as AJ or Porton, proved equally or less susceptible than BALB/c mice (T.J.G. Brooks, unpublished data). Previous workers have reported BALB/c mice as susceptible to virulent B. anthracis strain Vollum 1B, a toxin-producing and encapsulated strain when delivered by the subcutaneous route (LD50 6.6 spores per mouse).20 The difference in the MLD reported here compared with data by other workers may be due to bacterial strain variation.
The data obtained from this study suggested that the infection could be contained or eliminated if antibiotic treatment was started before a high bacterial load was reached in the exposed animals. Mice are very susceptible to anthrax given in this way, and the challenge reflects the ability of the antibiotics to control a septicaemic infection. Once the numbers of anthrax bacilli have reached high levels and elaborated significant amounts of toxin,21 survival is unlikely. However, in mice the presence of circulating encapsulated organisms alone can be sufficient to cause death even in the absence of toxin production.22
Applying Morris's formula, an estimated dose of 44 mg/kg for gatifloxacin and moxifloxacin and 55 mg/kg for ciprofloxacin was predicted.23 Concentrations of antibiotics determined in tissues and serum following administration of these doses however were lower than values predicted for man. Gatifloxacin at a dose of 100 mg/kg given twice daily gave a pharmacokinetic profile in BALB/c mice that resembled that seen with once-daily dosing in man based on the AUC.24 However, a dose of 100 mg/kg of moxifloxacin in mice did not give the predicted profile of that reported in man, although in the treatment of Mycobacterium tuberculosis, a dose of 100 mg/kg of moxifloxacin was previously shown to be effective.25,26 Therefore to allow direct comparisons between antibiotics, a standardized dose of 100 mg/kg was chosen in this study for all three antibiotics.
Gatifloxacin and moxifloxacin are 8-methoxy fluoroquinolones with broad-spectrum antimicrobial activity. The structures of these drugs are similar to that of ciprofloxacin in that they have the same cyclopropyl ring but differ at the C-7 position. Gatifloxacin has a 3-methyl piperazinyl ring and moxifloxacin a diazabicyclo group. These changes at C-7 increase their potency against Gram-positive organisms.27 Gatifloxacin and moxifloxacin have a long elimination half-life in humans (t1/2), which can prolong the duration of their bactericidal activity, making them attractive candidates for prophylaxis.27 Our MIC data show that both drugs are significantly more active than ciprofloxacin against B. anthracis in vitro.
The AUC and t1/2 values for gatifloxacin in conjunction with the relatively low MIC value may explain the improved performance of gatifloxacin. The MIC value of ciprofloxacin (0.25 mg/L) against B. anthracis Ames strain, in this study, was within the MIC range reported by Cavallo et al.5 The MIC of gatifloxacin (0.015 mg/L) against the Ames strain of B. anthracis in this study was within the MIC range reported by Drago et al.28 but more susceptible than the isolates tested by Cavallo et al. where the MIC was found to be 0.125 mg/L.5 This suggests that B. anthracis Ames strain is more susceptible to gatifloxacin compared to the strains tested by Cavallo et al. but has the same susceptibility to ciprofloxacin as other strains. However, both tests used to determine the MIC value gave virtually identical values and the Ames strain of B. anthracis used in this study was virulent for BALB/c mice.
The MIC of moxifloxacin (0.03 mg/L) in this study was comparable to that reported by previous studies against the Sterne strain and Russian vaccine strain ST-1 of B. anthracis.7
The recommended antibiotic treatment for B. anthracis infection relies on current knowledge of antibiotic resistance, animal studies and the possible number of people requiring treatment. Our results indicate that if antibiotic treatment commenced shortly after exposure, a long period of therapy was required. This was evident as a substantial number of deaths occurred in both the moxifloxacin and ciprofloxacin post-exposure treatment groups, either during therapy or within a week of completing the 14 day course. A delay in the start of antibiotic treatment therefore may substantially decrease the chance of survival. The treatment in this study was not given for a longer period, as this was an evaluation experiment to screen antibiotic efficacy over the initial period of infection.
A number of antibiotic treatments have been assessed against B. anthracis in the mouse model, non-human primates and guinea pigs.2931 However, as there is a lack of clinical trials in humans for prophylaxis and treatment of anthrax infection, the recommendations are based on in vivo data and on studies in laboratory animals and non-human primates. For prophylaxis of anthrax, CDC guidelines for bioterrorism suggest that ciprofloxacin is the drug of choice, but doxycycline may be used as an alternative to prevent the development of ciprofloxacin-resistant strains or if ciprofloxacin is contraindicated.1,32 The duration of post-exposure prophylaxis for anthrax-related infection is unclear. However, 60 days is recommended based mainly on animal studies of anthrax-related mortality and spore clearance post-exposure.1 The widespread use of ciprofloxacin as prophylaxis could lead to resistance in B. anthracis and other organisms.33 Treatment for respiratory anthrax is initially with intravenous ciprofloxacin or doxycycline before commencing these antibiotics orally.34,35
Gatifloxacin has been shown previously to be effective in increasing the survival of anthrax-infected mice.36 The results of our study suggest that gatifloxacin could be a potential alternative to ciprofloxacin for the prophylaxis and treatment of B. anthracis infection, and that at similar doses, moxifloxacin is comparable, or possibly inferior, to ciprofloxacin. However, these results should be interpreted in the light of differing drug kinetics and disease dynamics in mice and men and different antimicrobial activities. Further research is required.
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Acknowledgements |
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Footnotes |
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Present address. HPA Porton Down, Salisbury, Wiltshire SP4 OJG, UK.
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