1 Biomedical Sciences, Dstl Porton Down, Salisbury SP4 OJQ; 3 HPA Porton Down, Salisbury SP4 OJG; 2 Department of Medical Microbiology, Royal Free and University College Medical School, University College London, London, UK
Received 13 July 2004; returned 6 November 2004; revised 23 November 2004; accepted 30 December 2004
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Abstract |
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Methods: BALB/c mice were inoculated subcutaneously with 1.75 x 106 cfu of B. pseudomallei strain 576. Gatifloxacin, moxifloxacin and ciprofloxacin (100 mg/kg) were given orally at 12 hourly intervals for 14 days starting at 6 h, 7 days or 12 days post-challenge. Control mice did not receive antibiotic therapy.
Results: No regimen gave 100% protection. Prophylaxis was most effective when started 6 h post-challenge, with survival rates at 42 days for ciprofloxacin, gatifloxacin and moxifloxacin being 58%, 75% and 75%, respectively. For treatment started at day 7 post-challenge, survival rates were 17%, 11% and 44%, respectively. When antibiotic treatment was delayed until day 12 post-challenge, survival rates fell to 21%, 17% and 28%, respectively. Following dexamethasone treatment of survivors at 42 days post-challenge, relapses occurred in all treatment groups.
Conclusions: Fluoroquinolones do not provide good post-exposure protection against infection with B. pseudomallei. The newer agents moxifloxacin and gatifloxacin are not significantly better than ciprofloxacin for this purpose.
Keywords: melioidosis , murine , chemotherapy , fluoroquinolones
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Introduction |
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The disease has an acute or subacute form and also a chronic relapsing state with associated high mortality. Recrudescence during therapy or relapse after completion of apparently successful therapy is well recognized.2,3 Acute melioidosis may present as a localized or septicaemic infection, whereas chronic infection can remain asymptomatic for years but can reactivate from a latent focus as an acute localized infection when the host becomes immunocompromised.4
The efficacy of various antimicrobial agents, including chloramphenicol, co-trimoxazole, doxycycline, imipenem and ciprofloxacin, has been evaluated for the treatment of melioidosis.59 In humans, intravenous ceftazidime (with or without additional co-trimoxazole), co-amoxiclav, imipenem and cefoperazone/sulbactam have each been reported as effective treatments for acute disease, following randomized clinical trials.9 Meropenem has also been used successfully.10 Oral regimens comprising chloramphenicol, co-trimoxazole and doxycycline, or co-amoxiclav alone, have been shown to be effective for eradication therapy. Doxycycline and the fluoroquinolones ciprofloxacin and ofloxacin used as monotherapy for this indication had high failure rates, as did a combination of ciprofloxacin and azithromycin. There are few data from animal models to support the use of other regimens at present.8 The current recommended treatment for acute melioidosis infection is high-dose intravenous ceftazidime, or a carbapenem, for at least 1014 days, followed by oral eradication therapy.1013 B. pseudomallei is intrinsically resistant to many antimicrobial agents and despite having good intracellular penetration, quinolones such as ciprofloxacin have been disappointing in practice.7,14,15 However, the ability of quinolones, and newer fluoroquinolones in particular, to concentrate within phagocytes suggests they may prove useful for the treatment of melioidosis.
This study, therefore, compared the efficacy of ciprofloxacin with two of the latest generation fluoroquinolones, gatifloxacin and moxifloxacin, for the treatment of experimental melioidosis in an established BALB/c mouse model.
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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 Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989.
Two hundred and forty female BALB/c mice (Charles River Laboratories, Maidstone, UK) were randomized into cages containing six animals. Mice were allowed free access to water and rodent diet (Harlan Teklad, Oxon, UK). Challenge studies were carried out in a custom built ACDP animal containment level 3 rigid wall isolation unit (B & B Environmentals Ltd, Manchester, UK) complying to British Standard 5726. All mice were allowed to acclimatize to their environment for 7 days before any procedures were undertaken.
Preparation of challenge material
All bacteriological procedures were carried out in a Class III microbiological safety cabinet within an ACDP containment level 3 laboratory. Briefly, the challenge culture was prepared by recovering 6 Protect beads (TSC Ltd) of B. pseudomallei strain 576 into nutrient broth (Oxoid) and incubated at 37°C for 24 h. A 1 mL aliquot of this culture was inoculated into 9 mL nutrient broth and serially diluted in nutrient broth for animal challenges. The challenge dose of 1.75 x 106 cfu (100 µL) was administered by subcutaneous injection into the scruff of the neck and mice were observed for 56 days post-challenge.
Antimicrobial treatment regimens
Gatifloxacin (Bristol-Myers Squibb, USA), moxifloxacin (Bayer, Germany) and ciprofloxacin (Bayer, UK) were dissolved in sterile deionized water and filter sterilized, as described previously.16 Each group contained 24 animals. The antibiotic solutions were administered at a dose of 100 mg/kg twice daily (as described previously), at 12 hourly intervals orally according to one of three regimens; starting at 6 h, 7 days or 12 days post-challenge and continued for 14 days.16 Infected control mice were given sterile deionized water only for 14 days.
Dexamethasone treatment
Dexamethasone (5 mg once daily continuing for 7 days) was administered to all surviving mice at day 42 post-challenge by intraperitoneal injection.
Bacteriology
Additional groups of six mice per regimen were humanely culled at 14 and 28 days post-challenge. Control mice were not assessed beyond day 14, due to progressive illness. Spleens were aseptically removed, homogenized in nutrient broth and serial dilutions were plated out onto nutrient agar (bioMérieux, UK). Plates were incubated at 37°C for 72 h in air and the number of viable B. pseudomallei were determined.
MICs
MICs of ciprofloxacin, gatifloxacin and moxifloxacin were determined for the challenge strain of B. pseudomallei (strain 576) used in this study, using standard NCCLS guidelines.17 The MIC results were interpreted as the lowest concentration of antibiotic that showed no visible growth.
Statistical analysis
Proportions were compared using the 2 test. The geometric mean of B. pseudomallei cells isolated from mouse spleens in different treatment regimens was analysed by Student's t-test.
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Results |
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A delay in administration of antibiotic treatment resulted in increased numbers of B. pseudomallei within the spleen at day 28 (Table 1).
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MICs for B. pseudomallei strain 576 were 8.0, 0.5 and 0.5 mg/L of ciprofloxacin, gatifloxacin and moxifloxacin, respectively.
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Discussion |
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Therapeutic options for the treatment of melioidosis are limited as B. pseudomallei is resistant to most aminoglycosides, penicillins, first- and second-generation cephalosporins and most macrolides.18,19 However, the organism is susceptible in vitro to some third-generation cephalosporins, carbapenems and some fluoroquinolones, as well as co-amoxiclav, chloramphenicol, tetracyclines and co-trimoxazole.18,20 Ceftazidime is currently the first antibiotic of choice for the treatment of acute melioidosis, while either oral ciprofloxacin (despite moderate in vitro activity) or doxycycline is recommended for prophylaxis of those exposed in the event of a deliberate release in the UK.13 There are few recommendations for post-exposure prophylaxis in other circumstances, e.g. for laboratory workers, although recent US guidance recommends co-trimoxazole (with or without doxycycline).21
However, data on the efficacy of prophylactic regimens are very limited, including experimental data.8 Clinical experience with post-exposure prophylaxis is lacking, although there is considerable experience with oral eradication antibiotic therapy for prevention of relapse following successful treatment of acute disease. These studies suggest that the best regimens are either co-amoxiclav alone or a combination of chloramphenicol, doxycycline and co-trimoxazole, or possibly co-trimoxazole alone.5,22 Regimens containing ofloxacin, ciprofloxacin or doxycycline alone have been disappointing.7,14,23 However, the ability of newer fluoroquinolones to concentrate within phagocytes, plus their improved spectra of activity, makes them attractive candidates for further investigation.
Our MIC data indicate that B. pseudomallei strain 576 is significantly more susceptible to gatifloxacin and moxifloxacin than to ciprofloxacin (0.5 mg/L compared with to 8.0 mg/L). The MIC of ciprofloxacin (8.0 mg/L) in this study was within the MIC range previously reported for B. pseudomallei,20,24,25 although higher than that reported by Russell et al.8 The difference in the MIC reported here compared with data by other workers may be due to bacterial strain variation or differences in experimental technique. The MICs of gatifloxacin and moxifloxacin (0.5 mg/L) in this study for B. pseudomallei strain 576 are lower than those reported for the 71 isolates of B. pseudomallei tested by Ho et al.26 in which the MICs were reported to be 16 and 4 mg/L of gatifloxacin and moxifloxacin, respectively. To date, no other MIC data on gatifloxacin and moxifloxacin against B. pseudomallei are available.
Previous studies have shown BALB/c mice to be susceptible to B. pseudomallei infection and they are an appropriate model to study the acute form of melioidosis.2729 The nature of the infection in mice is dose-dependent, with lower doses generally leading to chronic infections. Higher challenge doses result in a more acute and lethal disease resembling the acute, severe, human form of melioidosis. In this study, we deliberately used a high challenge dose (106 cfu/mouse). It is, however, possible that this high challenge dose may have reduced the likelihood of successful prophylaxis.
Treatment initiated at 6 h post-challenge was used to simulate immediate post-exposure prophylaxis; at 7 days to simulate early treatment when the first indications of illness might appear, and at 12 days post-challenge when symptoms would be apparent.
The pharmacokinetic profiles in BALB/c mice, following administration of a dose of 55 mg/kg for ciprofloxacin and 44 mg/kg for gatifloxacin and moxifloxacin were lower than values predicted for humans.30 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 humans based on the AUC.31 However, a dose of 100 mg/kg of moxifloxacin in mice did not give the predicted profile of that reported in humans, although a dose of 100 mg/kg of moxifloxacin was previously shown to be effective in the treatment of Mycobacterium tuberculosis.32,33 To allow direct comparisons between antibiotics however, a standardized dose of 100 mg/kg was chosen in this study for all three antibiotics. As a result, it is possible that pharmacokinetic differences account for the apparent marginal benefit seen with moxifloxacin when started 7 days post-challenge.
The lowest levels of mortality were achieved with gatifloxacin and moxifloxacin when given at 6 h and moxifloxacin at day 7 post-challenge. Ciprofloxacin, gatifloxacin and moxifloxacin were all significantly more effective at preventing mortality compared with controls at 6 h post-challenge (P < 0.05), although there were no differences between the three antibiotics. Only moxifloxacin demonstrated any significant benefit compared with controls when started at day 7 (P < 0.05), although it was not statistically better than ciprofloxacin. There were no significant differences between any of the survival rates in any antibiotic treatment regimen or the control group when treatment commenced at day 12. These findings are consistent with previous work where ciprofloxacin was found to be ineffective in vivo in experimental melioidosis.8 In addition, clinical evidence has suggested that the use of quinolones for the treatment of human melioidosis is associated with a high incidence of relapse and treatment failure.14,34
Splenic colony counts were not significantly different between the groups, except at 14 days, when the organism was not recovered from any mice treated with gatifloxacin. This was no longer apparent by day 28. Furthermore, dexamethasone treatment induced significant rates of relapse in all antibiotic groups, and the organism was recovered from spleens in similar numbers from each group. When treatment was delayed until day 12 post-challenge, the rate of relapse was 100% for all antibiotics. These results indicate that none of these antibiotics was capable of eradicating B. pseudomallei even when administered after only 6 h. This is consistent with clinical reports of failure of eradication therapy with fluoroquinolones.7,14
Our results support previous evidence suggesting that ciprofloxacin alone should not be considered as a first choice antimicrobial for post-exposure prophylaxis or treatment of B. pseudomallei infection. In addition, our data indicate that the newer fluoroquinolones gatifloxacin and moxifloxacin do not offer significant advantages over ciprofloxacin for this purpose.
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Acknowledgements |
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