JAC Advance Access originally published online on January 12, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):674-678; doi:10.1093/jac/dkm527
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Original research |
Pre- and post-exposure prophylaxis of experimental Burkholderia pseudomallei infection with doxycycline, amoxicillin/clavulanic acid and co-trimoxazole
Defence Medical and Environmental Research Institute, DSO, National Laboratories, 27 Medical Drive, 117510 Singapore, Singapore
* Corresponding author. Tel: +65-6485-7255; Fax: +65-648-7262; E-mail: ssuppiah{at}dso.org.sg
Received 24 October 2007; returned 6 December 2007; revised 12 November 2007; accepted 11 December 2007
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Abstract |
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Objectives: Melioidosis, a potentially fatal disease of humans and animals, is caused by the Gram-negative bacterium, Burkholderia pseudomallei. There is no approved vaccine or effective prophylaxis. Given its potential as a bioterrorism agent and a cause of serious laboratory-acquired infection, we studied the efficacy of pre- and post-exposure oral antibiotic prophylaxis in BALB/c mice infected with aerosolized B. pseudomallei through the inhalational route.
Methods: Amoxicillin/clavulanic acid, doxycycline or co-trimoxazole was administered 48 h before infection as pre-exposure prophylaxis, orally, twice daily and continued up to 10 days post-challenge. In the post-exposure prophylaxis regimen, the oral antibiotics were administered twice daily, at 0, 10, 24 and 48 h and continued for 10 days. Survival of all animals was observed until 21 days.
Results: All infected control animals developed infection between 24 and 48 h, and died within 5 days. Animals receiving amoxicillin/clavulanic acid as pre-exposure prophylaxis succumbed to the disease at day 7, whereas those in the co-trimoxazole and doxycycline groups had survival rate of 100% and 80%, respectively, at day 21. As post-exposure prophylaxis, all antibiotics were not effective when treatment was initiated 48 h post-challenge. However, animals receiving co-trimoxazole had a 100% survival rate when the antibiotic was started 0, 10 and 24 h post-infection, and amoxicillin/clavulanic acid was the least effective.
Conclusions: Co-trimoxazole appears to be an effective oral antibiotic both as pre- and post-exposure prophylaxis to B. pseudomallei. Data derived from this study have important implications on the management of laboratory accidents or following an intentional release of B. pseudomallei, a potential bioterrorism agent.
Keywords: aerosol , melioidosis , antimicrobials , murine
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Introduction |
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Gram-negative Burkholderia pseudomallei is the aetiological agent of melioidosis, a potentially fatal disease in humans and animals. In Singapore, a high incidence of melioidosis cases was observed in early 2004 with a high mortality rate (40%).1 It is postulated that the inhalational route of infection may influence the severity of disease outcome.2 The management of melioidosis remains a challenge as the disease has both a high initial mortality rate and a high relapse rate, despite prolonged use of antimicrobials which appears highly active in vitro.3 The lack of effective antimicrobial agents and delay in diagnosis contribute to the overall severe outcome of the disease. Treatment with intravenous ceftazidime or imipenem has been reported to be effective if administered soon after the onset of symptoms.4 However, the efficacy of oral antibiotics for use as pre- and post-exposure prophylaxis is lacking. Oral form of therapy is the most convenient mode of drug administration for prophylaxis. In the absence of a vaccine, antibiotic prophylaxis for use in the context of preventing laboratory-acquired infection following an accidental inoculation or an intentional release of this potential biological agent could represent an important means of disease prevention. Others have shown that oral doxycycline/ciprofloxacin could prevent melioidosis in experimentally infected mice, with bacterium inoculated through the intraperitoneal route.5 Here, we have studied the efficacies of amoxicillin/clavulanic acid, doxycycline and co-trimoxazole as pre- and post-exposure prophylaxis at various time points in experimental B. pseudomallei aerosol infection in BALB/c mice, using a strain of B. pseudomallei (K96243) previously investigated in our laboratory for in vitro susceptibility using the above antimicrobials and found to be susceptible.6
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Materials and methods |
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Preparation of bacteria
B. pseudomallei clinical isolate (K96243) was first cultured on tryptone soya agar at 37°C for 24 h. Bacterial colonies were re-suspended in sterile PBS to achieve an optical density of 1.9 (equivalent to 109 cfu/mL) and diluted to 107 cfu/mL for generating aerosol. Bacterial suspensions were plated in duplicate for retrospective determination of actual number of viable bacteria delivered to the mice. Experimental protocols were reviewed and approved by DSO National Laboratories Biosafety Committee and all studies were carried out in a BSL3 laboratory.
Antibiotic solutions for the administration were prepared freshly each day by dissolving doxycycline powder in sterile PBS (40 mg/kg), amoxicillin/clavulanic acid (Augmentin) in de-ionized water (160 mg/kg), and co-trimoxazole (Septrin) in de-ionized water (40 mg/kg).
Delivery of B. pseudomallei into mice via the inhalational route
Animal studies were carried out in accordance with the Animal Care and Use Committee's guidelines, DSO National Laboratories. Female BALB/c mice aged 7–8 weeks were purchased from the Centre for Animal Resources of National University of Singapore. The mice were exposed to nose-only aerosol in an aerosolization chamber for 30 min. Aerosols were generated by three collision air jet nebulizers, each containing 20 mL (107 cfu/mL) of B. pseudomallei. Nebulization was performed for 10 min, after which 10 L of air was passed through an impinger containing 20 mL of PBS to determine the bacterial density in the aerosol. Control mice were subjected to aerosols of PBS. Good linear correlation between the bacterial concentrations (cfu/mL) and those in the aerosols in the chamber during nebulization (cfu/L air) was obtained (data not shown). High degree of reproducibility of the nebulization process was demonstrated. Three mice per group were sacrificed immediately post-aerosol exposure, and their lungs were homogenized, diluted and plated onto Ashdown agar plates to determine the actual amount of bacteria in the lung.
For pre-prophylaxis regimen, three groups of five mice each were treated with doxycycline, amoxicillin/clavulanic acid or co-trimoxazole orally, twice daily (0.1 mL) using a gavage tube. Mice in the control group were fed with sterile PBS. For the pre-prophylaxis regimen, the antimicrobials were initiated 48 h before challenge and continued for 10 days post-infection. In post-exposure prophylaxis, the animals were divided into five groups of five animals, each based on the time of drug initiation after aerosol challenge: Group I, control; Group II, immediate; Group III, 10 h; Group IV, 24 h; and Group V, 48 h. The treatment was continued for 10 days and the animals were observed for survival until 21 days. The bacterial loads were done separately using three mice per group. The mice were challenged with 10 times the established LD50 (data not shown) of B. pseudomallei through the inhalational route.
Survival curves were created using the method of Kaplan–Meier survival analysis and were calculated at 95% confidence interval for fractional survival at any particular time (GraphPad Prism software version 4.0, San Diego, CA, 2003). Bacterial load was analysed using Student's t-test.
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Results |
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Pre-exposure prophylaxis
Bacterial loads obtained from the lungs on day 0, 3 and 9 post-aerosol infection are summarized in Table 1. The actual inhaled dose was 1.25 x 103 cfu in the lung immediately after infection in the control mice and was similar in other treatment groups. On day 3, in the control group, bacteria had colonized the lungs and reached very high loads of 2.36 x 108 cfu, and all animals succumbed by day 5. In contrast, the mice treated with amoxicillin/clavulanic acid, doxycycline and co-trimoxazole had reduced bacterial loads of 2.33 x 107, 1.32 x 104 and 8.00 x 102 cfu, respectively. The use of doxycycline or co-trimoxazole is observed to significantly improve the clearance of B. pseudomallei from the lung of infected mice at day 3 (P < 0.007), and total clearance from the lungs was observed by day 9.
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The efficacies of doxycycline, amoxicillin/clavulanic acid and co-trimoxazole for pre-exposure prophylaxis are summarized in Figure 1. The control mice became sick within 24–48 h post-challenge, indicated by non-specific symptoms such as piloerection and hypo-activity with trembling. The disease symptoms in mice progressed rapidly, and first death was observed at 72 h post-challenge. All animals in the control group succumbed within 96 h post-challenge. No significant improvement in the survival rate was observed in mice pre-treated with amoxicillin/clavulanic acid. Few relapses did occur in mice treated with doxycycline when treatments ceased at day 10 (P < 0.0143). Co-trimoxazole was the most effective antibiotic with a 100% survival rate (P < 0.0027) in pre-exposure prophylaxis. Post-mortem examination of the surviving mice in both co-trimoxazole- and doxycycline-treated groups showed that lungs, liver and spleen were culture-negative for B. pseudomallei at day 21 post-infection.
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Post-exposure prophylaxis
Doxycycline, amoxicillin/clavulanic acid or co-trimoxazole was administered orally at 0, 10, 24 or 48 h post-challenge (Figure 2a–d). Mice in all groups of post-exposure prophylaxis with amoxicillin/clavulanic acid died within 8 days post-challenge. Survival rates at day 21 were 100% (P < 0.0035) for mice treated with both doxycycline and co-trimoxazole immediately after challenge (Figure 2a). However, when treatment was initiated at 10 and 24 h post-challenge, the survival rates of mice for doxycycline fall to 80% (P < 0.0128) and 60% (P < 0.0128), respectively (Figure 2b and c). Interestingly, 100% (P < 0.0016) survival was observed in mice treated with co-trimoxazole at 10 and 24 h post-infection (Figure 2b and c). Post-mortem examination on day 21 for surviving mice in both co-trimoxazole- and doxycycline-treated groups was culture-negative for B. pseudomallei in lung, liver and spleen, whereas those carried out on animals with observed relapse revealed splenomegaly and multiple small abscesses in the lungs and liver. However, they were not as severe as that seen in control animals (data not shown).
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Discussion |
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We evaluated the effect of three antimicrobials, doxycycline, co-trimoxazole and amoxicillin/clavulanic acid, for mass prophylaxis, using an aerosol animal model system, as this is the most likely route of inoculation in the event of a laboratory accident or a bioterrorism threat. The control of acute infection by doxycycline and co-trimoxazole clearly indicates the usefulness of these antibiotics in preventing acute disease and establishment of chronic disease. The few relapses encountered in the study using doxycycline could be attributed to insufficient duration of antimicrobial administration. Although doxycycline and ciprofloxacin are capable of penetrating into tissues at concentrations higher than corresponding serum concentrations,7 remissions were not seen in all cases.8
From this study, the time between exposure to the pathogen and successful prevention of infection, ‘the window period’ with antimicrobials, was identified to be <24 h following challenge. This clearly suggests that the infectious agent could be contained or eliminated if early antibiotic treatment was initiated following exposure to prevent the bacterial load in the host from culminating to lethal dosage. This is certainly true in clinical practice where delays in differential diagnosis of septicaemic melioidosis are cited as the cause for the high mortality of this form of the disease. Our study on pre-exposure prophylaxis identified a reduction in the bacterial load following treatment with amoxicillin/clavulanic acid, doxycycline or co-trimoxazole, compared with controls. This highlights the importance of starting the antibiotics as early as possible to prevent mortality and enhance remission rates. Although no animal study can replace controlled human clinical trials, data derived from this study provide useful information that could be extrapolated to human subjects.
Currently, ceftazidime is the first antibiotic of choice for the treatment of acute melioidosis,9 whereas either oral ciprofloxacin or doxycycline is recommended as prophylaxis for those exposed in the event of a deliberate release in the UK.10 In our study, the absence of relapse following antibiotic withdrawal and the sterility of organs and blood of surviving mice showed that co-trimoxazole completely cleared B. pseudomallei from the infected host. Therefore, it is evident that co-trimoxazole is more effective than doxycycline as pre- or post-exposure prophylaxis. A report published by the CDC recommended post-exposure prophylaxis with co-trimoxazole and doxycycline for a period of 3 weeks.11 However, our animal data showed clear evidence that 10 days of post-exposure prophylaxis with co-trimoxazole was sufficient to prevent infection. Co-trimoxazole appears to be more effective than doxycycline as a post-exposure prophylaxis. We have not investigated the mechanism to explain this difference in our study. However, we postulate that co-trimoxazole may have better intracellular penetration than doxycycline, which is important as B. pseudomallei is an intracellular pathogen. Currently, clinical experience with post-exposure prophylaxis is lacking. Data derived from animal studies would serve as a guideline in instituting the right antimicrobials to offer protection in the case of laboratory accidents or following an intentional release of B. pseudomallei, a potential bioterrorism agent.
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Funding |
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This study was funded by HQ Medical Corps of the Singapore Armed Forces.
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None to declare.
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Acknowledgements |
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We thank HQ Medical Corps of the Singapore Armed Forces for their support of this study.
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References |
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1 Liu Y, Loh JP, AW LT, et al. Rapid molecular typing of Burkholderia pseudomallei, isolated in an outbreak of melioidosis in Singapore in 2004, based on variable-number tandem repeats. Trans R Soc Trop Med Hyg (2006) 100:687–92.[CrossRef][Web of Science][Medline]
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4 Simpson AJ, Suputtamongkol Y, Smith MD, et al. Comparison of imipenem and ceftazidime as therapy for severe melioidosis. Clin Infect Dis (1999) 29:381–7.[Web of Science][Medline]
5
Russell P, Eley SM, Ellis J, et al. Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders. J Antimicrob Chemother (2000) 45:813–8.
6 Sivalingam SP, Sim SH, Aw LT, et al. Antibiotic susceptibility of fifty clinical isolates of Burkholderia pseudomallei from Singapore. J Antimicrob Chemother (2006) 55:1029–31.[CrossRef][Web of Science]
7 Van der Auwera P, Matsumoto T, Husson M. Intraphagocytic penetration of antibiotics. J Antimicrob Chemother (1998) 22:185–92.[CrossRef]
8
Steward J, Piercy T, Lever MS, et al. Comparison of gatifloxacin, moxifloxacin and ciprofloxacin for treatment of experimental Burkholderia pseudomallei infection. J Antimicrob Chemother (2005) 55:523–7.
9 Inglis TJ, Rodrigues F, Rigby P, et al. Comparison of the susceptibilities of Burkholderia pseudomallei to meropenem and ceftazidime by conventional and intracellular methods. J Antimicrob Chemother (2004) 48:2999–3005.[CrossRef]
10 Bossi P, Tegnell A, Baka A, et al. Bichat guidelines for the clinical management of glanders and melioidosis and bioterrorism-related glanders and melioidosis. Euro Surveill (2004) 9:E17–8.[Medline]
11 Centers for Disease Control Prevention (CDC). Laboratory exposure to Burkholderia pseudomallei-Los Angeles, California, 2003. MMWR Morb Mortal Wkly Rep (2004) 53:988–90.[Medline]
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