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Treatment of murine pneumonic Francisella tularensis infection with gatifloxacin, moxifloxacin or ciprofloxacin
International Journal of Antimicrobial Agents 27 (2006) 439–443
Treatment of murine pneumonic Francisella tularensis infection with gatifloxacin, moxifloxacin or ciprofloxacin J. Steward a,∗ , T. Piercy a , M.S. Lever a , A.J.H. Simpson a,b , T.J.G. Brooks a,c b
a DSTL Porton Down, Salisbury, Wiltshire SP4 OJQ, UK Department of Medical Microbiology, Royal Free and University College Medical School, University College London, London, UK c Centre for Emergency Preparedness and Response, HPA Salisbury, UK
Received 3 February 2006; accepted 9 February 2006
Abstract The efficacies of gatifloxacin and moxifloxacin were assessed in a BALB/c mouse model of pneumonic tularemia and compared with the efficacy of ciprofloxacin. The rate of relapse following dexamethasone treatment was also investigated. Mice were given 100 mg/kg of the antibiotic by oral administration twice daily for 14 days following an aerosol challenge. All three fluoroquinolones prevented disease during the treatment period, but significant failure rates occurred after the cessation of therapy. Both gatifloxacin and moxifloxacin were more effective than ciprofloxacin at reducing late mortality. Fluoroquinolones may therefore be considered useful candidates for the treatment of pneumonic tularemia. Crown Copyright © 2006 DSTL – published with the permission of the Controller of Her Majesty’s Stationery Office Keywords: Francisella tularensis; Aerosol; Quinolones; Mouse model
1. Introduction Francisella tularensis is the causative organism of the zoonotic disease tularemia. It is a small, fastidious, Gramnegative, facultative, intracellular coccobacillus. Type A strains are found in North America and cause a more severe form of the disease, with an untreated mortality rate of up to 30%, whilst type B strains cause a milder infection that may be unnoticed or undiagnosed [1]. Infection in humans is often associated with contact with infected rodents or lagomorphs and the infectious dose is very low [2]. In endemic areas, ticks, flies and mosquitoes also transmit the infection from the animal reservoir to humans. Other routes of entry include skin abrasions, cuts and the alimentary tract [3]. After an incubation period of 3–5 days, an acute febrile illness develops with malaise, headache and fatigue. Local lymph nodes become enlarged and may suppurate. The liver and spleen ∗
Corresponding author. Tel.: +44 1980 613 169; fax: +44 1980 613 284. E-mail address: [email protected] (J. Steward).
are usually infected, although hepatosplenomegaly may not be apparent until late in the illness. Disseminated disease is common in type A strain infections, but type B disease is often self-limiting. Type A strains can cause lethal infection in humans following exposure to infectious aerosols. As a result, and because of the low infectious dose, F. tularensis is considered to be a potential biological warfare or bioterrorism agent. Francisella tularensis is naturally resistant to ampicillin, and treatment with cephalosporins may lead to relapse. Streptomycin or gentamicin are considered to be the antibiotics of choice, although there is some evidence that fluoroquinolones are effective [2,4–8]. Experimental human studies have demonstrated that prophylactic tetracycline was effective in the prevention of pulmonary tularemia [9]. Ciprofloxacin has been used successfully against experimental tularemia and in the treatment of clinical infection [8,10,11]. Importantly, given the deliberate release potential, the development of streptomycin-resistant strains has been reported [12]. This study therefore sought to assess the in vivo efficacy of two of
0924-8579/$ – see front matter. Crown Copyright © 2006 DSTL – published with the permission of the Controller of Her Majesty’s Stationery Office doi:10.1016/j.ijantimicag.2006.02.006
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the latest-generation fluoroquinolones, gatifloxacin and moxifloxacin, in comparison with ciprofloxacin, as post-exposure prophylaxis against challenge with aerosolised F. tularensis strain Schu4 in BALB/c mice. 2. Materials and methods 2.1. Animals 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 female BALB/c mice (Charles River Laboratories) were randomised into cages containing five animals each. The animals were maintained within an Advisory Committee for Dangerous Pathogens (UK) animal containment level 3 rigid wall isolator complying with British Standard 5726. They were allowed to feed and drink ad libitum and subjected to a 12 h light/dark cycle. The animals were allowed to acclimatise for 7 days prior to challenge.
2.4. Dexamethasone treatment Dexamethasone (5 mg once daily continuing for 7 days) was administered to all surviving mice at Day 42 post exposure by intraperitoneal injection. 2.5. 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 χ2 test [16].
3. Results 3.1. Challenge dose Mice were exposed to an aerosol containing 1.5 × 104 CFU/mouse F. tularensis strain Schu4 as calculated using the Guyton formula [17].
2.2. Antibiotics and treatment regimens
3.2. Antibiotic efficacy study
Antibiotic solutions were prepared by dissolving ciprofloxacin (Bayer, Newbury, UK), gatifloxacin (Bristol Myers Squibb, New York, NY) and moxifloxacin (Bayer, Leverkusen, Germany) tablets in sterile deionised water as previously described [13,14]. All treatments were given at a dose of 100 mg/kg orally twice daily at 12-h intervals and continuing for 14 days. Antibiotic treatment commenced 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 non-treated control group was given sterile deionised water only, twice daily at 12-h intervals continuing for 14 days. Mice were then observed for up to 56 days post exposure.
All non-treated control mice had died by Day 4 post exposure. In the antibiotic treatment groups, all mice survived during the antibiotic treatment phase, but once this was completed animals started dying within 4 days in each group. Survival curves are shown in Fig. 1. All mice in the three groups treated with ciprofloxacin died within 7 days of cessation of therapy. Most of the deaths in the other antibiotic treatment groups also occurred within 7 days of ceasing treatment. For the group receiving antibiotics starting at 6 h post exposure, survival rates at Day 42 were 0%, 53% and 53% for ciprofloxacin, gatifloxacin and moxifloxacin treatment, respectively (Fig. 1a). Both gatifloxacin and moxifloxacin were significantly more effective than ciprofloxacin (P = 0.002). When treatment was delayed for 24 h post exposure, survival rates at Day 42 were 0%, 41% and 12%, respectively (Fig. 1b). Antibiotic treatment initiated at 48 h post exposure resulted in survival rates at Day 42 of 0%, 65% and 35%, respectively (Fig. 1c). Both gatifloxacin and moxifloxacin treatments significantly improved Day 42 survival compared with non-treated controls for all regimens (P < 0.01). Both treatments were also significantly better than ciprofloxacin at all time points, except for moxifloxacin in the 24-h treatment group only (Day 42 survival, 12% versus 0%; P = 0.47). Gatifloxacin was not significantly better than moxifloxacin in the 6-h and 48-h groups, but was significantly better in the 24-h group (Day 42 survival, 41% versus 12%; P = 0.03). For both gatifloxacin and ciprofloxacin, there were no statistically significant differences between the groups commencing therapy at the different time points. For moxifloxacin, there was no difference between therapy started at either 6 h or 48 h (P = 0.2), although there appeared to be an advantage for the 6-h group
2.3. Animal challenge For pneumonic infection studies, a Collison nebuliser containing 20 mL of F. tularensis strain Schu4 at a concentration of 4.32 × 109 colony-forming units (CFU)/mL and three drops of Antifoam 289 (Sigma, Poole, UK) was used to generate and deliver aerosol particles infectious to mice. The particle size of the aerosols produced in this manner is ca. 1–3 m in range. The aerosol was conditioned in a modified Henderson apparatus [15]. In a single experiment, all mice were placed in groups of 20 in a nose-only exposure chamber and exposed for 10 min to a dynamic aerosol. The concentration of F. tularensis in the air stream was determined by taking samples from the exposure chamber using an AGI30 impinger containing fresh modified cysteine partial hydrolysate broth and antifoam. Impinger samples were plated onto blood cysteine glucose agar and incubated for 3 days in air at 37 ◦ C before counting.
J. Steward et al. / International Journal of Antimicrobial Agents 27 (2006) 439–443
441
Fig. 1. Survival curves of BALB/c mice challenged with aerosolised Francisella tularensis Schu4 and treated with either ciprofloxacin (×), gatifloxacin () or moxifloxacin (). Antibiotics were given orally starting at (a) 6 h, (b) 24 h and (c) 48 h post exposure at 12-h intervals and continuing for 14 days. Non-treated controls () were given oral diluent at 12-h intervals and continuing for 14 days.
over the 24-h group (Day 42 survival, 53% versus 12%; P = 0.002). Following dexamethasone treatment, there was one further death in the 6-h moxifloxacin-treated group on Day 56 and one in the 24-h gatifloxacin-treated group on Day 48 post exposure.
4. Discussion In this study, the in vivo efficacies of gatifloxacin and moxifloxacin were assessed in a BALB/c mouse model of aerosolised tularemia Schu4 infection and compared with the efficacy of ciprofloxacin. Streptomycin and gentamicin are the preferred treatments for tularemia, although ciprofloxacin and doxycycline are alternative choices of therapy for mass casualties [2,6]. A number of antibiotic treatments have been assessed against F. tularensis, including imipenem, quinolones and chloramphenicol [4]. Previous studies have demonstrated that F. tularensis Schu4 is susceptible to ciprofloxacin, gatifloxacin and moxifloxacin, with minimum inhibitory concentrations of 0.063, 0.03 and 0.03 mg/L, respectively [8]. There have been several reports of successful treatment
in human tularemia infection using quinolones, including ciprofloxacin, levofloxacin and norfloxacin [4,10,18–20]. Ciprofloxacin also appeared to be equivalent to doxycycline in an animal model [8]. Limaye and Hooper [19] reviewed the English language literature of tularemia cases treated with quinolones and found that there was 100% successful treatment with no relapse within 12 months of follow-up in ten clinical cases. However, there have also been reports of relapse following ciprofloxacin therapy [21]. Evidence from Sweden also suggests that ciprofloxacin is a useful therapeutic option [22]. In this study, all the quinolones appeared to offer good protection against development of disease during the 14day treatment phase. However, all three antimicrobial agents were poor at eradicating the organism and thus preventing later disease, as there was high mortality in each treatment group once the course of antibiotics was completed. This was particularly true for ciprofloxacin, with 100% mortality in each of the three groups within 7 days of cessation of therapy. Gatifloxacin and moxifloxacin mortality rates were significantly lower but were still substantial. Gatifloxacin and moxifloxacin appeared to offer equivalent protective efficacy. Treatment started at 6, 24 or 48 h post exposure made no significant difference to mortality rates, with the exception of a
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single time point. Only two further deaths occurred after late treatment with dexamethasone, which suggests that organisms had been eradicated in surviving mice by Day 42 in most cases. Francisella tularensis is known to locate intracellularly and, as quinolones achieve high intracellular concentrations, it is unclear whether longer treatment regimens would have been more effective. The improved survival rates in gatifloxacin- and moxifloxacin-treated mice compared with ciprofloxacin-treated and non-treated controls suggest partial bactericidal activity but not complete elimination of F. tularensis. Extrapolation of these results to humans is difficult. The pharmacokinetic profiles in 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 man [23]. A dose of 100 mg/kg of gatifloxacin given twice daily gave a pharmacokinetic profile in BALB/c mice that resembled that seen with once-daily dosing in man based on the area under the concentration–time curve [24]. However, a dose of 100 mg/kg moxifloxacin in mice did not give the predicted profile of that reported in man, although a dose of 100 mg/kg moxifloxacin was previously shown to be effective in the treatment of Mycobacterium tuberculosis, Yersinia pestis and Bacillus anthracis [13,14,25,26]. However, in all our studies a standardised dose of 100 mg/kg was chosen for all three antibiotics to allow direct comparisons between antibiotics. Very few inhaled organisms of F. tularensis strain Schu4 are required to cause human disease [6]. Clinical symptoms in humans usually appear 3–5 days following exposure and, if left untreated, mortality can be as high as 30–60% following exposure to infectious aerosols [1,27,28]. This mortality rate can be significantly reduced with appropriate treatment. Previous studies have demonstrated that infection of mice with F. tularensis delivered as small particle aerosols produced an acute fatal disease and that the disease in mice resembled that of humans [3]. This was seen in our study, with F. tularensis delivered by the aerosol route causing a rapidly fatal disease in the non-treated control mice, with 100% mortality within 96 h post exposure. The findings of this study suggest that fluoroquinolones are effective at preventing the development of tularemia in mice following an aerosol challenge, but there is a significant failure rate once post-exposure prophylactic antibiotics have been withdrawn. Gatifloxacin and moxifloxacin both appeared to be more effective than ciprofloxacin in this study. Further in vivo data are required regarding the efficacy of fluoroquinolones for the prevention of pneumonic tularemia.
Acknowledgments The United Kingdom Ministry of Defence supported this work. The authors gratefully acknowledge the assistance of T. Stagg, D. Rogers and M. Brown. Some of the data were
presented at the ASM Biodefense Research Meeting, Baltimore, USA, March 2003.
References [1] Butler T. Plague and tularemia. Pediatr Clin North Am 1979;26:355–66. [2] Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biological weapon: medical and public health management. JAMA 2001;285:2763–73. [3] Conlan JW, Chen W, Shen H, Webb A, KuoLee R. Experimental tularemia in mice challenged by aerosol or intradermally with virulent strains of Francisella tularensis: bacteriologic and histopathologic studies. Microb Pathog 2003;34:239–48. [4] Enderlin G, Morales L, Jacobs RF, Cross JT. Streptomycin and alternative agents for the treatment of tularemia: review of the literature. Clin Infect Dis 1994;19:42–7. [5] Evans ME, Gregory DW, Schaffner W, McGee ZA. Tularemia: a 30-year experience with 88 cases. Medicine 1985;64:251–69. [6] Health Protection Agency. Interim guidelines for action in the event of a deliberate release: Tularaemia, 25 January 2006, version 2.0. London: HPA. Available at: www.hpa.org.uk/infections/topics az/ deliberate release/Tularaemia/PDFs/tularaemia guidelines.pdf [accessed 15 February 2006]. [7] Mason WL, Eigelsbach HT, Little SF, Bates JH. Treatment of tularemia, including pulmonary tularemia, with gentamicin. Am Rev Respir Dis 1980;121:39–45. [8] Russell P, Eley SE, Fulop MJ, Bell DL, Titball RW. The efficacy of ciprofloxacin and doxycycline against experimental tularaemia. J Antimicrob Chemother 1998;41:461–5. [9] Sawyer WD, Dangerfield HG, Hogge AL, Crozier D. Antibiotic prophylaxis and therapy of airborne tularemia. Bacteriol Rev 1966;30:542–8. [10] Arav-Boger R. Cat-bite tularemia in a seventeen-year-old girl treated with ciprofloxacin. Pediatr Infect Dis J 2000;19:583–4. [11] Scheel O, Reiersen R, Hoel T. Treatment of tularemia with ciprofloxacin. Eur J Clin Microbiol Infect Dis 1992;11:447–8. [12] Darling RG, Catlett CL, Huebner KD, Jarrett DG. Threats in bioterrorism. I: CDC category A agents. Emerg Med Clin North Am 2002;20:273–309. [13] Steward J, Lever MS, Simpson AJH, Sefton AM, Brooks TJG. Postexposure prophylaxis of systemic anthrax in mice and treatment with fluoroquinolones. J Antimicrob Chemother 2004;54:95–9. [14] Steward J, Lever MS, Russell P, et al. Efficacy of the latest fluoroquinolones against experimental Yersinia pestis. Int J Antimicrob Agents 2004;24:609–12. [15] Druett HA. A mobile form of the Henderson apparatus. J Hyg (Cambridge) 1969;67:37–48. [16] Petrie A, Watson P. Statistics for veterinary and animal science. London, UK: Blackwell Science; 2000. [17] Guyton AC. Measurement of the respiratory volumes of laboratory animals. Am J Physiol 1947;150:70–7. [18] Ik¨aheimo I, Syrj¨al¨a H, Karhukorpi J, Schildt R, Koskela M. In vitro antibiotic susceptibility of Francisella tularensis isolated from humans and animals. J Antimicrob Chemother 2000;46:287–90. [19] Limaye AP, Hooper CJ. Treatment of tularemia with fluoroquinolones: two cases and review. Clin Infect Dis 1999;29:922–4. [20] Syrj¨al¨a H, Schildt R, R¨ais¨ainen S. In vitro susceptibility of Francisella tularensis to fluoroquinolones and treatment of tularemia with norfloxacin and ciprofloxacin. Eur J Clin Microbiol Infect Dis 1991;19:68–71. [21] Chocarro A, Gonzalez A, Garcia I. Treatment of tularemia with ciprofloxacin. Clin Infect Dis 2000;31:623. [22] Johansson A, Berglund L, Sj¨ostedt A, Tarnvik A. Ciprofloxacin for treatment of tularemia. Clin Infect Dis 2001;33:267–8.
J. Steward et al. / International Journal of Antimicrobial Agents 27 (2006) 439–443 [23] Morris TH. Antibiotic therapeutics in laboratory animals. Lab Anim 1995;29:16–36. [24] Bristol-Myers Squibb. Gatifloxacin package insert. Princeton, NJ: Bristol-Myers Squibb; 2000. [25] Ji B, Lounis N, Maslo C, Truffot-Pernot C, Bonnafous P, Grosset J. In vitro and in vivo activities of moxifloxacin and clinafloxacin against Mycobacterium tuberculosis. Antimicrob Agents Chemother 1998;42:2066–9.
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[26] Miyazaki E, Miyazaki M, Chen JM, Chaisson RE, Bishai WR. Moxifloxacin (BAY12-8039), a new 8-methoxyquinolone, is active in a mouse model of tuberculosis. Antimicrob Agents Chemother 1999;43:85–9. [27] Jacobs RF. Tularemia. Adv Pediatr Infect Dis 1996;12:55– 69. [28] McCrumb FR. Aerosol infection of man with Pasteurella tularensis. Bacteriol Rev 1961;25:262–7.
Treatment of murine pneumonic Francisella tularensis infection with gatifloxacin, moxifloxacin or ciprofloxacin J. Steward a,∗ , T. Piercy a , M.S. Lever a , A.J.H. Simpson a,b , T.J.G. Brooks a,c b
a DSTL Porton Down, Salisbury, Wiltshire SP4 OJQ, UK Department of Medical Microbiology, Royal Free and University College Medical School, University College London, London, UK c Centre for Emergency Preparedness and Response, HPA Salisbury, UK
Received 3 February 2006; accepted 9 February 2006
Abstract The efficacies of gatifloxacin and moxifloxacin were assessed in a BALB/c mouse model of pneumonic tularemia and compared with the efficacy of ciprofloxacin. The rate of relapse following dexamethasone treatment was also investigated. Mice were given 100 mg/kg of the antibiotic by oral administration twice daily for 14 days following an aerosol challenge. All three fluoroquinolones prevented disease during the treatment period, but significant failure rates occurred after the cessation of therapy. Both gatifloxacin and moxifloxacin were more effective than ciprofloxacin at reducing late mortality. Fluoroquinolones may therefore be considered useful candidates for the treatment of pneumonic tularemia. Crown Copyright © 2006 DSTL – published with the permission of the Controller of Her Majesty’s Stationery Office Keywords: Francisella tularensis; Aerosol; Quinolones; Mouse model
1. Introduction Francisella tularensis is the causative organism of the zoonotic disease tularemia. It is a small, fastidious, Gramnegative, facultative, intracellular coccobacillus. Type A strains are found in North America and cause a more severe form of the disease, with an untreated mortality rate of up to 30%, whilst type B strains cause a milder infection that may be unnoticed or undiagnosed [1]. Infection in humans is often associated with contact with infected rodents or lagomorphs and the infectious dose is very low [2]. In endemic areas, ticks, flies and mosquitoes also transmit the infection from the animal reservoir to humans. Other routes of entry include skin abrasions, cuts and the alimentary tract [3]. After an incubation period of 3–5 days, an acute febrile illness develops with malaise, headache and fatigue. Local lymph nodes become enlarged and may suppurate. The liver and spleen ∗
Corresponding author. Tel.: +44 1980 613 169; fax: +44 1980 613 284. E-mail address: [email protected] (J. Steward).
are usually infected, although hepatosplenomegaly may not be apparent until late in the illness. Disseminated disease is common in type A strain infections, but type B disease is often self-limiting. Type A strains can cause lethal infection in humans following exposure to infectious aerosols. As a result, and because of the low infectious dose, F. tularensis is considered to be a potential biological warfare or bioterrorism agent. Francisella tularensis is naturally resistant to ampicillin, and treatment with cephalosporins may lead to relapse. Streptomycin or gentamicin are considered to be the antibiotics of choice, although there is some evidence that fluoroquinolones are effective [2,4–8]. Experimental human studies have demonstrated that prophylactic tetracycline was effective in the prevention of pulmonary tularemia [9]. Ciprofloxacin has been used successfully against experimental tularemia and in the treatment of clinical infection [8,10,11]. Importantly, given the deliberate release potential, the development of streptomycin-resistant strains has been reported [12]. This study therefore sought to assess the in vivo efficacy of two of
0924-8579/$ – see front matter. Crown Copyright © 2006 DSTL – published with the permission of the Controller of Her Majesty’s Stationery Office doi:10.1016/j.ijantimicag.2006.02.006
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J. Steward et al. / International Journal of Antimicrobial Agents 27 (2006) 439–443
the latest-generation fluoroquinolones, gatifloxacin and moxifloxacin, in comparison with ciprofloxacin, as post-exposure prophylaxis against challenge with aerosolised F. tularensis strain Schu4 in BALB/c mice. 2. Materials and methods 2.1. Animals 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 female BALB/c mice (Charles River Laboratories) were randomised into cages containing five animals each. The animals were maintained within an Advisory Committee for Dangerous Pathogens (UK) animal containment level 3 rigid wall isolator complying with British Standard 5726. They were allowed to feed and drink ad libitum and subjected to a 12 h light/dark cycle. The animals were allowed to acclimatise for 7 days prior to challenge.
2.4. Dexamethasone treatment Dexamethasone (5 mg once daily continuing for 7 days) was administered to all surviving mice at Day 42 post exposure by intraperitoneal injection. 2.5. 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 χ2 test [16].
3. Results 3.1. Challenge dose Mice were exposed to an aerosol containing 1.5 × 104 CFU/mouse F. tularensis strain Schu4 as calculated using the Guyton formula [17].
2.2. Antibiotics and treatment regimens
3.2. Antibiotic efficacy study
Antibiotic solutions were prepared by dissolving ciprofloxacin (Bayer, Newbury, UK), gatifloxacin (Bristol Myers Squibb, New York, NY) and moxifloxacin (Bayer, Leverkusen, Germany) tablets in sterile deionised water as previously described [13,14]. All treatments were given at a dose of 100 mg/kg orally twice daily at 12-h intervals and continuing for 14 days. Antibiotic treatment commenced 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 non-treated control group was given sterile deionised water only, twice daily at 12-h intervals continuing for 14 days. Mice were then observed for up to 56 days post exposure.
All non-treated control mice had died by Day 4 post exposure. In the antibiotic treatment groups, all mice survived during the antibiotic treatment phase, but once this was completed animals started dying within 4 days in each group. Survival curves are shown in Fig. 1. All mice in the three groups treated with ciprofloxacin died within 7 days of cessation of therapy. Most of the deaths in the other antibiotic treatment groups also occurred within 7 days of ceasing treatment. For the group receiving antibiotics starting at 6 h post exposure, survival rates at Day 42 were 0%, 53% and 53% for ciprofloxacin, gatifloxacin and moxifloxacin treatment, respectively (Fig. 1a). Both gatifloxacin and moxifloxacin were significantly more effective than ciprofloxacin (P = 0.002). When treatment was delayed for 24 h post exposure, survival rates at Day 42 were 0%, 41% and 12%, respectively (Fig. 1b). Antibiotic treatment initiated at 48 h post exposure resulted in survival rates at Day 42 of 0%, 65% and 35%, respectively (Fig. 1c). Both gatifloxacin and moxifloxacin treatments significantly improved Day 42 survival compared with non-treated controls for all regimens (P < 0.01). Both treatments were also significantly better than ciprofloxacin at all time points, except for moxifloxacin in the 24-h treatment group only (Day 42 survival, 12% versus 0%; P = 0.47). Gatifloxacin was not significantly better than moxifloxacin in the 6-h and 48-h groups, but was significantly better in the 24-h group (Day 42 survival, 41% versus 12%; P = 0.03). For both gatifloxacin and ciprofloxacin, there were no statistically significant differences between the groups commencing therapy at the different time points. For moxifloxacin, there was no difference between therapy started at either 6 h or 48 h (P = 0.2), although there appeared to be an advantage for the 6-h group
2.3. Animal challenge For pneumonic infection studies, a Collison nebuliser containing 20 mL of F. tularensis strain Schu4 at a concentration of 4.32 × 109 colony-forming units (CFU)/mL and three drops of Antifoam 289 (Sigma, Poole, UK) was used to generate and deliver aerosol particles infectious to mice. The particle size of the aerosols produced in this manner is ca. 1–3 m in range. The aerosol was conditioned in a modified Henderson apparatus [15]. In a single experiment, all mice were placed in groups of 20 in a nose-only exposure chamber and exposed for 10 min to a dynamic aerosol. The concentration of F. tularensis in the air stream was determined by taking samples from the exposure chamber using an AGI30 impinger containing fresh modified cysteine partial hydrolysate broth and antifoam. Impinger samples were plated onto blood cysteine glucose agar and incubated for 3 days in air at 37 ◦ C before counting.
J. Steward et al. / International Journal of Antimicrobial Agents 27 (2006) 439–443
441
Fig. 1. Survival curves of BALB/c mice challenged with aerosolised Francisella tularensis Schu4 and treated with either ciprofloxacin (×), gatifloxacin () or moxifloxacin (). Antibiotics were given orally starting at (a) 6 h, (b) 24 h and (c) 48 h post exposure at 12-h intervals and continuing for 14 days. Non-treated controls () were given oral diluent at 12-h intervals and continuing for 14 days.
over the 24-h group (Day 42 survival, 53% versus 12%; P = 0.002). Following dexamethasone treatment, there was one further death in the 6-h moxifloxacin-treated group on Day 56 and one in the 24-h gatifloxacin-treated group on Day 48 post exposure.
4. Discussion In this study, the in vivo efficacies of gatifloxacin and moxifloxacin were assessed in a BALB/c mouse model of aerosolised tularemia Schu4 infection and compared with the efficacy of ciprofloxacin. Streptomycin and gentamicin are the preferred treatments for tularemia, although ciprofloxacin and doxycycline are alternative choices of therapy for mass casualties [2,6]. A number of antibiotic treatments have been assessed against F. tularensis, including imipenem, quinolones and chloramphenicol [4]. Previous studies have demonstrated that F. tularensis Schu4 is susceptible to ciprofloxacin, gatifloxacin and moxifloxacin, with minimum inhibitory concentrations of 0.063, 0.03 and 0.03 mg/L, respectively [8]. There have been several reports of successful treatment
in human tularemia infection using quinolones, including ciprofloxacin, levofloxacin and norfloxacin [4,10,18–20]. Ciprofloxacin also appeared to be equivalent to doxycycline in an animal model [8]. Limaye and Hooper [19] reviewed the English language literature of tularemia cases treated with quinolones and found that there was 100% successful treatment with no relapse within 12 months of follow-up in ten clinical cases. However, there have also been reports of relapse following ciprofloxacin therapy [21]. Evidence from Sweden also suggests that ciprofloxacin is a useful therapeutic option [22]. In this study, all the quinolones appeared to offer good protection against development of disease during the 14day treatment phase. However, all three antimicrobial agents were poor at eradicating the organism and thus preventing later disease, as there was high mortality in each treatment group once the course of antibiotics was completed. This was particularly true for ciprofloxacin, with 100% mortality in each of the three groups within 7 days of cessation of therapy. Gatifloxacin and moxifloxacin mortality rates were significantly lower but were still substantial. Gatifloxacin and moxifloxacin appeared to offer equivalent protective efficacy. Treatment started at 6, 24 or 48 h post exposure made no significant difference to mortality rates, with the exception of a
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single time point. Only two further deaths occurred after late treatment with dexamethasone, which suggests that organisms had been eradicated in surviving mice by Day 42 in most cases. Francisella tularensis is known to locate intracellularly and, as quinolones achieve high intracellular concentrations, it is unclear whether longer treatment regimens would have been more effective. The improved survival rates in gatifloxacin- and moxifloxacin-treated mice compared with ciprofloxacin-treated and non-treated controls suggest partial bactericidal activity but not complete elimination of F. tularensis. Extrapolation of these results to humans is difficult. The pharmacokinetic profiles in 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 man [23]. A dose of 100 mg/kg of gatifloxacin given twice daily gave a pharmacokinetic profile in BALB/c mice that resembled that seen with once-daily dosing in man based on the area under the concentration–time curve [24]. However, a dose of 100 mg/kg moxifloxacin in mice did not give the predicted profile of that reported in man, although a dose of 100 mg/kg moxifloxacin was previously shown to be effective in the treatment of Mycobacterium tuberculosis, Yersinia pestis and Bacillus anthracis [13,14,25,26]. However, in all our studies a standardised dose of 100 mg/kg was chosen for all three antibiotics to allow direct comparisons between antibiotics. Very few inhaled organisms of F. tularensis strain Schu4 are required to cause human disease [6]. Clinical symptoms in humans usually appear 3–5 days following exposure and, if left untreated, mortality can be as high as 30–60% following exposure to infectious aerosols [1,27,28]. This mortality rate can be significantly reduced with appropriate treatment. Previous studies have demonstrated that infection of mice with F. tularensis delivered as small particle aerosols produced an acute fatal disease and that the disease in mice resembled that of humans [3]. This was seen in our study, with F. tularensis delivered by the aerosol route causing a rapidly fatal disease in the non-treated control mice, with 100% mortality within 96 h post exposure. The findings of this study suggest that fluoroquinolones are effective at preventing the development of tularemia in mice following an aerosol challenge, but there is a significant failure rate once post-exposure prophylactic antibiotics have been withdrawn. Gatifloxacin and moxifloxacin both appeared to be more effective than ciprofloxacin in this study. Further in vivo data are required regarding the efficacy of fluoroquinolones for the prevention of pneumonic tularemia.
Acknowledgments The United Kingdom Ministry of Defence supported this work. The authors gratefully acknowledge the assistance of T. Stagg, D. Rogers and M. Brown. Some of the data were
presented at the ASM Biodefense Research Meeting, Baltimore, USA, March 2003.
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