In vivo efficacy of fluoroquinolones against systemic tularaemia infection in mice

T. Piercy1,*, J. Steward1, M. S. Lever1 and T. J. G. Brooks1,2

1 Biomedical Sciences, Dstl Porton Down, Salisbury SP4 OJQ, UK; 2 HPA Porton Down, Salisbury SP4 OJG, UK


* Correspondence address. Room 14, Bldg 245, Dstl Porton Down, Salisbury, Wiltshire SP4 OJQ, UK. Tel: +44-1980-613221; Fax: +44-1980-613284; E-mail: tjpiercy{at}dstl.gov.uk

Received 3 May 2005; returned 25 June 2005; revised 9 September 2005; accepted 9 September 2005


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Objectives: The in vivo efficacy of ciprofloxacin, gatifloxacin and moxifloxacin were assessed in an experimental Francisella tularensis Schu S4 infection in the BALB/c mouse model.

Methods: Mice were given 100 mg/kg of antibiotic by oral administration twice daily commencing at 6, 24 or 48 h post-exposure and continued for 14 days post-exposure. All mice were challenged subcutaneously with 1 x 106 cfu F. tularensis Schu S4 and observed for a period of 56 days.

Results: Treatment initiated 6 h post-exposure resulted in 94, 100 and 100% survival for ciprofloxacin, gatifloxacin and moxifloxacin, respectively. When treatment was delayed until 24 h post-exposure the survival rates were ciprofloxacin 67%, gatifloxacin 96% and moxifloxacin 100%. Treatment initiated at 48 h post-exposure resulted in a significant reduction in the survival rate of the ciprofloxacin-treated mice, with 0% survival compared with 84 and 62% for gatifloxacin and moxifloxacin, respectively. Non-treated infected control mice died within 96 h post-exposure. Dexamethasone given at day 42 for 7 days to suppress the animals' immune system caused relapse in all of the treatment groups.

Conclusions: Both gatifloxacin and moxifloxacin were more effective at preventing mortality than ciprofloxacin and could be considered as alternative antibiotics in the treatment of systemic F. tularensis infection.

Keywords: chemotherapy , fluoroquinolones , murine , quinolones , tularaemia


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Francisella tularensis, the aetiological agent of tularaemia, was first described in Tulare County, California, in 1911 during the outbreak of a plague-like disease transmissible to man.1 Human disease occurs throughout the Northern Hemisphere and most frequently in Scandinavia, North America, Japan and Russia.2 There have also been cases throughout central and southern Europe, although less frequent in incidence.35

It was originally proposed that two distinct biotypes of tularaemia exist differing in virulence,6 geographical distribution and animal reservoir, Types A and B, Type A being the more virulent biotype. More recently, the species has been re-classified into four subspecies based on virulence and marked differences in 16S ribosomal DNA sequences; F. tularensis subsp. tularensis, holarctica, mediaasiatica and novicida,7,8 the most virulent of these being tularensis, incorporating all strains previously classified as type A. This highly virulent subspecies is found predominantly in the United States and Canada. The principal animal reservoir for F. tularensis is thought to be the cottontail rabbit (Sylvilagus spp.) and transmission to humans is usually by tick bites or direct contact. Before the introduction of effective antibiotic treatment, human infections caused by such strains resulted in mortality rates of up to 30%.911

Since 1947 when streptomycin was introduced it has traditionally been the drug of choice for the treatment of tularaemia,12 with other aminoglycosides such as gentamicin and amikacin showing highly bactericidal properties.13 Although the aminoglycosides offer good bactericidal properties and low relapse rates, their toxicity and necessity for parenteral administration do not make them ideal as a drug of choice for prophylactic treatment. Tetracycline, doxycycline or chloramphenicol can also be used as alternative treatments to streptomycin,14,15 but tetracycline and doxycycline treatment has reported relapse rates of up to 10%.10,16 There have also been reported high relapse rates when using chloramphenicol and it is considered an ineffective line of treatment.2,17 Others, however, have reported tetracycline to be as effective as streptomycin when a prolonged course of treatment was given.14,18

Quinolones offer new options for the treatment of tularaemia; ciprofloxacin has been shown to be effective against experimental in vivo tularaemia,19 with MIC values in the range of 0.01–0.1 mg/L achieved in some studies.20 Ciprofloxacin has also been used successfully in clinical treatment of various forms of type B tularaemia.2123 Other quinolones have shown promising results when tested in vitro on both natural and human isolates of F. tularensis subsp. tularensis,24 but as yet there is no experience with quinolones and the treatment of type A tularaemia.

This study was designed to examine the in vivo efficacy of ciprofloxacin compared with that of gatifloxacin and moxifloxacin, two of the latest generation quinolones.


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Animals

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.

Two hundred and forty female BALB/c mice aged between 6 and 8 weeks were obtained from Charles River Laboratories (Maidstone, Kent, UK) and randomized into cages of six animals. Each group contained 24 animals. The mice were allowed free access to water and rodent diet (Harlan Teklad, UK). 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 for 7 days before any procedures were undertaken.

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). Tablets were prepared by dissolving in sterile deionized water as previously described.25 BALB/c mice were 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). Sterile deionized water was given to the infected control mice.

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. The infected untreated control group was given diluent only, twice daily at 12 h intervals continuing for 14 days. Mice were observed for 56 days post-exposure.

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.

Preparation of challenge material

F. tularensis subsp. tularensis strain Schu S4 was used for the challenge experiment. All manipulations were carried out in a Class 3 microbiological safety cabinet complying with British Standard 5726. The median lethal dose that kills 50% (MLD50) of untreated animals has previously been demonstrated as 1 cfu by the subcutaneous route.19 Glycerol stocks of Schu S4 stored at –80°C were streaked onto blood cysteine glucose agar (BCGA) and incubated overnight at 37°C.26 The culture was checked for purity and sufficient colonies resuspended into sterile phosphate-buffered saline (PBS) to give an optical density (OD) at 590 nm equal to 0.2, this has been shown to equal ~1 x 109 cfu/mL. This was serially diluted in PBS to ~107 cfu/mL, all animals were challenged with 0.1 mL, equal to ~106 cfu by the subcutaneous route.

Bacteriological analysis

Six animals from each treatment group were culled at equivalent times after antibiotic treatment had ceased in order to assess tissue burden of organs. Liver and spleen were removed from each animal and homogenized in 2 mL Modified Cysteine Partial Hydrolysate (MCPH) broth, using a Medi-machine homogenizer (Becton Dickinson, UK). Ten-fold serial dilutions were carried out in MCPH broth using a 24-well tissue culture plate, 0.25 mL of each dilution was plated out in duplicate on BCGA and incubated at 37°C for 3 days. Colony counts were then taken for each sample to determine bacterial load.

Statistical analysis

The experimental design allowed a minimum group size of 18 to compare different antibiotic treatment and challenge regimens at Days 14 and 28 post-exposure. This gave an 80% power of detecting a difference of 50% in the effectiveness of treatment regimens with 95% confidence limits.27

MICs

The MICs of ciprofloxacin, gatifloxacin and moxifloxacin for the challenge strain of F. tularensis Schu S4 used in this study were determined using methods previously described.19 The antibiotic dilution series used gave a final concentration range from 0.008 to 64 mg/L.


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Antibiotic efficacy study

F. tularensis when delivered by the systemic route caused rapidly fatal disease within 96 h in non-treated, infected control mice. Figures 1Go3 show survival data up to day 42 post-exposure. Antibiotic treatment initiated 6 h post-exposure proved to be the most effective treatment regimen. There was 100, 100 and 94% survival for gatifloxacin, moxifloxacin and ciprofloxacin, respectively, at Day 42. Survival rates were significantly higher in animals treated with any of the three antibiotic regimens (P < 0.001, {chi}2 test) compared with untreated controls; however, there were no significant differences in the survival rates when the antibiotics were compared with each other (P > 0.05, {chi}2 test).



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Figure 1. Survivors of BALB/c mice challenged with F. tularensis Schu S4 by subcutaneous injection and treated with 100 mg/kg of ciprofloxacin (open circles), gatifloxacin (open squares) or moxifloxacin (open triangles). Antibiotics given orally at 12 h intervals commencing at 6 h post-exposure and continuing for 14 days. Untreated controls (crosses) were given diluent at 12 h intervals for 14 days.

 


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Figure 2. Survivors of BALB/c mice challenged with F. tularensis Schu S4 by subcutaneous injection and treated with 100 mg/kg of ciprofloxacin (open circles), gatifloxacin (open squares) or moxifloxacin (open triangles). Antibiotics given orally at 12 h intervals commencing at 24 h post-exposure and continuing for 14 days. Untreated controls (crosses) were given diluent at 12 h intervals for 14 days.

 


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Figure 3. Survivors of BALB/c mice challenged with F. tularensis Schu S4 by subcutaneous injection and treated with 100 mg/kg of ciprofloxacin (open circles), gatifloxacin (open squares) or moxifloxacin (open triangles). Antibiotics given orally at 12 h intervals commencing at 48 h post-exposure and continuing for 14 days. Untreated controls (crosses) were given diluent at 12 h intervals for 14 days.

 
Survival rates in groups where antibiotic treatment was commenced 24 h post-exposure were lower, with survival rates of 100, 96 and 67% for moxifloxacin, gatifloxacin and ciprofloxacin, respectively. Survival rates were again significantly higher in all three treatment regimens (P < 0.001, {chi}2 test) compared with untreated control mice; however, there was also a significant difference (P < 0.05, {chi}2 test) in the survival rates achieved with both gatifloxacin and moxifloxacin when compared with those achieved with ciprofloxacin.

Survival rates in groups where antibiotic treatment was commenced 48 h post-exposure were gatifloxacin 84%, moxifloxacin 62%; however, all ciprofloxacin-treated animals in this regimen were culled due to the severity of disease. Survival rates were poor when treatment was delayed by 48 h, with no significant difference in survival rates of mice in the ciprofloxacin treatment group (P > 0.05) and untreated control mice. There was, however, a difference in the survival rates achieved in both the gatifloxacin and moxifloxacin treatment groups (P < 0.001) compared with the untreated control mice. There was also no significant difference in survival rates achieved when comparing gatifloxacin with moxifloxacin (P > 0.05).

Bacteriological analysis

No growth was observed from any of the samples including the positive controls. Organs were frozen at –80°C prior to processing and plating out due to time constraints, and it is probable that the effects of freeze and thaw inactivated F. tularensis cells within organs. To check for quality of the BCGA plates, positive control cultures of Schu S4 were plated out in parallel to test samples. All positive control cultures grew after 3 days.

Mortality on administration of dexamethasone

There were high rates of mortality following dexamethasone treatment of survivors, starting on day 42; the data are summarized in Table 1.


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Table 1. Mortality rates after administration of dexamethasone, based on number of survivors at day 56

 
MICs

The MICs measured against F. tularensis Schu S4 in this study were 0.063, 0.03 and 0.03 mg/L for ciprofloxacin, gatifloxacin and moxifloxacin, respectively.


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This study aimed to assess the efficacies of ciprofloxacin, gatifloxacin and moxifloxacin in an experimental F. tularensis Schu S4 infection in BALB/c mice.

Bacteraemia may be common in early F. tularensis infection but is rarely detected, during initial stages of infection the organism infects and multiplies within macrophages spreading throughout the body via the haematogenous route. The bacteria cause cell death by apoptosis, leading to the formation of necrotic foci and granuloma within the liver, spleen and lymph nodes.28 Mortality then results from overwhelming organ damage attributed to these high levels of cell debris and bacteria.29 F. tularensis infection in BALB/c mice causes rapidly fatal infection, if left untreated causing death within 96 h in non-treated control mice.

Streptomycin is currently the antibiotic of choice for the treatment of human tularaemia;12,30 however, previous studies have shown ciprofloxacin to be effective against experimental tularaemia, and it has also been shown to be effective when used in cases of naturally occurring infection.18,19

Applying Morris's formula, an estimated dose of 44 mg/kg for gatifloxacin and moxifloxacin and 55 mg/kg for ciprofloxacin was predicted.31 The concentrations of antibiotics determined in tissues and serum following administration of these doses, however, was lower than values predicted for man. Gatifloxacin given at a dose of 100 mg/kg twice daily gave a pharmacokinetic profile in BALB/c mice that resembled that seen with a once daily dose of 400 mg in man based on AUC.32 Direct comparison between animals and man is difficult and accurate determination of pharmacokinetic profiles in mice, in our experience has wide margins of error. A standardized dose of 100 mg/kg was chosen, therefore, to allow direct comparison of all three antibiotics in this study.

There is evidence of relapse following withdrawal of antibiotic treatment in humans especially where tetracyclines have been used.14,17,18,22 Our study showed relapse at day 42, in all three treatment groups on the withdrawal of antibiotics, but not with all of the antibiotic treatment regimens. In the 6 h post-exposure group relapse was seen only in the ciprofloxacin-treated mice with low relapse rates, whereas 100% survival was achieved in both the gatifloxacin- and moxifloxacin-treated groups. The effectiveness of quinolones in the early treatment groups can be attributed to high intracellular concentrations, preventing the bacteria from penetrating intracellularly and establishing infection within the organs. In the 24 h post-exposure treatment group relapse was seen in both the ciprofloxacin- and gatifloxacin-treated mice with relapse rates of 33 and 4%, respectively, while there was still no relapse seen in the moxifloxacin-treated mice. When treatment was delayed by 48 h the effect on relapse rates was much greater with relapse occurring in all of the antibiotic-treated mice, this time ciprofloxacin was particularly ineffective with 100% relapse, gatifloxacin- and moxifloxacin-treated mice had relapse rates of 16 and 38%, respectively.

Dexamethasone treatment was given at day 42; this suppressed the immune system causing mortality of animals with latent infection. All groups were affected by dexamethasone administration; however, the largest percentage mortality was seen in the 48 h treatment groups, with high levels of mortality observed for gatifloxacin and moxifloxacin, the two treatment groups with remaining animals. Mortality in the 24 and 6 h treatment groups was observed again with all three antibiotics, however, to a lesser extent in the gatifloxacin- and moxifloxacin-treated mice. These data suggest partial bactericidal effects, but out of the three antibiotics tested in this study, none offered 100% effectiveness.

The data from this study suggest that delayed antibiotic treatment enabled the bacteria to establish infection within the organs, where it reached significantly high levels before the antibiotics were administered, therefore reducing the chance of survival. Despite quinolones penetrating intracellularly, some organisms were clearly protected to some extent in this environment, as seen with the relapse rates of F. tularensis following withdrawal of antibiotics. A number of other microorganisms are known to change virulence factors and other key metabolic processes in order to evade killing mechanisms and ensure persistence.33 It is possible that F. tularensis is able to adopt a similar strategy in order to avoid antibiotic activity. Higher doses of antibiotics may prevent this from occurring, however, in diseases such as melioidosis and brucellosis, relapse is often seen even after repeated high dosing.34,35

This study showed that gatifloxacin and moxifloxacin were better at increasing survival percentages than ciprofloxacin, when treatment was delayed, especially in the 48 h post-exposure treatment group.

Other studies have also shown ciprofloxacin to be particularly effective with MIC values ranging from 0.031 to 0.125 mg/L, when tested against a range of type B strains of tularaemia and compared with 10 other antimicrobial agents, with ciprofloxacin having the lowest MIC value across the board.36 Ciprofloxacin MIC results in this study (0.063 mg/L) were comparable to values reported previously, when testing a range of quinolones against eight isolates of F. tularensis subsp. tularensis.24 Previous work has also shown both gatifloxacin and moxifloxacin to have a more improved pharmacokinetic profile than ciprofloxacin.25 Owing to the improved pharmacokinetic profile and the lower MICs as demonstrated in this study, it is not therefore unexpected that both the new generation quinolones offer significantly higher survival rates when compared with ciprofloxacin.

Currently, ciprofloxacin has been used as an alternative to streptomycin in the treatment of less virulent type B strains of tularaemia.2123 This study, however, has shown it to be less effective against type A strains especially when compared with the two newer generation quinolones tested.

Gatifloxacin and moxifloxacin offered promising effectiveness in this study and could be considered as alternatives to treatments for tularaemia infection.


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None to declare.


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26. Hartley MG, Green M, Choules G et al. Protection afforded by heat shock protein 60 from Francisella tularensis is due to copurified lipopolysaccharide. Infect Immun 2004; 72 Suppl 7: 4109–13.[Abstract/Free Full Text]

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30. Finch RG, Greenwood D, Norrby SR et al. Aminoglycosides and aminocyclitols. In: Antibiotic and Chemotherapy, 8th edn. Edinburgh: Churchill Livingstone 2003; P155–84.

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