a Liverpool School of Tropical Medicine and Faculty of Veterinary Science, Liverpool; b Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool, UK
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Abstract |
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Introduction |
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The renewed awareness that filarial nematodes are infected with rickettsia-like intracellular bacteria has stimulated interest in them as chemotherapeutic targets.2 The bacteria are closely related to the Wolbachia endosymbionts of arthropods3 and have been detected in the majority of filarial nematodes analysed so far and in all developmental stages.2
Several studies have demonstrated sublethal effects of oxytetracycline on filarial worms and related that activity to effects on the Wolbachia.2 Thus, whilst tetracycline treatment of rodents infected with Litomosoides sigmodontis (Wolbachia infected) resulted in filarial growth retardation and infertility, penicillin known to be ineffective against rickettsial agents did not, nor did tetracycline treatment of rats infected with Acanthocheilonema vitae, which lacks Wolbachia.4 Importantly, a macrofilaricidal effect was observed in a bovine model of onchocerciasis. In cattle naturally infected with Onchocerca ochengi, intermittent treatment with oxytetracycline over a period of 6 months led to elimination of Wolbachia from the hypodermis of the worms, as demonstrated by electron microscopy, and to complete resolution of intradermal nodules and death of all adult worms and microfilariae 9 months post-treatment, suggesting that the bacteria were essential for worm survival.5
Although a human trial with onchocerciasis patients in Ghana, treated for 6 weeks with doxycycline, has shown promising results,6 much work still has to be done on determining the most active compounds against Wolbachia and in optimizing treatment regimens. Therefore, a relatively fast and simple in vitro assay to screen efficacies of antibiotics would benefit the design of future drug trials. The establishment of cell cultures of nematode Wolbachia has not yet proven possible but the closely related arthropod Wolbachia has been cultured in a continuous cell line of the mosquito Aedes albopictus.7 We have utilized this insect cell culture to monitor effects of antibiotics on Wolbachia by direct visualization of the bacteria, with the aim of deriving MICs and MBCs of these antibiotics. A range of antibiotics was chosen that were expected to vary in their activity against Wolbachia.
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Materials and methods |
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Five different antibiotics were tested. Stock solutions of doxycycline, oxytetracycline and rifampicin (all Sigma) in ethanol and benzyl penicillin (Brittannia Pharmaceuticals, Redhill, UK) and ciprofloxacin (Sigma) in water of 1.6, 1.6, 3.2, 12.8 and 2.0 mg/mL, respectively, were kept at 20°C and diluted and filtered before each use. The stock solutions were diluted with culture medium to give final concentrations in culture as shown in the Table. Test cultures were maintained with antibiotic for 14 days. Duplicate cultures were maintained for each concentration of antibiotic except for those treated with penicillin. Cultures were then maintained without antibiotics from day 14 to day 21. In addition, cell cultures were maintained that received either no antibiotic treatment except for routine inclusion in the culture medium of penicillin (100 U/mL) and streptomycin (100 mg/mL) (Life Technologies, Paisley, Scotland, UK)Wolbachia-positive controls, or continuous treatment with tetracycline hydrochloride (10 mg/L, Sigma)Wolbachia-negative controls (Figure
).
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The MIC was taken as the lowest concentration of antibiotic that achieved an average score of <1 on day 14. Similarly, the MBC was the lowest concentration of antibiotic with average scores <1 on day 21.
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Results |
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The MIC and MBC values (mg/L), respectively, were: doxycycline 0.0625, 0.25; oxytetracycline 4, 1; rifampicin 0.0625, 2; ciprofloxacin >8, >8; penicillin >256, >256. The MBC of oxytetracycline was lower than the MIC, which is probably due to the difficulty in some cytospins of determining categorically the absence of all bacteria. This also explains why not all negative control samples had scores of 0 and why scores did not always establish a dose-dependent effect of the antibiotics. Since the highest concentrations of ciprofloxacin and penicillin tested did not result in scores <1 these antibiotics are concluded to be ineffective against Wolbachia. The lowest concentration used for rifampicin (0.0625 mg/L) resulted in a score of <1 on day 14. The true MIC of rifampicin might, therefore, be <0.0625 mg/L.
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Discussion |
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The results described here indicate a greater activity of doxycycline against Wolbachia than oxytetracycline. This is encouraging in that oxytetracycline has been shown to be macrofilaricidal against O. ochengi;5 thus it is likely that doxycycline will prove at least as effective in vivo if used in an appropiate treatment regimen. The results are consistent with the efficacy of doxycycline against the Wolbachia in Onchocerca volvulus in humans.6 Differential activities of tetracyclines can be fully accounted for by differences in cellular uptake, which in turn is correlated with tetracycline lipophilicity.8 The more lipophilic doxycycline would therefore achieve higher intracellular concentrations and be more effective against the intracellular Wolbachia than the non-lipophilic oxytetracycline.
The activity of rifampicin against Wolbachia has not been reported previously, but Townson et al.9 found that this antibiotic reduced the recovery of Onchocerca lienalis microfilariae in a surrogate infection mouse system by 74% compared with controls, whilst oxytetracycline treatment led to a 56% reduction. The effects on the Wolbachia were not monitored in these studies.
The contraindications of the tetracyclines in children and pregnant women and the reservation of rifampicin for the treatment of tuberculosis are reasons for seeking other antibiotics with activities against Wolbachia. The proven effectiveness of ciprofloxacin against the spotted fever rickettsiae10 suggested that it might be efficacious against Wolbachia. However, doses of ciprofloxacin of up to 8 mg/L failed to clear the Wolbachia bacteria from the insect cell culture. Studies with L. sigmodontis in cotton rats treated with ciprofloxacin for 63 days showed no antifilarial effects of the treatment, and immunohistology and semiquantitative PCR indicated no significant depletion of the endosymbionts.11 It is thus unlikely that ciprofloxacin merits further study for activity against Wolbachia. The activity of ß-lactams and quinolones other than ciprofloxacin against Wolbachia could be examined in this system.
In conclusion, these results indicate the utility of this system and are in accordance with limited in vivo data on the antifilarial and anti-Wolbachia activity of different antibiotics. The insect cell line provides a simple in vitro means of comparing antibiotic efficacy before in vivo experiments against filaria-infected animals or humans.
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Acknowledgments |
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Notes |
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References |
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2 . Taylor, M. J. & Hoerauf, A. (1999). Wolbachia bacteria of filarial nematodes. Parasitology Today 15, 43742.[ISI][Medline]
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Hoerauf, A., Nissen-Pähle, K., Schmetz, C., Henkle-Dührsen, K., Blaxter, M. L., Büttner, D. W. et al. (1999). Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. Journal of Clinical Investigation 103, 118.
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11 . Hoerauf, A., Volkmann, L., Nissen-Pähle, K., Schmetz, C. & Autenrieth, I. (2000). Targeting of Wolbachia endobacteria in Litomosoides sigmodontis: comparison of tetracyclines with chloramphenicol, macrolides and ciprofloxacin. Tropical Medicine and International Health 5, 2759.
Received 26 October 2000; returned 13 December 2000; revised 5 January 2001; accepted 22 January 2001