In vitro activity of antimicrobial agents against the endosymbiont Wolbachia pipientis

P. G. Hermansa, C. A. Hartb and A. J. Treesa,*

a Liverpool School of Tropical Medicine and Faculty of Veterinary Science, Liverpool; b Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arthropod-transmitted (filarial) nematodes are important causes of disease in humans in tropical countries, yet no safe drug appropriate for mass delivery kills the adult worms. However, most filarial nematodes contain rickettsia-like bacteria of the genus Wolbachia, and related bacteria also occur in insects. There is increasing evidence that these bacteria have significant functions in the biology of filarial nematodes. They are thus important targets in the search for antifilarial drugs and experiments in animals and humans have suggested that antibiotic therapy has potential in treating filarial infections. To optimize future clinical trials there is a need for a fast and simple in vitro drug screen to compare drug efficacies against Wolbachia. In the absence of Wolbachia-infected nematode cell lines, we have utilized an Aedes albopictus insect cell line, naturally infected with Wolbachia, to test the activity of antimicrobial agents. Of the five antibiotics tested, doxycycline, oxytetracycline and rifampicin showed good activity (MICs of 0.0625, 4 and 0.0625 mg/L, respectively) whereas ciprofloxacin and penicillin were shown to have no effect.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Onchocerciasis (river blindness) and lymphatic filariasis are vector-borne, nematode diseases of significant public health importance and are major obstacles to socio-economic development. Control has been based on the mass application of chemotherapeutic agents and insecticides. Presently available anthelmintic drugs such as ivermectin kill only the microfilariae, and because of the longevity of the adult worms, treatment programmes may have to be sustained for up to 12 years.1 The availability of a safe and effective macrofilaricidal agent (i.e. lethal to adult worms) would greatly benefit current control efforts.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cell line of A. albopictus (Aa23) was kindly supplied by Dr S. L. O'Neill (Yale University, New Haven, CT, USA). Cultivation methods were used as described by O'Neill et al.7 and, briefly, were as follows. Cells were grown in 25 cm2 plastic tissue culture flasks containing 5 mL of growth medium at 26°C. The medium consisted of equal volumes of Schneider's insect medium and Mitsuhashi & Maramorosch (MM) insect medium (both from Sigma, St Louis, MO, USA) and was supplemented with 10% heat-inactivated fetal bovine serum. To prevent overgrowth, cells were passaged every 3–4 days by replacing the spent media, dislodging the cells with a scraper and seeding a new flask with 20% of the suspended cells.

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 TableGo. 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 (FigureGo).


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Table. Effects of antibiotics on Wolbachia in cell culture
 


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Figure. Cytospin preparations of an A. albopictus insect cell line, naturally infected with Wolbachia (Giemsa x1000). (a) Untreated (positive control); (b) treated continuously with tetracycline (10 mg/L) (negative control).

 
Cytospin slides were prepared as follows. Every 3–4 days, when cell cultures were passaged, cells not used for passage were resuspended in sterile phosphate-buffered saline, counted using a haemocytometer and adjusted to a final concentration of 2 x 105 cells/mL. Using a cytospin centrifuge, 250 µL of the cell suspension was added to a cytospin tube (5 mm diameter) mounted on a glass slide and centrifuged for 5 min at 160g. The slides were air-dried, fixed in methanol for 10 min and stained in 10% Giemsa solution for 7–10 min. The slides were numbered at random and the whole slide was examined by light microscopy (x1000), not knowing what treatment had been applied. They were scored according to the following scale: 0, no categorically identifiable Wolbachia bacteria present; 1, few Wolbachia bacteria present; 2, many Wolbachia bacteria present and as such comparable to the untreated control cultures.

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The scores of the Giemsa-stained cytospin slides are listed in the TableGo. Scores are average values based on two duplicate cultures except for the penicillin assay, which was not done in duplicate. Positive control slides were almost consistently given the maximum score of 2, indicating maximum numbers of Wolbachia present.

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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
With current interest in the use of antibiotics to treat filarial nematode infections through their activity on endosymbiotic Wolbachia, there is a need for a simple in vitro system to compare the efficacy of agents against the bacteria. Since Wolbachia cannot be cultured axenically and nematode cell culture has not been achieved, we have used a Wolbachia-infected insect cell line to compare drug activity. Phylogenetic analyses confirm that the filarial endosymbionts are closely related to arthropod Wolbachia3 so we hypothesize that relative drug efficacy data obtained with insect Wolbachia is likely to reflect relative activity against nematode Wolbachia.

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.


    Acknowledgments
 
We are very grateful to Dr Scott O'Neill, who provided the Wolbachia-infected Aedes albopictus cell line, and Dr M. Ono for helpful advice. This work was partially supported by the MACROFIL programme of WHO-TDR/OCP/APOC.


    Notes
 
* Correspondence address. Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. Tel: +44-151-708-9393; Fax: +44-151-709-3681; E-mail: trees{at}liverpool.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Plaisier, A. P., Alley, E. S., van Oortmarssen, G. J., Boatin, B. A. & Habbema, J. D. F. (1997). Required duration of combined annual ivermectin treatment and vector control in the onchocerciasis control programme in West Africa. Bulletin of the World Health Organization 75, 237–45.[ISI][Medline]

2 . Taylor, M. J. & Hoerauf, A. (1999). Wolbachia bacteria of filarial nematodes. Parasitology Today 15, 437–42.[ISI][Medline]

3 . Sironi, M., Bandi, C., Sacchi, L., Di Sacco, B., Damiani, G. & Genchi, C. (1995). Molecular evidence for a close relative of the arthropod endosymbiont Wolbachia in a filarial worm. Molecular and Biochemical Parasitology 74, 223–7.[ISI][Medline]

4 . 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, 11–8.[Abstract/Free Full Text]

5 . Langworthy, N. G., Renz, A., Meckenstedt, U., Henkle-Dührsen, K., Bronsvoort, M., Tanya, V. N. et al. (2000). Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship. Proceedings of the Royal Society of London Series B 267, 1–7.[ISI]

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9 . Townson, S., Siemienska, J., Hollick, L. & Hutton, D. (1999). The activity of rifampicin, oxytetracycline and chloramphenicol against Onchocerca lienalis and O. gutturosa. Transactions of the Royal Society of Tropical Medicine and Hygiene 93, 123–4.

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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, 275–9.

Received 26 October 2000; returned 13 December 2000; revised 5 January 2001; accepted 22 January 2001