The Environmental Pathogen Mycobacterium ulcerans Grows in Amphibian Cells at Low Temperatures

Mycobacterium ulcerans, the etiological agent of Buruli ulcers,is an environmental pathogen. We cultivated it in an amphibian(XTC-2) cell line that grows at 28°C. By counting of Ziehl-Neelsen-stainedmycobacteria and by quantitative PCR analysis, we found thatM. ulcerans multiplies rapidly in association with XTC-2 cells.Transmission electron microscopy demonstrated the presence ofintracellular M. ulcerans microorganisms. These data suggestan intracellular environmental niche, and we propose use ofXTC-2 cells for isolation of M. ulcerans from environmentalsources.

INTRODUCTION

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Introduction

Mycobacterium ulcerans is a slowly growing mycobacterium thatcauses necrotizing skin ulcers, named Buruli ulcers (1, 12,13). It is an environmental pathogen with worldwide distribution.The disease often occurs in people who live or work close torivers and stagnant bodies of water. Emergence of Buruli ulcerin Australia and Africa has been linked to modifications ofthe hydric environment (11). M. ulcerans has been detected bymolecular methods in stagnant water in Australia (11) as wellas in aquatic African insects (F. Portaels, P. Elsen, A. Guimaraes-Peres,P. A. Fonteyne, and M. W. Meyers, Letter, Lancet 353:986, 1999),suggesting a hydric reservoir. However, M. ulcerans is cultivablefrom clinical material on Lowenstein-Jensen medium but has notbeen isolated from environmental sources so far (3). M. ulceransis reported to be a strictly extracellular mycobacterium whichgrows best below 32°C. However, it is closely related toMycobacterium marinum (3, 7), another environmental pathogenwhich has been recently grown in fish cells (4). The purposeof our work was to culture M. ulcerans in a poikilothermic cellline to test whether M. ulcerans can grow intracellularly atlow temperatures.

Culture and kinetics in XTC2 cells.

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Culture and Kinetics in...

A clinical isolate of M. ulcerans was propagated on LowensteinJensen medium (BioMérieux, Marcy l'Etoile, France) (6)and adjusted to 10⁵ CFU/ml. It was aliquoted as 100 µlin tubes, which constitutes the inoculum that was used to infectXTC2 cells (average, 20 mycobacteria/cell) at 28°C as previouslyreported (8). Briefly, the XTC-2 cell line, derived from Xenopuslaevis, was used for culture by the shell vial centrifugationtechnique (8). Subconfluent cells were obtained by incubatingthe shell vials at 28°C for 48 h after they were injectedwith 50,000 cells in 1 ml of Leibowitz-15 medium with L-glutamineand L-amino acids (GIBCO, Rockville, Md.), 5% (vol/vol) fetalcalf serum, and 2% (vol/vol) tryptose phosphate (GIBCO). Afterinjection of the inoculum resuspended in 0.8 ml of medium, theshell vials were centrifuged at 700 x g for 1 h at 20°C,and the supernatant was discarded. After two washings in sterilephosphate-buffered saline, 1 ml of fresh medium was added tothe shell vials, which were incubated at 28°C. The cellculture medium was replaced every 7 days for up to 30 days.Cultures were sampled at days 0, 7, 15, 30, and 45.

M. ulcerans growth was evaluated by counting Ziehl-Neelsen-stainedmycobacteria in serial dilutions of infected cells. Experimentswere performed in triplicate. The numbers of mycobacteria were3.7 ± 2.1 at day 0, 5.8 ± 1.7 at day 7, 10.2 ±6.8 at day 15, 11.5 ± 5.7 at day 30, 29.6 ± 8.3at day 45, and 35.2 ± 7 at day 60, giving an estimateddoubling time of 10 days. M. ulcerans DNA was also quantifiedin the same cell samples using real-time PCR by the LightCycleramplification and detection system (Roche Diagnostics, Mannheim,Germany) and 0.5 pmol (final concentration) of the primers UF(5'-TGA GCT GAT CCA GGA CCA GA-3') and UR (5'-TGG TTC CGA AGAACT CCT TG-3'), targeting a 155-bp fragment of the M. ulceransrpoB gene (GenBank accession number AF057491) (5); quantitativePCR amplification was carried out with 45 cycles of denaturation(8 s at 95°C), annealing (5 s at 62°C), and extension(10 s at 72°C). Serial dilutions of M. ulcerans (10⁰ to10⁵ CFU/ml) in 0.01% Tween 20 (Sigma-Aldrich Chimie, Saint-QuentinFallavier, France)-sterile phosphate-buffered saline were lysedusing a French press device at 4-ton pressure and served asexternal standards in each run. The concentrations of mycobacterialDNA in coculture samples were calculated by comparing the cyclenumbers of the logarithmic linear phase of the samples withthe cycle numbers of the external standards. Noninoculated XTC-2cells were used as a negative control. Experiments were carriedout in triplicate. DNA quantification in cells gave an estimationof 5 copies/ml at day 0, 5 copies/ml at day 7, 15 copies/mlat day 15, 45 copies/ml at day 30, 70 copies/ml at day 45, and10² copies/ml at day 60. The DNA copy kinetics is comparableto the growth kinetics measured by staining.

Intracellular detection of M. ulcerans.

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Intracellular detection of m....

With Ziehl-Neelsen staining, the mycobacteria grown within XTC-2cells appeared as long, purple-red and intracellular bacteria(data not shown). They were often in small clusters. For electronmicroscopy, harvested infected cells were fixed in 2.5% glutaraldehydein 0.1 M cacodylate buffer (pH 7.2) containing 0.1 M sucrosefor 1 h at 4°C. After being washed in this buffer overnight,the cells were fixed in 1% osmium tetroxide in 0.1 M cacodylatebuffer for 1 h at room temperature and dehydrated by washingin increasing concentrations (25 to 100%) of acetone. The cellswere finally embedded in Araldite (Fluka, St. Quentin Fallavier,France), and thin sections were cut with an Ultracut microtome(Reichert-Leica, Marseille, France), stained with a saturatedsolution of methanol-uranyl acetate and lead nitrate with sodiumcitrate in water, and examined in a Morgagni 268 D electronmicroscope (Philipps, Limeil-Brevannes, France). The mycobacteriawere found in the cytoplasm (Fig. 1) but never in the nucleus.Dividing mycobacteria were occasionally observed.

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FIG. 1. Transmission electron microscopy of intracellular M. ulcerans in XTC-2 cells showing a dividing mycobacterium. The bar represents 5 µm.

The above findings show that M. ulcerans is able to grow inassociation with XTC2 cells. It is observed intracellularly,and the medium without cells did not support the growth of M.ulcerans (data no shown). Therefore, we believe that our datademonstrate that M. ulcerans grows in XTC2 cells. A key pointin our experiments was the availability of a cell line derivedfrom a poikilothermic, amphibian vertebrate, with which we couldcocultivate M. ulcerans at 28°C. This is not possible withthe murine macrophage cell line J-774, which was used by Rastogiet al. (9, 10), as these cells do not survive at 28°C formore than a few days (D. Raoult, unpublished data). In theirstudy, at 37°C, M. ulcerans caused massive lysis of macrophage-likecells as reported from histological examination of patients(13). Our data clearly indicate that M. ulcerans could be afacultative intracellular mycobacterium when grown at low temperatures.This is comparable to what is reported with M. marinum in afish monocyte cell line (CLC cell line) cultivated at 28°C(4). These data suggest an intracellular, environmental nichefor M. ulcerans.

FOOTNOTES

* Corresponding author. Mailing address: Unité des Rickettsies CNRS UPRES A 6020, Faculté de Médecine, IFR 48, Université de la Méditerranée, 27 Bd. Jean Moulin, 13385 Marseille, France. Phone: 33 (0)4 91 32 43 75. Fax: 33 (0)4 91 38 77 72. E-mail: didier.raoult{at}medecine.univ-mrs.fr.

REFERENCES

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