Mycobacterium ulcerans Persistence at a Village Water Source of Buruli Ulcer Patients

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Abstract
Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission. Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans which affects mainly children in West Africa. Although it is commonly believed that the infection originates from an environmental source, both the reservoir of M. ulcerans and the mode of transmission to human patients remain to be elucidated. Previous investigations indicated that transmission likely takes place away from the homes of patients. We therefore screened the farms as well as village and farm water locations of 46 laboratory confirmed BU patients of the Mapé Basin of Cameroon for the presence of M. ulcerans DNA by real-time PCR. In this analysis three positive village water locations were identified. By studying one of these locations in great detail we found that M. ulcerans DNA persists in underwater detritus in one section of the village water location even after all local cases had been treated. The detritus may represent a reservoir of M. ulcerans from where infection could take place through either direct contamination of skin lesions or through contamination or colonization of insect vectors.

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