Experimental Survival of Mycobacterium ulcerans in Watery Soil, a Potential Source of Buruli Ulcer

Abstract

Abstract. The reservoir of Mycobacterium ulcerans causing Buruli ulcer (BU) remains unknown. Here, sterilized watery soil was mixed with 2 × 10 6 colony-forming units (CFU)/g of M. ulcerans Agy99 or M. ulcerans ATCC 33728 and incubated in a microaerophilic atmosphere in the presence of negative controls. Both M. ulcerans strains survived in soil for 4 months with a final inoculum of 300-440 CFU/g. Further, three groups of five mice with and without footpad scarification were exposed to control soil or M. ulcerans-inoculated soil. Although no specific clinical and histopathological lesions were observed in control animals, red spots observed on 8/20 scarified feet in 8/10 challenged mice yielded inflammatory infiltrates and positive real-time polymerase chain reaction detection of M. ulcerans DNA in five mice. BU can be acquired as an inoculation infection with watery soil as a transient source of infection. These experimental observations warrant additional field observations. Moreover, the only environmental M. ulcerans isolate was recovered from a water strider in Benin. 7 These observations suggest a link between BU and stagnant and slow-flowing aquatic environments in West Africa. However, freshwater itself may not be the ultimate source of M. ulcerans. In particular, fishermen who are presumably in frequent contact with water have a lower prevalence of BU than other groups in endemic regions in Africa. 8,9 In addition, the M. ulcerans inoculum has been estimated to be 100-1,000 mycobacteria/mL in water, based on real-time polymerase chain reaction (RT-PCR) targeting IS2404.3 This estimated inoculum level is three to four times lower than the levels reported for major waterborne human pathogens such as Vibrio. 10We have recently shown that Mycobacterium tuberculosis, a Mycobacterium that is phylogenetically closely related to M. ulcerans, retains its infectivity after an extended stay in soil. 11 We therefore hypothesized that watery soil could be a source of M. ulcerans.To test the hypothesis, M. ulcerans ATCC 19423 TMT (strain Agy99) and M. ulcerans ATCC 33728 (both obtained from the American Type Culture Collection, Masassas, VA) were cultured in an incubator at 32°C on Middlebrook 7H10 Agar (Becton Dickinson, Le Pont de Claix, France), supplemented with OADC (oleic acid, bovine albumin, dextrose, and catalase) (Becton Dickinson). Natural soil was steam sterilized at 134°C for 15 minutes, and the sterility was then assessed by inoculating 2 g of steam-sterilized soil onto Middlebrook 7H10 Agar incubated at 32°C for 4 weeks. A 20-g sample of sterilized soil was inoculated and mixed with 2 × 10 6 colonyforming units (CFU)/g of each of the two M. ulcerans isolates, placed into 25-cm 2 cell culture flasks (Corning, Corning, NY) and thoroughly mixed. Three flasks were prepared for each M. ulcerans strain. The flasks were placed inside a transparent sterile plastic container where they were exposed to a microaerophilic atmosphere created using a CampyGen atmosphere generation system (Oxoid Ltd., Basingstoke, Un...

Buruli Ulcer, a Prototype for Ecosystem-Related Infection, Caused by Mycobacterium ulcerans

“…Accordingly, genomic analysis has suggested that M. ulcerans may reside inside one or several hosts (14), in agreement with previous observations (22). However, it was demonstrated in an experimental study that it can live as a free-living organism in its environmental niches, where it can survive for a long time despite its fragility under certain climatic conditions, such as solar light, temperature elevation, and UV light (14,29,37). As discussed above, these aspects have been poorly investigated, as the vast majority of field studies have relied upon molecular biology methods, which gave no clues regarding the viability of the detected mycobacteria.…”

“…In West Africa and Central Africa, outbreaks of Buruli ulcer in the 1980s were linked to man-made changes in the natural environment (34,35). More-recent studies have shown that in aquatic and swampy environments, M. ulcerans is detected in biofilms, soil, and aquatic insects (36)(37)(38)(39)(40).…”

Environmental and Behavioral Drivers of Buruli Ulcer Disease in Selected Communities Along the Densu River Basin of Ghana: A Case-Control Study

“…Agricultural activities, such as farming particularly in swampy areas, puts cases in constant contact with moist/watery soil. Tian and others recently showed that MU strains can survive in soil for 4 months suggesting that BU could be acquired through contact of open wounds with watery soil as a transient source of infection 37. Moreover, Aboagye and others recently isolated MU from soil in a BU-endemic community along the Densu river basin of Ghana 38.…”

The Immunology of Buruli Ulcer

“…Another debatable topic in BU is disease transmission. Although not the main focus of this chapter, some epidemiological records and experimental laboratory results point to environmental exposure as the main source for BU acquisition, namely through contact of injured skin with water bodies and through insect bites [25,26,113,[129][130][131][132][133]. However, some work in Africa, Asia and Australia additionally raised the possibility of host genetics playing a major part in BU development [92,[134][135][136][137].…”