A protocol for culturing environmental strains of the Buruli ulcer agent, Mycobacterium ulcerans

Abstract

Contaminations and fastidiousness of M. ulcerans may have both hamper isolation of strains from environmental sources. We aimed to optimize decontamination and culture of environmental samples to circumvent both limitations. Three strains of M. ulcerans cultured onto Middlebrook 7H10 at 30 °C for 20 days yielded a significantly higher number of colonies in micro-aerophilic atmosphere compared to ambient atmosphere, 5% CO2 and anaerobic atmosphere. In a second step, we observed that M. ulcerans genome uniquely encoded chitinase, fucosidase and A-D-GlcNAc-diphosphoryl polyprenol A-3-L-rhamnosyl transferase giving M. ulcerans the potential to metabolize chitine, fucose and N-acetyl galactosamine (NAG), respectively. A significant growth-promoting effect of 0.2 mg/mL chitin (p < 0.05), 0.01 mg/mL N-acetyl galactosamine (p < 0.05), 0.01 mg/mL fucose (p < 0.05) was observed with M. ulcerans indicating that NAG alone or combined with fucose and chitin could complement Middlebrook 7H10. Finally, the protocol combining 1% chlorhexidine decontamination with micro-aerophilic incubation on Middlebrook 7H10 medium containing chitin (0.2%), NAG (0.01%) and fucose (0.01%) medium and auto-fluorescence detection of colonies allowed for the isolation of one mycolactone-encoding strain from Thryonomys swinderianus (aulacode) feces specimens collected near the Kossou Dam, Côte d’Ivoire. We propose that incubation of chlorhexidine-decontaminated environmental specimens on Middlebrook 7H10-enriched medium under micro-aerophilic atmosphere at 30 °C may be used for the tentative isolation of M. ulcerans strains from potential environmental sources.

TranslocatingMycobacterium ulcerans: an experimental model

“…Our experimental results thus confirm that some wild rodents are digestively infectable with M. ulcerans, as it has been previously observed for wild agoutis in Côte d’Ivoire [7–8]. and for possums in Australia [10].…”

“…We have previously reported the molecular detection of M. ulcerans DNA in the digestive tract and the spleens of wild agoutis caught in Côte d’Ivoire: and its culture in one case [7–8] In the present work, we developed an experimental model of gastrointestinal infection in rats to confirm that our field observations were the result of probable gastrointestinal contamination of wild agoutis. We chose the rat model as the laboratory mammal closest to agouti, sharing its general morphology (in terms of size and weight), a herbivorous rodent diet and a body temperature of 37°C, which also makes it a relevant model of human infection [15].…”

“…Accordingly, it’s been reported that M. ulcerans is being cultivated from aquatic Hemiptera [6]. indeed M. ulcerans DNA has also been detected in some animals including Thryonomys swinderianus (here designed agoutis, the word commonly used in West Africa) [7–8] rabbits and rats, which have all in common to be rodent mammals [9]. Moreover, two isolates of M. ulcerans have been reported from possum feces collected in Melbourne, region, Australia although characterization and repository reference were not provided [10].…”

“…Furthermore, we recently reported the isolation of M. ulcerans from T. swinderianus feces collected in the vicinity of the Kossou Dam, Côte d’Ivoire, although we were unable to sub-culture this isolate [8]. These observations suggested that T. swinderianus , an herbivorous mammal rodent may contaminate its digestive tract by the oral route after eating M. ulcerans -contaminated food; possibly acting as a secondary source of infection for populations as it is catched as brush meat and eaten after unprotected, manual evisceration [7] Likewise, it has been suggested that the small terrestrial mammals Mastomys natalensis can play a potential role in the natural history of M. ulcerans [12].…”

Screening anti-infectious molecules againstMycobacterium ulcerans: a step towards decontaminating environmental specimens

“…In the present study, we developed a protocol that allowed M. ulcerans subculture after only four days of preculture in TRANS MUl followed by 30 days of incubation on TRANS MUg at 30°C. This protocol is now used for the tentative isolation and culture of M. ulcerans from environmental samples, including aulacode feces samples in which M. ulcerans has been previously isolated but not subcultured [24].…”