First Isolation of Mycobacterium ulcerans from an Aquatic Environment: The End of a 60-Year Search?

First Isolation of Mycobacterium ulcerans from an Aquatic Environment: The End of a 60-Year Search?

  • Tim Stinear, 
  • Paul D. R. Johnson
  • Published: March 26, 2008
  • DOI: 10.1371/journal.pntd.0000216

In a landmark paper in this issue of PLoS Neglected Tropical Diseases, Portaels et al. [1] describe the first isolation in pure culture of Mycobacterium ulcerans from an aquatic environment, ending a quest that began over 60 years ago when MacCallum and his Australian colleagues identified M. ulcerans as the causative agent of the ulcerative skin disease that later became known as Buruli ulcer [2]. This is a major achievement and will serve as the definitive reference point for scientists intent on revealing the ecology, environmental reservoir, and precise mode of transmission of M. ulcerans.

Buruli ulcer is a terrible, disfiguring disease of skin and soft tissue that may leave sufferers permanently disabled (Figure 1). Those most affected are children living in rural West and Central Africa, but the disease is known in more than 30 countries worldwide, and people of all ages and races are susceptible. In some highly endemic regions, Buruli ulcer is now more common than the two most notorious mycobacterial diseases, leprosy and tuberculosis (TB) [3].

Figure 1. River Offin in the Amansie West District of Ghana showing a typical environmental setting for a region of Buruli ulcer endemicity, and M. ulcerans infection of the elbow of a female patient from the same district.


Recently, the combination of the potent antimycobacterial drugs rifampicin and streptomycin has been shown to be able to kill the causative agent, M. ulcerans, in early nodular Buruli ulcer [4], and a new WHO protocol has been implemented in several endemic countries [5]. The new protocol is proving very effective, greatly reducing costs and often avoiding the need for surgery [6]. More research is required if we are to understand and control this potentially devastating disease.

Exactly how M. ulcerans is introduced into the skin of humans remains unknown, but unlike TB or leprosy, the infection is acquired directly or indirectly from the environment and not from contact with other patients. The highly focal epidemiology and association with swamps and slow-flowing water are hallmarks of Buruli ulcer (Figure 1), and these observations have led to 50 years of failed attempts by many research groups to try to cultivate M. ulcerans from a variety of environmental sources that has included bats, sand flies, rodents, fish, molluscs, vegetation, water, and soil [7][11]. And while isolation of M. ulcerans from clinical specimens is relatively straightforward, efforts to isolate the bacterium from the environment have been confounded by its slow growth, its predicted paucity (based on PCR) in the environment, and the complex, competing microbial flora in these sample types.

The discovery of the M. ulcerans−specific insertion sequence IS2404 in 1997 [12] and the subsequent development of molecular diagnostics for M. ulcerans was the catalyst for renewed efforts to find its environmental sources. The detection of IS2404 in carnivorous water bugs indicated that aquatic insects (among other things) might harbour the bacterium [13]. Laboratory-based feeding studies with M. ulcerans in Naucouris sp. have shown that the bacterium can colonize the salivary glands of these insects and also be transferred by biting a mammalian (mouse) host to cause disease [14]. These observations led Portaels et al. to try and culture M. ulcerans directly from homogenates of five aquatic insects captured from a Buruli ulcer–endemic region of Benin. In a complicated and patience-testing process spanning more than 2 years and involving classical mycobacterial culture techniques and monitoring of cultures by IS2404 PCR, followed by three rounds of blind passaging through mice and then further culture, the team were finally able to obtain a single M. ulcerans isolate from a water strider (Gerris sp.). Phenotypic analysis showed this isolate produced the same polyketide toxin as clinical isolates and was fully virulent for mice. Most importantly, molecular characterization confirmed it was M. ulcerans and not another recently reported M. ulcerans–like mycobacterium [15]. Molecular studies also revealed a novel single nucleotide polymorphism in the 16S rRNA gene of this strain, proving that the result was not a laboratory artefact caused by contamination. The same mutation has since been found in clinical M. ulcerans isolates recovered from patients in the same region of Benin, suggesting a common origin for environmental and patient isolates. Portaels and colleagues were the first to link insects with M. ulcerans [13], and in Australia, two recent papers have taken her observation one step further by demonstrating M. ulcerans DNA in mosquitoes and establishing that mosquito exposure increases the odds of Buruli ulcer in humans [16],[17]. However, whether insects transmit M. ulcerans to humans or just mark its presence in the environment has yet to be definitively established.

The research presented in this paper represents considerable technical prowess, and is the result of knowledge accrued over many decades of M. ulcerans research. It does not end the quest to establish the environmental source of M. ulcerans but provides encouragement (or perhaps discouragement depending on your natural disposition) and an opportunity for future studies to also attempt M. ulcerans isolation from the environment. It will only be through the analysis of many different M. ulcerans isolates from the environment, in the context of solid epidemiological and ecological data, that the lingering and critical questions surrounding the reservoirs and modes of transmission of M. ulcerans will finally be resolved. Much work remains to be done.


  1. 1. Portaels F, Meyers WM, Ablordey A, Castro AG, Chemlal K, et al. (2008) First cultivation and characterization of Mycobacterium ulcerans from the environment. PLoS Negl Trop Dis 2: e178. doi:10.1371/journal.pntd.0000178.
  2. 2. MacCallum P, Tolhurst J, Buckle G, HA S (1948) A new mycobacterial infection in man. J Path Bacteriol 60: 93–122.
  3. 3. Debacker M, Aguiar J, Steunou C, Zinsou C, Meyers WM, et al. (2004) Mycobacterium ulcerans disease (Buruli ulcer) in rural hospital, Southern Benin, 1997–2001. Emerg Infect Dis 10: 1391–1398.
  4. 4. Etuaful S, Carbonnelle B, Grosset J, Lucas S, Horsfield C, et al. (2005) Efficacy of the combination rifampin-streptomycin in preventing growth of Mycobacterium ulcerans in early lesions of Buruli ulcer in humans. Antimicrob Agents Chemother 49: 3182–3186.
  5. 5. Anonymous (2006) Provisional guidelines for antibiotic treatment of Buruli ulcer. Geneva: World Health Organization.
  6. 6. Chauty A, Ardant MF, Adeye A, Euverte H, Guédénon A, et al. (2007) Promising clinical efficacy of streptomycin-rifampin combination for treatment of Buruli ulcer (Mycobacterium ulcerans disease). Antimicrob Agents Chemother 51: 4029–4035.
  7. 7. Barker DJP, Clancey JK, Rao SK (1972) Mycobacteria on vegetation in Uganda. East Afr Med J 49: 667–671.
  8. 8. Buckle G (1972) Notes on Mycobacterium ulcerans. Aust N Z J Surg 41: 320–323.
  9. 9. Portaels F (1973) [Environmental mycobacteria in Lower Zaire]. Ann Soc Belg Med Trop 53: 373–387.
  10. 10. Stanford JL, Paul RC (1973) A preliminary report on some studies of environmental mycobacteria. Ann Soc Belg Med Trop 53: 389–393.
  11. 11. Ross BC, Johnson PD, Oppedisano F, Marino L, Sievers A, et al. (1997) Detection of Mycobacterium ulcerans in environmental samples during an outbreak of ulcerative disease. Appl Environ Microbiol 63: 4135–4138.
  12. 12. Ross BC, Marino L, Oppedisano F, Edwards R, Robins-Browne RM, et al. (1997) Development of a PCR assay for rapid diagnosis of Mycobacterium ulcerans infection. J Clin Microbiol 35: 1696–1700.
  13. 13. Portaels F, Elsen P, Guimares-Peres A, Fonteyne PA, Meyers WM (1999) Insects in the transmission of Mycobacterium ulcerans infection [letter]. Lancet 353: 986.
  14. 14. Marsollier L, Robert R, Aubry J, Saint Andre JP, Kouakou H, et al. (2002) Aquatic insects as a vector for Mycobacterium ulcerans. Appl Environ Microbiol 68: 4623–4628.
  15. 15. Yip MJ, Porter JL, Fyfe JA, Lavender CJ, Portaels F, et al. (2007) Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor. J Bacteriol 189: 2021–2029.
  16. 16. Johnson PD, Azuolas J, Lavender CJ, Wishart E, Stinear TP, et al. (2007) Mycobacterium ulcerans in mosquitoes captured during outbreak of Buruli ulcer, southeastern Australia. Emerg Infect Dis 13: 1653–1660.
  17. 17. Quek TY, Athan E, Henry MJ, Pasco JA, Redden-Hoare J, et al. (2007) Risk factors for Mycobacterium ulcerans infection, southeastern Australia. Emerg Infect Dis 13: 1661–1666.