Distribution of Mycobacterium ulcerans in buruli ulcer endemic and non-endemic aquatic sites in Ghana

Abstract

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer.

Figure 1. Buruli ulcer on the left limb following surgical debridement.

Also shown is joint contracture of the left wrist from scarring caused by Buruli ulcer.

Figure 2. Comparison of IS2404 and ER PCR for detection of M. ulcerans.

Lanes represent serial dilutions of M. ulcerans 1615 from 10⁷ to 10⁻⁴ CFU detected using probes for IS2404 (A) or ER (B). Lanes are labeled 1: 1KB ladder; 2: water blank for DNA extraction; 3: water blank for PCR; 4: M. marinum 1218; 5: M. ulcerans 1615 10⁷ CFU; 6: M. ulcerans 1615 10⁶ CFU; 7: M. ulcerans 1615 10⁵ CFU; 8: M. ulcerans 1615 10⁴ CFU; 9: M. ulcerans 1615 10³ CFU; 10: M. ulcerans 1615 10² CFU; 11: M. ulcerans 1615 10¹ CFU; 12: M. ulcerans 1615 1 CFU; 13: M. ulcerans 1615 10⁻¹ CFU; 14: M. ulcerans 1615 10⁻² CFU; 15: M. ulcerans 1615 10⁻³ CFU; 16: M. ulcerans 1615 10⁻⁴ CFU; 17: M. ulcerans 1615 positive control for PCR.

Figure 3. Sites sampled 2004–2006.

Endemicity is based on human incidence of disease defined at the community level from data obtained from the Ghana Ministry of Health. **Location is approximate and endemicity is based on district level disease incidence data (GPS coordinates not available).

Figure 4. Collection of bacterial biofilms on glass slides from aquatic environments.

(A) Slide 1, Adigon: 3 weeks, slide 2, Adigon: 14 weeks, slide 3, Amasaman: 3 weeks, slide 4, Amasaman: 14 wks. (B) 1, Acid-fast stain of bacilli found on a slide in which VNTR profiling matched M. liflandii. 2, Acid-fast stain of bacilli found on a slide in which VNTR profiling matched M. ulcerans. Acid-fast bacilli shown are representative of those found on most collected slides.

Figure 5. A timecourse of VNTR profiles of M. ulcerans or other mycolactone producing mycobacteria from biofilm slide samples collected from a Buruli ulcer endemic and non-endemic aquatic site.

(Top) Percent of ER-positive biofilm slides with M. ulcerans or MPM VNTR profiles at 21d, 42, and 98d from A-Adigon (non-endemic). (Bottom) Percent ER-positive biofilm slides collected at 21d, 42d, and 98d from Amasaman (endemic).