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

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.

Figure 1.

Histogram of changes in Mycobacterium ulcerans inoculum in soil. Blue boxes, M. ulcerans Agy99; red boxes, M. ulcerans ATCC 33728; x axis, time (days), note that x axis was broken after D90 to change the y axis scale; left-hand y axis and right-hand y axis, M. ulcerans inoculum (colony-forming units).

Figure 2.

Clinical appearance of hind footpads in (A) scarified group A mice exposed to Mycobacterium ulcerans Agy99 illustrating skin lesions, (B) scarified group B mice exposed to M. ulcerans ATCC 33728, and (C) non-scarified control group C with no lesions.

Figure 3.

Histological observation of mouse footpads from (A) group A (exposed to Mycobacterium ulcerans Agy99-inoculated soil) mice and (B) group C mice (negative control). In the group A mice, but not in the group C mice, we observed an abundance of inflammatory infiltrate composed of neutrophils and foamy macrophages (Giemsa stain, original magnification ×100).