Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Abstract

BackgroundThe reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.Methodology/Principal FindingsWe investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).Conclusion/SignificanceThis study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…In their protozoan hosts, "hidden" mycobacteria might find easier opportunities to infect vertebrate end hosts, multiplying within protozoans to escape immune reactions (480). This ability to persist within amoebae has been widely documented (481)(482)(483). The internalization of infectious agents inside other parasites represents an evolutionary strategy for survival that may sometimes enhance pathogenesis or transmissibility (480).…”

Mycobacterium ulcerans Disease (Buruli Ulcer): Potential Reservoirs and Vectors

“…Underwater detritus at a localized position of the water hole was repeatedly tested positive for M. ulcerans-specific DNA sequences over a period of more than 2 years after all local BU cases had been treated [45], suggesting that M. ulcerans has adapted to survive in a restricted niche environment. Several studies have indicated that M. ulcerans is unlikely to be free-living and may persist in the environment as a commensal, associated with another protective organism such as amoeboid protozoa or inhabitants of biofilms in stagnant water bodies [46][47][48]. Characteristic genome signatures of M. ulcerans including (1) the acquisition of foreign DNA such as pMUM, IS2404, and IS2606, (2) the proliferation of IS elements, (3) extensive gene loss through pseudogene formation, and (4) genome downsizing, provide further evidence that M. ulcerans is adapting to a new, probably relatively protected and stable ecological niche [11•, 49, 50].…”

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice

“…ulcerans have been shown to survive in free living amoeba [27]. Studies by Gryseels et al [22] and Amissah et al [28] have also confirmed that M . ulcerans can survive in Acanthamoeba spps.…”