Investigating the role of free-living amoebae as a reservoir for Mycobacterium ulcerans

Abstract

Background: The 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 findings: We 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/significance: This 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.

Figure 1. Distribution of the IS2404 target and intracellular mycobacteria from the sampling sites.

Figure 2. Proportions of FLA isolated from specimens, IS2404 detected in FLA cultures, mycobacteria isolated from an intracellular source over the sampling period (October 2008–July 2009) and BU notification rate until four months after the sampling period.

Error bars on the left Y-axis represent 95% confidence interval (CI) of the proportions of isolated FLA (68.3±1.66), IS2404 detected in FLA cultures (3.5±0.37) and intracellular mycobacteria (21.49±0.95) sampled monthly from environmental specimens (n = 539) for the specified period. Error bars on the right Y-axis represent the 95% CI of BU notification rate of the 5 endemic communities (0.08±0.04) with a population size of 6,296.

Figure 3. (A) Percentage of fluorescing bacteria present following growth at room temperature for 7 days in AC buffer supplemented with 150 µg/ml amikacin.

(B) Summary of flow cytometry data of A. castelanii infected with fluorescing (JKD8083) and non-fluorescing (04126204) M. ulcerans for 28 days. The mean percentage and SD of 3 biological repeats of fluorescent trophozoites at a MOI of 1 are shown. (C) Confocal microscopy demonstrating the intracellular location of M. ulcerans 3 hours post-infection in a DAPI stained trophozoite. Scale bar indicates 10 µm. (D–F) Representative FACS plots indicating forward scatter (x-axis) and side scatter (y-axis) following 7 days of co-incubation of amoebae alone (D), M. ulcerans JKD8083 (E) and M. ulcerans 04126204 (F). Lower panels show the gated trophozoites and FL1 fluorescence vs counts. Numbers refer to percentage of gated trophozoites fluorescing.

Figure 4. Transmission electron microscopy showing uninfected (A) cysts and (B) trophozoites as well as the intravacuolar location of M. ulcerans (arrows) within infected, intact trophozoites (C, D) 3 h, (E, F) 24 h, (G, H) 48 h and (I) cysts 3 weeks post infection.

(J) is a composite image showing a cyst 22 days post infection by fluorescence microscopy.