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

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