Single nucleotide polymorphism typing of Mycobacterium ulcerans reveals focal transmission of buruli ulcer in a highly endemic region of Ghana

Abstract

Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted.

Figure 1. SNP typing analysis of African M. ulcerans isolates.

A M. ulcerans isolates from the Densu river basin of Ghana were analyzed at 65 SNP loci (Primer IDs) by real-time PCRs. Base exchanges relative to the reference sequence of strain Agy99 were registered as 1 (grey). Allele matches with Agy99 were recorded as 0 (white). 5 haplotypes in addition to haplotype 1 (Agy99) could be distinguished on the basis of 14 SNP loci. B SNP typing results of strains from a second BU endemic area of Ghana as well as from additional African countries carried out with the set of SNP assays developed by whole genome sequencing of Ghanaian isolates. AW: Amansie West; Ga: strains from the Densu river basin; IC: Ivory Coast; T: Togo; B: Benin; C: Democratic Republic of Congo; A: Angola.

Figure 2. Geographical distribution of African M. ulcerans clades.

Map of West-Africa, showing the distribution and SNP haplotypes of three African M. ulcerans clades. Clade 1: yellow; clade 2: green; clade 3: blue. AW: Amansie West; Ga: strains from the Densu river basin; IC: Ivory Coast; T: Togo; B: Benin; C: Democratic Republic of Congo; A: Angola. A neighbor-joining tree shows sub-grouping of detected haplotypes from the Densu river basin together with the only strain from Togo into clade 1, strains from AW together with strain Agy99 and strain 1 from the Ivory Coast into clade 2 and all other strains from additional African countries into clade 3 (scale: number of differences at the SNP loci tested).

Figure 3. Neighbor-joining tree of 75 Ghanaian M. ulcerans isolates.

75 M. ulcerans isolates were aligned based on their SNP type (scale: number of differences at the SNP loci tested). HT  =  haplotype.

Figure 4. Geographical distribution of M. ulcerans haplotypes.

Map of the Densu river basin, showing the homes of patients from whom the strains have been isolated between 2001 and 2006 (colored dots). Haplotypes 2 (black), 3 (white), 4 (yellow), 6 (purple), 7 (dark blue), 8 (light blue), 9 (dark green), 10 (light green) are unevenly distributed, whereas haplotype 5 (red) co-localizes with all other haplotypes. The background map was created using elevation data from the Shuttle Radar Topography Mission (SRTM). Water bodies were classified using optical data from Landsat ETM and radar data from TerraSAR-X.