Single nucleotide polymorphisms on the road to strain differentiation in Mycobacterium ulcerans

Abstract

The genomic fine-typing of strains of Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.

FIG. 1.

Sequence variation in haplotype-specific IS2404 elements. IS2404 elements that are unique for, and confined to, the shown haplotypes were amplified with site-specific primers located outside of the ISEs, and SNPs were determined by sequencing. Only sites that differ in the aligned nucleotides (European Bioinformatics Institute ClustalW2) are shown in the sequence panels (JalView, version 2.4) (9) from the 1,362-bp (RD9), 1,366-bp, and adjacent regions (RD11) and from 1,497 bp (RD1) combined with 1,822 bp (RD12) to yield 3,319 bp of concatenated chromosomal ISE fragments, including their flanking regions. SNP position numbers in RD1 and RD12 are given according to the M. ulcerans Agy99 sequence, with 1 corresponding to position 3313231 in RD1 and 1498 to position 4326896 in RD12. SNP position numbers in RD9 and RD11 are given according to the M. marinum M sequence, with 1 corresponding to position 4348015 in RD9 and 1 corresponding to position 3108032 in RD11. For the 79 sequenced M. ulcerans isolates of the African/Australian haplotype, 11 various ISE-SNP types (1 to 11) were identified, as indicated in the sequence panel. MRCA, most recent common ancestor.

FIG. 2.

Phylogenetic and epidemiological patterns of the ISE-SNP types in the classical lineage. Identified SNPs were used to create a neighbor-joining tree (using percent identity) with JalView, version 2.4 (9), for visualization of their phylogenetic relationships. Clusters were assigned ISE-SNP type numbers (1 to 11, corresponding to Fig. 1). The geographic origins of the African strains are shown on the map (created using HealthMapper, version 4.3.1, software [http://www.who.int/health_mappping/tools/healthmapper/en/index html] with the permission of the WHO Geographic Information Systems department). Frequencies of the respective ISE-SNP type per geographic sample size are indicated in brackets for each country or region. Genetic distances (legend) are reflected by the lengths of the branches. GH, Ghana; IC, Ivory Coast; DRC, Democratic Republic of Congo; AN, Angola; BEN, Benin; PNG, Papua New Guinea; AU, Australia; MAL, Malaysia.