Insertion sequence element single nucleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa

Abstract

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

FIG 1

Sequence variation in two haplotype-specific concatenated IS2404 elements: MUL_2990 (RD1) and MUL_3871 (RD12). Only variable nucleotides in the aligned sequences are shown for all 28 ISE-SNP types. SNP position numbers are given according to the scheme described by Käser et al. (28), with position 1 corresponding to position 3313231 in RD1 and 1498 to position 4326896 in RD12, according to the Agy99 bacterial reference chromosome.

FIG 2

(A) The geographical distribution of African M. ulcerans. The location of residence of the individual BU patients at the time of clinical visit was retrospectively correlated with the ISE-SNP typing results. ISE-SNP types represented by only one clinical isolate or specimen are depicted as numbers, while more common ISE-SNP types are color coded. (B) The uneven distribution of ISE-SNP types over the different greater hydrological drainage basins of West Africa.

FIG 3

(A) Neighbor-joining tree showing the phylogenetic relationships between the 28 currently known ISE-SNP types of M. ulcerans, with haplotype ISE-SNP 28 from Papua New Guinea as an outgroup. Bootstrap values (if >70%) for the neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) analyses are indicated at the nodes as NJ/ML/MP. ISE-SNP types belonging to the pan-African clade and the Gabonese/Cameroonian clade are highlighted in gray and red, respectively. (B) Geographical distribution of the pan-African clade and the Gabonese/Cameroonian clade. The location of residence of the individual BU patients at the time of clinical visit was retrospectively correlated with the ISE-SNP typing results.

FIG 4

Phylogenetic network showing patterns of descent among the 28 currently know ISE-SNP types of M. ulcerans in relation to their geographic origin. The network was derived by using the median joining algorithm after processing the data with the reduced median method as implemented within Network v.4.6.1.0. Each circle represents a unique ISE-SNP type, and the size of the circle is proportional to the number of individuals sharing that type. Numbers in boxes represent the number of mutational steps (if not given, then there was a single mutational step). Positions at which mutations occurred are given in Fig. 1. Color codes represent the country of origin, as shown in the key.