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.

Insertion Sequence Element Single Nucleotide Polymorphism Typing Provides Insights into the Population Structure and Evolution of Mycobacterium ulcerans across Africa

“…This resulted in 28 ISE-SNP types, of which 23 were found on the African continent. Sixteen of these were newly identified types, while the other seven corresponded to the ISE-SNP types described by Käser et al (28). The Papua New Guinean ISE-SNP type 28, used in the phylogenetic analyses as an outgroup, was also a novel type.…”

“…Isolates ITM_5150, ITM_5151 ITM_940511, ITM_940512, ITM_960658, ITM_940662, ITM_970359, and ITM_970680 were included in the panel to validate the redesigned assays. These isolates were also included in the panel of Käser et al (28) and gave identical genotypes as with the redesigned assays. As our collection of African M. ulcerans isolates did not represent all countries and their different regions where BU is endemic to the same extent, owing to the low sensitivity of culture, we fine-tuned the technique for application directly on clinical specimens by adjusting individual PCR component concentrations and optimizing the thermal PCR profile.…”

“…The PCR design was challenging, as only haplotypespecific copies of IS2404 (MUL_2990 and MUL_3871) were to be amplified and because the relevant regions were of considerable size (1,730 bp for RD1 and 1,905 bp for RD12). Although we reduced the size of the amplicons in both assays (by 299 bp for RD1 and by 48 bp for RD12), they still contained all the variable nucleotide positions described by Käser et al (28). PCR mixtures contained 1.0 U of HotStarTaq polymerase (Qiagen, Hilden, Germany), 3.0 l 10ϫ PCR buffer, 6.0 l Qsolution, 1.5 mM MgCl 2 , 200 M each deoxynucleoside triphosphate, and 0.5 M each primer in a total volume of 30 l. PCRs were carried out on a Biometra TProfessional thermal cycler under the following conditions: an initial denaturation step of 15 min at 95°C, followed by 40 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 65°C (RD1) or 70°C (RD12), and elongation for 2 min at 72°C, and ending with a final elongation step of 10 min at 72°C.…”

“…Sequences were trimmed to an equal length, and all currently known ISE-SNP types (including 5 ISE-SNP types from Papua New Guinea, Australia, and Malaysia) (28) were added to this data set. We mapped SNPs according to the Agy99 bacterial reference chromosome (GenBank accession no.…”

Lack of Insertional-Deletional Polymorphism in a Collection of Mycobacterium ulcerans Isolates from Ghanaian Buruli Ulcer Patients

“…We conclude that on a regional geographic scale, differentiation within these largely monomorphic local clones might be possible through identification of SNPs. First evidence for a SNP-based genetic discrimination between and within African countries has already been provided (31).…”