Barcoding Identifies Biclonal Mycobacterium ulcerans Buruli Ulcer, Côte d'Ivoire.

Mycobacterium ulcerans, an environmental opportunistic pathogen, causes necrotic cutaneous and subcutaneous lesions, named Buruli ulcers, in tropical countries. PCR-derived tests used to detect M. ulcerans in environmental and clinical samples do not allow one-shot detection, identification, and typing of M. ulcerans among closely related Mycobacterium marinum complex mycobacteria. We established a 385-member M. marinum/M. ulcerans complex whole-genome sequence database by assembling and annotating 341 M. marinum/M. ulcerans complex genomes and added 44 M. marinum/M. ulcerans complex whole-genome sequences already deposited in the NCBI database. Pangenome, core genome, and single-nucleotide polymorphism (SNP) distance-based comparisons sorted the 385 strains into 10 M. ulcerans taxa and 13 M. marinum taxa, correlating with the geographic origin of strains. Aligning conserved genes identified one (proline-proline-glutamate) gene sequence to be species and intraspecies specific, thereby genotyping the 23 M. marinum/M. ulcerans complex taxa. PCR sequencing of the gene correctly genotyped nine M. marinum/M. ulcerans complex isolates among one M. marinum taxon and three M. ulcerans taxa in the African taxon (T2.4). Further, successful gene PCR sequencing in 15/21 (71.4%) swabs collected from suspected Buruli ulcer lesions in Côte d'Ivoire exhibited positive M. ulcerans 2404 real-time PCR and identified the M. ulcerans T2.4.1 genotype in eight swabs and M. ulcerans T2.4.1/T2.4.2 mixed genotypes in seven swabs. gene sequencing could be used as a proxy for whole-genome sequencing for the one-shot detection, identification, and typing of clinical M. ulcerans strains, offering an unprecedented tool for identifying M. ulcerans mixed infections. We describe a new targeted sequencing approach that characterizes the gene to disclose the simultaneous presence of different variants of a single pathogenic microorganism. This approach has direct implications on the understanding of pathogen diversity and natural history and potential therapeutic implications when dealing with obligate and opportunistic pathogens, such as Mycobacterium ulcerans presented here as a prototype.