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

Abstract

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 PPE (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 PPE 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 PPE gene PCR sequencing in 15/21 (71.4%) swabs collected from suspected Buruli ulcer lesions in Côte d'Ivoire exhibited positive M. ulcerans IS2404 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. PPE 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. IMPORTANCE We describe a new targeted sequencing approach that characterizes the PPE 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.

Keywords: Buruli ulcer; Mycobacterium marinum; Mycobacterium ulcerans; PPE gene; genotyping; whole-genome sequencing.

FIG 1

Phylogenetic tree of 385 strains of the M. marinum/M. ulcerans complex showing the different detailed taxa. An unclassified strain is represented by a blue star. FastTree v2.1.11 on the NGphylogeny website was used to create a phylogenetic tree using default parameters.

FIG 2

Phylogenetic tree of the PPE (proline-proline-glutamate; GenBank PPE gene ID for Mycobacterium ulcerans Agy99: ABL03116.1) gene extracted from 384 genomes of strains of M. ulcerans and M. marinum clades showing the different characterized taxa. An unclassified strain is represented by a blue star, and the SRR6346364 isolate is represented by a red star. FastTree v2.1.11 on the NGphylogeny website was used to create a phylogenetic tree using default parameters.

FIG 3

Alignment profile of the extracted PPE (proline-proline-glutamate; GenBank PPE gene ID for Mycobacterium ulcerans Agy99: ABL03116.1) gene from genomes showing the different sizes of this gene based on taxa of M. ulcerans and M. marinum.

FIG 4

Partial PPE (proline-proline-glutamate; GenBank PPE gene ID for Mycobacterium ulcerans Agy99: ABL03116.1) gene sequence-based phylogenetic tree of 9 IHU clinical strains (IHU 5 to 13) and 15 clinical samples from Côte d'Ivoire (CI) with the extracted PPE gene from genomes of several strains as the reference for each taxon; background label yellow: IHU5 = CSURQ0185, IHU6 = CSURP7741, IHU7 = CSURP9954, IHU8 = CSURP9950, IHU9 = M. marinum B, IHU10 = CSURP9949, IHU11 = CSURP9951, IHU12 = CSURP9952, S13 = CSURP9953; background label red: CI. FastTree v2.1.11 on the NGphylogeny website was used to create a phylogenetic tree using defaults parameters.