Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

Abstract

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Fig. 1

Correlation between bacillary index and successful sequencing. The content of M. leprae DNA in sequencing libraries derived from human skin biopsies was determined and found to be proportional to the bacillary index (not available for all samples). Empty circles are samples that were not included in the study due to insufficient genome coverage. Sample count and sequencing success rates are given at the top of each category

Fig. 2

Geographic distribution of the M. leprae samples used in this study. World map shows the number of registered cases of leprosy per 10,000 population (prevalence rates) in 2015 as reported by the World Health Organization (http://apps.who.int/neglected_diseases/ntddata/leprosy/leprosy.html). Blue numbers indicate ancient M. leprae strains

Fig. 3

Phylogeny of M. leprae. a Maximum parsimony tree of 154 genomes of M. leprae. The tree is drawn to scale, with branch lengths representing number of substitutions. M. lepromatosis was used as outgroup. Bootstrap values (500 replicates) are shown next to the branches. Dots indicate protein-changing mutations in the corresponding gene as given in Table 1. b Bayesian phylogenetic tree of 146 genomes of M. leprae calculated with BEAST 2.4.4. Hypermutated samples with mutations in the nth gene were excluded from the analysis. The tree is drawn to scale, with branch lengths representing years of age. Samples were binned according to geographic origin as given in the legend. Posterior probabilities for each node are shown in gray. Location probabilities of nodes were inferred by the Discrete Phylogeny model

Fig. 4

Mutations of M. leprae genes associated with antimicrobial resistance. Triangles point to the location of the mutation in the protein. Black triangles indicate known resistance-conferring mutations identified in this study that are situated in the drug resistance determining regions (DRDR): D dapsone, Q quinolone, R rifampicin. Orange border means the mutation was found to be homoplasic. Triangle size reflects the number of isolates from this study harboring the mutation, ranging from 1 to 17. Frameshifts and premature stop codons are in turquoise. Substitutions predicted to have an impact on the biological function of the protein are in bold. Proteins are drawn to scale

Fig. 5

Timeline of the leprosy treatment and emergence of drug resistance in the XDR strains. Mutated genes conferring resistance to the corresponding drugs are shown in red. Arrows span from the onset of disease to the end of treatment. Horizontal lines show the period when a drug was given. Dotted lines mean irregular treatment. CAM chloramphenicol, CLO clofazimine, DDS dapsone, DPT thiambutosine (diphenylthiourea), ETO ethionamide, INH isoniazid, KAN kanamycin, LVX levofloxacin, MIN minocycline, OFX ofloxacin, PTO protionamide, RIF rifampicin, SMP sulfamethoxypyridazine, SPX sparfloxacin, STR streptomycin, TZA thiozamin