Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities

Abstract

Mycetoma is a neglected tropical chronic granulomatous inflammatory disease of the skin and subcutaneous tissues. More than 70 species with a broad taxonomic diversity have been implicated as agents of mycetoma. Understanding the full range of causative organisms and their antibiotic sensitivity profiles are essential for the appropriate treatment of infections. The present study focuses on the analysis of full genome sequences and antibiotic inhibitory concentration profiles of actinomycetoma strains from patients seen at the Mycetoma Research Centre in Sudan with a view to developing rapid diagnostic tests. Seventeen pathogenic isolates obtained by surgical biopsies were sequenced using MinION and Illumina methods, and their antibiotic inhibitory concentration profiles determined. The results highlight an unexpected diversity of actinomycetoma causing pathogens, including three Streptomyces isolates assigned to species not previously associated with human actinomycetoma and one new Streptomyces species. Thus, current approaches for clinical and histopathological classification of mycetoma may need to be updated. The standard treatment for actinomycetoma is a combination of sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Most tested isolates had a high IC (inhibitory concentration) to sulfamethoxazole/trimethoprim or to amoxicillin alone. However, the addition of the β-lactamase inhibitor clavulanic acid to amoxicillin increased susceptibility, particularly for Streptomyces somaliensis and Streptomyces sudanensis. Actinomadura madurae isolates appear to have a particularly high IC under laboratory conditions, suggesting that alternative agents, such as amikacin, could be considered for more effective treatment. The results obtained will inform future diagnostic methods for the identification of actinomycetoma and treatment.

Fig 1. Extracts from the 16S rRNA phylogenies of Streptomyces (full figure in S1 Fig and https://itol.embl.de/shared/1MX60mtB0Ohk3) inferred using iqtree2 and the GTR+F+R5 model showing the placement of isolates (green dots) with related rRNA sequences from the ezBioCloud 16S rRNA database (blue), soil isolates from Sudan (yellow dots [48]) and isolates collected by Sengupta, Goodfellow and Hamid (NCBI accessions EU544231.1 to EU544241.1 from human actinomycetoma (purple) and from donkey withers (lilac).

A red dot on branches indicates ultrafast bootstrap support >95. Triangles are used to represent collapsed clades.

Fig 2

The 16S rRNA phylogeny of Actinomadura showing the placement of isolates (green) with related rRNA sequences from the ezBioCloud database (blue; pink for known pathogens). Inferred using iqtree2 and the GTR+F+R4 model. A black dot on branches indicates ultrafast bootstrap support >95. All isolates form a clade with Actinomadura madurae and Actinomadura darangshiensis, though support for this grouping is low.

Fig 3

Tree of single copy orthologs belonging to the GTDB bac120 dataset, from the genomes of Streptomyces related isolates from the Mycetoma Research Centre (green), their relatives according to ANI in GTDB (with the closest relatives indicated in blue) and genomes from type strains of species typically associated with actinomycetoma (black). A red dot on branches indicates ultrafast bootstrap support >95. Triangles are used to represent collapsed clades. The tree was inferred in iqtree2 using the C20 model. The full figure is available at https://itol.embl.de/shared/1MX60mtB0Ohk3.

Fig 4

Pairwise comparison of Average Nucleotide Identity between the genomes of Isolates from the Mycetoma Research Centre (red) and their closest relatives in GTDB (all genomes from GTDB with an ANI >90 with any single isolate genome or the genome of type strains). The heatmap is ordered based on hierarchical clustering (Ward, Euclidean distance). Black lines delineate species boundaries based on ANI > 95%. The genomes of the reference species are from type strains.

Fig 5

Tree of single copy orthologs that belong to the GTDB bac120 dataset from the genomes of Actinomadura related isolates from the Mycetoma Research Centre (green), their relatives according to ANI in GTDB (with the closest relatives indicated in blue) and genomes from type strains of species typically associated with actinomycetoma (black). Additionally, the type strain genome of any organism previously isolated from mycetoma patients and present in the GTDB is highlighted (pink). A red dot on branches indicates ultrafast bootstrap support >95. The tree was inferred in iqtree2 using the C20 model.

Fig 6. The distribution of genome sizes (in Mb) of all species within the Streptomyces genus (as defined by GTDB) for which a high quality genome is available (see materials and methods).

The mean genome size for the genus is 8.45Mb. The genomes of all S. sudanensis and S. somaliensis related isolates are amongst the smallest genomes in the genus.

Fig 7. Comparative antibiotic susceptibility profiles of actinomycetoma isolates based on a disk diffusion assay.

Halo size was recorded in cm. Yellow low IC. Blue high IC. The isolates were grouped using hierarchical clustering based on their IC profiles using the default parameters of heatmap2 in R.