Genomic characterization of the C. tuberculostearicum species complex, a prominent member of the human skin microbiome

Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on the skin is incompletely characterized and underrepresented in public databases. We investigated the distribution of Corynebacterium species on the skin and expanded the existing genome reference catalog. We used extant V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity across human skin. Corynebacterium tuberculostearicum, recently proposed to belong to a species complex, is frequently found on human skin. We identified two distinct C. tuberculostearicum ribotypes (A and B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence: ribotype A is distributed across all skin sites, while B is found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five individuals across seven skin sites. We generated five closed C. tuberculostearicum genomes and determined that they are largely syntenic and carry a diversity of methylation patterns, plasmids, and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1,806 genes and a pangenome size of 5,451 genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from skin metagenomes. We demonstrated differential growth phenotypes of C. tuberculostearicum ribotypes A and B on rich and skin-like media, suggesting functional differences. Finally, while the genomes from this study fall within the C. tuberculostearicum species complex, we propose that ribotype B isolates constitute a putative new species.IMPORTANCE Amplicon sequencing data combined with isolate whole genome sequencing have expanded our understanding of Corynebacterium on the skin. Healthy human skin is colonized by a diverse collection of Corynebacterium species, but Corynebacterium tuberculostearicum predominates on many skin sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex, including a potentially new species we term Corynebacterium hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomic analysis, and growth in skin-like media. This isolate collection and high-quality genome resource set the stage for developing engineered strains for both basic and translational clinical studies.