The microbiome and host immune system maintain a dynamic homeostatic equilibrium at the skin interface. Prior studies have shown that the skin microbiome is profoundly altered in immunodeficient conditions. Patients with idiopathic CD4 lymphopenia, a rare clinical syndrome with an obscure cause, and people living with HIV are 2 etiologically distinct groups of individuals with CD4 T-cell lymphopenia. We conducted shotgun metagenomic sequencing, metagenome assembly, and read-based mapping to examine the multi-kingdom taxonomic diversity of skin microbiomes in patients with idiopathic CD4 lymphopenia and people living with HIV who were followed longitudinally before and after initiation of antiretroviral therapy. Compared with healthy individuals, the skin microbiomes of patients with idiopathic CD4 lymphopenia and antiretroviral therapy-naïve people living with HIV showed greater inter-individual variation and higher relative abundances of eukaryotic viruses. Both patient groups carried pathogenic microbes, including high-oncogenic-risk human papillomaviruses and dermatophytes such as Trichophyton rubrum, which were rarely seen in healthy controls. In people living with HIV, high-oncogenic-risk human papillomaviruses persisted after 2 months of antiretroviral therapy but were mostly cleared after 14 months. The loss of peripheral blood CD4 T-cells was associated with shifts in the skin microbiome and a relative expansion of pathogenic microbes. Investigating microbiome dynamics during immunodeficiency and subsequent immune reconstitution provides additional insights into host-microbial interactions.

Candida auris is an opportunistic, multidrug-resistant yeast with a high capacity for human skin colonization in healthcare settings, which can lead to subsequent infections with high mortality rates. Despite the recent emergence of at least four distinct clades at the global scale, little remains known about how C. auris is so adept at growing on skin and the key genes and pathways it utilizes to metabolize the scarce nutrients available. Here, we identify the roles that conventional and alternative carbon metabolism genes and metabolic pathways have in facilitating C. auris growth through laboratory-based experiments and bioinformatics analyses. In artificial skin-like media, all four clades of C. auris were more capable of growing than Candida albicans SC5314, a clinically relevant counterpart. By investigating the differential regulation of C. auris when growing in skin-like media as compared to rich fungal media, we uncovered hundreds of genes in multiple metabolic pathways. To further test the mechanisms of these metabolic pathways, we deleted several non-essential gene candidates including FOX2 (B9J08_002847), CAT2 (B9J08_000010), and ICL1 (B9J08_003374). The mutant strains all exhibited abrogated growth in skin-like media and demonstrated nutrient preferences that differed from the wild type. Thus, we propose a model of how C. auris has the capacity to metabolize nutrients that are available on skin by optimizing its metabolic profile. Targeting these metabolic pathways to mitigate C. auris growth on skin is a potential avenue to explore in controlling the spread of this emerging human fungal pathogen.IMPORTANCECandida auris is an emerging fungal pathogen with human skin as its primary site of colonization and subsequent transmission. Here, we show the importance of conventional and alternative carbon metabolism for the ability of C. auris to grow in artificial skin-like media. This knowledge provides a better understanding of C. auris metabolism and sheds light on genes and pathways that could be targeted to interfere with persistent skin colonization.

Abstract Antimicrobial resistance (AMR) is a health threat disproportionately affecting nursing home (NH) residents. Surveillance and infection control in NHs are restricted to nares or perirectal cultures, overlooking skin colonization and multidrug-resistant organisms (MDROs) not recovered by selective media. Here, within the PROTECT trial NCT03118232, we show, that NH residents’ skin serves as a reservoir of transmissible MDROs. We analyzed 207 groin and axilla swabs from 38 residents across 15 California NHs using metagenomics, culturing, and genome sequencing. Culture detected MDROs in 10 of 38 residents (26.3%), including 4 (10.5%) with ESBL-producing Escherichia coli sequence type (ST)131/ST648 and 7 (18.4%) with methicillin-resistant Staphylococcus aureus . Skin microbiome analysis by metagenome-assembled genomes identified broader MDRO colonization, including 27 (71.1%) with E. coli ST93, 14 (36.8%) with Staphylococcus epidermidis ST2, 16 (42.1%) with Proteus mirabilis , 7 (18.4%) with Providencia stuartii , 7 (18.4%) with Enterococcus faecalis , and 5 (13.2%) with Pseudomonas aeruginosa . Colonization persisted after bathing. Clonal E. coli ST93 was shared by 27 residents across 9 facilities, and 5 resident pairs carried clonally related strains of ≥2 MDRO species, suggesting polymicrobial transmission. We confirmed skin as a reservoir of MDROs, utilizing metagenomics to detect colonization and transmission pathways, supporting AMR surveillance in long-term care.

Cytotoxic T-lymphocyte-associated protein 4 deficiency (CTLA4-D) is an inborn error of immunity (IEI) caused by heterozygous mutations, and characterized by immune cell infiltration into the gut and other organs, leading to intestinal disease, immune dysregulation and autoimmunity. While regulatory T-cell dysfunction remains central to CTLA4-D immunopathogenesis, mechanisms driving disease severity and intestinal pathology are unknown but likely involve intestinal dysbiosis. We determined whether the intestinal microbiome and metabolome could distinguish individuals with severe CTLA4-D and identify biomarkers of disease severity. The genera Veillonella and Streptococcus emerged as biomarkers that distinguished CTLA4-D from healthy cohorts from both the National Institutes of Health (NIH) Clinical Center, USA (NIH; CTLA-D, n = 32; healthy controls, n = 16), and a geographically distinct cohort from the Center for Chronic Immunodeficiency (CCI) of the Medical Center - University of Freiburg, Germany (CCI; CTLA4-D, n = 25; healthy controls, n = 24). Since IEIs in general may be associated with perturbations of the microbiota, a disease control cohort of individuals with common variable immunodeficiency (CVID, n = 20) was included to evaluate for a CTLA4-D-specific microbial signature. Despite common IEI-associated microbiome changes, the two bacterial genera retained their specificity as biomarkers for CTLA4-D. We further identified intestinal microbiome and metabolomic signatures that distinguished patients with CTLA4-D having severe vs. mild disease. Microbiome changes were associated with distinct stool metabolomic profiles and predicted changes in metabolic pathways. These differences were impacted by the presence of gastrointestinal manifestations and were partially reversed by treatment with abatacept and/or sirolimus. Loss of intestinal microbial diversity and dysbiosis causing metabolomic changes was observed in CTLA4-D. Albeit some of these features were shared with CVID, the distinct changes associated with CTLA4-D highlight the fact that IEI-associated microbiome changes likely reflect the underlying immune dysregulation. Identified candidate intestinal microbial and metabolic biomarkers distinguishing individuals with CTLA4-D based on severity should be studied prospectively to determine their predictive value, and investigated as potential therapeutic ta.

The human skin microbiome is intricately intertwined with host immunity. While studies have elucidated microbial influences on immunity, understanding how immune alterations modulate this equilibrium remains limited. We investigated the dual impact of immune deficiency and hematopoietic stem cell transplantation (HSCT) on the skin microbiome in 24 patients with dedicator of cytokinesis 8 (DOCK8) deficiency, a rare inborn error of immunity. Analyzing 590 metagenomic and 534 16S rDNA sequencing samples across eight skin sites, we observed disrupted microbiota pre-HSCT (median eukaryotic viruses 67.6% vs. 0.04% in controls), with extremely diverse human papillomaviruses (HPVs) and polyomaviruses-including oncogenic viruses. Specific bacterial species markedly changed, including decreased Staphylococcus aureus post-HSCT. DNA eukaryotic viruses dramatically decreased (79.7% ± 28.3% to 4.9% ± 8.6%; p < 0.01) 12 months post-HSCT. Recovered microbial communities remained relatively stable through 1-year follow-up with clearance of oncogenic HPV and no convergence with transplant donors. These results highlight the immune system's critical role in restoring microbial balance and skin health.

Antimicrobial resistance (AMR) is a global public health threat that disproportionately affects vulnerable populations, including nursing home (NH) residents. Surveillance and control in NHs are resource-limited and typically restricted to perirectal cultures, overlooking both skin colonization and multidrug-resistant organisms (MDROs) not recovered by selective media. Here we show, within the cluster-randomized Project PROTECT trial ( NCT03118232 ), that residents' skin serves as a major reservoir of transmissible MDROs. We analyzed 207 groin and axilla swabs from 38 residents across 15 California NHs using shotgun metagenomics, selective culturing, and isolate genome sequencing. Culture detected MDROs in 10 of 38 residents (26.3%), including extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli ST131/ST648 in 4 (10.5%) and methicillin-resistant Staphylococcus aureus in 7 (18.4%). In contrast, metagenome-assembled genomes identified broader MDRO colonization, including multidrug-resistant E. coli ST93 in 27 residents (71.1%), methicillin-resistant Staphylococcus epidermidis ST2 in 14 (36.8%), Proteus mirabilis in 16 (42.1%), Providencia stuartii in 7 (18.4%), Enterococcus faecalis in 7 (18.4%), and Pseudomonas aeruginosa in 5 (13.2%). Colonization persisted after bathing. Clonal E. coli ST93 (<=30 SNPs) was shared by 27 residents across 9 facilities, and 5 resident pairs (13.2%) carried clonally related strains of >=2 MDRO species, consistent with polymicrobial transmission. Our findings demonstrated the skin as a persistent reservoir of MDROs and the importance of metagenomic surveillance to uncover hidden colonization and transmission pathways, underscoring the need to expand AMR monitoring in long-term care.

Recent outbreaks of multidrug-resistant fungi infecting human skin emphasize the importance of understanding fungal pathophysiology and spread. In efforts to address health concerns with various Indigenous Peninsular Malaysians (Orang Asli [OA]), tinea imbricata-a Trichophyton concentricum fungal skin infection-emerged as a particular concern. We investigated the etiology and transmission of tinea imbricata by culturing, testing antifungal sensitivities, and sequencing T. concentricum isolates in remote OA villages. Among regionally conserved isolates, we identified the emergence of terbinafine-resistant T. concentricum microbiologically and genomically. Investigating the skin microbiomes of 82 Indigenous OA, we found unique microbiota and lower relative abundances of bacterial commensals (Cutibacterium acnes, Staphylococcus epidermidis) among OA versus Malaysian and US urban populations, emphasizing how understudied populations provide unprecedented knowledge on host-microbiome co-evolution. These findings provide valuable insights into clinical, microbiological, and genomic features of chronic fungal skin infections, offering the potential to inform strategies to address drug resistance and effective therapy.

Antimicrobial resistance is a public health threat associated with increased morbidity, mortality and financial burden in nursing homes and other healthcare settings1. Residents of nursing homes are at increased risk of pathogen colonization and infection owing to antimicrobial-resistant bacteria and fungi. Nursing homes act as reservoirs, amplifiers and disseminators of antimicrobial resistance in healthcare networks and across geographical regions2. Here we investigate the genomic epidemiology of the emerging, multidrug-resistant human fungal pathogen Candida auris in a ventilator-capable nursing home. Coupling strain-resolved metagenomics with isolate sequencing, we report skin colonization and clonal spread of C. auris on the skin of nursing home residents and throughout a metropolitan region. We also report that most Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Entobacter species (ESKAPE) pathogens and other high-priority pathogens (including Escherichia coli, Providencia stuartii, Proteus mirabilis and Morganella morganii) are shared in a nursing home. Integrating microbiome and clinical microbiology data, we detect carbapenemase genes at multiple skin sites on residents identified as carriers of these genes. We analyse publicly available shotgun metagenomic samples (stool and skin) collected from residents with varying medical conditions living in seven other nursing homes and provide additional evidence of previously unappreciated bacterial strain sharing. Taken together, our data suggest that skin is a reservoir for colonization by C. auris and ESKAPE pathogens and their associated antimicrobial-resistance genes.

We report a case of Acanthamoeba infection in an HCT recipient with steroid-refractory GVHD. We highlight the multiple challenges that free-living ameba infections present to the clinician, the clinical laboratory, transplant infectious disease for review, hospital epidemiology if nosocomial transmission is considered, and public health officials, as exposure source identification can be a significant challenge. Transplant physicians should include Acanthamoeba infections in their differential diagnosis of a patient with skin, sinus, lung, and/or brain involvement.

ABSTRACT The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8 + T cell response pre-emptively, in the absence of an infection 1 . However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel β-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high titers of antibody under conditions of physiologic colonization, including a robust IgA response in the nasal mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

Corynebacterium species are globally ubiquitous in human nasal microbiota across the lifespan. Moreover, nasal microbiota profiles typified by higher relative abundances of Corynebacterium are often positively associated with health. Among the most common human nasal Corynebacterium species are C. propinquum, C. pseudodiphtheriticum, C. accolens, and C. tuberculostearicum. To gain insight into the functions of these four species, we identified genomic, phylogenomic, and pangenomic properties and estimated the metabolic capabilities of 87 distinct human nasal Corynebacterium strain genomes: 31 from Botswana and 56 from the United States. C. pseudodiphtheriticum had geographically distinct clades consistent with localized strain circulation, whereas some strains from the other species had wide geographic distribution spanning Africa and North America. All species had similar genomic and pangenomic structures. Gene clusters assigned to all COG metabolic categories were overrepresented in the persistent versus accessory genome of each species indicating limited strain-level variability in metabolic capacity. Based on prevalence data, at least two Corynebacterium species likely coexist in the nasal microbiota of 82% of adults. So, it was surprising that core metabolic capabilities were highly conserved among the four species indicating limited species-level metabolic variation. Strikingly, strains in the U.S. clade of C. pseudodiphtheriticum lacked genes for assimilatory sulfate reduction present in most of the strains in the Botswana clade and in the other studied species, indicating a recent, geographically related loss of assimilatory sulfate reduction. Overall, the minimal species and strain variability in metabolic capacity implies coexisting strains might have limited ability to occupy distinct metabolic niches. IMPORTANCE:Pangenomic analysis with estimation of functional capabilities facilitates our understanding of the full biologic diversity of bacterial species. We performed systematic genomic, phylogenomic, and pangenomic analyses with qualitative estimation of the metabolic capabilities of four common human nasal Corynebacterium species, along with focused experimental validations, generating a foundational resource. The prevalence of each species in human nasal microbiota is consistent with the common coexistence of at least two species. We identified a notably high level of metabolic conservation within and among species indicating limited options for species to occupy distinct metabolic niches, highlighting the importance of investigating interactions among nasal Corynebacterium species. Comparing strains from two continents, C. pseudodiphtheriticum had restricted geographic strain distribution characterized by an evolutionarily recent loss of assimilatory sulfate reduction in U.S. strains. Our findings contribute to understanding the functions of Corynebacterium within human nasal microbiota and to evaluating their potential for future use as biotherapeutics.

Strains of two novel Corynebacterium species were cultured from samples of human nostrils and skin collected in the United States and Botswana. These strains demonstrated growth on Columbia Colistin-Nalidixic Acid agar with 5% sheep blood and in liquid media (brain heart infusion and tryptic soy broth) supplemented with Tween 80, a source of the fatty acid oleic acid. Cells were Gram-positive, non-spore-forming, non-motile bacilli that showed catalase but not oxidase activity. Major fatty acids in both of these species were 18:1 ω9c (oleic acid), 16:0 (palmitic acid), and 18:0 (stearic acid). Analysis of the 16S ribosomal RNA gene sequences identified these strains as belonging to the genus Corynebacterium (family Corynebacteriaceae). Whole-genome sequencing revealed that these strains formed distinct branches on a phylogenomic tree, with C. tuberculostearicum being the closest relative but with average nucleotide identities of < 95% relative to all previously described species. These results indicate that these strains represent novel species of Corynebacterium, for which we propose the names Corynebacterium hallux sp. nov., with the type strain CTNIH22T (=ATCC TSD-435T=DSM 117774T), and Corynebacterium nasorum sp. nov., with the type strain KPL3804T (=ATCC TSD-439T=DSM 117767T). We also describe the characteristics of two strains isolated from human nasal passages that are members of the recently named species Corynebacterium yonathiae.

The draft genome of Mucor velutinosus NIH1002, a 2011 isolate from a case of disseminated disease, was sequenced using PacBio long-read and HiSeq short-read technologies. The genome has 43 contigs, an N50 of 2.65 Mb, and 13,295 protein-coding genes. It is the most complete M. velutinosus genome to date.

Abstract Background Widespread CHG bathing to prevent infection has raised concerns about potential skin microbiome perturbations and depletion of commensal microbiota. Methods A prospective repeated measures cross-over study in NH residents/hospital patients evaluated the impact of CHG vs routine soap bathing on the nose/skin microbiome. Participants underwent serial visits during distinct CHG and routine soap phases (Figure) involving nares, axilla, groin, and finger/hand swabs processed for MDROs, CHG concentration (skin sites only), and bacterial 16S rRNA V1-3 gene sequencing (Illumina MiSeq). Sequences were processed with DADA2 and analyzed with PhyloSeq and Vegan packages in R to calculate microbial diversity and composition. Results We enrolled 30 participants (20 NH, 10 hospital). Mean age was 62y, 52% female. Mean CHG concentration was 1736 µg/mL (range: 0-20000) during CHG visits vs 17.5 µg/mL (range: 0-1250) during routine soap visits. MDRO prevalence was lower during CHG visits (NH: 19%, hospital: 9%) vs routine soap visits (NH: 47%, hospital: 23%). 555 samples from 30 participants were adequate for microbiome analyses. CHG did not affect overall alpha diversity of microbial communities on skin (Figure), but proteobacteria were notably higher in NH vs hospital participants (P< 0.001). Because gram-negative bacteria often exhibit higher MICs to CHG than gram-positive bacteria, we assessed whether CHG concentration in NHs was associated with greater relative abundance of proteobacteria using generalized linear mixed models clustered by person. In NHs, CHG concentration was not associated with relative abundance of skin proteobacteria, although body mass index ≥30 (14% higher abundance, P=0.01) and stool/urine incontinence were (13% higher, P=0.01). Compared to the axilla, the groin had 35% higher relative abundance of proteobacteria (P< 0.001); fingers/hand samples had 9% lower (P=0.01). Nose/Skin Taxonomic Diversity Not Impacted by CHG Bathing Timeline and alpha diversity using the Shannon Diversity Index of axilla, groin, finger/hand, and nares samples during CHG versus routine soap bathing phases. Each dot represents a sample. There were no significant differences in alpha diversity between CHG and routine soap phases. Conclusion CHG bathing reduced MDRO prevalence but did not impact skin or nasal microbial alpha diversity in hospital or NH participants. Overall, NH residents had notably higher relative abundance of skin proteobacteria vs hospital patients. This finding was not associated with CHG concentration. Rather, proteobacteria appeared enriched in NH residents with obesity or incontinence. Disclosures Gabrielle Gussin, MS, Medline Industries, Inc: Conducted studies where participating hospitals/nursing homes received cleaning & antiseptic product|Xttrium Laboratories: Conducted studies where participating hospitals & nursing homes received antiseptic bathing product Raveena D. Singh, MA, Medline Industries, Inc: Conducted studies where participating hospitals/nursing homes received cleaning & antiseptic product|Xttrium Laboratories: Conducted studies where participating hospitals & nursing homes received antiseptic bathing product Raheeb Saavedra, AS, Medline Industries, Inc: Conducted studies where participating hospitals/nursing homes received cleaning & antiseptic product|Xttrium Laboratories: Conducted studies where participating hospitals & nursing homes received antiseptic bathing product Connie Nguyen, n/a, Xttrium Laboratories: Conducted studies where participating hospitals & nursing homes received antiseptic bathing product Robert Pedroza, BS, Medline Industries, Inc: Conducted studies where participating hospitals/nursing homes received cleaning & antiseptic product Chase Berman, BS, Medline Industries, Inc: Conducted studies where participating hospitals/nursing homes received cleaning & antiseptic product Susan S. Huang, MD MPH, Medline Industries, Inc: Conducted studies whereby participating nursing homes and hospital patients received cleaning & antiseptic products|Xttrium Laboratories: Conducted studies where participating nursing homes and hospital patients received antiseptic products

Human skin is stably colonized by a distinct microbiota that functions together with epidermal cells to maintain a protective physical barrier. Staphylococcus , a prominent genus of the skin microbiota, participates in colonization resistance, tissue repair, and host immune regulation in strain-specific manners. To unlock the potential of engineering skin microbial communities, we aim to characterize the diversity of this genus within the context of the skin environment. We reanalyzed an extant 16S rRNA amplicon dataset obtained from distinct body sites of healthy volunteers, providing a detailed biogeographic depiction of staphylococcal species that colonize our skin. S. epidermidis , S. capitis, and S. hominis were the most abundant staphylococcal species present in all volunteers and were detected at all body sites. Pan-genome analysis of isolates from these three species revealed that the genus-core was dominated by central metabolism genes. Species-restricted-core genes encoded known host colonization functions. The majority (~68%) of genes were detected only in a fraction of isolate genomes, underscoring the immense strain-specific gene diversity. Conspecific genomes grouped into phylogenetic clades, exhibiting body site preference. Each clade was enriched for distinct gene sets that are potentially involved in site tropism. Finally, we conducted gene expression studies of select isolates showing variable growth phenotypes in skin-like medium. In vitro expression revealed extensive intra- and inter-species gene expression variation, substantially expanding the functional diversification within each species. Our study provides an important resource for future ecological and translational studies to examine the role of shared and strain-specific staphylococcal genes within the skin environment.

ABSTRACT Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A & B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates 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 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species. IMPORTANCE Amplicon sequencing data combined with isolate whole genome sequencing has expanded our understanding of Corynebacterium on the skin. Human skin is characterized by a diverse collection of Corynebacterium species but C. tuberculostearicum predominates many 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 which we are calling C. hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomics and growth in skin-like media. This isolate collection and high-quality genome resource sets the stage for developing engineered strains for both basic and translational clinical studies. Microbiomes are shaped by taxa that are both characteristic to those sites and functionally important to that community. The genus Corynebacterium is one such taxa for the human skin and nares. Foundational studies using 16S rRNA gene sequencing and shotgun metagenomics by our lab (1, 2) and others (3) have established Corynebacterium as common members of the skin microbiome. While Corynebacterium have been positively correlated with the resolution of dysbiosis associated with eczema flares (4), the importance of the Corynebacterium spp. is less defined for skin disease severity in primary immune deficient patients (5, 6). Corynebacterium spp. are predominant members of the human aerodigestive tract microbiome (nares, oral cavity and respiratory tract) (3) and participate in microbe-microbe interactions with members of nasal microbiome (7, 8). Corynebacterium have been shown to engage with the host immune system, specifically C. accolens -promoted IL23-dependent inflammation in mice on a high-fat diet (9). C. bovis and C. mastiditis have been shown to predominate the microbiome of a ADAM10-deficient mouse model (10) as well as an ADAM17-deficient mouse model of eczema (11). Finally, C. tuberculostearicum has been shown to induce inflammation in human epidermal keratinocyte cell cultures (12). These studies establish Corynebacterium spp. as key members of the skin microbiome capable of both microbe-microbe and microbe-host interactions. A critical resource for understanding the biology of Corynebacterium on the skin is a robust collection of complete reference genomes, including isolates collected from a variety of individuals and body sites. Previously published genome collections from skin- or nares-resident species include Staphylococcus epidermidis (13), Cutibacterium acnes (14) and the recent comparative analysis of Dolosigranulum pigrum (15). Of note, while emerging bioinformatic methods and pipelines are now being employed to extract nearly-complete genomes (MAGs) from metagenomic assemblies of skin samples (16), MAGs are not yet a substitute for genomes from cultured isolates to understand strain level or pangenomic diversity. In addition to functional prediction, comparative genomics is increasingly being used to augment conventional microbiological methods to define or redefine taxonomic boundaries (17, 18), as well as describe the full extent of diversity within these boundaries (19). A pangenome, which encompasses the complete set of genes present within a set of genome sequences, enables the characterization of gene-level heterogeneity within a taxonomic group. The pangenome is commonly subdivided into the ‘core’ genome, referring to genes present in all strains, and the ‘accessory’ or ‘dispensable’ genome, referring to those present in only one or some isolates. (The accessory pangenome can be further subdivided to reflect a wider range of gene uniqueness, e.g. singletons.) Thorough characterization of taxa is limited by the availability of representative and high-quality genome assemblies. Unfortunately, with the exceptions of clinically relevant Corynebacterium spp. ( e.g. , C. diphtheriae , C. striatum and C. pseudotuberculosis ), the genus is inadequately sequenced, with 75% of species having fewer than six genomes. This includes common skin-associated species like C. tuberculostearicum with just five unique isolate genomes, only two of which are from skin. This work seeks first to characterize the distribution of Corynebacterium across 14 skin sites from 23 healthy volunteers. The second goal of this work focuses on what we identify as the predominant skin Corynebacterium species, C. tuberculostearicum . We have sequenced 23 distinct C. tuberculostearicum strains (n=40 genomes before dereplication), a five-fold increase in the number of publicly available, unique genomes (n=5). In addition to short-read assemblies, we generated five complete genomes which, along with the type strain (DSM44922), demonstrate that C. tuberculostearicum genomes are largely syntenic and carry a number of methylation systems as well as a CRISPR/Cas system. Genes from the C. tuberculostearicum genomes in our collection fall into 5451 gene clusters comprising the species pangenome. This expanded pangenome, as compared to existing public references, improved the mapping of C. tuberculostearicum metagenomic reads from unrelated healthy volunteers. In addition, we have identified a distinct C. tuberculostearicum clade that is highly enriched on the feet that may represent a new species, tentatively designated Corynebacterium hallux .

Atopic dermatitis (AD) is a multifactorial, chronic relapsing disease associated with genetic and environ-mental factors. Among skin microbes, Staphylococcus aureus and Staphylococcus epidermidis are associ-ated with AD, but how genetic variability and staphylococcal strains shape the disease remains unclear. We investigated the skin microbiome of an AD cohort (n = 54) as part of a prospective natural history study using shotgun metagenomic and whole genome sequencing, which we analyzed alongside publicly available data (n = 473). AD status and global geographical regions exhibited associations with strains and genomic loci of S. aureus and S. epidermidis. In addition, antibiotic prescribing patterns and within-household transmission between siblings shaped colonizing strains. Comparative genomics determined that S. aureus AD strains were enriched in virulence factors, whereas S. epidermidis AD strains varied in genes involved in interspecies interactions and metabolism. In both species, staphylococcal interspecies genetic transfer shaped gene content. These findings reflect the staphylococcal genomic diversity and dynamics associated with AD.