Combined metagenomic- and culture-based approaches to investigate bacterial strain-level associations with medication-controlled mild-moderate atopic dermatitis.

Background: The skin microbiome is disrupted in atopic dermatitis (AD). Existing research focuses on moderate to severe, unmedicated disease. Objective: We sought to investigate metagenomic- and culture-based bacterial strain-level differences in mild, medicated AD and the effects these have on human keratinocytes (HKs). Methods: Skin swabs from anterior forearms were collected from 20 pediatric participants (11 participants with AD sampled at lesional and nonlesional sites and 9 age- and sex-matched controls). Participants had primarily mild to moderate AD and maintained medication use. Samples were processed for microbial metagenomic sequencing and bacterial isolation. Isolates identified as Staphylococcus aureus were tested for enterotoxin production. HK cultures were treated with cell-free conditioned media from representative Staphylococcus species to measure barrier effects. Results: Metagenomic sequencing identified significant differences in microbiome composition between AD and control groups. Differences were seen at the species and strain levels for Staphylococci, with S aureus found only in participants with AD and differences in Staphylococcus epidermidis strains between control and AD swabs. These strains showed differences in toxin gene presence, which was confirmed in vitro for S aureus enterotoxins. The strain from the participant with the most severe AD produced enterotoxin B levels more than 100-fold higher than the other strains (P < .001). Strains also displayed differential effects on HK metabolism and barrier function. Conclusions: Strain-level differences in toxin genes from Staphylococcus strains may explain varying effects on HK, with S aureus and non-aureus strains negatively affecting viability and barrier function. These differences are likely important in AD pathogenesis.

The effect of anatomic location on porcine models of burn injury and wound healing.

Porcine models are frequently used for burn healing studies; however, factors including anatomic location and lack of standardised wound methods can impact the interpretation of wound data. The objectives of this study are to examine the influence of anatomical locations on the uniformity of burn creation and healing in porcine burn models. To optimise burn parameters on dorsal and ventral surfaces, ex vivo and in situ euthanized animals were first used to examine the location-dependence of the burn depth and contact time relationship. The location-dependent healing in vivo was then examined using burn and excisional wounds at dorsal, ventral, caudal and cranial locations. Lactate dehydrogenase (LDH) and H&E were used to assess burn depth and wound re-epithelialization. We found that burn depth on the ventral skin was significantly deeper than that of the dorsal skin at identical thermal conditions. Compared with burns created ex vivo, burns created in situ immediately post-mortem were significantly deeper in the ventral location. In live animals, 2 out of 12 burn wounds were fully re-epithelialized after 14 days in contrast to complete re-epithelialization of all excisional wounds. Among the burn wounds, those at the cranial-dorsal site exhibited faster healing than at the caudal-dorsal site. This study showed that anatomical location is an important consideration for the consistency of burn depth creation and healing. These data support symmetric localization of treatment and control for comparative assessment of burn healing in porcine models to prevent misinterpretation of results and increase the translatability of findings to humans.

The porcine skin microbiome exhibits broad fungal antagonism.

The skin and its microbiome function to protect the host from pathogen colonization and environmental stressors. In this study, using the Wisconsin Miniature Swine™ model, we characterize the porcine skin fungal and bacterial microbiomes, identify bacterial isolates displaying antifungal activity, and use whole-genome sequencing to identify biosynthetic gene clusters encoding for secondary metabolites that may be responsible for the antagonistic effects on fungi. Through this comprehensive approach of paired microbiome sequencing with culturomics, we report the discovery of novel species of Corynebacterium and Rothia. Further, this study represents the first comprehensive evaluation of the porcine skin mycobiome and the evaluation of bacterial-fungal interactions on this surface. Several diverse bacterial isolates exhibit potent antifungal properties against opportunistic fungal pathogens in vitro. Genomic analysis of inhibitory species revealed a diverse repertoire of uncharacterized biosynthetic gene clusters suggesting a reservoir of novel chemical and biological diversity. Collectively, the porcine skin microbiome represents a potential unique source of novel antifungals.

Key aspects of papillomavirus infection influence the host cervicovaginal microbiome in a preclinical murine papillomavirus (MmuPV1) infection model.

Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome.IMPORTANCEHuman papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome.

Upper respiratory microbial communities of healthy populations are shaped by niche and age.

Background: Alterations in upper respiratory microbiomes have been implicated in shaping host health trajectories, including by limiting mucosal pathogen colonization. However, limited comparative studies of respiratory microbiome development and functioning across age groups have been performed. Herein, we perform shotgun metagenomic sequencing paired with pathogen inhibition assays to elucidate differences in nasal and oral microbiome composition and functioning across healthy 24-month-old infant (n=229) and adult (n=100) populations. Results: We find that beta diversity of nasal and oral microbiomes varies with age, with nasal microbiomes showing greater population-level variation compared to oral microbiomes. Infant microbiome alpha diversity was significantly lower across nasal samples and higher in oral samples, relative to adults. Accordingly, we demonstrate significant differences in genus- and species-level composition of microbiomes between sites and age groups. Antimicrobial resistome patterns likewise varied across body sites, with oral microbiomes showing higher resistance gene abundance compared to nasal microbiomes. Biosynthetic gene clusters encoding specialized metabolite production were found in higher abundance across infant oral microbiomes, relative to adults. Investigation of pathogen inhibition revealed greater inhibition of gram-negative and gram-positive bacteria by oral commensals, while nasal isolates had higher antifungal activity. Conclusions: In summary, we identify significant differences in the microbial communities inhabiting nasal and oral cavities of healthy infants relative to adults. These findings inform our understanding of the interactions impacting respiratory microbiome composition and functioning, with important implications for host health across the lifespan.

What is slough? Defining the proteomic and microbial composition of slough and its implications for wound healing.

Slough is a well-known feature of non-healing wounds. This pilot study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Ten subjects with slow-to-heal wounds and visible slough were enrolled. Aetiologies included venous stasis ulcers, post-surgical site infections and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analysed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound aetiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation and immune processes. To predict variables associated with wound healing, protein, microbial and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anaerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterise wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilised biomarker.

skDER: microbial genome dereplication approaches for comparative and metagenomic applications.

skDER (https://github.com/raufs/skDER) combines recent advances to efficiently estimate average nucleotide identity (ANI) between thousands of microbial genomes by skani1 with two low-memory methods for genomic dereplication. The first method implements a dynamic algorithm to determine a concise set of representative genomes. This approach is well-suited for selecting reference genomes to align metagenomic reads onto for tracking strain presence across related microbiome samples. This is because fewer representative genomes should alleviate the concern that reads belonging to the same strain get falsely partitioned across closely related genomes. The other method, which uses a greedy approach, is better suited for use in comparative genomics, where users might be overwhelmed with the high number of genomes available for certain taxa and aim to reduce redundancy and, therefore, computational requirements for downstream analytics. This method selects a larger number of representative genomes to comprehensively sample the pangenome space for the taxon of interest. To further aid usage for comparative genomics studies, skDER also features an option to automatically download genomes classified as a particular species or genus in the Genome Taxonomy Database2-4 and we provide precomputed representative genomes for commonly studied bacterial taxa5.

zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters.

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous or homologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of protein-encoding ortholog groups for individual genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of statistics for each inferred ortholog group. These programs are showcased through application to: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-genetic insights of two common BGCs in a fungal species, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.

The dynamic balance of the skin microbiome across the lifespan.

For decades research has centered on identifying the ideal balanced skin microbiome that prevents disease and on developing therapeutics to foster this balance. However, this single idealized balance may not exist. The skin microbiome changes across the lifespan. This is reflected in the dynamic shifts of the skin microbiome's diverse, inter-connected community of microorganisms with age. While there are core skin microbial taxa, the precise community composition for any individual person is determined by local skin physiology, genetics, microbe-host interactions, and microbe-microbe interactions. As a key interface with the environment, the skin surface and its appendages are also constantly exchanging microbes with close personal contacts and the environment. Hormone fluctuations and immune system maturation also drive age-dependent changes in skin physiology that support different microbial community structures over time. Here, we review recent insights into the factors that shape the skin microbiome throughout life. Collectively, the works summarized within this review highlight how, depending on where we are in lifespan, our skin supports robust microbial communities, while still maintaining microbial features unique to us. This review will also highlight how disruptions to this dynamic microbial balance can influence risk for dermatological diseases as well as impact lifelong health.

Sweat and Sebum Preferences of the Human Skin Microbiota.

The microorganisms inhabiting human skin must overcome numerous challenges that typically impede microbial growth, including low pH, osmotic pressure, and low nutrient availability. Yet the skin microbiota thrive on the skin and have adapted to these stressful conditions. The limited nutrients available for microbial use in this unique niche include those from host-derived sweat, sebum, and corneocytes. Here, we have developed physiologically relevant, synthetic skin-like growth media composed of compounds present in sweat and sebum. We find that skin-associated bacterial species exhibit unique growth profiles at different concentrations of artificial sweat and sebum. Most strains evaluated demonstrate a preference for high sweat concentrations, while the sebum preference is highly variable, suggesting that the capacity for sebum utilization may be a driver of the skin microbial community structure. In particular, the prominent skin commensal Staphylococcus epidermidis exhibits the strongest preference for sweat while growing equally well across sebum concentrations. Conversely, the growth of Corynebacterium kefirresidentii, another dominant skin microbiome member, is dependent on increasing concentrations of both sweat and sebum but only when sebum is available, suggesting a lipid requirement of this species. Furthermore, we observe that strains with similar growth profiles in the artificial media cluster by phylum, suggesting that phylogeny is a key factor in sweat and sebum use. Importantly, these findings provide an experimental rationale for why different skin microenvironments harbor distinct microbiome communities. In all, our study further emphasizes the importance of studying microorganisms in an ecologically relevant context, which is critical for our understanding of their physiology, ecology, and function on the skin. IMPORTANCE The human skin microbiome is adapted to survive and thrive in the harsh environment of the skin, which is low in nutrient availability. To study skin microorganisms in a system that mimics the natural skin environment, we developed and tested a physiologically relevant, synthetic skin-like growth medium that is composed of compounds found in the human skin secretions sweat and sebum. We find that most skin-associated bacterial species tested prefer high concentrations of artificial sweat but that artificial sebum concentration preference varies from species to species, suggesting that sebum utilization may be an important contributor to skin microbiome composition. This study demonstrates the utility of a skin-like growth medium, which can be applied to diverse microbiological systems, and underscores the importance of studying microorganisms in an ecologically relevant context.

Comparative Genomic and Metagenomic Investigations of the Corynebacterium tuberculostearicum Species Complex Reveals Potential Mechanisms Underlying Associations To Skin Health and Disease.

Corynebacterium are a diverse genus and dominant member of the human skin microbiome. Recently, we reported that the most prevalent Corynebacterium species found on skin, including Corynebacterium tuberculostearicum and Corynebacterium kefirresidentii, comprise a narrow species complex despite the diversity of the genus. Here, we apply high-resolution phylogenomics and comparative genomics to describe the structure of the C. tuberculostearicum species complex and highlight genetic traits which are enriched or depleted in it relative to other Corynebacterium. Through metagenomic investigations, we also find that individual species within the complex can associate with specific body sites. Finally, we discover that one species from the complex, C. kefirresidentii, increases in relative abundance during atopic dermatitis flares, and show that most genomes of this species encode a colocalized set of putative virulence genes. IMPORTANCE Corynebacterium are commonly found bacteria on the human skin. In this study, we perform comparative genomics to gain insight into genetic traits which differentiate a phylogenetically related group of Corynebacterium, the Corynebacterium tuberculostearicum species complex, that includes the most prevalent species from the genus in skin microbiomes. After resolving the presence of distinct species within the complex, we applied metagenomic analysis to uncover biogeographic associations of individual species within the complex with specific body sites and discovered that one species, commonly found in the nares of individuals, increases in abundance across multiple body sites during atopic dermatitis flares.

IWII Wound Infection in Clinical Practice consensus document: 2022 update.

ABSTRACT: Wound infection is a major challenge for clinicians globally, with accurate and timely identification of wound infection being critical to achieving clinical and cost-effective management, and promotion of healing. This paper presents an overview of the development of the International Wound Infection Institute (IWII)'s 2022 Wound Infection in Clinical Practice consensus document. The updated document summarises current evidence and provides multidisciplinary healthcare providers with effective guidance and support on terminology, paradigms related to biofilm, identification of wound infection, wound cleansing, debridement and antimicrobial stewardship. Integral to the update is revision of wound infection management strategies which are incorporated within the IWII's Wound Infection Continuum (IWII-WIC) and management plan. The aim of the 2022 IWII consensus document update was to provide an accessible and useful clinical resource in at least six languages, incorporating the latest evidence and current best practice for wound infection and prevention. Dissemination techniques for the consensus are discussed and highlighted.

Forgotten fungi: the importance of the skin mycobiome.

The mosaic ecosystems of microbes that live on our skin encompass not only bacteria but also fungi, microeukaryotes, and viruses. As the second most prevalent group, unique fungal communities are found across the dry, moist, and oily microenvironments of human skin, and alterations of these communities are largely driven by changes in skin physiology throughout an individual's lifespan. Fungi have also been associated with infection and dermatological disorders, resulting from the disrupted balance between fungal-bacterial networks on the skin. Mechanisms of colonization resistance toward fungi in the skin microbiome of animals have advanced our understanding in conservation strategies, yet in the human skin, the fungal microbiome (mycobiome) remains vastly unexplored. Here, we review recent studies on the role of fungi in the skin microbiome, emphasizing how fungal-bacterial interactions at the skin surface play an important ecological function in vertebrate hosts.

Ankle brachial indices and anaerobes: is peripheral arterial disease associated with anaerobic bacteria in diabetic foot ulcers?

Background: Lower extremity amputations from diabetic foot ulcers (DFUs) are rebounding, and new biomarkers that predict wound healing are urgently needed. Anaerobic bacteria have been associated with persistent ulcers and may be a promising biomarker beyond currently recommended vascular assessments. It is unknown whether anaerobic markers are simply a downstream outcome of peripheral arterial disease (PAD) and ischemia, however. Here, we evaluate associations between two measures of anaerobic bacteria-abundance and metabolic activity-and PAD. Methods: We built a prospective cohort of 37 patients with baseline ankle brachial index (ABI) results. Anaerobic bacteria were measured in two ways: DNA-based total anaerobic abundance using 16S rRNA gene amplicon sequencing and resulting summed relative abundance, and RNA-based metabolic activity based on bacterial read annotation of metatranscriptomic sequencing. PAD was defined three ways: PAD diagnosis, ABI results, and a dichotomous definition of mild ischemia (versus normal) based on ABI values. Statistical associations between anaerobes and PAD were evaluated using univariate odds ratios (ORs) or Spearman's correlations. Results: Total anaerobe abundance was not significantly associated with PAD diagnosis, ABI results, or mild ischemia (ORPAD = 0.47, 95% CI = 0.023-7.23, p = 0.60; Spearman's correlation coefficientABI = 0.24, p = 0.17; ORmild ischemia = 0.25, 95% CI = 0.005-5.86, p = 0.42). Anaerobic metabolic activity was not significantly associated with PAD diagnosis, ABI results, or mild ischemia (ORPAD = 1.99, 95% CI = 0.17-21.44, p = 0.57; Spearman's correlation coefficientABI = 0.12, p = 0.52; ORmild ischemia = 0.90, 95% CI = 0.03-15.16, p = 0.94). Conclusion: Neither anaerobic abundance nor metabolic activity was strongly associated with our three definitions of PAD. Therefore, anaerobic bacteria may offer additional prognostic value when assessing wound healing potential and should be investigated as potential molecular biomarkers for DFU outcomes.

Cobamide Sharing Is Predicted in the Human Skin Microbiome.

The skin microbiome is a key player in human health, with diverse functions ranging from defense against pathogens to education of the immune system. While recent studies have begun to shed light on the valuable role that skin microorganisms have in maintaining the skin barrier, a detailed understanding of the complex interactions that shape healthy skin microbial communities is limited. Cobamides, the vitamin B12 class of cofactor, are essential for organisms across the tree of life. Because this vitamin is only produced by a limited fraction of prokaryotes, cobamide sharing is predicted to mediate community dynamics within microbial communities. Here, we provide the first large-scale metagenomic assessment of cobamide biosynthesis and utilization in the skin microbiome. We show that while numerous and diverse taxa across the major bacterial phyla on the skin encode cobamide-dependent enzymes, relatively few species encode de novo cobamide biosynthesis. We show that cobamide producers and users are integrated into the network structure of microbial communities across the different microenvironments of the skin and that changes in microbiome community structure and diversity are associated with the abundance of cobamide producers in the Corynebacterium genus, for both healthy and diseased skin states. Finally, we find that de novo cobamide biosynthesis is enriched only in Corynebacterium species associated with hosts, including those prevalent on human skin. We confirm that the cofactor is produced in excess through quantification of cobamide production by human skin-associated species isolated in the laboratory. Taken together, our results reveal the potential for cobamide sharing within skin microbial communities, which we hypothesize mediates microbiome community dynamics and host interactions. IMPORTANCE The skin microbiome is essential for maintaining skin health and function. However, the microbial interactions that dictate microbiome structure, stability, and function are not well understood. Here, we investigate the biosynthesis and use of cobamides, a cofactor needed by many organisms but only produced by select prokaryotes, within the human skin microbiome. We found that while a large proportion of skin taxa encode cobamide-dependent enzymes, only a select few encode de novo cobamide biosynthesis. Further, the abundance of cobamide-producing Corynebacterium species is associated with skin microbiome diversity and structure, and within this genus, de novo biosynthesis is enriched in host-associated species compared to environment-associated species. These findings identify cobamides as a potential mediator of skin microbiome dynamics and skin health.

Modeling Candida auris Colonization on Porcine Skin Ex Vivo.

Candida auris spreads person to person in hospitals and other healthcare facilities. The heightened capacity for C. auris to colonize skin contributes to the difficulty in eradicating this drug-resistant and deadly pathogen in nosocomial settings. Models for the study of C. auris skin colonization are critical for understanding this virulence trait. In light of the similarities between the skin properties of humans and pigs, pigs represent an ideal model for the investigation of skin-C. auris interactions. Here, we describe how to utilize porcine skin for ex vivo studies of C. auris colonization.

vRhyme enables binning of viral genomes from metagenomes.

Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, few approaches exist for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identify nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. To refine bins, vRhyme utilizes unique features of viral genomes, namely a protein redundancy scoring mechanism based on the observation that viruses seldom encode redundant genes. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

Two-for-one: Dual host-microbe functions of S. epidermidis Sph.

The skin microbiome is essential for skin function, yet the mechanisms responsible are only beginning to be uncovered. In this issue of Cell Host & Microbe, Zheng et al. demonstrate that a Staphylococcus epidermidis sphingomyelinase has a mutually beneficial role in supporting the skin barrier and promoting S. epidermidis colonization.