Qi Zheng

The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome

ABSTRACT Viruses make up a major component of the human microbiota but are poorly understood in the skin, our primary barrier to the external environment. Viral communities have the potential to modulate states of cutaneous health and disease. Bacteriophages are known to influence the structure and function of microbial communities through predation and genetic exchange. Human viruses are associated with skin cancers and a multitude of cutaneous manifestations. Despite these important roles, little is known regarding the human skin virome and its interactions with the host microbiome. Here we evaluated the human cutaneous double-stranded DNA virome by metagenomic sequencing of DNA from purified virus-like particles (VLPs). In parallel, we employed metagenomic sequencing of the total skin microbiome to assess covariation and infer interactions with the virome. Samples were collected from 16 subjects at eight body sites over 1 month. In addition to the microenviroment, which is known to partition the bacterial and fungal microbiota, natural skin occlusion was strongly associated with skin virome community composition. Viral contigs were enriched for genes indicative of a temperate phage replication style and also maintained genes encoding potential antibiotic resistance and virulence factors. CRISPR spacers identified in the bacterial DNA sequences provided a record of phage predation and suggest a mechanism to explain spatial partitioning of skin phage communities. Finally, we modeled the structure of bacterial and phage communities together to reveal a complex microbial environment with a Corynebacterium hub. These results reveal the previously underappreciated diversity, encoded functions, and viral-microbial dynamic unique to the human skin virome. IMPORTANCE To date, most cutaneous microbiome studies have focused on bacterial and fungal communities. Skin viral communities and their relationships with their hosts remain poorly understood despite their potential to modulate states of cutaneous health and disease. Previous studies employing whole-metagenome sequencing without purification for virus-like particles (VLPs) have provided some insight into the viral component of the skin microbiome but have not completely characterized these communities or analyzed interactions with the host microbiome. Here we present an optimized virus purification technique and corresponding analysis tools for gaining novel insights into the skin virome, including viral “dark matter,” and its potential interactions with the host microbiome. The work presented here establishes a baseline of the healthy human skin virome and is a necessary foundation for future studies examining viral perturbations in skin health and disease. To date, most cutaneous microbiome studies have focused on bacterial and fungal communities. Skin viral communities and their relationships with their hosts remain poorly understood despite their potential to modulate states of cutaneous health and disease. Previous studies employing whole-metagenome sequencing without purification for virus-like particles (VLPs) have provided some insight into the viral component of the skin microbiome but have not completely characterized these communities or analyzed interactions with the host microbiome. Here we present an optimized virus purification technique and corresponding analysis tools for gaining novel insights into the skin virome, including viral “dark matter,” and its potential interactions with the host microbiome. The work presented here establishes a baseline of the healthy human skin virome and is a necessary foundation for future studies examining viral perturbations in skin health and disease.

Skin Microbiome Surveys Are Strongly Influenced by Experimental Design.

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provides more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e., gastrointestinal) and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource and cost intensive, provides evidence of a communitys functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This study highlights the importance of experimental design for downstream results in skin microbiome surveys.

Temporal Stability in Chronic Wound Microbiota Is Associated With Poor Healing

Microbial burden of chronic wounds is believed to play an important role in impaired healing and the development of infection-related complications. However, clinical cultures have little predictive value of wound outcomes, and culture-independent studies have been limited by cross-sectional design and small cohort size. We systematically evaluated the temporal dynamics of the microbiota colonizing diabetic foot ulcers, a common and costly complication of diabetes, and its association with healing and clinical complications. Dirichlet multinomial mixture modeling, Markov chain analysis, and mixed-effect models were used to investigate shifts in the microbiota over time and their associations with healing. Here we show, to our knowledge, previously unreported temporal dynamics of the chronic wound microbiome. Microbiota community instability was associated with faster healing and improved outcomes. Diabetic foot ulcer microbiota were found to exist in one of four community types that experienced frequent and nonrandom transitions. Transition patterns and frequencies were associated with healing time. Exposure to systemic antibiotics destabilized the wound microbiota, rather than altering overall diversity or relative abundance of specific taxa. This study provides evidence that the dynamic wound microbiome is indicative of clinical outcomes and may be a valuable guide for personalized management and treatment of chronic wounds.

Commensal microbiota modulate gene expression in the skin

BackgroundThe skin harbors complex communities of resident microorganisms, yet little is known of their physiological roles and the molecular mechanisms that mediate cutaneous host-microbe interactions. Here, we profiled skin transcriptomes of mice reared in the presence and absence of microbiota to elucidate the range of pathways and functions modulated in the skin by the microbiota.ResultsA total of 2820 genes were differentially regulated in response to microbial colonization and were enriched in gene ontology (GO) terms related to the host-immune response and epidermal differentiation. Innate immune response genes and genes involved in cytokine activity were generally upregulated in response to microbiota and included genes encoding toll-like receptors, antimicrobial peptides, the complement cascade, and genes involved in IL-1 family cytokine signaling and homing of T cells. Our results also reveal a role for the microbiota in modulating epidermal differentiation and development, with differential expression of genes in the epidermal differentiation complex (EDC). Genes with correlated co-expression patterns were enriched in binding sites for the transcription factors Klf4, AP-1, and SP-1, all implicated as regulators of epidermal differentiation. Finally, we identified transcriptional signatures of microbial regulation common to both the skin and the gastrointestinal tract.ConclusionsWith this foundational approach, we establish a critical resource for understanding the genome-wide implications of microbially mediated gene expression in the skin and emphasize prospective ways in which the microbiome contributes to skin health and disease.

Evolutionary and functional implications of hypervariable loci within the skin virome

Localized genomic variability is crucial for the ongoing conflicts between infectious microbes and their hosts. An understanding of evolutionary and adaptive patterns associated with genomic variability will help guide development of vaccines and antimicrobial agents. While most analyses of the human microbiome have focused on taxonomic classification and gene annotation, we investigated genomic variation of skin-associated viral communities. We evaluated patterns of viral genomic variation across 16 healthy human volunteers. Human papillomavirus (HPV) and Staphylococcus phages contained 106 and 465 regions of diversification, or hypervariable loci, respectively. Propionibacterium phage genomes were minimally divergent and contained no hypervariable loci. Genes containing hypervariable loci were involved in functions including host tropism and immune evasion. HPV and Staphylococcus phage hypervariable loci were associated with purifying selection. Amino acid substitution patterns were virus dependent, as were predictions of their phenotypic effects. We identified diversity generating retroelements as one likely mechanism driving hypervariability. We validated these findings in an independently collected skin metagenomic sequence dataset, suggesting that these features of skin virome genomic variability are widespread. Our results highlight the genomic variation landscape of the skin virome and provide a foundation for better understanding community viral evolution and the functional implications of genomic diversification of skin viruses.

Topical antimicrobial treatments can elicit shifts to resident skin bacterial communities and reduce colonization by Staphylococcus aureus competitors

ABSTRACT The skin microbiome is a complex ecosystem with important implications for cutaneous health and disease. Topical antibiotics and antiseptics are often employed to preserve the balance of this population and inhibit colonization by more pathogenic bacteria. However, despite their widespread use, the impact of these interventions on broader microbial communities remains poorly understood. Here, we report the longitudinal effects of topical antibiotics and antiseptics on skin bacterial communities and their role in Staphylococcus aureus colonization resistance. In response to antibiotics, cutaneous populations exhibited an immediate shift in bacterial residents, an effect that persisted for multiple days posttreatment. By contrast, antiseptics elicited only minor changes to skin bacterial populations, with few changes to the underlying microbiota. While variable in scope, both antibiotics and antiseptics were found to decrease colonization by commensal Staphylococcus spp. by sequencing- and culture-based methods, an effect which was highly dependent on baseline levels of Staphylococcus. Because Staphylococcus residents have been shown to compete with the skin pathogen S. aureus, we also tested whether treatment could influence S. aureus levels at the skin surface. We found that treated mice were more susceptible to exogenous association with S. aureus and that precolonization with the same Staphylococcus residents that were previously disrupted by treatment reduced S. aureus levels by over 100-fold. In all, the results of this study indicate that antimicrobial drugs can alter skin bacterial residents and that these alterations can have critical implications for cutaneous host defense.

HmmUFOtu: An HMM and phylogenetic placement based ultra-fast taxonomic assignment and OTU picking tool for microbiome amplicon sequencing studies

Culture-independent analysis of microbial communities frequently relies on amplification and sequencing of the prokaryotic 16S ribosomal RNA gene. Typical analysis pipelines group sequences into operational taxonomic units (OTUs) to infer taxonomic and phylogenetic relationships. Here, we present HmmUFOtu, a novel tool for processing microbiome amplicon sequencing data, which performs rapid per-read phylogenetic placement, followed by phylogenetically informed clustering into OTUs and taxonomy assignment. Compared to standard pipelines, HmmUFOtu more accurately and reliably recapitulates microbial community diversity and composition in simulated and real datasets without relying on heuristics or sacrificing speed or accuracy.

Antiseptic Agents Elicit Short-Term, Personalized, and Body Site–Specific Shifts in Resident Skin Bacterial Communities

Despite critical functions in cutaneous health and disease, it is unclear how resident skin microbial communities are altered by topical antimicrobial interventions commonly used in personal and clinical settings. Here we show that acute exposure to antiseptic treatments elicits rapid but short-term depletion of microbial community diversity and membership. Thirteen subjects were enrolled in a longitudinal treatment study to analyze the effects of topical treatments (i.e., ethanol, povidone-iodine, chlorhexidine, and water) on the skin microbiome at two skin sites of disparate microenvironment: forearm and back. Treatment effects were highly dependent on personalized and body site-specific colonization signatures, which concealed community dynamics at the population level when not accounted for in this analysis. The magnitude of disruption was influenced by the identity and abundance of particular bacterial inhabitants. Lowly abundant members of the skin microbiota were more likely to be displaced, and subsequently replaced, by the most abundant taxa prior to treatment. Members of the skin commensal family Propionibactericeae were particularly resilient to treatment, suggesting a distinct competitive advantage in the face of disturbance. These results provide insight into the stability and resilience of the skin microbiome, while establishing the impact of topical antiseptic treatment on skin bacterial dynamics and community ecology.