The heterogeneous wound microbiome varies with wound care pain, dressing type, and inflammatory gene expression.

Wound dressing changes are essential procedures for wound management. However, ~50% of patients experience severe pain during these procedures despite the availability of analgesic medications, indicating a need for novel therapeutics that address underlying causes of pain. Along with other clinical factors, wound pathogens and inflammatory immune responses have previously been implicated in wound pain. To test whether these factors could contribute to severe pain during wound dressing changes, we conducted an exploratory, cross-sectional analysis of patient-reported pain, inflammatory immune responses, and wound microbiome composition in 445 wounds at the time of a study dressing change. We profiled the bacterial composition of 406 wounds using 16S ribosomal RNA amplicon sequencing and quantified gene expression of 13 inflammatory markers in wound fluid using quantitative real-time polymerase chain reaction (qPCR). Neither inflammatory gene expression nor clinically observed inflammation were associated with severe pain, but Corynebacterium and Streptococcus were of lower relative abundance in wounds of patients reporting severe pain than those reporting little or no pain. Wound microbiome composition differed by wound location, and correlated with six of the inflammatory markers, including complement receptor C5AR1, pro-inflammatory cytokine interleukin (IL)1ß, chemokine IL-8, matrix metalloproteinase MMP2, and the antimicrobial peptide encoding cathelicidin antimicrobial peptide. Interestingly, we found a relationship between the wound microbiome and vacuum-assisted wound closure (VAC). These findings identify preliminary, associative relationships between wound microbiota and host factors which motivate future investigation into the directional relationships between wound care pain, wound closure technologies, and the wound microbiome.

Longitudinal evaluation of the cutaneous and rectal microbiota of German shepherd dogs with perianal fistulas undergoing therapy with ciclosporin and ketoconazole.

BACKGROUND: Perianal fistulas are painful ulcers or sinus tracts that disproportionately affect German shepherd dogs and are proposed as a spontaneous animal model of fistulising Crohn's disease. OBJECTIVES: To characterise the rectal and cutaneous microbiota in German shepherd dogs with perianal fistulas and to investigate longitudinal shifts with lesion resolution during immunomodulatory therapy. ANIMALS: Eleven German shepherd dogs with perianal fistulas and 15 healthy German shepherd dogs. MATERIALS AND METHODS: Affected dogs were evaluated and swabbed at three visits, 30 days apart, while undergoing treatment with ciclosporin and ketoconazole. Healthy German shepherd dogs were contemporaneously sampled. Sites included the rectum, perianal skin and axilla. The microbiome was evaluated following sequencing of the V4 hypervariable region of the 16S ribosomal RNA (rRNA) gene. RESULTS: Alpha diversity was not significantly different between healthy and affected dogs at each of the three body sites (p > 0.5), yet rectal and perianal beta diversities from affected dogs differed significantly from those of healthy dogs at Day 0 (p = 0.004). Rectal and perianal relative abundance of Prevotella spp. increased and perianal Staphylococcus spp. relative abundance decreased in affected dogs over time, coincident with lesion resolution. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in lesional cutaneous and rectal microbiota occur in German shepherd dogs with perianal fistulas and shift over time with lesion resolution during immunomodulatory therapy. Further investigations of the role of cutaneous and enteric microbiota in the pathogenesis of perianal fistulas, and whether manipulation of microbial populations may ameliorate disease, are needed.

Microbiota instruct IL-17A-producing innate lymphoid cells to promote skin inflammation in cutaneous leishmaniasis.

Innate lymphoid cells (ILCs) comprise a heterogeneous population of immune cells that maintain barrier function and can initiate a protective or pathological immune response upon infection. Here we show the involvement of IL-17A-producing ILCs in microbiota-driven immunopathology in cutaneous leishmaniasis. IL-17A-producing ILCs were RORγt+ and were enriched in Leishmania major infected skin, and topical colonization with Staphylococcus epidermidis before L. major infection exacerbated the skin inflammatory responses and IL-17A-producing RORγt+ ILC accumulation without impacting type 1 immune responses. IL-17A responses in ILCs were directed by Batf3 dependent CD103+ dendritic cells and IL-23. Moreover, experiments using Rag1-/- mice established that IL-17A+ ILCs were sufficient in driving the inflammatory responses as depletion of ILCs or neutralization of IL-17A diminished the microbiota mediated immunopathology. Taken together, this study indicates that the skin microbiota promotes RORγt+ IL-17A-producing ILCs, which augment the skin inflammation in cutaneous leishmaniasis.

Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence.

Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed, but how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glial­ILC3­epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably, ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit, revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals.

Insights into the skin microbiome of sickle cell disease leg ulcers.

Leg ulcers are estimated to occur in 1%-10% of North American patients with sickle cell disease (SCD). Their pathophysiology remains poorly defined, but as with other chronic wounds, it is hypothesised that the microbial milieu, or microbiome, contributes to their healing and clinical outcomes. This study utilises 16S ribosomal RNA (rRNA) gene sequencing to describe, for the first time, the microbiome of the SCD leg ulcer and its association with clinical factors. In a cross-sectional analysis of 42 ulcers, we recovered microbial profiles similar to other chronic wounds in the predominance of anaerobic bacteria and opportunistic pathogens including Staphylococcus, Corynebacterium, and Finegoldia. Ulcers separated into two clusters: one defined by predominance of Staphylococcus and smaller surface area, and the other displaying a greater diversity of taxa and larger surface area. We also find that the relative abundance of Porphyromonas is negatively associated with haemoglobin levels, a key clinical severity indicator for SCD, and that Finegoldia relative abundance is negatively associated with CD19+ B cell count. Finally, ratios of Corynebacterium:Lactobacillus and Staphylococcus:Lactobacillus are elevated in the intact skin of individuals with a history of SCD leg ulcers, while the ratio of Lactobacillus:Bacillus is elevated in that of individuals without a history of ulcers. Investigations of the skin microbiome in relation to SCD ulcer pathophysiology can inform clinical guidelines for this poorly understood chronic wound, as well as enhance broader understanding about the role of the skin microbiome in delayed wound healing.

The otic microbiota and mycobiota in a referral population of dogs in eastern USA with otitis externa.

BACKGROUND: Canine otitis externa (OE) is a common inflammatory disease that is frequently complicated by secondary bacterial and/or yeast infections. The otic microbial population is more complex than appreciated by cytological methods and aerobic culture alone. HYPOTHESIS/OBJECTIVES: Differences in bacterial and fungal populations of the external ear canal will correlate with specific cytological and culture-based definitions of bacterial and Malassezia otitis. ANIMALS: Forty client-owned dogs; 30 with OE and 10 with healthy ears. METHODS AND MATERIALS: Prospective study comparing cytological samples, aerobic bacterial cultures and culture-independent sequencing-based analyses of the external ear canal. Subjects with OE included 10 dogs with only cocci [≥25/high power field (HPF)] on cytological evaluation and culture of Staphylococcus spp.; 10 dogs with rods (≥25/HPF) and exclusive culture of Pseudomonas aeruginosa; 10 dogs with only yeast on cytological results morphologically compatible with Malassezia spp. (≥5/HPF). RESULTS: Staphylococcus was the most abundant taxa across all groups. Ears cytologically positive for cocci had decreased diversity, and all types of OE were associated with decreased fungal diversity compared to controls. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytological and culture-based assessment of the ear canal is not predictive of the diverse microbiota of the ear canal in cases of Pseudomonas or Malassezia otitis. Less abundant bacterial taxa in cases of staphylococcal OE are worth scrutiny for future biological therapy.

Human macrophage response to microbial supernatants from diabetic foot ulcers.

Diabetic foot ulcers (DFUs) are a major clinical problem exacerbated by prolonged bacterial infection. Macrophages, the primary innate immune cells, are multifunctional cells that regulate diverse processes throughout multiple phases of wound healing. To better understand the influence of microbial species on macrophage behavior, we cultured primary human monocyte-derived macrophages from four donors for 24 hours in media conditioned by bacteria and fungi (Pseudomonas aeruginosa, Corynebacterium amycolatum, Corynebacterium striatum, Staphylococcus aureus, Staphylococcus simulans, and Candida albicans) isolated from the DFUs of six patients. The effects of these microbe-derived signals on macrophage behavior were assessed by measuring the gene expression of a panel of 25 genes related to macrophage phenotype, angiogenesis, bacterial recognition, and cell survival, as well as secretion of two inflammatory cytokines using NanoString multiplex analysis. Principal component analysis showed that macrophage gene expression and protein secretion were affected by both microbial species as well as human donor. S. simulans and C. albicans caused up-regulation of genes associated with a proinflammatory (M1) phenotype, and P. aeruginosa caused an increase in the secretion of the proinflammatory cytokine and M1 marker tumor necrosis factor-alpha (TNFα). Together, these results suggest that macrophages respond to secreted factors from microbes by up-regulating inflammatory markers, and that the effects are strongly dependent on the monocyte donor. Ultimately, increased understanding of macrophage-microbe interactions will lead to the development of more targeted therapies for DFU healing.

The intersection of microbiome and host at the skin interface: genomic- and metagenomic-based insights.

The past two decades have been marked by a surge in research to understand the microbial communities that live in association with the human body, in part stimulated by affordable, high-throughput DNA sequencing technology. In the context of the skin, this Perspective focuses on the current state of genomic- and metagenomic-based host-microbe research and future challenges and opportunities to move the field forward. These include elucidating nonbacterial components of the skin microbiome (i.e., viruses); systematic studies to address common perturbations to the skin microbiome (e.g., antimicrobial drugs, topical cosmetic/hygienic products); improved approaches for identifying potential microbial triggers for skin diseases, including species- and strain-level resolution; and improved, clinically relevant models for studying the functional and mechanistic roles of the skin microbiome. In the next 20 years, we can realistically expect that our knowledge of the skin microbiome will inform the clinical management and treatment of skin disorders through diagnostic tests to stratify patient subsets and predict best treatment modality and outcomes and through treatment strategies such as targeted manipulation or reconstitution of microbial communities.

The microbiota of traumatic, open fracture wounds is associated with mechanism of injury.

Open fractures are characterized by disruption of the skin and soft tissue, which allows for microbial contamination and colonization. Preventing infection-related complications of open fractures and other acute wounds remains an evolving challenge due to an incomplete understanding of how microbial colonization and contamination influence healing and outcomes. Culture-independent molecular methods are now widely used to study human-associated microbial communities without introducing culture biases. Using such approaches, the objectives of this study were to (1) define the long-term temporal microbial community dynamics of open fracture wounds and (2) examine microbial community dynamics with respect to clinical and demographic factors. Fifty-two subjects with traumatic open fracture wounds (32 blunt and 20 penetrating injuries) were enrolled prospectively and sampled longitudinally from presentation to the emergency department (ED) and at each subsequent inpatient or outpatient encounter. Specimens were collected from both the wound center and adjacent skin. Culture-independent sequencing of the 16S ribosomal RNA gene was employed to identify and characterize microbiota. Upon presentation to the ED and time points immediately following, sample collection site (wound or adjacent skin) was the most defining feature discriminating microbial profiles. Microbial composition of adjacent skin and wound center converged over time. Mechanism of injury most strongly defined the microbiota after initial convergence. Further analysis controlling for race, gender, and age revealed that mechanism of injury remained a significant discriminating feature throughout the continuum of care. We conclude that the microbial communities associated with open fracture wounds are dynamic in nature until eventual convergence with the adjacent skin community during healing, with mechanism of injury as an important feature affecting both diversity and composition of the microbiota. A more complete understanding of the factors influencing microbial contamination and/or colonization in open fractures is a critical foundation for identifying markers indicative of outcome and deciphering their respective contributions to healing and/or complication.

Severe pain during wound care procedures: A cross-sectional study protocol.

AIM: The aim of this study is to: (a) develop and evaluate a model to predict severe pain during wound care procedures (WCPs) so that high-risk patients can be targeted for specialized dressings and preventive pain control; and (b) identify biological factors associated with severe pain during WCPs so that novel pain control strategies can be developed. BACKGROUND: Wound care procedures such as dressing changes can cause moderate to severe pain in 74% of patients, with nearly half (36%) of all patients experiencing severe pain (rated as 8-10 on a 10-point numeric rating scale) during dressing change. Additionally, clinicians have little direction with current guidelines regarding pain control during WCPs including the selection of the appropriate advanced wound dressings and the appropriate use of analgesics. DESIGN: This is a cross-sectional study. METHODS: The National Institute of Nursing Research approved and funded the study June of 2015 and the appropriate Institutional Review Board approved all study protocols prior to funding. Study enrolment is underway at the University of Iowa Hospitals and Clinics with a target of 525 participants. Potential participants must be adults (21+ years) and have a nonburn, nondiabetic foot, full-thickness wound. The research team performs a one-time study dressing change on enrolled participants and collects all study data. DISCUSSION: This study will allow the development of a tool for clinicians to use to predict severe pain during WCPs and identify biological factors significantly associated with severe pain during WCPs.

Topical Antimicrobial Treatments Can Elicit Shifts to Resident Skin Bacterial Communities and Reduce Colonization by Staphylococcus aureus Competitors.

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.

AlignerBoost: A Generalized Software Toolkit for Boosting Next-Gen Sequencing Mapping Accuracy Using a Bayesian-Based Mapping Quality Framework.

Accurate mapping of next-generation sequencing (NGS) reads to reference genomes is crucial for almost all NGS applications and downstream analyses. Various repetitive elements in human and other higher eukaryotic genomes contribute in large part to ambiguously (non-uniquely) mapped reads. Most available NGS aligners attempt to address this by either removing all non-uniquely mapping reads, or reporting one random or "best" hit based on simple heuristics. Accurate estimation of the mapping quality of NGS reads is therefore critical albeit completely lacking at present. Here we developed a generalized software toolkit "AlignerBoost", which utilizes a Bayesian-based framework to accurately estimate mapping quality of ambiguously mapped NGS reads. We tested AlignerBoost with both simulated and real DNA-seq and RNA-seq datasets at various thresholds. In most cases, but especially for reads falling within repetitive regions, AlignerBoost dramatically increases the mapping precision of modern NGS aligners without significantly compromising the sensitivity even without mapping quality filters. When using higher mapping quality cutoffs, AlignerBoost achieves a much lower false mapping rate while exhibiting comparable or higher sensitivity compared to the aligner default modes, therefore significantly boosting the detection power of NGS aligners even using extreme thresholds. AlignerBoost is also SNP-aware, and higher quality alignments can be achieved if provided with known SNPs. AlignerBoost's algorithm is computationally efficient, and can process one million alignments within 30 seconds on a typical desktop computer. AlignerBoost is implemented as a uniform Java application and is freely available at https://github.com/Grice-Lab/AlignerBoost.

Complement deficiency promotes cutaneous wound healing in mice.

Wound healing is a complex homeostatic response to injury that engages numerous cellular activities, processes, and cell-to-cell interactions. The complement system, an intricate network of proteins with important roles in immune surveillance and homeostasis, has been implicated in many physiological processes; however, its role in wound healing remains largely unexplored. In this study, we employ a murine model of excisional cutaneous wound healing and show that C3(-/-) mice exhibit accelerated early stages of wound healing. Reconstitution of C3(-/-) mice with serum from C3(+/+) mice or purified human C3 abrogated the accelerated wound-healing phenotype. Wound histology of C3(-/-) mice revealed a reduction in inflammatory infiltrate compared with C3(+/+) mice. C3 deficiency also resulted in increased accumulation of mast cells and advanced angiogenesis. We further show that mice deficient in the downstream complement effector C5 exhibit a similar wound-healing phenotype, which is recapitulated in C5aR1(-/-) mice, but not C3aR(-/-) or C5aR2(-/-) mice. Taken together, these data suggest that C5a signaling through C5aR may in part play a pivotal role in recruitment and activation of inflammatory cells to the wound environment, which in turn could delay the early stages of cutaneous wound healing. These findings also suggest a previously underappreciated role for complement in wound healing, and may have therapeutic implications for conditions of delayed wound healing.

The preadolescent acne microbiome: A prospective, randomized, pilot study investigating characterization and effects of acne therapy.

BACKGROUND/OBJECTIVES: Acne, a common pediatric disease, tends to be more comedonal in preadolescents, whereas older individuals are more likely to have inflammatory lesions in addition to comedones. Thus the microbiome of preadolescents may be different. In this pilot study we aimed to characterize the preadolescent acne microbiome, compare the microbiome in preadolescents with and without acne, and investigate changes in the microbiome after topical treatment with benzoyl peroxide or a retinoid in a small cohort of preadolescents. METHODS: Participants were 7-10 years of age with (intervention group) or without (control group) acne and were recruited during routine outpatient dermatology visits. Baseline questionnaires, physical examination, and pore strip application were performed for all participants. Intervention group participants were randomized to receive topical therapy with benzoyl peroxide 5% gel or cream or tretinoin 0.025% cream. Participants with acne were followed up 8-10 weeks later and pore strip application was repeated. RESULTS: Preadolescents with acne were colonized with a greater diversity of cutaneous bacteria than controls and the most commonly identified bacterium was Streptococcus. The number of bacterial species and phylogenetic diversity decreased after treatment with benzoyl peroxide and tretinoin. CONCLUSION: The predominant bacteria in microbiome studies of adult acne is Propionibacterium, whereas in this pediatric population we saw a lot of Streptococcus bacteria. After treatment, the microbiomes of intervention group participants more closely resembled those of control group participants.

Interactions between host factors and the skin microbiome.

The skin is colonized by an assemblage of microorganisms which, for the most part, peacefully coexist with their hosts. In some cases, these communities also provide vital functions to cutaneous health through the modulation of host factors. Recent studies have illuminated the role of anatomical skin site, gender, age, and the immune system in shaping the cutaneous ecosystem. Alterations to microbial communities have also been associated with, and likely contribute to, a number of cutaneous disorders. This review focuses on the host factors that shape and maintain skin microbial communities, and the reciprocal role of microbes in modulating skin immunity. A greater understanding of these interactions is critical to elucidating the forces that shape cutaneous populations and their contributions to skin homeostasis. This knowledge can also inform the tendency of perturbations to predispose and/or bring about certain skin disorders.

Complement modulates the cutaneous microbiome and inflammatory milieu.

The skin is colonized by a plethora of microbes that include commensals and potential pathogens, but it is currently unknown how cutaneous host immune mechanisms influence the composition, diversity, and quantity of the skin microbiota. Here we reveal an interactive role for complement in cutaneous host-microbiome interactions. Inhibiting signaling of the complement component C5a receptor (C5aR) altered the composition and diversity of the skin microbiota as revealed by deep sequencing of the bacterial 16S rRNA gene. In parallel, we demonstrate that C5aR inhibition results in down-regulation of genes encoding cutaneous antimicrobial peptides, pattern recognition receptors, and proinflammatory mediators. Immunohistochemistry of inflammatory cell infiltrates in the skin showed reduced numbers of macrophages and lymphocytes with C5aR inhibition. Further, comparing cutaneous gene expression in germ-free mice vs. conventionally raised mice suggests that the commensal microbiota regulates expression of complement genes in the skin. These findings demonstrate a component of host immunity that impacts colonization of the skin by the commensal microbiota and vice versa, a critical step toward understanding host-microbe immune mutualism of the skin and its implications for health and disease. Additionally, we reveal a role for complement in homeostatic host-microbiome interactions of the skin.

The human microbiome: our second genome.

The human genome has been referred to as the blueprint of human biology. In this review we consider an essential but largely ignored overlay to that blueprint, the human microbiome, which is composed of those microbes that live in and on our bodies. The human microbiome is a source of genetic diversity, a modifier of disease, an essential component of immunity, and a functional entity that influences metabolism and modulates drug interactions. Characterization and analysis of the human microbiome have been greatly catalyzed by advances in genomic technologies. We discuss how these technologies have shaped this emerging field of study and advanced our understanding of the human microbiome. We also identify future challenges, many of which are common to human genetic studies, and predict that in the future, analyzing genetic variation and risk of human disease will sometimes necessitate the integration of human and microbial genomic data sets.

Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.

Atopic dermatitis (AD) has long been associated with Staphylococcus aureus skin colonization or infection and is typically managed with regimens that include antimicrobial therapies. However, the role of microbial communities in the pathogenesis of AD is incompletely characterized. To assess the relationship between skin microbiota and disease progression, 16S ribosomal RNA bacterial gene sequencing was performed on DNA obtained directly from serial skin sampling of children with AD. The composition of bacterial communities was analyzed during AD disease states to identify characteristics associated with AD flares and improvement post-treatment. We found that microbial community structures at sites of disease predilection were dramatically different in AD patients compared with controls. Microbial diversity during AD flares was dependent on the presence or absence of recent AD treatments, with even intermittent treatment linked to greater bacterial diversity than no recent treatment. Treatment-associated changes in skin bacterial diversity suggest that AD treatments diversify skin bacteria preceding improvements in disease activity. In AD, the proportion of Staphylococcus sequences, particularly S. aureus, was greater during disease flares than at baseline or post-treatment, and correlated with worsened disease severity. Representation of the skin commensal S. epidermidis also significantly increased during flares. Increases in Streptococcus, Propionibacterium, and Corynebacterium species were observed following therapy. These findings reveal linkages between microbial communities and inflammatory diseases such as AD, and demonstrate that as compared with culture-based studies, higher resolution examination of microbiota associated with human disease provides novel insights into global shifts of bacteria relevant to disease progression and treatment.