The skin as our outermost epithelial barrier is home to a diverse community of microbiota comprised of bacteria, fungi, viruses, and even microeukaryotes. These communities have coevolved with humans to use the unique resources provided by the skin and its appendages. In doing so they also provide essential roles in fortifying the skin barrier. One layer of skin defense is the microbial barrier itself, wherein the healthy skin microbiota provide colonization resistance against pathogenic organisms. Furthermore, the skin microbiome shapes the skin immune barrier by promoting immune tolerance of commensals, homeostatic immunity, and immune recognition of pathogens (Nakatsuji et al. 2021a; Swaney and Kalan 2021; Zhang et al. 2022).

      The acidic pH of the skin acts as a chemical barrier against pathogen colonization. The acidification of the skin surface is due in part to hydrolysis of sebum lipids by skin commensals (Schmid-Wendtner and Korting 2006; Juntachai et al. 2009). A proper physical skin barrier is dependent on the tightly regulated maintenance of stratified epithelia, which is also regulated by commensal skin microbiota (Uberoi et al. 2021).

      A barrier breach destroys the existing cutaneous microbiota and also creates a nutrient-rich niche ripe for opportunism, whether of existing microbes or those encountered in the environment. Resolving a skin barrier breach requires a carefully orchestrated series of events to achieve wound closure and/or reepithelialization. Here, we will examine how microbes influence this series of events, including inflammation, pathogen clearance, and reepithelialization, during delayed wound healing.

     Clinically, delayed wound healing is a looming concern as the United States population is increasingly obese, diabetic, and aging. Chronic wounds affect over eight million people in the United States each year (Sen 2021). These wounds present a significant cost to the healthcare system, with the United States spending between 28 and 96 billion annually on the management of nonhealing wounds (Nussbaum et al. 2018). The burden on patients should also not be overlooked, as chronic wounds often lead to severe pain, immobility, social isolation, morbidity, and mortality (Margolis et al. 2011; Frykberg and Banks 2015; Armstrong et al. 2017; Järbrink et al. 2017). Despite the magnitude of this problem, there has been limited progress in the development of effective targeted treatments for nonhealing wounds. Thus, there is an urgent need to identify new therapeutic interventions to improve wound healing. One such target is the skin microbiota, which is an important component of the wound environment. Studying the role of the microbiome and mechanisms in delayed healing may shed light on novel targets for microbial-based therapies.

      In this review, we discuss the advancement of methods to measure and survey the wound microbiota, as well as the themes that have emerged from microbiome surveys across many types of chronic wounds. We will also examine microbial mechanisms of impaired healing that influence all stages of wound healing. Finally, we close on beneficial roles for microbes in wound healing and our perspectives of how these roles may be leveraged to treat delayed healing and improve clinical outcomes.

TOOLS TO SURVEY THE WOUND MICROBIOME

     Accurate and precise methods for the identification of microbial community members are needed to begin to understand their role in wound healing. Until recently, techniques to survey the wound microbiota were primarily dependent on culture-based methods (Robson and Heggers 1969). These methods typically involve swabbing of the wound tissue or collection of debridement tissue and subsequent quantitative bacterial culture. The clinically recommended standard for wound swabbing is the Levine technique (Levine et al. 1976; Cross 2014). Using this method, a swab is rotated over a defined area of the wound with enough pressure to express tissue fluid. This technique has been shown to capture similar bioburden as cultures of wound tissue (Gardner et al. 2006) and is relatively noninvasive. However, like all techniques of specimen collection, the accuracy of the method is dependent on wound bed preparation, area of the wound sampled, and duration of sampling.

      Culture-based methods rely on plating wound specimens on culture media that support growth of putative wound pathogens (Fig. 1). The results of these methods are biased in their detection of microorganisms that are easily isolated and readily grown under standard laboratory culture conditions. Therefore, they underestimate microbes that rely on a community lifestyle for their growth and survival (e.g., biofilms) and fastidious microbes (e.g., anaerobes) (Han et al. 2011). Fastidious and biofilm-forming bacteria comprise important components of the wound microbiome, as discussed further in this review.

      In light of these shortcomings, it is unsurprising that in the absence of clinical infection, culture-dependent surveys of the microbiome fail to predict outcomes of diabeticfoot ulcers (DFUs), a common type of nonhealing wound (Gardner et al. 2014). Even when examining several aspects of wound bioburden (e.g., microbial load, diversity, and presence of likely pathogens), there were no associations with healing time. These results support the current Infectious Diseases Society of America (IDSA) guidelines that DFUs without clinical signs of infection should not be treated with antibiotics (Lipsky et al. 2012). Despite these recommendations, there is still high prevalence of antibiotic use for treatment of chronic wounds without signs of infection (Howell-Jones et al. 2006). This practice of improper antibiotic prescriptions leads to increased antibiotic resistance among wound microbiota. Indeed, antibiotic treatment has been correlated with changes to the wound microbiome in composition (Price et al. 2009) and an enrichmentfor genes encoding for antibiotic resistance (Kalan et al. 2019). Given these shortcomings of culture-based surveys and their impact on management of nonhealing wounds, additional techniques are needed to comprehensively profile the wound microbiota.

      Next-generation sequencing-based, cultureindependent methods developed over the past few decades have increased resolution for studies of the polymicrobial wound community (Fig. 1). One such method that has become increasingly affordable and accessible is sequencing of the 16S ribosomal RNA (rRNA) gene, which is found in all prokaryotic genomes. This gene has both conserved and hypervariable regions, where the conserved segments allow for universal polymerase chain reaction (PCR) amplification and the hypervariable regions act as signatures that enable taxonomic classification and identification. Similarly, fungal wound communities may be profiled by targeting portions of their ribosomal RNA genes for PCR and sequencing (Kalan and Grice 2018). After sequencing, bioinformatic approaches are used to cluster highly similar sequences into operational taxonomic units (OTUs) and subsequently assign taxonomic classifications to these OTUs (Jo et al. 2016). Further analysis can then be performed to study several dimensions of wound bioburden such as species richness and diversity (Grogan et al. 2019).

      Culture-independent 16S rRNA gene sequencing performed on DFUs has shown more comprehensive estimates of wound bioburden than traditional culture techniques, which underestimate bacterial load and species richness (Gardner et al. 2013). Furthermore, this genomic approach was the first to demonstrate that wound bioburden is associated with clinical factors (Gardner et al. 2013), which was failed to be shown with quantitative cultures. One shortcoming of amplicon-based sequencing methods is limited taxonomic resolution. Genus-level resolution is typical for 16S rRNA gene sequencing, whereas species-level resolution is possible only if using customized databases (Meisel et al. 2016). Species-level distinction becomes important for genera residing on the skin such as Staphylococcus, where pathogens (Staphylococcus aureus) are closely related to skin commensals (e.g., Staphylococcus epidermidis).

      Greater resolution of the microbiota is afforded by next-generation techniques like shotgun metagenomic sequencing. In this method, all DNA present in a sample is subjected to shotgun sequencing (Fig. 1). Therefore, a true community-wide survey of all microorganisms, including viruses, fungi, and bacteria, can be performed (Oh et al. 2014). Importantly, this technique also reveals the functional potential of the community constituents. Using reference gene databases, genetic enrichment for metabolic pathways, virulence genes, and other pathways/ processes of interest can be analyzed. For example, shotgun metagenomic sequencing performed on longitudinal DFU samples revealed higher levels of biofilm metabolic activity in nonhealing wounds (Kalan et al. 2019). Additionally, shotgun metagenomics captures strain-level variation across multiple kingdoms of the microbiota (Oh et al. 2014). In the same study of DFUs, strain-level variation in S. aureus was associated with differential healing outcomes, with certain strains found primarily in nonhealing wounds (Kalan et al. 2019). These findings mirrored those in another dermatological disease, atopic dermatitis (AD), where strain-level heterogeneity in S. aureus clinical isolates was correlated with differences in disease severity and immune responses (Byrd et al. 2017).

      Since culture-independent methods detect DNA, it is not possible to distinguish live versus dead microorganisms. Thus, a caveat is that these techniques may survey the history of the microbiome versus a snapshot in time. Ideally, for research purposes, a combinatorial approach is taken to sample wound microbiota, where highthroughput sequencing-based methods are coupled to targeted cultures. Future innovations to surveying the microbiome include implementation of metatranscriptomic methods that could simultaneously profile host and microbe (Westermann et al. 2012), providing enhanced insight into what the microbes are doing and how they are affecting the host wound tissue.

MEMBERS OF THE WOUND MICROBIOME

      Chronic, nonhealing wounds can develop from various types of injury and in the setting of underlying conditions. These factors are likely to influence the different types of microbes that are able to initially colonize the wound. Age and duration of the wound are also likely factors to consider, as well as anatomical location, which would influence wound perfusion and skin regenerative potential. With these important variables in mind, here we focus on the major themes that have emerged in the study of wound microbiomes while highlighting the different types of chronic wounds and how they select for unique populations of microbes (Fig. 2).

      DFUs are a common type of chronic wound that affect ∼25% of those with diabetes, often resulting in lower extremity amputation, exorbitant health care costs, and significant morbidity and mortality (Armstrong et al. 2017). S. aureusis typically the most abundant microbe recovered from DFUs (Citron et al. 2007; Gardner et al. 2013). However, this is not unique to DFUs, as all different types of skin wounds and infections are associated with S. aureus, which causes the majority of skin and soft tissue infections (Moran et al. 2006; Olaniyi et al. 2017). Beyond Staphylococcus, another genera of the Firmicutes phylum that is commonly found in DFUs is Streptococcus. β-Hemolytic Streptococci are pathogens commonly cultured from DFUs (Bowler et al. 2001). In a study of 401 patients with infected DFUs, 41% were culture positive for Streptococcus species, with the most common being Streptococcus agalactiae (Citron et al. 2007).

      Culture-independent 16S rRNA gene sequencing has been particularly instrumental in revealing that anaerobic bacteria are found at a higher prevalence in DFUs than culture methods alone would have predicted (Dowd et al. 2008; Gardner et al. 2013). Anaerobic bacteria can be fastidious and require specialized culture conditions for both transport and isolation. In DFU, abundance of anaerobic bacteria was found to be positively correlated with ulcer depth in a study of 52 uninfected neuropathic wounds (Gardner et al. 2013). Commonly identified anaerobic bacteria in this study and culture-dependent studies include Anaerococcus, Finegoldia, Peptoniphilus, Prevotella, and Porphyromonas (Citron et al. 2007; Malone et al. 2017b). These anaerobes can be present in mixed communities with aerobic bacteria. It is important to identify anaerobic species in the wound environment as they have been associated with worse healing outcomes (Sloan et al. 2019; Min et al. 2020; Verbanic et al. 2020); furthermore, clearance of anaerobes with sharp debridement improves wound healing outcomes (Kalan et al. 2019).

      Burn wounds also compromise the skin barrier and leave patients especially susceptible to wound contamination and infection, in part due to an immunocompromised status, prolonged hospitalization, and other generalized effects (Tiwari 2012). Gram-negative bacteria, such as Pseudomonas aeruginosa, play a major pathogenic role in burn wounds (Azzopardi et al. 2014). A meta-analysis of studies investigating burn wound infections performed by Azzopardi et al. found that the most common gram-negative pathogens are P. aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp., Proteus spp., and Escherichia coli. A longitudinal 16S rRNA gene-sequencing study of the cutaneous microbiome in burn wound patients from hospital admission until 28 d postinjury showed that skin commensals (i.e., Cutibacterium acnes, S. epidermidis) were depleted and that microbes that ended up causing infection were generally present in the microbiome prior to detection of infection (Lima et al. 2021).

      Pressure ulcers (PUs) are another type of chronic wound that also have significant associated morbidity and healthcare costs and are found in ∼20% of long-term stay patients (Zulkowski et al. 2005; Stroupe et al. 2011). PUs have been found to have higher abundance of strict anaerobes, such as Peptoniphilis, Peptococcus, and Finegoldia, than DFUs (Dowd et al. 2008). Similar to the environment of DFUs, PU were found to be polymicrobial in nature and also highly variable between different patients (Smith et al. 2010). A study on how PU microbiota evolve over time found that the presence of the genera Finegoldia and Anaerococcus were associated with worse outcomes (Dunyach-Remy et al. 2021). In contrast, the genus Corynebacterium was found to be associated with improved wound evolution. These studies demonstrate the interindividual variability in chronic wound bioburden, indicating that treatments that target the wound microbiota would need to account for the patient’s unique microbial community.

      Venous leg ulcers (VLUs) are prevalent in elderly individuals, affecting ∼1% of Americans and are a result of venous insufficiency, usually in the lower extremities (Margolis et al. 2011). Thus, factors required for wound healing and immune defense are unable to reach the wound due to poor circulation in VLU patients. The few studies that have examined the microbiome of VLU have been small and inconclusive, identifying taxa that inhabit other types of wounds including anaerobes, S. aureus, and P. aeruginosa (Thomsen et al. 2010; Tuttle et al. 2011).

      Patients with sickle cell disease (SCD) are particularly prone to ulceration of the legs, in part due to venous incompetence, mechanical obstruction by sickled blood cells, and anemia (Minniti et al. 2010). Like other chronic wounds, it is thought that the microbiome influences SCD ulcer healing. Recently, 16S rRNA gene sequencing on SCD ulcers was performed to profile the colonizing microbiota. The most commonly identified taxa in this study are similar to those found in other chronic wound types, such as Staphylococcus, Corynebacterium, and Finegoldia (Byeon et al. 2021). Associations were found between relative abundances of bacterial taxa and SCD clinical factors, indicating that further research is needed to understand how to target the SCD ulcer microbiome for clinical therapies. Culture-dependent and -independent investigations have together revealed in greater detail the major microbial players across different types of chronic wounds (Fig. 2). Almost all evidence points to a polymicrobial wound community; nonetheless, identifying the problematic players in the community remains a challenge. Studies to examine mechanisms of how individual microbes interact with the wound environment and with each other can shed light on their roles in a polymicrobial setting. Recent developments in this area are discussed in the next two sections.

BAD BUGS: HOW MICROBES SURVIVE AND THRIVE IN THE WOUND

      A primary goal of characterizing the wound microbiome community members is leveraging this information to predict clinical outcomes and apply more precise therapies. In this way, distinguishing benign colonization from pathogenic bioburden early is a key priority. When there is failure to clear pathogenic bacterial colonization—which often occurs in the setting of chronic wounds—these bacteria can further replicate and invade surrounding tissue, leading to local infection and subsequent systemic infection. Thus, pathogenic bioburden leads to poor outcomes such as osteomyelitis and amputation (Gardner and Frantz 2008). In wound settings, there are many host factors that lead to poor bacterial clearance, such as impaired immune responses and endothelial dysfunction. Here we will focus on mechanisms that allow microbes to survive, proliferate, and thrive in the wound environment (Fig. 3).

      Microbes in wounds will often form biofilms, polymicrobial communities that promote bacterial survival. Previous studies found that ∼60%–80% of chronic wounds contain biofilms (James et al. 2008; Malone et al. 2017a). A recent study investigated the presence of biofilms in 65 DFU tissue samples using multifaceted detection including scanning electron microscopy and fluorescent in situ hybridization. They found that 100% of the samples contained biofilms, supporting the view that biofilms are ubiquitous in DFUs, and likely other chronic wound types (Johani et al. 2017). The longitudinal stability of chronic wound microbiota has been associated with poor outcomes (Loesche et al. 2017; Tipton et al. 2017; Sloan et al. 2019), suggestive of a bio- film-like setting that would protect the community from disruption. Shotgun metagenomic sequencing studies of DFU are also suggestive, where poor DFU outcomes were associated with an enrichment of biofilm formation genes in the metagenome (Kalan et al. 2019). There can be cross-kingdom interactions in biofilms, where fungi provide a surface for bacterial attachment (Kalan et al. 2016). These fungal-bacterial biofilms have been shown to have increased virulence compared to the bacterial or fungal species alone (Fox et al. 2014; Cheong et al. 2021).

      In the biofilm setting, microbial cells are embedded in extracellular polymers such as polysaccharides and DNA, which form a structural barrier and enhance adherence. Bacteria grown in biofilms have different properties compared to bacteria grown planktonically, such as metabolism, structure, and gene expression (López et al. 2010). For example, S. aureus biofilms have been shown to cause reduced viability, increased apoptosis, and altered cytokine production of keratinocytes compared to planktonic S. aureus treatment (Kirker et al. 2009; Secor et al. 2011). Biofilm phase bacteria can also be more resistant to immune cell killing; a study of biofilm phase S. epidermidis demonstrated increased survival in macrophages compared to their planktonic phase (Spiliopoulou et al. 2012). Antibiotic treatment is often ineffective in clearing biofilms, and the minimum inhibitory concentration of commonly used antimicrobials is much higher for biofilms than planktonic bacteria (Stewart and Costerton 2001).

      A further argument for judicious use of antibiotics when considering the wound microbiome is that misuse can lead to antimicrobial resistance and further depletion of therapeutic options. In the skin microbiome, shotgun metagenomic sequencing was used to study the longterm effects of systemic antibiotics. Antibiotic treatment, particularly with doxycycline and trimethoprim/sulfamethoxazole, promoted emergence of resistance and enrichment of genes involved in gene mobilization (Jo et al. 2021). In burn wounds, P. aeruginosa, S. aureus, and A. baumannii isolates cultured from wound infections are often found to be resistant to many commonly used antibiotics (Gong et al. 2014; Ronat et al. 2014; van Langeveld et al. 2017). The prevalence of methicillin-resistant S. aureus (MRSA) isolates cultured from DFUs is of increasing concern. A prospective study of cultured bacteria from DFUs of 84 patients found that roughly 50% of S. aureus isolates were MRSA, and MRSA colonization was significantly associated with wound infection (Tentolouris et al. 2006). A high proportion of Staphylococcal strains isolated from DFUs were found to be multiresistant to antibiotics, including commonly used antibiotics such as erythromycin and ciprofloxacin (Mottola et al. 2016). These results were supported by a shotgun metagenomic study of DFUs, which found that antibiotic resistance genes were widespread, and some isolates even had resistance genes for >10 classes of antibiotics (Kalan et al. 2019).

      Host immune responses often fail to clear bacterial infection in the setting of chronic wounds, which leads to sustained inflammation and incomplete wound healing. This is due in part to bacterial virulence mechanisms of immune evasion. As previously discussed, S. aureus is prevalent and abundant in many wound types and is an efficient pathogen with many mechanisms to avoid immune-mediated killing. One such example is the ability of S. aureus to create an intracellular niche within the epidermis. During wounding, there is increased expression of Perforin-2 (P2), which is an antimicrobial peptide (AMP) with activity against intracellular bacteria (Strbo et al. 2019). However, S. aureus suppresses P2 expression, leading to accumulation of intracellular S. aureus, and subsequent inflammasome activation and pyroptosis. This S. aureus accumulation and increase in pyroptosis was correlated with worse healing outcomes in patients with DFUs (Pastar et al. 2021). Another inflammatory response of wound healing that is dysregulated by S. aureus is microRNA-15B-5P expression. S. aureus induces expression of this microRNA in DFUs, which leads to repression of DNA repair and deregulation of inflammatory responses (Ramirez et al. 2018).

      Bacterial virulence mechanisms that allow host evasion are oftentimes encoded on bacteriophage or plasmids, mobile genetic elements that facilitate rapid microbial adaptation to their environment, particularly under conditions of stress. In P. aeruginosa–infected human wounds, a filamentous bacteriophage was found to be associated with wound duration and out come. Strains of P. aeruginosa that harbored the bacteriophage had increased fitness in the wound setting, whereby the phage suppressed bacterial clearance by immune cells through subversion of the antiviral immune responses (Sweere et al. 2019).

THE WOUND MICROBIOME IN A NEW LIGHT: BENEFICIAL MICROBES

      Wound microbiota research and clinical wound care typically focus on “neutralizing” microbial pathogens. Most approaches to countering pathogens rely on antibiotic therapies that are nonspecific and broad spectrum, and do not consider the existence of commensal wound microbiota that could suffer collateral damage. In unwounded skin, microbial interactions are rarely pathogenic in nature. Indeed, a symbiotic, homeostatic relationship exists between commensal microbiota and the skin epithelium. There is building evidence that during disruptions of epithelial homeostasis, such as wounding, commensal bacteria can actually promote several components of the host repair response. Here we highlight several of these processes and the mechanisms whereby microbes enhance skin wound healing (Fig. 3).

      Skin commensals promote immune cell recruitment and activation, which is needed for successful wound closure. In particular, the skin microbiota has been shown to promote bacteria-specific lymphocyte subsets. These commensal-specific T cells then migrate to the skin to promote barrier function, such as enhanced antimicrobial defense (Naik et al. 2015). Skin commensals like S. epidermidis have been shown to induce T cells that have distinct gene signatures of tissue repair effector functions, such as tissue remodeling and angiogenesis (Linehan et al. 2018). When injury occurs, these commensal-specific T cells are poised for repair, and can promote wound healing in S. epidermidis colonized mice (Harrison et al. 2019). Skin commensals also promote the development of another subset of T cells, mucosal-associated invariant T (MAIT) cells. MAIT cells are innate-like lymphocytes that are tissue-resident in the skin. Early in life, commensals imprint on MAIT cells by producing vitamin B metabolites. Subsequently, these lymphocytes can be activated by skin commensals and promote tissue repair and wound healing (Constantinides et al. 2019).

      Commensal skin microbiota are also important for driving innate immune responses during healing. For example, commensals initiate a type I interferon (IFN) response through recruitment of IFN-producing dendritic cells. The IFN response stimulates fibroblasts and macrophages to release healing-related growth factors. Thus, skin microbiota trigger a type I IFN response that accelerates wound healing (Di Domizio et al. 2020). In a model of healing and regeneration known as wound-induced hair follicle neogenesis (WIHN), specific pathogen-free mice were found to have higher levels of healing than germ-free mice. Interestingly, this study found that skin bacteria—even S. aureus—promoted regeneration through IL1-β-dependent MyD88 signaling (Wang et al. 2021).

      Another aspect of the host innate immune response that is activated during wounding and enhanced by skin commensals is antimicrobial defense. Commensals can promote epidermal production of AMPs. A lipopeptide from S. epidermidis was shown to promote AMP expression in keratinocytes, and treatment of mice with these molecules prevented infection by group A Streptococcus and S. aureus (Lai et al. 2010; Li et al. 2013). In addition to promoting innate host defense, skin-resident bacteria produce antimicrobials themselves. Because epithelial microbiota exist in resource-restricted environments, they naturally develop competition strategies with bacteria that occupy the same niche. AMPs produced by coagulase-negative Staphylococci (CoNS), common inhabitants of the skin, selectively killed and decreased in vivo colonization by S. aureus (Nakatsuji et al. 2017). A human commensal Staphylococcus lugdunensis was reported to produce a novel antibiotic that prevents S. aureus colonization (Zipperer et al. 2016). These naturally evolved antagonistic mechanisms may be useful for countering pathogenic colonization during skin disease. Bacteriotherapy for AD, using S. hominis A9, was recently reported in a phase I clinical trial to significantly decrease S. aureus burden and improve disease activity of the skin (Nakatsuji et al. 2021b). These studies demonstrate that the skin microbiome is an underexplored reservoir of novel and promising antimicrobial therapies, which are desperately needed given the widespread problem of antibiotic resistance.

      As previously discussed, biofilms commonly form in chronic wound settings, increase resistance to antibiotic therapy, and are difficult to disrupt. However, there is recent evidence that skin microbiota can inhibit biofilms of pathogenic species. Certain S. epidermidis strains secrete a serine protease that inhibits formation and disrupts existing S. aureus biofilms (Iwase et al. 2010). Short chain fatty acids derived from C. acnes inhibits formation and increases antibiotic sensitivity of S. epidermidis biofilms (Nakamura et al. 2020). Microbe–microbe in teractions have also been shown to influence bacterial virulence. Corynebacterium striatum and S. aureus often coexist in the skin and wound environment. Interestingly, exposure of S. aureus to C. striatum shifts the transcriptome and fitness S. aureus from a pathogen to commensal-like state (Ramsey et al. 2016). Relatedly, skin commensal CoNS isolates were found to produce autoinducing peptides that repress agrquorum sensing of S. aureus and prevent S. aureus–mediated damage of the skin (Williams et al. 2019).

      Reepithelialization of the epidermis is crucial for proper healing, and this process is often deficient in chronic wounds. In a murine model of superficial barrier damage and wound healing, skin commensal bacteria were found to promote barrier repair and integrity through activation of keratinocyte aryl hydro carbon receptor (AHR) (Uberoi et al. 2021). These studies also found that precolonization with skin commensals prevented barrier damage and S. aureus infection during epicutaneous sensitization, an effect that was dependent on AHR. Future studies are needed to understand the microbial mediators of enhanced epidermal repair and how these signals may be different between pathogens and commensals.

      Together, these studies demonstrate that the skin microbiota can act as an adjuvant for the host wound healing response. However, most current clinical wound care guidelines focus on depletion of all wound bioburden. Therefore, further studies are needed to better parse apart the mechanisms of host–microbial interactions that lead to repair versus infection.

CONCLUDING REMARKS

      Our understanding of the wound microbiome has burgeoned in recent decades due in part to technical advances. Traditional culture-based methods gave us the first insights into the bacterial species that typically inhabit various wound types. However, next generation sequencing methods have afforded more comprehensive in vestigations into microbial wound inhabitants. Amplicon-based sequencing methods illuminate the community structure and diversity of wound microbiota. Shotgun metagenomic sequencing elucidates the functional capacity and strain-level variation contained in a microbial community. As these studies become more affordable and feasible to implement, cutting-edge techniques—such as metatranscriptomics—are simultaneously being implemented to study the skin microbiota.

      Diverse etiologies cause different wound types, such as DFUs, burn wounds, and PUs. Despite the diversity in their underlying causes and body sites, the microbial communities of these wound types share several common themes. A major theme that has emerged from wound microbiome studies is that S. aureus is abundant and prevalent across many wound types. Additionally, anaerobic bacteria such as Finegoldia and Anaerococcus are underestimated by culture methods but are prevalent in many wound types and often associated with worse outcomes. Another commonality is the polymicrobial nature of wound communities, with interactions even spanning across kingdoms.

      Given the polymicrobial nature of wound microbiota, it is important to distinguish between “good” and “bad” actors in the wound community. In terms of pathogens, these bacteria have adapted several virulence mechanisms that allow them to outcompete the other members of the microbiome and flourish in the wound bed. Biofilm formation, antibiotic resistance, and evasion of host immune responses are commonly employed virulence mechanisms that often lead to wound infection and poor outcomes. In contrast, several recent studies have shed light on commensal bacteria that are beneficial to wound healing. Microbial-based mechanisms of improved wound healing include increased recruitment of lymphocytes with healing effector functions, enhanced innate immune repair and antimicrobial defenses, and improved epithelial barrierrecovery.

     Future studies will need to integrate these evolving methods to study how the wound microbial community interacts with each other and the host. Addressing the lack in effective therapies to manage chronic nonhealing wounds, these studies have the potential to uncover novel and accessible targets of treatment that lie within the wound microbiome.

ACKNOWLEDGMENTS

      We thank the members of the Grice laboratory for their underlying contributions and our funding sources that make this work possible. Support to E.A.G. includes grants from the National Institutes of Health (R01NR015639, R01AI143790, R01AR079856), the Linda Pechenik Montague Investigator Award, and awards from the Burroughs Welcome Fund (PATH Award) and the Dermatology Foundation (Sun Pharma Research Award). E.K.W. is supported by a fellowship from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (F31AR079852). All figures were created with BioRender.

REFERENCES

1. Armstrong DG, Boulton AJM, Bus SA. 2017. Diabetic foot ulcers and their recurrence.N Engl J Med 376: 2367–2375. doi:10.1056/NEJMra1615439

2. Azzopardi EA, Azzopardi E, Camilleri L, Villapalos J, Boyce DE, Dziewulski P, Dickson WA, Whitaker IS. 2014. Gram negative wound infection in hospitalised adult burn patients-systematic review and metanalysis. PLoS ONE 9: e95042. doi:10.1371/journal.pone.0095042

3. Bowler PG, Duerden BI, Armstrong DG. 2001. Wound microbiology and associated approaches to wound management. Clin Microbiol Rev 14: 244–269. doi:10.1128/CMR .14.2.244-269.2001

4. Byeon J, Blizinsky KD, Persaud A, Findley K, Lee J, Buscetta AJ, You S, Bittinger K, Minniti CP, Bonham VL, et al. 2021. Insights into the skin microbiome of sickle cell disease leg ulcers. Wound Repair Regen 29: 801–809. doi:10.1111/wrr.12924

5. Byrd AL, Deming C, Cassidy SKB, Harrison OJ, Ng WI, Conlan S, Belkaid Y, Segre JA, Kong HH. 2017. Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis. Sci Transl Med 9: eaal4651. doi:10.1126/scitranslmed.aal4651

6. Cheong JZA, Johnson CJ, Wan H, Liu A, Kernien JF, Gibson ALF, Nett JE, Kalan LR. 2021. Priority effects dictate community structure and alter virulence of fungal-bacterial biofilms. J 15: 2012–2027. doi:10.1038/s41396-021-00901-5

7. Citron DM, Goldstein EJC, Merriam CV, Lipsky BA,Abramson MA. 2007. Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol 45: 2819–2828. doi:10.1128/ JCM.00551-07

8. Constantinides MG, Link VM, Tamoutounour S, Wong AC, Perez-Chaparro PJ, Han SJ, Chen YE, Li K, Farhat S, Weckel A, et al. 2019. MAIT cells are imprinted by the microbiota in early life and promote tissue repair. Science 366: eaax6624. doi:10.1126/science.aax6624

9. Cross HH. 2014. Obtaining a wound swab culture specimen. Nursing (Lond) 44: 68–69. doi:10.1097/01.NURSE .0000446645.33489.2e

10. Di Domizio J, Belkhodja C, Chenuet P, Fries A, Murray T, Mondéjar PM, Demaria O, Conrad C, Homey B, Werner S, et al. 2020. The commensal skin microbiota triggers type I IFN–dependent innate repair responses in injured skin. Nat Immunol 21: 1034–1045. doi:10.1038/s41590- 020-0721-6

11. Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA, Wolcott RD. 2008. Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol 8: 43. doi:10.1186/1471-2180-8-43

12. Dunyach-Remy C, Salipante F, Lavigne JP, Brunaud M, Demattei C, Yahiaoui-Martinez A, Bastide S, Palayer C, Sotto A, Gélis A. 2021. Pressure ulcers microbiota dynamics and wound evolution. Sci Rep 11: 18506. doi:10.1038/ s41598-021-98073-x

13. Fox EP, Cowley ES, Nobile CJ, Hartooni N, Newman DK, Johnson AD. 2014. Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures. Curr Biol 24: 2411–2416. doi:10 .1016/j.cub.2014.08.057

14. Frykberg RG, Banks J. 2015. Challenges in the treatment of chronic wounds. Adv Wound Care 4: 560–582. doi:10 .1089/wound.2015.0635

15. Gardner SE, Frantz RA. 2008. Wound bioburden and infection-related complications in diabetic foot ulcers. Biol Res Nurs 10: 44–53. doi:10.1177/1099800408319056

16. Gardner SE, Frantz RA, Saltzman CL, Hillis SL, Park H, Scherubel M. 2006. Diagnostic validity of three swab techniques for identifying chronic wound infection. Wound Repair Regen 14: 548–557. doi:10.1111/j.1743-6109.2006 .00162.x

17. Gardner SE, Hillis SL, Heilmann K, Segre JA, Grice EA. 2013. The neuropathic diabetic foot ulcer microbiome is associated with clinical factors. Diabetes 62: 923–930. doi:10.2337/db12-0771

18. Gardner SE, Haleem A, Jao YL, Hillis SL, Femino JE, Phisitkul P, Heilmann KP, Lehman SM, Franciscus CL. 2014. Cultures of diabetic foot ulcers without clinical signs of infection do not predict outcomes. Diabetes Care 37: 2693–2701. doi:10.2337/dc14-0051

19. Gong Y, Chen J, Liu C, Zhang C, Luo X, Peng Y. 2014. Comparison of pathogens and antibiotic resistance of burn patients in the burn ICU or in the common burn ward. Burns 40: 402–407. doi:10.1016/j.burns.2013.07 .010

20. Grogan MD, Bartow-McKenney C, Flowers L, Knight SAB, Uberoi A, Grice EA. 2019. Research techniques made simple: profiling the skin microbiota. J Invest Dermatol 139: 747–752.e1. doi:10.1016/j.jid.2019.01.024

21. Han A, Zenilman JM, Melendez JH, Shirtliff ME, Agostinho A, James G, Stewart PS, Mongodin EF, Rao D, Rickard AH, et al. 2011. The importance of a multifaceted approach to characterizing the microbial flora of chronic wounds. Wound Repair Regen 19: 532–541. doi:10 .1111/j.1524-475X.2011.00720.x

22. Harrison OJ, Linehan JL, Shih HY, Bouladoux N, Han SJ, Smelkinson M, Sen SK, Byrd AL, Enamorado M, Yao C, et al. 2019. Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury. Science 363: eaat6280. doi:10.1126/science.aat6280

23. Howell-Jones RS, Price PE, Howard AJ, Thomas DW. 2006. Antibiotic prescribing for chronic skin wounds in primary care. Wound Repair Regen 14: 387–393. doi:10.1111/j .1743-6109.2006.00144.x

24. Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, Agata T, Mizunoe Y. 2010. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature 465: 346–349. doi:10 .1038/nature09074

25. James GA, Swogger E, Wolcott R, Pulcini ED, Secor P, Sestrich J, Costerton JW, Stewart PS. 2008. Biofilms in chronic wounds. Wound Repair Regen 16: 37–44. doi:10.1111/j .1524-475X.2007.00321.x

26. Järbrink K, Ni G, Sönnergren H, Schmidtchen A, Pang C, Bajpai R, Car J. 2017. The humanistic and economic burden of chronic wounds: a protocol for a systematic review. Syst Rev 6: 15. doi:10.1186/s13643-016-0400-8

27. Jo JH, Kennedy EA, Kong HH. 2016. Research techniques made simple: bacterial 16S ribosomal RNA gene sequencing in cutaneous research. J Invest Dermatol 136: e23–e27. doi:10.1016/j.jid.2016.01.005

28. Jo JH, Harkins CP, Schwardt NH, Portillo JA, Zimmerman MD, Carter CL, Hossen MA, Peer CJ, Polley EC, Dartois V, et al. 2021. Alterations of human skin microbiome and expansion of antimicrobial resistance after systemic antibiotics. Sci Transl Med 13: 8077. doi:10.1126/scitransl med.abd8077

29. Johani K, Malone M, Jensen S, Gosbell I, Dickson H, Hu H, Vickery K. 2017. Microscopy visualisation confirms multi-species biofilms are ubiquitous in diabetic foot ulcers. Int Wound J 14: 1160–1169. doi:10.1111/iwj.12777

30. Juntachai W, Oura T, Murayama SY, Kajiwara S. 2009. The lipolytic enzymes activities of Malassezia species. Med Mycol 47: 477–484. doi:10.1080/13693780802314825

31. Kalan L, Grice EA. 2018. Fungi in the wound microbiome. Adv Wound Care 7: 247–255. doi:10.1089/wound.2017 .0756

32. Kalan L, Loesche M, Hodkinson BP, Heilmann K, Ruthel G, Gardner SE, Grice EA. 2016. Redefining the chronic wound microbiome: fungal communities are prevalent, dynamic, and associated with delayed healing. MBio 7:e01058-16. doi:10.1128/mBio.01058-16

33. Kalan LR, Meisel JS, Loesche MA, Horwinski J, Soaita I, Chen X, Uberoi A, Gardner SE, Grice EA. 2019. Strain and species-level variation in the microbiome of diabetic wounds is associated with clinical outcomes and therapeutic efficacy. Cell Host Microbe 25: 641–655.e5. doi:10 .1016/j.chom.2019.03.006

34. Kirker KR, Secor PR, James GA, Fleckman P, Olerud JE, Stewart PS. 2009. Loss of viability and induction of apo ptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro. Wound Repair Regen 17: 690– 699. doi:10.1111/j.1524-475X.2009.00523.x

35. Lai Y, Cogen AL, Radek KA, Park HJ, MacLeod DT, Leichtle A, Ryan AF, Di Nardo A, Gallo RL. 2010. Activation of TLR2 by a small molecule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections. J Invest Dermatol 130: 2211–2221. doi:10.1038/jid.2010.123

36. Levine NS, Lindberg RB, Mason ADJ, Pruitt BAJ. 1976. The quantitative swab culture and smear: a quick, simple method for determining the number of viable aerobic bacteria on open wounds. J Trauma 16: 89–94. doi:10 .1097/00005373-197602000-00002

37. Li D, Lei H, Li Z, Li H, Wang Y, Lai Y. 2013. A novel lipopeptide from skin commensal activates TLR2/CD36- p38 MAPK signaling to increase antibacterial defense against bacterial infection. PLoS ONE 8: e58288. doi:10 .1371/journal.pone.0058288

38. Lima KM, Davis RR, Liu SY, Greenhalgh DG, Tran NK. 2021. Longitudinal profiling of the burn patient cutaneous and gastrointestinal microbiota: a pilot study. Sci Reports 11: 10667. doi:10.1038/s41598-021-89822-z

39. Linehan JL, Harrison OJ, Han SJ, Byrd AL, Vujkovic-Cvijin I, Villarino AV, Sen SK, Shaik J, Smelkinson M, Tamoutounour S, et al. 2018. Non-classical immunity controls microbiota impact on skin immunity and tissue repair. Cell 172: 784–796.e18. doi:10.1016/j.cell.2017.12.033

40. Lipsky BA, Berendt AR, Cornia PB, Pile JC, Peters EJG, Armstrong DG, Deery HG, Embil JM, Joseph WS, Karchmer AW, et al. 2012. 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clin Infect Dis 54: e132–e173. doi:10.1093/cid/cis346

41. Loesche M, Gardner SE, Kalan L, Horwinski J, Zheng Q, Hodkinson BP, Tyldsley AS, Franciscus CL, Hillis SL, Mehta S, et al. 2017. Temporal stability in chronic wound microbiota is associated with poor healing. J Invest Dermatol 137: 237–244. doi:10.1016/j.jid.2016.08.009

42. López D, Vlamakis H, Kolter R. 2010. Biofilms. Cold Spring Harb Perspect Biol 2: a000398. doi:10.1101/cshperspect .a000398

43. Malone M, Bjarnsholt T, McBain AJ, James GA, Stoodley P, Leaper D, Tachi M, Schultz G, Swanson T, Wolcott RD. 2017a. The prevalence of biofilms in chronic wounds: a systematic review and meta-analysis of published data. J Wound Care 26: 20–25. doi:10.12968/jowc.2017.26.1.20

44. Malone M, Johani K, Jensen SO, Gosbell IB, Dickson HG, Hu H, Vickery K. 2017b. Next generation DNA sequencing of tissues from infected diabetic foot ulcers. EBioMed 21: 142–149. doi:10.1016/j.ebiom.2017.06.026

45. Margolis DJ, Malay DS, Hoffstad OJ, Leonard CE, MaCurdy T, Tan Y, Molina T, de Nava KL, Siegel KL. 2011. Economic burden of diabetic foot ulcers and amputations: data points #3. Agency for Healthcare Research and Quality (US), Rockville, MD.

46. Meisel JS, Hannigan GD, Tyldsley AS, SanMiguel AJ, Hodkinson BP, Zheng Q, Grice EA. 2016. Skin microbiome surveys are strongly influenced by experimental design. J Invest Dermatol 136: 947–956. doi:10.1016/j.jid.2016.01 .016

47. Min KR, Galvis A, Nole KLB, Sinha R, Clarke J, Kirsner RS, Ajdic D. 2020. Association between baseline abundance of peptoniphilus, a gram-positive anaerobic coccus, and wound healing outcomes of DFUs. PLoS ONE 15: e0227006. doi:10.1371/journal.pone.0227006

48. Minniti CP, Eckman J, Sebastiani P, Steinberg MH, Ballas SK. 2010. Leg ulcers in sickle cell disease. Am J Hematol 85: 831–833. doi:10.1002/ajh.21838

49. Moran GJ, Krishnadasan A, Gorwitz RJ, Fosheim GE, McDougal LK, Carey RB, Talan DA. 2006. Methicillinresistant S. aureus infections among patients in the emergency department. N Engl J Med 355: 666–674. doi:10 .1056/NEJMoa055356

50. Mottola C, Semedo-Lemsaddek T, Mendes JJ, Melo-Cristino J, Tavares L, Cavaco-Silva P, Oliveira M. 2016. Molecular typing, virulence traits and antimicrobial resistance of diabetic foot staphylococci. J Biomed Sci 23: 33. doi:10 .1186/s12929-016-0250-7

51. Naik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, Conlan S, Himmelfarb S, Byrd AL, Deming C, et al. 2015. Commensal–dendritic-cell interaction specifies a unique protective skin immune signature. Nature 520: 104–108. doi:10.1038/nature14052

52. Nakamura K, O’Neill AM, Williams MR, Cau L, Nakatsuji T, Horswill AR, Gallo RL. 2020. Short chain fatty acids produced by Cutibacterium acnes inhibit biofilm formation by Staphylococcus epidermidis. Sci Reports 10: 1–12.

53. Nakatsuji T, Chen TH, Narala S, Chun KA, Two AM, Yun T, Shafiq F, Kotol PF, Bouslimani A, Melnik AV, et al. 2017. Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis. Sci Transl Med 9: eaah4680. doi:10 .1126/scitranslmed.aah4680

54. Nakatsuji T, Cheng JY, Gallo RL. 2021a. Mechanisms for control of skin immune function by the microbiome. Curr Opin Immunol 72: 324–330. doi:10.1016/j.coi.2021 .09.001

55. Nakatsuji T, Hata TR, Tong Y, Cheng JY, Shafiq F, Butcher AM, Salem SS, Brinton SL, Rudman Spergel AK, Johnson K, et al. 2021b. Development of a human skin commensal microbe for bacteriotherapy of atopic dermatitis and use in a phase 1 randomized clinical trial. Nat Med 27: 700– 709. doi:10.1038/s41591-021-01256-2

56. Nussbaum SR, Carter MJ, Fife CE, DaVanzo J, Haught R, Nusgart M, Cartwright D. 2018. An economic evaluation of the impact, cost, and Medicare policy implications of chronic nonhealing wounds. Value Heal 21: 27–32. doi:10.1016/j.jval.2017.07.007

57. Oh J, Byrd AL, Deming C, Conlan S, NISC Comparative Sequencing Program, Kong HH, Segre JA. 2014. Biogeography and individuality shape function in the human skin metagenome. Nature 514: 59–64. doi:10.1038/na ture13786

58. Olaniyi R, Pozzi C, Grimaldi L, Bagnoli F. 2017. Staphylococcus aureus–associated skin and soft tissue infections: anatomical localization, epidemiology, therapy and potential prophylaxis. Curr Top Microbiol Immunol 409: 199–227.

59. Pastar I, Sawaya AP, Marjanovic J, Burgess JL, Strbo N, Rivas KE, Wikramanayake TC, Head CR, Stone RC, Jozic I, et al. 2021. Intracellular Staphylococcus aureus triggers pyroptosis and contributes to inhibition of healing due to perforin-2 suppression. J Clin Invest 131: e133727. doi:10 .1172/JCI133727

60. Price LB, Liu CM, Melendez JH, Frankel YM, Engelthaler D, Aziz M, Bowers J, Rattray R, Ravel J, Kingsley C, et al. 2009. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota. PLoS ONE 4: e6462. doi:10.1371/journal.pone .0006462

61. Ramirez HA, Pastar I, Jozic I, Stojadinovic O, Stone RC, Ojeh N, Gil J, Davis SC, Kirsner RS, Tomic-Canic M. 2018. Staphylococcus aureus triggers induction of miR-15B-5P to diminish DNA repair and deregulate inflammatory response in diabetic foot ulcers. J Invest Dermatol 138: 1187–1196. doi:10.1016/j.jid.2017.11.038

62. Ramsey MM, Freire MO, Gabrilska RA, Rumbaugh KP, Lemon KP. 2016. Staphylococcus aureus shifts toward commensalism in response to Corynebacterium species. Front Microbiol 7: 1230. doi:10.3389/fmicb.2016.01230

63. Robson MC, Heggers JP. 1969. Bacterial quantification of open wounds. Mil Med 134: 19–24. doi:10.1093/ milmed/134.1.19

64. Ronat JB, Kakol J, Khoury MN, Berthelot M, Yun O, Brown V, Murphy RA. 2014. Highly drug-resistant pathogens implicated in burn-associated bacteremia in an Iraqi burn care unit. PLoS ONE 9: e101017. doi:10.1371/jour nal.pone.0101017

65. Schmid-Wendtner MH, Korting HC. 2006. The pH of the skin surface and its impact on the barrier function. Skin Pharmacol Physiol 19: 296–302. doi:10.1159/000094670

66. Secor PR, James GA, Fleckman P, Olerud JE, McInnerney K, Stewart PS. 2011. Staphylococcus aureus biofilm and planktonic cultures differentially impact gene expression, MAPK phosphorylation, and cytokine production in human keratinocytes. BMC Microbiol 11: 143. doi:10.1186/ 1471-2180-11-143

67. Sen CK. 2021. Human wound and its burden: updated 2020 compendium of estimates. Adv Wound Care 10: 281–292. doi:10.1089/wound.2021.0026

68. Sloan TJ, Turton JC, Tyson J, Musgrove A, Fleming VM, Lister MM, Loose MW, Sockett RE, Diggle M, Game FL, et al. 2019. Examining diabetic heel ulcers through an ecological lens: microbial community dynamics associated with healing and infection. J Med Microbiol 68: 230–240. doi:10.1099/jmm.0.000907

69. Smith DM, Snow DE, Rees E, Zischkau AM, Hanson JD, Wolcott RD, Sun Y, White J, Kumar S, Dowd SE. 2010. Evaluation of the bacterial diversity of pressure ulcers using bTEFAP pyrosequencing. BMC Med Genomics 3: 1–12. doi:10.1186/1755-8794-3-41

70. Spiliopoulou AI, Kolonitsiou F, Krevvata MI, Leontsinidis M, Wilkinson TS, Mack D, Anastassiou ED. 2012. Bacterial adhesion, intracellular survival and cytokine induction upon stimulation of mononuclear cells with planktonic or biofilm phase Staphylococcus epidermidis. FEMS Microbiol Lett 330: 56–65. doi:10.1111/j.1574-6968.2012 .02533.x

71. Stewart PS, Costerton JW. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358: 135–138. doi:10.1016/ S0140-6736(01)05321-1

72. Strbo N, Pastar I, Romero L, Chen V, Vujanac M, Sawaya AP, Jozic I, Ferreira ADF, Wong LL, Head C, et al. 2019. Single cell analyses reveal specific distribution of anti-bacterial molecule perforin-2 in human skin and its modulation by wounding and Staphylococcus aureus infection. Exp Dermatol 28: 225–232. doi:10.1111/exd.13870

73. Stroupe KT, Manheim L, Evans CT, Guihan M, Ho C, Li K, Cowper-Ripley D, Hogan TP, St. Andre JR, Huo Z, et al. 2011. Cost of treating pressure ulcers for veterans with spinal cord injury. Top Spinal Cord Inj Rehabil 16: 62–73. doi:10.1310/sci1604-62

74. Swaney MH, Kalan LR. 2021. Living in your skin: microbes, molecules, and mechanisms. Infect Immun 89: e00695- 20. doi:10.1128/IAI.00695-20

75. Sweere JM, Van Belleghem JD, Ishak H, Bach MS, Popescu M, Sunkari V, Kaber G, Manasherob R, Suh GA, Cao X, et al. 2019. Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection. Science 363: eaat9691. doi:10.1126/science.aat9691

76. Tentolouris N, Petrikkos G, Vallianou N, Zachos C, Daikos GL, Tsapogas P, Markou G, Katsilambros N. 2006. Prevalence of methicillin-resistant Staphylococcus aureus in infected and uninfected diabetic foot ulcers. Clin Microbiol Infect 12: 186–189. doi:10.1111/j.1469-0691.2005 .01279.x

77. Thomsen TR, Aasholm MS, Rudkjøbing VB, Saunders AM, Bjarnsholt T, Givskov M, Kirketerp-Møller K, Nielsen PH. 2010. The bacteriology of chronic venous leg ulcer examined by culture-independent molecular methods. Wound Repair Regen 18: 38–49. doi:10.1111/j.1524-475X.2009.00561.x

78. Tipton CD, Mathew ME, Wolcott RA, Wolcott RD, Kingston T, Phillips CD. 2017. Temporal dynamics of relative abundances and bacterial succession in chronic wound communities. Wound Repair Regen 25: 673–679. doi:10 .1111/wrr.12555

79. Tiwari VK. 2012. Burn wound: how it differs from other wounds? Indian J Plast Surg 45: 364–373. doi:10.4103/ 0970-0358.101319

80. Tuttle MS, Mostow E, Mukherjee P, Hu FZ, Melton-Kreft R, Ehrlich GD, Dowd SE, Ghannoum MA. 2011. Characterization of bacterial communities in venous insufficiency wounds by use of conventional culture and molecular diagnostic methods. J Clin Microbiol 49: 3812–3819. doi:10.1128/JCM.00847-11

81. Uberoi A, Bartow-McKenney C, Zheng Q, Flowers L, Campbell A, Knight SAB, Chan N, Wei M, Lovins V, Bugayev J, et al. 2021. Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor. Cell Host Microbe 29: 1235–1248.e8. doi:10.1016/j.chom.2021.05.011

82. van Langeveld I, Gagnon RC, Conrad PF, Gamelli RL, Martin B, Choudhry MA, Mosier MJ. 2017. Multiple-drug resistance in burn patients: a retrospective study on the impact of antibiotic resistance on survival and length of stay. J Burn Care Res 38: 99–105. doi:10.1097/BCR .0000000000000479

83. Verbanic S, Shen Y, Lee J, Deacon JM, Chen IA. 2020. Microbial predictors of healing and short-term effect of debridement on the microbiome of chronic wounds. NPJ Biofilms Microbiomes 6: 21. doi:10.1038/s41522-020-0130-5

84. Wang G, Sweren E, Liu H, Wier E, Alphonse MP, Chen R,Islam N, Li A, Xue Y, Chen J, et al. 2021. Bacteria induce skin regeneration via IL-1β signaling. Cell Host Microbe 29: 777–791.e6. doi:10.1016/j.chom.2021.03.003

85. Westermann AJ, Gorski SA, Vogel J. 2012. Dual RNA-seq of pathogen and host. Nat Rev Microbiol 10: 618–630. doi:10 .1038/nrmicro2852

86. Williams MR, Costa SK, Zaramela LS, Khalil S, Todd DA, Winter HL, Sanford JA, O’Neill AM, Liggins MC, Nakatsuji T, et al. 2019. Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis. Sci Transl Med 11: 8329. doi:10.1126/ scitranslmed.aat8329

87. Zhang C, Merana GR, Harris-Tryon T, Scharschmidt TC. 2022. Skin immunity: dissecting the complex biology of our body’s outer barrier. Mucosal Immunol 15: 551–561. doi:10.1038/s41385-022-00505-y

88. Zipperer A, Konnerth MC, Laux C, Berscheid A, Janek D, Weidenmaier C, Burian M, Schilling NA, Slavetinsky C, Marschal M, et al. 2016. Human commensals producing a novel antibiotic impair pathogen colonization. Nature 535: 511–516. doi:10.1038/nature18634

89. Zulkowski K, Langemo D, Posthauer ME. 2005. Coming to consensus on deep tissue injury. Adv Skin Wound Care 18: 28–29. doi:10.1097/00129334-200501 000-00013

This article is excerpted from the 《The Wound Microbiome》 by Wound World.