Malassezia: A Commensal, Pathogen, and Mutualist of Human and Animal Skin.

Identified in the late nineteenth century as a single species residing on human skin, Malassezia is now recognized as a diverse genus comprising 18 species inhabiting not only skin but human gut, hospital environments, and even deep-sea sponges. All cultivated Malassezia species are lipid dependent, having lost genes for lipid synthesis and carbohydrate metabolism. The surging interest in Malassezia results from development of tools to improve sampling, culture, identification, and genetic engineering, which has led to findings implicating it in numerous skin diseases, Crohn disease, and pancreatic cancer. However, it has become clear that Malassezia plays a multifaceted role in human health, with mutualistic activity in atopic dermatitis and a preventive effect against other skin infections due to its potential to compete with skin pathogens such as Candida auris. Improved understanding of complex microbe-microbe and host-microbe interactions will be required to define Malassezia's role in human and animal health and disease so as to design targeted interventions.

A 3D-printed transepidermal microprojection array for human skin microbiome sampling.

Skin microbiome sampling is currently performed with tools such as swabs and tape strips to collect microbes from the skin surface. However, these conventional approaches may be unable to detect microbes deeper in the epidermis or in epidermal invaginations. We describe a sampling tool with a depth component, a transepidermal microprojection array (MPA), which captures microbial biomass from both the epidermal surface and deeper skin layers. We leveraged the rapid customizability of 3D printing to enable systematic optimization of MPA for human skin sampling. Evaluation of sampling efficacy on human scalp revealed the optimized MPA was comparable in sensitivity to swab and superior to tape strip, especially for nonstandard skin surfaces. We observed differences in species diversity, with the MPA detecting clinically relevant fungi more often than other approaches. This work delivers a tool in the complex field of skin microbiome sampling to potentially address gaps in our understanding of its role in health and disease.

The human pathobiont Malassezia furfur secreted protease Mfsap1 regulates cell dispersal and exacerbates skin inflammation.

Malassezia form the dominant eukaryotic microbial community on the human skin. The Malassezia genus possesses a repertoire of secretory hydrolytic enzymes involved in protein and lipid metabolism which alter the external cutaneous environment. The exact role of most Malassezia secreted enzymes, including those in interaction with the epithelial surface, is not well characterized. In this study, we compared the expression level of secreted proteases, lipases, phospholipases, and sphingomyelinases of Malassezia globosa in healthy subjects and seborrheic dermatitis or atopic dermatitis patients. We observed upregulated gene expression of the previously characterized secretory aspartyl protease MGSAP1 in both diseased groups, in lesional and non-lesional skin sites, as compared to healthy subjects. To explore the functional roles of MGSAP1 in skin disease, we generated a knockout mutant of the homologous protease MFSAP1 in the genetically tractable Malassezia furfur. We observed the loss of MFSAP1 resulted in dramatic changes in the cell adhesion and dispersal in both culture and a human 3D reconstituted epidermis model. In a murine model of Malassezia colonization, we further demonstrated Mfsap1 contributes to inflammation as observed by reduced edema and inflammatory cell infiltration with the knockout mutant versus wildtype. Taken together, we show that this dominant secretory Malassezia aspartyl protease has an important role in enabling a planktonic cellular state that can potentially aid in colonization and additionally as a virulence factor in barrier-compromised skin, further highlighting the importance of considering the contextual relevance when evaluating the functions of secreted microbial enzymes.

Azole resistance mechanisms in pathogenic M. furfur.

Malassezia are emerging fungal pathogens causing opportunistic skin and severe systemic infection. Nosocomial outbreaks are associated with azole resistance and understanding of the underlying mechanisms are limited to knowledge from other fungal species. Herein, we identified distinct antifungal susceptibility patterns in 26 Malassezia furfur isolates derived from healthy and diseased individuals. A Y67F CYP51 mutation was identified in five isolates of M. furfur However, this mutation alone was insufficient to induce reduce azole susceptibility in the wild type strain. RNA-seq and differential gene analysis of healthy and disease derived strains exposed to clotrimazole in vitro identified several key metabolic pathways and transporter proteins which are involved in reduce azole susceptibility. The pleiotropic drug transporter PDR10 was the single most highly upregulated transporter gene in multiple strains of M. furfur after azole treatment and increased expression of PDR10 is associated with reduced azole susceptibility in some systemic disease isolates of M. furfur Deletion of PDR10 in a pathogenic M. furfur strain with reduced susceptibility reduced MIC values to the level of that in susceptible isolates. The current dearth of antifungal technologies, globally emerging multi-azole resistance, and broad agriculture and consumer care use of azoles means improved understanding of the mechanisms underlying intrinsic and acquired azole resistance in Malassezia is crucial for development of antibiotic stewardship and antifungal treatment strategies.

Comparative analysis of Malassezia furfur mitogenomes and the development of a mitochondria-based typing approach.

Malassezia furfur is a yeast species belonging to Malasseziomycetes, Ustilaginomycotina and Basidiomycota that is found on healthy warm-blooded animal skin, but also involved in various skin disorders like seborrheic dermatitis/dandruff and pityriasis versicolor. Moreover, Malassezia are associated with bloodstream infections, Crohn's disease and pancreatic carcinoma. Recent advances in Malassezia genomics and genetics have focused on the nuclear genome. In this work, we present the M. furfur mitochondrial (mt) genetic heterogenicity with full analysis of 14 novel and six available M. furfur mt genomes. The mitogenome analysis reveals a mt gene content typical for fungi, including identification of variable mt regions suitable for intra-species discrimination. Three of them, namely the trnK-atp6 and cox3-nad3 intergenic regions and intron 2 of the cob gene, were selected for primer design to identify strain differences. Malassezia furfur strains belonging to known genetic variable clusters, based on AFLP and nuclear loci, were assessed for their mt variation using PCR amplification and sequencing. The results suggest that these mt regions are excellent molecular markers for the typing of M. furfur strains and may provide added value to nuclear regions when assessing evolutionary relationships at the intraspecies level.

Effect of zinc pyrithione shampoo treatment on skin commensal Malassezia.

Malassezia restricta and Malassezia globosa are lipid dependent commensal yeasts associated with dandruff. Antifungal actives such as zinc pyrithione are commonly used in antidandruff shampoos, although their efficacy is not clearly demonstrated. In this study, we assessed the efficacy of antifungal treatments on scalp Malassezia via a combination of culturomic and genomic detection methods. Zinc pyrithione inhibited Malassezia growth at low minimum inhibitory concentrations (MICs). In a longitudinal pilot study, quantitative polymerase chain reaction (qPCR) analysis showed a decrease in M. restricta on the scalp after zinc pyrithione treatment. These findings validate the antifungal efficacy of zinc pyrithione as a dandruff treatment. LAY ABSTRACT: Malassezia yeasts are associated with dandruff and seborrheic dermatitis. Zinc pyrithione is effective against Malassezia growth in vitro and when tested on human skin as a shampoo. These findings will be useful for investigating the role of Malassezia in skin microbiome intervention studies.

Wanted, dead and alive: Why a multidisciplinary approach is needed to unlock hair treatment potential.

Human recorded history is littered with attempts to improve the perceived appearance of scalp hair. Throughout history, treatments have included both biological and chemical interventions. Hair "quality" or "perceived appearance" is regulated by multiple biological intervention opportunities: adding more hairs by flipping follicles from telogen to anagen, or delaying anagen follicles transiting into catagen; altering hair "apparent amount" by modulating shaft diameter or shape; or, in principle, altering shaft physical properties changing its synthesis. By far the most common biological intervention strategy today is to increase the number of hairs, but to date this has proven difficult and has yielded minimal benefits. Chemical intervention primarily consists of active material surface deposition to improve shaft shine, fibre-fibre interactions and strength. Real, perceptible benefits will best be achieved by combining opportunity areas across the three primary sciences: biology, chemistry and physics. Shaft biogenesis begins with biology: proliferation in the germinative matrix, then crossing "Auber's Critical Line" and ceasing proliferation to synthesize shaft components. Biogenesis then shifts to oxidative chemistry, where previously synthesized components are organized and cross-linked into a shaft. We herein term the crossing point from biology to chemistry as "The Orwin Threshold." Historically, hair biology and chemistry have been conducted in different fields, with biological manipulation residing in biomedical communities and hair shaft chemistry and physics within the consumer care industry, with minimal cross-fertilization. Detailed understanding of hair shaft biogenesis should enable identification of factors necessary for optimum hair shaft production and new intervention opportunities.

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis.

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.

Malassezia ecology, pathophysiology, and treatment.

Malassezia are lipid dependent basidiomycetous yeasts that inhabit the skin and mucosa of humans and other warm-blooded animals, and are a major component of the skin microbiome. They occur as skin commensals, but are also associated with various skin disorders and bloodstream infections. The genus currently comprises 17 species and has recently been assigned its own class, Malasseziomycetes. Importantly, multiple Malassezia species and/or genotypes may cause unique or similar pathologies and vary in their antifungal susceptibility. In addition to culture-based approaches, culture-independent methods have added to our understanding of Malassezia presence and abundance and their relationship to pathogenicity. Moreover, these novel approaches have suggested a much wider-spread presence, including other human body parts and even other ecosystems, but their role in these arenas requires further clarification. With recent successful transformation and genetic engineering of Malassezia, the role of specific genes in pathogenesis can now be studied. We suggest that characterizing the metabolic impact of Malassezia communities rather than species identification is key in elucidation of pathophysiological associations. Finally, the increasing availability of genome sequences may provide key information aiding faster diagnostics, and understanding of the biochemical mechanisms for Malassezia skin adaptation and the design of future drugs.

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin.

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin's carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.

Hair Shaft Formation and Mitochondrial Bioenergetics: Combining Biology, Chemistry, and Physics.

Research into biological manipulation of hair "quality" has ebbed and waned but today is in a resurgence. Hair appearance is regulated by multiple intervention opportunities-adding more hairs; increasing hair "amount" by modulating shaft diameter or shape; or, in principle, by altering shaft physical properties by changing its synthesis. It is likely that improved benefits may be achieved by combining multiple areas-minimizing follicle loss and miniaturization, maximizing shaft production, and treating the existing shaft. A previously overlooked opportunity is follicle metabolism: building "better" hairs. Hair production is energy intensive, and it is known that follicle metabolism influences shaft diameter. Multiphoton microscopy enables metabolic investigation of live, growing, human, hair follicles. This allows definition of multiple "zones" with vastly different metabolism: proliferation-where keratinocytes proliferate and migrate into specialized layers; production-proliferation ceases, and synthesis and patterning begin; construction and elongation-the structural framework is seeded and cells extend to create the nascent fiber; and maturation-gradual hardening and transformation into mature shaft. Recent investigations into the transition from construction to maturation reinforce this as a key developmental threshold, where shaft production transforms from a biologically driven into a biochemically driven process. We now name this "Orwin's transition."

A Malassezia pseudoprotease dominates the secreted hydrolase landscape and is a potential allergen on skin.

Malassezia globosa is abundant and prevalent on sebaceous areas of the human skin. Genome annotation reveals that M. globosa possesses a repertoire of secreted hydrolytic enzymes relevant for lipid and protein metabolism. However, the functional significance of these enzymes is uncertain and presence of these genes in the genome does not always translate to expression at the cutaneous surface. In this study we utilized targeted RNA sequencing from samples isolated directly from the skin to quantify gene expression of M. globosa secreted proteases, lipases, phospholipases and sphingomyelinases. Our findings indicate that the expression of these enzymes is dynamically regulated by the environment in which the fungus resides, as different growth phases of the planktonic culture of M. globosa show distinct expression levels. Furthermore, we observed significant differences in the expression of these enzymes in culture compared to healthy sebaceous skin sites. By examining the in situ gene expression of M. globosa's secreted hydrolases, we identified a predicted aspartyl protease, MGL_3331, which is highly expressed on both healthy and disease-affected dermatological sites. However, molecular modeling and biochemical studies revealed that this protein has a non-canonical active site motif and lacks measurable proteolytic activity. This pseudoprotease MGL_3331 elicits a heightened IgE-reactivity in blood plasma isolated from patients with atopic dermatitis compared to healthy individuals and invokes a pro-inflammatory response in peripheral blood mononuclear cells. Overall, our study highlights the importance of studying fungal proteins expressed in physiologically relevant environments and underscores the notion that secreted inactive enzymes may have important functions in influencing host immunity.

Retrospective review of medication-related incidents at a major teaching hospital and the potential mitigation of these incidents with electronic prescribing and medicines administration.

OBJECTIVES: To describe the frequency of the different types of medication-related incidents that caused patient harm, or adverse consequences, in a major teaching hospital and investigate whether the likelihood of these incidents occurring would have been reduced by electronic prescribing and medicines administration (EPMA). METHODS: A retrospective review of harmful incidents (n=387) was completed for medication-related reports at the hospital between 1 September 2020 and 31 August 2021. Frequencies of different types of incidents were collated. The potential for EPMA to have prevented these incidents was assessed by reviewing DATIX reports and additional information, including results of any investigations. RESULTS: The largest proportion of harmful medication incidents were administration related (n=215, 55.6%), followed by incidents classified as 'other' and 'prescribing'. Most incidents were classified as low harm (n=321, 83.0%). EPMA could have reduced the likelihood of all incidents which caused harm by 18.6% (n=72) without configuration, and a further 7.5% (n=29) with configuration where configuration refers to adapting the software's functionality without supplier input or development. For 18.4% of the low-harm incidents (n=59) and 20.3% (n=13) of the moderate-harm incidents, EPMA could reduce the likelihood of the incident occurring without configuration. Medication errors most likely to be reduced by EPMA were due to illegibility, multiple drug charts or missing drug charts. CONCLUSION: This study found that administration incidents were the most common type of medication-related incidents. Most of the incidents (n=243, 62.8%) could not be mitigated by EPMA in any circumstance, even with connectivity between technologies. EPMA has the potential to prevent certain types of harmful medication-related incidents, and further improvements could be achieved with configuration and development.

Reducing Therapeutic Duplication in Inpatient Medication Orders.

BACKGROUND: Therapeutic duplication, the presence of multiple agents prescribed for the same indication without clarification for when each should be used, can contribute to serious medical errors. Joint Commission standards require that orders contain clarifying information about when each order should be given. In our system, as needed (PRN) acetaminophen and ibuprofen orders are major contributors to therapeutic duplication. OBJECTIVE: The objective of this study is to design and evaluate effectiveness of clinical decision support (CDS) to reduce therapeutic duplication with acetaminophen and ibuprofen orders. METHODS: This study was done in a pediatric health system with three freestanding hospitals. We iteratively designed and implemented two CDS strategies aimed at reducing the therapeutic duplication with these agents: (1) interruptive alert prompting clinicians for clarifying PRN comments at order entry and (2) addition of discrete "first-line" and "second-line" PRN reasons to orders. Therapeutic duplications were measured by manual review of orders for 30-day periods before and after each intervention and 6 months later. RESULTS: Therapeutic duplications decreased from 1,485 in the 30 days prior to the first alert implementation to 818 in the 30 days after but rose back to 1,208 in the 30 days prior to the second intervention. After discrete reasons were added to the order, therapeutic duplication decreased to 336 in the immediate 30 days and 6 months later remained at 277. Alerts firing rates decreased from 76.0 per 1,000 PRN acetaminophen or ibuprofen orders to 42.9 after the second intervention. CONCLUSION: Interruptive alerts may reduce therapeutic duplication but are associated with high rates of user frustration and alert fatigue. Leveraging discrete PRN reasons for "first line" and "second line" produced a greater reduction in therapeutic duplication as well as fewer interruptive alerts and less manual entry for providers.

Pharmacist Metrics in the Pediatric Intensive Care Unit: an Exploration of the Medication Regimen Complexity-Intensive Care Unit (MRC-ICU) Score.

INTRODUCTION: The medication regimen complexity-intensive care unit (MRC-ICU) score has been developed and validated as an objective predictive metric for patient outcomes and pharmacist workload in the adult critically ill population. The purpose of this study was to explore the MRC-ICU and other workload metrics in the pediatric ICU (PICU). METHODS: This study was a retrospective cohort of pediatric ICU patients admitted to a single institution -between February 2, 2022 - August 2, 2022. Two scores were calculated, including the MRC-ICU and the pediatric Daily Monitoring System (pDMS). Data were extracted from the electronic health record. The primary outcome was the correlation of the MRC-ICU to mortality, as measured by Pearson -correlation -coefficient. Additionally, the correlation of MRC-ICU to number of orders was evaluated. Secondary -analyses explored the correlation of the MRC-ICU with pDMS and with hospital and ICU length of stay. RESULTS: A total of 2,232 patients were included comprising 2,405 encounters. The average age was 6.9 years (standard deviation [SD] 6.3 years). The average MRC-ICU score was 3.0 (SD 3.8). For the primary outcome, MRC-ICU was significantly positively correlated to mortality (0.22 95% confidence interval [CI 0.18 - 0.26]), p<0.05. Additionally, MRC-ICU was significantly positively correlated to ICU length of stay (0.38 [CI 0.34 - 0.41]), p<0.05. The correlation between the MRC-ICU and pDMS was (0.72 [CI 0.70 - 0.73]), p<0.05. CONCLUSION: In this pilot study, MRC-ICU demonstrated an association with existing prioritization metrics and with mortality and length of ICU stay in PICU population. Further, larger scale studies are required.

Therapeutic targeting of vascular malformation in a zebrafish model of hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) causes arteriovenous malformations (AVMs) in multiple organs to cause bleeding, neurological and other complications. HHT is caused by mutations in the BMP co-receptor endoglin. We characterised a range of vascular phenotypes in embryonic and adult endoglin mutant zebrafish and the effect of inhibiting different pathways downstream of Vegf signalling. Adult endoglin mutant zebrafish developed skin AVMs, retinal vascular abnormalities and cardiac enlargement. Embryonic endoglin mutants developed an enlarged basilar artery (similar to the previously described enlarged aorta and cardinal vein) and larger numbers of endothelial membrane cysts (kugeln) on cerebral vessels. Vegf inhibition prevented these embryonic phenotypes, leading us to investigate specific Vegf signalling pathways. Inhibiting mTOR or MEK pathways prevented abnormal trunk and cerebral vasculature phenotypes, whereas inhibiting Nos or Mapk pathways had no effect. Combined subtherapeutic mTOR and MEK inhibition prevented vascular abnormalities, confirming synergy between these pathways in HHT. These results indicate that the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of Vegf signalling. Combined low-dose MEK and mTOR pathway inhibition could represent a novel therapeutic strategy in HHT.

Scaling adult doses of antifungal and antibacterial agents to children.

My general pharmacokinetic scaling theory is discussed for the important matter of determining pediatric dosing for existing and new therapeutic drugs when optimal, or near-optimal, dosing for adults is known. The basis for the scaling is the requirement of a time-scaled likeness of the free-drug concentration time histories of children and adults. Broad categories of single and periodic dosing are considered. The former involves the scaling of dosage, and the latter involves both the dosage and schedule. The validity of the scaling relations is demonstrated by using measurements from previously reported clinical trials with adults and children (with ages generally 1 year or older) for the relatively new antifungal agent caspofungin and for the relatively new antibacterial agent linezolid. Standard pharmacodynamic effectiveness criteria are shown to be satisfied for the scaled dosage and schedule for children to the same extent that they are for the referenced adult. Consideration of scaling from adults to children is discussed for the case of new agents where no pediatric data are available and needed parameters are determined from in vitro measurements and preclinical animal data. A connection is also made between the allometric representation of clearance data and the dosing formulas. Limitations of the scaling results for infants because of growth and maturational matters are discussed. The general conclusion from this work is that the scaling theory does indeed have application to pediatric dosing for children, for both confirmation and refinement of present practice and guidance in pediatric treatment with new therapeutic agents.

Multiple Hybridization Events Punctuate the Evolutionary Trajectory of Malassezia furfur.

Malassezia species are important fungal skin commensals and are part of the normal microbiota of humans and other animals. However, under certain circumstances these fungi can also display a pathogenic behavior. For example, Malassezia furfur is a common commensal of human skin and yet is often responsible for skin disorders but also systemic infections. Comparative genomics analysis of M. furfur revealed that some isolates have a hybrid origin, similar to several other recently described hybrid fungal pathogens. Because hybrid species exhibit genomic plasticity that can impact phenotypes, we sought to elucidate the genomic evolution and phenotypic characteristics of M. furfur hybrids in comparison to their parental lineages. To this end, we performed a comparative genomics analysis between hybrid strains and their presumptive parental lineages and assessed phenotypic characteristics. Our results provide evidence that at least two distinct hybridization events occurred between the same parental lineages and that the parental strains may have originally been hybrids themselves. Analysis of the mating-type locus reveals that M. furfur has a pseudobipolar mating system and provides evidence that after sexual liaisons of mating compatible cells, hybridization involved cell-cell fusion leading to a diploid/aneuploid state. This study provides new insights into the evolutionary trajectory of M. furfur and contributes with valuable genomic resources for future pathogenicity studies. IMPORTANCEMalassezia furfur is a common commensal member of human/animal microbiota that is also associated with several pathogenic states. Recent studies report involvement of Malassezia species in Crohn's disease, a type of inflammatory bowel disease, pancreatic cancer progression, and exacerbation of cystic fibrosis. A recent genomics analysis of M. furfur revealed the existence of hybrid isolates and identified their putative parental lineages. In this study, we explored the genomic and phenotypic features of these hybrids in comparison to their putative parental lineages. Our results revealed the existence of a pseudobipolar mating system in this species and showed evidence for the occurrence of multiple hybridization events in the evolutionary trajectory of M. furfur. These findings significantly advance our understanding of the evolution of this commensal microbe and are relevant for future studies exploring the role of hybridization in the adaptation to new niches or environments, including the emergence of pathogenicity.

The adult microbiome of healthy and otitis patients: Definition of the core healthy and diseased ear microbiomes.

Otitis media (OM) and externa (OE) are painful, recurrent ear conditions. As most otitis publications focus on the bacterial content of childhood ears, there remains a dearth of information regarding the adult ear microbiome including both bacteria and fungi. This study compares the outer ear microbiome of healthy adults to adults affected by OE and OM using both intergenic-transcribed-spacer (ITS) and 16S-rDNA sequencing. The adult ear core microbiome consists of the prokaryote Cutibacterium acnes and the eukaryotic Malassezia arunalokei, M. globosa, and M. restricta. The healthy ear mycobiome is dominated by Malassezia and can be divided into two groups, one dominated by M. arunalokei, the other by M. restricta. Microbiome diversity and biomass varied significantly between healthy and diseased ears, and analyses reveal the presence of a potential mutualistic, protective effect of Malassezia species and C. acnes. The healthy ear core microbiome includes the bacteria Staphylococcus capitis and S. capitis/caprae, while the diseased ear core is composed of known bacterial and fungal pathogens including Aspergillus sp., Candida sp., Pseudomonas aeruginosa, S. aureus, and Corynebacterium jeikeium. The data presented highlight the need for early detection of the cause of otitis to direct more appropriate, efficient treatments. This will improve patient outcomes and promote improved antimicrobial stewardship.