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The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

NIH has acknowledged and committed to ending structural racism. The framework for NIH's approach, summarized here, includes understanding barriers; developing robust health disparities/equity research; improving its internal culture; being transparent and accountable; and changing the extramural ecosystem so that diversity, equity, and inclusion are reflected in funded research and the biomedical workforce.

Recovering genomes from shotgun metagenomic sequence data allows detailed taxonomic and functional characterization of individual species or strains in a microbial community. Retrieving these metagenome-assembled genomes (MAGs) involves seven stages. First, low-quality bases, along with adapter and host sequences, are removed. Second, overlapping sequences are assembled to create longer contiguous fragments. Third, these fragments are clustered based on sequence composition and abundance. Fourth, these sequence clusters, or bins, undergo rounds of quality assessment and refinement to yield MAGs. The optional fifth stage is dereplication of MAGs to select representatives. Next, each MAG is taxonomically classified. The optional seventh stage is assessing the fraction of diversity that has been recovered. The output of this protocol is draft genomes, which can provide invaluable clues about uncultured organisms. This protocol takes ~1 week to run, depending on computational resources available, and requires prior experience with high-performance computing, shell script programming and Python. This protocol recovers prokaryotic genomes from short-read metagenomic data. De novo assembly and binning are used to reconstruct metagenome-assembled genomes, which after quality assessment can provide insights into uncultured bacterial species.

Background The human intestinal microbiome is a complex community that contributes to host health and disease. In addition to normal microbiota, pathogens like carbapenem-resistant Enterobacteriaceae may be asymptomatically present. When these bacteria are present at very low levels, they are often undetectable in hospital surveillance cultures, known as occult or subclinical colonization. Through the receipt of antibiotics, these subclinical pathogens can increase to sufficiently high levels to become detectable, in a process called outgrowth. However, little is known about the interaction between gut microbiota and Enterobacteriaceae during occult colonization and outgrowth. Results We developed a clinically relevant mouse model for studying occult colonization. Conventional wild-type mice without antibiotic pre-treatment were exposed to Klebsiella pneumoniae but rapidly tested negative for colonization. This occult colonization was found to perturb the microbiome as detected by both 16S rRNA amplicon and shotgun metagenomic sequencing. Outgrowth of occult K. pneumoniae was induced either by a four-antibiotic cocktail or by individual receipt of ampicillin, vancomycin, or azithromycin, which all reduced overall microbial diversity. Notably, vancomycin was shown to trigger K. pneumoniae outgrowth in only a subset of exposed animals (outgrowth-susceptible). To identify factors that underlie outgrowth susceptibility, we analyzed microbiome-encoded gene functions and were able to classify outgrowth-susceptible microbiomes using pathways associated with mRNA stability. Lastly, an evolutionary approach illuminated the importance of xylose metabolism in K. pneumoniae colonization, supporting xylose abundance as a second susceptibility indicator. We showed that our model is generalizable to other pathogens, including carbapenem-resistant Escherichia coli and Enterobacter cloacae. Conclusions Our modeling of occult colonization and outgrowth could help the development of strategies to mitigate the risk of subsequent infection and transmission in medical facilities and the wider community. This study suggests that microbiota mRNA and small-molecule metabolites may be used to predict outgrowth-susceptibility.

Although systemic antibiotics are critical in controlling infections and reducing morbidity and mortality, overuse of antibiotics is presumed to contribute to negative repercussions such as selection of antimicrobial-resistant organisms and collateral damage to commensal microbes. In a prospective, randomized study of four clinically relevant antibiotic regimens [doxycycline (20mg or 100mg), cephalexin, or trimethoprim/sulfamethoxazole], we investigated microbial alterations on skin after administration of systemic antibiotics to healthy human volunteers. Samples from different skin and oral sites as well as stool were collected before, during, and up to 1 year after antibiotic use, and shotgun metagenomic sequencing was performed. Taxonomic analysis showed that subjects receiving doxycycline 100mg and trimethoprim/sulfamethoxazole (TMP/SMX) exhibited greater changes to their skin microbial communities, as compared to those receiving other regimens, or untreated controls. Oral and stool microbiota also demonstrated fluctuations after antibiotics. Bacterial culturing in combination with whole-genome sequencing revealed specific emergence, expansion, and persistence of antibiotic-resistant staphylococci harboring tetK or tetL and dfrC or dfrG genes in all subjects who received doxycycline 100mg or TMP/SMX, respectively. Last, analysis of metagenomic data revealed an increase of genes involved in gene mobilization, indicating stress responses of microbes to antibiotics. Collectively, these findings demonstrate direct, long-lasting effects of antibiotics on skin microbial communities, highlighting the skin microbiome as a site for the development and persistence of antibiotic resistance and the risks of overprescribing.

Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene (IL2RG), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.).

Antibiotics are a modifiable iatrogenic risk factor for the most common human nosocomial fungal infection, invasive candidiasis, yet the underlying mechanisms remain elusive. We found that antibiotics enhanced the susceptibility to murine invasive candidiasis due to impaired lymphocyte-dependent IL-17A- and GM-CSF-mediated antifungal immunity within the gut. This led to non-inflammatory bacterial escape and systemic bacterial co-infection, which could be ameliorated by IL-17A or GM-CSF immunotherapy. Vancomycin alone similarly enhanced the susceptibility to invasive fungal infection and systemic bacterial co-infection. Mechanistically, vancomycin reduced the frequency of gut Th17 cells associated with impaired proliferation and RORγt expression. Vancomycin's effects on Th17 cells were indirect, manifesting only in vivo in the presence of dysbiosis. In humans, antibiotics were associated with an increased risk of invasive candidiasis and death after invasive candidiasis. Our work highlights the importance of antibiotic stewardship in protecting vulnerable patients from life-threatening infections and provides mechanistic insights into a controllable iatrogenic risk factor for invasive candidiasis.

Candida auris is a human fungal pathogen classified as an urgent threat to the delivery of health care due to its extensive antimicrobial resistance and the high mortality rates associated with invasive infections. Global outbreaks have occurred in health care facilities, particularly, long-term care hospitals and nursing homes. Skin is the primary site of colonization for C. auris. To accelerate research studies, we developed microbiome sequencing protocols, including amplicon and metagenomic sequencing, directly from patient samples at health care facilities with ongoing C. auris outbreaks. We characterized the skin mycobiome with a database optimized to classify Candida species and C. auris to the clade level. While Malassezia species were the predominant skin-associated fungi, nursing home residents also harbored Candida species, including C. albicans, and C. parapsilosis. Amplicon sequencing was concordant with culturing studies to identify C. auris-colonized patients and provided further resolution that distinct clades of C. auris are colonizing facilities in New York and Illinois. Shotgun metagenomic sequencing from a clinical sample with a high fungal bioburden generated a skin-associated profile of the C. auris genome. Future larger scale clinical studies are warranted to more systematically investigate the effects of commensal microbes and patient risk factors on the colonization and transmission of C. auris. IMPORTANCE Candida auris is a human pathogen of high concern due to its extensive antifungal drug resistance and high mortality rates associated with invasive infections. Candida auris skin colonization and persistence on environmental surfaces make this pathogen difficult to control once it enters a health care facility. Residents in long-term care hospitals and nursing homes are especially vulnerable. In this study, we developed microbiome sequencing protocols directly from surveillance samples, including amplicon and metagenomic sequencing, demonstrating concordance between sequencing results and culturing.

Candida auris is a fungal pathogen of high concern due to its ability to cause healthcare-associated infections and outbreaks, its resistance to antimicrobials and disinfectants and its persistence on human skin and in the inanimate environment. To inform surveillance and future mitigation strategies, we defined the extent of skin colonization and explored the microbiome associated with C. auris colonization. We collected swab specimens and clinical data at three times points between January and April 2019 from 57 residents (up to ten body sites each) of a ventilator-capable skilled nursing facility with endemic C. auris and routine chlorhexidine gluconate (CHG) bathing. Integrating microbial-genomic and epidemiologic data revealed occult C. auris colonization of multiple body sites not targeted commonly for screening. High concentrations of CHG were associated with suppression of C. auris growth but not with deleterious perturbation of commensal microbes. Modeling human mycobiome dynamics provided insight into underlying alterations to the skin fungal community as a possible modifiable risk factor for acquisition and persistence of C. auris. Failure to detect the extensive, disparate niches of C. auris colonization may reduce the effectiveness of infection-prevention measures that target colonized residents, highlighting the importance of universal strategies to reduce C. auris transmission. Longitudinal skin site sampling of residents in a skilled nursing facility sheds light on persistent niches of the emerging fungal pathogen Candida auris.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory Tcells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S.epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

BACKGROUND: Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation. METHODS: We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions. RESULTS: The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses. CONCLUSIONS: This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.)

BackgroundThe human intestinal microbiome is a complex community that contributes to host health and disease. In addition to normal microbiota, pathogens like carbapenem-resistant Enterobacteriaceae may be asymptomatically present. When these bacteria are present at very low levels, they are often undetectable in hospital surveillance cultures, known as occult or subclinical colonization. Through the use of antibiotics, these subclinical pathogens can increase to sufficiently high levels to become detectable, in a process called outgrowth. However, little is known about the interaction between gut microbiota and Enterobacteriaceae during occult colonization and outgrowth.ResultsWe developed a clinically relevant mouse model for studying occult colonization. Conventional wild-type mice without antibiotic pre-treatment were exposed to K. pneumoniae but rapidly tested negative for colonization. This occult colonization was found to perturb the microbiome as detected by both 16S rRNA amplicon and shotgun metagenomic sequencing. Outgrowth of occult K. pneumoniae was induced either by a four-antibiotic cocktail or by individual receipt of ampicillin, vancomycin or azithromycin, which all reduced overall microbial diversity. Notably, vancomycin was shown to trigger K. pneumoniae outgrowth in only a subset of exposed animals (outgrowth-susceptible). To identify factors that underlie outgrowth susceptibility, we analyzed microbiome-encoded gene functions and were able to classify outgrowth-susceptible microbiomes using pathways associated with mRNA stability. Lastly, an evolutionary approach illuminated the importance of xylose metabolism in K. pneumoniae colonization, supporting xylose abundance as a second susceptibility indicator. We showed that our model is generalizable to other pathogens, including carbapenem-resistant Escherichia coli and Enterobacter cloacae.ConclusionsThis study suggests that microbiota mRNA and small-molecule metabolites may be used to predict outgrowth-susceptibility. Our modeling of occult colonization and outgrowth could help the development of strategies to mitigate the risk of subsequent infection and transmission in medical facilities and the wider community.

Previous cross-sectional studies have shown that skin microbiomes in adults are distinct from those in children. However, the human skin microbiome in individuals as they sexually mature has not been studied as extensively. We performed a prospective, longitudinal study to investigate the puberty-associated shifts in skin microbiota. A total of 12 healthy children were evaluated every 6–18 months for up to 6 years. Using 16S ribosomal RNA (V1–V3) and internal transcribed spacer 1 amplicon sequencing analyzed with Divisive Amplicon Denoising Algorithm 2, we characterized the bacterial and fungal communities of five different skin and nares sites. We identified significant alterations in the composition of skin microbial communities, transitioning toward a more adult microbiome, during puberty. The microbial shifts were associated with Tanner stages (classification method for the degree of sexual maturation) and showed noticeable sex-specific differences. Over time, female children demonstrated a predominance of Cutibacterium with decreasing diversity. Among fungi, Malassezia predominated at most skin sites in more sexually mature subjects, which was more pronounced in female children. The higher relative abundances of these lipophilic taxa—C. acnes and M. restricta—were strongly associated with serum sex hormone concentrations with known influence on sebaceous gland activity. Taken together, our results support the relationship between sexual maturation, skin physiology, and the skin microbiome.

Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting β-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.

The composition of the skin microbiota varies widely among individuals when sampled at the same body site. A key question is which molecular factors determine strain-level variability within sub-ecosystems of the skin microbiota. Here, we used a genomics-guided approach to identify an antibacterial biosynthetic gene cluster in Cutibacterium acnes (formerly Propionibacterium acnes), a human skin commensal bacterium that is widely distributed across individuals and skin sites. Experimental characterization of this biosynthetic gene cluster resulted in identification of a new thiopeptide antibiotic, cutimycin. Analysis of individual human skin hair follicles revealed that cutimycin contributed to the ecology of the skin hair follicle microbiota and helped to reduce colonization of skin hair follicles by Staphylococcus species.

Human skin functions as a physical barrier to foreign pathogen invasion and houses numerous commensals. Shifts in the human skin microbiome have been associated with conditions ranging from acne to atopic dermatitis. Previous metagenomic investigations into the role of the skin microbiome in health or disease have found that much of the sequenced data do not match reference genomes, making it difficult to interpret metagenomic datasets. We combined bacterial cultivation and metagenomic sequencing to assemble the Skin Microbial Genome Collection (SMGC), which comprises 622 prokaryotic species derived from 7,535 metagenome-assembled genomes and 251 isolate genomes. The metagenomic datasets that we generated were combined with publicly available skin metagenomic datasets to identify members and functions of the human skin microbiome. The SMGC collection includes 174 newly identified bacterial species and 12 newly identified bacterial genera, including the abundant genus 'Candidatus Pellibacterium', which has been newly associated with the skin. The SMGC increases the characterized set of known skin bacteria by 26%. We validated the SMGC metagenome-assembled genomes by comparing them with sequenced isolates obtained from the same samples. We also recovered 12 eukaryotic species and assembled thousands of viral sequences, including newly identified clades of jumbo phages. The SMGC enables classification of a median of 85% of skin metagenomic sequences and provides a comprehensive view of skin microbiome diversity, derived primarily from samples obtained in North America.

Recovering genomes from shotgun metagenomic sequence data allows detailed taxonomic and functional characterization of individual species or strains in a microbial community. Retrieving these metagenome-assembled genomes (MAGs) involves seven stages. First, low-quality bases, along with adapter and host sequences, are removed. Second, overlapping sequences are assembled to create longer contiguous fragments. Third, these fragments are clustered based on sequence composition and abundance. Fourth, these sequence clusters, or bins, undergo rounds of quality assessment and refinement to yield MAGs. The optional fifth stage is dereplication of MAGs to select representatives. Next, each MAG is taxonomically classified. The optional seventh stage is assessing the fraction of diversity that has been recovered. The output of this protocol is draft genomes, which can provide invaluable clues about uncultured organisms. This protocol takes ~1 week to run, depending on computational resources available, and requires prior experience with high-performance computing, shell script programming and Python.

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infections. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota’s production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota’s composition and promotes pathogen invasion. Together, this work reveals a process, akin to the immune system’s capacity for training, by which the host can deploy taurine as a prebiotic to nourish and educate the microbiota, promoting its resistance to infection.

Late-onset sepsis (LOS) is thought to result from systemic spread of commensal microbes from the intestines of premature infants. Clinical use of probiotics for LOS prophylaxis has varied owing to limited efficacy, reflecting an incomplete understanding of relationships between development of the intestinal microbiome, neonatal dysbiosis and LOS. Using a model of LOS, we found that components of the developing microbiome were both necessary and sufficient to prevent LOS. Maternal antibiotic exposure that eradicated or enriched transmission of Lactobacillus murinus exacerbated and prevented disease, respectively. Prophylactic administration of some, but not all Lactobacillus spp. was protective, as was administration of Escherichia coli. Intestinal oxygen level was a major driver of colonization dynamics, albeit via mechanisms distinct from those in adults. These results establish a link between neonatal dysbiosis and LOS, and provide a basis for rational selection of probiotics that modulate primary succession of the microbiome to prevent disease.

The human microbiome has captured the attention of both researchers and the lay press. Given its emerging role in health and disease, it is imperative that studies are communicated well so that in turn the public does not misinterpret the findings.

Building upon the knowledge garnered from investigations utilizing traditional culturing methods, advances in sequencing technologies have catalyzed a revolution in studying human-associated microbes: bacteria, fungi and viruses. Skin microbiome research in healthy individuals and patients with dermatologic disorders has provided insights into the complexity and biogeography of human skin microbes. The continual developments in sequencing and analyses will provide increasingly sophisticated tools to interrogate human-associated microbes, ultimately to improve our understanding of health and disease.

Background Disease flares of established atopic dermatitis (AD) are generally associated with a low-diversity skin microbiota and Staphylococcus aureus dominance. The temporal transition of the skin microbiome between early infancy and the dysbiosis of established AD is unknown. Methods We randomly selected 50 children from the Cork Babies After SCOPE: Evaluating the Longitudinal Impact Using Neurological and Nutritional Endpoints (BASELINE) longitudinal birth cohort for microbiome sampling at 3 points in the first 6 months of life at 4 skin sites relevant to AD: the antecubital and popliteal fossae, nasal tip, and cheek. We identified 10 infants with AD and compared them with 10 randomly selected control infants with no AD. We performed bacterial 16S ribosomal RNA sequencing and analysis directly from clinical samples. Results Bacterial community structures and diversity shifted over time, suggesting that age strongly affects the skin microbiome in infants. Unlike established AD, these patients with infantile AD did not have noticeably dysbiotic communities before or with disease and were not colonized by S aureus . In comparing patients and control subjects, infants who had affected skin at month 12 had statistically significant differences in bacterial communities on the antecubital fossa at month 2 compared with infants who were unaffected at month 12. In particular, commensal staphylococci were significantly less abundant in infants affected at month 12, suggesting that this genus might protect against the later development of AD. Conclusions This study suggests that 12-month-old infants with AD were not colonized with S aureus before having AD. Additional studies are needed to confirm whether colonization with commensal staphylococci modulates skin immunity and attenuates development of AD.

Background:Candida auris is a highly transmissible healthcare-associated pathogen that can cause severe infection as well as long-lasting colonization. C. auris is often resistant to the antifungals that are commonly used to treat Candida infections, which may lead to clinical failure. Therefore, healthcare facilities must identify the organism quickly and implement strict precautions to prevent its spread. In 2019, the NIH Clinical Center instituted C. auris admission screening among its high-risk patient populations. Methods: Patients admitted to the NIH Clinical Center, a 200-bed research hospital, were identified on admission as having been hospitalized outside the United States in the prior 6 months. Admission screening began in August 2019. In September 2019, due to evolving regional epidemiology, we expanded surveillance criteria to include patients housed in any healthcare facility in the District of Columbia, Maryland, and Virginia metro area in the previous 6 months. Screening was performed as routine clinical care, and therefore did not require written informed consent. Swabs were obtained from nares, axilla and groin, with subsequent addition of mouth and toe web (BD ESwabs). Patients were placed on empiric contact isolation for at least 48 hours and concurrently screened for carbapenemase-producing organisms. Swabs were cultured on CHROMagar Candida and in Sabouraud dextrose broth with 10% NaCL and 50 mg/L chloramphenicol and gentamicin, and incubated for 14 days at 30°C and 40°C, respectively. Positive broth tubes were subcultured onto CHROMagar Candida. C. auris was identified by MALDI-TOF MS and ITS sequence analysis. Susceptibility testing was performed using Sensititre YeastOne Colorimetric assay. Whole-genome sequencing was used to identify clonal designations and genetic relatedness of isolates. Results: Since August 2019, 1 to 2 patients per week have been screened for C. auris. As of November 2019, 1 of 15 patients screened on admission grew C. auris from a groin swab. The patient, who had been hospitalized abroad, was found to be cocolonized with blaNDM-1+ E. coli and K. pneumoniae. Subsequent screening of other patients on the same ward identified no evidence of spread. Admission surveillance is ongoing. Conclusions: Healthcare-associated outbreaks can originate from C. auris–colonized patients. Admission surveillance of high-risk patients is intended to prevent transmission from undetected reservoirs. Our sampling of multiple sites, though laborious, may add to the data on C. auris colonization. Future plans include incorporating molecular testing and streamlining geographic criteria. C. auris admission screening has already identified one colonized patient, and will continue as a new and important patient safety measure at our hospital.Funding: NoneDisclosures: None

Growing interest in host-fungal interactions has implications for human health and disease

Understanding the skin mycobiome (fungal communities) is important because both commensal and pathogenic fungi can drive cutaneous disease depending on host status and body sites, including the scalp, feet, and groin. Interestingly, age may also affect skin fungal infections as certain dermatophytoses (i.e., tinea capitis) are more frequent in children than adults. We previously described the skin mycobiomes in healthy adults, showing lipophilic fungi Malassezia predominate in most skin sites. Because children have less sebaceous skin before puberty, we compared the fungal communities of primary clinical samples from healthy children and adults, based on sequencing of a fungal phylogenetic marker. Although Malassezia predominated on the trunk, head, and arm skin of adults (age 18-39), children (age14) had more diverse fungal communities, for example, Eurotiomycetes, which includes common dermatophytes. Species-level classification showed that Malassezia globosa predominated in children. Collectively, our findings indicate that prepubertal skin is colonized by diverse fungi, whereas adult skin is predominantly obligatory lipophilic Malassezia, suggesting that fungal communities on skin profoundly shift during puberty. Mycobiome shifts during puberty are likely due to alterations in sebaceous gland activation and sebum composition. This study provides a foundational framework for studies investigating interactions between fungi, skin, and pediatric dermatophytosis.

Antibiotic-resistant bacteria are a serious threat to medically fragile patient populations. The spread of antibiotic resistance through plasmid-mediated mechanisms is of grave concern as it can lead to the conversion of endogenous patient-associated strains to difficult-to-treat pathogens., Antibiotics, which are used both to prevent and to treat infections, are a mainstay therapy for lifesaving procedures such as transplantation. For this reason, and many others, increased antibiotic resistance among human-associated pathogens, such as the carbapenem-resistant Enterobacteriaceae species, is of grave concern. In this study, we report on a hematopoietic stem cell transplant recipient in whom cultures detected the emergence of carbapenem resistance and spread across five strains of bacteria that persisted for over a year. Carbapenem resistance in Citrobacter freundii, Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae was linked to a pair of plasmids, each carrying the Klebsiella pneumoniae carbapenemase gene (blaKPC). Surveillance cultures identified a carbapenem-susceptible strain of Citrobacter freundii that may have become resistant through horizontal gene transfer of these plasmids. Selection of a multidrug-resistant Klebsiella pneumoniae strain was also detected following combination antibiotic therapy. Here we report a plasmid carrying the blaKPC gene with broad host range that poses the additional threat of spreading to endogenous members of the human gut microbiome.

The composition of the skin microbiome varies widely among individuals sampled at the same body site. A key question is which molecular factors determine strain-level variability within sub-ecosystems of the skin. We used a genomics-guided approach to identify an antibacterial biosynthetic gene cluster in Cutibacterium acnes (formerly Propionibacterium acnes) that is widely distributed across individuals and skin sites. Experimental characterization of this cluster identified of a new thiopeptide antibiotic, cutimycin. Analysis of individual human skin hair follicles revealed that cutimycin plays a role in the chemical ecology of follicular microbiota and in colonization resistance against Staphylococcus species. ONE SENTENCE SUMMARY: Cutimycin, a thiopeptide antibiotic produced by a widespread skin commensal, reduces Staphylococcus colonization of human follicles.

Summary Candida auris is an emerging multi-drug-resistant human fungal pathogen. C. auris skin colonization results in environmental shedding, which underlies hospital transmissions, and predisposes patients to subsequent infections. We developed a murine skin topical exposure model for C. auris to dissect risk factors for colonization and to test interventions that might protect patients. We demonstrate that C. auris establishes long-term residence within the skin tissue compartment, which would elude clinical surveillance. The four clades of C. auris, with geographically distinct origins, differ in their abilities to colonize murine skin, mirroring epidemiologic findings. The IL-17 receptor signaling and specific arms of immunity protect mice from long-term C. auris skin colonization. We further determine that commonly used chlorhexidine antiseptic serves as a protective and decolonizing agent against C. auris. This translational model facilitates an integrated approach to develop strategies to combat the unfolding global outbreaks of C. auris and other skin-associated microbial pathogens.

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.

Carbapenemase-producing organisms have spread worldwide, and infections with these bacteria cause significant morbidity. Horizontal transfer of plasmids carrying genes that encode carbapenemases plays an important role in the spread of multidrug-resistant Gram-negative bacteria. Here we investigate parameters regulating conjugation using an Escherichia coli laboratory strain that lacks plasmids or restriction enzyme modification systems as a recipient and also using patient isolates as donors and recipients. Because conjugation is tightly regulated, we performed a systematic analysis of the transfer of Klebsiella pneumoniae carbapenemase ( bla KPC )-encoding plasmids into multiple strains under different environmental conditions to investigate critical variables. We used four bla KPC -carrying plasmids isolated from patient strains obtained from two hospitals: pKpQIL and pKPC-47e from the National Institutes of Health, and pKPC_UVA01 and pKPC_UVA02 from the University of Virginia. Plasmid transfer frequency differed substantially between different donor and recipient pairs, and the frequency was influenced by plasmid content, temperature, and substrate, in addition to donor and recipient strain. pKPC-47e was attenuated in conjugation efficiency across all conditions tested. Despite its presence in multiple clinical species, pKPC_UVA01 had lower conjugation efficiencies than pKpQIL into recipient strains. The conjugation frequency of these plasmids into K. pneumoniae and E. coli patient isolates ranged widely without a clear correlation with clinical epidemiological data. Our results highlight the importance of each variable examined in these controlled experiments. The in vitro models did not reliably predict plasmid mobilization observed in a patient population, indicating that further studies are needed to understand the most important variables affecting horizontal transfer in vivo .

Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the bla KPC-2 gene in one isolate prompted inclusion of non- Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes.

Human-associated microbiota form and stabilize communities based on interspecies interactions. We review how these microbe-microbe and microbe-host interactions are communicated to shape communities over a human’s lifespan, including periods of health and disease. Modeling and dissecting signaling in host-associated communities is crucial to understand their function, and will open the door to therapies that prevent or correct microbial community dysfunction to promote health and treat disease.

Studies in patients with genetic defects can provide unique insights regarding the role of specific genes and pathways in humans. Patients with defects in the Th17/IL-17 axis, such as patients harboring loss-of-function STAT3 mutations (autosomal-dominant hyper IgE syndrome; AD-HIES) present with recurrent oral fungal infections. Our studies aimed to comprehensively evaluate consequences of STAT3 deficiency on the oral commensal microbiome. We characterized fungal and bacterial communities in AD-HIES in the presence and absence of oral fungal infection compared with healthy volunteers. Analyses of oral mucosal fungal communities in AD-HIES revealed severe dysbiosis with dominance of Candida albicans (C. albicans) in actively infected patients and minimal representation of health-associated fungi and/or opportunists. Bacterial communities also displayed dysbiosis in AD-HIES, particularly in the setting of active Candida infection. Active candidiasis was associated with decreased microbial diversity and enrichment of the streptococci Streptococcus oralis (S. oralis) and S. mutans, suggesting an interkingdom interaction of C. albicans with oral streptococci. Increased abundance of S. mutans was consistent with susceptibility to dental caries in AD-HIES. Collectively, our findings illustrate a critical role for STAT3/Th17 in the containment of C. albicans as a commensal organism and an overall contribution in the establishment of fungal and bacterial oral commensal communities.

Human microbiome studies have revealed the intricate interplay of host immunity and bacterial communities to achieve homeostatic balance. Healthy skin microbial communities are dominated by bacteria with low viral representation1-3, mainly bacteriophage. Specific eukaryotic viruses have been implicated in both common and rare skin diseases, but cataloging skin viral communities has been limited. Alterations in host immunity provide an opportunity to expand our understanding of microbial-host interactions. Primary immunodeficient patients manifest with various viral, bacterial, fungal, and parasitic infections, including skin infections4. Dedicator of cytokinesis 8 (DOCK8) deficiency is a rare primary human immunodeficiency characterized by recurrent cutaneous and systemic infections, as well as atopy and cancer susceptibility5. DOCK8, encoding a guanine nucleotide exchange factor highly expressed in lymphocytes, regulates actin cytoskeleton, which is critical for migration through collagen-dense tissues such as skin6. Analyzing deep metagenomic sequencing data from DOCK8-deficient skin samples demonstrated a notable increase in eukaryotic viral representation and diversity compared with healthy volunteers. De novo assembly approaches identified hundreds of novel human papillomavirus genomes, illuminating microbial dark matter. Expansion of the skin virome in DOCK8-deficient patients underscores the importance of immune surveillance in controlling eukaryotic viral colonization and infection.

The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization of blaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with blaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings., IMPORTANCE Carbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and mortality associated with these resistant Gram-negative bacteria. Horizontal plasmid transfer spreads the resistance mechanism to new bacteria, and understanding the plasmid ecology of the hospital environment can assist in the design of control strategies to prevent nosocomial infections. A 5-year genomic and epidemiological survey was undertaken to study the CPOs in the patient-accessible environment, as well as in the plumbing system removed from the patient. This comprehensive survey revealed a vast, unappreciated reservoir of CPOs in wastewater, which was in contrast to the low positivity rate in both the patient population and the patient-accessible environment. While there were few patient-environmental isolate associations, there were plasmid backbones common to both populations. These results are relevant to all hospitals for which CPO colonization may not yet be defined through extensive surveillance.

Functioning as the exterior interface of the human body with the environment, skin acts as a physical barrier to prevent the invasion of foreign pathogens while providing a home to the commensal microbiota. The harsh physical landscape of skin, particularly the desiccated, nutrient-poor, acidic environment, also contributes to the adversity that pathogens face when colonizing human skin. Despite this, the skin is colonized by a diverse microbiota. In this Review, we describe amplicon and shotgun metagenomic DNA sequencing studies that have been used to assess the taxonomic diversity of microorganisms that are associated with skin from the kingdom to the strain level. We discuss recent insights into skin microbial communities, including their composition in health and disease, the dynamics between species and interactions with the immune system, with a focus on Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus.

Background Emollients are a mainstay of treatment in atopic dermatitis (AD), a disease distinguished by skin bacterial dysbiosis. However, changes in skin microbiota when emollients are used as a potential AD preventative measure in infants remain incompletely characterized. Results We compared skin barrier parameters, AD development, and bacterial 16S ribosomal RNA gene sequences of cheek, dorsal and volar forearm samples from 6-month-old infants with a family history of atopy randomized to receive emollients (n = 11) or no emollients (controls, n = 12). The emollient group had a lower skin pH than the control group. The number of bacterial taxa in the emollient group was higher than in the control group at all sites. The Streptococcus salivarius proportion was higher in the emollient versus control groups at all sites. S. salivarius proportion appeared higher in infants without AD compared to infants with AD. A decrease in S. salivarius abundance was further identified in a separate larger population of older children demonstrating an inverse correlation between AD severity at sampling sites and S. salivarius proportions. Conclusions The decreased skin pH and the increased proportion of S. salivarius after long-term emollient use in infants at risk for developing AD may contribute to the preventative effects of emollients in high-risk infants.

Background vSNF patients are at high risk of colonization and infection with C. auris. CHG bathing has been used as an intervention to reduce nosocomial transmission of multi-drug-resistant organisms, but its effect on C. auris is unclear. Methods We studied a 70-bed ventilator ward in a 300-bed vSNF in Chicago, IL with a high prevalence of C. auris and established CHG bathing. Swab samples were collected from patients for culture, microbiome analysis, and CHG skin concentration testing (Table 1). Results We collected 2,467 samples (950 culture, 950 microbiome, 567 CHG) from 57 patients during 2 surveys conducted January–March 2019. Forty-six (81%) patients had C. auris cultured from ≥1 body site. Mean (±SD) age was 59 (±14) years, 40% were women, 70% were African American, mean (±SD) Charlson score was 3 (±2). Patients colonized with C. auris were more likely to be mechanically ventilated (50% vs. 0%, P < 0.001), have a gastrostomy tube (78% vs. 27%, P < 0.001) or have urinary catheter (72% vs. 23%, P = 0.01) than noncolonized patients. Frequency of C. auris isolation varied among 10 body sites tested (P < 0.001); colonization of anterior nares (41%) and hands (40%) was detected most often (Figure 1). By ITS1 analysis, all isolates were members of the C. auris South American clade. Skin microbiome sequencing confirmed culture Results. While Malassezia is the dominant genera observed in healthy volunteers and patients in this vSNF, C. auris was observed to dominate the fungal community of multiple skin sites, including nares, hands, inguinal, toe web (Figure 2). Other Candida spp. were also identified on the skin of patients in the current study, but at lower relative abundance. CHG was detected on skin of 52 (91%) patients (median CHG concentration 19.5 µg/mL; IQR 4.9–78.1 µg/mL). In a mixed-effects model controlling for body site and multiple measurements per patient, odds of C. auris detection by culture were less at CHG concentrations ≥625 µg/mL than at lower concentrations (Figure 3; OR 0.25, 95% CI: 0.10–0.66; P = 0.005). Conclusion Frequent C. auris colonization of vSNF patients’ anterior nares and hands suggests that nasal decolonization and patient hand hygiene are potential options to reduce C. auris transmission. High concentrations of CHG may be needed to suppress C. auris on skin. Disclosures All Authors: No reported Disclosures.

Human skin, the body’s largest organ, functions as a physical barrier to bar the entry of foreign pathogens, while concomitantly providing a home to myriad commensals. Over a human’s life span, keratinized skin cells, immune cells, and microbes all interact to integrate the processes of maintaining skin’s physical and immune barrier under homeostatic healthy conditions and also under multiple stresses, such as wounding or infection. In this Review, we explore the intricate interactions of microbes and immune cells on the skin surface and within associated appendages to regulate this orchestrated maturation in the context of both host physiological changes and environmental challenges.