Clostridioides difficile infection is associated with differences in transcriptionally active microbial communities.

Clostridioides difficile infection (CDI) is responsible for around 300,000 hospitalizations yearly in the United States, with the associated monetary cost being billions of dollars. Gut microbiome dysbiosis is known to be important to CDI. To the best of our knowledge, metatranscriptomics (MT) has only been used to characterize gut microbiome composition and function in one prior study involving CDI patients. Therefore, we utilized MT to investigate differences in active community diversity and composition between CDI+ (n = 20) and CDI- (n = 19) samples with respect to microbial taxa and expressed genes. No significant (Kruskal-Wallis, p > 0.05) differences were detected for richness or evenness based on CDI status. However, clustering based on CDI status was significant for both active microbial taxa and expressed genes datasets (PERMANOVA, p ≤ 0.05). Furthermore, differential feature analysis revealed greater expression of the opportunistic pathogens Enterocloster bolteae and Ruminococcus gnavus in CDI+ compared to CDI- samples. When only fungal sequences were considered, the family Saccharomycetaceae expressed more genes in CDI-, while 31 other fungal taxa were identified as significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) associated with CDI+. We also detected a variety of genes and pathways that differed significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) based on CDI status. Notably, differential genes associated with biofilm formation were expressed by C. difficile. This provides evidence of another possible contributor to C. difficile's resistance to antibiotics and frequent recurrence in vivo. Furthermore, the greater number of CDI+ associated fungal taxa constitute additional evidence that the mycobiome is important to CDI pathogenesis. Future work will focus on establishing if C. difficile is actively producing biofilms during infection and if any specific fungal taxa are particularly influential in CDI.

Selection of ethanol tolerant strains of Candida albicans by repeated ethanol exposure results in strains with reduced susceptibility to fluconazole.

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol-a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

Dynamics of the Gut Mycobiome in Patients With Ulcerative Colitis.

BACKGROUND & AIMS: Intestinal fungi have been implicated in the pathogenesis of ulcerative colitis (UC). However, it remains unclear if fungal composition is altered during active versus quiescent disease. METHODS: We analyzed clinical and metagenomic data from the Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease (SPARC IBD), available via the IBD Plexus Program of the Crohn's & Colitis Foundation. We evaluated the fungal composition of fecal samples from 421 patients with UC during clinical activity and remission. Within a longitudinal subcohort (n = 52), we assessed for dynamic taxonomic changes across alterations in clinical activity over time. We examined if fungal amplicon sequence variants and fungal-bacterial relationships were altered during activity versus remission. Finally, we classified activity in UC using a supervised machine learning random forest model trained on fungal abundance data. RESULTS: During clinical activity, the relative abundance of genus Candida was increased 3.5-fold (P-adj < 1 × 10-4) compared with during remission. Patients with longitudinal reductions in clinical activity demonstrated parallel reductions in Candida relative abundance (P < .05). Candida relative abundance correlated with Parabacteroides diastonis, Faecalibacterium prausnitzii, and Bacteroides dorei relative abundance (P < .05) during remission; however, these correlations were disrupted during activity. Fungal abundance data successfully classified patients with active or quiescent UC (area under the curve ∼0.80), with Candida relative abundance critical to the success of the model. CONCLUSIONS: Clinical activity in UC is associated with an increased relative abundance of Candida, cross-sectionally and dynamically over time. The role of fecal Candida as a target for therapeutics in UC should be evaluated.

Selection of Ethanol Tolerant Strains of Candida albicans by Repeated Ethanol Exposure Results in Strains with Reduced Susceptibility to Fluconazole.

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol-a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

Pre-colonization with the fungus Candida glabrata exacerbates infection by the bacterial pathogen Clostridioides difficile in a murine model.

The contributions of commensal fungi to human health and disease are not well understood. Candida species such as C. albicans and C. glabrata are opportunistic pathogenic fungi and common colonizers of the human intestinal tract. They have been shown to affect the host immune system and interact with the gut microbiome and pathogenic microorganisms. Therefore, Candida species could be expected to play important ecological roles in the host gastrointestinal tract. Previously, our group demonstrated that pre-colonization of mice with C. albicans protected them against lethal C. difficile infection (CDI). Here, we show that mice pre-colonized with C. glabrata succumbed to CDI more rapidly than mice that were not pre-colonized suggesting an enhancement in C. difficile pathogenesis. Further, when C. difficile was added to pre-formed C. glabrata biofilms, an increase in matrix and overall biomass was observed. These effects were also shown with C. glabrata clinical isolates. Interestingly, the presence of C. difficile increased C. glabrata biofilm susceptibility to caspofungin, indicating potential effects on the fungal cell wall. Defining this intricate and intimate relationship will lead to an understanding of the role of Candida species in the context of CDI and novel aspects of Candida biology. IMPORTANCE Most microbiome studies have only considered the bacterial populations while ignoring other members of the microbiome such as fungi, other eukaryotic microorganisms, and viruses. Therefore, the role of fungi in human health and disease has been significantly understudied compared to their bacterial counterparts. This has generated a significant gap in knowledge that has negatively impacted disease diagnosis, understanding, and the development of therapeutics. With the development of novel technologies, we now have an understanding of mycobiome composition, but we do not understand the roles of fungi in the host. Here, we present findings showing that Candida glabrata, an opportunistic pathogenic yeast that colonizes the mammalian gastrointestinal tract, can impact the severity and outcome of a Clostridioides difficile infection (CDI) in a murine model. These findings bring attention to fungal colonizers during CDI, a bacterial infection of the gastrointestinal tract.

Characterization of the Effects of Candida Gastrointestinal Colonization on Clostridioides difficile Infection in a Murine Model.

The role of fungal colonizers of the gastrointestinal tract during disease states is not well understood. Antibiotic treatment renders patients highly susceptible to infection by the bacterial pathogen C. difficile while also leading to blooms in fungal commensals, setting the stage for trans-kingdom interactions. Here, we describe a murine model of Candida gastrointestinal colonization coupled to a C. difficile infection (CDI) model, the measurement of CFU of both organisms, and collection of cecum and colon contents for the purpose of quantifying C. difficile toxin production. Additionally, we describe how to induce and purify C. difficile spores.

Minimal Effects of Medium-Chain Triglyceride Supplementation on the Intestinal Microbiome Composition of Premature Infants: A Single-Center Pilot Study.

Compared to term infants, the microbiota of preterm infants is less diverse and often enriched for potential pathogens (e.g., members of the family Enterobacteriaceae). Additionally, antibiotics are frequently given to preterm infants, further destabilizing the microbiota and increasing the risk of fungal infections. In a previous communication, our group showed that supplementation of the premature infant diet with medium-chain triglyceride (MCT) oil reduced the fungal burden of Candida spp. in the gastrointestinal tract. The objective of this study was to determine whether MCT supplementation impacts the bacterial component of the microbiome. Pre-term infants (n = 17) receiving enteral feedings of either infant formula (n = 12) or human milk (n = 5) were randomized to MCT supplementation (n = 9) or no supplementation (n = 8). Fecal samples were taken at randomization and prior to MCT supplementation (Week 0), on days 5-7 (Week 1) and day 21 (Week 3). After DNA extraction from samples, the QIIME2 pipeline was utilized to measure community diversity and composition (genera and phyla). Our findings show that MCT supplementation did not significantly alter microbiota diversity or composition in the gastrointestinal tract. Importantly, there were no significant changes in the family Enterobacteriaceae, suggesting that MCT supplementation did not enrich for potential pathogens. MCT holds promise as a therapeutic intervention for reducing fungal colonization without significant impact on the bacterial composition of the host gastrointestinal tract.

Gut Microbiome Dysbiosis in Alcoholism: Consequences for Health and Recovery.

Since the mid 1980's, the impact of gastrointestinal (GI) microbiome changes during alcohol use disorder has been an area of significant interest. This work has resulted in the identification of specific changes in the abundance of certain members of the GI microbiome and the role these changes play in a variety of alcohol related disorders (i.e. alcoholic liver disease). Interestingly, some findings suggest a possible role for the GI microbiome in alcohol addiction or withdrawal. Unfortunately, there is a significant gap in knowledge in this area. Here we describe differences in the GI microbiome of alcoholic and non-alcoholic individuals and discuss the possible impact of microbes on the gut-brain axis, which could impact alcohol related behaviors (i.e. addiction). Understanding the role of the GI microbiome in alcohol related disorders will potentially lead to the development of successful microbiome-targeted therapeutics to help mitigate these disorders.

Bypass of Dfi1 Regulation of Candida albicans Invasive Filamentation by Iron Limitation.

Candida albicans filamentation, the ability to convert from oval yeast cells to elongated hyphal cells, is a key factor in its pathogenesis. Previous work has shown that the integral membrane protein Dfi1 is required for filamentation in cells grown in contact with a semisolid surface. Investigations into the downstream targets of the Dfi1 pathway revealed potential links to two transcription factors, Sef1 and Czf1. Sef1 regulates iron uptake and iron utilization genes under low-iron conditions, leading us to hypothesize that there exists a link between iron availability and contact-dependent invasive filamentation. In this study, we showed that Sef1 was not required for contact-dependent filamentation, but it was required for wild-type (WT) expression levels of a number of genes during growth under contact conditions. Czf1 is required for contact-dependent filamentation and for WT levels of expression of several genes. Constitutive expression and activation of either Sef1 or Czf1 individually in a dfi1 null strain resulted in a complete rescue of the dfi1 null filamentation defect. Because Sef1 is normally activated in low-iron environments, we embedded WT and dfi1 null cells in iron-free agar medium supplemented with various concentrations of ferrous ammonium sulfate (FAS). dfi1 null cells embedded in media with a low concentration of iron (20 µM FAS) showed increased filamentation in comparison to mutant cells embedded in higher concentrations of iron (50 to 500 µM). WT cells produced filamentous colonies in all concentrations. Together, the data indicate that Dfi1, Czf1, Sef1, and environmental iron regulate C. albicans contact-dependent filamentation. IMPORTANCE Candida albicans is an opportunistic pathogen responsible for a larger proportion of candidiasis and candidemia cases than any other Candida species. The ability of C. albicans cells to invade and cause disease is linked to their ability to filament. Despite this, there are gaps in our knowledge of the environmental cues and intracellular signaling that triggers the switch from commensal organism to filamentous pathogen. In this study, we identified a link between contact-dependent filamentation and iron availability. Over the course of tissue invasion, C. albicans cells encounter a number of different iron microenvironments, from the iron-rich gut to iron-poor tissues. Increased expression of Sef1-dependent iron uptake genes as a result of contact-dependent signaling will promote the adaptation of C. albicans cells to a low-iron-availability environment.

Debaryomyces, the Achilles heel of wound repair.

In a recent Science paper, Jain et al. (2021) discover that a fungus contributes to delayed wound healing in mice and is enriched in inflamed tissue from Crohn's disease patients. This culprit is not a well-known pathogen, but cheese yeast Debaryomyces hansenii, highlighting the importance of further studying fungi-host interactions.

Decreased Ecological Resistance of the Gut Microbiota in Response to Clindamycin Challenge in Mice Colonized with the Fungus Candida albicans.

The mammalian gut microbiota is a complex community of microorganisms which typically exhibits remarkable stability. As the gut microbiota has been shown to affect many aspects of host health, the molecular keys to developing and maintaining a "healthy" gut microbiota are highly sought after. Yet, the qualities that define a microbiota as healthy remain elusive. We used the ability to resist change in response to antibiotic disruption, a quality we refer to as ecological resistance, as a metric for the health of the bacterial microbiota. Using a mouse model, we found that colonization with the commensal fungus Candida albicans decreased the ecological resistance of the bacterial microbiota in response to the antibiotic clindamycin such that increased microbiota disruption was observed in C. albicans-colonized mice compared to that in uncolonized mice. C. albicans colonization resulted in decreased alpha diversity and small changes in abundance of bacterial genera prior to clindamycin challenge. Strikingly, co-occurrence network analysis demonstrated that C. albicans colonization resulted in sweeping changes to the co-occurrence network structure, including decreased modularity and centrality and increased density. Thus, C. albicans colonization resulted in changes to the bacterial microbiota community and reduced its ecological resistance.IMPORTANCECandida albicans is the most common fungal member of the human gut microbiota, yet its ability to interact with and affect the bacterial gut microbiota is largely uncharacterized. Previous reports showed limited changes in microbiota composition as defined by bacterial species abundance as a consequence of C. albicans colonization. We also observed only a few bacterial genera that were significantly altered in abundance in C. albicans-colonized mice; however, C. albicans colonization significantly changed the structure of the bacterial microbiota co-occurrence network. Additionally, C. albicans colonization changed the response of the bacterial microbiota ecosystem to a clinically relevant perturbation, challenge with the antibiotic clindamycin.

Colonization with the commensal fungus Candida albicans perturbs the gut-brain axis through dysregulation of endocannabinoid signaling.

Anxiety disorders are the most prevalent mental health disorder worldwide, with a lifetime prevalence of 5-7 % of the human population. Although the etiology of anxiety disorders is incompletely understood, one aspect of host health that affects anxiety disorders is the gut-brain axis. Adolescence is a key developmental window in which stress and anxiety disorders are a major health concern. We used adolescent female mice in a gastrointestinal (GI) colonization model to demonstrate that the commensal fungus Candida albicans affects host health via the gut-brain axis. In mice, bacterial members of the gut microbiota can influence the host gut-brain axis, affecting anxiety-like behavior and the hypothalamus-pituitary-adrenal (HPA) axis which produces the stress hormone corticosterone (CORT). Here we showed that mice colonized with C. albicans demonstrated increased anxiety-like behavior and increased basal production of CORT as well as dysregulation of CORT production following acute stress. The HPA axis and anxiety-like behavior are negatively regulated by the endocannabinoid N-arachidonoylethanolamide (AEA). We demonstrated that C. albicans-colonized mice exhibited changes in the endocannabinoidome. Further, increasing AEA levels using the well-characterized fatty acid amide hydrolase (FAAH) inhibitor URB597 was sufficient to reverse both neuroendocrine phenotypes in C. albicans-colonized mice. Thus, a commensal fungus that is a common colonizer of humans had widespread effects on the physiology of its host. To our knowledge, this is the first report of microbial manipulation of the endocannabinoid (eCB) system that resulted in neuroendocrine changes contributing to anxiety-like behavior.

The gut, the bad and the harmless: Candida albicans as a commensal and opportunistic pathogen in the intestine.

Candida albicans is a regular member of the intestinal microbiota in the majority of the human population. This underscores C. albicans' adaptation to life in the intestine without inducing competitive interactions with other microbes, or immune responses detrimental to its survival. However, specific conditions such as a dysbalanced microbiome, a suppression of the immune system, and an impaired intestinal barrier can predispose for invasive, mostly nosocomial, C. albicans infections. Colonization of the intestine and translocation through the intestinal barrier are fundamental aspects of the processes preceding life-threatening systemic candidiasis. Insights into C. albicans' commensal lifestyle and translocation can thus help us to understand how patients develop candidiasis, and may provide leads for therapeutic strategies aimed at preventing infection. In this review, we discuss the commensal lifestyle of C. albicans in the intestine, the role of morphology for commensalism, the influence of diet, and the interactions with bacteria of the microbiota.

Lipid Species in the GI Tract are Increased by the Commensal Fungus Candida albicans and Decrease the Virulence of Clostridioides difficile.

Prior antibiotic treatment is a risk factor for Clostridioides difficile infection (CDI); the commensal gut microbiota plays a key role in determining host susceptibility to the disease. Previous studies demonstrate that the pre-colonization of mice with a commensal fungus, Candida albicans, protects against a lethal challenge with C. difficile spores. The results reported here demonstrate that the cecum contents of antibiotic-treated mice with C. albicans colonization contained different levels of several lipid species, including non-esterified, unsaturated long-chain fatty acids compared to non-C. albicans-colonized mice. Mice fed olive oil for one week and challenged with C. difficile spores showed enhanced survival compared to PBS-fed mice. The amount of olive oil administered was not sufficient to cause weight gain or to result in significant changes to the bacterial microbiota, in contrast to the effects of a high-fat diet. Furthermore, the direct exposure of C. difficile bacteria in laboratory culture to the unsaturated fatty acid oleic acid, the major fatty acid found in olive oil, reduced the transcription of genes encoding the toxins and reduced the survival of bacteria in the post-exponential phase. Therefore, the effects of C. albicans on the metabolite milieu contributed to the attenuation of C. difficile virulence.

On Commensalism of Candida.

Candida species are both opportunistic fungal pathogens and common members of the human mycobiome. Over the years, the main focus of the fungal field has been on understanding the pathogenic potential and disease manifestation of these organisms. Therefore, understanding of their commensal lifestyle, interactions with host epithelial barriers, and initial transition into pathogenesis is less developed. In this review, we will describe the current knowledge on the commensal lifestyle of these fungi, how they are able to adhere to and colonize host epithelial surfaces, compete with other members of the microbiota, and interact with the host immune response, as well as their transition into opportunistic pathogens by invading the gastrointestinal epithelium.

In Vitro and In Vivo Study of Amphotericin B Formulation with Quaternized Bioreducible Lipidoids.

Invasive fungal infections are well-known causes of morbidity and mortality in immunocompromised patients. Amphotericin B (AmB) is a polyene fungicidal agent with excellent properties of the broad antifungal spectrum, high activity, and relatively rare drug resistance. However, significant toxicities limit the clinical application of AmB and its conventional formulation AmB deoxycholate (Fungizone). Here we investigated nanoparticle formulations of AmB using synthetic biodegradable lipidoids and evaluated their stability, in vitro antifungal efficacy, and in vivo toxicity and pharmacokinetics. We found that the AmB formulated using a mixture of quaternized lipidoid (Q78-O14B) and DSPE-PEG2000 has the size around 70-100 nm and is stable during storage. The formulation showed no hemotoxicity to red blood cells (RBCs) in vitro. It also possesses the highest antifungal activity (in vitro) and lowest toxicity (both in vitro and in vivo). These metrics are significantly superior to the commercial antifungal product Fungizone. Meanwhile, AmB/Q78-O14B-P exhibited prolonged blood circulation in comparison to Fungizone in vivo. In AmB/Q78-O14B-P formulation, AmB was still detectable in the liver, spleen, and lung tissues with a concentration above the minimum inhibitory concentrations 72 h after low-dose intravenous injection. Based on these results, AmB in lipidoid nanoparticle formulation may produce sustained antifungal activity against blood-borne and systemic organ infections. Moreover, the new AmB formulation showed low nephrotoxicity and hepatotoxicity in rats even at high doses, allowing a dramatically wider and safer therapeutic window than Fungizone. This method provides a means to develop much needed antifungal agents that will be more therapeutically efficacious, more affordable (than AmBisome), and less toxic (than Fungizone) for the treatment of systemic fungal infections.

A novel genetic circuitry governing hypoxic metabolic flexibility, commensalism and virulence in the fungal pathogen Candida albicans.

Inside the human host, the pathogenic yeast Candida albicans colonizes predominantly oxygen-poor niches such as the gastrointestinal and vaginal tracts, but also oxygen-rich environments such as cutaneous epithelial cells and oral mucosa. This suppleness requires an effective mechanism to reversibly reprogram the primary metabolism in response to oxygen variation. Here, we have uncovered that Snf5, a subunit of SWI/SNF chromatin remodeling complex, is a major transcriptional regulator that links oxygen status to the metabolic capacity of C. albicans. Snf5 and other subunits of SWI/SNF complex were required to activate genes of carbon utilization and other carbohydrates related process specifically under hypoxia. snf5 mutant exhibited an altered metabolome reflecting that SWI/SNF plays an essential role in maintaining metabolic homeostasis and carbon flux in C. albicans under hypoxia. Snf5 was necessary to activate the transcriptional program linked to both commensal and invasive growth. Accordingly, snf5 was unable to maintain its growth in the stomach, the cecum and the colon of mice. snf5 was also avirulent as it was unable to invade Galleria larvae or to cause damage to human enterocytes and murine macrophages. Among candidates of signaling pathways in which Snf5 might operate, phenotypic analysis revealed that mutants of Ras1-cAMP-PKA pathway, as well as mutants of Yak1 and Yck2 kinases exhibited a similar carbon flexibility phenotype as did snf5 under hypoxia. Genetic interaction analysis indicated that the adenylate cyclase Cyr1, a key component of the Ras1-cAMP pathway interacted genetically with Snf5. Our study yielded new insight into the oxygen-sensitive regulatory circuit that control metabolic flexibility, stress, commensalism and virulence in C. albicans.

The role of fungi in C. difficile infection: An underappreciated transkingdom interaction.

Novel culture independent technologies have further elucidated the composition of the human mycobiome, though the role of fungi in human health and disease remains largely unknown. Recent studies have suggested conflicting roles for fungi in the gastrointestinal tract, underscoring the complexity of the interactions between the mycobiome, its bacterial counterpart, and the host. One key example is the observation that fungal taxa are overrepresented in patients with Clostridioides difficile infection (CDI), suggesting a role for fungi in this disease. Recent studies in murine models have demonstrated the ability of the commensal fungus Candida albicans to alter the course of CDI, supporting the notion that fungi play a role in this infection. This review summarizes current data on fungi and CDI, and shows that views of the dysbiotic state that is central to the pathogenesis of CDI are incomplete without consideration of the mycobiome.

A Synthetic System That Senses Candida albicans and Inhibits Virulence Factors.

Due to a limited set of antifungals available and problems in early diagnosis, invasive fungal infections caused by Candida species are among the most common hospital-acquired infections with staggering mortality rates. Here, we describe an engineered system able to sense and respond to the fungal pathogen Candida albicans, the most common cause of candidemia. In doing so, we identified hydroxyphenylacetic acid (HPA) as a novel molecule secreted by C. albicans. Furthermore, we engineered E. coli to be able to sense HPA produced by C. albicans. Finally, we constructed a sense-and-respond system by coupling the C. albicans sensor to the production of an inhibitor of hypha formation, thereby reducing filamentation, virulence factor expression, and fungal-induced epithelial damage. This system could be used as a basis for the development of novel prophylactic approaches to prevent fungal infections.