In Vitro and In Vivo Evaluation of the Antifungal Activity of APX001A/APX001 against Candida auris.

Candida auris is an emerging multidrug-resistant yeast that has been responsible for invasive infections associated with high morbidity and mortality. C. auris strains often demonstrate high fluconazole and amphotericin B MIC values, and some strains are resistant to all three major antifungal classes. We evaluated the susceptibility of 16 C. auris clinical strains, isolated from a wide geographical area, to 10 antifungal agents, including APX001A, a novel agent that inhibits the fungal protein Gwt1 (glycosylphosphatidylinositol-anchored wall transfer protein 1). APX001A demonstrated significantly lower MIC50 and MIC90 values (0.004 and 0.031 µg/ml, respectively) than all other agents tested. The efficacy of the prodrug APX001 was evaluated in an immunocompromised murine model of disseminated C. auris infection. Significant efficacy (80 to 100% survival) was observed in all three APX001 treatment groups versus 50% survival for the anidulafungin treatment group. In addition, APX001 showed a significant log reduction in CFU counts in kidney, lung, and brain tissue (1.03 to 1.83) versus the vehicle control. Anidulafungin also showed a significant log reduction in CFU in the kidneys and lungs (1.5 and 1.62, respectively) but did not impact brain CFU. These data support further clinical evaluation of this new antifungal agent.

Evaluation of the efficacy of rezafungin, a novel echinocandin, in the treatment of disseminated Candida auris infection using an immunocompromised mouse model.

Background: Multiple cases of Candida auris infection have been reported with high mortality rates owing to its MDR nature. Rezafungin (previously CD101) is a novel echinocandin with enhanced stability and pharmacokinetics that achieves high plasma drug exposure and allows for once weekly dose administration. Objectives: Evaluate the efficacy of rezafungin in the treatment of disseminated C. auris infection using a mouse model of disseminated candidiasis. Methods: Mice were immunosuppressed 3 days prior to infection and 1 day post-infection. On the day of infection, mice were inoculated with 3 × 107C. auris blastospores via the tail vein. Mice were randomized into four groups (n = 20): rezafungin at 20 mg/kg, amphotericin B at 0.3 mg/kg, micafungin at 5 mg/kg and a vehicle control. Treatments were administered 2 h post-infection. Rezafungin was given additionally on days 3 and 6 for a total of three doses, while the remaining groups were treated every day for a total of seven doses. Five mice from each group were sacrificed on days 1, 4, 7 and 10 of the study. Kidneys were removed from each mouse to determine the number of cfu for each respective day. Results: Rezafungin had significantly lower average log10 cfu/g of tissue compared with amphotericin B- and vehicle-treated mice on all days when kidneys were harvested. Additionally, rezafungin-treated mice had significantly lower average log10 cfu/g of tissue compared with micafungin-treated mice on day 10. Conclusions: Our findings show that rezafungin possesses potent antifungal activity against C. auris in a disseminated model of candidiasis.

Polymeric Nanofiber/Antifungal Formulations Using a Novel Co-extrusion Approach.

We report the successful implementation of a novel melt co-extrusion process to fabricate ca. 1 µm diameter fibers of poly(caprolactone) (PCL) containing the antifungal compound clotrimazole in concentrations between 4 and 8 wt%. The process involves co-extrusion of a clotrimazole-loaded PCL along with poly(ethylene oxide) (PEO) as a co-feed, with subsequent removal of PEO to isolate PCL-clotrimazole fibers. In vitro tests of the clotrimazole-containing fibers against the fungus Aspergillus fumigatus, Candida albicans, and Trichophyton mentagrophytes strains demonstrated good antifungal activity which was maintained for more than 3 weeks. An in vivo study using a mouse model showed the lowest tissue fungal burden for PCL-clotrimazole when compared to a PCL-only patch and untreated controls. Comparative studies were conducted with clotrimazole-containing PCL fibers fabricated by electrospinning. Our data showed that the co-extruded, clotrimazole-containing fibers maintain activity for longer times vs. electrospun samples. This, coupled with the much higher throughput of the co-extrusion process vs. electrospinning, renders this new approach very attractive for the fabrication of drug-releasing polymer fibers.

Indole-3-acetic acid synthesized through the indole-3-pyruvate pathway promotes Candida tropicalis biofilm formation.

We previously found that the elevated abundance of the fungus Candida tropicalis is positively correlated with the bacteria Escherichia coli and Serratia marcescens in Crohn's disease patients and the three pathogens, when co-cultured, form a robust mixed-species biofilm. The finding suggests that these three pathogens communicate and promote biofilm formation, possibly through secretion of small signaling molecules. To identify candidate signaling molecules, we carried out a metabolomic analysis of the single-species and triple-species cultures of the three pathogens. This analysis identified 15 metabolites that were highly increased in the triple-species culture. One highly induced metabolite was indole-3-acetic acid (IAA), which has been shown to induce filamentation of certain fungi. We thus tested the effect of IAA on biofilm formation of C. tropicalis and demonstrated that IAA promotes biofilm formation of C. tropicalis. Then, we carried out isotope tracing experiments using 13C-labeled-tryptophan as a precursor to uncover the biosynthesis pathway of IAA in C. tropicalis. The results indicated that C. tropicalis synthesizes IAA through the indole-3-pyruvate pathway. Further studies using inhibitors of the indole-3-pyruvate pathway are warranted to decipher the mechanisms by which IAA influences biofilm formation.

Intestinal Stem Cell Niche Defects Result in Impaired 3D Organoid Formation in Mouse Models of Crohn's Disease-like Ileitis.

Intestinal epithelial barrier dysfunction is a risk factor in the pathogenesis of Crohn's disease (CD); however, no corrective FDA-approved therapies exist. We used an enteroid (EnO)-based system in two murine models of experimental CD, SAMP1/YitFc (SAMP) and TNFΔARE/+ (TNF). While severely inflamed SAMP mice do not generate EnOs, "inflammation-free" SAMP mice form EnO structures with impaired morphology and reduced intestinal stem cell (ISC) and Paneth cell viability. We validated these findings in TNF mice concluding that inflammation in intestinal tissues impedes EnO generation and suppressing inflammation by steroid administration partially rescues impaired formation in SAMP mice. We generated the first high-resolution transcriptional profile of the SAMP ISC niche demonstrating that alterations in multiple key pathways contribute to niche defect and targeting them may partially rescue the phenotype. Furthermore, we correlated the defects in formation and the rescue of EnO formation to reduced viability of ISCs and Paneth cells.

Effects of a Novel Probiotic Combination on Pathogenic Bacterial-Fungal Polymicrobial Biofilms.

Dysbiosis of the gut microbiome has been implicated in inflammatory bowel diseases. We have shown that levels of Candida tropicalis, along with those of Escherichia coli and Serratia marcescens, are significantly elevated in Crohn's disease (CD) patients. Here, we evaluated the ability of a novel probiotic to prevent and treat polymicrobial biofilms (PMB) formed by C. tropicalis with E. coli and S. marcescens Since Candida albicans has been reported to be elevated in CD patients, we investigated the interactions of C. albicans with these bacterial species in biofilm formation. We determined whether the interaction between Candida spp. and bacteria is specific by using Trichosporon inkin and Saccharomyces fibuligera as comparators. Additionally, the effects of probiotics on C. albicans germination and biofilm formation were determined. To determine the ability of the probiotic to prevent or treat mature biofilms, probiotic filtrate was added to the PMB at early (prevention) and mature (treatment) phases. Biofilm thickness and architecture were assessed by confocal scanning laser microscopy. The effects of the probiotic on germination were evaluated in the presence of serum. Exposure of C. tropicalis PMB to probiotic filtrate reduced biofilm matrix, decreased thickness, and inhibited hyphal formation. We showed that C. albicans or C. tropicalis formed significantly thicker PMB than control biofilms, indicating that this interaction is Candida specific. Treatment with probiotic filtrate inhibited C. albicans germination and prevented/treated C. albicans PMB. The designed probiotic may have utility in the management of biofilm-associated gastrointestinal diseases such as Crohn's and colorectal cancer.IMPORTANCE The effects of diversity of the gut microbiome on inflammation have centered mainly on bacterial flora. Recent research has implicated fungal species and their interactions with other organisms in the inflammatory process. New ways to restore microbial balance in the gut are being explored. Our goal was to identify beneficial probiotic strains that would antagonize these fungal and bacterial pathogens that are elevated in the inflamed gut, and which also have antibiofilm activity. Fungus-bacterium correlation analysis allowed us to identify candidate probiotic species that can antagonize microbial pathogens, which we subsequently incorporated into a novel probiotic formulation. Amylase, which is known to have some antibiofilm activity, was also added to the probiotic mixture. This novel probiotic may have utility for the management of inflammatory bowel diseases by disrupting polymicrobial biofilm formation.

Fungal Translocation Is Associated with Immune Activation and Systemic Inflammation in Treated HIV.

The mechanisms causing HIV-associated immune activation remain incompletely understood. Alteration of intestinal integrity with subsequent translocation of bacterial products appears to play an important role; however, little is known about the impact of fungal translocation. We assessed the effect of fungal translocation and its association with immune activation in people living with HIV (PLWH) compared with uninfected controls. We measured serum levels of ß-D-glucan (BDG) and anti-Saccharomyces cerevisiae antibodies (ASCA) immunoglobulin G (IgG) and immunoglobulin A (IgA) and markers of systemic inflammation and immune activation in virally suppressed PLWH on antiretroviral therapy (ART) and uninfected controls. T-test and Mann-Whitney tests were used to compare markers by HIV status and correlation and regression analyses were used to assess associations of fungal translocation markers with markers of inflammation. One hundred seventy-six participants were included (128 HIV+ and 48 HIV-); 72% male, 65% African American, median age was 50 years, and CD4 was 710 cells/cm3. Levels of BDG tended to be lower in HIV+ when compared with controls (p = .05). No significant difference in levels of ASCA IgG and IgA was seen between groups (p > .75). There was a significant correlation between BDG and several markers of inflammation and immune activation in PLWH, not seen in uninfected controls. In contrast, no correlations were seen between levels of ASCA IgG and IgA with inflammatory markers. PLWH on ART do not have higher levels of BDG or ASCA when compared with uninfected controls, however, the association found between BDG and several inflammation markers suggests a potential role of fungal translocation in the heightened immune activation seen in treated HIV.

The mycobiome in HIV.

PURPOSE OF REVIEW: There are a limited number of studies investigating the association between the microbiome and HIV. Although the majority of these published investigations have focused on the role of the bacterial community (bacteriome) in this setting, a handful of studies have also characterized the role of the mycobiome in HIV-infected individuals. This review will summarize the most recent reports pertaining to the role of the fungal community in HIV. RECENT FINDINGS: Differences in the composition of the oral and respiratory mycobiome in HIV-infected individuals compared with uninfected individuals have been reported. SUMMARY: Our review shows that studies investigating the role of the mycobiome in the setting of HIV are severely lacking. With recent advances in our understanding of the composition of the human microbiome, investigations into the role of the bacteria and fungus comprising the overall microbiota and how the two interact to influence each other and the host is crucial.

The mycobiome: Role in health and disease, and as a potential probiotic target in gastrointestinal disease.

The human gastrointestinal (GI) tract is home to trillions of microorganisms, some beneficial and others potentially harmful. Recent advances in science have allowed us to identify the multitude of organisms inhabiting the GI tract and parse out those that play a role in inflammatory bowel disease (IBD). Unfortunately, most research has focused on studying only the bacteria while ,overlooking a key player, fungus. In order to address this issue, we have focused our efforts on studying the fungal community in the GI tract known as the mycobiome. We found that patients with Crohn's disease (CD) tend to have much higher levels of the fungus Candida tropicalis compared to their healthy family members, as well as two bacteria, Escherichia coli and Serratia marcescens. Furthermore, we showed that these three organisms worked together to form robust biofilms capable of exacerbating intestinal inflammation. Herein, we discuss the role of the mycobiome in health and disease, and highlight the importance of maintaining balance of the GI microbiota. Additionally, taking into consideration recent next generation sequencing data, we provide insight into potentially new therapeutic approaches in the treatment of IBD through the use of antifungals and/or probiotics aimed at establishing and maintaining a healthy balance of the GI total microbial community including fungi and bacteria.

Clinical Effects of Gamma-Radiation-Resistant Aspergillus sydowii on Germ-Free Mice Immunologically Prone to Inflammatory Bowel Disease.

We report and investigated a case of inadvertent contamination of 125 mice (housed in two germ-free positive-pressurized isolators) with emerging human and coral pathogen Aspergillus sydowii. The infected mice correspond to genetic line SAMP1/YitFc, which have 100% immune predisposition to develop Crohn's disease-like spontaneous pathologies, namely, inflammatory bowel disease (IBD). Pathogen update based on a scoping review of the literature and our clinical observations and experimentation are discussed. The unwanted infection of germ-free mice (immunologically prone to suffer chronic inflammation) with human pathogen A. sydowii resulted in no overt signs of clinical disease over 3-week exposure period, or during DSS-induced colitis experiments. Results and observations suggest that A. sydowii alone has limited clinical effect in immunocompromised germ-free mice or that other commensal microbial flora is required for Aspergillus-associated disease to occur.