In vitro determination of the susceptibility of Malassezia furfur biofilm to different commercially used antimicrobials.

Malassezia furfur is a yeast known as the etiological agent of seborrheic dermatitis. We evaluated the action of five different antimicrobials (amphotericin B, chloramphenicol, ketoconazole, fluconazole, and nystatin) on inhibiting biofilm formation and removing biofilm already formed by M. furfur. The assays were carried out using the microdilution method, and scanning electron microscopy images were used to analyze the biofilm structure. According to the results obtained, the percentage of inhibition was higher for chloramphenicol, followed by ketoconazole, nystatin, and amphotericin B. Regarding the eradication of the biofilm formed, the highest percentage was chloramphenicol, followed by ketoconazole and nystatin. Amphotericin B did not affect biofilm eradication, whereas fluconazole did not cause significant changes inhibiting or removing M. furfur biofilm. Therefore, except for fluconazole, all evaluated antimicrobials had inhibiting effects on the biofilm of M. furfur, either in its formation and/or eradication. Although the results achieved with chloramphenicol have been highlighted, further in vitro and in vivo studies are still needed in order to include this antimicrobial in the therapy of seborrheic dermatitis due to its toxicity, especially to the bone marrow.

A Bacillus velezensis strain shows antimicrobial activity against soilborne and foliar fungi and oomycetes.

Biological control uses naturally occurring antagonists such as bacteria or fungi for environmentally friendly control of plant pathogens. Bacillus spp. have been used for biocontrol of numerous plant and insect pests and are well-known to synthesize a variety of bioactive secondary metabolites. We hypothesized that bacteria isolated from agricultural soil would be effective antagonists of soilborne fungal pathogens. Here, we show that the Delaware soil isolate Bacillus velezensis strain S4 has in vitro activity against soilborne and foliar plant pathogenic fungi, including two with a large host range, and one oomycete. Further, this strain shows putative protease and cellulase activity, consistent with our prior finding that the genome of this organism is highly enriched in antifungal and antimicrobial biosynthetic gene clusters. We demonstrate that this bacterium causes changes to the fungal and oomycete hyphae at the inhibition zone, with some of the hyphae forming bubble-like structures and irregular branching. We tested strain S4 against Magnaporthe oryzae spores, which typically form germ tubes and penetration structures called appressoria, on the surface of the leaf. Our results suggest that after 12 hours of incubation with the bacterium, fungal spores form germ tubes, but instead of producing appressoria, they appear to form rounded, bubble-like structures. Future work will investigate whether a single antifungal molecule induces all these effects, or if they are the result of a combination of bacterially produced antimicrobials.

Disseminated phaeohyphomycosis due to Cladophialophora bantiana in an immunocompetent child.

A 3-year-old boy presented with acute headache, vomiting and right focal clonic seizures without history of fever, joint pain or altered sensorium. Neuroimaging showed multifocal contrast enhancing lesions with significant perilesional edema. CECT chest and abdomen showed multiple variable sized nodules in the lungs and hypodense lesion in liver with mesenteric lymphadenopathy. There was persistent eosinophilia with maximum upto 35 %. Liver biopsy and brain biopsy revealed Cladophialophora bantiana. He was treated with IV liposomal amphotericin and voriconazole for 6 weeks with repeat neuroimaging showing more than 50 % resolution of the intracranial lesions. He was transitioned to oral combination of flucytosine and voriconazole. At 14 months follow-up, he remained symptom free with complete radiological resolution of the lesions and no eosinophilia. High suspicion, an aggressive approach in obtaining microbiological diagnosis and timely combination antifungal therapy may give satisfactory outcome without surgery.

Successful treatment of cutaneous protothecosis with fluconazole: A case report and epidemiology study of Prototheca infection in China.

BACKGROUND: Protothecosis is an infection of humans and animals caused by a rare conditionally pathogenic fungus (prototheca). It can occur in immunocompromised or normal patients. AIMS: To describe the epidemiology of prototheca infection in China. METHODS: We report a case of successful treatment of cutaneous protothecosis with fluconazole and analyzed the epidemiological characteristics, risk factors, clinical manifestations, diagnosis, treatment and prognosis of prototheca infections in China. RESULTS: We describe this case and 29 cases of prototheca infections in China. At present, Prototheca wickerhamii (Pw) infection is the most common infection in China, and single or combined itraconazole is the preferred treatment. CONCLUSIONS: These results provide detailed information and relevant clinical treatment strategies for the diagnosis and treatment of protothecosis in China.

Pharmacokinetics, safety, and tolerability of fosmanogepix IV to oral switch and multiple IV doses in healthy participants.

Fosmanogepix [FMGX, APX001; active form: manogepix (MGX), APX001A] is a first-in-class, intravenous (IV)/oral antifungal currently being evaluated for invasive fungal disease treatment. Data from two phase 1, placebo-controlled studies [IV-oral switch (study 1) and multiple IV doses (study 2)] evaluating FMGX tolerability, and pharmacokinetics (PK) are presented. Healthy adults (study 1: 18-65 years; study 2: 18-55 years) were eligible (randomized 3:1 to FMGX: placebo). Eleven participants completed study 1. In study 2, 51 participants (48 planned + 3 replacement) were enrolled in six cohorts (8 participants each; 34 completed the study). In study 1, overall MGX systemic exposures were comparable from day 1 to day 42 of dosing; steady-state plasma concentrations were achieved in ≤24 h following two IV loading doses (1,000 mg) and exposures maintained after switching [IV (600 mg) to daily oral doses (800 mg)]. FMGX was safe and well-tolerated. In study 2, FMGX IV doses (loading doses twice daily/maintenance doses once daily; 3-h infusion) of 1,500/900 mg (cohort A), 900/900 mg (cohort B), and 1,000/900 mg (cohort C: with ondansetron) were not well-tolerated; most participants reported nausea and infrequent vomiting. FMGX IV doses of 1,000/750 mg (cohort D), 1,000/850 mg (cohort E), and 1,000/900 mg (cohort F: ondansetron prn) were relatively better tolerated. Steady-state systemic exposures were achieved between days 2 and 4. All cohorts had similar geometric mean (GM) concentrations during maintenance dosing and similar GM PK parameters. Dosing regimen evaluated in study 1 was safe, well-tolerated, and may be used for future clinical evaluations.

Evolutionary trends in antifungal resistance: a meta-analysis.

The present paper includes a meta-analysis of literature data on 318 species of fungi belonging to 34 orders in their response to 8 antifungal agents (amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole, posaconazole, terbinafine, and voriconazole). Main trends of MIC results at the ordinal level were visualized. European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute (CLSI) clinical breakpoints were used as the staff gauge to evaluate MIC values ranging from resistance to susceptibility, which were subsequently compared with a phylogenetic tree of the fungal kingdom. Several orders (Hypocreales, Microascales, and Mucorales) invariably showed resistance. Also the basidiomycetous orders Agaricales, Polyporales, Sporidiales, Tremellales, and Trichosporonales showed relatively high degrees of azole multi-resistance, while elsewhere in the fungal kingdom, including orders with numerous pathogenic and opportunistic species, that is, Onygenales, Chaetothyiales, Sordariales, and Malasseziales, in general were susceptible to azoles. In most cases, resistance vs susceptibility was consistently associated with phylogenetic distance, members of the same order showing similar behavior. IMPORTANCE: A kingdom-wide the largest set of published wild-type antifungal data comparison were analyzed. Trends in resistance in taxonomic groups (monophyletic clades) can be compared with the phylogeny of the fungal kingdom, eventual relationships between fungus-drug interaction and evolution can be described.

Modeling of intravenous caspofungin administration using an intestine-on-chip reveals altered Candida albicans microcolonies and pathogenicity.

Candida albicans is a commensal yeast of the human intestinal microbiota that, under predisposing conditions, can become pathogenic and cause life-threatening systemic infections (candidiasis). Fungal-host interactions during candidiasis are commonly studied using conventional 2D in vitro models, which have provided critical insights into the pathogenicity. However, microphysiological models with a higher biological complexity may be more suitable to mimic in vivo-like infection processes and antifungal drug efficacy. Therefore, a 3D intestine-on-chip model was used to investigate fungal-host interactions during the onset of invasive candidiasis and evaluate antifungal treatment under clinically relevant conditions. By combining microbiological and image-based analyses we quantified infection processes such as invasiveness and fungal translocation across the epithelial barrier. Additionally, we obtained novel insights into fungal microcolony morphology and association with the tissue. Our results demonstrate that C. albicans microcolonies induce injury to the epithelial tissue by disrupting apical cell-cell contacts and causing inflammation. Caspofungin treatment effectively reduced the fungal biomass and induced substantial alterations in microcolony morphology during infection with a wild-type strain. However, caspofungin showed limited effects after infection with an echinocandin-resistant clinical isolate. Collectively, this organ-on-chip model can be leveraged for in-depth characterization of pathogen-host interactions and alterations due to antimicrobial treatment.

In vitro studies into establishing therapeutic bioequivalence of complex topical products: Weight of evidence.

Over the past decade, topically applied drug products have experienced extraordinary price increases, due to the shortage of multisource generic drug products. This occurrence is mainly related to the underlying challenges evolved in topical bioequivalence documentation. Although there has been continuing regulatory efforts to present surrogate in vitro methods to clinical endpoint studies, there is still a continued need for cost- and time-efficient alternatives that account for product specificities. Hence, this work intended to expose bioequivalence assessment issues for complex topical formulations, and more specifically those related with product efficacy guidance. As a model drug and product, a bifonazole 10 mg/g cream formulation was selected and two different batches of the commercially available Reference Product (RP) were used: RP1 that displayed lower viscosity and RP4 which presented high, but not the highest, viscosity. In vitro human skin permeation testing (IVPT) was carried out and the results were evaluated by means of the traditional bioequivalence assessment approach proposed by the EMA, as well as by the Scaled Average Bioequivalence assessment approach proposed by the FDA. Based on previous experience, there was an expectation of a high level of variability in the results, thus alternative methods to evaluate local drug skin availability were developed. More specifically, an infected skin disease model, where ex vivo human skin was infected and ATP levels were used as a biological marker for monitoring antifungal activity after product application. The results showed that permeation equivalence could not be supported between the different RP batches. In contrast, this statistical difference between the formulation batches was not indicated in the disease model. Nevertheless, in pivotal IVPT studies, the lowest permeant formulation (RP4) evidenced a higher antifungal in vitro activity as reported by the lower levels of ATP. A critical appraisal of the results is likewise presented, focusing on an outlook of the real applicability of the regulatory guidances on this subject.

Breaking-Down Barriers: Proposal of Using Cellulose Biosynthesis Inhibitors and Cellulase Enzyme as a Novel Treatment Modality for Vision Threatening Pythium Insidiosum Keratitis.

Pythium insidiosum, an Oomycete, causes severe keratitis that endangers vision. Its clinical, morphological, and microbiological characteristics are often indistinguishable from those of fungal keratitis, earning it the moniker "parafungus". Distinctive clinical hallmarks that set it apart from other forms of keratitis include radial keratoneuritis, tentacles, marginal infiltration, and a propensity for rapid limbal spread. The therapeutic approach to Pythium keratitis (PK) has long been a subject of debate, and topical and systemic antifungals and antibacterials have been tried with limited success. Approximately 80% of these eyes undergo therapeutic keratoplasty to salvage the eye. Hence, there is a need to innovate for alternative and better medical therapy to safeguard these eyes. The resistance of Pythium to standard antifungal treatments can be attributed to the absence of ergosterol in its cell wall. Cell walls of plants and algae have cellulose as an essential constituent. Cellulose imparts strength and structure and acts as the "skeleton" of the plant. Fungal and animal cell walls typically lack cellulose. The cellular architecture of Pythium shares a similarity with plant and algal cells through the incorporation of cellulose within its cell wall structure. Inhibitors targeting cellulose biosynthesis (CBI), such as Indaziflam, Isoxaben, and Quinoxyphen, serve as critical tools for elucidating the pathways of cellulose synthesis. Furthermore, the enzymatic action of cellulase is instrumental for the extraction of proteins and DNA. To circumvent this issue, we hypothesize that CBI's and cellulase enzymes can act on the Pythium cell wall and may effectively treat PK. The available literature supporting the hypothesis and proof of concept has also been discussed. We have also discussed these drugs' molecular mechanism of action on the Pythium cell wall. We also aim to propose how these drugs can be procured and used as a potential medical management option for this devastating entity.

Antifungal Use in Perforated Peptic Ulcer Disease: A Western Australian Perspective.

Background Perforated peptic ulcer disease has a high mortality rate, and there is consensus regarding the use of antifungals in the management of immunocompromised patients; however, there is variability in the utilization of antifungals in the non-immunocompromised cohort. This study aims to describe the current practice related to the use of antifungals in perforated peptic ulcer disease in Western Australia and to determine the peri-operative morbidity and mortality in the immunocompromised and non-immunocompromised cohort receiving antifungals. Methods Medical records of patients who underwent surgical repair of perforated peptic ulcer in all Western Australian tertiary hospitals between January 1, 2010, and December 31, 2017, were reviewed retrospectively. Data regarding pre-operative patient factors such as age, gender, and comorbidities, post-operative outcomes such as intra-abdominal sepsis/bleeding, peri-operative antifungal prescription, and abundance of fungal growth on intra-operative samples were collected. Results The study included 359 patients. The antifungal prescription was variable. An American Society of Anesthesiologists (ASA) score of 3 or more, presence of pre-operative shock and acidosis, and level of abundance of fungal growth on intra-operative samples were associated with antifungal prescription. Amongst the non-immunocompromised cohort, receiving antifungals was associated with higher morbidity. Conclusion The use of antifungals for patients with perforated peptic ulcer disease was variable. An ASA score of 3 or greater and pre-operative shock and acidosis are pre-operative factors predisposing patients to receiving antifungals. There was no difference in morbidity or mortality amongst immunocompromised patients regardless of antifungal prescription or non-prescription. However, in the non-immunocompromised cohort, those who received antifungals had a higher morbidity compared to those who did not.

Insights into the anti-Candida albicans properties of natural phytochemicals: An in vitro and in vivo investigation.

Invasive candidiasis, attributed to Candida albicans, has long been a formidable threat to human health. Despite the advent of effective therapeutics in recent decades, the mortality rate in affected patient populations remains discouraging. This is exacerbated by the emergence of multidrug resistance, significantly limiting the utility of conventional antifungals. Consequently, researchers are compelled to continuously explore novel solutions. Natural phytochemicals present a potential adjunct to the existing arsenal of agents. Previous studies have substantiated the efficacy of phytochemicals against C. albicans. Emerging evidence also underscores the promising application of phytochemicals in the realm of antifungal treatment. This review systematically delineates the inhibitory activity of phytochemicals, both in monotherapy and combination therapy, against C. albicans in both in vivo and in vitro settings. Moreover, it elucidates the mechanisms underpinning the antifungal properties, encompassing (i) cell wall and plasma membrane damage, (ii) inhibition of efflux pumps, (iii) induction of mitochondrial dysfunction, and (iv) inhibition of virulence factors. Subsequently, the review introduces the substantial potential of nanotechnology and photodynamic technology in enhancing the bioavailability of phytochemicals. Lastly, it discusses current limitations and outlines future research priorities, emphasizing the need for high-quality research to comprehensively establish the clinical efficacy and safety of phytochemicals in treating fungal infections. This review aims to inspire further contemplation and recommendations for the effective integration of natural phytochemicals in the development of new medicines for patients afflicted with C. albicans.

A comprehensive guide to assess gut mycobiome and its role in pathogenesis and treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an immune mediated chronic inflammatory disorder of gastrointestinal tract, which has underlying multifactorial pathogenic determinants such as environmental factors, susceptibility genes, gut microbial dysbiosis and a dysregulated immune response. Human gut is a frequent inhabitant of complex microbial ecosystem encompassing bacteria, viruses, parasites, fungi and other microorganisms that have an undisputable role in maintaining balanced homeostasis. All of these microbes interact with immune system and affect human gut physiology either directly or indirectly with interaction of each other. Intestinal fungi represent a smaller but crucial component of the human gut microbiome. Besides interaction with bacteriome and virome, it helps in balancing homoeostasis between pathophysiological and physiological processes, which is often dysregulated in patients with IBD. Understanding of gut mycobiome and its clinical implications are still in in its infancy as opposed to bacterial component of gut microbiome, which is more often focused. Modulation of gut mycobiome represents a novel and promising strategy in the management of patients with IBD. Emerging mycobiome-based therapies such as diet interventions, fecal microbiota transplantation (FMT), probiotics (both fungal and bacterial strains) and antifungals exhibit substantial effects in calibrating the gut mycobiome and restoring dysbalanced immune homeostasis by restoring the core gut mycobiome. In this review, we summarized compositional and functional diversity of the gut mycobiome in healthy individuals and patients with IBD, gut mycobiome dysbiosis in patients with IBD, host immune-fungal interactions and therapeutic role of modulation of intestinal fungi in patients with IBD.

Fusariosis in burn patients: A systematic review of case reports.

Burns can cause skin damage, facilitating the entry of fungi and other microorganisms into the body, leading to infections. Fusarium is a fungus capable of infecting individuals with burn injuries. Diagnosing and treating Fusarium infections in burn patients can be challenging due to the manifestation of nonspecific symptoms. This study aims to investigate case reports and case series from published literature describing Fusarium infection in burned patients, in order to assess treatment regimens, clinical outcomes, and make recommendations for future management. We conducted searches on Web of Science, PubMed, ScienceDirect, and Medline for all case reports and case series containing keywords 'Burn', 'Burns', 'Burned', 'Fusarium', or 'Fusariosis' in the title or abstract. All burn patients who developed Fusarium fungal infections between January 1974 and March 2023 were included in the study. Demographic and clinical data were analyzed retrospectivity. The final analysis incorporates 24 case reports encompassing a total of 87 burn patients with Fusarium infection. Patient ages ranged from one to 85 years, with the majority being male (53%). The median percentage of burn surface area was 78%, and the skin in the face, upper limbs, and lower limbs were the most commonly infected sites. Fungal infections appeared around 10 days after the burn injury on average. The majority of the patients were identified through culture or histopathology. The Fusarium dimerum species complex, which was found in nine patients, was the most frequently identified Fusarium species complex. Amphotericin B was the most preferred treatment drug, followed by voriconazole, and 62% of patients underwent debridement. In our study, 23 patients (37%) died from fungal infections. Implementing early and effective treatment protocols targeting Fusarium spp. in burn treatment units can significantly reduce mortality rates. It is critical to enhance the understanding of fusariosis epidemiology and emphasize the importance of maintaining a high clinical suspicion for this condition in burn patients.

Characterization of the Fusarium circinatum biofilm environmental response role.

The capacity to form biofilms is a common trait among many microorganisms present on Earth. In this study, we demonstrate for the first time that the fatal pine pitch canker agent, Fusarium circinatum, can lead a biofilm-like lifestyle with aggregated hyphal bundles wrapped in extracellular matrix (ECM). Our research shows F. circinatum's ability to adapt to environmental changes by assuming a biofilm-like lifestyle. This was demonstrated by varying metabolic activities exhibited by the biofilms in response to factors like temperature and pH. Further analysis revealed that while planktonic cells produced small amounts of ECM per unit of the biomass, heat- and azole-exposed biofilms produced significantly more ECM than nonexposed biofilms, further demonstrating the adaptability of F. circinatum to changing environments. The increased synthesis of ECM triggered by these abiotic factors highlights the link between ECM production in biofilm and resistance to abiotic stress. This suggests that ECM-mediated response may be one of the key survival strategies of F. circinatum biofilms in response to changing environments. Interestingly, azole exposure also led to biofilms that were resistant to DNase, which typically uncouples biofilms by penetrating the biofilm and degrading its extracellular DNA; we propose that DNases were likely hindered from reaching target cells by the ECM barricade. The interplay between antifungal treatment and DNase enzyme suggests a complex relationship between eDNA, ECM, and antifungal agents in F. circinatum biofilms. Therefore, our results show how a phytopathogen's sessile (biofilm) lifestyle could influence its response to the surrounding environment.

Therapeutic drug monitoring of antifungal therapies: do we really need it and what are the best practices?

INTRODUCTION: Despite advancements, invasive fungal infections (IFI) still carry high mortality rates, often exceeding 30%. The challenges in diagnosis, coupled with limited effective antifungal options, make managing IFIs complex. Antifungal drugs are essential for IFI management, but their efficacy can be diminished by drug-drug interactions and pharmacokinetic variability. Therapeutic Drug Monitoring (TDM), especially in the context of triazole use, has emerged as a valuable strategy to optimize antifungal therapy. AREAS COVERED: This review provides current evidence regarding the potential benefits of TDM in IFI management. It discusses how TDM can enhance treatment response, safety, and address altered pharmacokinetics in specific patient populations. EXPERT OPINION: TDM plays a crucial role in achieving optimal therapeutic outcomes in IFI management, particularly for certain antifungal agents. Preclinical studies consistently show a link between therapeutic drug levels and antifungal efficacy. However, clinical research in mycology faces challenges due to patient heterogeneity and the diversity of fungal infections. TDM's potential advantages in guiding Echinocandin therapy for critically ill patients warrant further investigation. Additionally, for drugs like Posaconazole, assessing whether serum levels or alternative markers like saliva offer the best measure of efficacy is an intriguing question.

Risk Factors for Invasive Fungal Infection in Lung Transplant Recipients on Universal Antifungal Prophylaxis.

Background: Many centers use universal antifungal prophylaxis after lung transplant, but risk factors for invasive fungal infection (IFI) in this setting are poorly described. Methods: This retrospective, single-center cohort study including 603 lung transplant recipients assessed risk factors for early (within 90 days of transplant) invasive candidiasis (IC) and invasive mold infection (IMI) and late (90-365 days after transplant) IMI using Cox proportional hazard regression. Results: In this cohort, 159 (26.4%) patients had 182 IFIs. Growth of yeast on donor culture (hazard ratio [HR], 3.30; 95% CI, 1.89-5.75) and prolonged length of stay (HR, 1.02; 95% CI, 1.01-1.03) were associated with early IC risk, whereas transplantation in 2016 or 2017 (HR, 0.21; 95% CI, 0.06-0.70; HR, 0.25; 95% CI, 0.08-0.80, respectively) and female recipient sex (HR, 0.53; 95% CI, 0.30-0.93) were associated with reduced risk. Antimold therapy (HR, 0.21; 95% CI, 0.06-0.78) was associated with lower early IMI risk, and female donor sex (HR, 0.40; 95% CI, 0.22-0.72) was associated with lower late IMI risk. Recent rejection was a risk factor for late IMI (HR, 1.73; 95% CI, 1.02-2.95), and renal replacement therapy predisposed to early IC, early IMI, and late IMI (HR, 5.67; 95% CI, 3.01-10.67; HR, 7.54; 95% CI, 1.93-29.45; HR, 5.33; 95% CI, 1.46-19.49, respectively). Conclusions: In lung transplant recipients receiving universal antifungal prophylaxis, risk factors for early IC, early IMI, and late IMI differ.

Whole genome analysis of Bacillus velezensis 160, biological control agent of corn head smut.

Corn head smut is a disease caused by the fungus Sporisorium reilianum. This phytosanitary problem has existed for several decades in the Mezquital Valley, an important corn-producing area in central Mexico. To combat the problem, a strain identified as Bacillus subtilis 160 was applied in the field, where it decreased disease incidence and increased crop productivity. In this study, the sequencing and analysis of the whole genome sequence of this strain were carried out to identify its genetic determinants for the production of antimicrobials. The B. subtilis 160 strain was found to be Bacillus velezensis. Its genome has a size of 4,297,348 bp, a GC content of 45.8%, and 4,174 coding sequences. Comparative analysis with the genomes of four other B. velezensis strains showed that they share 2,804 genes and clusters for the production of difficidin, bacillibactin, bacilysin, macrolantin, bacillaene, fengycin, butirosin A, locillomycin, and surfactin. For the latter metabolite, unlike the other strains that have only one cluster, B. velezensis 160 has three. A cluster for synthesizing laterocidine, an antimicrobial reported only in Brevibacillus laterosporus, was also identified. IMPORTANCE: In this study, we performed sequencing and analysis of the complete genome of the strain initially identified as Bacillus subtilis 160 as part of its characterization. This bacterium has shown its ability to control corn head smut in the field, a disease caused by the basidiomycete fungus Sporisorium reilianum. Analyzing the complete genome sequence not only provides a more precise taxonomic identification but also sheds light on the genetic potential of this bacterium, especially regarding mechanisms that allow it to exert biological control. Employing molecular and bioinformatics tools in studying the genomes of agriculturally significant microorganisms offers insights into the development of biofungicides and bioinoculants. These innovations aim to enhance plant growth and pave the way for strategies that boost crop productivity.

Natural compounds from freshwater mussels disrupt fungal virulence determinants and influence fluconazole susceptibility in the presence of macrophages in Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen responsible for fatal infections, especially in patients with a depressed immune system. Overexposure to antifungal drugs due to prolonged treatment regimens and structure-similar applications in agriculture have weakened the efficacy of current antifungals in the clinic. The rapid evolution of antifungal resistance urges the discovery of new compounds that inhibit fungal virulence determinants, rather than directly killing the pathogen, as alternative strategies to overcome disease and reduce selective pressure toward resistance. Here, we evaluated the efficacy of freshwater mussel extracts (crude and clarified) against the production of well-defined virulence determinants (i.e., thermotolerance, melanin, capsule, and biofilm) and fluconazole resistance in C. neoformans. We demonstrated the extracts' influence on fungal thermotolerance, capsule production, and biofilm formation, as well as susceptibility to fluconazole in the presence of macrophages. Additionally, we measured the inhibitory activity of extracts against commercial peptidases (family representatives of cryptococcal orthologs) related to fungal virulence determinants and fluconazole resistance, and integrated these phenotypic findings with quantitative proteomics profiling. Our approach defined distinct signatures of each treatment and validated a new mechanism of anti-virulence action toward the polysaccharide capsule from a selected extract following fractionation. By understanding the mechanisms driving the antifungal activity of mussels, we may develop innovative treatment options to overcome fungal infections and promote susceptibility to fluconazole in resistant strains. IMPORTANCE: As the prevalence and severity of global fungal infections rise, along with an increasing incidence of antifungal resistance, new strategies to combat fungal pathogens and overcome resistance are urgently needed. Critically, our current methods to overcome fungal infections are limited and drive the evolution of resistance forward; however, an anti-virulence approach to disarm virulence factors of the pathogen and promote host cell clearance is promising. Here, we explore the efficacy of natural compounds derived from freshwater mussels against classical fungal virulence determinants, including thermotolerance, capsule production, stress response, and biofilm formation. We integrate our phenotypic discoveries with state-of-the-art mass spectrometry-based proteomics to identify mechanistic drivers of these antifungal properties and propose innovative avenues to reduce infection and support the treatment of resistant strains.