Ludmilla Tonani

Reconstructed human skin microbiome models represent human-relevant New Approach Methodologies (NAMs) for investigating host–microorganism interactions and evaluating microbiome-modulating ingredients. However, microbiome-compatible reconstructed skin models remain poorly standardized and require further characterization to support their broader application. In this study, we developed reconstructed human skin models colonized with defined combinations of Malassezia furfur, Staphylococcus epidermidis, and Cutibacterium acnes to establish experimental conditions representative of preserved and disrupted tissue homeostasis associated with seborrheic dermatitis. The models were characterized by histology, scanning electron microscopy, Grocott–Gomori staining, coproporphyrin III-associated autofluorescence, host gene expression, and cytokine secretion. Among the tested microbial combinations, the M. furfur + S. epidermidis model preserved tissue morphology and exhibited host-response markers comparable to those of uncolonized skin, whereas the M. furfur + C. acnes model induced moderate epidermal alterations together with increased expression of the aryl hydrocarbon receptor (AHR) and human β-defensin 2 (HBD2), and was selected as the challenge model for treatment evaluation. The platform was subsequently applied to assess the effects of α-glucan oligosaccharide (AGO), an antimicrobial preservative, and empty or AGO-loaded cupuaçu butter nanoparticles. AGO-loaded nanoparticles significantly reduced AHR expression, whereas all treatments reduced HBD2 expression. Among the cytokines evaluated, only the antimicrobial preservative significantly decreased IL-6 secretion. Collectively, these findings demonstrate that the reconstructed human skin microbiome platform can discriminate among distinct host responses to defined microbial communities and detect treatment-specific biological effects, supporting its application as a human-relevant in vitro model for mechanistic studies and preclinical evaluation of microbiome-modulating cosmetic ingredients.

Fungi are a rich source of chemical diversity for bioprospecting, yet most of their metabolic potential remains unexplored. This study investigated the metabolic profile of phytopathogenic fungi of the genus Fusarium , isolated from fruits with high protease content, such as papaya and pineapple. Comparative metabolomics was used to evaluate how culture substrates and co-cultivation with the endophyte Neofusicoccum ribis modulate secondary metabolite production. Molecular Networking analysis (GNPS2) revealed an impressive diversity of 173 annotated metabolites, including one previously undescribed compound. Metabolite production was highly dependent on the substrate and fungal strain, showing a strong correlation between phylogeny and metabolic profiles. Several extracts displayed significant inhibitory activity against the protease papain, with some co-cultivation combinations further enhancing this effect. These findings highlight how environmental conditions and microbial interactions activate silent biosynthetic pathways, confirming these fungi as promising sources for bioactive molecule discovery. Graphical Abstract

Salmonella Infantis ( S. Infantis) is a zoonotic and globally distributed serovar. Despite its clinical relevance, information regarding its pathogenic potential is scarce. This study evaluated the pathogenic potential of S. Infantis isolated from foods, humans, and veterinary sources in Brazil between 2013 and 2018 using genomic and phenotypic approaches. A total of 80 S. Infantis genomes were screened for virulence genes and Salmonella pathogenicity islands (SPIs). Phenotypically, 25 selected strains were analyzed for analyses of biofilm formation, invasion in Caco‐2 cells, survival in human macrophages, and in vivo infection in Galleria mellonella. A total of 9 SPIs and 150 virulence genes associated with fimbrial/nonfimbrial adherence, macrophage induction, magnesium uptake, regulation, siderophores, and type three secretion systems were detected. The 25 strains tested showed strong (4%), moderate (56%) or weak (40%) biofilm formation. A 6.1 log ₁₀ CFU/mL average rate was found for invasion in Caco‐2 and survival in U937 macrophages. In G. mellonella , 56% of the strains were highly virulent or virulent. In conclusion, S. Infantis from diverse sources in Brazil have a high pathogenic potential, evidenced by high rates of Caco‐2 invasion and intramacrophage survival, capacity to form biofilms and to cause infection in vivo that are associated with highly frequent and conserved virulence genes and SPIs.

The fungi Fusarium guttiforme and Phytophthora palmivora were cultivated in four different media (Potato Dextrose Agar, Czapek, rice, and ISP2) and co‐cultured to stimulate fungal interactions and enhance secondary metabolite production. Promising extracts were fractionated, yielding compounds such as the iron complex of fusaric acid ( 1 ), magnesium complex of fusaric acid ( 2 ), haematocin ( 4 ), fusarinolic acid ( 7 ), and cyclonerodiol ( 8 ). These compounds exhibited significant papain inhibitory activity, with compounds 2 and 4 showing IC ₅₀ values below 20 µM. Notable anti‐ Trypanosoma cruzi activity was also observed, particularly for compounds 2 (50% inhibitory concentration [IC ₅₀ ] = 12.19 µM) and 4 (IC ₅₀ = 13.32 µM). Additionally, antifungal activity against Candida spp. was detected, with compound 2 and the magnesium complex of 9,10‐dehydrofusaric acid complex ( 3 ) showing MICs of 50 µg/mL. This study highlights the potential of fungal co‐cultivation and variation in culture medium as effective strategies for discovering novel bioactive protease inhibitors and antiparasitic agents.

Fungal secondary metabolites display remarkable chemical diversity and biological potential, with applications in agriculture and pharmaceuticals. This study isolated and characterized bioactive metabolites from two phytopathogenic fungi, Fusarium falciforme (from papaya) and Aspergillus terreus (from pineapple), assessing their antifungal and cysteine protease‐inhibitory activities. The compounds hymeglusin, fusaridioic acid A, and butyrolactone I exhibited significant antifungal effects, with hymeglusin (IC ₅₀ 22.12 µg/mL) inhibiting A. terreus and butyrolactone I (IC ₅₀ 39.72 µg/mL) inhibiting F. falciforme . These metabolites also strongly inhibited papain, with IC ₅₀ values of 6.24 µM (butyrolactone I), 9.38 µM (hymeglusin), and 28.58 µM (fusaridioic acid A). Butyrolactone I exhibited the highest antitrypanosomal activity (IC ₅₀ 24.72 µM), highlighting its antiparasitic potential. These findings emphasize the biotechnological significance of fungal metabolites for antifungal, protease‐inhibitory, and antiparasitic applications while also shedding light on their ecological role in host–pathogen interactions.

Sarocladium spp. are filamentous fungi commonly associated with plant diseases and only rarely cause hyalohyphomycosis in humans. Immunosuppressed patients are at risk for this infection, which typically presents with skin and subcutaneous lesions that may eventually disseminate to internal organs. This study reports a case of a man in intensive care following SARS-CoV-2 infection. During hospitalization, he developed neutropenia, persistent fever, and a cavitary lung lesion. Sarocladium spp. was isolated from blood cultures, and the patient was treated with voriconazole, leading to a successful cure. To our knowledge, this is the first reported case of Sarocladium implicatum infection in a COVID-19 patient, underscoring the importance of monitoring opportunistic fungal infections in immunocompromised individuals, particularly during epidemics and pandemics.

Onychomycoses are nail infections that require prolonged therapy and have high recurrence rates. Dermatophytes are the main etiological agents of these infections, followed by yeasts and non-dermatophyte filamentous fungi. The limited antifungal arsenal used to treat onychomycosis and the change in the susceptibility profile of these agents contribute to the chronicity and recalcitrant profile of infections. The present study aimed to determine the antifungal activity of extracts obtained from pure and mixed cultures of Candida parapsilosis, Trichophyton mentagrophytes, and Trichophyton rubrum. Additionally, in vivo toxicity tests with Galleria mellonella and time-kill assays were carried out. The susceptibility profiles of dermatophytes were determined using a microdilution technique with minimum inhibitory concentrations (MICs) between 250 and 8000 µg/mL. The time-kill assay, compared to growth control, resulted in the death of dermatophytes within 48 h. No toxicity of the extracts was detected in experiments with Galleria mellonella larvae under the test conditions. The extracts of pure and mixed cultures of Candida parapsilosis and dermatophytes present antifungal activity against T. mentagrophytes and T. rubrum. Isolating and identifying compounds in the extracts may allow the development of new therapeutic approaches to control fungal infections.

The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives.

Disseminated fusariosis is treated with amphotericin B and voriconazole. To determine adequate therapy, the minimal inhibitory concentration (MIC) is used. However, MIC analysis is based on visual observation and requires a long period of fungal incubation. The measure of the minimal profile change concentration (MPCC) using MALDI-TOF MS is a quick spectral method that has presented good results in determining the antimicrobial resistance of yeasts. However, there is a lack of information on filamentous fungi. In the present work, 13 Fusarium spp. clinical isolates and two reference strains were used. MIC was obtained according to the M38-A2 protocol of the Clinical Laboratory Standards Institute, while MPPC was obtained following the initial steps of the M38-A2 protocol. Both Biotyper and the Rstudio environment were used to analyze mass spectra. For some fungal strains, the data obtained from the software MALDI Biotyper Compass 4.1 led to fuzzy heatmaps resulting in difficult interpretation, while heatmaps obtained using Rstudio tools generated better MPCC resolutions. Herein, 86.6% of the AMB MPCC values were highly correlated with the gold-standard AMB MIC. MALDI-TOF MS is a prominent tool used to determine MPCCs quicker, cost-effectively, and more accurately for Fusarium spp. strains. However, better statistical analyses could help measure the technique’s limit detection.