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Aspergillus flavus is a medically relevant fungus, particularly in tropical regions. Although its aflatoxin production and thermotolerance are well documented, its biofilm-forming ability has received less attention, despite being a key factor in the virulence of A. flavus as an opportunistic pathogen, which can significantly impact therapeutic outcomes. To investigate the influence of temperature on the growth and biofilm formation of an A. flavus isolate, we compared it on solid media with the reference strain A. flavus ATCC 22546 and documented morphological changes during conidial germination. We examined biofilm formation in both strains across different temperatures and evaluated the susceptibility of this A. flavus isolate to antifungal agents in both planktonic and biofilm form. Our results showed that the temperature can promote conidiation on solid media. Radial growth was highest at 28 °C, while the conidial count and density were favored at higher temperatures. Moreover, we determined that 37 °C was the optimal temperature for conidial germination and biofilm formation. We described four distinct phases in A. flavus biofilm development-initiation (0-12 h), consolidation (12-24 h), maturation (24-48 h), and dispersion (48-72 h)-with the notable presence of conidial heads at 42 °C. Carbohydrates and proteins constitute the primary components of the extracellular matrix. We observed an abundance of lipid droplets within the hyphae of the MMe18 strain biofilm. The mature biofilms demonstrated reduced susceptibility to amphotericin B and itraconazole, requiring higher inhibitory concentrations for both antifungals compared with their planktonic counterparts.

Pyogenic liver abscesses (PLAs) are serious infections in which doctors often fail in identifying the causative agent due to microbiological limitations. These limitations in detecting uncommon pathogens complicate the treatment and recovery. Molecular techniques, like massive sequencing, enable the detection of uncommon pathogens and highlight the shortcomings of traditional cultures. The aim of this work was to characterise the bacterial composition of PLAs through massive sequencing of the V3-V4 hypervariable region of the 16S rRNA gene in cases where conventional culture methods were negative. Purulent material was collected from three patients with PLAs at Hospital Juárez de México. Classical and molecular microbiological cultures were performed in parallel. Metagenomic DNA was extracted and massively sequenced ( 16S rRNA gene) using the Illumina MiSeq platform. A bioinformatic analysis was performed to determine the diversity at six different taxa levels and the relative abundances. The culture methods were not sufficient to detect the causative agent of the PLAs. However, the massive sequencing revealed the causative agents of the monomicrobial and polymicrobial infectious foci, with Gardnerella vaginalis , Lactobacillus iners , and Prevotella timonensis as the dominant bacteria. The massive sequencing revealed the presence of unusual pathogens that traditional culture failed to detect. There is an immediate need for molecular or comprehensive microbiological culture techniques to search for unusual bacteria in the diagnosis of PLAs.

Infections pose a risk for patients undergoing hematopoietic stem cell (HSC) transplants due to their immunosuppression, making them susceptible to opportunistic infections. Therefore, understanding the composition of the aerobiome in this area is vital. The aim of this study was to characterize the aerobiome in an HSC transplant area, evaluating the impact of infrastructure and health personnel operations on air contamination. The environmental parameters and aerobiome of the HSC transplant area at Hospital Juárez de México were quantified over one year. Finally, a double-entry Vester matrix was constructed to classify problems according to their degree of causality. The abundance and taxonomic diversity of the aerobiome were dependent on seasonality, environmental factors, and high-efficiency filtration. Gram-positive bacteria predominated, followed by fungi and Gram-negative bacteria. ANOVA revealed significant differences in the bacterial aerobiome but not in the fungal aerobiome among the transplant rooms. Clinically, fungi such as Aspergillus fumigatus , Alternaria spp., Cladosporium spp., and Penicillium spp. were identified. ESKAPE bacteria typing revealed clonal dispersion. Finally, the Vester matrix highlighted critical problems associated with contamination due to the absence of HEPA filtration and non-adherence in patient management practices. HEPA filtration and positive pressure are essential to improve the air quality and reduce the microbiological load. However, the control areas will depend on patient management and routine activities, such as entry protocols in controlled areas.

This work reports the modification in the homogeneity of ablation effects with the assistance of nonlinear optical phenomena exhibited by C. albicans ATCC 10231, forming a biofilm. Equivalent optical energies with different levels of intensity were irradiated in comparative samples, and significant changes were observed. Nanosecond pulses provided by an Nd:YAG laser system at a 532 nm wavelength in a single-beam experiment were employed to explore the photodamage and the nonlinear optical transmittance. A nonlinear optical absorption coefficient -2 × 10 -6 cm/W was measured in the samples studied. It is reported that multiphotonic interactions can promote more symmetric optical damage derived by faster changes in the evolution of fractional photoenergy transference. The electrochemical response of the sample was studied to further investigate the electronic dynamics dependent on electrical frequency, and an electro-capacitive behavior in the sample was identified. Fractional differential calculations were proposed to describe the thermal transport induced by nanosecond pulses in the fungi media. These results highlight the nonlinear optical effects to be considered as a base for developing photothermally activated phototechnology and high-precision photodamage in biological systems.

Microorganisms showed unique mechanisms to resist and detoxify harmful metals in response to pollution. This study shows the relationship between presence of heavy metals and plant growth regulator compounds. Additionally, the responses of Rhodotorula mucilaginosa YR29 isolated from the rhizosphere of Prosopis sp. growing in a polluted mine jal in Mexico are presented. This research carries out a phenotypic characterization of R. mucilaginosa to identify response mechanisms to metals and confirm its potential as a bioremediation agent. Firstly, Plant Growth-Promoting (PGP) compounds were assayed using the Chrome Azurol S (CAS) medium and the Salkowski method. In addition, to clarify its heavy metal tolerance mechanisms, several techniques were performed, such as optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) supplemented with assorted detectors. Scanning transmission electron microscopy (STEM) was used for elementary mapping of the cell. Finally, yeast viability after all treatments was confirmed by confocal laser scanning microscopy (CLSM). The results have suggested that R. mucilaginosa could be a PGP yeast capable of triggering Pb 2+ biosorption (representing 22.93% of the total cell surface area, the heavy metal is encapsulated between the cell wall and the microcapsule), and Pb 2+ bioaccumulation (representing 11% of the total weight located in the vacuole). Based on these results, R. mucilaginosa as a bioremediation agent and its wide range of useful mechanisms for ecological purposes are highlighted., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Invasive infections caused by filamentous fungi have increased considerably due to the alteration of the host's immune response. Aspergillus terreus is considered an emerging pathogen and has shown resistance to amphotericin B treatment, resulting in high mortality. The development of fungal biofilm is a virulence factor, and it has been described in some cases of invasive aspergillosis. In addition, although the general composition of fungal biofilms is known, findings related to biofilms of a lipid nature are rarely reported. In this study, we present the identification of a clinical strain of A. terreus by microbiological and molecular tools, also its in vitro biofilm development capacity: (i) Biofilm formation was quantified by Crystal Violet and reduction of tetrazolium salts assays, and simultaneously the stages of biofilm development were described by Scanning Electron Microscopy in High Resolution (SEM-HR). (ii) Characterization of the organizational structure of the biofilm was performed by SEM-HR. The hyphal networks developed on the surface, the abundant air channels created between the ECM (extracellular matrix) and the hyphae fused in anastomosis were described. Also, the presence of microhyphae is reported. (iii) The chemical composition of the ECM was analyzed by SEM-HR and CLSM (Confocal Laser Scanning Microscopy). Proteins, carbohydrates, nucleic acids and a relevant presence of lipid components were identified. Some structures of apparent waxy appearance were highlighted by SEM-HR and backscatter-electron diffraction, for which CLSM was previously performed. To our knowledge, this work is the first description of a lipid-type biofilm in filamentous fungi, specifically of the species A. terreus from a clinical isolate., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In Mexico, millions of tons of mining wastes are deposited in the open pit. Their content in potentially toxic elements (PTE) represents an environmental risk. In the tailings, pioneer plant communities are established, associated with a determined diversity of fungi; plants, and fungi are fundamental in the natural rehabilitation of mining wastes. The objective was to evaluate the impact of the natural establishment of two plant species on the microbial activity, on the composition of the fungal community, and on the mitigation of the effect of PTE in a contaminated mine tailing. In a tailing, we selected three sites: one non-vegetated; one vegetated by Reseda luteola, and one vegetated by Asphodelus fistulosus. In the substrates, we conducted a physical and chemical characterization; we evaluated the enzymatic activity, the mineralization of the carbon, and the concentration of PTE. We also determined the fungal diversity in the substrates and in the interior of the roots, and estimated the accumulation of carbon, nitrogen, phosphorus and PTE in plant tissues. The tailings had a high percentage of sand; the non-vegetated site presented the highest electric conductivity, and the plant cover reduced the concentration of PTE in the substrates. Plants increased the carbon content in tailings. The enzymatic activities of β-glucosidase and dehydrogenase, and the mineralization of carbon were highest at the site vegetated with A. fistulosus. Both plant species accumulated PTE in their tissues and exhibited potential in the phytoremediation of lead (Pb), cadmium (Cd), and copper (Cu). Fungal diversity was more elevated at the vegetated sites than in the bare substrate. Ascomycota prevailed in the substrates; the substrates and the plants shared some fungal taxa, but other taxa were specific. The plant coverage and the rhizosphere promoted the natural attenuation and a rehabilitation of the extreme conditions of the mining wastes, modulated by the plant species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

As a consequence of industrial mining activity, high volumes of tailings are scattered around Mexico. Frequently, tailings contain heavy metals (HM) which entail threats against all organisms. The aim of this research was to identify plants and root fungal endophytes in polymetallic polluted tailings with the potential to be used in strategies of bioremediation. Four deposits of mine wastes, situated in a semi-arid region near urban and semi-urban populations, and agricultural areas, were studied. The physical and chemical characteristics of substrates, accumulation of HM in plant tissues, root colonization between arbuscular mycorrizal (AMF) and dark septate endophyte (DSE) fungi, and the identification of DSE fungi isolated from the roots of two plant species were studied. Substrates from all four sites exhibited extreme conditions: high levels in sand; low water retention; poor levels in available phosphorus and nitrogen content; and potentially toxic levels of lead (Pb), cadmium (Cd), and zinc (Zn). The native plants Lupinus campestris, Tagetes lunulata, and Cerdia congestiflora, as well as the exotic Cortaderia selloana and Asphodelus fistulosus, demonstrated a relevant potential role in the phytostabilization and/or phytoextraction of Pb, Cd, and Zn, according to the accumulation of metal in roots and translocation to shoots. Roots of eleven analyzed plant species were differentially co-colonized between AMF and DSE fungi; the presence of arbuscules and microsclerotia suggested an active physiological interaction. Fourteen DSE fungi were isolated from the inner area of roots of T. lunulata and Pennisetum villosum; molecular identification revealed the predominance of Alternaria and other Pleosporales. The use of native DSE fungi could reinforce the establishment of plants for biological reclamation of mine waste in semi-arid climate. Efforts are needed in order to accelerate a vegetation practice of mine wastes under study, which can reduce, in turn, their potential ecotoxicological impact on organisms, human populations, and agricultural areas., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Coinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus , can form a biofilm. However, mixed fungal-bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection., Objective: To determine the capacity of biofilm formation during this fungal-bacterial interaction on primary limbo-corneal fibroblast monolayers., Results: The biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited., Conclusion: This is the first report of mixed fungal-bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal-bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ramírez-Granillo, Bautista-Hernández, Bautista-De Lucío, Magaña-Guerrero, Domínguez-López, Córdova-Alcántara, Pérez, Martínez-Rivera and Rodríguez-Tovar.)

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

Background: Infections caused by Candida species and Staphylococcus aureus are associated with biofilm formation. C. albicans-S. aureus interactions are synergistic due to the significant increase in mixed biofilms and improved resistance to vancomycin of S. aureus . C. glabrata and S. aureus both are nosocomial pathogens that cause opportunistic infections in similar host niches. However, there is scarce information concerning the interaction between these last microorganisms. Results: The relationship between C. glabrata and S. aureus was evaluated by estimating the viability of both microorganisms in co-culture of planktonic cells and in single and mixed biofilms. An antagonistic behavior of S. aureus and their cell-free bacterial supernatant (CFBS) toward C. glabrata , both in planktonic form and in biofilms, was demonstrated. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) images showed yeast cells surrounded by bacteria, alterations in intracytoplasmic membranes, and non-viable blastoconidia with intact cell walls. Concomitantly, S. aureus cells remained viable and unaltered. The antagonistic activity of S. aureus toward C. glabrata was not due to cell-to-cell contact but the presence of CFBS, which causes a significant decrement in yeast viability and the formation of numerous lipid droplets (LDs), reactive oxygen species (ROS) accumulation, as well as nuclear alterations, and DNA fragmentation indicating the induction of an apoptotic mechanism. Conclusion: Our results demonstrate that the S. aureus CFBS causes cell death in C. glabrata by an apoptotic mechanism.

Signaling systems allow microorganisms to sense and respond to different stimuli through the modification of gene expression. The phosphorelay signal transduction system in eukaryotes involves three proteins: a sensor protein, an intermediate protein and a response regulator, and requires the transfer of a phosphate group between two histidine-aspartic residues. The SLN1-YPD1-SSK1 system enables yeast to adapt to hyperosmotic stress through the activation of the HOG1-MAPK pathway. The genetic sequences available from Saccharomyces cerevisiae were used to identify orthologous sequences in Candida glabrata, and putative genes were identified and characterized by in silico assays. An interactome analysis was carried out with the complete genome of C. glabrata and the putative proteins of the phosphorelay signal transduction system. Next, we modeled the complex formed between the sensor protein CgSln1p and the intermediate CgYpd1p. Finally, phosphate transfer was examined by a molecular dynamic assay. Our in silico analysis showed that the putative proteins of the C. glabrata phosphorelay signal transduction system present the functional domains of histidine kinase, a downstream response regulator protein, and an intermediate histidine phosphotransfer protein. All the sequences are phylogenetically more related to S. cerevisiae than to C. albicans. The interactome suggests that the C. glabrata phosphorelay signal transduction system interacts with different proteins that regulate cell wall biosynthesis and responds to oxidative and osmotic stress the same way as similar systems in S. cerevisiae and C. albicans. Molecular dynamics simulations showed complex formation between the response regulator domain of histidine kinase CgSln1 and intermediate protein CgYpd1 in the presence of a phosphate group and interactions between the aspartic residue and the histidine residue. Overall, our research showed that C. glabrata harbors a functional SLN1-YPD1-SSK1 phosphorelay system.

Background: Biofilms are a highly structured consortia of microorganisms that adhere to a substrate and are encased within an extracellular matrix (ECM) that is produced by the organisms themselves. Aspergillus fumigatus is a biotechnological fungus that has a medical and phytopathogenic significance, and its biofilm occurs in both natural and artificial environments; therefore, studies on the stages observed in biofilm formation are of great significance due to the limited knowledge that exists on this specific topic and because there are multiple applications that are being carried out., Results: Growth curves were obtained from the soil and clinical isolates of the A. fumigatus biofilm formation. The optimal conditions for both of the isolates were inocula of 1 × 10 6 conidia/mL, incubated at 28 °C during 24 h; these showed stages similar to those described in classic microbial growth: the lag, exponential, and stationary phases. However, the biofilms formed at 37 °C were uneven. The A. fumigatus biofilm was similar regardless of the isolation source, but differences were presented according to the incubation temperature. The biofilm stages included the following: 1) adhesion to the plate surface (4 h), cell co-aggregation and exopolymeric substance (EPS) production; 2) conidial germination into hyphae (8-12 h), development, hyphal elongation, and expansion with channel formation (16-20 h); and 3) biofilm maturation as follows: mycelia development, hyphal layering networks, and channels formation, and high structural arrangement of the mycelia that included hyphal anastomosis and an extensive production of ECM (24 h); the ECM covered, surrounded and strengthened the mycelial arrangements, particular at 37 °C. In the clinical isolate, irregular fungal structures, such as microhyphae that are short and slender hyphae, occurred; 4) In cell dispersion, the soil isolate exhibited higher conidia than the clinical isolate, which had the capacity to germinate and generate new mycelia growth (24 h). In addition, we present images on the biofilm's structural arrangement and chemical composition using fluorochromes to detect metabolic activity (FUNI) and mark molecules, such as chitin, DNA, mannose, glucose and proteins., Conclusions: To our knowledge, this is the first time that, in vitro, scanning electronic microscopy (SEM) images of the stages of A. fumigatus biofilm formation have been presented with a particular emphasis on the high hyphal organization and in diverse ECM to observe biofilm maturation.

A case of disseminated infection caused by Penicillium chrysogenum in a 10-year-old boy with a history of Henoch-Schönlein purpura and proliferative glomerulonephritis, treated with immunosuppressors, is reported herein. The patient had a clinical picture of 2 weeks of fever that did not respond to treatment with broad-spectrum antibiotics and amphotericin B. Computed tomography imaging showed diffuse cotton-like infiltrates in the lungs, hepatomegaly, mesenteric lymphadenopathy, and multiple well-defined round hypodense lesions in the spleen. His treatment was changed to caspofungin, followed by voriconazole. One month later, a splenic biopsy revealed hyaline septate hyphae of >1μm in diameter. Fungal growth was negative. However, molecular analysis showed 99% identity with P. chrysogenum. A therapeutic splenectomy was performed, and treatment was changed to amphotericin B lipid complex and caspofungin. The patient completed 2 months of treatment with resolution of the infection. P. chrysogenum is a rare causative agent of invasive fungal infections in immunocompromised patients, and its diagnosis is necessary to initiate the appropriate antifungal treatment., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Yeasts were quantified and isolated from the rhizospheres of 5 plant species grown at 2 sites of a Mexican region contaminated with arsenic, lead, and other heavy metals. Yeast abundance was about 10(2) CFU/g of soil and 31 isolates were obtained. On the basis of the phylogenetic analysis of 26S rRNA and internal transcribed spacer fragment, 6 species were identified within the following 5 genera: Cryptococcus (80.64%), Rhodotorula (6.45%), Exophiala (6.45%), Trichosporon (3.22%), and Cystobasidium (3.22%). Cryptococcus spp. was the predominant group. Pectinases (51.6%), proteases (51.6%), and xylanases (41.9%) were the enzymes most common, while poor production of siderophores (16.1%) and indole acetic acid (9.67%) was detected. Isolates of Rhodotorula mucilaginosa and Cystobasidium sloffiae could promote plant growth and seed germination in a bioassay using Brassica juncea. Resistance of isolates by arsenic and heavy metals was as follows: As(3+) ≥ 100 mmol/L, As(5+) ≥ 30 mmol/L, Zn(2+) ≥ 2 mmol/L, Pb(2+) ≥ 1.2 mmol/L, and Cu(2+) ≥ 0.5 mmol/L. Strains of Cryptococcus albidus were able to reduce arsenate (As(5+)) into arsenite (As(3+)), but no isolate was capable of oxidizing As(3+). This is the first study on the abundance and identification of rhizosphere yeasts in a heavy-metal- and arsenic-contaminated soil, and of the reduction of arsenate by the species C. albidus.

Background: Microorganisms of different species interact in several ecological niches, even causing infection. During the infectious process, a biofilm of single or multispecies can develop. Aspergillus fumigatus and Staphyloccocus aureus are etiologic agents that can cause infectious keratitis. We analyzed in vitro single A. fumigatus and S. aureus, and mixed A. fumigatus-S. aureus biofilms. Both isolates were from patients with infectious keratitis. Structure of the biofilms was analyzed through microscopic techniques including scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal, and fluorescence microscopy (CLSM) in mixed biofilm as compared with the single A. fumigatus biofilm., Results: To our knowledge, this is the first time that the structural characteristics of the mixed biofilm A. fumigatus-A. fumigatus were described and shown. S. aureus sharply inhibited the development of biofilm formed by A. fumigatus, regardless of the stage of biofilm formation and bacterial inoculum. Antibiosis effect of bacterium on fungus was as follows: scarce production of A. fumigatus biofilm; disorganized fungal structures; abortive hyphae; and limited hyphal growth; while conidia also were scarce, have modifications in their surface and presented lyses. Antagonist effect did not depend on bacterial concentration, which could probably be due to cell-cell contact interactions and release of bacterial products. In addition, we present images about the co-localization of polysaccharides (glucans, mannans, and chitin), and DNA that form the extracellular matrix (ECM). In contrast, single biofilms showed extremely organized structures: A. fumigatus showed abundant hyphal growth, hyphal anastomosis, and channels, as well as some conidia, and ECM. S. aureus showed microcolonies and cell-to-cell bridges and ECM., Conclusions: Herein we described the antibiosis relationship of S. aureus against A. fumigatus during in vitro biofilm formation, and report the composition of the ECM formed.

Worldwide, dermatophytoses represent a high percentage of all superficial mycoses. The most frequently isolated dermatophyte is Trichophyton rubrum. Solanum chrysotrichum is a vegetal species widely used in Mexican traditional medicine to treat skin infections; its extract has been used to formulate an herbal medicinal product that is used successfully to treat Tinea pedis. Spirostanic saponin SC-2 from S. Chrysotrichum possesses high activity against dermatophytes. The present study reports the ultrastructural changes observed by means of transmission electron microscopy (TEM) in clinical isolates of T. rubrum, T. mentagrophytes, and Microsporum gypseum induced by saponin SC-2. Strains were grown in RPMI 1640 containing SC-2 (1600 microg/mL). Fungi were harvested at 6, 12, 24, and 48 h; controls without SC-2 were included. T. mentagrophytes was the most susceptible to the SC-2 saponin, followed by M. gypseum, while T. rubrum was the most resistant. The main alterations caused by the SC-2 saponin were as follows: i) loss of cytoplasmic membrane continuity; ii) organelle degradation; iii) to a lesser extent, irreversible damage to the fungal wall; and iv) cellular death., (Georg Thieme Verlag KG Stuttgart, New York.)

Mexican traditional medicine uses Solanum chrysotrichum to treat fungi-associated dermal and mucosal illness; its methanolic extract is active against dermatophytes and yeasts. Different spirostanic saponins (SC-2-SC-6) were identified as the active molecules; SC-2 was the most active in demonstrating a fungicidal effect against Candida albicans and non-albicans strains. The aim of the present study was to compare the clinical (elimination of signs and symptoms) and mycological effectiveness (negative mycological studies) of an S. chrysotrichum herbal medicinal product (Sc-hmp), standardized in 1.89 mg of SC-2, against ketoconazole (400 mg) in the topical treatment of cervical and/or vaginal infection by Candida. Both treatments (vaginal suppositories) were administered daily during 7 continuous nights. The study included 101 women (49 in the experimental group) with a confirmed clinical condition and positive mycological studies (direct examination and/or culture) of Candida infection. Basal conditions did not show differences between the groups; a moderate clinical picture was present in 62% of the cases, direct examination was positive in 69%, and the culture was positive with C. albicans predominating (65%). At the end of the administration period, both treatments demonstrated 100% tolerability, and clinical cure in 57.14% of S. chrysotrichum-treated cases and in 72.5% of ketoconazole-treated cases (p = 0.16), as well as 62.8% and 97.5% of mycological effectiveness, respectively (p = 0.0 001). We conclude that, at the doses used, Sc-hmp exhibits the same clinical effectiveness as ketoconazole, but with lower percentages of mycological eradication. Additional clinical studies with Sc-hmp are necessary, with increasing doses of SC-2, for improving the clinical and mycological effectiveness.

Solanum chrysotrichum is utilized in traditional Mexican medicine for the treatment of mycotic skin infections. Several microbiological studies have provided evidence of its antifungal activity against dermatophytes and yeasts. S. chrysotrichum saponins have been identified as a group of compounds with antifungal activity and saponin SC-2 has demonstrated to be the most active. Previous clinical studies have shown the therapeutic effectiveness of S. chrysotrichum-derived saponin-standardized herbal products in the treatment of Tinea pedis and Pityriasis capitis. There is no previous evidence of the activity of these saponins against Candida non-albicans species, or fluconazole- and ketoconazole-resistant Candida strains. The present study reports the biological activity of the SC-2 saponin (inhibitory concentration [IC (50)] and minimum fungicide concentration [MFC]), against 12 Candida strains of clinical significance ( C. albicans, five strains; C. glabrata and C. parapsilosis, two; C. krusei, C. lusitaniae and C. tropicalis, one), including some fluconazole (Fluco)- and ketoconazole (Keto)-resistant clinical isolates. In addition, SC-2-associated microstructural alterations were reported in four of the above-mentioned Candida species. Seven strains had IC (50) of 200 microg/mL for SC-2, 400 microg/mL was found in four strains, and 800 microg/mL for a sole C. glabrata strain. Susceptibility to SC-2 saponin was as follows: C. albicans = C. lusitaniae > C. krusei > C. glabrata. The MFC was 800 microg/mL for the majority of strains (nine), 400 microg/mL for C. albicans (two strains) and C. lusitaniae. The ultrastructural Candida changes originated by SC-2 included the following: 1) damage on cytoplasmic membrane and organelles; 2) changes in cell wall morphology and density, with separation of cytoplasmatic membrane from cell wall and disintegration of the latter; and 3) total degradation of cellular components and death. Changes were manifested from 6 h of incubation, reaching their maximum effect at 48 h. In conclusion, the saponin SC-2 possesses fungicide and fungistatic activity on different Candida albicans and non- albicans species (including some azole-resistant strains) with IC (50) values of 200 microg/mL (in Fluco-susceptible strains) and of 400 - 800 mug/mL (in Fluco-resistant strains). Additionally, we observed by transmission electron microscopy (TEM) that saponin SC-2 causes severe changes in all fungal cell membranes, and to a lesser degree on the cell wall.

In the present work the genetic transformation and the expression of gene markers in transgenic Pisolithus tinctorius are reported. The ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this association (more than 80% of gymnosperms are associated with these fungi), its study from both basic and applied viewpoints is relevant. We have transformed this fungus with reporter genes and analyzed their expression in its saprophytic state. Genetic transformation was performed by microprojectile bombardment and Agrobacterium-mediated transformation. This last method proved to be the more efficient. Southern analysis of biolistic-transformed fungi revealed the random integration of the transgene into the genome. The accumulation of the transcript of the reporter gene was demonstrated by RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia confirmed the expression of the reporter gene. This is the first report on the stable transformation and expression of GFP in the ectomycorrhizal fungus P. tinctorius.