Multiphotonic Ablation and Electro-Capacitive Effects Exhibited by Candida albicans Biofilms.

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.

Rhodotorula mucilaginosa YR29 is able to accumulate Pb2+ in vacuoles: a yeast with bioremediation potential.

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 Pb2+ biosorption (representing 22.93% of the total cell surface area, the heavy metal is encapsulated between the cell wall and the microcapsule), and Pb2+ 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.

Lipid-Like Biofilm from a Clinical Brain Isolate of Aspergillus terreus: Quantification, Structural Characterization and Stages of the Formation Cycle.

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.

Microbial Warfare on Three Fronts: Mixed Biofilm of Aspergillus fumigatus and Staphylococcus aureus on Primary Cultures of Human Limbo-Corneal Fibroblasts.

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.

Pioneer plant species and fungal root endophytes in metal-polluted tailings deposited near human populations and agricultural areas in Northern Mexico.

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.

Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light.

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.

The phosphorelay signal transduction system in Candida glabrata: an in silico analysis.

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.

Analysis and description of the stages of Aspergillus fumigatus biofilm formation using scanning electron microscopy.

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 × 106 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.

Disseminated penicilliosis due to Penicillium chrysogenum in a pediatric patient with Henoch-Schönlein syndrome.

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.

Candida glabrata survives and replicates in human osteoblasts.

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.

Isolation and characterization of yeasts associated with plants growing in heavy-metal- and arsenic-contaminated soils.

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.

Ultrastructural changes on clinical isolates of Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum caused by Solanum chrysotrichum saponin SC-2.

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.

Exploratory study on the clinical and mycological effectiveness of a herbal medicinal product from Solanum chrysotrichum in patients with Candida yeast-associated vaginal infection.

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.

Mycological and electron microscopic study of Solanum chrysotrichum saponin SC-2 antifungal activity on Candida species of medical significance.

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.