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.

Molecular detection of candida species from hospitalized patient's specimens. / Detección molecular de especies de Candida en especímenes de pacientes hospitalizados.

Objective: To identify the most frequent Candida species in specimens from patients hospitalized in different medical centers of Mexico City, with suspected fungal infection. Methods: Specimens were grown on Sabouraud dextrose agar at 28°C for 72 h. In addition, DNA was extracted. Isolates were grown on CHROMagar Candida™, at 37°C for 48 h. The molecular identification was performed by polymerase chain reaction (PCR) using primers specific for four species. Results: Eighty one specimens were processed and included: bronchial lavage, pleural, cerebrospinal, peritoneal, ascites and bile fluids; blood, sputum, bone marrow, oro-tracheal cannula and ganglion. By culture, 30 samples (37%) were positive, and by PCR, 41 (50.6%). By PCR, the frequency of species was: Candida albicans 82.9%, Candida tropicalis 31.7%, Candida glabrata 24.4%, and Candida parapsilosis 4.9%. In 34.1% of specimens a species mixture was detected suggesting a co-infection: Two species in five specimens (C. albicans-C tropicalis and C. albicans-C glabrata), and three species in three specimens (C. albicans-C. glabrata-C. tropicalis). Conclusions: The PCR is an useful tool for detection the most common Candida species causing infection in hospitalized patients, it avoids the requirement of culture weather we start from clinical specimen and it favors the early diagnosis of invasive candidiasis.

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.

Antibiosis interaction of Staphylococccus aureus on Aspergillus fumigatus assessed in vitro by mixed biofilm formation.

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.

Parasitic polymorphism of Coccidioides spp.

BACKGROUND: Coccidioides spp. is the ethiological agent of coccidioidomycosis, an infection that can be fatal. Its diagnosis is complicated, due to that it shares clinical and histopathological characteristics with other pulmonary mycoses. Coccidioides spp. is a dimorphic fungus and, in its saprobic phase, grows as a mycelium, forming a large amount of arthroconidia. In susceptible persons, arthroconidia induce dimorphic changes into spherules/endospores, a typical parasitic form of Coccidioides spp. In addition, the diversity of mycelial parasitic forms has been observed in clinical specimens; they are scarcely known and produce errors in diagnosis. METHODS: We presented a retrospective study of images from specimens of smears with 15% potassium hydroxide, cytology, and tissue biopsies of a histopathologic collection from patients with coccidioidomycosis seen at a tertiary-care hospital in Mexico City. RESULTS: The parasitic polymorphism of Coccidioides spp. observed in the clinical specimens was as follows: i) spherules/endospores in different maturation stages; ii) pleomorphic cells (septate hyphae, hyphae composed of ovoid and spherical cells, and arthroconidia), and iii) fungal ball formation (mycelia with septate hyphae and arthroconidia). CONCLUSIONS: The parasitic polymorphism of Coccidioides spp. includes the following: spherules/endospores, arthroconidia, and different forms of mycelia. This knowledge is important for the accurate diagnosis of coccidioidomycosis. In earlier studies, we proposed the integration of this diversity of forms in the Coccidioides spp. parasitic cycle. The microhabitat surrounding the fungus into the host would favor the parasitic polymorphism of this fungus, and this environment may assist in the evolution toward parasitism of Coccidioides spp.

Combined therapy with amphotericin B and caspofungin in an experimental model of disseminated histoplasmosis.

OBJECTIVE: To assess the effect of amphotericin B and caspofungin, as well as their combinations in the therapy of experimental disseminated histoplasmosis. MATERIAL AND METHODS: BALB/c mice were intraperitoneally infected with four different strains of Histoplasma capsulatum and given to antifungal treatments. The response to intraperitoneal therapy with amphotericin B (0.5, 1.0, and 2.0 mg/kg of body weight) or caspofungin (10 mg/kg of body weight) and their combinations, was evaluated by the quantification of yeast colony-forming units (CFU) per gram of spleen or lung, from each animal. Additionally, the pathogen was monitored histopathologically in the excised organs. Data were analyzed with the Kruskall-Wallis and Tukey tests. RESULTS: Caspofungin was more effective than amphotericin B in reducing the CFU/ g. A synergistic effect was observed when caspofungin (10 mg/ kg) was combined with amphotericin B (0.5 or 1.0 mg/kg). Significant differences in CFU values, H = 119.78 (P = 0.00001), were found among the treatment groups. However, statistical analyses did not reveal significant differences, H = 2.837 (P = 0.428), in the therapeutic responses with the four H. capsulatum strains tested. CONCLUSION: Combined therapy with amphotericin B and caspofungin could represent an alternative treatment to be explored in severe human histoplasmosis.