BackgroundBiofilms 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.ResultsGrowth 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.ConclusionsTo 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.

BackgroundMicroorganisms 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.ResultsTo 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.ConclusionsHerein we described the antibiosis relationship of S. aureus against A. fumigatus during in vitro biofilm formation, and report the composition of the ECM formed.

BackgroundCoinfections 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.ObjectiveTo determine the capacity of biofilm formation during this fungal–bacterial interaction on primary limbo-corneal fibroblast monolayers.ResultsThe 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.ConclusionThis 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.

Invasive infections caused by lamentous fungi have increased considerably due to the alteration of the host's immune response. Recently, Aspergillus terreus is considered an emerging pathogen and has shown resistance to amphotericin B treatment, resulting in high mortality. The development of fungal bio lm is a virulence factor, and it has been described in some cases of invasive aspergillosis. In addition, although the general composition of fungal bio lms is known, ndings related to bio lms of a lipid nature are rarely reported. In this study, we present the identi cation of a clinical strain of A. terreus by microbiological and molecular tools, also its in vitro bio lm development capacity: i) Bio lm formation was quanti ed by Crystal Violet (CV) and reduction of tetrazolium salts assays (MTT), and simultaneously the stages of bio lm development were described by Scanning Electron Microscopy in High Resolution (SEM-HR). ii) Characterization of the organizational structure of the bio lm 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 identi ed. Some structures of apparent waxy appearance were highlighted by SEM-HR and backscatterelectron diffraction, for which CLSM was previously performed. To our knowledge, this work is the rst description of a lipid-type bio lm in lamentous fungi, speci cally of the species A. terreus from a clinical isolate.

In response to pollution, microorganisms showed unique mechanisms to resist and detoxify harmful metals. 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. Then, 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. Likewise, scanning transmission electron microscopy (STEM) was used for elementary mapping of the cell. Further, 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.