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.


Introduction
The Aspergillus group is made up of over 350 species. They are found in the soil as saprobes, but also cause diseases in plants, animals, and humans. This fungus demonstrates a series of adaptation mechanisms contained in its genome, which allow it to survive as a saprobiont and as a successful pathogen in human beings [1,2]. The genus Aspergillus has approximately 250 species, including about 30 species that are infectious agents in humans; the main species causing invasive aspergillosis (IA) are A. fumigatus, A. flavus, A. niger, A. terreus, and A. versicolor [3,4]. Specifically, in the case of A. terreus, which is integrated in the Terrei section, it is scarcely isolated from hosts that are undergoing some type of aspergillosis [5]. Epidemiologically, a clinical incidence of 4% of all IA due to this emerging fungus has been reported [6]. Likewise, the prevalence of IA caused by A. terreus is estimated to be between 15 and 23% in patients with hematologic malignancies [7]. In addition, the mortality rate is of concern since the immunosuppression factors of the patients are determinant for the invasive form of infection by this fungal species. At the same time, the clinical isolated frequently show resistance to first choice antifungal drugs (such as amphotericin B); moreover, the patients with a severe and prolonged neutropenia state show the absence of a therapeutic response with high mortality rate (51-86%) [6][7][8]. Biofilm development for certain species of genus Aspergillus (e.g., A. fumigatus, A. flavus, A. niger, A. terreus, A. brasiliensis) has been reported to be a virulence factor favoring establishment and persistence of infection in the host [9][10][11][12]. In the development and establishment of the biofilm, the differential expressions of genes implicated in the production of the extracellular matrix (ECM) have been reported [13], as well as during the conidial morphogenesis, which involves the change of the conidia into germinating tubes with an apical grown for the creation of hyphal networks. The biofilm allows the fungus to interact with the host, increasing the resistance to antifungal drugs (in part associated with the functionality of the efflux pumps). In addition, the study of biofilm formation allows to analyze and propose the development of the pathogenesis of aspergillus infection, focusing mainly on possible therapeutic targets to control the infectious process, together with the ability of the fungus to evade the host immune response [7,10].
In the present work, biofilm formation was analyzed and quantified by Crystal Violet (CV) and reduction of tetrazolium salts (MTT) assays. Simultaneously, the stages of biofilm development and characterization of the organizational structure of the biofilm were performed by SEM-HR (Scanning Electron Microscopy in High Resolution). Furthermore, the hyphal networks developed on the surface, the abundant air channels created between the extracellular matrix and the fused hyphae in anastomosis were described. Also, the presence of microhyphae rarely reported in filamentous fungi biofilm is reported. Moreover, by using SEM-HR and backscatter-electron diffraction, some structures of apparent waxy appearance were highlighted. Finally, the study was complemented by the analysis of the chemical composition of the ECM by Confocal Laser Scanning Microscopy (CLSM); proteins, carbohydrates, nucleic acids and a relevant presence of lipid components were identified. Data obtained on the chemical composition of the ECM are of special interest in filamentous fungi, since there are no reports on lipid synthesis in biofilms. Knowledge of the biofilm of A. terreus has multiple applications from the medical, agricultural, and biotechnological point of view. Therefore, this study opens a new research topic related to the virulent factors and possible treatments of and emerging pathogen [9,10,13].

Biological Material
A clinical strain of Aspergillus terreus, was isolated from a pediatric patient with cerebral aspergillosis in the Mycology Laboratory of the Hospital Infantil de México ''Dr. Federico Gómez'' (HIMFG). The isolate was cultured and preserved on Potato Dextrose Agar (PDA) medium (BD Bioxon, Mexico) at 37°C.

Microbiological and Molecular Identification
The microbiological identification of A. terreus was carried out following the description made by Pasqualotto and Larone [14,15]. Colonial morphology was obtained on PDA and Sabourad Dextrose Agar (SDA), both cultures were incubated at 28 and 37°C for 1-7 days. In the description of microscopic morphology, the characteristics of the species were evaluated from a microculture incubated for 2 weeks on PDA medium.
Regarding molecular identification, deoxyribonucleic acid (DNA) was obtained through the method described by Rodríguez-Tovar et al. [16], from an A. terreus culture developed in a PDA medium, incubated during 7 days at 37°C. The DNA was quantified (Nanodrop 2000 ThermofisherÒ), adjusting the concentration to 100 ng/lL. Subsequently, the rRNA's ITS (Internal Transcript Spacer) molecular marker and the RNA polymerase II (RPBII) subunits were amplified by polymerase chain reaction (PCR) (MAXY-GENÒ brand thermocycler). The two molecular markers were amplified using the initiators described mainly for the Aspergillus species [17][18][19]. The reaction mixture was carried out for a final volume of 50 lL using TaqPol 5 U/lL (ThermofisherÒ), MgCl 2 3 mM, dNTP mixture 0.2 mM, 20 pmol of each initiator and sterile distilled water. The amplification protocol included a first denaturation cycle at 95°C for 5 min, followed by 30 cycles at 95°C/1 min, 55°C/1 min, and 72°C/1 min, with a final polymerization cycle at 72°C for 10 min. The products were sequence by MACROGENÒ. The sequences were analyzed with the SeaView program, and a concatenated phylogenetic tree was built using the MEGA 7.0 program to obtain the taxonomic position of the isolate.

Formation and Quantification of A. terreus in vitro Biofilm by CV and MTT Assays
The biofilm was developed in polystyrene microplates (Nunk Roskilde, Denmark). Conidia were extracted by the method described by Mowat [20] to obtain the biofilm, which were then suspended in a Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Waltham, MA, USA) supplemented with 2% glucose to a final concentration of 4 9 10 5 conidia/mL [21]. The biofilm was developed adding 200 lL of the conidia suspension to each of the wells in the 96-well plates, which were incubated at 37°C for 4 h (adherence phase). The supernatant was eliminated with the purpose of removing the non-adhered cells, and 200 lL of fresh RPMI medium were added. The plates were incubated during 24, 48, 72, and 96 h at 37°C. The culture medium was eliminated and 200 lL of the phosphate regulation solution 1X (PBS) were added to wash the wells. The quantification of the biofilm was carried out in accordance with the method described by Christensen [22] and modified by Ramírez-Granillo et al., [21] using 0.005% Crystal Violet. The excess colorant was removed from the wells, which were washed with sterile distilled water and air-dried at room temperature. Finally, the colorant adhered to the biofilm was dissolved in 200 lL of acetic acid (JT Baker, Phillipsburg, NJ, USA) at 33% (v/v). The solution was transferred to 96-well clean microplates and the absorbance was read at 595 nm in a spectrophotometer (Mulstiskan Ascent Thermo Labsystems, AIE, Waltham, MA, USA). The absorbance values are proportional to the amount of biofilm developed. This data was evaluated by the respective statistic methods described in the following paragraphs. To quantify the biofilm developed by determining its metabolic activity, the technique described by Walencka et al. [23], modified by Ramírez-Granillo et al., was used [11,24,25]. The biofilm was developed in 96-well polystyrene plates with a flat bottom, as described previously. Also, 150 lL of PBS 1X and 50 lL of the tetrazolium salts reagent at 0.3% MTT [Bromide 3-(4,5dimethylthiazol-2)-2,5-diphenyltetrazolium bromide] (SIGMAÒ, St. Louis, MO, USA) were added to each well. The plates were incubated during 2 h at 37°C. Then, the MTT reagent was removed and 150 lL of DMSO (Riedel-de Haën TM , Seelze, Germany) and 25 lL of glycine buffer 0.1 M at pH 10.2 (SIGMAÒ, St. Louis, MO, USA) were added. The plates were incubated during 15 min at room temperature stirring softly; the optical density was read with an ELISA reader (Multiskan Ascent Thermo Labsystems; AIE, Waltham, MA, USA) at 570 nm. The well with PBS 1X was used as a reagent blank. In this assay, twelve repetitions of three individual experiments were carried out, and the corresponding statistical analysis was performed with the data obtained.
Biofilm Analysis Through Scanning Electron Microscopy in High Resolution (SEM-HR) The biofilm was developed as described previously, and adjusted to 12-well polystyrene plates, incubated at 37°C during 24, 48, 72 and 96 h. The SEM samples were processed in accordance with the method described by Bozzola and Russell, along with the protocols used by Vázquez-Nin and Echeverría [26,27]. The biofilms were washed with PBS 1X and fixed with 2% glutaraldehyde (Electron Microscopy SciencesÒ, Washington, PA.) for 2 h. A postfixing was carried out with 1% osmium tetroxide (Electron Microscopy SciencesÒ, Washington, PA.) for 2 h. Then, the intact biofilm was recovered from the bottom of the 12-well plates. The samples were dehydrated with 10-90% ethanol during 10 min for each solution, respectively, and thrice with absolute ethanol for 10 min. The humidity in the biofilms was eliminated with a dryer, and they were dried to a critical point with hexamethyldisilazane (Electron Microscopy SciencesÒ, Washington, PA, USA). The samples were covered with a gold/palladium alloy during 400 s at 15,000 kV and 10 lA; and were observed in a scanning electron microscope (JEOL, Tokyo, Japan) in the Nanosciences and Micro Nanotechnologies Center at the IPN.

Biofilm Structural Composition Analysis through CLSM
For the Confocal Laser Scanning Microscopy, the biofilms were developed as described previously.

Microbiological and Molecular Identification
The clinical isolate was identified as the Aspergillus terreus filamentous fungus. The molecular identification was performed with the analysis of sequences obtained from the ITS fragment and the RPBII subunit as molecular marker using species closely related to the genus Aspergillus. The identity of the fungal isolate was confirmed with the concatenated phylogenetic tree for A. terreus species (Fig. 1a) and is related to section Terrei. The characteristics of the A. terreus species in a PDA medium were described. The radial colony was observed with a phenotype typical of the species (granular texture, flat surface with production of white pigment at the periphery and eventually darkening to brown). On the reverse side plate, a brown pigment, and a yellow pigment diffusible to the medium were formed (Fig. 1b). Similarly, at the microscopic level, the characteristic structures of A.
terreus were observed. The morphology of the A. terreus conidial head was subspherical, and two series of phialides originate from the conidiophore at an angle of approximately 180°, with a chain arrangement of columnar conidia. Aleurioconidia were not observed at in vitro culture (Fig. 1c). Furthermore, the effect of incubation temperature (28 and 37°C) and the composition of the culture medium (SDA or PDA) on fungal growth were analyzed. Regarding the diameter of colonies developed at 28°C, no significant differences were observed between PDA and SDA medium (Online Resource 1a). Likewise, at 37°C, a statistically significant difference was observed in the PDA medium (Online Resource 1b) in the diameter of fungal colonies during a 7 day incubation period.
Quantification of A. terreus in vitro biofilm.
The ability to develop biofilm in vitro for A. terreus clinical isolate, under different growth conditions (temperature, conidial concentration, and incubation time) was evaluated. The kinetics of biofilm showed that the optimum temperature and the highest biofilm production occurred at 37°C with a concentration of 4 9 10 5 conidia/mL (results not shown). Quantification of the A. terreus biofilm was performed by Christensen-Crystal Violet (CV-biomass) and tetrazolium salt reduction (MTT-metabolic activity) methods described by Walencka [23]. Additionally, the characteristics of the biofilm of the filamentous fungus were described by SEM-HR (Fig. 2). By CV method, growth kinetics data were shown with the highest The phylogenetic tree of maximum likelihood elaborated shows the relation of the molecular markers for filamentous fungi that were used (ITS and RPBII), with some sequences of Aspergillus species obtained by BLAST. b Microbiological identification.
A. terreus was grown on PDA at 378C/48 h; Colonial morphology: powdery texture, cinnamon-brown color, front (left) and back (right). c Microscopy observation: Conidial head, metula and phialides with conidium emerging in columnar form (100x; lactophenol cotton blue stain) amount of biofilm developed at 72 h (AU [ 2.0) (Fig. 2a). The highest metabolic activity of the fungus was detected at 24 h of in vitro biofilm development (AU [ 0.7) (Fig. 2b). Correlating the structure of the A. terreus in vitro biofilm by SEM-HR with the amount evaluated, the typical characteristics of filamentous fungal biofilm (abundant hyphal growth, development of air channels and anastomosis) were visualized (Fig. 2c). First, a hyphal network was observed at 24 h with ECM extensively covering the colonized surface, according to CV analysis (AU \ 2.0), and high metabolic activity (AU [ 0.7). Then, the number of hyphae decreased along with the presence of ECM at 48 h, like what was quantified previously (CVM, AU [ 1.5; MTT, AU \ 0.6). After 72 h of incubation, biofilm fields with a high biomass density were observed (AU [ 2.0), and no surface on which the fungus adhered was evident. Also, the metabolic activity of this biofilm formation time was elevated (MTT, AU \ 0.8). During the 96 h period, a decrease in the fungal population was observed, with some remnants of the biofilm developing (Fig. 2c). The quantification of the biomass at this time was determined with a value of AU&0.4 and a MTT value of AU [ 0.3.

Stages of A. terreus Biofilm Formation
The establishment during the first hours of the biofilm of the filamentous fungus has been described as adhesion processes, cell aggregation, exopolymeric substance (EPS) production and development of biofilm formation (Fig. 3a). For each stage of the fungal biofilm, the presence of typical characteristics at previously quantified times was described ( Table 1). The biofilm in vitro formation cycle, after the first hours of establishment, for the clinical isolate of A. terreus was described by SEM-HR as shown below:

Early Maturation Stage (24 h)
The formed biofilm was observed to initiate the early maturation phase with predominant hyphal networks forming anastomosis, and abundant air channels between the produced ECM (Fig. 3b). In addition, only during this phase were structures called microhyphae visualized with a diameter of &1 lm and a shorter length compared to a standard size hypha ([ 10 lm). Several scattered microhyphae with catenular organization adjacent to the ECM and fused hyphae were observed in the biofilm (Fig. 3c).

Depletion Stage (48 h)
At this stage, different organizations of the ECM were observed. First, a condensed porous structure with embedded hyphae was visualized (Fig. 3d), along with structural arrangements resembling a coral of waxy aspect (Fig. 3e). In another field, a porous ECM with Fig. 3 Stages of the biofilm formation cycle of the clinical brain isolate of A. terreus. The biofilm was grown in RPMI at 37°C. The micrographs were made by SEM-HR. (a) Establishment during the first hours of the filamentous fungal biofilm was described as processes of adhesion, cell aggregation, EPS production and development of biofilm formation. 24 h) During the early maturation stage, the presence of anastomosis (arrowhead) and the formation of canals (arrow) were observed (b, 9 1000). Also, evidence of the presence of microhyphae (Mh) was observed in the developed hyphae (c, 9 5000). 48 h) In depletion phase, a wider ECM with embedded hyphae (arrowhead) was present (d, 9 1000). In some fields, a type of ECM (arrow) with both condensed and porous type consistency was visible. In addition, some artifacts with an apparently waxy consistency were observed (e, 9 10,000). In another field, a porous-type ECM with globular structures at the periphery was observed (f, 9 1000; g, 9 5000). 72 h) At late maturation stage, we observed the highest ECM formation (h, 9 1000). In the same field was evidenced the presences of a condensed ECM and film ECM (i, 9 5000). While in other fields porous ECM was detected (j, 9 5000). Anastomosis (arrow); fungal channels (arrowhead). 96 h) The dispersion phase showed a significant decrease in biofilm formation. Accumulation of exopolymeric substances was also observed (k, 9 1000). An amorphous substance covered by a layer of ECM coating the hyphae was evident (l, 9 10,000). In some fields, sparse remains of conidia were found (m, 9 1000), which were coated with exopolymeric material adjacent to the hyphal growth of the fungus (n, 9 10,000). Conidia (c); hyphae (h); exopolymeric substances (EPS) adjacent hyphae (Fig. 3f), and globular arrangements were observed at the periphery of the ECM (Fig. 3g). Notably, a decreasing fungal biomass with few canals and anastomoses was observed.

Late Maturation Stage (72 h)
At this stage, extracellular complexes with a condensed ECM and embedded organized hyphae were observed massively covering the abiotic surface (Fig. 3h). In addition, several air channels and stable anastomosed hyphae were observed among the fungal biofilm (Fig. 3i). Also, further biofilm development was shown with prominent porous ECM formations covering the developed hyphae (Fig. 3j).

Dispersion Stage (96 h)
A weak biofilm with low biomass and wide spaces between hyphae was observed. The deficit of ECM production was evident, as well as the scarce presence of aerial canals (Fig. 3k). In some sample fields, ECM residues were observed with fragments of sectioned hyphae enveloped by a layer of EPS secreted by the filamentous fungus (Fig. 3l). During this stage, several asexual structures (conidia) were observed aggregated or isolated (Fig. 3m, n).

Analysis of the Biofilm Structural Composition of a Clinical Isolate of A. terreus by CLSM and SEM-HR
The composition of EPS composing the ECM of the biofilm in vitro was detected by CLSM. At 24 and 48 h, glucose and mannose residues were observed in CW-and Con-A-labeled hyphae. These cells were metabolically active, as they overlapped with the staining corresponding to FUN 1. Blue halo cells labeled with Flamingo for proteins were also observed (Data not shown). The most representative results were obtained at 72 and 96 h. In general, the micrographs analyzed (Fig. 4a, b); an intense mark was detected along the hyphal wall, related to the chitin (CW-green halo). Likewise, together with this signal, several halos were detected in the hyphae related to the secretion of molecules of lipid nature (Nile red-pink halo). At 72 h (Fig. 4a), we observed a moderate secretion of lipids in the in vitro biofilm development kinetics, compared to 96 h in which showed a higher amount of these molecules (Fig. 4b). Furthermore, the co-localization of carbohydrate and lipid labeling molecules was noted in the biofilm of A. terreus (Fig. 4c).
SEM-HR micrographs of the biofilm at 96 h were analyzed showing evidence of fields exhibiting faceted bodies (similar to a polyhedral structure) (Fig. 4d). These foreign bodies were denominated waxy ECM (Fig. 4e), due to their appearance and texture. Further studies will be able to define if their origin is indeed lipidic; but, under the microscope, it was observed to be secreted by the hyphae of the To determine that this fungal biofilm structure was derived from the hyphae, SEM-HR analysis with backscatter-electron diffraction was performed. Finally, crystallographic structures of the material were shown to be part of the waxy ECM of the fungus secreted by the hyphae (Fig. 4f).

Discussion
In recent years, interest in A. terreus has been related to its role as opportunistic pathogen causing invasive aspergillosis (IA) in immunocompromised patients, such as individuals with chronic neutropenia cancer, bone marrow transplanted and those undergoing immunosuppressive therapy [5]. Success of IA has been related to antifungal resistance such as azoles, currently used as the treatment of choice in these mycoses. An amphotericin B may be a therapeutic option in contrast to the negative outcome with the use of azoles for IA. However, recent studies have shown an increase in amphotericin B resistance of Aspergillus species between 2010 and 2020. After A. fumigatus, the species with the highest amphotericin B Fig. 4 Lipid detection of in vitro biofilm in a clinical brain isolate of A. terreus. The biofilm was cultured in RPMI at 37°C. Lipid analysis of the fungal biofilm was performed by CLSM and SEM-HR with backscatter-electron diffraction. CLSM) The lipid was detected outside the hyphae at about 72 h after biofilm formation (a, 40x). Compared to the earlier period, at 96 h an increased presence of lipids was observed in the peripheral hyphae (b, 40x). Further, in magnified detail, lipid clusters were observed in coexistence with carbohydrates bound to an ECM (c, 63x). Calcofluor white (chitin-green halo) and Nile red (lipid-pink halo) were used as fluorochrome markers. SEM-HR) The waxy ECM was surrounded by hyphae (d, 1000x), and at higher magnification; a faceted form with waxy consistency secreted by the hyphae could be discerned (e, 5000x). SEM with backscatter-electron diffraction was used to differentiate the waxy material originating from the ECM secreted by the fungus (f, 2200x). Molecular co-localization of lipids (white arrow); presence of ECM (white arrowheads); hyphae (H); and conidia (C) resistance reported in Asia was A. terreus with a prevalence of 40.4%, followed by Europe at 40.1% and the Americas at 25.1% [29,30]. Additionally, it has been reported that the highest number of cases of A. terreus resistant to amphotericin B occurs in Asian countries such as India [31,32]. Regarding the clinical forms of IA, central nervous system (CNS) aspergillosis is rare worldwide [33][34][35]. The main causative agents of aspergillosis are A. fumigatus and A. flavus [36,37]. Although, the mortality rate in patients acquiring this fungal infection by A. terreus was increased relative to Aspergillus species mentioned above [30,38]. CNS aspergillosis is frequently reported in Asian countries such as India and Saudi Arabia [14,39].
The clinical strain used in this study was isolated by the Hospital Infantil de México ''Dr. Federico Gómez''. The identity of the fungal agent was corroborated as A. terreus. The origin of the clinical isolate is from a pediatric patient with CNS aspergillosis. Although this is not a clinical report, it is important to mention that, to our knowledge, there are scarce reports of this IA caused by A. terreus in Mexico [37,40].
Filamentous fungi of the genus Aspergillus are described as saprophytic microbes or opportunistic pathogens. These biological behaviors are conditioned by multifactorial interactions such as host type, host immune status, fungal inoculum concentration, conidial diameter, climatic conditions and the expression of virulence factors of each Aspergillus species [1,20,41]. Specifically for A. terreus, some virulence factors such as the ability to develop a biofilm have been described [2,[41][42][43][44][45][46][47], although it has not been described in detail at the quantitative and structural level. The development of a biofilm is a common process among filamentous fungi for their establishment, survival, and colonization in nature, as well as in specific hosts [48,49]. The data obtained in this research have made possible to describe the stages of in vitro biofilm formation by A. terreus. It was shown that around 24 h an early maturation occurs, and around 48 and 72 h respectively, a decrease in the biofilm formed with a statistically representative later elevation was shown. Likewise, it is possible to link a similar behavior to the logarithmic phase and the lag phase (adaptation) in conventional microbial growth kinetics, like that described for A. fumigatus biofilm [50][51][52][53]. The decrease in fungal biofilm formation between 24 and 48 h (Fig. 2a, b) could be considered a stress caused by the incubation temperature at 37°C (for A. terreus efficient colonial growth was determined at 28°C) (Online Resource 1a). Between 48 and 72 h, the absorbance units increased in relation to the biofilm concentration. Possible because a process of co-aggregation of planktonic cells on the adherent layer of sessile hyphae was manifested. This behavior was shown in micrographs with abundant hyphae embedded in abundant ECM (Fig. 2c). Likewise, the lowest biofilm development was detected at 96 h. This decrease in metabolic activity of A. terreus may be related to the dispersion phase described in filamentous fungi [50,54]. The dispersal phase allows recolonization of other surfaces given the release of propagules, either hyphae or conidia, to restart the cycle. At the same time, cell death is imminent given the extensive development time; therefore, there is a decrease in fungal cell number and metabolic activity in the biofilm [54][55][56].
Our research group has characterized previously the biofilm of A. fumigatus [21,50]. The composition and characteristics of the biofilm formed by an ocular clinical isolate and an isolate from nature are rather similar, but the main difference detected is that A. terreus biofilm has a high lipid content detected by backscattered-electron diffraction and confirmed by Nile red staining. The clinical relevance of this high lipid content and its ecological advantages should be addressed in future research.
Otherwise, the architecture of fungal biofilms of Aspergillus species, except for A. fumigatus, has been described in a discrete manner, and particularly in A. terreus there are no reports detailing the structure. In this study, the morphology of the biofilm of A. terreus was analyzed by SEM at different incubation times. Initially, abundant biomass (hyphae embedded in the ECM and germinating conidia) was observed in the 24 h micrographs. This behavior was complementary to the result obtained by MTT assay (high fungal metabolic activity). In the fungal biofilm of A. terreus, structures known as microhyphae formed along anastomosed hyphae were observed at 24 h (Fig. 3b, c). In other Aspergillus species microhyphae have been described only in the biofilm of A. fumigatus [50]. Other reports of microhyphae have been related to infections in plant tissues caused by endophytic fungi such as Ophiostoma ulmi, Phialocephala fortinii and Fusarium oxysporum [57][58][59]. In human pathogens there are no reports demonstrating the existence of microhyphae, except for a clinical strain of A. fumigatus [50]. There is an article mentioning the name of microhyphae in mycetoma infections caused by actinobacteria, but it refers to the description of microsyphonate mycelium [60]. The role of microhyphae has not been explained for Aspergillus genus, the immediate background has been with plant models and, different propagules. In A. terreus, other modified asexual structures (phialidic and globular accessory conidia) have been reported, attributing to them an invasion effect on the infected host, in combination with other fungal virulence factors [44].
At 48 h, a decrease in biomass and metabolic activity of the fungus was detected, comparable with SEM micrographs (Fig. 3d, f); at the same time, two types of extracellular matrix were observed: condensed-porous ECM (Fig. 3e) and vesicular ECM (Fig. 3g). Several types of ECM have been described in the in vitro biofilm model of A. fumigatus, such as porous-type ECM, dense-type ECM and film-type ECM. The cause of the presence of these ECM was related to the antibiosis manifested by the fungusbacteria interaction with S. aureus [21]. For the diversity of ECM structures in A. terreus, probably it is related to the lipids produced in the biofilm. A waxy like matrix appearance of condensed and porous ECM was observed (Fig. 3e).
Likewise, in the micrographs obtained at 72 h, in comparison with the quantitative analysis obtained from highest biomass production, the characteristics of biofilm patterns were verified ( Fig. 2c; Fig. 3h). An ECM with abundant embedded mycelium was observed, and in the same fields several types of extracellular matrix: condensed ECM, film-like ECM (Fig. 3i) and vesicular ECM (Fig. 3j). In particular, a comparison of film-like ECM of A. terreus showed similar characteristics as those described for the biofilm of A. fumigatus [21]. Once described, the biofilm of A. terreus at 72 h of incubation was compared with stage V of the filamentous fungal biofilm model proposed by Harding et al. [54]. And it also relates to the maturation stage of the A. fumigatus biofilm model that was observed by González-Ramirez et al. [50]. Furthermore, this result is also similar to that reported by Wuren et al., since despite having different culture conditions; they detected a mature biofilm between 24 and 72 h [47].
During 96 h of biofilm development, sparse hyphae were observed by SEM, comparable to a decrease in absorbance units statistically verified by CV and MTT when quantifying the biofilm (Fig. 2). Although the characteristics of a functional fungal biofilm (hyphal anastomosis, canals, and ECM) were maintained at 96 h, it can be assumed to corresponds this phase of dispersion ( Fig. 3k; m). This deduction was derived from the phase VI proposed in the fungal biofilm model and dispersal phase of A. fumigatus leading to the shedding of reproductive structures, either hyphae or conidia [50,54]. The restart of the biofilm production cycle during this phase explains the decrease of mycelium in several fields, even though it is possible to observe growing conidia in this final phase of the fungal biofilm ( Fig. 3m; n).
Afterwards, to know the chemical composition of ECM, CLSM technique was used on the in vitro biofilm of A. terreus at 72 and 96 h, to detect chitin and lipids. All the most representative signals were observed in the biofilms at these incubation times when labeled with the specific fluorochromes as calcofluor white (green halo); it was repeated for both incubation times. Moreover, lipids were labeled with Nile Red (purple halo). The signal was highest at 96 h and co-localized with the green halos of chitin ( Fig. 4a; b). Previous studies have reported the role of carbohydrates in the biofilm structure, whose function is linked to providing stability and integrity due to hydrophilic polysaccharides that maintain the hydration of the biofilm, preventing desiccation and capturing water from the medium in the biofilm. Probably, this function is also carried out in the biofilm of A. terreus [10,21,56,61,62]. The lipid content among the structural components of the ECM was unexpected, as these constituents have not been described in filamentous fungal biofilms. Therefore, the detection of lipids with the fluorochrome Nile Red was shown that the biofilm is formed of significant amounts of these molecules given the intensity and number of homogeneous purple halos tagged practically in all hyphae ( Fig. 4a; b; c). Prior to this, other microscopy techniques were used to elucidate the existence of lipids in the biofilm, as proved by CLSM. While analyzing a few SEM micrographs of fungal biofilm, specifically at 96 h, wax-like icosahedral faceted structures were observed adjacent to the hyphae ( Fig. 4d; e). To analyze the crystallographic patterns of some of the biofilm components, the backscatter electron diffraction technique was used [63,64]. The hyphae and waxy ECM showed a similar crystallographic pattern, so the fungus was considered to secrete this exopolymeric material (Fig. 4f). The production of these polyhedral wax-like structures on the surface hyphae and as secreted lipids, was also detected with CLSM. While these results from microscopic analysis are a first approximation of lipid composition in filamentous fungal biofilms, there is also evidence for the synthesis of lipid molecules in A. terreus. From the finding of the description of lipid components in the ECM, a relationship can be made with respect to the studies of A. terreus that have been performed at the biotechnological level to produce lovastatin [competitive inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMG-CoA)] [52,65,66]. This enzyme was involved in the synthesis of mevalonate, an intermediate metabolite in the synthesis of ergosterol (main components of the fungal membrane) and cholesterol. Thus, since lovastatin has a lipid origin (statin family), it could be related to the ergosterol synthesis pathway [5,42]. Presumably, this metabolic pathway is also involved with gene up-regulation during ergosterol biosynthesis, as reported for A. terreus. This negative regulation, in which ERG5, ERG6 and ERG25 genes are overexpressed and lead to ergosterol deficiency in the cell membrane, triggers the modification of the target site where amphotericin B acts and, consequently, antifungal resistance is manifested [30].
In conclusion, the most important aspects observed during the development of the in vitro biofilm of A. terreus were the following: (i) the stages of biofilm development were described; (ii) the organizational structure of the biofilm was characterized, reporting the presence of microhyphae, previously unreported for this species; and (iii) the chemical composition of the ECM was analyzed, demonstrating a relevant content of lipid components. 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. Additionally, this study is first to show the identification of a clinical isolate of A. terreus, responsible for cerebral aspergillosis in our country, with the ability to develop a biofilm in vitro.