The genus Aspergillus consists of four sections: Fumigati (A. fumigatus), Nigri (A. niger), Terrei (A. terreus) and Flavi (A. flavus). Currently, there are ≈16 species accepted in section Terrei [8, 20, 21, 55]. In recent years, medical interest in A. terreus has been related to its role as an emerging opportunistic pathogen causing invasive aspergillosis (IA) in immunocompromised patients, such as individuals with severe neutropenia for prolonged periods, cancer patients, bone marrow transplant recipients and those undergoing immunosuppressive therapy [55]. The largest number of cases of A. terreus resistant to amphotericin B treatment is reported from India [21, 22]. Regarding the clinical forms of IA, central nervous system (CNS) aspergillosis is rare in Mexico. While worldwide, the main causative agents of aspergillosis are A. fumigatus and A. flavus [23, 24]. Epidemiologically, IA worldwide presents incomplete data that vary according to the study region. During the last few years, an increase in IA has been reported along with cases in immunocompromised patients [24], and at the same time, the mortality rate in patients acquiring this fungal infection has increased, although the incidence in recent decades has decreased [25].
CNS aspergillosis is commonly reported in Middle Eastern countries with dry climates (India and Saudi Arabia) [8, 26]. Additionally, cases of this clinical presentation caused by A. terreus are rare [27–29].
The clinical strain used in this study was isolated by the Hospital Infantil de México "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 aspergilosis. 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 [24, 64].
Filamentous fungi of the genus Aspergillus are described as saprobes or opportunistic pathogens, and these 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, 13, 30]. Specifically for A. terreus, some virulence factors have been described [2, 30–35]. Additionally, A. terreus is related to its ability to develop a biofilm [30, 36], 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. Additionally, the importance of the study of fungal in vitro biofilm provides information that can be applied in fields such as clinical practice, organ, and tissue engineering or in the study of infectious processes. In the industrial field, some studies indicate that biofilms adhere to pipes and inert surfaces, causing economic losses in manufacturing processes [37, 38]. The data obtained in this research have made it possible to describe the stages of in vitro biofilm formation. It was shown that around 24 hours an early maturation occurs, and around 48 and 72 hours 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 [41–44]. The decrease in fungal biofilm formation between 24 and 48 hours (Fig. 2a; Fig. 2b) 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 hours, the absorbance units increased in relation to the biofilm concentration. The reason is 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 hours. This decrease in metabolic activity of A. terreus may be related to the dispersion phase described in filamentous fungi [40, 41]. 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 [40, 45, 46].
Otherwise, the architecture of fungal biofilms of Aspergillus species, except for A. fumigatus, has been described in a discrete manner. Particularly, in A. terreus there are no reports detailing the structure of its biofilm. 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-hour 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, Fig. 3c). The occurrence of these structures is usually rare. In the case of A. terreus, no microhyphae have been found, but for other Aspergillus species only microhyphae have been described in the biofilm of A. fumigatus [41]. Other reports on microhyphae in which these fungal structures predominate have been related to infections in plant tissues caused by some species of endophytic fungi such as Ophiostoma ulmi, Phialocephala fortinii and Fusarium oxysporum [58, 59, 60]. In human pathogens there are no reports demonstrating the existence of microhyphae, except for the role mentioned above on a clinical strain of A. fumigatus [41]. There has been an article mentioning the name of microhyphae in mycetoma infections caused by actinomycetes, but it refers to the description of microsyphonate mycelium [61]. However, the role of microhyphae has not been explained for the genus Aspergillus, apart from plant models. 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. Future studies should focus on defining their role in the metabolism of the fungus [33].
Outstanding biofilm findings were also observed at 48 hours, accompanied by a decrease in biomass and metabolic activity of the fungus, comparable with SEM micrographs (Fig. 3d, 3f); 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 fungus-bacteria interaction with S. aureus [15]. 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 hours, 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). Particularly, a comparison of film-like ECM of A. terreus showed similar characteristics to those described for the biofilm of A. fumigatus [15]. 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. [40]. And it also relates to the maturation stage of the A. fumigatus biofilm model that was observed by González-Ramirez et al. [41]. 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 hours [36].
During 96 hours 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 hours, it can be assumed that this is the dispersal phase of the A. terreus biofilm (Fig. 3k; Fig. 3m). This deduction was derived from the phase VI proposed in the fungal biofilm model and the dispersal phase of A. fumigatus in which the shedding of reproductive structures, either hyphae or conidia, occurs [40, 41]. The restart of the biofilm production cycle during this phase explains the decrease of mycelium in several fields, although it is possible to observe developing conidia during this final phase of the fungal biofilm (Fig. 3m; Fig. 3n).
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 hours and co-localized with the green halos of chitin (Fig. 4a; Fig. 4b). 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 [6, 15, 46–48]. The lipid content among the structural components of the ECM in A. terreus 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; Fig. 4b; Fig. 4c). Prior to this, other microscopy techniques were used to elucidate the existence of lipids in the biofilm, as proved by CLSM. While analyzing some SEM micrographs of the fungal biofilm, specifically at 96 hours, wax-like icosahedral faceted structures (referred to as ECM wax) were observed adjacent to the hyphae (Fig. 4d; Fig. 4e). To analyze the crystallographic patterns of some of the biofilm components, the backscatter electron diffraction technique was used. Under this microscopic analysis, the reasoning is focused on detecting some type of reflective effect in the faceted structure that differs from the bio-organic crystalline pattern of the fungus; in the case of a non-hyphal component, such as a metal [49, 50]. Finally, the hyphae and the waxy ECM showed a similar crystallographic pattern, so it was considered that the fungus secreted this exopolymeric material (Fig. 4f). Presumably, the production of these polyhedral structures showing a waxy appearance is related to the scarcity of hyphae on the surface and the secreted lipid composition (also detected with CLSM). Similarly, the presence of poor lipids was reported when a condensed and porous ECM was observed at 48 hours (Fig. 3d-3g). Although these results of microscopic analysis are a first approximation to the lipid composition in filamentous fungal biofilms, and should be studied in detail, there are also evidences of the synthesis of molecules of this nature in these fungal species. Based on this finding of the description of lipid constituents in the ECM, it should be mentioned that A. terreus was used at the biotechnological level to produce lovastatin [competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA)]. 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 [31, 43, 51, 52, 55]. Currently, this could be the answer to the increase in lipid production at 96 h of incubation of the A. terreus biofilm in this work.
This study presents a series of findings, which are important given the scope of applications that can be handled in the short and long period in the clinical, epidemiological, pharmaceutical, and microbiological fields. These perspectives can be focused on the search for therapeutic targets, biological control and containment for a fungal agent that has been rarely isolated in México and is beginning to manifest itself in several clinical cases of this filamentous fungus on a worldwide scale [51, 53, 54]. Finally, 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. Moreover, this study is the first to show the identification of a clinical isolate of A. terreus, the cause of cerebral aspergillosis in our country, as well as to demonstrate the ability of this isolate to develop an in vitro biofilm and the characterization of them.