Saccharide sources do not influence the biofilm formation in Scedosporium/Lomentospora species

Abstract

Scedosporium and Lomentospora species are ubiquitous saprophytic filamentous fungi that emerged as human pathogens with impressive multidrug-resistance profile. The ability to form biofilm over several biotic and abiotic surfaces is one of the characteristics that contributes to their resistance patterns against almost all currently available antifungals. Herein, we have demonstrated that Scedosporium apiospermum, Scedosporium minutisporum, Scedosporium aurantiacum and Lomentospora prolificans were able to form biofilm, in similar amounts, when conidial cells were incubated in a polystyrene substrate containing Sabouraud medium supplemented or not with different concentrations (2%, 5% and 10%) of glucose, fructose, sucrose and lactose. Likewise, the glucose supplementation of culture media primarily composed of amino acids (SCFM, synthetic cystic fibrosis medium) and salts (YNB, yeast nitrogen base) did not modulate the biofilm formation of Scedosporium/Lomentospora species. Collectively, the present data reinforce the ability of these opportunistic fungi to colonize and to build biofilm structures under different environmental conditions.

Introduction

Scedosporium and Lomentospora species are widely distributed filamentous fungi that emerged as etiologic agents of localized and disseminated diseases in immunocompromised and immunocompetent individuals. Fungal ball and mycetoma are usual clinical manifestations caused by these fungi, resembling a classical biofilm structure (Mello et al., 2019).

Biofilm is considered a microbial community adhered to a biotic/abiotic surface covered by a complex extrapolymeric substance, which confers resistance to environmental stresses (Flemming et al., 2016). Scedosporium and Lomentospora species form biofilm structures over different substrates, including polystyrene, glass, catheters and lung epithelial cells, which present a typical multidrug-resistance profile (Mello et al., 2016, 2018; Rollin-Pinheiro et al., 2017). However, little is known about the environmental conditions that interfere with the biofilm formation in these fungi. In this respect, it is well-established that bacterial/yeast biofilms are influenced by available nutrients, particularly carbohydrates (Jahid et al., 2013; Waldrop et al., 2014; Pereira et al., 2015).

Objective

The aim of the present study was to evaluate the influence of monosaccharides (glucose and fructose) and disaccharides (sucrose and lactose) on the ability of S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans conidial cells to form biofilm structures over a polystyrene surface containing different nutritional culture media.

Methods

Fungi

Scedosporium apiospermum (RKI07_0416) was provided by Dr. Bodo Wanke (Brazil), S. minutisporum (FMR4072), S. aurantiacum (FMR8630) and L. prolificans (FMR3569) were given by Dr. Josep Guarro (Spain). Fungi were maintained in Sabouraud medium for 7 days. Conidia were obtained as previously described (Mello et al. 2016).

Biofilm assay

Conidia (10⁶ cells) were placed to interact (72 h/37˚C) with 96-well polystyrene plates containing Sabouraud supplemented with different concentrations (0%, 2%, 5% and 10%) of glucose, fructose, sucrose and lactose. Synthetic cystic fibrosis medium (SCFM) and yeast nitrogen base (YNB) added or not with 2% glucose were also tested. Biofilm parameters (biomass, metabolic activity and extracellular matrix) and scanning electron microscopy (SEM) were evaluated as previously published (Mello et al. 2016).

Statistics

All experiments were performed in triplicate, in three independent experimental sets. Data were expressed as mean ± standard deviation. Results were evaluated by two-way ANOVA followed by Tukey’s test.

Results

Initially, we analyzed the biofilm formation of Scedosporium/Lomentospora species when cultivated in Sabouraud, a classic culture medium for fungal studies, supplemented or not with different concentrations of monosaccharides (glucose and fructose) and disaccharides (sucrose and lactose). In all fungal species tested, no significant differences were observed regarding the biomass, cellular metabolic activity and production of extracellular matrix comparing the biofilm formed in the absence and presence of soluble carbohydrates, which was also independent of the concentration used (Figs. 1 and 2).

Figure 1. Biofilm formation by S. apiospermum (Sap), S. minutisporum (Sm), S. aurantiacum (Sau) and L. prolificans (Lp) under different glucose concentrations. Conidia (10⁶) were placed to interact with polystyrene for 72 h in Sabouraud containing different glucose concentrations (0%, 2%, 5% and 10%) at 37˚C. Subsequently, in order to assess biofilm formation, the following parameters were spectrophotometrically inspected: biomass was measured in a methanol-fixed biofilm using crystal violet dye (590 nm) as well as extracellular matrix and metabolic activity (viability) were quantified in non-fixed biofilm by safranin incorporation (530 nm) and XTT metabolization (492 nm), respectively. Scanning electron microscopy images of the fungal mature biofilms formed in polystyrene containing Sabouraud supplemented with 2% of glucose were also shown.

Figure 2. Biofilm formation by S. apiospermum (Sap), S. minutisporum (Sm), S. aurantiacum (Sau) and L. prolificans (Lp) under different saccharide sources. Conidia (10⁶) were placed to interact with polystyrene for 72 h at 37˚C in Sabouraud supplemented with different concentrations (0%, 2%, 5% and 10%) of fructose (A), sucrose (B) and lactose (C). Then, the systems were processed in order to detect the fungal biomass by incorporation of crystal violet in methanol-fixed biofilms at 590 nm.

Posteriorly, two additional culture media were selected based on their chemical composition, SCFM (rich in amino acids, which mimics the cystic fibrosis sputum) and YNB (rich in salts), to evaluate the influence of glucose on fungal biofilm formation. Nevertheless, no differences on biofilm biomasses of S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans were found taking into consideration the media with and without glucose supplementation (Fig. 3).

Figure 3. Biofilm formation by S. apiospermum (Sap), S. minutisporum (Sm), S. aurantiacum (Sau) and L. prolificans (Lp) in different culture media. Conidia (10⁶) were placed to interact with polystyrene containing SCFM or YNB media supplemented or not with 2% of glucose for 72 h at 37˚C. Posteriorly, the systems were processed in order to detect the fungal biomass by incorporation of crystal violet in methanol-fixed biofilms at 590 nm.

Discussion

The ability to adapt to different physicochemical conditions is a central vein in the virulence arsenal of pathogens, because it reflects their ability to colonize several natural environments and anatomic sites of human body. Scedosporium/Lomentospora conidia easily adapt and germinate in a wide range of pH, temperature and available nutrients (Mello et al. 2016). In addition, these fungal species can adapt to distinct types and concentrations of saccharides, forming a similar amount of biofilm under all tested experimental conditions. Similarly, glucose concentration did not affect the Candida albicans biofilm formation (Kolecka et al., 2015). Conversely, Candida parapsilosis was able to respond to stress caused by glucose (at 10%) by up-regulating the genes related to biofilm formation, which culminated to a more robust biofilm formation with a higher concentration of carbohydrates and β-(1,3)-glucan in the extracellular matrix composition (Pereira et al., 2015).

Conclusions

The present findings indicate that soluble carbohydrates (mono- and disaccharides), at different concentrations, were not able to modulate the capacity of S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans to form biofilm on an inert surface containing distinct culture media (Sabouraud, SCFM or YNB). These data can corroborate the high adaptive capacity of these fungi, reflecting in their metabolic plasticity.