Antimicrobial and cytotoxic activity of fungal mycelial extracts from aquatic environments in the Amazon

Fungi are a prolific source of biologically active metabolites, including a wide range of clinically important drugs. Therefore, this study aims to evaluate the antimicrobial and cytotoxic activity of secondary metabolites extracted from fungal mycelia isolated from freshwater samples in the state of Amazonas. Mycelial extracts from 12 fungal were used, extracted with MeOH/AcOEt (1:1) according to the criteria established by Souza et al. (2004). For antimicrobial activity, the extracts were tested against the pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus feacalis, Candida albicans and C. tropicalis. To identify the minimum inhibitory dosage (MID) the microdilution method was used. To perform the cytotoxicity assay, the VERO strain (ATCC® CCL81TM) was used. The assays were determined by the Alamar Blue method according to Ahmed et al. (1994). The tested extracts did not show antibacterial activity. Five extracts (41.7%), obtained from the fungi Aspergillus 1283, Chrysoporther 1169, Diaporther – 1203, Fusarium – 1085, and Trichoderma, showed antifungal activity against C. albicans. Diaporther extract (8.3%) 1203 was active against C. tropicalis. In the cytotoxicity assay, 58.3% of the evaluated extracts showed no significant toxic effect. Five extracts, Cladosporium 1135, Chrysoporther 1169, Research, Society and Development, v. 10, n. 10, e273101018795, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18795 2 Cytospora 1098, Fusarium 1085, and Talaromyces 1244, showed cytotoxic potential, exhibiting viability lower than 70%. The results obtained suggest that mycelial extracts of fungi isolated from water samples from the Amazon region have potential against yeasts of medical interest. Only two of the active extracts were revealed potentially cytotoxic.


Introduction
Antimicrobial resistance is a major public health problem worldwide and jeopardizes the effectiveness of preventing and treating an increasing number of infectious diseases caused by viruses, bacteria, fungi, and parasites according to the Pan American Health Organization (PAHO, 2020).
The need to control emerging diseases or resistant strains requires the constant search for new sources of antimicrobial compounds with new action mechanisms, not only for human use but also for use in livestock (Santos et al., 2015;Premjanu & Jaynthy, 2015). Antimicrobials are natural or synthetic compounds capable of inhibiting or killing microorganisms through specific interactions with their targets, regardless of the compound source or its class (Davies & Davies, 2010).
Currently, natural products and their derivatives are still the most important resources for new drugs discovery (Ayoub et al., 2014;Atanasov et al., 2021). Fungi are considered an important source of biologically active secondary metabolites, including a wide variety of clinically important drugs (Hoeksma et al., 2019). Fungi have a chemical and biological rich diversity. Historically they have given rise to many new drugs and may hold the key to dealing with emerging antimicrobial resistances.
Fungi are excellent producers of hydrolytic enzymes, biofuels, organic acids, polysaccharides, and secondary metabolites, such as antibiotics, anticancer drugs, hypocholesterolemic agents, immunosuppressants, among other (Sanchez & Demain, 2017), which make them attractive for the discovery of new bioactive molecules with antimicrobial properties.
Given the economic and industrial importance of fungi, the aquatic environment is an important place to expand the discovery of new fungal species and new biological properties (Heo et al., 2019). According to Wurzbacher et al. (2016), freshwater ecosystems are neglected and harbor high microbial diversity. Magwaza et al. (2017), points out that freshwater fungi produce a wide variety of antimicrobial metabolites, which help them to compete against other microorganisms.
Considering the high fungi biodiversity estimates and the notion that only a small fraction of them have been biologically tested for active compounds production, it is evident that there is a great number of bioactive compounds yet to be discovered (Hoeksma et al., 2019). The isolation and identification of new fungal strains are the first steps towards the discovery of new metabolites with antimicrobial activities. This is more imperious for the Amazon region, which has one of the richest biodiversity on the planet but still has insufficient studies to cover all this diversity (Oliveira et al., 2021). In this context, we present an evaluation of antimicrobial and cytotoxic activities of extracts from mycelia of fungi isolated from freshwater samples in the Amazonas state.

Fungal material
In this study, 12 fungal strains isolated from freshwater samples collected from aquatic environments in the State of Amazonas were used. The fungal isolates were obtained from the collection of microorganisms at the Laboratory of Bioassays and Microorganisms of the Amazon of the Federal University of Amazonas (LabMicrA/UFAM), registered under the SisGen (National System for the Management of Genetic Heritage and Traditional Knowledge Associated) with the number: AD64E07. All the strains were previously identified up to genus level by LabMicrA through macro and micro morphological analysis of their structures and by rDNA sequence analysis with an internal transcribed spacer (ITS1-5.8S-ITS2).

Production of fungal extracts
Fungi were reactivated in Petri plates containing PDA+L culture medium (potato, dextrose, and agar, with 0.2% yeast extract), cultivated at 26 ºC for eight days. After this period, five fragments of 1 cm 2 from each fungus culture were inoculated into a 1000 mL Erlenmeyer containing 300 mL of PD+L liquid culture medium under sterile conditions (Souza et al., 2004) and maintained at 26°C, in static mode and in the absence of light.
The cultivation time of each fungus was determined until the glucose level in the culture was consumed. Then, the mycelium was separated from the culture medium by filtration on Whatman paper, nº 4. The mycelial mass obtained was covered with a mixture of ethyl acetate and methanol 1:1 (v/v) and kept in maceration for 48 hours, followed by filtration. This process of extraction was repeated more twice every 24 hours. Afterward, the extracts were combined and concentrated in a rotary evaporator at 45 °C. The samples obtained were kept in a desiccator with activated silica to obtain dry mycelial extracts.
After reactivation of the pathogens, a pure culture of each strain was transferred to a test tube containing 5 mL of SB broth and BHI broth and kept at 36 ± 1 °C for 24 hours. Then, the concentrations of the colonies to be used in the tests was adjusted to achieve 2.5 x 10 3 CFU/mL for yeast (CLSI, 2002) and 5 x 10 5 CFU/mL for bacteria (CLSI, 2003). The samples of the fungi mycelial extracts were weighed and solubilized at a concentration of 2 mg/mL (stock solution) in 10% dimethyl sulfoxide (DMSO).
For negative control, 10% DMSO solution was utilized, and positive control was performed using antibiotics at a concentration of 2 mg/mL, tetracycline for E. coli and P. aeruginosa, ampicillin for S. aureus and E. feacalis, and nystatin for C. albicans and C. tropicalis.
To determine the antimicrobial activity, a screening of the fungal extracts was initially carried out to verify their effectiveness against the tested pathogens. The screening was performed in 96-well Elisa microplates, where it was added in triplicate, 100 μL of the culture medium in doubled concentration (2x), 100 μL of the stock extract solution (2 mg/mL), and 10 μL of the pathogen cells suspension to be assayed, reaching a final pathogen concentration of 2.5 x 10 3 for yeasts and 5 x 10 5 for bacteria. Finally, the plates were incubated at 36 ± 1 °C for 24 hours. After incubation, 10 µl of 1% of a revelator, NBT (Nitroblue Tetrazolium) for fungal and TTC (2,3,5-2,3,5-Triphenyltetrazolium Chloride) for bacteria were added to all wells.
When microbial growth occurs, the color in the well changes.
With the positive results of the screening, the successive dilution test was performed to determine the minimum inhibitory dosage (MID). In an Elisa plate were added in triplicate, 100 µL of the extract to the first well, with 100 µL of the culture medium in doubled concentration (2x). After homogenization, 100 µL was transferred from the first well to the subsequent one (B1, B2, B3), successively performing the dilutions in each well. The extract concentrations obtained after serial dilutions resulted in eight concentrations ranging from 1 to 0,0078125 mg/mL (CLSI, 2012). The same serial dilution procedure was performed for the positive and negative controls.
Subsequently, 10 µL of the pathogen cells suspension was added to all wells to achieve the same concentration as in the screening assay above. The plates were incubated at 36 ± 1°C for 24 hours. After this period, 10 µL of 1% revelators were added: NBT in wells with fungal inoculum and TTC in wells with bacterial inoculum. As a control, 10 μL of the tested pathogen cells suspension were inoculated on a plate with a solid medium to verify the microbial growth or its absence, being defined as fungicidal or bactericidal the complete absence of microbial growth and fungistatic or bacteriostatic the reduction of microbial growth.

In vitro cytotoxicity assay
To perform the cytotoxicity assay, the fungal extracts were solubilized in 0.5% dimethylsulfoxide (DMSO) and tested in eight concentrations, from 50 to 0.39 µg/mL. The VERO strain (ATCC® CCL-81TM) utilized was acquired from the American Type Culture Collection, grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco), and supplemented with 10% inactivated fetal bovine serum (Gibco) and penicillin (50 μg/ml). All tests were performed in triplicate.
Assays were determined by the Alamar Blue method according to Ahmed et al. (1994). The cells suspension was plated at a concentration of 1.0 x 10 4 cells/well in 96 well Elisa plates and treated with extracts at the determined concentrations, totalizing a final volume of 200 µL. The plates were kept in a CO 2 incubator for 24 hours with 5% CO 2 at 37 ºC. After this period, 10 µL of 0.4% resazurin (diluted 1:20) was added to each well and the Alamar Blue™ (Sigma-Aldrich) metabolization time of 2 hours was awaited. Fluorescence was monitored in a microplate reader (GloMax® Explorer) with emission of wavelengths between 580 and 640 nm and an excitation of 520 nm.
The cell growth was used as a positive control, and 0.1% DMSO was used as a negative control. The percentage of cell viability was calculated according to the formula: %Viability= Ft x 100/Fb, where Ft= (cell fluorescence + medium + substance + resazurin) and Fb= (cell fluorescence + medium + resazurin).
The result evaluation was performed according to the criteria established by ISO (2009) -the lower the % viability value, the higher the cytotoxic potential of the tested material. If the viability is less than 70%, the tested sample has cytotoxic potential.

Results and Discussion
For the screening assay, all twelve mycelial extracts were tested for antimicrobial activity against six pathogens -two fungi (C. albicans -CC 001 and C. tropicalis -CC 002) and four bacteria (S. aureus -S 007, E. coli -E 004, P. aeruginosa -P 004, and E. feacalis -E 002). No extract showed antibacterial activity and five, representing 41.7% of the tested extracts showed antifungal activity. The active extracts come from five different fungal genera (Diaporther, Chrysoporther, Aspergillus, Trichoderma, and Fusarium) ( Table 1).  For the minimum inhibitory dosage (MID) assay, it was observed that five extracts showed fungistatic activity against C. albicans, three at a concentration of 1 mg/mL (Chrysoporther -1169, Diaporther -1203, and Fusarium -1085) and two at a concentration of 0,25 mg/mL (Aspergillus -1283 and Trichoderma -1136). The extract of Diaporther -1203 also had fungistatic activity against C. tropicalis at a concentration of 1 mg/mL and the extract of Aspergillus -1283 showed fungicidal activity against C. albicans at a concentration of 1 mg/mL (Table 2). Aspergillus
Diaporthe -1203 mycelial extract did not show activity against bacterial pathogens. However, it was the only extract with activity against more than one pathogen, showing fungistatic activity against C. albicans and C. tropicalis. In the study by Dos Reis et al. (2019), Diaporthe extracts also showed antifungal potential, inhibiting the growth of different Candida species.
Different results were found by Moreira et al. (2020), in a study in which the fungus Diaporthe showed promising antimicrobial activity against S. aureus and E. coli but no activity against C. albicans. Fungal species of the Diaporthe genus are commonly studied for their ability to generate metabolites with diverse biotechnological applications (Flores et al., 2013;Moreira et al., 2020). Yenn et al. (2017) isolated a new compound (3-hydroxy-5-methoxyhex-5-ene-2,4-dione) with fungicidal activity against C. albicans from a Diaporthe strain.
Aspergillus -1283 mycelial extract showed the best performance in the antimicrobial test, despite also not presenting antibacterial activity. The strain demonstrated fungistatic and fungicidal activity against C. albicans. The results obtained are similar to those found by Sakhri et al. (2019), in which an Aspergillus crebe strain exhibited only antifungal activity when evaluated against the pathogens: E. coli, P. aeruginosa, C. albicans, and C. glabrata. Aspergillus genus has a worldwide distribution and contain a large number of species, which stands out for producing a range of natural products with biological activities (Lotfy et al., 2018), such as asperlicin, echinocandin B, and fumagillin isolated from A. alliaceus, A. nidulans, and A.
coli and S. aureus (Nirma et al., 2015); and Fusarium species produced metabolites with activities against microbial pathogens (Sibero et al., 2019). Besides, from F. larvarum, an antifungal compound active against C. albicans and a wide range of pathogenic fungi was isolated, called Parafungin (Harvey et al., 2015). Research, Society andDevelopment, v. 10, n. 10, e273101018795, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18795 7 There is a growing demand for new antibacterial and antifungal compounds due to the constant increase in the number of people with health problems caused by pathogenic microorganisms resistant to existing antimicrobials (Xu et al., 2015;Sanchez & Demain, 2017). Fungal infections are considered a serious complication of immunosuppression and are associated with substantial utilization of health services and high mortality rates (Sharma & Chowdhary, 2017).
Microorganisms of the Candida genus are the main agents that cause fungal infections in the bloodstream, known as candidemia. It causes mortality rates varying from 15 to 35% and increases the period and costs of hospitalization, making the disease a serious public health problem, especially in developing countries (Canela et al., 2021). C. albicans and C. tropicalis are found in the microbiota of the reproductive and gastrointestinal mucosa, living symbiotically in about 50 -70% of healthy individuals (Megri et al., 2020). However, these strains can become pathogenic through changes in the environment or in individuals with weakened immune systems (De Barros et al., 2018). C. albicans is known as the most prevalent cause of candidemia and C. tropicalis is the second most prevalent species in Brazil (Wille et al., 2013;Arastehfar et al., 2020).
According to Kholoujini et al. (2019) there are a small number of studies carried out in search of compounds with antifungal activity compared to the number of studies investigating antibacterial compounds. Considering the increase of fungal diseases, it is relevant to perform more research with this emphasis. As they are considered an important source of bioactive compounds, fungal have been important sources in the pursuit for new antibacterial and antifungal compounds (Sathi et al., 2015;Xu et al., 2015;De Medeiros et al., 2018).
The cytotoxicity assay against VERO cells, according to the criteria established by ISO (2009), showed that 58.3% of the extracts evaluated in this study had no significant toxic effect on cell viability (Figure 1). Of the twelve fungal mycelial extracts, five (41.7%) showed cytotoxic potential, with cell viability less than 70%. They belong to the strains Cladosporium -1135, Chrysoporther -1169, Cytospora -1098, Fusarium -1085 and Talaromyces -1244. Controls: positive (Cell) and negative (DMSO). Source: Authors. Saravanakumar et al. (2018) emphasizes that metabolites extracted from fungi in some cases can be naturally toxic, so it is essential to investigate the toxicity of extracts of fungal origin, considering that one of the most important steps during the development of a new drug is the absence of cellular cytotoxicity (Kholoujini et al., 2019). In the present study, considering the five active fungal mycelial extracts only Chrysoporther -1169 and Fusarium -1085 showed cytotoxic potential against VERO cells. The other active extracts in the antimicrobial assay showed no cytotoxicity.

Conclusion
The present study revealed that mycelial extracts from five fungal strains isolated from water samples from the Amazon region, present antimicrobial activity against C. albicans, being one of them also active against C. tropicalis. Among them, only two strains showed cytotoxic potential on VERO cells. The data generated in this study contribute to the knowledge of the antimicrobial and cytotoxic activities of fungi obtained from water samples from the Amazon region. However, further studies aimed at the isolation and identification of bioactive compounds are needed. through the Pró-Amazônia: Biodiversity and Sustainability project, and the Oswaldo Cruz Foundation -Leônidas & Maria Deane Institute -Manaus/AM.