Anti-Staphylococcal, Anti-Candida, and Free-Radical Scavenging Potential of Soil Fungal Metabolites: A Study Supported by Phenolic Characterization and Molecular Docking Analysis

Staphylococcus and Candida are recognized as causative agents in numerous diseases, and the rise of multidrug-resistant strains emphasizes the need to explore natural sources, such as fungi, for effective antimicrobial agents. This study aims to assess the in vitro anti-staphylococcal and anti-candidal potential of ethyl acetate extracts from various soil-derived fungal isolates. The investigation includes isolating and identifying fungal strains as well as determining their antioxidative activities, characterizing their phenolic substances through HPLC analysis, and conducting in silico molecular docking assessments of the phenolics’ binding affinities to the target proteins, Staphylococcus aureus tyrosyl-tRNA synthetase and Candida albicans secreted aspartic protease 2. Out of nine fungal species tested, two highly potent isolates were identified through ITS ribosomal gene sequencing: Aspergillus terreus AUMC 15447 and A. nidulans AUMC 15444. Results indicated that A. terreus AUMC 15447 and A. nidulans AUMC 15444 extracts effectively inhibited S. aureus (concentration range: 25–0.39 mg/mL), with the A. nidulans AUMC 15444 extract demonstrating significant suppression of Candida spp. (concentration range: 3.125–0.39 mg/mL). The A. terreus AUMC 15447 extract exhibited an IC50 of 0.47 mg/mL toward DPPH radical-scavenging activity. HPLC analysis of the fungal extracts, employing 18 standards, revealed varying degrees of detected phenolics in terms of their presence and quantities. Docking investigations highlighted rutin as a potent inhibitor, showing high affinity (−16.43 kcal/mol and −12.35 kcal/mol) for S. aureus tyrosyl-tRNA synthetase and C. albicans secreted aspartic protease 2, respectively. The findings suggest that fungal metabolites, particularly phenolics, hold significant promise for the development of safe medications to combat pathogenic infections.


Introduction
Soil contains a diverse range of microorganisms, including fungi, which are the most abundant producers of potentially beneficial biologically active metabolites [1].Out of all the bioactive compounds that have been isolated from biological sources, more than 38% of the metabolites that are biologically active come from fungi [2].It has been reported that fungi are potent producer of diverse bioactive metabolites, with their secondary metabolism comprising polysaccharides [3], enzymes [4,5], lipids and fatty acids [6], and low molecular-weight byproducts that are produced as an adaptation for specific functions in nature [7].These metabolites may also include alkaloids, flavonoids, benzopyranones, phenolic acids, tannins, quinones, saponins, terpenoids, steroids, tetralones, xanthones, and many others [8].The identified metabolites showed varied bioactivities with anticancer [9], antioxidative [10,11], antifungal [12,13], immunosuppressive [14], antiprotozoal [15], antiviral [16], and antibacterial efficiencies [17].However, harmful influences can be caused by mycotoxins produced by some types of fungi [18,19].Due to their unique metabolism, fungi are able to produce a wide range of useful secondary metabolites, which increases their potential for drug development [20].
Phenolics are compounds that include one or more phenol units and are primarily found in plants, although they can also be found in bacteria and fungi [21].For example, tyrosol and p-hydroxyphenylacetamide isolated from the extract of the endophytic fungus Coriolopsis rigida showed potent antioxidative activity [22].The significance of phenolic compounds like vanillic and caffeic acids in potentially overcoming resistance in multidrug-resistant bacteria like methicillin-resistant Staphylococcus aureus (MRSA) has been highlighted in a numerous research studies [23,24] elucidating the hopeful impact of phenolic acids in suppressing the emergence of new resistant strains [25].Flavonoids and isoflavonoids are other promising antimicrobial mediators that target many microbial cells and reduce the virulence particularity of drug-resistant strains [26].
The ability of Candida species to cause infections ranging from superficial to potentially life-threatening is well documented [27,28].This variability in infection severity reflects the complexity and adaptability of these pathogenic organisms.Candida albicans is the most widespread species, while the ratio of non-albicans species has grown over the past few decades, comprising C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis [29][30][31].Candida albicans is a polymorphic and pathogenic fungal species causing enormous damage to humans, including biofilm formation, and oral, vaginal, and skin infections in immunedeficient patients [32,33].Candida albicans can grow together with S. aureus [34] and other diverse bacteria or fungi in oral as well as in skin/wound infections [35].Staphylococcus aureus serves as a commensal bacterium in a significant portion (approximately 30%) of the human population [36].It commonly colonizes the skin and is associated with infections in wounds and deep tissues [37].The bacterium has demonstrated remarkable genetic adaptability, leading to the emergence of various drug-resistant strains that display resistance to existing antimicrobial medications.This resistance poses a challenge to current treatment options [38].
Staphylococcus aureus is frequently found alongside C. albicans in systemic bloodstream infections, with over 25% of candidemia cases estimated to be polymicrobial, whereas systemic Staphylococcus infections without Candida are less prevalent [39,40].The emergence of azole-resistant C. albicans isolates has prompted various suggested approaches to address this issue.Similarly, effectively treating drug-resistant S. aureus poses a significant challenge in the field of medicine [41].Growing fungal resistance limits their convenient therapeutic efficacies, thus making the treatment of fungal infection disease more intractable [42].MRSA strains are intrinsically resistant to β-lactams and rapidly develop resistance to multiple antimicrobial drug classes [43].The widespread use of antibiotics has led to the prominent problem of microbial pathogens developing drug resistance, underscoring the urgent need to explore new antibacterial and antifungal agents that are both safe for patients and capable of efficiently eliminating pathogens to promote wound healing [44].
The genetic plasticity of C. albicans is directly reflected in its metabolic diversity and its ability to produce a wide range of virulence factors, such as secreted aspartyl proteases (SAPs), which cleave the extracellular matrix and fluid-phase proteins to induce tissue damage and promote infection [33,45,46].Secreted aspartic proteinase 2 (SAP2) represents an important virulence factor for vaginal infection, which is released by the yeast cells and causes damage to the reconstituted human epithelium [47,48].As a result, inhibiting the enzyme's active center using phytochemicals would reduce the severity of the enzyme's virulence [32].Staphylococcus aureus tyrosyl-tRNA synthetase belongs to the group of aminoacyl-tRNA synthetases, which are responsible for catalyzing the covalent binding of amino acids to their corresponding tRNA by generating charged tRNAs and which play an important role in the production of proteins [49].The structural differences between bacterial and eukaryotic aaRSs are a unique feature that allows the consideration of tyrosyl-tRNA synthetase as a good therapeutic target for the prevention of bacterial infection [50,51].Thus, inhibition of this enzyme influences cell proliferation due to its vital role in the biosynthesis of proteins [52].
Progress in analytical chemistry, computational tools, and drug discovery research has facilitated the creation of fungal-derived antimicrobial compounds.These compounds exhibit potential therapeutic effects and can be utilized independently or in combination therapies to manage challenging pathogens that are resistant to traditional treatments [53].Moreover, structure-based drug design plays a pivotal role [54].Molecular docking is performed to check the reliability of compounds in protein-binding sites [55].The present study focused on the in vitro antimicrobial potency of soil fungal extracts, which are rich in phenolic constituents, from different S. aureus and Candida species isolates.HPLC analysis of phenolic compounds as well as their in silico analysis in comparison with S. aureus tyrosyl-tRNA synthetase and C. albicans secreted aspartic protease 2 will be conducted to evaluate the interactions of these bioactive compounds and to provide clues for the therapeutic targeting of the enzymes' active sites.

Sample Collection and Fungal Isolation
Seven soil samples were randomly collected from Ha'il, (27.8942 • N, 42.6832 • E), Saudi Arabia, during December 2022, from the superficial layer of the soil with a maximum depth of 10 cm and transported in sterile plastic bags to the laboratory.Mycological analysis was achieved by the dilution-plate method [56] and Czapek's agar medium was utilized for the isolation and purification of fungal isolates.

Identification of Fungal Isolates
The most bioactive fungal isolates were identified based on their macromorphology and microscopic features using the key references of Raper and Fennell [57], Moubasher [58] and Domsch et al. [59]; these were then deposited into the Assiut University Mycological Center (AUMC) culture collection with an institutional number.

Fermentation and Extraction of Fungal Metabolites
The fermentation process for the fungal isolates was performed in autoclaved flasks containing solid rice inoculated with 1 mL of the fungal spore suspension (10 7 spore/mL), and then incubated at 28 ± 1 • C for thirty days [60].The fermented medium was extracted twice by ethyl acetate (EtOAc) (Analytical Grade, Alpha Chemika, Mumbai, India) [61], filtered over Na 2 SO 4 anhydrous (AL-Nasr Chemicals Co., Cairo, Egypt), and concentrated by a vacu-rotavapor [62].

Test Microorganisms
Test microbial strains and isolates were obtained from the culture collection at the Botany and Microbiology Department, Faculty of Science, Assiut, Al-Azhar University, Egypt.The tested Staphylococcus aureus strains included S. aureus ATCC 6538, enterotoxigenic S. aureus AZHAR2 (MF563554) [63], S. aureus AUHL123 [64], S. aureus BLBM 112, and S. aureus MLBM 117, which were grown on nutrient agar medium (NA) at 37  [65], and these were grown on Sabouraud dextrose agar medium (SDA) for 2 days at 25 • C.

Agar Well-Diffusion Method
The preliminary anti-staphylococcal and anti-candidal activities of fungal EtOAc crude extracts were screened by the well-diffusion method [66] on NA and SDA, respectively.One hundred mg of each fungal extract was dissolved in 1 mL of dimethyl sulfoxide (DMSO) (MilliporeSigma-St.Louis, MI, USA).Wells of 8 mm diameter were made in the media plates, pre-inoculated with the staphylococcal and candidal spore suspension (10 5 CFU/mL), filled with 100 µL of the extract (10% w/v), and then incubated for 24 h at 37 • C (bacteria) or and at 28 • C for 2 days (Candida).The diameter of the inhibition zone around the well was measured in millimeters (mm) using vernier calipers.Chloramphenicol (Chemical Industries Development Company, Cairo, Egypt) (0.1% w/v) was utilized in the anti-staphylococcal test as a positive control, while 0.1% w/v fluconazole (Pfizer, Egypt) was used for candida.DMSO was used as a negative control, and all experiments were performed three times.Extracts that showed positive activities were subjected to minimum inhibitory concentration (MIC) determination.

Determination of Minimum Inhibitory Concentration
A microdilution assay combined with p-iodonitrotetrazolium chloride (INT) (Merck, Darmstadt, Germany) at 0.2 mg/mL was used to determine the MIC values of the active EtOAc fungal extracts on the tested S. aureus.A 96-well microtiter plate was filled with a final volume of 200 µL in each well, comprising 100 µL of 10 5 CFU/mL and 100 µL of 2-fold serially diluted fungal extract in nutrient broth, which was incubated at 37 • C for 24 h.The final fungal extract concentration, using two-fold serial dilution, in each well was 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, and 0.098 mg/mL, respectively.Positive and negative controls (wells with and without chloramphenicol, 2-fold serially diluted) and blank (noninoculated wells) were included.After incubation, 40 µL of INT was added to the wells and set aside for 30 min at 37 • C, and then the MICs of the extracts were determined.Lall et al. defined the MIC as the lowest concentration at which INT was reduced to formazan due to mitochondrial dehydrogenase in the bacterial cell, which results in a color change from yellow to purple [67].The minimum bactericidal concentration (MBC) was assessed by streaking 50 µL from each well of the determined MIC and higher concentrations to NA plates, and these were then examined after incubation for one day at 37 • C.
Candida spp.cell suspension was adjusted to 10 5 cells/mL in sterile Sabouraud dextrose broth, and then 100 µL of this was added to a 96-well microtiter plate containing 100 µL aliquots of 2-fold serially diluted fungal extract in Sabouraud dextrose broth.Positive (wells with fluconazole, 2-fold serially diluted) and negative (wells without any treatment) controls as well as blank (wells with uninoculated medium) were established.The plates were incubated at 30 • C for 48 h, and then the MIC values were recorded visually, based on the growth of the microorganism.The MIC was defined as the lowest concentration at which no visible growth was established [68].The minimum fungicidal concentration (MFC) was confirmed by transferring 50 µL from the wells of the previously assessed MIC and of higher concentrations to SDA plates and then observed after incubation for two days at 30 • C.

Free-Radical-Scavenging Assay
The radical-scavenging assay was performed by using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) (MilliporeSigma-St.Louis, USA) based on the procedures described in [69].Several concentrations of fungal extracts ranging between 10 and 0.1 mg/mL were prepared, and 0.2 mL of each extract was added to 1.8 mL of 0.1 mM methanolic DPPH (negative control).These were measured spectrophotometrically after 30 min 517 nm and compared with the blank.To determine the scavenging capacity, the following formula was carried out: where A0 represents the absorbance of the negative control and A is the absorbance of the tested extract.The IC 50 was derived by intercalation from the linear regression exploration.

Brine-Shrimp Lethality Assay
Fungal extract cytotoxicity was assessed on Artemia salina larvae by the addition of 100 µL from each extract dilution to tubes containing 0.9 mL seawater with 10 living larvae for 1 day.After that, the number of vital larvae in each tube was counted and the average mortality percentage for each extract was calculated along with the LC 50 (extract concentration which caused 50% larval mortality) [70].

PCR Amplification and Nucleotide Sequence Analysis
As described by Mohamed et al. [72], the internal transcribed spacer (ITS) region of the tested strain was amplified using the specific universal primers ITS-1 (5 ′ -TCC GTA GGT GAA CCT GCG G-3 ′ ) and ITS-4 (5 ′ -TCC TCC GCT TAT TGA TAT GC-3 ′ ), while the PCR was performed as described previously by Hassan et al. [71].The obtained sequences were aligned with the BLAST search tool from NCBI to reveal any similarities.The BioEdit software program (version No. 7.2.5) was employed to check and analyze the ITS sequences, and the search for homology was achieved by comparison with strains from sequencing databases using the BLAST search algorithm of GenBank (http://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 13 May 2023)).

Determination of Total Phenolic and Flavonoid Content
The total phenolic content (TPC) was assessed spectrophotometrically three times in accordance with Suleria et al. [73], with the procedure modified by adding a 0.5 mL volume (1% w/v) of the extract to Folin-Ciocalteu's phenol reagent (MilliporeSigma-St.Louis, CA, USA) (0.5 mL) and 1 mL of sodium carbonate (10% w/v) and measuring at 750 nm after 1 h against the blank.The TPC was expressed as gallic acid equivalents (mg/g) through the standardization curve equation.Assessment of the total flavonoid content (TFC) was conducted spectrophotometrically as reported by Quettier-Deleu et al. [74] by mixing a 0.5 mL extract (1% w/v) with 1 mL of ethanolic aluminum chloride (2% w/v) (AL-Nasr Chemicals Co., Egypt) and measured after 10 min at 430 nm against the blank.The TFC was calculated as quercetin equivalents (mg/g) by employing the standardization curve equation.

Molecular Docking Simulations with Target Proteins
The docking experiments were carried out using the PyRx software version 0.9 [76].The inhibition of S. aureus tyrosyl-tRNA synthetase (PDB ID: 1JIJ) [77] and C. albicans secreted aspartic protease 2 (PDB ID: 3PVK) [78] by the eighteen detected compounds were assessed by evaluating their ligand-protein binding patterns and interactions with enzymes retrieved from the Protein Data Bank (http://www.rcsb.org/pdb(accessed on 20 June 2023)).The protein targets were prepared for docking by removing unnecessary water molecules.The active site for interactions was selected as the complex inhibitor ligand site.Further, the detected compounds were justified, and a virtual ligand database was generated.The docking scores were recorded by a rigid receptor-flexible ligand-docking procedure and 2D and 3D interaction figures were generated by BIOVIA Discovery Studio (v21.1.0.20298) [76,79].

Data Analysis
All experiments were carried out three times.Data were presented as the mean ± SD and established by analysis of variance (one-way ANOVA) using the SPSS software, version 16 (IBM, Armonk, NY, USA), as being below the 0.05 level of significance.

Antimicrobial Activity
As depicted in Table 2, the investigation into the anti-staphylococcal effects of diverse fungal extracts at a concentration of 100 mg/mL demonstrated notable efficacy against the tested S. aureus.Notably, among the S. aureus isolates, including ATCC 6538, AUHL123, and MLBM 117, the ethyl acetate (EtOAc) extracts derived from A. terreus AUMC 15447 and A. nidulans AUMC 15444 exhibited significant potency.For AUMC 15447, the inhibition zone diameters were 22.33 mm, 24.67 mm, and 23.00 mm, respectively, compared with the control.Similarly, for AUMC 15444, the inhibition zone diameters were 23.33 mm, 19.67 mm, and 20.33 mm, respectively, demonstrating substantial inhibitory activity compared with the control.On the contrary, some extracts exhibited diverse levels of activity, including instances of no inhibitory effects.3).In contrast, the remaining extracts displayed varying or no inhibition against the tested Candida species.It is noteworthy that certain fungal extracts exhibited antimicrobial efficacy against both Staphylococcus and Candida species, while others were effective against only one of these pathogens.

Determination of the Minimum Inhibitory Concentration (MIC) of Extracts
The MIC, minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) of the bioactive fungal extracts were assessed utilizing the microdilution assay.For S. aureus, the MIC and MBC were determined employing INT, and the results showed a great variation among the values in the range of the tested concentrations (100 to 0.39 mg/mL).Both A. terreus AUMC 15447 and A. nidulans AUMC 15444 extracts showed the most significant MIC values against Staphylococcus isolates in the range between 25 and 0.39 mg/mL, followed by extracts from T. harzianum AUMC 15440 and A. terreus AUMC 15448 (Table 4).It was noted that the obtained MFC values were represented by the concentrations preceding the MIC.The determined MIC and MFC of extracts against the Candida spp.revealed the high potency of the A. nidulans AUMC 15444 extracts, where the MIC range was between 3.125 and 0.39 mg/mL, while the MFC was in the range between 6.25 and 0.78 mg/mL (Table 5).

Antioxidative and Cytotoxic Activities of Extracts
The results illustrated that the lowest IC 50 value (0.47 mg/mL) in the DPPH antioxidant assay was afforded by A. terreus AUMC 15447 followed by that of the A. terreus AUMC 15448 EtOAc extract (0.58 mg/mL), while the highest IC 50 value (42.87 mg/mL) was scored by T. harzianum AUMC 15443.Regarding the Artemia cytotoxicity assay, the best (lowest LC 50 value) of 1.18 mg/mL was recorded by the T. harzianum AUMC 15440 extract followed by the A. terreus AUMC 15448 extract (1.29 mg/mL), whereas the highest LC 50 values (2.01 and 2.0 mg/mL) were presented by extracts from T. harzianum AUMC 15443 and A. terreus AUMC 15447, respectively (Table 6).

Molecular Identification of the of Most Potent Isolates
Identification of the most biopotent isolates was performed molecularly by sequencing the ITS region, and then sequences were subjected to a BLAST search of the NCBI database.The isolates were confirmed as Aspergillus nidulans AUMC 15444 (GenBank accession no.OR064351) and Aspergillus terreus AUMC 15447 (GenBank accession no.OR064355).Nucleotide comparison of the ITS regions among A. nidulans AUMC 15444 strain and other similar strains recaptured from the NCBI showed 99.62-99.81%identity and 99-100% coverage with several strains of the same species.Penicillium chrysogenum was included as an outgroup strain (Figure 1a).Meanwhile, A. terreus AUMC 15447 showed 99.67-100% identity and 98-100% coverage with numerous strains of the same species comprising the type strain A. terreus ATCC1012 with GenBank accession no.NR_131276.Aspergillus ochraceus was included as an outgroup strain (Figure 1b).

Evaluation of Total Phenolics and Flavonoids
The results proved that the A. terreus AUMC 15447 EtOAc extract had the highest content of phenolics (138.30mg/g) and flavonoids (72.09 mg/g), while the A. terreus AUMC 15448 EtOAc extract showed good phenolic and flavonoid content (116.54 and 53.60 mg/g, respectively).The lowest content of phenolics was noted in the extracts of P. novae-zeelandiae AUMC 15442 and T. harzianum AUMC 15443 (20.60 and 25.81 mg/g, respectively), and the lowest flavonoid content in A. aureolatus AUMC 15441 and P. novae-zeelandiae AUMC 15442 (17.43 and 18.48 mg/g, respectively) (Table 7).Data are presented as the mean ± SD, and values associated with superscripts differ significantly at p < 0.05.

Flavonoid and Phenolic HPLC Profile of Extracts
HPLC analysis for profiling the flavonoid and phenolic chemical constituents of EtOAc extracts of the derived fungal isolates was performed.Standards utilized in the analysis, according to their retention time, included chlorogenic acid, gallic acid, catechin, methyl gallate, caffeic acid, syringic acid, rutin, ellagic acid, p-coumaric acid, vanillin, cinnamic acid, ferulic acid, daidzein, naringenin, quercetin, apigenin, kaempferol, and hesperetin, as shown in Table 8 and Supplementary Figures S1-S9

In Silico Molecular Docking of Identified Compounds
According to the HPLC analysis, the diverse extracts were found to contain approximately 18 phenolic compounds, as detailed in Table 8.Docking studies were then conducted to assess the binding affinities of these compounds as potential antimicrobials against both S. aureus and C. albicans.The ranking of binding interactions for each compound with the respective target proteins was based on factors such as the lowest energy and lowest RMSD (Root-Mean-Square Deviation).It is noteworthy that a lower binding energy score indicates better stability in the protein-ligand binding interaction [80].

Molecular Docking Simulation against S. aureus Tyrosyl-tRNA Synthetase
The exploration of molecular simulation outcomes for the identified molecules against S. aureus tyrosyl-tRNA synthetase indicated diverse affinities for the enzyme, ranging from −16.43 kcal/mol (rutin) to −7.30 kcal/mol (cinnamic acid), suggesting their potential as inhibitors (Table 9).Rutin, chlorogenic acid, and ellagic acid emerged as the top-scoring compounds, exhibiting pose scores of −16.43 (RMSD = 1.32 Å), −13.94 (RMSD = 1.40 Å), and −13.89 (RMSD = 0.55 Å) kcal/mol, respectively.Notably, the highest-affinity compound, rutin, demonstrated hydrogen-bond interactions with Cys 37 and Asp 40 amino acid residues, as revealed in Figure 2. Additionally, the 2D and 3D interaction models illustrated the involvement of hydrophobic interactions with the Ala 39, Pro 53, and Phe 54 amino acid residues.Also, the interactions of rutin with the enzyme include an acidic interaction with Asp 40, 80, and 195 as well as basic interactions with the Lys 84 and Arg 88 amino acid residues.Other polar interactions for chlorogenic and ellagic acids are shown in Figures 3 and 4.       Concerning the binding affinity of the investigated compounds against the C. albicans secreted aspartic protease 2 (Table 9), the compounds rutin, ferulic acid, and kaempferol achieved the highest binding energy scores of −12.3505, −11.1575, and −10.9706 kcal/mol, respectively.The binding mode of rutin showed HB binding with the Asp 86, Asp 120, and Thr 222 amino acid residues (Figure 5).It is noteworthy that hydrophobic interactions are involved in the binding of rutin with the Ile 30, 119, 123, and Val 12 amino acid Concerning the binding affinity of the investigated compounds against the C. albicans secreted aspartic protease 2 (Table 9), the compounds rutin, ferulic acid, and kaempferol achieved the highest binding energy scores of −12.3505, −11.1575, and −10.9706 kcal/mol, respectively.The binding mode of rutin showed HB binding with the Asp 86, Asp 120, and Thr 222 amino acid residues (Figure 5).It is noteworthy that hydrophobic interactions are involved in the binding of rutin with the Ile 30, 119, 123, and Val 12 amino acid residues.Other polar interactions for ferulic acid and kaempferol are shown in Figures 6 and 7.

Discussion
Fungi are a prolific source of bio-effective secondary metabolites, including those with antimicrobial efficiency, which have been developed into significant pharmaceuticals.Endophytic fungi have been documented as having the capacity to generate novel antibacterial, antifungal, antiviral, anti-inflammatory, antitumor, and antimalarial compounds.These bioactive natural products are eliciting substantial interest from biologists and natural product chemists [81].Numerous diseases caused by S. aureus, ranging from mild skin infections to more serious and potentially fatal diseases, accompanied by the emergence of multi-resistant strains, have increased the demand for new antibacterial agents [53].Limited availability, in addition to the side effects of efficient antifungal agents, are some of the issues faced with C. albicans infections [82].Consequently, it is imperative to develop new drugs by exploring alternate sources such as terrestrial and

Discussion
Fungi are a prolific source of bio-effective secondary metabolites, including those with antimicrobial efficiency, which have been developed into significant pharmaceuticals.Endophytic fungi have been documented as having the capacity to generate novel antibacterial, antifungal, antiviral, anti-inflammatory, antitumor, and antimalarial compounds.These bioactive natural products are eliciting substantial interest from biologists and natural product chemists [81].Numerous diseases caused by S. aureus, ranging from mild skin infections to more serious and potentially fatal diseases, accompanied by the emergence of multi-resistant strains, have increased the demand for new antibacterial agents [53].Limited availability, in addition to the side effects of efficient antifungal agents, are some of the issues faced with C. albicans infections [82].Consequently, it is imperative to develop new drugs by exploring alternate sources such as terrestrial and marine environments for novel medications [17,83].
In the current investigation, nine EtOAc extracts were obtained from soil fungal isolates belonging to six different fungal species, namely T. harzianum, A. aureolatus, A. nidulans, A. terreus, P. crustosum, and P. novae-zeelandiae, which exerted anti-staphylococcal and anticandidal influences against tested species of Staphylococcus and Candida.The resultant anti-staphylococcal activities of the different fungal extracts at 100 mg/mL revealed that most of these extracts exhibited efficiency against the tested S. aureus species.Aspergillus terreus, A. nidulans, and T. harzianum extracts exhibited the highest activity, with MIC values ranging between 25 and 0.39 mg/mL.On the other hand, the A. nidulans extract revealed a high potency, with a MIC of 3.125-0.39mg/mL against Candida spp.The Aspergillus terreus MK-1 ethyl acetate extract showed antagonistic activity against S. aureus and C. albicans, with inhibition zones of 45 and 30 mm, respectively [84].Phupiewkham et al. [85] reported that T. harzianum TS3 and TS12 filtrates exert a positive control effect against S. aureus ATCC25923.A crude extract from T. harzianum exhibited the highest zone of inhibition of 26 mm against methicillin-resistant S. aureus [86].A Trichoderma harzianum extract had an MIC of 50 µg/mL against S. aureus [87].An Aspergillus nidulans ethyl acetate extract against an oral isolate of C. albicans (CA 09) showed a growth inhibitory effect, with an 18.4 mm diameter inhibition zone and an MIC value of 250 µg/mL [88].External secondary metabolites in an n-butanol extract from Aspergillus terreus var.africans exhibited a 22 and 19 mm diameter inhibition zone against C. albicans and C. glabrata, respectively [12].The ability of C. albicans to transform into a hyphal state and adhere to tissue surfaces is one of the main causes for its severe pathogenicity [88].
Staphylococcus aureus exerted resistance to gentamicin and chloramphenicol while presenting susceptibility to amoxicillin and vancomycin, with MICs of 0.1 and 0.015 mg/mL, respectively [89].Antibacterial agents such as ampicillin, amoxicillin, chloramphenicol, and cefotaxime had MIC values of 39, 39, 16, and 1 µg/mL, respectively, against S. aureus ATCC 11632 [90].However, ampicillin exhibited MICs of 2.5 and 10 µg/mL on S. aureus ATCC 25923 and S. aureus TN2A, respectively [52].Al Halteet et al. [65], in their study to determine the antifungal susceptibility of 24 Candida species, indicated that C. krusei MN419370 exhibited resistance to all the tested antifungal agents (fluconazole, voriconazole, caspofungin, micafungin, amphotericin B, and flucytosine), while C. krusei MN419388 was resistant only to fluconazole and flucytosine.Meanwhile, 2 out of 18 C. albicans strains showed resistance to all antifungal agents.On the other hand, three C. glabrata strains were susceptible to all tested antifungal agents.
The utilization of the microbroth dilution method using microtiter plates has emerged as the technique of choice for assessing drug susceptibility testing due to its low sample needs, affordability, and high throughput rate [91].The p-iodonitrotetrazolium (INT) was chosen as an indicator because of its preparation in ethanol that, as an antiseptic, makes it appropriate for reducing the contamination risk during test procedures [92].Violet INT is a tetrazolium dye precursor that, upon reduction, constitutes a purple formazan whose intensity is directly proportional to the number of live cells.The reduction of INT is due to the mitochondrial enzymatic activity within living cells [67].
According to the results, the A. terreus AUMC 15447 extract exhibited the highest phenolic content of 138.30 mg/g, a flavonoid content of 72.09 mg/g, and an IC 50 of 0.47 mg/mL by the anti-DPPH radical-scavenging assay, followed by the A. terreus AUMC 15448 extract.The ethyl acetate extract fraction from A. terreus LS01 showed an IC 50 of 19.91 µg/mL in the in vitro anti-DPPH radical-scavenging assay [93].A total phenolic content of 122.475 mg/g was detected in an A. terreus-18 EtOAc extract with the highest DPPH scavenging activity of 80.4% inhibition [94].Chandra and Arora [95] determined a phenolic content (20.4 and 16.7 mg/mL) and DPPH scavenging efficiency (86.8 and 65.8%) in A. terreus-1 and -2 soil isolates, respectively, after fermentation on sugarcane bagasse.Aspergillus nidulans ST22 showed total phenolic and flavonoid amounts of 0.1413 and 0.01162 mg/mL, respectively, and a 34.17% antioxidative capacity by a hydroxyl radical-scavenging assay [96].Goncalves and Pombeiro-Sponchiado [97] reported the ability of melanin from A. nidulans MEL1 to scavenge the oxidants HOCl and H 2 O 2 .Al-Askar [98] established total phenols of 53.30 µg/mL in a T. harzianum 1-SSR filtrate.Tavares et al. [99] evaluated a TPC of 8 mg/g and an EC 50 value of 25.41 µg/mL for free radical DPPH in an ethyl acetate extract of A. aureolatus CML2964 isolated from caves, while Ahmed and Al-Shamary [100] determined a phenolic content of 51 µg/mL in the soil A. niger B1b strain.In our results, T. harzianum AUMC 15440 recorded an LC 50 of 1.18 mg/mL in the brine-shrimp cytotoxicity assay, while the A. terreus AUMC 15448 extract showed an LC 50 of 1.29 mg/mL.The biotoxicity of the A. terreus var.africanus crude ethyl acetate extract showed a slight toxicity against Artemia salina (LC 50 = 1500 µg/mL) [101].
The most effective fungal isolates were subjected to phylogenetic analysis by ITS region sequencing, which molecularly identified them as A. nidulans AUMC 15444 and A. terreus AUMC 15447.The ITS regions represent important efficient markers for confirming fungal strain identification at the species level [102].Molecular identification of Aspergillus strains at the species level using ITS region sequencing was presented as an efficient substitute approach for their accurate identification [103].
HPLC analysis for the detection of phenolic compounds in fungal EtOAc extracts with reference standards showed great variation in the types and quantities of detected constituents among the fungal extracts.In the same context, Hassane et al. [10,11] detected a variety of phenolic and flavonoid compounds in fungal extracts belonging to different genera of Aspergillus, Penicillium, Chaetomium, and Rhizopus.Abdel-Wareth and Ghareeb [104] identified 22 phenolic and flavonoid compounds (cinnamic acid, 3,4,5-methoxy cinnamic acid, α-coumaric acid, p-coumaric acid, coumarin, pyrogallol, gallic acid, methyl gallate, ellagic acid, benzoic acid, 4-amino-benzoic acid, catechin, epicatechin, protocatechuic acid, vanillic acid, ferulic acid, isoferulic acid, chlorogenic acid, caffeic acid, resveratrol, and salicylic acid) in Penicillium implicatum and Aspergillus niveus extracts from fresh-water isolates by RP-HPLC/DAD analysis, where pyrogallol and e-vanillic were the major phenolics detected in the extracts, respectively.Abdel-Aty et al. [105] optimized the production of p-coumaric, apigenin, and kaempferol using SSF of Trichoderma reesei with a TPC of 30 mg/g.The diorcinol phenolic was detected and isolated from A. nidulans MCCC 3A00050 [106].Shevelev et al. [44] reported the in vivo efficacy of polyphenols, including resveratrol, dihydroquercetin, and dihydromyricetin, in the treatment of infected wounds with S. aureus ATCC 25923 and C. albicans NCTC 2625.Kepa et al. [107] demonstrated the promised efficiency of caffeic acid with MICs (256-1024 µg/mL) against MR S. aureus, clinical isolates, and reference strains derived from wound infections.
Moreover, advancements in computational chemistry have enabled the development of some fungal-derived anti-pathogenic compounds with potential therapeutic influences in controlling health-threatening microbes [53].The docking results of detected phenolics against the S. aureus tyrosyl-tRNA synthetase and the C. albicans secreted aspartic protease 2 found that rutin had the lower binding energy score, which conferred on it better proteinligand binding stability [108].Rutin and rutin-gentamicin act as pro-oxidants against Pseudomonas aeruginosa through oxidative stress by inducing reactive oxygen species generation, which leads to cell death [109].Docking of flavonoids-saltillin, taxifolin, and 6-methoxyflavone-from the A. nidulans chloroform extract displayed good binding interactions with the C. albicans growth regulator N-myristoyltransferase [88].
Naturally derived substances are a prime contender for treating microbial infections and restricting the spread of drug-resistant strains because of their capacity to interact with the microbial cell through a variety of antimicrobial processes [110,111].Phenolic acids and their derivatives showed antimicrobial effects by influencing the solubility of microbial membranes, rupturing the cellular membranes and allowing vital intracellular components to outflow, thus resulting in death of the microbial cell [23,112,113].The compounds' capacity to pass through the cytoplasmic membrane and acidify the cytoplasm is dependent on the number of hydroxyl, methoxy, and carboxyl groups in addition to the saturation state of the alkyl side chain [114].Lou et al. [115] evaluated p-coumaric acid's antimicrobial efficacy in triggering bacterial cell death by disturbing the cell membrane and the interaction with genomic DNA.Wu et al. [26] reported that flavonoids and their isomers represent a valuable anti-pathogenic mediator by targeting a variety of pathogens and suppressing virulence properties in drug-resistant microbial strains.Their capacity to impede microbial cell energy metabolism, nucleic acid synthesis, complex formation with the bacterial cell wall, and cytoplasmic membrane function, are the mechanisms linked to their bacteriostatic actions [25,116].

Conclusions
Ethyl acetate (EtOAc) extracts from the fungal isolates exhibited notable anti-staphylococcal and anti-candidal efficacy, with A. terreus AUMC 15447 and A. nidulans AUMC 15444 strains demonstrating particularly high efficiency, as determined through microdilution assays combined with INT for assessing antimicrobial efficacy.Additionally, the assayed extracts displayed considerable phenolic and flavonoid contents, along with significant IC 50 values for antioxidative activity and LC 50 values in Artemia cytotoxicity assays.HPLC analysis revealed the presence of various phenolics in different concentrations within the fungal EtOAc extracts.Molecular simulations of these detected phenolics highlighted the high affinity of rutin with the S. aureus tyrosyl-tRNA synthetase and the C. albicans secreted aspartic protease 2, indicating inhibitory properties.This research suggests the potential for bioprospecting anti-staphylococcal and anti-candidal bio-active metabolites from fungi, providing a sustainable source for safe medication.

2. 7 .
Phylogenetic Analysis of the Most Potent Isolates 2.7.1.Isolation of Genomic DNA Total DNA was extracted from the pure fungal culture, which had been cultivated in Czapeks' broth for 5 days at 28 • C, using the Norgen Plant/Fungi DNA Isolation Kit (Sigma, Thorold, ON, Canada) and preserved at −20 • C [71]. .

Table 1 .
Bioactive fungal species isolated from different soil samples.

Table 2 .
Anti-staphylococcal activity of the fungal extracts at a concentration of 100 mg/mL.Data are presented as the mean ± SD, and values associated with superscripts differ significantly, with p < 0.05.Regarding the anti-candidal effects of the fungal extracts, the EtOAc extract from A. nidulans AUMC 15444 stood out as the most effective, demonstrating inhibition zone diameters of 20.67 mm, 19.67 mm, 22.33 mm, 20.00 mm, and 24.00 mm against various tested Candida species.Additionally, T. harzianum AUMC 15440 showed significant activity against both C. albicans ATCC 10231 and C. albicans MLBM 73 compared with the control (Table

Table 3 .
Anti-candidal activity of the fungal extracts at a concentration of 100 mg/mL.
R, resistant.Data are represented as the mean ± SD, and values associated with superscripts differ significantly, with p < 0.05.

Table 4 .
Anti-staphylococcal activity assay and MIC and MBC (mg/mL) determination of the tested fungal extracts.

Table 5 .
Anti-candidal activity assay and MIC and MBC (mg/mL) determination of the tested fungal extracts.

Table 6 .
Antioxidative activities (IC 50 values) by the DPPH assay and the brine-shrimp lethality assay (LC 50 values) of ethyl acetate extracts from the investigated fungal isolates.
Data are presented as the mean ± SD, and values associated with superscripts differ significantly at p < 0.05.

Table 7 .
Total phenolic and flavonoid content of ethyl acetate extracts from the investigated fungal isolates.

Table 9 .
Pose score results of detected compound interactions with target proteins.

Table 9 .
Pose score results of detected compound interactions with target proteins.