40 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Isolation and Identification of Aspergillus niger Causes Otomycosis and Treatment by Phenolic Extract of Agaricus bisporus Sarah Ali Hussein 1* and Rusol Mohammed AL Bahrani 2 1,2 Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. Corresponding author * Received: 4 July 2023 Accepted: 21 November 2023 Published: 20 July 2025 doi.org/10.30526/38.3.3644 Abstract This study aimed to isolate and characterize fungus Aspergillus niger and treated by crude phenolic extract from Agaricus bisporus mushroom that contains a bioactive compound to treat fungi. Thirty samples were taken of suffered patients with otomycosis. Phenol extract was performed by HPLC technology, Molecular diagnosis of fungal isolation by PCR technique. Results show that the microscopic and culture examination on sabouraud dextrose agar medium showed that the fungal isolates A. niger had a percentage of (60%). The molecular diagnosis of fungal isolates using the polymerase chain reaction technique. HPLC showed being present of six biologically active substances in A. bisporus. Active substance (phenols) was extracted from the fungus A. bisporus, and the concentrations were prepared (6.25, 12.5, 25 mg/ml) and then tested against A. niger fungus using the assessment of antifungal activity, the antifungal activity of the extracts and the inhibition of fungal growth were calculated individually. The outcomes demonstrated that the extract's inhibitory activity Phenolic against A. niger a concentration of 6.25 mg/ml showed lowest inhibition activity, the diameter of the colony was (1.63 ± 0.18cm), while the concentration of 25 mg/ml was the maximum of inhibitory activity, the diameter of the colony, was (1.23±0.03 cm) compared to the control (8.67 ± 0.33 cm). Keywords: Aspergillus niger, Otomycosis, Agaricus bisporus, Phenolic, HPLC technique 1. Introduction Otomycosis is a fungal infection of the external auditory canal that can be superficial, subacute, or chronic. It occasionally has consequences that affect the middle and inner ear. Prickling is the most common symptom of fungal-induced otomycosis (1). The most widespread variety of Aspergillus is Aspergillus niger. Aspergillus niger derives its name from the Latin aspergillum, meaning "holy water sprinkler," due to its resemblance to a sprinkler under a microscope. Black mold can frequently be caused by it. Additionally, it has milder opportunistic traits than other species, giving it the nickname "black mold," and it is more likely to cause pneumonia in impaired people. Some strains of Aspergillus niger produce mycotoxins (2). A capable microbial cell factory for the synthesis of organic acids is Aspergillus niger (3). The Aspergillus genus contains more than 340 distinct species (4). https://orcid.org/0009-0000-3762-6275 mailto:sara.ali1202a@sc.uobaghdad.edu.iq https://orcid.org/0009-0009-3314-4928 mailto:Rusol.jasim@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-3762-6275 mailto:sara.ali1202a@sc.uobaghdad.edu.iq https://orcid.org/0009-0009-3314-4928 mailto:Rusol.jasim@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-3762-6275 mailto:sara.ali1202a@sc.uobaghdad.edu.iq https://orcid.org/0009-0009-3314-4928 mailto:Rusol.jasim@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-3762-6275 mailto:sara.ali1202a@sc.uobaghdad.edu.iq https://orcid.org/0009-0009-3314-4928 mailto:Rusol.jasim@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-3762-6275 mailto:sara.ali1202a@sc.uobaghdad.edu.iq https://orcid.org/0009-0009-3314-4928 mailto:Rusol.jasim@sc.uobaghdad.edu.iq https://orcid.org/0009-0000-3762-6275 mailto:sara.ali1202a@sc.uobaghdad.edu.iq https://orcid.org/0009-0009-3314-4928 mailto:Rusol.jasim@sc.uobaghdad.edu.iq IHJPAS. 2025, 38(3) 41 Most instances of aspergillosis occur in immunocompromised patients (5). Extrapulmonary aspergillosis includes keratitis and sinusitis (6). These infections, however, are less serious and frequently treatable with antifungal medications. Here, we describe a rare instance of Aspergillus niger otomycosis-related left central tympanic membrane perforation. Citric acid has been manufactured industrially using the filamentous fungus Aspergillus niger; current output exceeds 2 million tonnes annually (7). Aspergillus niger is popular due to its simplicity in cultivation and tolerance of common industrial fermentation conditions (8). An ideal organism for use in industries. In addition to its current applications, Aspergillus niger also has the ability to create succinic and other important compounds (9) and contains significant quantities of oxalic and citric acids (10). Plants produce phenolic chemicals primarily to aid in their development, growth, and defense. During the interactions between the plant's biotic and abiotic stresses, these aromatic benzene ring molecules play a crucial role. They are a crucial component of the secondary metabolites found in plants and are crucial for a number of physiological and mechanical processes. They might serve as pathogen and insect repellents as well as attractants for helpful organisms (11). One of the most popular edible mushrooms worldwide, Agaricus bisporus has a long history of being beneficial for people's health. People commonly consume the mushroom as a food item. It is also known as a champignon, button mushroom, or white mushroom. Agaricus bisporus has a lot of metabolites and other biologically active substances, such as indole, phenolic compounds, fatty acids, sterols, statins, vitamins, trace elements, minerals, and simple sugars or carbohydrates (12). 2. Materials and Methods 2.1. Sample Collection Thirty samples of Aspergillus niger from different ages and genders were collected for the period between December 2022 and March 2023. The samples were cultured on SDA media for the purpose of isolation and the initial diagnosis of pathogenic fungi. 2.2. Identification of Aspergillus niger In this investigation, Aspergillus niger was identified based on its morphology (13). This identification relied on the subsequent: Colonial characteristics (color, significant pigment) and colony retreats (color, significant pigment). Microscopic structure (microconidia and macroconidia: their shape, arrangement, and hyphal structures). Multiple Lactophenol cotton blue was used to show spores made up of big, septate macroconidia and microscopic, single- celled microconidia on samples from distinct fungal development sites. 2.3. Preparation of Phenolic compounds Phenolic compounds were extracted using ultrasound from a homogenized plant sample (3 g) using an ethanol/water (70/30) solvent. The extraction process was placed for an hour at room temperature utilizing an Ultrasonic Bath (USA). 5 mL of the liquid extract was used to determine the extraction yield after filtering. In Slovenia, the solvent was extracted using a rotary evaporator while under vacuum, and it was then dried at 40°C to a consistent mass. In order to prevent oxidative damage until analysis, dry extracts were kept in the glass bottles at 4°C. By using a Zorbax Eclipse Plus-C18-OSD, Chemstation, and a SYKAMN HPLC chromatographic system equipped with a UV detector, reversed-phase HPLC analysis was used to quantify specific phenolic chemicals in a 4.6mm column, 25 cm. The gradient elution method used a column with an ambient temperature of 30 o C (14). IHJPAS. 2025, 38(3) 42 2.4. Determination of total phenolic compounds. Using a typical Folin-Ciocalteu reagent, the total amount of phenolic components in the ethanolic extract was calculated. 100 l of the extract, 500 l of the Folin-Ciocalteu reagent (Merck, Germany), and 1.5 ml of 20% sodium carbonate made up the reaction mixture. After being blended using a vortex mixer, the sample was finally diluted with distilled water to a final amount of 10 ml. After a two-hour process, the 765 nm absorbance was measured, and the phenolic content was estimated using a calibration curve created with gallic acid (Sigma- Aldrich, Germany). Gallic acid equivalent (GAE) was used to express the total amount of phenolic compounds per gram of dry weight (15). 2.5. Preparation of different concentrations of plant extracts Phenols extracts were prepared by dissolving a certain weight of each sample extract according to the concentrations (6.25, 12.5, 25) mg/ml of sample extracts that were prepared using the dilution equation following (16): (C1V1 = C2V2) 2.6. Determination of Minimal Inhibitory Concentration of Agaricus bisporus extract on isolated Otomycosis: The appropriate concentrations (6.25, 12.5, 25 mg/ml) of phenol extract were obtained by making different volumes, which were then separately combined with 100 ml of SDA (Sabouraud Dextrose Agar). For each concentration, three replicates were created. The extract and SDA combination is added to Petri plates and allowed to solidify while being kept sterile. millimeters of mycelial growth from a 7-day fungal culture were placed in the middle of each plate at 28°C, and the inoculated plates were incubated. fungal growths After seven days, measured diameters were taken and, each concentration's antifungal activity was determined by measuring the growth inhibition (17). 3. Results 3.1. Chromatography Laboratory HPLC The result of extracting the compounds included Agaricus bisporus mushroom phenolic compounds by HPLC technology as shown in Figure (1). Figure 1. The detection of the active compounds of Agaricus bisporus extract by the HPLC technology. IHJPAS. 2025, 38(3) 43 HPLC results showed the presence of six (6) biologically active compounds in Agaricus bisporus. Present phenolic compounds were estimated by HPLC analysis, and the antifungal characteristics of phenolic acids Several studies against fungi have also reported their presence, as well as human pathogenic fungi (18,19). The findings showed that Apigenin has the greatest concentration of total phenols. Major phenolic compounds were: Apigenin (62.15 ppm), Gallic acid (60.25 ppm), Caffeic acid (23.55ppm), Kaempferol (24.12 ppm), Ferulic acid (40.25 ppm) and Rutin (22.58 ppm), where total phenolic content (mg Gallic / 100 gm) (112.05) (Table 1). Table 1. Total phenolic content, Otomycosis was diagnosed, namely Aspergillus niger, with a percentage of (60%) as in Table (2). Table 2. Frequency and percentage of Otomycosis isolated from patients. Otomycosis No Percentage (%) Aspergillus niger 30 60.00 Aspergillus niger was the most prevalent fungus species isolate that causes Otomycosis globally (20, 21, 22). The most common appearance was Aspergillus niger (60%) (Figure 2). The current study showed that the gender factor affected the extent of infection (Table 3). The rate of infection for the age of ≤ 10 years is in Table (4). Table 3. Distribution of patients with Otomycosis according to gender. Otomycosis species No Male No. (%) Female No. (%) P-value Aspergillus niger 30 16 (53.33%) 14 (46.67%) 0.782 NS Table 4. Distribution of patients with Otomycosis according to age. Clinical type Age (year) 10-20 21-30 31-40 41-50 Total Aspergillus niger 3 10 10 7 30 A B Figure 2. Aspergillus species grew on SDA after seven days of incubation at 28±2°C. (A): Top view: Aspergillus niger. (B): microscopic feature of Aspergillus niger (40 X). Name Con ( ppm ) Name Con ( ppm ) Apigenin 62.15 Gallic acid 60.25 Caffeic acid 23.55 Kaempferol 24.12 Ferulic acid 40.25 Rutin 22.58 Total phenolic content ( mg Gallic / 100 gm ) 112.05 IHJPAS. 2025, 38(3) 44 3.2. .Identification of the fungi by PCR PCR (polymerase chain reaction) has been effective in detecting a wide range of fungi and could be an effective diagnostic tool. This outcome is comparable to those of earlier research on the identification of fungus using sequencing analysis of the ITS region, which provides faster, more accurate, and more reliable diagnosis at the species and subspecies levels compared to traditional laboratory methods (23) (Table 5 and Figure 3 ). Table 5. Ribosomal RNA gene(ITS). Gene : 5.8S ribosomal RNA gene(ITS) No. of sample Type of substitution Location Nucleotide Source Sequence ID with compare Identities 1 Transversion 310 G\T Aspergillus niger ID: MT582749.1 99% Transition 385 C\T Figure 3. PCR product the band size .The product was electrophoresis on 1.5% agarose at 5 volt/cm2. 1x TBE buffer for 1:30 hours. M: DNA ladder (100). The active substance (phenols) was extracted from the fungus Agaricus bisporus, and the concentrations were prepared (6.25, 12.5, 25 mg/ml) and then tested against the Aspergillus niger fungus using the evaluation of the extracts' antifungal activity and the calculation of the amount of fungal growth inhibition. The results demonstrated that the extract phenolic inhibitory activity against Aspergillus niger was lowest at a concentration of 6.25 mg/ml, with a diameter of inhibition of (1.63±0.18 cm), and highest at a concentration of 12.5 mg/ml, with a diameter of inhibition of (1.43 ± 0.07 cm), and highest at a concentration of 25 mg/ml, with a diameter of inhibition of (1.23 ± 0.03 cm), compared to the control (8.67 ±0.33 cm) (Table 6 and Figures 4 and 5). Table 6. Effect of Agaricus bisporus mushroom crude phenolic extract at different concentrations on fungal growth or colony on SDA at 28±2 °C. Concentration (mg/ml) Mean ± SE Fungal diameter(cm) of Aspergillus niger Control 8.67 ±0.33 a 6.25 mg/ml 1.63 ±0.18 b 12.5 mg/ml 1.43 ±0.07 b 25.00 mg/ml 1.23 ±0.03 b LSD 0.634 ** P-value 0.0001 ** (P≤0.01). https://www.ncbi.nlm.nih.gov/nucleotide/MT582749.1?report=genbank&log%24=nuclalign&blast_rank=1&RID=XHU9U9ND016 IHJPAS. 2025, 38(3) 45 Figure 4. The effect of crude phenol extract for Agaricus bisporus mushroom on Aspergillus niger growth. Figure 5. Effect of Agaricus bisporus mushroom phenolic extract at different concentrations on fungal growth or colony on SDA at 28±2 °C//Aspergillus niger. 4. Discussion Aspergillus niger is one of the most widespread species; they may flourish even in nutrient-poor conditions. To prevent the spread of spores, Aspergillus niger and other filamentous fungus must be handled carefully (24). Ear fungi can develop as a result of spore dust contamination of the ear canals. The quantity of proteins and carbohydrates, humidity, and temperature in the human auditory canal create an excellent habitat for the growth of these fungi (25). Immune-compromised individuals (26). The ear canal is thought to be primarily colonized by Aspergillus (27). According to our study, males are more likely than females to contract a fungus (53% of males and 46% of females, respectively). Risk factors include being exposed to tainted water, plucking at one's ears frequently, and persistent otitis media. It results in symptoms like tinnitus, hearing loss, otalgia, otorrhea, and fullness in the ears (28, 29). The edible fungus known as Agaricus bisporus, sometimes known as the white mushroom, is widely used throughout the world. Results showed the inhibitory activity of the phenol extract against Aspergillus niger, and a concentration of 25 mg/ml was most effective, as the diameter of the inhibition was (1.23 ± 0.03 cm). 5. Conclusion In this study, Aspergillus niger was identified as the most common agent of otomycosis. and accurate identification of Aspergillus niger by morphological characteristics. The results showed the inhibitory activity of the phenolic extract against Aspergillus niger and that a concentration of 25 mg/ml was most efficient. 6.25 mg/ml 12.50 mg/ml 25 mg/ml Control IHJPAS. 2025, 38(3) 46 Acknowledgment Many thanks to the Department of Biology, College of Education for Pure Sciences, Ibn-AL Haitham, University of Baghdadfor facilitating the work of the practice partsin thisarticle. Conflict of Interest The authors declare that they have no conflicts of interest. Funding No funding. References 1. Khan A, Jain SK. Fungal otomycosis in swimmers. Int J Life Sci Bioeng. 2019;6:1–8. 2. Mokobi F. Aspergillus niger—An overview [Internet]. 2021. 3. Laothanachareon T, Asin-Garcia E, Volkers RJM, Tamayo-Ramos JA, Martins dos Santos VAP, Schaap PJ. Identification of Aspergillus niger aquaporins involved in hydrogen peroxide signaling. J Fungi. 2023;9(4):499. https://doi.org/10.3390/jof9040499 4. Park HS, Jun SC, Han KH, Hong SB, Yu JH. Diversity, application, and synthetic biology of industrially important Aspergillus fungi. Adv Appl Microbiol. 2017;100:161–202. https://doi.org/10.1016/bs.aambs.2017.03.001. 5. Baddley JW, Thompson GR, Chen SC, White PL, Johnson MD, Nguyen MH, Hoenigl M, Warris A, Kontoyiannis DP, Armstrong-James D. Coronavirus disease 2019–associated invasive fungal infection. Open Forum Infect Dis. 2021;8(12):ofab510. 6. Arastehfar A, Carvalho A, van de Veerdonk FL, Jenks JD, Koehler P, Krause R, Cornely OA, Hoenigl M. COVID-19 associated pulmonary aspergillosis (CAPA)—from immunology to treatment. J Fungi. 2020;6(2):91. https://doi.org/10.3390/jof6020091. 7. Ciriminna R, Meneguzzo F, Delisi R, Pagliaro M. Citric acid: emerging applications of key biotechnology industrial product. Chem Cent J. 2017;11:22. https://doi.org/10.1186/s13065-017- 0248-3. 8. Meyer V, Wu B, Ram AF. Aspergillus as a multi purpose cell factory: current status and perspectives. Biotechnol Lett. 2011;33:469–476. https://doi.org/10.1007/s10529-010-0500-0. 9. Yang L, Henriksen MM, Hansen RS, Lübeck M, Vang J, Andersen JE, Lübeck PS. Metabolic engineering of Aspergillus niger via ribonucleoprotein based CRISPR–Cas9 system for succinic acid production from renewable biomass. Biotechnol Biofuels. 2020;13:124. https://doi.org/10.1186/s13068-020-01708-9. 10. Tian D, Wang L, Hu J, Zhang L, Zhou N, Xia J, Wu J, Wang Y. A study of P release from Fe P and Ca P via the organic acids secreted by Aspergillus niger. J Microbiol. 2021;59(9):819–826. https://doi.org/10.1007/s12275-021-1079-5 11. Pratyusha S. Phenolic compounds in plant development and defense. In: Plant Stress Physiology: Perspectives in Agriculture. 2022:125. 12. Muszyńska B, Kała K, Rojowski J, Grzywacz A, Opoka W. Composition and Biological Properties of Agaricus bisporus Fruiting Bodies- a Review. Polish J Food Nut Sci. 2017;67(3):173-81. https://doi.org/10.1515/pjfns-2016-0032. 13. Tille PM, Forbes BA. Bailey & Scott's diagnostic microbiology. 13th ed. St. Louis (MO): Elsevier; 2014. 14. Mladenović JD, Mašković PZ, Pavlović RM, Radovanović BC, Aćamović Đoković G, Cvijović MS. Antioxidant activity of ultrasonic extracts of leek Allium porrum L. Hem Ind. 2011;65(4):473–477. https://doi.org/10.2298/HEMIND1104473M. 15. Laouini SE, Ouahrani MR. Phytochemical screening, in vitro antioxidant and antibacterial activity of Rumex vesicarius L. extract. Sci Study Res Chem Chem Eng Biotechnol Food Ind. 2017;18(4):367–376. https://doi.org/10.3390/jof9040499 https://doi.org/10.1016/bs.aambs.2017.03.001 https://doi.org/10.3390/jof6020091 https://doi.org/10.1186/s13065-017-0248-3 https://doi.org/10.1186/s13065-017-0248-3 https://doi.org/10.1007/s10529-010-0500-0 https://doi.org/10.1186/s13068-020-01708-9 https://doi.org/10.1515/pjfns-2016-0032 https://doi.org/10.2298/HEMIND1104473M IHJPAS. 2025, 38(3) 47 16. Ayim TP, Yoriyo KP, Ayim P, Ayim JO, Ombu. Biosynthesis and characterisation of silver (Ag) nanoparticles using Tagetes erecta leaves and its larvicidal efficacy against Anopheles mosquitoes. 2023. https://doi.org/10.56892/bima.v7i01.391 17. Wang SY, Wu CL, Chu FH, Chien SC, Kuo YH, Shyur LF, Chang ST, Chu FH. Chemical composition and antifungal activity of essential oil isolated from Chamaecyparis formosensis Matsum wood. Holzforschung. 2005;59(3):295–299. https://doi.org/10.1515/HF.2005.049 18. Aguirre-Joya JA, Pastrana-Castro L, Nieto-Oropeza D, Ventura-Sobrevilla J, Rojas-Molina R, Aguilar CN. The physicochemical, antifungal and antioxidant properties of a mixed polyphenol- based bioactive film. Heliyon. 2018;4(12):e01047. https://doi.org/10.1016/j.heliyon.2018.e01047 19. Elansary HO, Szopa A, Kubica P, Ekiert H, Ali HM, Elshikh MS, Abdel-Salam EM, El-Esawi M, El-Ansary DO. Bioactivities of traditional medicinal plants in Alexandria. Evid Based Complement Alternat Med. 2018;2018:1463579. https://doi.org/10.1155/2018/1463579 20. Agarwal P, Devi LS. Otomycosis in a rural community attending a tertiary care hospital: assessment of risk factors and identification of fungal and bacterial agents. J Clin Diagn Res. 2017;11(6):DC14. https://doi.org/10.7860/JCDR/2017/26547.10135. 21. Aneja KR, Sharma C, Joshi R. Fungal infection of the ear: a common problem in the north eastern part of Haryana. Int J Pediatr Otorhinolaryngol. 2010;74(6):604–607. 22. Hagiwara S, Tamura T, Satoh K, Kamewada H, Nakano M, Shinden S, Kamata H, Wada N. The molecular identification and antifungal susceptibilities of Aspergillus species causing otomycosis in Tochigi, Japan. Mycopathologia. 2019;184:13–21. https://doi.org/10.1007/s11046-019-00333-y. 23. Naseif TS, Mohammed AJ, Abbas HS. Molecular identification of the dermatophytes causing tinea diseases using ITS sequencing analysis. Med Leg Update. 2020;20(4):256–260. 24. Schuster E, Dunn-Coleman N, Frisvad JC, Van Dijck PW. On the safety of Aspergillus niger – a review. Appl Microbiol Biotechnol. 2002;59:426–435. 25. Barati B, Okhovvat SA, Goljanian A, Omrani M. Otomycosis in central Iran: a clinical and mycological study. Iran Red Crescent Med J. 2011;13(12):873–876. 26. Viswanatha B, Sumatha D, Vijayashree MS. Otomycosis in immunocompetent and immunocompromised patients: comparative study and literature review. Ear Nose Throat J. 2012;91(3):114–121. 27. Jia X, Liang Q, Chi F, Cao W. Otomycosis in Shanghai: aetiology, clinical features and therapy. Mycoses. 2012;55(5):404–409. https://doi.org/10.1111/j.1439-0507.2012.02216.x. 28. Chen Q, Chu H, Tao Y, Peng L, Zhou L, Liu L, Wang Y, Zhao J. A comparison of triamcinolone acetonide econazole cream and nystatin suspension in treatment of otomycosis. Laryngoscope. 2021;131(5):E1640–E1646. https://doi.org/10.1002/lary.29224. 29. Zhou ZQ, Xiao J, Fan HX, Yu Y, He RR, Feng XL, Liu Y, Zhang L. Polyphenols from wolfberry and their bioactivities. Food Chem. 2017;214:644–654. https://doi.org/10.1016/j.foodchem.2016.07.091. https://doi.org/10.56892/bima.v7i01.391 https://doi.org/10.1515/HF.2005.049 https://doi.org/10.1016/j.heliyon.2018.e01047 https://doi.org/10.1155/2018/1463579 https://doi.org/10.7860/JCDR/2017/26547.10135 https://doi.org/10.1007/s11046-019-00333-y https://doi.org/10.1111/j.1439-0507.2012.02216.x https://doi.org/10.1002/lary.29224 https://doi.org/10.1016/j.foodchem.2016.07.091