Antimicrobial Activities of Fungi Derived from the Gorgonian Sea Fan Annella Sp

Indian Journal of Geo Marine Sciences Vol.45 (11), November 2016, pp. 1491-1498

Antimicrobial activities of fungi derived from the gorgonian sea fan Annella sp. & their metabolites

Sita Preedanon1,2, Souwalak Phongpaichit1*, Jariya Sakayaroj2, Vatcharin Rukachaisirikul3, Nanthaphong Khamthong3, Kongkiat Trisuwan4 & Sakanan Plathong5 1Department of Microbiology and Natural Products Research Center of Excellence, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand 2National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Rd., KhlongNueng, KhlongLuang, PathumThani 12120, Thailand 3Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand 4Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. 5Centre for Biodiversity of Peninsular Thailand, Department of Biology, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand *[E-mail: [email protected]]

Received 10 February 2013; revised 22 July 2013

One hundred and sixty-three fungi isolated from gorgonian sea fan Annella sp. were screened for antimicrobial activity against human pathogens. Forty-seven percents of the isolates produced antimicrobial metabolites against at least one pathogen with minimum inhibitory concentrations (MIC) ranged from 1 to 1,280 µg mL-1. Crude extracts from 16%, 21%, 18% and 23% of the isolates inhibited standard strain of Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli and Pseudomonas aeruginosa, respectively. Eighteen percents of the isolates displayed antifungal activity against Microsporum gypseum while only 3% inhibited Cryptoccocus neoformans and Candida albicans. Potential isolates were identified by morphological and molecular characteristics. Twenty-nine pure compounds isolated from ten isolates were evaluated for antimicrobial activity. Griseofulvin from Nigrospora sp. PSU-F13 exhibited the best activity against M. gypseum (MIC 2 µg mL-1). The result indicates that sea fan-derived fungi are a potential source of antimicrobial agents.

[Keywords: Sea fan-derived fungi, antibacterial, antifungal, molecular identification, griseofulvin]

Introduction samples. Our preliminary study revealed that this Infectious diseases caused by many resistant group of fungi was diverse and the crude extracts strains of bacteria and fungi pose an increasing from culture broths and cells exhibited threat to public health worldwide1-4. Therefore, antimicrobial activities. In connection with our there is an urgent need to find novel sources of ongoing research for antimicrobial substances antimicrobial drugs. Natural products from from fungi, more sea fan samples were collected marine microorganisms remain the most to obtain more sea fan-derived fungi and all were important sources for drug discovery. Marine screened for their ability to produce antibacterial environment can be a prolific resource for the and antifungal substances against potential human isolation of less exploited microorganisms5. pathogens. Fungi, that produced an inhibition Marine-derived fungi are a rich source of with low minimum inhibitory concentration structurally novel and biologically active (MIC) values, were then identified based on their metabolites6-7. Most of the studies have been from morphology and the analysis of the Internal fungi isolated from sediments or sponges6-10. Transcribed Spacer (ITS) of rDNA. Bioactive Recently, diversity and antimicrobial activities of compounds from some selected fungi were also fungi derived from gorgonians from the South isolated and identified. China Sea were reported11-12. Following the 2004 tsunami in southern Thailand, a high incidence of Materials and Methods an unknown epizootic infection was noted in the Sea fan samples were collected by scuba fallen gorgonian sea fan population13 and a divers at Mu KoSimilan National Park, PhangNga number of fungi were isolated from tissue province (8°39′ 09″ N 97°38′ 27″ E) and from the 1492 INDIAN J. MAR. SCI., VOL. 45, NO. 11, NOVEMBER 2016

coastline in SuratThani province (9°8′ 0″ N using a gradient system of ethyl acetate-light 99°19′ 54″ E) in southern Thailand. They were petroleum to yield dechlorogriseofulvin22-24 in 18.2 kept within sea water in a Ziploc bag and mg as a white solid. Structures of the isolated transferred to the Mycology Laboratory at Prince compounds were identified by spectroscopic of Songkla University, Thailand for fungal methods14-24. isolation. Sea fan samples were identified as The following potentially pathogenic test Annella sp. by C. Benzies and S. Plathong from strains were used: Staphylococcus aureus ATCC the Coral Reef and Benthos Research Unit, Center 25923 and a clinical isolate of methicillin- for Biodiversity of Peninsular Thailand (CBIPT), resistant Staphylococcus aureus (MRSA) SK1, as Department of Biology, Prince of Songkla representatives of Gram-positive bacteria, University, Thailand. Escherichia coli ATCC 25922 and Pseudomonas Twenty sea fan samples were collected from aeruginosa ATCC 27853, as Gram-negative Mu KoSimilan and six samples were from representatives, Candida albicans ATCC 90028 SuratThani. Twenty-four fragments of 1 cm2were and Cryptococcus neoformans ATCC 90112, as randomly cut from each sample. Each fragment representatives of yeasts, and a clinical isolate of was individually surface-sterilized in 70% ethanol Microsporum gypseum SH-MU-4, as a for 1 min and rinsed with sterile distilled water representative of a filamentous fungus. for another 1 min. Sample fragments were placed MICs of the BE, CH and CE crude extracts on sea water agar, corn meal agar and water agar against bacteria and yeasts were obtained using supplemented with 50 mg L-1 streptomycin and the agar microdilution method25. Briefly, fungal penicillin G antibiotics to suppress bacterial crude extracts dissolved in dimethyl sulfoxide growth and incubated at 25 oC until fungal (DMSO) were mixed with melted Mueller-Hinton outgrowth from the samples was observed. Pure agar for bacteria and Sabouraud dextrose agar for cultures were obtained from hyphal tip isolation. yeasts in the ratio of 1:10 to give the final Each fungal isolate was checked for purity and concentrations of 0.5-1,280 µg mL-1. 100 L of transferred to new media. All isolates were kept each concentration was dropped into a well on in 20% glycerol at -80oC. each of three 96-well microtiter plates. One Fungal strains were each inoculated into 300 microliter of bacterial or yeast suspension mL potato dextrose broth in a 500 mL Erlenmeyer containing approximately 104 colony-forming flask and incubated at room temperature for 3 units (CFU) was then dropped onto the agar plus weeks under stationary conditions. Broth culture fungal extract already in the wells. Plates were filtrates were extracted three times with ethyl incubated at 35oC for 16-24 h for bacteria and C. acetate (EtOAc), evaporated under vacuum to albicans and 48 h for C. neoformans. MICs were obtain the crude broth extracts (BE). Fungal recorded by reading the lowest fungal extract mycelia were extracted with methanol followed concentration that showed no growth. by hexane or EtOAc to obtain a crude hexane A microbroth dilution CLSI 38-A method26 extract of mycelia (CH) and a crude ethyl acetate was performed against M. gypseum. Equal extract of mycelia (CE) after evaporation. volumes of a suspension of conidia (4x103 Selected extracts were purified using conidia mL-1) were added to each test dilution to chromatographic techniques to obtain compounds make a final concentrations of 0.5-200 µg mL-1 in of various types. The purification procedures for triplicate. Plates were incubated at 25oC for 72 h. compounds obtained from the fungi PSU-F514, The lowest concentration of fungal extract that F1115, F1215, F1416, F1817, F2218, F4419, F10020 inhibited mycelial growth was recorded as MIC. and F13521 have been previously described. For DMSO was used as solvent control. the fungus PSU-F13, the BE extract (941.4 mg) Vancomycin and gentamicin were used as was subjected to Sephadex LH-20 column standard antibacterial agents and amphotericin B chromatography (CC) using methanol (MeOH) as and miconazole were used as standard antifungal an eluent to afford three fractions. The second agents. fraction (91.7 mg) was crystallized in 50% MeOH Selected fungi that produced significant in dichloromethane (CH2Cl2) to yield antimicrobial activity were identified based on griseofulvin22-23 in 21.0 mg as a white solid. morphological characteristics and the analysis of Filtrate was further purified by silica gel CC gel the DNA sequences of the ITS regions of their PREEDANON et al.: ANTIMICROBIAL ACTIVITIES OF FUNGI 1493

rRNA gene using primers ITS1/ITS4 and 2). Between 10 to 16% of the extracts were active ITS5/ITS427. A BLAST search was used to search against all four strains of bacteria and a fungus, for the most closely matched sequence in the M. gypseum, but only 2% inhibited the pathogenic GenBank database28. Fungal sequences from this yeasts C. albicans and C. neoformans. The ethyl study and other related sequences were multiply acetate broth extracts produced the most positive aligned using BioEdit 7.0.529 and the alignments samples (BE, 53%) followed by the ethyl acetate were adjusted manually where necessary to mycelial extracts (CE, 26%) and the hexane maximize alignments. Phylogenetic relationships mycelial extracts (CH, 21%). were estimated using PAUP* v4.0b1030. ITS The most potent extract from this study was sequences of these active fungi were submitted to the BE from Nigrospora sp. PSU-F13. It GenBank for retrieving their accession numbers. exhibited the strongest antifungal activity against M. gypseum with an MIC value comparable to the Results standard antifungal drug miconazole of only 1 µg A total of 163 fungal isolates were obtained mL-1. Nigrospora sp. PSU-F13 also exhibited a from 26 sea fan samples. Three hundred and broad spectrum activity against both Gram- thirty-seven crude extracts were tested against positive and Gram-negative bacteria and M. various human pathogens. Results are shown in gypseum whereas Fusarium sp. PSU-F135 Tables 1-2. Overall, 108 extracts (32%) from 77 inhibited all the tested fungi. In addition, isolates (47%) exhibited antimicrobial activity to Fusarium sp. PSU-F63 was most active against at least one test microorganism with MIC values strains of S. aureus (MIC 10-20 µg mL-1), that ranged from 1 to 1280 µg mL-1. For some Scolecobasidium sp. PSU-F77 against E. coli isolates, more than one extract was active (Table (MIC 40 µg mL-1), Annulohypoxylonnitens PSU-

Table 1–Antimicrobial activity and MIC ranges of crude extracts from sea fan-derived fungi. Test microorganism Sa MRSA Ec Pa Ca Cn Mg % Active extracts 16.6 12.6 10.6 13.9 2.0 2.3 14.9 (n=337) % Active isolates 21.5 16.0 17.8 22.7 3.1 3.1 17.8 (n=163) MIC (µg mL-1) 10-1280 8-320 40-1280 10-1280 80-640 20-640 1-1280 MIC, minimum inhibitory concentration; Sa, Staphylococcus aureus ATCC 25923; MRSA, methicillin-resistant Staphylococcus aureus SK1; Ec, Escherichia coli ATCC 25922; Pa, Pseudomonas aeruginosa ATCC 27853; Ca, Candida albicans ATCC 90028; Cn, Cryptococcus neoformans ATCC 90112; Mg, Microsporum gypseum SH-MU-4

F151 against P. aeruginosa (MIC 10 µg mL-1), Compounds from extracts of ten selected fungi Fusarium sp. PSU-F135 and Aspergillus which showed strong antimicrobial activity or aculeatus PSU-F78 against C. albicans (MIC 80 interesting MNR patterns were isolated and their µg mL-1) and Cladosporium sp. PSU-F22 against structures elucidated. However, only compounds C. neoformans (MIC 20 µg mL-1). with sufficient amounts were tested for Among 37 potential sea fan-derived fungi, 34 antimicrobial activity against some test strains. belonged to 14 genera in the phylum Ascomycota Results are shown in Table 3. Among 29 on the basis of morphological and molecular compounds tested, griseofulvin produced by characteristics: Nigrospora (7 isolates), Nigrospora sp. PSU-F13 exhibited the best Cladosporium (5), Fusarium/Gibberella (4), antifungal acitivity against M. gypseum with MIC Aspergillus (4), Scolecobasidium (3), of 2 µg mL-1 which was in the same range as its Trichoderma (3), Alternaria (1), crude extract. In addition, the fungus PSU-F13 Annulohypoxylon (1), Bipolaris (1), Curvularia produced dechlorogriseofulvin, another antifungal (1), Hypoxylon(1), Penicillium (1), Phialophora compounds (MIC 32 µg mL-1). The remaining (1) and Xylaria (1) as shown in Table 2. Three tested compounds were moderately to weakly isolates were unidentified. active (MIC 64-200 µg mL-1). 1494 INDIAN J. MAR. SCI., VOL. 45, NO. 11, NOVEMBER 2016

Discussion terrestrial fungi enter the marine environment by In this study we screened a large number of run-off from the land and there comes into contact sea fan-derived fungi for their ability to produce with sea fan colonies. antimicrobial metabolites. Almost half of the Various types of compounds were isolated isolates exhibited antimicrobial activity. from ten active sea fan-derived fungi including Furthermore, 36 active extracts had low MIC anthraquinone, cyclohexene, cyclopentanone, values (<100 µg mL-1). Surprisingly, the number cytochalasin, diketopiperazine, epoxydon, of active extracts against Gram-negative bacteria lactone, modiolide, naphthoquinone, phenol, and is in the same range as those active against Gram- pyrone derivatives. Among them, griseofulvin positive bacteria. Most of the reported marine- isolated from Nigrospora sp. PSU-F13 exhibited derived fungal metabolites were inhibitory the most potent antifungal activity against M. towards Gram-positive bacteria11, 31-32. The most gypseum. Griseofulvin is an antifungal drug that potent extracts having anti-E.coli and anti-P. has been used to treat tineas, skin fungal aeruginosa from this present study were the BE infections. It has been produced by Penicillium from Scolecobasidium sp. PSU-F77 (MIC 40 µg spp.35, Aspergillus oryzae36, Nigrospora oryzae37 mL-1) and CH from Annulohypoxylon nitens PSU- and Xylaria sp.38 Interestingly, most of the F151 (MIC 10 µg mL-1). This result indicates that extracts were more active than individual sea fan-derived fungi can be a good source of compounds. These results indicate the synergistic antibacterial substances in particular against effect of many constituents in the extract. Gram-negative bacteria. Therefore, partially purified extract may serve as A wide variety of fungal genera have been an important ingredient for drug development. isolated from the sea fan genus Annella sp. They Many novel antimicrobial and other bioactive belong to the Ascomycota, and mainly are metabolites were isolated from our sea fan- anamorphic and dematiaceous in nature. These derived fungi. This clearly confirms that sea fan- frequently found fungal genera have an airborne derived fungi are a source of possible and terrestrial origin which is in accordance with antimicrobial and novel compounds. other studies11-12, 33-34. It is possible that these

Table 2–Potential sea fan-derived fungi with strong antimicrobial activity Code Morphology Molecular identification Extract MIC (µg mL-1) PSU- (accession no.) Sa MRSA Ec Pa Ca Cn Mg F2 Nigrospora Nigrospora sp. BE ------128 (EU714382) F4 Bipolaris Pleosporales sp. BE ------128 (JQ026212) F5 NS Nigrospora sp. BE 64 128 (EF564154) CE - - - 160 - - - F6 Trichoderma Trichoderma aureoviride BE ------32 (EU714383) F7 Fusarium Gibberella sp. BE ------32 (EU714384) F11 Nigrospora ND BE ------64 F12 Nigrospora ND BE ------32 F13 Nigrospora Ascomycete sp. BE 64 64 128 128 - - 1 (JQ026214) F14 NS Fusarium sp. BE ------16 (EU714385) F18 NS Nigrospora sp. BE ------<8 (EU714386) PREEDANON et al.: ANTIMICROBIAL ACTIVITIES OF FUNGI 1495

Table 2–(continued) Code Morphology Molecular identification Extract MIC (µg mL-1) PSU- (accession no.) Sa MRSA Ec Pa Ca Cn Mg F22 NS Curvularia affinis BE ------32 (EU714387) F32 NS Cladosporium sp. BE ------32 (EU714388) F33 Aspergillus Aspergillus versicolor BE ------32 (EU191029) F36 Aspergillus Aspergillus versicolor BE 128 128 - - - - 32 (EU191030) F44 Penicillium ND BE 64 - 128 128 - - 8 F46 NS Unidentified BE ------16 ascomycete (EU714389) F54 Alternaria Alternaria sp. BE ------32 (EU714390) F63 NS Fusarium sp. CE 10 20 - - - - - (EU714391) F69 NS Scolecobasidium sp. BE 160 ------(EU714392) F76 Phialophora ND BE - - 160 - - - - F77 NS Scolecobasidium sp. BE - - 40 - - - - (EU714393) F78 Aspergillus aculeatus CE 80 - - - - - 20 (EU714394) CH - - - 80 80 - - F80 NS Xylariales sp. CH 160 160 - - - - - (EU714395) F95 Trichoderma Trichoderma aureoviride BE 80 80 - - - - - (EU714396) CE ------40 F97 Trichoderma ND BE 80 160 - - - - - CE - 160 - - - - 40 F98 Cladosporium Cladosporium CH - - - 80 - - - cladosporioides (EU714397) F100 NS Xylaria sp. CE 80 80 - - - - 40 (EU714398) CH ------40 F101 Cladosporium ND BE - - 160 - - - - CE 40 40 - 160 - 20 - F105 NS Nigrospora sp. CE - - - 160 - - - (EU714399) F118 NS Scolecobasidium sp. CE - - - 20 - - - (EU714400) F125 Cladosporium ND BE - - 160 - - - - F128 Cladosporium Cladosporium sp. CH - - - 80 - - - (EU714401)

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Table 2–(continued) Code Morphology Molecular identification Extract MIC (µg mL-1) PSU- (accession no.) Sa MRSA Ec Pa Ca Cn Mg F135 Fusarium Fusariumsp, BE 80 - - - 80 80 80 (EU714402) CE 80 80 - - - - - F151 ND Annulohypoxylon CH - - - 10 - - - nitens (EU714403) F154 Aspergillus ND BE 80 80 20 - - - CH - - 80 - - - - F156 Fusarium Fusarium sp. CE - 20 - - 160 - - (EU714404) F159 NS Hypoxylon sp. BE - - 80 - - - - (EU714405) Standard drug Vancomycin 0.5 1 Gentamicin 1 0.5 Amphotericin B 0.25 1 Miconazole 1 MIC, minimum inhibitory concentration; Sa, Staphylococcus aureus ATCC 25923; MRSA, methicillin-resistant Staphylococcus aureus SK1; Ec, Escherichia coli ATCC 25922; Pa, Pseudomonas aeruginosa ATCC 27853; Ca, Candida albicans ATCC 90028; Cn, Cryptococcus neoformans ATCC 90112; Mg, Microsporum gypseum SH-MU-4; BE, extract from culture broth with ethyl acetate; CE, extract from fungal cell with ethyl acetate; CH, extract from fungal cell with hexane; Bold letter, the best extract against each test microorganism; ND, not determined; NS, non-sporulating, -, no activity at 200 µg mL-1 (MIC >200 µg mL-1)

Table 3–Antimicrobial activities of pure compounds isolated from sea fan-derived fungi. Fungus Compounds MIC (µg mL-1) Sa MRSA Cn Mg Nigrospora sp. Nigrospoxydon A 64 >128 ND ND 14 PSU-F5 (+)-Epoxydon 128 >128 ND ND Nigrospora sp. Nigrosporanene A - - ND ND 15 PSU-F11 Nigrosporanene B - - ND ND Tyrosol - - ND ND Nigrospora sp. 4-Hydroxybenzoic acid - - ND ND 15 PSU-F12 Aplysiopsene D - - ND ND 3-Isochromanone - - ND ND (-)-Drimenin - - ND ND Diketopiperazine derivative - - ND ND Nigrospora sp. Griseofulvin ND ND ND 2 PSU-F13 Dechlorogriseofulvin ND ND ND 32 Fusarium sp. 9- 128 128 ND - PSU-F1416 Hydroxydihydrodesoxybostrycin Austrocortirubin - 128 ND - Nigrospora sp. Nigrosporapyrone A 128 128 ND ND PSU-F1817

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Table 3–(continued). Fungus Compounds MIC (µg mL-1) Sa MRSA Cn Mg Curvularia Curvularic acid - - ND ND affinis PSU-F2218 Stagonolide - - ND ND Penicillium sp. (+)-Brefeldin A ND ND ND 64 PSU-F4419 Xylaria sp. Xylarisin 200 200 ND ND PSU-F10020 (R)-(-)-Mellein methyl ether 200 200 ND ND (R)-(-)-5-Carboxymellein 200 200 ND ND (R)-(-)-5-Hydroxymethylmellein 200 200 ND ND 6-[(1R)-1-Hydroxypentyl]-4- 200 200 ND ND methoxy-2H-pyran-2-one (2E,4S)-2,4-Dimethyloct-2- 200 200 ND ND enoic acid Piliformic acid 200 200 ND ND Fusarium sp. Fusarnaphthoquinone B - - - 128 PSU-F13516 2,3-Dihydro-5-hydroxy-8- 128 200 - - methoxy-2,4-dimethylnaphtho- [1,2-b]furan-6,9-dione Fusarubin 128 - 64 64 Anhydrofusarubin 128 - - - MIC, minimum inhibitory concentration; Sa, Staphylococcus aureus ATCC 25923; MRSA, methicillin-resistant Staphylococcus aureus SK1; Cn, Cryptococcus neoformans ATCC 90112; Mg, Microsporum gypseum SH-MU-4; -, no activity at 200 g mL-1 (MIC > 200 g mL-1); ND, not determined because of insufficient amount

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