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Preliminary study of antibacterial activity from Indonesian marine-derived endophytic fungi.

INTRODUCTION

Marine natural products are known as sources of bioactive compounds. Recently, marine-related resources including plants, sponges, corals, alga and fungi have become the promising sources of biologically active products for developments new drugs [6][13]. One of the sources of novel new drugs are marine endophytic fungi [1]. Marine endophytic fungi live on the surface and inner healthy tissue of their hosts, such as marine tress, invertebrates [2][25]. Many marine fungal strains are isolated, screened, and reported to produce antibacterial metabolites belonging to alkaloids, terpenoids, steroids, phenolic compounds and other chemical structures [13][25].

The resistance drugs in human pathogenic bacteria have caused infectious diseases for people. The increase of antimicrobial resistance to antibiotics has led to a search for new therapeutic agents. Although the active constituents may occur in lower concentrations, endophytic fungi may be the better sources of new and effective antibacterial compounds than synthetic drugs to combat infectious diseases [6]. For example, the marine fungus Fasciatispora nypae had antimicrobial activity for Escherchia coli, Bacillus subtilis, Staphyloccous aureus, Candida albicans, Schizosaccharomyces pombe [24]. The seaweeds endophytic fungi Penicillium citrinum against pathogen bacteria Vibrio parahaemolyticus, V. cholera and Klebsiella sp [15]. Fungi Trichoderma sp, Fusarium sp, Alternaria alternata and Penicillium sp that isolated from seagrass, seaweeds and invertebrates have strong antimicrobial activity against ten human pathogenic microorganisms [18][19][25]. Fungi Aspergillus sp from coral reefs and sponges showed selective antibacterial activity against eight bacterial strains with MIC values between 1.25 and 20.0 [micro]M [8].

The marine endophytic microorganisms from marine plants and invertebrates has not been well studied and explored in Indonesia. Thus, the biodiversity of marine fungi and their antibacterial potential need to be studied. The researches goal were to isolate endophytic fungi from seaweeds, sea-worms, corals and sponges, to investigate their antibacterial activity against pathogen bacteria and to analyze phytochemical compounds of active metabolites and to identify the potential fungi.

MATERIALS AND METHODS

Marine organisms collection:

Specimens of marine sponge, seaweeds, sea-worm and coral reef were collected from Pameungpeuk Sea, Garut, West Java, Indonesia. Specimens were transferred directly to cooler bags containing seawater. Then, the specimens were processes immediately for isolation endophytic fungi in laboratory.

Marine endophytic fungi isolation:

Marine specimens were rinsed three times with sterile artificial seawater and disinfected with 70% ethanol. The seaweed and other invertebrates were cut aseptically into small pieces (2 x 2 cm) using a sterile dissection razor and plated on Potato Dextrose Agar (PDA) (Himedia) medium and Corn Meal Agar (CMA)(Oxoid) medium supplemented with 100 mg/L Streptomycin. The plates were incubated at room temperature for 1-2 weeks until the morphology of fungi could be distinguished. Then, each isolate was picked up and transferred into PDA medium. Pure cultures were maintenance in PDA slants for further study.

Selection of pathogenic bacteria:

Totally, seven pathogenic bacteria species were selected for antibacterial activity. Bacterial species such as, Escherchia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Shigella dysenteriae, Clostridium sporogenes, and Salmonella parathypi A were obtained from the laboratory of Biopharma, The Vaccine Manufacture, Bandung, Indonesia.

Fungi fermentation and extraction:

The endophytic fungus was cultivated on Potato Dextrose Agar plates at 25[degrees]C for 7-14 days. Three pieces (0.5 x 0.5 cm) of mycelia agar plugs were inoculated into 1L PDB and incubated at room temperature under agitation 150 rpm for 14 days. After this, the secondary metabolites of the fungus was separated from mycelia by vacuum filtration. The metabolites was extracted with ethyl acetate (EA extract) and ethanol (EtOH extract). Biomass was dried in oven and extracted with ethanol (Bio.EtOH extract). All the solutions were evaporated using rotary vacuum evaporator at 50[degrees]C. The crude extracts were tested for antibacterial activity and phytochemical screening.

In vitro antibacterial activity:

Amount of 20mg/mL of fungal extract were tested for antibacterial activity. Pathogenic bacteria were spread on Mueller Hinton Agar (Himedia) plates. Endophytic extracts (10 [micro]l) were pipetted onto sterile paper discs (6 mm diameter, Oxoid) and placed onto the surface of inoculated agar plates. Gentamicin sulphate (10 [micro]g, Oxoid) was used as the positive control. Plates were incubated at 37[degrees]C for 48 h [4]. Antibacterial activity of fungus extract was expressed as the diameter of the inhibition zone (mm) for three days observation.

Minimum Inhibitory Concentration(MIC) analysis:

The crude extracts that showed antimicrobial activity at 20 mg/mL were further assessed for obtaining their MICs using the same method. The concentrations of crude extracts were performed from 10mg/mL to 0.078 mg/mL. The lowest concentration of extract that inhibited growth of bacteria was recorded as the MIC. The strong MIC activities were recognized with concentration of extract < 10 [micro]g/ml [19].

Phytochemical analysis:

Phytochemical screening were performed to detect the presence of steroids, saponins, alkaloids, flavonoids, tannins, phenolic compounds, and terpenoids based on Kokate and Harborne method [5][7][22].

Molecular identification of fungi:

The DNA of selected fungi were conducted to identify based on the sequence of Internal Transcribe Spacer (ITS) regions as potential DNA barcodes for fungi [16]. DNA was isolated from mycelia and the ITS region was amplified by Polymerase Chain Reaction (PCR) with ITS1 and ITS4 primers. The PCR product was purified using the QIAquick PCR Purification kit (Qiagen). BigDye terminator cycle sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystem) was used and the product was purified with the AutoSEQ G-50 Kit (Qiagen). The sequence obtained analysed using Basic Local Alignment Search Tool nucleotide (BLAST)n homology search. ClustalX was used for sequence-alignment. Phylogenetic analysis was constructed using the Neighbour Joining (NJ) method with Kimura two parameters [9].

Results:

Marine endophytic fungi-associated with marine organisms:

A total of 12 endophytic fungi were isolated from seaweed (6 isolates), coral reefs (1 isolate), sea-worm (2 isolates) and sponge (3 isolates) using two medium. PDA medium and CMA medium obtained 9 (SF1, SF2, SF3, SwF4, SwF5, SweF7, SweF8, SweF10, SweF11, CrF12) and 3 (SweF6, SweF9, SweF10) fungi, respectively. All fungi showed various colony (Fig. 1).

[FIGURE 1 OMITTED]

Antibacterial activity of endophytic fungi:

All crude extracts of 12 endophytic were selected for screening of antimicrobial activity against human pathogens by disc diffusion method in three days observation. Totally, 3 microorganisms (SweF7, SweF8 and CrF11) did not produce antibacterial metabolites. The biomass of all fungi also did not produce antibacterial substances. The number of 9 potential microbes produced selective bioactive metabolites for pathogens from ethyl acetate and ethanol extracts. The moderate to strong antibacterial activity with diameter of zone inhibition [greater than or equal to] 18 mm were described in Table 1., performed in Fig 2. and conducted to obtain the MICs.

Table 1. described that six marine endophytic (SF1, SF3, SwF5, SweF9, SweF10 and SweF11) showed the strong antibacterial activity with diameter of inhibition zone about 18-25 mm and MICs range 20-0.078mg/mL. A number of extracts showed the permanent clear zones for three days. Some extracts could not sustain the clear zone towards the growth of bacteria on second and third day of observation. The marine fungi SweF9, SweF10 and SweF11 produced selective antibacterial substance for bacteria such as E. coli, C. sporogenes, and S. thypi A. The ethyl acetate and ethanol extracts of fungus SweF9 showed MICs for E.coli (0.078 mg/mL), C. sporogenes (0.313 mg/mL) and S.thypi A (0.625 mg/mL). The MICs of ethyl acetate of fungi SweF10 and SweF11 for S.thypi A and E.coli were 0.313 mg/mL respectively.

[FIGURE 2 OMITTED]

Phytochemical compounds of active extracts:

The active extracts were subjected to phytochemical screening. All ethyl acetate extracts from isolates SF1, SF3, SwF5, SweF9, SweF10 and SweF11 contained alkaloid and terpenoid. All the ethanol extracts of fungi obtained phenolic compounds. Some extracts contained the same phytochemical compounds as described in Table 2.

Molecular identification of selected endophytic fungi

The fungi SF1, SF3, SwF5, SweF9, SweF10 and SweF11 were the selected microorganisms for strong antibacterial activity. Based on macroscopic features, fungi SF3, SwF5, SweF9 and SweF11 were Aspergillus spp. Molecular identification showed that SF1 and SweF10 were Collectotrichum spp. The genetic relationships among ITS regions of marine fungi Aspergillus spp and Collectotrichum spp were shown in Fig 3 and Fig 4.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

In recent study, Aspergillus fungi were the most fungi associated with marine organisms. Fungi SweF9 and SweF11 were A. elegans with 92% and 97% identity, respectively. Fungi SF3 and SwF5 were closely related to A. nidulans, the similarity was 97%. Endophytic SF1 and SweF10 were closed related with Collectotrichum sp 3393 and C.inarcatum, with > 92% maximum identity.

Discussions:

Marine endophytic fungi have been isolated from sponge, sea-worm, coral reefs and seaweeds from Pameungpeuk Sea, Garut, West Java, Indonesia. A total of 12 isolated fungi grow well in PDA without adding NaCl. Some isolated fungi could not grow and isolate in PDA or CMA medium. As the marine organisms associated fungi come from marine environment, they required an alkaline pH and vitamins for their growth [10]. PDA plates could remove the adherent organisms from mycelia and store at 4[degrees]C for futher studies [14]. The pure cultures in PDA slants were used as working cultures and stock cultures.

In this study, marine fungi were obtained from sponge (3), sea-worm (2), seeweeds (7) and coral reefs (1). A total of 9 fungi produced antibacterial metabolites, but only 5 fungi (SF1, SF3, SwF5, Swe9F, SweF10 and SweF11) produced selective metabolites with strong activity. The strong activity of metabolites were shown with diameter of inhibition about 18-25 mm that sustain in three day observation. A inhibition zone of [greater than or equal to] 20 mm is described as the strong activity, and weak to moderate antibacterial activity have inhibition zone of [less than or equal to] 20 mm [11]. The fungi SF1 only showed moderate activity on B.subtilis with diameter zone 20 mm for three days and MICs was 20 mg/mL. Fungi SF3 described the moderate activity from ethyl acetate and ethanol extracts for E. coli, C. sporogenes, S. thypi A, S. aureus, B. subtilis and S. dysenteriae. The ability of ethanol extract was decreased to eliminate the growth of E. coli and S. dysenteriae for second and third day, it showed the clear zone was grown by the bacteria. Fungi SwF5 only obtained moderate activity on S. dysenteriae, and strong activity on B. subtilis and S. aureus with inhibition diameter 23-25 mm. Endophytic SweF9 showed moderate activity (E. coli, B.subtilis, C. sporogenes, S. thypi A) and strong activity (S. dysenteriae) with MICs range 2.5-0.078 mg/mL. The ethyl acetate of SweF10 and SweF11 could not sustain to eliminate E. coli, S. dysenteriae, S. thypi A and B.subtilis growth with MICs 5-0.313mg/mL. This research showed that marine fungi produced selective substances for pathogen bacteria only from ethanol and ethyl acetate extracts.

Ethanol extracts from biomassa of fungi fermentation did not produced antibacterial metabolites at all. A suitable solvent and partial extraction method for obtaining bioactive compounds are needed to explore in order to obtain the active and sensitive extracts [14]. Subsequently, culture conditions and incubation times are varied for fungal strain to produce bioactive compounds, and needed to further study [24].

Based on phytochemical screening, the active extracts of selected fungi contained steroid, saponin, phenolic compounds, terpenoid, alkaloids, tannin and flavonoid. Some researches show that antibacterial compounds contain major bioactive alkaloids, polyketides, terpenoids, isoprenoid and non-isoprenoid compounds, quinones, and phenol isolated from marine fungi [3][17]. Terpenoids and polyketides are the most purified antimicrobial secondary metabolites from endophytic, while flavonoids and lignans are rare [12]. The selected marine fungi mostly contained alkaloids, terpenoid and phenol compounds.

ITS sequence and phylogenetic tree supported by 1000 bootstrap replicates revealed fungi were highly similar to species Collectotrichum (SF1, SweF10), Aspergillus elegans (SweF9, SweF11) and A.nidulans (SF3, SwF5). The fungi were new strains for Collectotrichum and Aspergillus with same properties as sources of antibacterial agents. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of antimicrobial secondary metabolites between fungi (Mousa, 2013). Some studies show that Aspergillus clavutus, A. nidulans, A. sydowii, A. versicolor, A. granulosus isolated from sponge have antibacterial properties for E.coli, P.aeruginosa and S.aureus [9][10][23]. Aspergillus spp and Collectotrichum spp have been isolated from seaweeds Fucus seratus [26] and Turbinaria spp [20]. The metabolites have been well characterized into specific bacterial agents, such as desmethylnomifensine, circundatin, and methylaveratin [21]. Seaweed-associated microbes provide unique and novel metabolites of unprecedented structures, with antibacterial activities [18].

Present study shows Aspergillus nidulans strains, A. elegans strains, Collectotrichum sp and C. inarcatum strains are marine fungi associated with marine organisms. For the first time, fungi A. elegans and C. inarcatum reveal the secondary metabolites as new drug with broad spectrum for B. subtilis, S. aureus, and S. dysenteriae. The fungi also contain important compounds in pharmaceutical applications, such as alkaloid, terpenoid, and phenolic compounds. The compounds may present more novel bioactive natural products or potential molecules in future.

Conclusion:

Based on macroscopic features and ITS region rDNA gene analysis, six selected marine endophytic fungi were belong to Aspergillus spp and Collectotrichum spp. The fungi SF3, SwF5, SweF9 and SweF11 have an indication as a new strain of Aspergillus with < 98% maximum identity. While, endophytic SF1 and SweF10 were closely related to Collectotrichum sp and C. inarcatum with 93% and 97% maximum identity, respectively. Based on in vitro assayed, the selected fungi are potential to produce pharmaceutical compounds through new tools and techniques.

ARTICLE INFO

Article history:

Received 10 November 2015

Accepted 22 December 2015

Available online 30 December 2015

ACKNOWLEDGMENTS

We thank to Indonesia Institute of Sciences for facility provided and Dr. Zalinar Udin for the encouragement and guidance on this research. This research was supported by DIPA grant 2015.

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Yoice Srikandace and Desak Sri Gede Andayani

Indonesian Institute of Sciences, Research Center for Chemistry, Cisitu-Sangkuriang, Bandung 40135, Indonesia)

Corresponding Author: Yoice Srikandace, Indonesian Institute of Sciences, Research Center for Chemistry, Cisitu- Sangkuriang, Bandung 40135, Indonesia)
Table 1: Antibacterial activity and MICs value of active extracts

Fungi    Inhibition zone (mm):   Pathogen        Active    MICs
         day1/day2/day3          Bacteria        Extract   (mg/mL)

SF1      20/20/20                B.subtilis      EA        20
SF3      18/18/18                E.coli          EA        1.25
         20/20/20                C.sporogenes    EA        2.5
         20/20/20                S.thypi A       EA        1.25
         18/15/13                E.coli          EtOH      20
         18/18/18                S.aureus        EtOH      20
         20/20/20                B.subtilis      EtOH      10
         18/16/14                S.dysenteriae   EtOH      20
         20/20/20                S.thypi A       EtOH      20
SwF5     25/25/25                B.subtilis      EA        20
         23/23/23                S.aureus        EtOH      20
         25/25/25                B.subtilis      EtOH      20
         18/18/18                S.dysenteriae   EtOH      10
SweF9    20/18/18                E.coli          EA        0.078
         20/20/20                B.subtilis      EA        1.25
         20/20/20                C.sporogenes    EA        0.313
         20/18/18                E.coli          EtOH      1.25
         25/25/25                S.dysenteriae   EtOH      2.50
         20/20/20                C.sporogenes    EtOH      1.25
         18/18/10                S.thypi A       EtOH      0.625
SweF10   18/15/13                E.coli          EA        1.25
         18/13/10                S.dysenteriae   EA        5
         20/20/20                S.thypi A       EA        0.313
SweF11   20/15/13                E.coli          EA        0.313
         20/16/13                B.subtilis      EA        1.25
         18/12/10                S.dysenteriae   EA        1.25

Table 2: Phytochemical screening of active extracts

Fungi    Phytochemical compounds

         Ethyl acetate extract   Ethanol extract

SF1      Alkaloid, terpenoid,    Steroid, saponin
         flavonoid, saponin

SF3      Alkaloid, terpenoid,    Phenolic compounds,
         phenolic compounds      terpenoid, saponin,
                                 steroid

SwF5     Alkaloid, terpenoid,    Alkaloid, flavonoid,
         phenolic compounds,     phenolic compounds,
         tannin                  saponin

SweF9    Alkaloid, terpenoid,    Alkaloid, terpenoid,
         phenolic compounds      phenolic compounds,
                                 tannin

SweF10   Alkaloid, terpenoid,    Phenolic compound,
         tannin, saponin         tannin

SweF11   Alkaloid, terpenoid,    Terpenoid, tannin,
         phenolic compounds      saponin
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Author:Srikandace, Yoice; Andayani, Desak Sri Gede
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9INDO
Date:Dec 1, 2015
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