725 Mada Triandala (Antibacterial).cdr ANTIBACTERIAL ACTIVITY OF RED PIGMENT ISOLATED FROM COASTAL ENDOPHYTIC FUNGI AGAINST MULTI- DRUG RESISTANT BACTERIA 1,2 3 4 MADA TRIANDALA SIBERO , RITA SAHARA , NUR SYAFIQOH 3,5*and KUSTIARIYAH TARMAN 1Department of Coastal Resources Management, Graduate School, Diponegoro University, Semarang 50725, Indonesia 2Laboratory of Tropical Marine Biotechnology, Integrated Laboratory of Diponegoro University, Semarang 50269, Indonesia 3Department of Aquatic Products Technology, Faculty of Fisheries and Marine Science, Institut Pertanian Bogor, Bogor 16680, Indonesia 4 Department of Microbiology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Bogor 16680, Indonesia 5 Center for Coastal and Marine Resources Studies, Institut Pertanian Bogor, Bogor 16680, Indonesia Received 18 November 2016/Accepted 1 June 2017 ABSTRACT Multidrug-resistant (MDR) bacteria infections become a serious problem for these several decades. To solve this issue, finding of new antibiotics candidate in an urgency. Natural pigment is known to has biological activity against pathogenic bacteria. Coastal fungi are unexplored source of natural pigment to fight MDR bacteria. This research was aimed to isolate coastal endophytic fungi from smooth ant plant (Hydophytum formicarum), to screen endophytic fungi which produce red pigment, to extract the red pigment, to determine antibacterial activity of the red pigment and to identify the coastal endophytic fungi producing the red pigment. In this study, 7 fungi were isolated as endophytic fungi from H. formicarum. There were 3 isolates which produced extracellular pigment i.e. RS 1A which produced red pigment, RS 3 produced black pigment and RS 6A produced yellow pigment. Our study focused on red pigment which is produced by endophytic fungus strain RS 1A. The yield of red pigment was 8.8657% (w/w). This study showed that red pigment had antibacterial activity against Escherichia coli, Acinetobacter baumannii and Proteus mirabilis strain MDR. Judging from molecular and morphological identification, the endophytic fungus strain RS 1A was identified as Aspergillus versicolor. Keywords: Antibacterial, endophytic fungi, MDR, pigment INTRODUCTION Bacteria having resistance to several antibiotics used to treat the infections are referred to as Multidrug-Resistant (MDR) bacteria (Cornaglia 2009; Magiorakos et al. 2012). MDR is a serious problem for medical world and public health. Several bacteria already reported as MDR are Escherichia coli Staphylococcus Acinetobacter , spp., baumanni Proteus mirabilis and (Ahmed et al. 2015; Korytny et al. 2016; Panda et al. 2016). Therefore, it is urgent to find new antibiotics. As a megabiodiversity and maritime country, Indonesia has many coastal resources which are potential to be sources of new antibiotics against MDR bacteria. Among coastal resources, smooth ant plant ( ) is an neglected Hydnophytum formicarum source for new antibiotic candidate. H. formicarum is a member of myrmecophytes plant which provides nesting cavities for ants (Lok & Tan 2009; Defossez . 2009). This plant was et al found as epiphytic plant in sp. Avicennia (mangrove plant) in Sorong, Papua. The utilization of epiphytic plant as source of * Corresponding author: kustya@gmail.com BIOTROPIA 4 2 7 161 172 Vol. 2 No. , 201 : - DOI: 10.11598/btb.201 .2 . .7 4 2 725 161 bioact ive compounds i s h indered by environmental issues. Exploration of endophytic fungi is expected to overcome these issues. Information related to the biodiversity of endophytic fungi from is Hydnophytum formicarum rarely found. The first report about associated fungi from genus was reported in Hyndophytum 1911 (Defossez . 2009). Therefore, our study et al will add more information about endophytic fungi from .Hydnophytum formicarum Coastal endophytic fungi are commonly reported as potential source of bioactive compounds. However, there is rare report regarding its anti-multidrug resistant bacteria activity. Endophytic fungi refer to microfungi which live inside plant tissues without causing any symptoms to the host (Thirunavukkarasu . et al 2015; Sibero . 2016a). In addition, Kusari . et al et al (2013) stated that endophytic fungi could produce the same or similar bioactive compounds like its hosts. Endophytic fungi are known to produce metabolites with various biological activities et al et al et al (Meng . 2015; Wong . 2015; Cao . 2016; et al et alRahaweman . 2016; Sibero . 2016a). In contrary, fungal pigment is a neglected metabolite even though several researchers reported its bioactivity as antibacterial agent (Geweely 2011; et al et alMani . 2015; Patil . 2015). Our study was aimed to isolate coastal endophytic fungi from smooth ant plant ( . ), to screen H formicarum endophytic fungi which produce red pigment, to extract the red pigment, to determine antibacterial activity of the red pigment and to identify the coastal endophytic fungi producing the red pigment. MATERIALS AND METHODS Sampling Preparation Smooth ant plant (H. formicarum) was collected by Dr Kustiariyah Tarman, as epiphytic plant on Avicennia sp. in mangrove forest located in Sorong, West Papua Province (Fig. 1). The domatia part (hollow structure part) of smooth ant plant was collected by cutting the plant from the host plant using metal cutter. The plant was then put inside zipped plastic bag. The sample was taken to the Laborator y of Aquat ic Microorganisms, Department of Aquatic Products Technology, Faculty of Fisheries and Marine Science, Institut Pertanian Bogor for isolating the endophytic fungi. Fungal Isolation, Cultivation and Pigment Screening Endophytic fungal isolation was carried out using surface sterilization method (Kjer et al. 2010). H. formicarum was cut approximately 1 cm 2 Figure 1 Hydnophytum formicarum as epiphytic plant on Avicennia sp. (mangrove tree) found in mangrove forest in Sorong, West Papua Province 162 BIOTROPIA Vol. 24 No. 2, 2017 disk. The extracts concentrations were 50 µg/mL, 100 µg/mL, 250 µg/mL, 350 µg/mL and 500 µg/mL. Antimicrobial susceptibility disk for TM Amoxicillin (AML) 10 µg (Oxoid ) was used as positive control, while acetone was used as negative control. The bacteria turbidity standard was equivalent to a 0.5 McFarland. The bacteria were inoculated on Muller Hinton Agar (MHA) using cotton swab with rotation inoculation. After that, the positive control was placed in the middle, while the negative control and paper disks with extracts were placed around the positive control by forming a circular pattern and o incubated at 36 – 37 C for 24 h. The results of antibacterial assay were analyzed using factorial Analysis of Variance (ANOVA) with SPSS software. The confidence interval was 95%, while the significant difference was analyzed using Duncan test. Morphology Observation Slide culture method was performed for fungus cultivation with modifications (Qiu et al. 2005; Sibero et al. 2016b; Sibero et al. 2017). PDA media was prepared and cut approximately 2 × 2 cm and placed on sterilized object glass. The 2 mycelia were inoculated on each side of the PDA and a cover glass was placed on the PDA, then put into sterilized petri dish for incubation. After 3 days, each side of PDA had been overgrown by the fungus and the mycelia already covered the inner side of the cover and object glass. The PDA was then removed, while the object and cover glasses were observed under a compound microscope. Morphological characteristics of the fungus were compared to Huh et al. (2013), Visagie et al. (2014) and Ama (2016). Molecular Identification Chelex method with several modifications was used for DNA extraction (Sibero . 2017). The et al red pigmented fungus was cultured on PDA for 7 days. Mycelium of the red pigmented fungus was taken and put into Eppendorf microtube, added with 100 µL ddH O and 1, 000µL of 0.5% 2 saponin, then kept for overnight. After that, the mixture was centrifuged (12,000 rpm, 10 minutes o at 5 C). Supernatant were discarded, then 100 µL ddH O and 50 µL of 20% chelax 100 were added. 2 o The mixture was kept on water bath (80 C, 10 minutes). In the first 5 minutes, the mixture was and washed with distilled water followed by 70% ethanol (EtOH) for 1 minute and then re-washed with distilled water. After that, the pieces of samples were placed on Potato Dextrose Agar (PDA) without the addition of antibiotics and were incubated at 28 C until growth was initiated. o During isolation, a petri dish with PDA was left open as environmental control. After 7 days, there were several fungi growth on the PDA media. The fungus which grown nearby the sample but not found in environmental control petri dish was separated and placed into new PDA media as single colony. Each single colony was cultivated on PDA for 7 days at room temperature (27 – 28 C). Every day the color change of media o was observed. The fungus producing red pigment in PDA media was used for this research. Pigment Extraction Pigment was extracted using solid liquid extraction (Manikkam et al. 2015; Sibero et al. 2016b). PDA media were separated from the mycelia using sterilized metal cutter. The PDA media were then weighed and chopped until the segments became smaller and placed into Erlenmeyer flasks. A quantity of 100 mL acetone was poured into the flasks and shaken using shaker for 24 hours at 27 C. The contents of the o flasks were filtered through filter paper (Macherey-Nagel 640d·Ø 1125 mm). The filtrates were concentrated using rotary evaporator (30 – 33 C, 30 minutes). The yield was obtained o according to the following formula: Antibacterial Activity Antibacterial assay was conducted based on Sibero et al. (2016b) with several modifications. The pigment extracts were used to test against clinical MDR gram-negative bacteria, including Acinetobacter baumannii, Escherichia coli and Proteus mirabilis with two replications. These bacteria were clinical isolates and considered as MDR strains from RSUP Dr Kariadi (Dr Kariadi General Hospital Medical Center) and Rumah Sakit Nasional Diponegoro (Diponegoro National Hospital), both located in Semarang, Central Java Province, Indonesia. Gram negative bacteria were orefreshed on MacConkey for 24 hours at 37 C. Antibacterial assay was carried out using paper % Yield = x 100% Yield PDA weight 163 Antibacterial activity of red pigment isolated from coastal endophytic fungi – Sibero et al. mixed using vortex. The final mixture was o centrifuged (12,000 rpm, 10 minutes at 5 C). The o supernatant was taken and stored at -20 C. The Internal Transcribed Spacer (ITS) region of fungus were amplified by PCR using universal primers internal transcribed spacer (ITS) 1 (5'-TCC GTA GGT GAA CCT GCG G-3') as forward and ITS 4 (5'-TCC TCC GCT TAT TGA TAT GC-3') as reverse (Sibero . 2017). PCR mixture consisted of et al GoTaq Green Master Mix Promega (12.5 µL), primer ITS 1 (1 µL), primer ITS 4 (1 µL), DNA extract (0.5 µL) and ddH O (10 µL). The PCR 2 reaction condition consisted of preheat at 95 C for 3 o minutes, denaturation at 95 C for 1 minute, o annealing (gradient from 51.4 C to 67.4 C) for 1 o o minute, extension at 72 C for 1 minute, final o extention at 72 C for 7 minutes and holding o temperature at 16 C. Denaturation, annealing and o extension stages were performed for 30 cycles. The PCR product was loaded in agarose gel (1%) electro- phoresis. The product of electrophoresis was visualized by UVI Doc HD5 (UVITEC Cambridge). PCR product which performed clear DNA band was used for DNA sequencing. This stage was conducted at 1 Base Laboratories Sdn Bhd, st Malaysia. The sequence was compared and aligned by Basic Local Alignment Search Tool (BLAST), then analyzed to its homology to other fungi obtained from Gene Bank. This research used neighbor-joining for statistical method, bootstrap method for test of phylogeny with number of bootstrap replications was 1,000. The phylogenetic analysis was constructed using MEGA 7 software package (Tamura . 2011).et al RESULTS AND DISCUSSION Endophytic Fungi from . H formicarum There were seven fungi isolates which were successfully isolated from H. formicarum (Fig. 2). Each fungus had different macroscopic characteristics based on colony forms, mycelia colors and reverse media colors. Biodiversity of culturable fungi is influenced by nutrient content in media and isolation method (Kjer et al. 2010; Toma & Abdulla 2013). Surface sterilization is the most important procedure in endophytic fungal isolation. The aim of surface sterilization using ethanol 70% is to eliminate spores and other microbial contaminants attached to the sample surface (Kjer et al. 2010). Environmental control petri dish is also important to minimize the possibility of improper isolation. Fungi grown in environmental control petri dish and sample petri dishes were suspected as contaminant. From endophytic fungal isolation, there were seven fungal isolates obtained (Fig. 2). Each fungus ha s d i f f e r en t mor pho log i c a l characteristics. Among the seven endophytic fungi isolated, there were three fungal isolates produced extracellular pigment. They were RS 1A which produced red pigment, RS 3 produced black pigment and RS 6A produced yellow pigment. Pigment production was shown by the color change of the medium. Sibero et al. a(2016 ) successfully characterized black pigment from RS 3 as melanin. Figure 2 Endophytic fungal isolates obtained from Hydnophytum formicarum grown on PDA a) RS 1A; b) RS 1B; c) RS 2A; d) RS 2B; e) RS 3; f) RS 6A; g) RS 6B 164 BIOTROPIA Vol. 24 No. 2, 2017 a. c.b. d. e. g.f. Nutrient content in media has important role in fungal pigments. Mugesh et al. (2014) stimulated biopigment production of several endophytic fungi by diversifying growth media. As a result, the MECV01 fungus isolate produced red biopigment on Czapek-Dox Agar (CDA) and Czapek-Dox Yeast Autolysate Agar (CYA). On Malt Glucose Yeast Peptone Agar (MGYP) and Yeast Glucose Trace (YGT) media, the MECV01 fungus isolate produced yellowish red biopigment. Mugesh et al. (2014) stated that carbon source (sucrose) and trace elements gave impact to growth and pigment production. Our study focused on red pigment needed in many industries such as food, textile and cosmeceutical industries. Based on macroscopic observation, fungus RS 1A had green colony and o grew well at room temperature (28 C). Green color was produced by the colony, while the mycelia had white color. Fungus RS 1A produced extracellular red pigment since the fourth day of obeing on PDA media at room temperature (28 C). Production of red pigment increased and the pigment was released not only to the PDA media, but also to the mycelia (Fig. 3). Red pigment from this fungus was judged as extracellular pigment, because it was released outside the cells. Several fungi produced extracellular pigments and known to have biological activity (Dong & Yao 2012; Mani et al. 2015; a; Sibero et al. 2016b). Antibacterial Activity of Red Pigment Produced from Fungus RS 1A Extracellular pigment is extracted from media because it is spread outside the cells (Xiong et al. 2015; Akilandeswari & Pradeep 2016). Pigment production depends on several conditions, such as nutrient in media, light intensity, pH, trace elements, temperature and agitation (Mugesh et al. 2014; Bühler et al. 2015; Patil et al. 2015; Shi et al. 2015). This pigment was extracted directly from solid media and performed by maceration method with acetone. The yield of red pigment from fungus RS 1A was 8.8657 % (% w/w). Organic solvent such as methanol, acetone, chloroform and ethyl acetate are commonly used to extract natural pigment (Robinson et al. 2014; Vora et al. 2015). The pigment was tested against several MDR bacteria. Results of antibacterial assay are presented in Table 1 and Figure 4. Enterobacteriaceae members are known as gram negative pathogenic bacteria such as E. coli, Proteus, Salmonella and Shigella (Shaikh et al. 2015; Dutta et al. 2016; CLSI 2016). These pathogenic bacteria cause urinary tract infection, nosocomial infection, blood stream infection and meningitis, causing death to human (Harrish et al. 2015; Shaikh et al. 2015; Iqbal et al. 2016). Based on the result of antibacterial assay, red pigment had activity to combat clinical MDR gram-negative bacteria. The best antibacterial activity was performed at concentration 500 µg/mL against MDR E. coli with inhibition zone of 19.8±1.13 mm. We highlighted the diameter of inhibition zone of Amoxicillin against E. coli and P. mirabilis. Amoxicillin had inhibition zone ≤ 13 mm. Therefore, according to CLSI (2016) these bacteria were resistant to Amoxicillin. Wong et al. (2013) and Dutta et al. (2016) successfully isolated and characterized E. coli from hospital and P. mirabilis from chicken carcasses as MDR which Figure 3 Fungus RS 1A after ten days of cultivation on PDA 165 Antibacterial activity of red pigment isolated from coastal endophytic fungi – Sibero et al. Note: Data presented are mean±SD Values at the same column followed by the same letters are not significantly different at p < 0.05 Values with * shared letters denote significant difference at p < 0.05 Figure 4 Inhibition zones of red pigment produced from fungus RS 1A against MDR bacteria: (a) Acinetobacter baumannii; (b) Escherichia coli; (c) Proteus mirabilis MDR bacteria Concentration of red pigment extract (µg/mL) Inhibition zone (mm) Acinetobacter baumannii 50 4.0±0.42a 100 4.25±0.35a 250 4.6±0.00a 350 5.25±0.63ab 500 6.75±0.91ab Amoxicillin 10 µg 12.00±0.00c* Escherichia coli 50 5.80±0.28a 100 6.35±0.49a 250 7.05±0.49a 350 7.45±0.07a 500 19.8±1.13c* Amoxicillin 10 µg 10.60±0.00b Proteus mirabilis 50 5.15±2.1a 100 6.15±0.63ab 250 6.55±0.35ab 350 6.85±0.21ab 500 9.15±0.21b Amoxicillin 10 µg 12.00±0.00c* Table 1 Antibacterial activity from fungal red pigment RS 1A against MDR bacteria 166 BIOTROPIA Vol. 24 No. 2, 2017 resistant to Amoxicillin using CLSI standard. Somwanshi and Bodhankar (2015) reported that the endophytic fungi had antibacterial activity against MDR human pathogens such as Escherichia coli, Acinetobacter baumannii, Salmonella typhi and Klebsiella pneumoniae. On the other hand, Sibero et al. (2017) used marine fungi against E. coli strain MDR. In addition, Zhao et al. (2016) reported that Monascus pigment had activity against E. coli with MIC 2.5 mg/mL. Fungus Identification Fungus identification was carried out through molecular and microscopic observation approaches. For molecular identification, deter minat ion of opt imum anneal ing temperature was conducted at temperature range from 51.4 C to 67.4 C in PCR. o o Annealing temperature has important role on the success of PCR products. If the annealing temperature is too low, it will cause the amplification of non-specific DNA fragments. If the annealing temperature is too high, it will reduce the purity of PCR product (Rychlik et al. 1990). Figure 5 shows the result of PCR products visualization in agarose gel. DNA of fungus RS 1A was well amplified from 51.40 to 60.2 C. Temperature of 51.40 C o o showed the brightest band in visualization. The Figure 5 PCR products visualization from determination of annealing temperature Figure 6 A cladiogram resulted from neighbor-joining tree of fungus RS 1A obtained from the ITS rDNA sequences analysis Aspergillus oerlinghausensis NR 138362.1 Aspergillus waksmanii NR 135040.1 Aspergillus vitricola KT809136.1 Aspergillus vadensis AY585549.1 Aspergillus luppii NR 137477.1 Aspergillus polyporicola NR 137471.1 Aspergillus leporis NR 135327.1 Aspergillus pseudotamarii NR 135329.1 Aspergillus caelatus NR 135326.1 Aspergillus tamarii NR 135325.1 Aspergillus pulvericola NR 135453.1 Aspergillus occultus NR 135454.1 Aspergillus subramanianii NR 135385.1 Aspergillus salwaensis NR 135455.1 Aspergillus westlandensis NR 135451.1 RS IA Aspergillus versicolor NR 131277.1 Microascus verrucosus NR 132950.1 100 50 99 31 87 58 100 34 42 50 68 100 76 83 99 167 Antibacterial activity of red pigment isolated from coastal endophytic fungi – Sibero et al. annealing temperature of fungal DNA is varied and depended on the species, primers and PCR instrument. Other research successfully amplified fungal DNA with annealing temperature of 40, 47 and 54 (Choo et al. 2015; Kramer et al. 2016; o C Krishnan et al. 2016). According to the homology comparison, fungus RS 1A was closed to several strains of Aspergillus versicolor. This fungus had 99% nucleotide similarity to . strain NR A versicolor 131277.1 which was done by Haugland et al. (2016). Phylogenetic relationship of this fungus is shown in Figure 6. The result of microscopic morphological characterization of fungus RS 1A is shown in Figure 7. Based on the microscopic morphology characterization, fungus RS 1A had smooth conidiophore without any branches, biseriate phiallides and produced round conidia. This fungus produced unique Peniciliium -like conidiophore (Fig.7c). A. versicolor is known to produce Penicillium-like conidiophores. This conidiophore had phialides which attached to stipe. These conidiophores are vegetative hyphae with very short stipe. The production of conidia using Penicillium like-head was faster than the production of conidia by Aspergillus-head (Klich 1993; Ama 2016). The production of this unique conidiophore usually causes misidentification for morphological characterization. Ama (2016) successfully showed the production of Penicillium- like head started in the first day of incubation. A. versicolor has been reported as an endophytic and associatic fungi in plants and animals (Zhuang et al. 2011; Hawas et al. 2012). Hawas et al. (2012) isolated new metabolite name Isorhodoptilometrin-1-methyl ether from endophytic A. versicolor with antibacterial activity against Bacillus cereus, B. subtilis and Staphylococcus aureus. In 2013, a new alkaloid named Asperverin was isolated from an algicolous A. versicolor. In addition, A. versicolor has been reported as endophytic fungi in Paris polyphylla var. yunnanensis and produced new butyrolactones versicolactones E-F (Zhou et al. 2016). Yan et al. (2016) reported this fungus was isolated from the mud of deep water in South China Sea and had antioxidant property. Wang et al. (2017) discovered a new antimicrobial compound from deep sea sediment which was A. versicolor named 2-(dimethoxymethyl)- Figure 7 Microscopic morphology of fungus RS 1A identified as Aspergillus versicolor (Note: a. Mycelia; b. Conidiophore; c. Penicillium-like conidiophore with conidia) 168 BIOTROPIA Vol. 24 No. 2, 2017 a b c 1-hydroxyanthracene-9,10-dione. Several studies proved that A. versicolor produced pigments (Jurjevic et al. 2012; Ama 2016). Red pigment from A. versicolor has been discussed since 1960s. In 1967, Hamasaki et al. (1967) reported three anthraquinoid pigments with red color. Three years later, Hatsuda et al. (1969) isolated a new orange red pigment named versiconol (Fig. 8). CONCLUSIONS This research successfully isolated seven endophytic fungi from epiphytic plant H. formicarum, from which only fungus RS 1A produced the red pigment. 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