Antibacterial Activity of Semi Purified Extract of Marine-Derived Trichoderma reesei PDSP 5.7 using Bioguided Fractionation Method

Bioguided fractionation method is commonly used to obtained targeted fraction with certain biological activity. In this study, bioguided fractionation method using HPLC was applied to obtained antibacterial fractions from marine-derived fungus Trichoderma reesei PDSP 5.7. The result shows that fraction from mycelium and medium extract had antibacterial activity against ESBL E. coli and S. enterica ser. Typhi with a range of inhibition zone was 1.35±0.15 to 8.82±0.22 mm 2 . From 32 fractions of each extract, the mycelial extract had 7 active fractions, 3 active fractions from broth medium which extracted using 1-butanol and 5 active fractions from ethyl acetate extract. This study indicated that crude extract of fungus T. reesei PDSP 5.7 was more potential as the source of antibacterial agents rather than the crude extract that obtained from its broth medium.


INTRODUCTION
Trichoderma is reported as an outstanding genus regarding to its ability to adapt in various ecological condition. This fungus commonly isolated from terrestrial and marine environment (Korkmaz et al., 2017;Awad et al., 2018;Greco et al., 2018;Sibero et al., 2018). Several of them were reported from extreme environment. Sing et al. (2018) isolated Trichoderma velutinum from cold habitat soil within temperature range of -5 to 2 o C while three species of Trichoderma were isolated from heavy metal polluted soil (Tansengco et al.,2018). With this wide niche, Trichoderma has been widely studied as source of natural products. A total of 578 compounds have been reported from Trichoderma with various biological activities (DNP, 2020). Several important chemical substances such as cyclopentenone derivatives, diterpenes, polyketides, and polypeptide were produced by this genus (Pang et al., 2017;Yamada et al., 2017;Zhou et al., 2017;Singh et al., 2018).Among all type of bioactive compounds that have been reported, Trichoderma is well known as a potential producer of polypeptide such as peptaibols.Peptaibols is defined as a group of bioactive peptides containing 5 to 20 unusual amino acids, which synthesized by a nonribosomal biosynthesis pathway (Szekereset al.,2005;Daniel and Filho, 2007). Plenty studies reported antibacterial properties of these peptides. Three new peptaibols were isolated from T. arundinaceum MSX70741 by Rivera-Chávezet al. (2017). In early 2018, Singh et al. (2018) published four novel lipovelutibols (A-D)from T. velutinum containing six amino acids with leucinol at the C-terminus and a fatty acyl moiety (n-octanoyl) at its N-terminus. Several compounds from Trichoderma were performed antibacterial activity against pathogenic microorganisms. Khamthong et al. (2012) isolated coniothranthraquinone 1 and emodin from T. aureoviride PSU-F95. These compounds were reported as a potential antibacterial agent to inhibit methicillin-resistant S. aureus (MRSA) with MIC values of 8 and 4 μg/mL. Moreover, five new fungal polyketide and two known analogs were isolated from T. koningiopsis QA-3 by Shi et al. (2017). They mentioned that among seven compounds, 1,6-di-epi-koninginin A and trichoketide A showed antibacterial activity against human pathogen E. coli. Adelin et al.
(2017) isolated a new antibiotic compound named EA-2081 was isolated from T. atroviridae with MIC values 32 to 128 μg/mL against human pathogens strain ATCC.
Production of antimicrobial compounds by Trichoderma has a high possibility of beingapplied in the antibiotic industry. However, the human pathogen has evolved to resistant to several groups of antibiotics, known as multidrugresistant organisms (MDRO). Every year, the infections caused by MDR bacteria areincreasing significantly due to the overuse of antimicrobial agents (Munita and Arias 2016). However, most of the antibiotic properties of bioactive compounds from Trichoderma were tested against ATCC bacteria. Therefore, it is interesting tostudy antibacterial compounds to inhibit MDR bacterial of Trichoderma.
Microorganisms produce secondary metabolites as a response to environmental stresses and threats. Ecologically, spongeassociated microorganisms produce bioactive compounds to protect themselves and its host from pathogens or predators (Taylor et al., 2007;Brinkmann et al., 2017). Moreover, as a filter feeder animal and it is continuously exposed to an array of microorganisms from the sea water, sponge obtains both of threat and opportunity for surviving. Sea water could bring harmful microorganisms such as biofilm forming bacteria and pathogenic bacteria. On the other hand, it also contains other microorganisms which may contribute to establishing symbiotic relationship to overcome the threat from harmful microbes. Selvin (2009) proved this theory by reporting the role of Streptomyces MSI051 in the protection of host sponge Dendrilla nigra against fouling processes. Streptomyces MSI051 showed strong inhibition against biofilm-forming bacteria Pseudoalteromonas sp. and Thalassomonas sp. The ability of sponge-associated microorganisms to produce antibacterial compounds to protect themselves and the host has been applied to produce antimicrobial compounds to inhibit human pathogen (Siberoet al., 2016). As a potential source of antimicrobial compounds, sponge-associated microorganisms such as fungi is a very important to be explored to find new candidate of antibiotic against MDR bacteria (Sibero et al., 2019).
Indonesia has broadmarine environment as an origin of potential sponge associated fungi. Due its potential, sponge-associated Trichoderma has been explored as an antibiotic compounds producer. As a result, several species of marinederived Trichoderma have been reported from Indonesian marine sponges (Sibero et al.,, 2016;Sibero et al., 2017;Trianto et al., 2018). In addition, the previous study showed the ability of crude extract from sponge-associated fungus T. reesei PDSP 5.7 inhibited clinical pathogenic multidrug resistant extended spectrum βlactamase (ESBL) E. coli, MRSA, S. enterica ser. Typhi, and S. haemoliticus.The crude extract had widest antibacterial against S. enterica ser. Typhi then followed by ESBL E. coli after cultivated for 15 days. The crude extract had wider inhibition zone than Amoxicillin+Clavulanic acid against S. enterica ser. Typhi (Sibero et al., 2018). It shows that marine derived Trichoderma is a potential source of new antibiotic agent against MDR bacteria. Our current study was aimed to obtain and characterize the fractions with antibacterial activity against clinical pathogenic bacteria ESBL E. coli and S. enterica ser. Typhi strain MDR using bioguided fractionation method.

MATERIAL AND METHODS
This research utilized marine derived Trichoderma reesei PDSP 5.7 with accession number MG547722.1. This fungus was isolated from sponge Cinachyrella sp. PPD.SP.05 which collected from Pandang Island, North Sumatra (Sibero et al., 2018). Fungus was revived on Malt Extract Agar (MEA) HiMedia for 7 days and incubated at 25 o C.
Clinical pathogenic ESBL Escherichia coli, and Salmonella enterica ser. Typhi strain multidrug-resistant (MDR) wasobtained from General Hospital Dr. Kariadi, Semarang, Central Java. These bacteria were isolated from the patient and confirmed as MDR.
Fungus T. reesei PDSP 5.7 was recultivated on Malt Extract Broth (MEB) Difco TM for 15 days in static condition (stand culture) at room temperature (25 o C). After cultivation, fungal mycelium and broth was separated using filter paper (Advantec Ø 125 mm). Mycelium wasdried at 37 o C then extracted using 1-butanol. Fungal metabolites in broth were extracted using a single extraction method with 1-butanol (Wako, Japan)and ethyl acetate (Wako, Japan). Solvent was added into fungal broth (2:1) then agitated using a shaker (200 rpm) for 1 h after that it was separated by orbital centrifugation (6000G, 15 min, 4 o C). Solvent was evaporated at 35 o C.
For the antibacterial assay, each fraction was dissolved in 50 µL DMSO then sonicated for 1 min. MDR clinical pathogenic ESBL Escherichia coli and Salmonella enterica ser. Typhi were refreshed on MacConkey HiMedia for 24 h before assay. For the assay, pathogens were diluted to be 0.5 McFarland in physiological saline solution, then mixed in Meuller Hinton Agar (MHA) Difco TM and poured into a square disc. Approximately 10 µL of each fraction was injected into a paper disc (Advantec Ø 6 mm), then placed onto MHA and incubated for 24 h at 32 o C. The presence of inhibition zone indicated the antibacterial activity.
Data of antibacterial activity were analyzed statistically using PASW Statistic software package for Windows with confidence interval 95% (P<0.05).

RESULT AND DISCUSSION
The microbial secondary metabolite could be secreted to the environment (extracellular metabolites) or kept inside the cell (intracellular metabolites). Most of the extracellular metabolites were produced to protect the producer (Arumugam et al., 2014). This study extracted fungal intracellular metabolites from mycelium and extracellular metabolites from the broth medium.Fungus Trichoderma reesei PDSP 5.7 has been studied to has antibacterial activity against several multidrug-resistant bacteria. Crude extract from this fungus performed the strongest antibacterial activity against S. enterica ser. Typhi with inhibition zone value was 14.72±0.07 mm 2 ,while Amoxicillin + Clavulanic acid had 12.20±0.28 mm 2 (Sibero et al., 2018). In this study, fungal secondary metabolites were extracted from broth medium using 1-butanol and ethyl acetate, while mycelial bioactive was extracted using 1-butanol. We tried to figure out the profile of fungal extract from mycelium and broth in different solvents. However, the application of crude extract for drug development is not suggested, so the isolation of single antibacterial compounds is highly recommended. To obtain pure compound with antibacterial property, bioguided fractionation and isolation method is commonly applied (Lo et al., 2004;Pieters dan Vlietinck 2005). This research performed bioguided fractionation using HPLC. The crude extract was fractionated to 32 fractions based on the retention times. Each fraction was tested against ESBL E. coli and S. enterica ser. Typhi strain MDR. The result of the antibacterial assay is presented in Table 1.
In Table 1, fractions from mycelial extract showed antibacterial activity against the tested pathogenic bacteria. ESBL E. coli was inhibited by fr. 20, 21, and 22 with widest inhibition zone was exhibited by fr. 21 (8.00±0.00 mm) mm while S. enterica ser. Typhi was inhibited by fr. 18, 19, 20, 21, 22, 23 and 25 with the widest inhibition zone performed also by fr. 21 (8.82±0.22 mm). Fungal extract from broth medium which extracted using 1-butanol had active fractions at retention time 20, 21 and 22 against both of tested bacteria. The strongest antibacterial activity was performed by fr. 21 with inhibition zone 6.05±0.05 mm against ESBL E. coli and 8.95±0.05 mm against S. enterica ser.   According to Figure 2, the mycelial extract of T. reesei PDSP 5.7 had 6 active fractions with 10 major peaks. In fractions 18, 19, 22, 23 and 25 we found one peak for each, while fraction 20 contained 3 peaks and fraction 21 had 2 peaks.
Fractions 20 and 21 showed stronger antibacterial activity rather than other fractions. We highlighted it with two hypotheses whether it was caused by synergic effect of compounds in each fraction or there was a major compound with great antibacterial activity. Moreover, Figure 2 shows that peaks in fraction 21b (Figure 2) had 1200 mAU which considered as main antibacterial compounds. In addition, it also needs to emphasize that a single peak does not indicate a single compound in the fraction. Figure 2 shows most of the peaks have a similar pattern. Although it had a similar UV pattern, it does not indicate the same compound in the fractions. Several compounds such as chrysophanol, pachybasin, sorbicillin andergosterolwere reported as mycelial intracellular metabolite from Trichoderma (Tarus    et al., 2003;Reino et al., 2008).Further, the inhibition zones, HPLC chromatogram and UV spectrum of active fractions from medium extract using 1-butanol are shown by Figure 3-4 while medium extract using ethyl acetate are shown by Figure 5-6. The result showed that both of mycelia and medium extract contained antimicrobial compounds that inhibited ESBL E. coli and S. enterica ser. typhi strain MDR. This similar result also reported by Malhadas et al. (2017). They found that fungal extract from mycelium and broth medium could inhibit pathogenic bacteria in a certain concentration. Figure 3 and Figure 5 show the inhibition zones of fungal extract from broth medium in a different solvent. The extract obtained from 1-butanol performed antibacterial property against both of tested bacteria at fraction 20, 21 and 22 while the extract from ethyl acetated inhibited the bacteria at fraction 20, 21, 22, 22, 23 and 24. Both extracts showed the widest inhibition zone at fraction 20 and 21 (Table 1). Figures 4 and 6 show that 1-butanol extract had fewer peaks than ethyl acetate extract. This result was correlated to the profile of each extract. Fraction 20 and 21 from ethyl acetate extract consisted of 5 peaks, while fractions from 1-butanol extract consisted of 3 peaks.
Besides, all extract had active fractions in similar retention time. According to HPLC condition, we considered the fraction as semipolar to non polar because it appeared when the concentration of solvent around 79-83%. Most of the active fractions had peaked with similar UV patterns. However, the similarity of the UV pattern is caused by the presence of similar functional group in the active compounds. In this study, we consider that ethyl acetate able to extract more compounds from broth medium rather than 1-butanol. Based on the inhibition zone from fungal fractions, we propose that fungus T. reesei PDSP 5.7 is a good candidate as an antibiotic producer to overcome infection of ESB E. coli and S. enterica ser. Typhi strain MDR.

CONCLUSION
Fraction from mycelium and medium extract of marine-derived fungus T. reesei PDSP 5.7 had antibacterial activity against ESBL E. coli and S. enterica ser. Typhi with range of inhibition zone was 1.35±0.15 to 8.82±0.22 mm. Mycelial extract had activity at fractions 18,19,20,21,22,23 and 25, active fractions from broth medium which extracted using 1-butanol were 20, 21 and 22 while active fractions from ethyl acetate extract were 20, 21, 22, 23 and 24. Most of the active fractions had similar UV absorption patterns.