Biological Activity of Lasiodiplodia pseudotheobromae IBRL OS- 64 Extracts, an Endophytic Fungus Isolated from Medicinal Herb, Ocimum sanctum Against Foodborne Diarrhea-Caused Bacteria

Food poisoning illness occurs as a result of the consumption of foods contaminated with bacteria and this is considered as one of the most causes of sickness and death in developing countries.1 There are several symptoms regarding food poisoning illness including fever, diarrhea, vomiting, dehydration and abdominal cramps.2 Although food poisoning causes discomfort to human, it is one of condition that should not be taken for granted as it could cause death, especially when dehydration happens.3 Generally, foodborne diseases have been associated with the consumption of foods such as processed meat, beef, soft cheese, poultry, eggs and other related foods.4 Most of food poisoning reports are associated with bacterial contamination especially Gram-negative bacteria such as E. coli,5 P. mirabilis,6 Y. enterocolitica,7 and Gram-positive bacteria including Bacillus cereus,8 B. mojavensis and B. subtilis.9 The ability of the foodborne bacteria such as Bacillus cereus to produce emetic toxin and enterotoxins is the most important cause of foodborne poisoning.10


INTRODUCTION
Food poisoning illness occurs as a result of the consumption of foods contaminated with bacteria and this is considered as one of the most causes of sickness and death in developing countries. 1 There are several symptoms regarding food poisoning illness including fever, diarrhea, vomiting, dehydration and abdominal cramps. 2 Although food poisoning causes discomfort to human, it is one of condition that should not be taken for granted as it could cause death, especially when dehydration happens. 3 Generally, foodborne diseases have been associated with the consumption of foods such as processed meat, beef, soft cheese, poultry, eggs and other related foods. 4 Most of food poisoning reports are associated with bacterial contamination especially Gram-negative bacteria such as E. coli, 5 P. mirabilis, 6 Y. enterocolitica, 7 and Gram-positive bacteria including Bacillus cereus, 8 B. mojavensis and B. subtilis. 9 The ability of the foodborne bacteria such as Bacillus cereus to produce emetic toxin and enterotoxins is the most important cause of foodborne poisoning. 10 Nowadays, the emergence of a multidrug-resistant strain of foodborne bacteria has become a worldwide human health problem and food safety. For instance, Brunelle 11 reported the multidrugresistant Salmonella enterica Serovar typhimurium isolated from clinical samples originally cultured from cattle. They found that six isolates were resistant to chloramphenicol, streptomycin, ampicillin, and tetracycline. Kiessling 12 reported the multidrug-resistant phenomenon occurred due to the uncontrolled use of chemical antimicrobial preservatives that induced the appearance of more microbial strains resistant towards classics antimicrobial agents. Besides that, excessive amounts of antibiotics used for human therapy, farm animals and aquaculture industry resulted in the selection of bacterial resistance to multiple drugs. 13 The bacterial multidrug resistance might be generated by two mechanisms which are the accumulation of multiple genes for resistance to a single drug in resistance (R) plasmids and expression of genes that coding multidrug efflux pumps which extruding a wide range of drugs. Besides that, consumer's demand has increased for the foods with long shelf-life but with the presence of no or minimum chemical food additives. 14 Thus, there has been a growing demand among customers for natural preservatives and additives in processed foods. 15 Therefore, the discovery of natural drugs and food preservatives has become popular among researchers.
Endophytic fungi are one of the well-known sources of natural products. Fungal endophytes are defined as mitosporic or meiosporic ascomycetes that asymptomatically live in the internal tissue of plants and they usually colonize the healthy and living tissue through quiescent infections. 16 The mutual relationship between endophytic fungi and their hosts could impose several effects on the formulation of bioactive substances that can benefit humans. 17 Lasiodiplodia pseudotheobromae is a member of Botryosphaeriaceae that is known to cause stem cankers, dieback and stem-end rot to crops worldwide especially mango. 18 This species is reported to possess several pharmaceutical potentials including cytotoxic 19 and antimicrobial 20 activities. Some of its bioactive compounds are mellein, 2-phenylethanol, cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA) and indole-3-carbaldehyde. 21 Therefore, the current study was designed to investigate the antibacterial activity of the ethyl acetate crude extract of endophytic fungus, Lasiodiplodia pseudotheobromae IBRL OS-64 against foodborne bacteria.

Endophytic fungus culture and maintenance
The endophytic fungus, Lasiodiplodia pseudotheobromae IBRL OS-64 was provided by the Industrial Biotechnology Research Laboratory (IBRL), School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia. The fungal isolate was cultured and maintained on potato dextrose agar (PDA) nourished with powdered host plant (2 g/L) and stored at 4 o C. To ensure its purity and viability, the isolate was subcultured once a month on sterile fresh media.

Test microorganisms
The Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Yersinia enterocolitica and Acinetobacter anitratus cultures were provided by the IBRL, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia. The bacterial cultures were grown on nutrient agar (NA), incubated at 37°C for 24 hours and stored at 4 o C.

Culture medium
Yeast extract sucrose (YES) broth (Merck, Germany) supplemented with host plant water extract was used as a fermentative medium and the culture medium was prepared according to procedures described by Taufiq and Darah. 22 Fermentation and extraction Two mycelial plugs of 3-d-old of endophytic fungal culture were introduced into 250 mL Erlenmeyer flasks containing 100 mL YES broth. The cultures were then incubated at 30 o C under a static condition in the dark for 16 days. The fermentative broth and fungal biomass were separated out using filter papers (Whatman, No.1). The filtered broth was then extracted thrice with an equal volume of ethyl acetate (1:1; v/v). The upper organic phase was collected and concentrated using a rotary evaporator. The concentrated mixture was left to dryness in a fume hood to obtain ethyl acetate crude paste. The crude paste was then extracted and fractionated with different polarity solvents including hexane, dichloromethane, ethyl acetate, and butanol. Each solvent extracts were left to dryness and the paste was weighed.

Disk diffusion assay
The assay was carried out according to the method described by NCCLS. 23 The fungal extracts from different solvents were dissolved in 50% methanol (w/v). Prior to that, the microbial inoculums were swabbed on the dried surface of the Muller Hinton agar (MHA) using sterile cotton swabs. An amount of 20 µL of the extracts was impregnated to the sterile Whatman antibiotic disk and then placed on the surface of the previously seeded medium. Twenty percent of methanol was used as a negative control whilst chloramphenicol (30 µg/mL) was set as a positive control. The plates were then inversely incubated at 37 o C for 24 hours. The diameter of the inhibition zone formed surrounding the disc was measured and recorded. The experiments were carried out in triplicates on different occasions.

MIC and MBC determinations
The MIC value of the fungal crude extract was determined using broth microdilution assay recommended by NCLLS. 23 A two-fold dilution of the fungal extract was prepared in sterile Muller Hinton Broth (MHB) medium and 100 µl of the extract was dispensed into each well of the microtiter plate. On the other hand, 100 µl of bacterial inoculum at approximately 1 × 10 6 CFU/ml was added into each of the wells for a final volume of 200 µl and the final concentration of bacteria in each well was 5 × 10 5 CFU/ml. Chloramphenicol was used as a positive control and 5% methanol was set as a negative control. The MIC value was recorded as the lowest concentration of the crude extract that capable to inhibit the visible growth of test bacteria after the overnight incubation period. MBC value of the fungal crude extract was subsequently determined upon the reading of MIC value. An amount of 0.1 mL of sample in clear wells at concentrations greater than the defined MIC was streaked on a fresh MHA and the procedures were repeated for the next wells in ascending extract concentrations. The inoculated plate was incubated at 37ºC overnight. The MBC was observed and recorded as the lowest concentration of fungal crude extract that resulted in a reduction of 99.9% bacterial growth relative to the growth control.

Scanning electron microscopy (SEM)
Bacterial samples, fixation and dehydration process were prepared according to the method described by Taufiq and Darah. 22 The mixture was then incubated at 37 o C, 150 rpm for 36 hours. At every 12 h of the time interval, the mixture was harvested and centrifuged to obtain the bacterial cell pellet. The pellet was then undergone the primary fixation, post-fixation and dehydration process. The dried bacterial cells were mounted on a specimen stub using conductive tape and coated with 5 -10 nm gold using a sputter coater machine (Fison SC-515, UK) before SEM viewing process. The specimen was then viewed under a scanning electron microscope (Leica Cambridge, S-360, UK).

Statistical analysis
All the experiments were performed in triplicate (n = 3) and the experimental data were expressed as mean ± standard deviation (SD). The data were analyzed by means of the One Way ANOVA using SPSS 15.0 and Duncan test was used to access the differences between means. The results were considered statistically significant if p < 0.05. Table 1 shows the extraction yield obtained from the solvent partitioning method. From the one gram of crude paste used, 0.01 g of hexane extract (0.69%) with colorless paste was obtained. Subsequently, an amount of 0.74 g of dichloromethane extracts which equivalent to 52.86% with brownish paste was obtained. Then, the ethyl acetate extract obtained was 0.32 g (22.86%) with beige-colored paste. Finally, 0.13 g of butanolic extract (9.29%) with yellowish colored paste was obtained. As shown in Table 2, all the test bacteria were susceptible to the ethyl acetate extract with inhibition zone in the range of 15.0 mm to 26.0 mm. Among Gram-positive bacteria, B. subtilis (26.0±1.0 mm) was more susceptible to the extract compared to B. cereus (21.0±1.0 mm) and S. aureus (20.4±0.6 mm). For Gram-negative bacteria, the extract exhibited higher antibacterial activity against Y. enterocolitica compared to E. coli and A. anitratus with an inhibition zone of 20.1±0.6 mm. The results revealed that the Gram-positive bacteria were more susceptible compared to the Gram-negative bacteria. Besides that, all test bacteria were resistant to the dichloromethane extract except Y. enterocolitica with inhibition zone in the value of 8.6 mm. This finding revealed that there might be more than a single bioactive compound in the extract that can inhibit bacterial cells. However, none of the test bacteria were susceptible to hexane and butanolic extract and this indicated that most of the bioactive compounds produced by L. pseudotheobromae IBRL OS-64 were mid-polar compounds since they can be extracted by midpolar solvents. Morphological changes of extract-treated cells Figure 1 shows the SEM micrograph of the Bacillus cereus cells after treatment at the MBC value. Figure 1a shows the control of bacterial cells without any treatment and it shows typical normal cells of B. cereus, with rugose and rod-shaped structure. Figure 1b exhibits the bacterial cells after treated with the extract and the cells become irregular in shape and some of them were crumpled losing of their rod shape. Figure 2 shows the photomicrographs of the untreated (control) and ethyl acetate extract-treated Yersinia enterocolitica cells at MIC value. Figure 2a exhibits the normal structure of Gram-negative bacteria with rod-shape and rugose morphology. The binary fission of the bacterial cells also occurred in the control sample (indicated by black arrow). However, the bacterial cells undergo several abnormalities after treated with the extract. The formation of shrunken, distorted cells with cavities and cell debris was observed on the majority of the cells. The cavity observed on the surface of the most bacteria cells finally led to cell lysis and death beyond repair.

DISCUSSION
The choice of solvent for extraction depends on the purpose of studying the extract. 3 In the present study, the organic solvents with different polarity ranges were selected for extraction to gain the diverse type of bioactive compounds with antibacterial activity. According to Gu,24 liquid-liquid extraction including aqueous two-phase and solvent partitioning is very useful procedures in bio-separation. Four types of organic solvents with different polarities were used in this study which was hexane, dichloromethane, ethyl acetate, and butanol. Abarca-Vagas, 25 reported the polarity of the organic solvents used for the extraction process plays a vital role in the efficacy and efficiency of bioactive compounds with pharmacological activities. Hexane is one of the organic solvents that frequently used for extraction and it can dissolve non-polar compounds such as lipid, wax, and lignin. 26 Meanwhile, dichloromethane and ethyl acetate are used to extract more lipophilic and hydrophilic compounds, respectively. 27 Besides that, butanol which is more polar organic solvents frequently used for extraction of polar compounds such as flavonoid glycosidase. 28,29 The liquid-liquid partitioning of ethyl acetate crude paste of L. pseudotheobromae IBRL OS-64 revealed that organic solvent, dichloromethane was able to dissolve the highest extract paste with the value of 0.74 g/L or 52.86%. Similar observation also reported by Tong 30 that mentioned dichloromethane gave the highest production of crude extract paste of Penicillium minioluteum ED24 with a value of 1.52 g/L. Meanwhile, the least yield obtained was the hexane extract with a percentage of 0.69. The results were in agreement with Mabiki 31 who stated the extraction process with hexane yielded a low amount of extract. This might be due to the low amount of non-polar compounds in the ethyl acetate crude extract since hexane was mainly to dissolve more non-polar compounds such as wax, lipid, and fat. 32 Hexane wash also eliminated unwanted metabolites such as fatty acid that not of interest in natural product screening. 33 According to Franco, 34 the solubility and polarity of different organic solvents used in the extraction process lead to variation in extraction yield. Begtrup and Larsen 35 suggested that polar organic solvents including dichloromethane and ethyl acetate are suitable in extracting various bioactive compounds since they are easily evaporated and dried due to low boiling point. The finding also showed that the colorless solution was observed in the hexane extract whereas brown color was observed in the dichloromethane extract. Besides that, the beige and pale yellow color was observed in the ethyl acetate and butanol extracts, respectively. The variation of extract coloration might be due to the capability of the organic solvents to dissolve compounds with different polarities. Due to the close association between endophytes and their host, natural products from fungal endophytes might comprise of a wide variety of bioactive compounds with several chemical classes and functional groups. Wright 36 postulated that natural products contain a variety of chemical functionalities and the changes in functionality have affected the polarity of the compounds. Therefore, the presence of bioactive compounds in natural products and their activities were affected by the type of organic solvent used for the extraction process. 37 Taufiq, et al.: Biological Activity of Lasiodiplodia pseudotheobromae IBRL OS-64 Extracts, an Endophytic Fungus Isolated from Medicinal Herb, Ocimum sanctum Against Foodborne Diarrhea-Caused Bacteria The present results showed that the antibacterial activity of the fungal isolate depends on the efficiency and polarity of the organic solvents. The current study revealed that ethyl acetate, which is the mid-polar solvent is the best organic solvent to extract bioactive compounds with antibacterial potential from the fungal extract paste. The result was in agreement with Avinash 38 who revealed that ethyl acetate extract of the fungal endophyte, Curvularia lunata demonstrated good activity against all the test pathogenic bacteria including Staphylococcus aureus and Salmonella thypi. According to Tong,39 this phenomenon indicates that the antimicrobial compounds were semi-polar and could be extracted using a mid-polar solvent such as ethyl acetate and dichloromethane. However, hexane and butanol extract showed no antibacterial activity against all the test bacteria in the present study. A similar observation was reported by Yenn 40 whereby none of the antimicrobial compounds were present in hexane fraction (non-polar) and butanol (highly polar) fractions of the endophytic fungal extract, Phomopsis sp. ED2 isolated from Orthosiphon stamineus Benth.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are frequently used by researchers to confirm resistance and determine the in vitro activity of the new antibiotics as well as determining their MIC's breakpoint. According to Andrew,41 MIC is defined as the lowest concentration of a certain antibiotic that will inhibit the growth of a microorganism after an overnight incubation period, whereas the MBC is the lowest concentration of an antimicrobial that will prevent the growth of a microorganism after subculture onto antibiotic free-media. The present study revealed that the MBC values for all the test bacteria were significantly higher than their MIC's indicating that the crude extract was concentrationdependent in inhibiting the growth of test bacteria. Tong 39 postulated that the higher MBC value compared to MIC value indicating the extract concentration would have to be increased to significantly kill the microbial cells rather than inhibiting their growth. Furthermore, the crude extract of L. pseudotheobromae IBRL OS-64 was observed to possess a bactericidal effect towards test bacteria due to the low MBC/ MIC ratio. According to Levison,42 antimicrobial drugs can be usually considered as bactericidal if the MBC value, not more four-fold higher than their MIC value. The current results also suggested that the fungal crude extract exhibited a strong antibacterial effect on Gram-positive bacteria as compared to Gram-negative bacteria. These findings are in agreement with the previous study by Marasini 43 who claimed that MIC values of plant extract against Gram-positive bacteria are significantly low than Gram-negative bacteria. They suggested that this phenomenon may due to the different nature of cell walls for both types of bacteria and the existence of the efflux system in Gram-negative that facilitates them in enhancing their resistant towards antibiotic agents. Generally, the Gram-negative bacteria possess an extra outer membrane that consists of the asymmetric distribution of the lipids with phospholipids and lipopolysaccharide located in the inner and outer leaflets that could act as an additional barrier that restricts the movement of a foreign substance through the cell. 44 This component is absent in the Gram-positive bacteria, but their cell wall contains lipoteichoic acids (LTA) that are unique and essential structure to the cells. However, the component could be good drug targets to bioactive compounds 45  This phenomenon could be due to some alterations occurred in the cell membrane permeability or may be leaking in the cell wall. As for Gram-positive bacteria, some cavities and dents were observed on the treated cell. A similar observation reported by Asnani 46 who studied the morphology of B. cereus cells exposed to water and ethyl acetate fraction of Spondias pinnata leaves under SEM. Hartmann 47 suggested the formation of holes, craters, and dents on the surface of bacterial cells indicated a failure or a mechanism rupture of the cell wall and membrane. The Gram-negative bacteria cells expose to fungal extract showed wrinkled, crumpled or shrunken cell surface and the cells had lost their original rod shape compared to control. There were some holes formed on the cell wall indicated the cell leakage. Similarly, Fu 48 demonstrated a Gram-negative bacterium, Y. enterocolitica exposed to endogenous peptide β-casein 197 showed disorganized cell structures with wrinkled surface upon SEM observation. According to Hyde, 49 the morphological changes of the bacterial cell treated with antibiotics occur when the antimicrobial agent attacked the cell membrane. The bioactive compound from the extract might target cell surface structure and permeabilized the bacterial membrane and any disruption in cell wall integrity leads to the complete damage of bacterial cells. 50 Armas 51 revealed that the morphological alterations of bacterial surface including roughening, blebbing with cellular debris and formation of holes indicated a membrane-damage activity by the antimicrobial compounds. According to Chaparro, 52 the aggregations and abnormal cell shapes with surface changes might be attributed to the leakage of cellular contents. Besides that, Al-Adham 53 postulated the crumpled cell and formation of hole may indicate the loss of cellular materials and organelles from the cell cytoplasm that will lead to cell death. Previous studies proposed some mechanisms that might be contributed to the dysfunction of bacteria cells. Guimaraes 54 reported the changes in the ion channels such as Na + and K + in the cell membrane might increase the permeability of the cell and thus release the vital intracellular constituents. Meanwhile, the bioactive compounds such as β-lactam able to disrupt the synthesis of peptidoglycan which is the main component of bacterial cell walls and hence, leading to cell death. 55 Besides that, antibiotics such as chloramphenicol and tetracyclines are able to inhibit protein biosynthesis by targeting the 50S or 30S subunit of the bacterial ribosome. 56 However, the mode of action of the ethyl acetate extract of L. pseudotheobromae IBRL OS-64 against test bacteria remains unknown but it is believed that the extract may target the bacterial cell wall and ignite some alterations causing collapsed cells and finally led to cell death.

CONCLUSION
The findings revealed that the ethyl acetate crude extract of L. pseudotheobromae IBRL OS-64 has a great potential as an antibacterial agent to treat infectious diseases caused by a range of pathogenic bacteria. The results could form the basis for further studies to develop endophytic fungal extracts as antibiotics and evaluate them against a wide range of bacterial strains.