Isolation and Characterization of a Novel Endophytic Streptomyces SUK 06 with Antimicrobial Activity from Malaysian Plant

Streptomyces which is characterized as Gram positive, filamentous bacteria with high G+C contents was formerly found as saprophyte that dwells in the soil. It`s an organism that produces a variety of metabolite particularly antimicrobial that was used either in medicinal therapies or as research tool. Extensive research have been carried out in the last decades to isolate Streptomyces from soil and while still yielding new drugs, the numbers of novel compound have been dwindling (Palaez, 2006). In order to avoid the redundancy of finding the same compound and to overcome the emerging of new drug resistance in many bacterial pathogens, new untapped source of bioactive products must be discovered. It is now obvious that these filamentous bacteria exist as endophytes within the living tissues of certain higher plants (Ezra et al., 2004; Castillo et al., 2006). The complex interaction has gained our interest because it can reveal new source of novel drugs which can be used in either agriculture or medicinal field. (Cao et al., 2004; Castillo et al., 2003, 2002; Strobel, 2002; Strobel et al., 2005).

A fast and accurate method has been widely used to identify newly isolated endophytes using 16S rDNA gene sequence analysis. This technique is important to identify organisms which are slow-growing, fastidious and where identification by conventional methods is time consuming along with subjective interpretation such as identifying Streptomycetes sp. Castillo et al. (2003) had succeeded in identifying Streptomyces sp. NRRL 30566, which produced novel antibiotic Kakadumycins using this method. When used with previously described phenotypic data, the addition of information from 16S rRNA can be applied to reestablish existing bacteria taxonomy (Kim and Goodfellow, 2002).

Previous report showed that plants representing families were present during the era of the ancient Gondwanaland, yielded numerous novel bioactive endophytic Streptomycetes (Castillo et al., 2007). Thus, it appears likely that other biologically active Streptomycete isolates exist in plants throughout the world. In this study, isolation of endophytic Streptomycetes have been carried out from the ethno botanical plants that hold feature to healing wounds, skin infection, or curing fever. This study illustrates the details on the isolation and bioactivities of this novel endophytic Streptomyces SUK 06 and showed that some of the bioactive products were extractable with organic solvents.

Plants selection and location: Thottea grandiflora stem and some root samples were collected from the province of Bukit Bauk in Terengganu State situated at the east coast of peninsular Malaysia. This plant was traditionally used as postpartum tonic, cure fever and malarial infection (Mat-Salleh and Latiff, 2002) A few portions of the stems with length of approximately 10 cm was cut, tagged and stored in a clean plastic bag. A specimen voucher number also given and submitted to Universiti Kebangsaan Malaysia (UKM) botanical collection.

Isolating endophytic Streptomycete sp.: Each plant stem samples were subjected to surface sterilization treatment to eliminate the contaminating microflora. This procedure includes sequential immersion of the stems in 70% alcohol for 5 min, 0.9% NaOCl for 10 min and 95% alcohol for 30 sec (Zin et al., 2007b). Using aseptic technique the bark of the stems was removed and the inner stem tissues were cut into small pieces sized 0.5x0.5 cm. The tissue was then placed onto Starch Casein Agar (SCA) surfaces which have been supplemented with 50 μg mL^-1 Cycloheximide, 50 μg mL^-1 Nystatin and 20 μg mL^-1 Nalidixic acid. The petri dish was sealed and incubated at 28°C. Observation was done for a few weeks and bacterial growth on the agar or from stem tissue was re-cultured on International Streptomyces Project medium (ISP 2) agar for morphology and pigment observation. The organism appearing as actinomycetes, yielding an earthy odor in culture, seemed as likely candidates as potential streptomycetes. It was cultured on nutrient agar and stored in sterile 15% glycerol water at -70°C. It was kept in the living microbe collection at UKM and labeled as SUK 06.

Microscopy observation: Microscopy observation was done using light and Scanning Electron Microscopy (SEM). Light microscopy observation was carried out to observe the morphology of SUK 06 using Gram staining and SEM for observation of spore morphology on agar. The colony of SUK 06 was fixed in 10% (v/v) Formalin Phosphate Buffered and after washing with PBS, it was then dehydration in serial ascending of alcohol concentration from 50% to absolute alcohol (15 min for each concentration except for absolute alcohol in which the dehydration was done twice). The samples were then processed by procedures described by Zin et al. (2007a). It was then critical-point dried, gold coated and images were recorded using Philips XL 30 SEM.

DNA extraction of Streptomyces SUK 06: For DNA extraction Streptomyces SUK 06 was cultured in 10 mL nutrient broth for 4 to 7 day so that satisfactory amount of bacteria cells was obtained. The whole culture was centrifuged at 10,000 g for 20 min and the supernatant was completely discarded. The DNA extraction protocol was based on Conn and Franco (2004), The DNA collected was dissolved in 50 μL sterile water and stored at -20°C until further use.

Amplification and sequencing 16S rRNA of Streptomyces SUK 06: The extracted DNA was quantified using spectrophotometer at A₂₆₀. PCR amplification was based on Coombs and Franco (2003) with modification which using primers 27f (5-AGAGTTTGATCMTGGCTCAG-3) and 1492r (5-TACGGYTACCTTGTTACGA3) (First Base Lab, Selangor, Malaysia). It was carried out in 50 μL mixture comprising 5 μL 27 f (10 pmol), 5 μL 1492r (10 pmol), 5 μL 10X PCR buffer, 1 μL DNTP 10 mM, 3 μL MgCl₂ 25 mM, 1 μL 5U μL Taq polymerase, 5 μL template DNA and 25 μL sterile H₂O. The temperature profile for PCR were: 94°C for 8 min; followed by 30 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 2 min and then final elongation at 72°C for 10 min. The PCR products were purified by using a Wizard PCR Prep DNA purification kit (Promega). After purification, cloning of amplified 16S rDNA was carried out using Invitrogen pCR®8/GW/TOPO®TA Cloning® kit. The procedure employed was based on manufacturer instruction. Plasmid extraction from transformant was done using miniprep plasmid extraction method from Sambrook et al. (1989). The plasmid then sequenced bidirectional 27f, 704f (5-GTAGCGGTGAAATGCGTAGA-3), 765r (5-CTGTTTGCTCCCCACGCTTTC-3) and 1492r primers (First Base Lab, Selangor, Malaysia). The sequence obtained were analyzed using CLC Free Workbench 4.0 software and compared to the other streptomycete gene sequences in GenBank by using the BLAST software (blastn) at the National Center of Biotechnology Information (NCBI) web site (http://www.ncbi.nih.gov/).

Bioassay of Streptomyces SUK6: Streptomyces SUK 06 was grown onto nutrient agar for 14 days. Then aseptically, a plug with diameter of 1 cm of the grown Streptomyces SUK 06 was transferred onto another nutrient agar which has been previously plated with test bacteria. The test bacteria used were Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis, Enterococus fecalis, Bacillus cereus, Salmonella aberdeen NCTC74, Escherichia coli NCTC 11601, Pleisiomonas shigelloides and MRSA ATCC 700699. The test fungi were as followed: Fusarium solani, Aspergillus fumigatus, Pythium ultimum, Thrichoderma, Phytophthora erythroseptica and Geothrichum candidum. The inhibition zones were observed for 24 and 48 h in which Oxacillin was used as a positive control.

Fermentation, extraction and assay procedures: A few blocks of ISP 2 containing Streptomyces SUK 06 were inoculated into 30 mL of ISP 4 broth and incubated at room temperature 28°C for 7 days and eventually transferred onto 300 mL nutrient broth and incubated in rotary shaker for 21 days at room temperature (Zin et al., 2007b). Culture filtrates were extracted with three half-volume of ethyl acetate. The organic phase was pooled followed by evaporation using rotavapor (model Eyela Rotary Vacuum Evaporator N-N series) at 40°C. The crude extract was dissolved in methanol and was then used for bioactivity against test bacteria and fungi. Known amounts of the dried extract taken up in methanol were then placed directly onto a spot on a PDA plate, dried in the hood and then the test fungi was placed 1.0 cm away from the test spot and eventually evaluated for growth by measuring hyphal development in contrast to the control plate. The data was expressed as percentage of linear growth as compared to the control and the test was performed in triplicate. The method is repeated for bacteria assay but the plate was carefully plated with test bacteria after adding the extract. It was incubated at 37°C for 24 h and zone of inhibition zone was measured.

Endophytic Streptomyces SUK 06 from Thottea grandiflora: Streptomyces sp. designated as SUK 06 was obtained from the stem of Thottea grandiflora after 2 weeks incubation on SCA. The macroscopic characterization was as followed: substrate mycelium emerged after four days incubation and formation of mycelium on the 6th day. The aerial formation was white in color then gradually changed into grey. The elevated colony appeared to be fine edge, chalky, dry, irregular folding and produced some brown pigmentation (Fig. 1). The observation under light microscope showed branching filament and Gram positive bacteria (Fig. 2). SEM observation revealed that the spore ornamentation was spiral (Spira) with the diameter sized ranged 0.5 um to 0.7 μm (Fig. 3) and this is not a common occurrence among endophytic Streptomycetes from previously reported by Castillo et al. (2005). Molecular analysis of 16S rRNA showed 98% similarity with Streptomyces misawanensis, Streptomyces fulvoviolaceus and Streptomyces ginsengisoli (Table 1).

Fig. 1:	Colony of Streptomyces SUK 06 on ISP2 agar

Fig. 2:	Streptomyces SUK 06 under light microscope (1000X)

Table 1:	Identification of Streptomyces SUK 06 based on partial sequencing of 1455 bp region of 16S rRNA gene sequence and other related Streptomyces in Gene Bank

Fig. 3:	Scanning electron microscopy of Streptomyces SUK 06, spores (A) and hyphae (B)

Table 2:	Inhibitory activity of the ethyl acetate extracts of Streptomyces SUK 06 broth culture against various human-pathogenic bacterium

Bioassay of Streptomyces SUK 06: Biological activity of several endophytic Streptomycetes against the fungal test organisms has been reported but none was lethal to either Gram-positive or Gram negative bacteria (Zin et al., 2007b). In this study, Streptomyces SUK 06 was active against Gram positive bacterium rather than Gram negative bacterium. The strongest activity was towards MRSA ATCC 700699 and to the rest of the tested organisms, can be graded as susceptible to intermediate susceptibility (Table 2). Using Oxacillin as a positive control, the antibacterial test showed that the crude extract has at least equivalent activity to that of Oxacillin. In comparison of the effects towards Bacillus subtilis, the same inhibition effect which was intermediately susceptible was observed between Oxacillin and crude extract, whereas for Bacillus cereus, the Oxacillin produced smaller inhibition zone. The crude extract however, gave a larger inhibition zone that can be classified as susceptible. Interestingly, test against Pseudomonas aeruginosa, using crude extract did produce inhibition zone as large as 20 mm despite the fact that oxacillin did not produce any inhibition. When tested against Pleisiomonas shigelloides and MRSA, the crude extract produced a smaller inhibition zone compared to oxacillin but it can still be concluded as susceptible. Results against fungi

Table 3:	Inhibitory activity of the ethyl acetate extract of Streptomyces SUK 06 broth culture against various plant-pathogenic fungi

showed that the strongest activity was towards Fusarium solani (Table 3). It is well known that Fusarium sp. is an agent that infects plants and causing root rot disease. For example Fusarium solani causes brown root rot on peanuts plants (Rojo et al., 2007) and Fusarium oxysporum causes fusarium wilt on banana (Cao et al., 2004) and these infections may cause low yield of crops. A lot of research has been done to find a good antagonistic agent for Fusarium sp. using various species of bacteria (Lim et al., 1991; Li et al., 2005; Pal et al., 2001). In this study, the crude extract of SUK6 was found to be capable of inhibiting Fusarium solani and this could be used as an indicator for its uses or further development of Streptomyces SUK6 as a biocontrol for infections due to Fusarium sp. This is of importance as Malaysia is an agroeconomic country seeking for environmental-friendly approach to control pathogen infection on economically valued crops such as banana. As for now, preliminary test revealed the potential of the extracts to inhibit several pathogenic bacteria and a few plant pathogenic fungi. Relationship between endophyte SUK 06 and the host Thottea grandiflora cannot be established at a current phase. In the meantime several reports on other endophyte using other plants has covered that the endophyte contributes to host survival against environmental hardiness, promotes production of growth hormone and also increase resistance again other pathogenic microbes (Selosse et al., 2004; Strobel, 2003; Tokala et al., 2002; Coombs and Franco, 2003; Cao et al., 2003). On the other hand, this study will consequently serve as prelude for more comprehensive study on the properties of the bioactive product yielded by Streptomyces SUK 06. Further investigation on the biological and chemical properties to ascertain if it may have the potential to be used in biological control or as new drugs for medicinal usage is underway.

This study was supported by IRPA Grant 09-020210050 EAR under RMK8 by the Malaysian Ministry of Science Technology and Innovation. Authors are grateful to Prof Gary Strobel from Montana State University, USA for providing the plant fungi samples.