Phongsopitanun et al., 2014 49 Original Article TJPS The Thai Journal of Pharmaceutical Sciences 38 (1), January - March 2014: 1-56 Identification and antimicrobial activity of Streptomyces strains from Thai mangrove sediment Wongsakorn Phongsopitanun1, Khanit Suwanborirux2, 3 and Somboon Tanasupawat1, 3 1Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Bangkok 10330, Thailand Abstract Two actinomycete strains, D2-1 and D2-2, were isolated from mangrove sediment sample collected from Samut Song Kram province, Thailand. On the screening of antimicrobial activity, the crude extracts of fermentation broth of both strains exhibited activities against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 while only the crude extract of strain D2-1 exhibited activity against Candida albicans ATCC 10231. Both strains were identified as the genus Streptomyces based on polyphasic taxonomy. They contained LL-DAP in cell wall peptidoglycan, MK-9 (H6) and MK-9 (H8) as the major menaquinone and contained iso-C16:0, anteiso-C15:0, iso-C15:0, iso-C14:0, C 17:0 cyclo, C 16:0 and anteiso-C17:0 as major cellular fatty acids. The 16S rRNA gene sequences analysis revealed that strains D2-1 and D2-2 are closely related to T Streptomyces iranensis HM35 (99.57 %) and Streptomyces sundarbansensis (99.78 %) respectively. The strain D2-1 was identified as S. iranensis and strain D2-2 was S. sundarbansensis. Key Words: Actinomycetes, Antimicrobial activity, Mangrove sediment, Polyphasic taxonomy, Streptomyces Introduction Verrucosispora isolated from mangrove sediments and from the mangrove swamps have been studied for diversity The mangrove environment is highly rich in the as well as antimicrobial activity [2-7]. In Thailand, organic matters consistent with high sulfur and nitrogen Streptomyces isolates from mangrove soils collected in which can be used by the living microorganisms [1]. Samut Prakan and Samut Song Kram provinces, the inner Actinomycetes in genera Streptomyces, Micromonospora, gulf of Thailand were reported [8]. Screening for the Microbispora, Nocardia, Nonomuraea, Actinoplanes, actinomycete species is an important aspect as there is a Actinomadura, Pseudonocardia, Rhodococcus and remarkable source for the production of diverse bioactive metabolites that possess pharmaceutically relevant biological activities [9]. Correspondence to: Somboon Tanasupawat, Department of Biochemistry and Microbiology, Faculty of Pharmaceutical In our investigation of actinomycetes distributed in Sciences, Chulalongkorn University, Bangkok 10330, Thailand mangrove forest soils along the inner gulf of Thailand, the Tel.: +66 2218 8376, e-mail: [email protected] actinomycete isolates from mangrove sediment in Samut Song Khrarm province were isolated, screened for Academic Editor: Pithi Chanvorachote antimicrobial activity and identified based on the www.pharm.chula.ac.th/tjps TJPS 2014, 38 (1): 49-56 50 Phongsopitanun et al., 2014 phenotypic and chemotaxonomic characteristics including the agar disc diffusion method [13]. The indicator bacteria 16S rRNA gene sequencing. mentioned above were cultivated on Mueller-Hinton agar (MHA, Difco) at 37 oC for 24 h while the yeast, Candida Materials and Methods albicans ATCC 27853 was cultivated on Sabouraud’s dextrose agar (SDA, Difco) at 30 oC for 48 h. The Sample collection and isolation of actinomycetes. inhibitory zones (mm) were measured and recorded. Mangrove sediment samples were collected from Klong Kone mangrove forest in Samut Song Khram province, Identification methods o Thailand, and preserved at 4 C before the isolation Phenotypic characteristics. The isolated process. The isolation of actinomycetes was done by the actinomycetes were cultivated on ISP2 agar medium at standard serial dilution method. The diluted soil suspension 30 oC for 14 days. The morphology of spores was observed of 1:100 and 1:1000 (0.1 ml) were spreaded on starch- by using a JEOL JSM-5410LV scanning electron casein nitrate agar (SCA) medium containing 15 g/ml microscope at Scientific and Technological Research novobiocin and 25 g/ml nystatin, and incubated at 30 °C Equipment Centre, Chulalongkorn University. To for 7 days [10]. After incubation, the single colony of determine their cultural characteristics, the actinomycete actinomycetes was selected by the morphology of colony isolates were grown on various ISP medium [11] at 30 oC and purified on Yeast extract-Malt extract agar (YM) for 14 days. The color of aerial and substrate mycelia (Internationnal Streptomyces project medium No. 2, ISP 2 including soluble pigment were determined by using the medium) [11]. NBS/IBCC color system. The hydrolysis of starch, gelatin, skimmed milk peptonization and nitrate reduction were Screening of antimicrobial activity. Primary screening of determined by standard methods [14-15]. Utilization of antimicrobial activity was performed on ISP 2 agar plate. carbon sources was determined by using ISP medium 9 The strains of Escherichia coli ATCC 25922, supplemented with 1% of the carbon source [11]. The Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis temperature 30, 37, 45 and 50 oC, and pH 4.0, 4.5, 5.0, ATCC 6633, Staphylococcus aureus ATCC 6538 and 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0 for growth were Candida albicans ATCC 10231 were used as indicator determined on the ISP 2 agar plates. strains as previously reported [12]. All indicator Chemotaxonomic characteristics. Freeze-dried cells microorganisms were cultivated on Mueller-Hinton agar were used for all chemotaxonomic analysis. The isomers of (MHA, Difco) slants at 37 oC for 24 h, except for the diaminopimelic acid on cell wall peptidoglycan were yeast strain that was cultivated on Sabouraud’s dextrose determined by TLC as described by Komagata and Suzuki agar (MHA, Difco) slant at 30 oC for 24 h. The [16]. The menaquinone was extracted by using chloroform: antimicrobial producing actinomycetes exhibited inhibitory methanol (2:1) [17] and analyzed by using HPLC distance against microorganisms tested. The inhibitory equipped with C18 reversed phase column (4.6 by 150 distances (mm) were measured and recorded. On the mm, Nacalai Tesque, Kyoto, Japan). Cellular fatty acid secondary screening, a loopful of acitnomycete spores on analysis of cells cultivated on ISP 2 agar medium for 4 ISP 2 agar medium at 30 oC for 7 days, was inoculated into days at 30 ºC was performed by Chromatography (GLC) 100 ml of ISP 2 broth and cultivated on a rotary shaker according to the instructions of the Microbial Identification (200 rpm) at 30 oC for 5 days and then 1 ml of the System (MIDI) Sherlock version 6.0 [18] with the inoculum was transferred to ISP 2 broth with 0.3 % CaCO3 RTSBA6 MIDI database (Faculty of Science, King and cultivated on a rotary shaker (200 rpm) at 30 oC for 14 Mongkut' Institute of Technology Ladkrabang). days. After incubation, the actinomycete mycelia were 16S rRNA sequence and phylogenetic tree analysis. separated from the culture broth by using the refrigerated The DNA of selected actinomycetes was extracted from centrifuge (7,000 rpm, 4 oC) for 15 minutes. cell grown on ISP2 broth at 30 oC for 4 days [19-20]. 16S The fermentation broth of strains was partition with rRNA gene was amplified by using universal primer, 27F ethyl acetate by using separatory funnel. The upper layer, (5’-GAGTTTGATCCTGGCTCAG-3’) and 1530R (5’- ethyl acetate part, were separated and then evaporated by a GTTACCTTGTTACGACTT-3’) [21]. The 16S rRNA rotary evaporator to dryness (crude ethyl acetate extract). gene was sequenced (Macrogen, Korea) by using universal The lower layer, water part, was further used for partition primer [22]. The BLAST analysis was analyzed on the with iso-butanol and the iso-butanol part, upper layer, was EzTaxon-e database [23]. The phylogenetic analysis was evaporated to dryness (crude iso-butanol extract). The constructed by the neighbour-joining method [24] on the actinomycete mycelia of cells were extracted by adding the MEGA 5.2 software. The GenBank/EMBL/DDBJ methanol to the cell mycelium for 48 h at room accession number for the 16S rRNA gene sequence of temperature. Then the mycelia of cells were separated from strains D2-1 and D2-2 are AB889543 and AB889543, the solvent by using filter paper and methanol part was respectively. evaporated to dryness (crude methanol extract). All crude extracts were determined the antimicrobial activity using www.pharm.chula.ac.th/tjps TJPS 2014, 38 (1): 49-56 Phongsopitanun et al., 2014 51 Results and Discussion strain D2-1 exhibited activities against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Two actinomycete strains, D2-1 and D2-2, were Escherichia coli ATCC 25922, Pseudomonas aeruginosa isolated from the sediment sample. The morphology of ATCC 27853, Candida albicans ATCC 27853 while strain isolate D2-1 exhibited the spiral spore type with rough D2-2 exhibited activities against both Gram-positive and surface while isolate D2-2 exhibited long spore chain with Gram-negative bacteria but not Candida albicans ATCC smooth surface (Figure 1). The upper colonial appearance 10231. of D2-1 on ISP 2 was dark grayish yellowish brown and On the secondary screening, the ethyl acetate extract D2-2 was yellowish white. The cultural characteristics on of the isolate D2-1 showed activities against ISP 3, ISP 4, and ISP 6 of both isolates were shown in Staphylococcus aureus ATCC 25923, Bacillus subtilis Table 1. Strain D2-1 grew at 4.5-9.0 while strain D2-2 ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas grew at 5.0-9.0. They grew at 30-45 oC and grew optimally aeruginosa ATCC 27853, Candida albicans ATCC 10231 at pH 6-8 and at 30-37 oC. They hydrolysed gelatin and (Table 4) while isolate D2-2 showed weak antimicrobial starch and utilized fructose, glucose and mannitol as activity as shown in Table 4.