Identification and antimicrobial activity of Micromonospora strains from Thai peat swamp forest soils

© 2018 Chitti Thawai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Somboon Tanasupawat; Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Wangmai, Bangkok 10330, Thailand. E-mail: Somboon.T @ chula.ac.th Identification and antimicrobial activity of Micromonospora strains from Thai peat swamp forest soils

Peat swamp forest is a very special type of the evergreen forests that occurs in fresh-water marshy land (Posa et al., 2011) and was an interested source for soil sampling because it was different from the normal soil.Nowadays, Micromonospora strains from tropical peat swamp forests are still limited in isolation and the research on taxonomy and antibiotic production of them have so far received little attention.Thus, the attempt to sampling the soil samples in the unique sources is focused.In this study, Micromonospora strains isolated from peat swamp forest soils in the southern areas of Thailand were characterized and identified by both classical and molecular techniques, and the investigation of antimicrobial activity is also performed.

Isolation and identification of isolates
Micromonospora strains were isolated from 11 soil samples (peat, muck) collected from peat swamp forest in Narathiwat, Patthaloong and Yhala provinces, the southern areas of Thailand.The soil suspension was diluted to a ten folds dilution series (10 −1 to 10 −3 ).The aliquots (100 µL) were spread onto Starch-Casein Nitrate agar (SCA) plates supplemented with antibiotics, nystatin, novobiocin, and tetracycline (Brock et al., 1993), and were incubated at 30°C for 7-21 days.The moist, pale yellow, orange, brown and black colonies of isolates were picked up and streaked for purification on ISP2 agar plates at 30°C for 7-21 days.The single colony was cultivated on ISP2 agar slants at 30°C for 14 days.The soil samples were dried under room temperature for a week.Dried soil (1 g) was put into a test tube which then 2.5 mL of distilled water was added and shaked for 30 sec.The soil solution was measured for pH value.
The morphological characteristics of Micromonospora isolates were determined by using simple inclined coverslip technique (Williams and Cross, 1971).Cultural characteristics were studied on the colors of mature substrate mycelium, spore, and diffusible pigment using crosshatch streak (Shirling and Gottlieb, 1966).The strains were cultivated on yeast extract malt extract agar (International Streptomyces Project, ISP2 medium) at 30°C for 21 days.The color of the upper and reverse surface mycelium and the soluble pigment color were observed by using the NBS/ISCC colour system (Kelly, 1964).Spore was observed by light microscope or scanning electron microscope.Utilization of various carbon sources and several biochemical tests were determined using standard methods as recommended by Shirling andGottlieb (1966) andArai (1975).NaCl tolerance was tested on ISP2 agar plates supplemented with 1.5, 3, 4, 5, 6 or 7% (w/v) NaCl.Growth at 30,40,and 45°C,at pH 4,4.5,5,6,7,and 8 were performed on ISP2 agar plates, incubated for 7-14 days.Standard thin-layer chromatography (TLC) procedures were used to determine the isomers of diaminopimelic acid and sugars in whole-cell hydrolysates (Staneck and Roberts, 1974).The N-acyl type of muramic acids in the cell wall was determined as described by Uchida and Aida (1984).Menaquinones were extracted as reported by Collins et al. (1977) and analysed using high-performance liquid chromatography (HPLC).Cellular fatty acids methyl esters were prepared as described by Sasser (1990) and were analysed using gas chromatography according to the instructions of the Sherlock Microbial Identification System (MIDI).Polar lipids were extracted and analysed by twodimensional TLC (Minnikin et al. 1984).

16S rRNA gene sequence and phylogenetic analyses
The genomic DNA of Micromonospora isolates was extracted from the cultures grown in yeast extract-glucose broth on a rotary shaker as described by Tamaoka and Komagata (1984).The DNA G+C content was determined by reversed phase HPLC (Tamaoka and Komagata, 1984).The 16S rRNA gene was amplified using the universal primers 27F [5'-AGAGTTTGATC(AC) TGGCTCAG-3'] and 1492R [5'-ACGG(CT)TACCTTG TTACGACTT-3'] as described by Weisburg et al. (1991).The PCR products were sequenced using universal primers (Lane, 1991).The closest phylogenetic neighbours were identified by BLAST searches using the EzBioCloud database (www.ezbiocloud.net/)(Yoon et al., 2017).The 16S rRNA gene sequence was manually verified and multiple-aligned with selected sequences.Phylogenetic tree was generated using the neighbour-joining (Saitou and Nei, 1987) in the MEGA 6.0 software (Tamura et al., 2013).Gaps and ambiguous nucleotides were completely eliminated before the calculations.Evolutionary distances among the strains were analysed based on Kimura's two-parameter method (Kimura, 1980) for neighbour-joining tree.The confidence values of nodes were evaluated by the bootstrap resampling method with 1000 replications (Felsenstein, 1985).

Screening of antimicrobial activity of isolates
Each isolate was cultured in 50 mL of seed medium conposing of glucose (0.4%), yeast extract (0.4%), malt extract (1.0%), pH 7.3 (in a 250 mL Erlenmeyer flask) and cultivated on shaker (200 rpm) at room temperature for 4 days.One percent of seed culture was transferred into 200 mL of production medium which comprised glucose (0.4%), yeast extract (0.4%), malt extract (1.0%), and CaCO 3 (0.1%), pH 7.3, incubated on a rotary shaker (200 rpm) at room temperature for 10 days.The antimicrobial activity of the isolated fractions was examined by the agar disc diffusion method (Acar and Goldstein, 1991) against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Kocuria rhizophila ATCC 9341, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 10231 on Mueller-Hinton agar plates at 37°C for 24 h, but the yeast strain was cultivated on Sabouraud's dextrose agar plates at 30°C for 24 h.

CONCLUSION
As part of the research on screening of actinomycete strains from soils in peat swamp forests collected in Narathiwat, Pattaloong and Yala provinces, thirteen isolates including, M. narathiwatensis strains were distributed in Narathiwat, Patthaloong, Yala provinces, the strain of Micromonospora chalcea was found in Yala, Micromonospora humi, Micromonospora aurantiaca strains were distributed in Narathiwat and Patthaloong provinces, while Micromonospora maritima strain was distributed in Patthaloong province.Two isolates in Group 4 expressed antimicrobial activity against Bacillus subtilis ATCC 6633 and Kocuria rhizophila ATCC 9341.This evidence showed that the new soil sources are useful for the investigation of new microorganisms and their secondary metabolites.

FINANCIAL SUPPORT AND SPONSORSHIP
This study was supported by the Research Grant of Faculty of Pharmaceutical Sciences, Chulalongkorn University and the scholarship from the Royal Golden Jubilee Ph.D. Program to C. T.

CONFLICT OF INTERESTS
There are no conflicts of interest.

Fig. 1 :
Fig. 1: Neighbour-joining tree based on distance analysis representing relationship between the partial 16S rRNA gene sequences of 13 Micromonospora isolates and the nearest relatives obtained from EzTaxon.

Table 1 :
Isolate number, sources of isolation, pH of soil, cultural characteristics, similarity (%) of 16S rRNA gene sequence similarity and identification of Micromonospora strains.

Table 3 :
Cellular fatty acid profiles of Miromonospora strains.