|Year : 2019 | Volume
| Issue : 4 | Page : 195-199
Potential antimicrobial properties of streptomyces isolated from Sammuk Mountain soil, Chonburi Province, Thailand
Paranee Sripreechasak1, Anan Athipornchai2
1 Department of Biotechnology, Faculty of Science, Burapha University, Bangsaen, Chonburi, Thailand
2 Department of Chemistry, Center of Excellence for Innovation in Chemistry, Faculty of Science, Burapha University, Bangsaen, Chonburi, Thailand
|Date of Web Publication||1-Oct-2019|
Dr. Anan Athipornchai
Department of Chemistry, Center of Excellence for Innovation in Chemistry, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131
Source of Support: None, Conflict of Interest: None
An infection of pathogenic microorganisms can create a big problem for human health. This has triggered the need for discovery and development of antibiotic drugs with altered modes of action. Approximately 45% of antibiotic drugs are derived from Streptomyces, which are the most commonly isolated actinomycete genera. The present investigation aimed to search for and study antimicrobial-producing actinomycetes isolated from soil samples from Sammuk Mountain, Chonburi province, Thailand. A total of 50 isolates from six soil samples were obtained and their actinomycetes were better isolated using humic acid-vitamin agar medium (64.0%) than starch casein nitrate agar medium (36.0%). In addition, the secondary metabolites produced from 13 isolates (26.0%) exhibited a broad spectrum of antimicrobial activity against Gram-positive bacteria and yeast. Thus, Sammuk Mountain soil is an important source of antibiotic-producing actinomycetes.
Keywords: Actinomycetes, antibiotic, antimicrobial activity, multidrug resistant
|How to cite this article:|
Sripreechasak P, Athipornchai A. Potential antimicrobial properties of streptomyces isolated from Sammuk Mountain soil, Chonburi Province, Thailand. J Adv Pharm Technol Res 2019;10:195-9
|How to cite this URL:|
Sripreechasak P, Athipornchai A. Potential antimicrobial properties of streptomyces isolated from Sammuk Mountain soil, Chonburi Province, Thailand. J Adv Pharm Technol Res [serial online] 2019 [cited 2021 Jan 20];10:195-9. Available from: https://www.japtr.org/text.asp?2019/10/4/195/268454
| Introduction|| |
Diseases caused by infections of pathogenic microorganisms such as diarrhea, gastrointestinal and urogenital diseases, and wound contamination are increasing and becoming a problem for human health because of the emergence of multidrug-resistant pathogens. It has been estimated that the multidrug-resistant Gram-positive and Gram-negative bacteria such as Enterobacteriaceae (carbapenem) and Enterococcus (vancomycin) together with Salmonella More Details enterica, Staphylococcus aureus(methicillin), Pseudomonas aeruginosa, Clostridium difficile, and Escherichia More Details coli could cause up to 10 million deaths/annum by 2050. This has triggered the need for the discovery and development of antibiotic drugs with altered modes of action. Natural products, including plants, microorganisms, and their compounds, have been utilized to treat and cure several diseases such as diarrhea, cancer, diabetes, Alzheimer's, anti-inflammatory, analgesic and antipyretic solutions, and as alternatives for hormone replacement therapy., In particular, microorganisms have an ability to produce a wide variety of bioactive metabolites, especially antibiotic agents. It has been recently reported that over 10,000 bioactive natural antibiotics such as penicillin, tetracycline, gentamicin, vancomycin, and pimaricin obtained from microbes were produced by actinomycetes.,, These are an extensive and diverse group of Gram-positive aerobic bacteria usually grow by filament formation, and they are one of the major microbial populations present in soils. Moreover, approximately 45% of these agents derived from Streptomyces which are the most commonly isolated actinomycete genera. Therefore, actinomycetes play an important natural resources role for producing new antimicrobial agents. In 2014, Sripreechasak et al. reported the isolation and identification of Streptomyces strains which were isolated from 13 soil samples collected around the Angthong Islands National Park, Thailand. All isolated Streptomyces were screened and evaluated for antimicrobial activity. All of these strains showed good inhibitory activity against Bacillus subtilis ATCC 6633, Kocuria rhizophila ATCC 9341, Mucor racemosus IFO 4581, Candida albicans KF1, E. coli NIHJ KB213, and Xanthomonas campestris pv. oryzae KB88. They also reported the isolation of two new naphthoquinones and 17 known metabolites from Streptomyces sp. BCC71188 isolated from soil. Some of the isolated compounds showed strong antimicrobial activities such as antimalarial, anti-tuberculosis and antibacterial activities, and cytotoxicity against MCF-7, KB, and NCI-H187. In an attempt to address the serious public health problem of the infectious disease, the present investigation aimed to search for and study antimicrobial-producing actinomycetes from soil samples from Sammuk Mountain, Chonburi province, Thailand.
| Materials and Methods|| |
Sample processing and isolation of actinomycetes
Six soil samples were collected around Sammuk Mountain (SM1-6), Chonburi province, Thailand. The samples were dried by heating at 100°C for 1 h, suspended in basic lauryl-sulfate buffer solution and heated at 60°C for 5 min. Serial 10-fold dilutions of the suspension were prepared and 0.1 mL of 10−2, 10−3 and 10−4 dilutions were spread on the surface of starch casein nitrate agar (SCN agar: starch 0.1%, sodium caseinate 0.03%, KNO30.2%, and agar 1.5%, pH 7.0) and humic acid-vitamin agar (HV agar: humic acid 0.1%, Na2 HPO40.05%, KCl 0.17 g, MgSO4.7H2O 0.005%, FeSO4.7H2O 0.001 g, CaCl20.1%, B-Vitamins including 0.05% each of thiamine-hydrochloride, riboflavin, niacin, pyridoxine, capantothenate, inositol, p-aminobenzoic acid, and 0.025% of biotin and agar 1.8% g, pH 7.4). Each agar plate was supplemented with antibiotics, 50 mg/L of cycloheximide and 20 mg/L of nalidixic acid. The different colonies were picked up after incubation at room temperature for 14 days, and these plates were streaked for further purification on yeast extract-malt extract agar (International Streptomyces Project (ISP) medium no. 2). The pure isolates were observed for their cultural characteristics after cultivation on ISP 2 agar (yeast extract 4.0%, malt extract 10.0%, dextrose 4.0%, and agar 20.0%, pH 7.3) at 28°C for 2 weeks. All pure isolates were kept in 20% (v/v) glycerol solution at −20°C until further use.
Screening of antimicrobial activity of pure isolates
The pure isolates were screened for antimicrobial activity against six microorganisms, including B. subtilis ATCC 6633, Micrococcus luteus ATCC 9341, S. aureus ATCC 25925, P. aeruginosa ATCC 27853, C. albicans ATCC 10231, and E. coli ATCC 25922 using the perpendicular streak method on ISP 2 agar medium., The results were conducted in triplicate and averaged. All pathogenic microorganisms were kindly supplied by Prof. Dr. Somboon Tanasupawat, Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Science, Chulalongkorn University.
Morphological characterization and identification of active isolates
All pure isolates were inoculated on ISP 2 media and incubated for 14 days at room temperature. Colony morphology was observed under a microscope and color, aerial and substrate mycelium were noted., The 16S rRNA gene was amplified using the primers described by Takahashi et al. The polymerase chain reaction products were checked by agarose gel electrophoresis and directly sequence using a BigDye ® Terminator V3.1 cycle sequencing kit (Applied Biosystems), according to the manufacturer's instructions.
Antimicrobial metabolites production of pure isolates
Each isolate was cultured in ISP 2 medium and shaken (150 rpm) at room temperature for 3 days. Two percent of the ISP 2 culture was transferred into 200 mL of new ISP 2 medium and also cultivated on a shaker for 9 days. The cultured broth was then evaluated for antimicrobial activity using an agar well diffusion assay., All analyses in this study were performed in three replicates.
| Results and Discussion|| |
Isolation and characterization of the isolates
As shown in the supporting information [Table S1], a total of 50 isolates obtained from six soil samples collected from Sammuk Mountain were studied. Actinomycetes cultures were isolated more using HV agar medium (64.0%) than SCN agar medium (36.0%). Thus, HV agar medium was found to be suitable for isolating actinomycetes from these soil samples. [Table 1] summarizes the morphological patterns of the active isolates actinomycetes. All of the isolates growth was found to be in good condition and showed the color appearance of individual isolates. The 16S rDNA sequence was generated for most potent isolates. Comparison of this nucleotide sequence with members of actinomycetes clearly showed that these strains belong to the genus Streptomyces.
Antimicrobial activity of the isolates
On screening all of the isolates for antimicrobial activity, using the perpendicular streak method on ISP 2 agar medium, 13 isolates (26.0%) showed strong inhibitory activity against human pathogens [Table 2]. The isolate SM2-HV8 showed good inhibition against C. albicans with the inhibition zone being 15.0 ± 0.00 mm, whereas SM6-SCN5 showed inhibitory activity against Gram-negative bacteria, P. aeruginosa (8.0 ± 1.00 mm).
|Table 2: Antimicrobial activity of the isolates using the perpendicular streak method|
Click here to view
The isolates were then fermented in ISP 2 medium to produce the secondary metabolites. The cultured broths were evaluated for their antimicrobial activity using an agar well diffusion assay and the results are shown in [Table 3]. Thirteen isolates (26.0%) out of 50 actinomycetes exhibited a broad spectrum of antimicrobial activity against Gram-positive bacteria. The isolates SM1-HV5 and SM1-SCN8 specifically inhibited against C. albicans with the inhibition zone being 8.7 ± 0.58 and 10.0 ± 1.00 mm, respectively.
|Table 3: Antimicrobial activity of secondary metabolites produced from actinomycetes|
Click here to view
In addition, all isolates could not inhibit Gram-negative bacteria, P. aeruginosa and E. coli. As shown in [Figure 1], all isolates showed moderate-to-high inhibitory activity to M. luteus (79.92%), B. subtilis (69.23%) and S. aureus (46.15%), whereas their isolates showed moderate activity against C. albicans (15.38%). The isolates obtained from HV agar medium were more active than from SCN agar medium, as shown in [Figure 2]. Thus, actinomycetes isolated from HV agar medium could be suitable for producing bioactive metabolites against Gram-positive bacteria. This result was confirmed by Hayakawa and Nonomura  who found that HV agar medium supported adequate growth and good sporulation for these actinomycetes because it contained soil humic acid as the sole source of carbon and nitrogen.
|Figure 1: Antimicrobial activity of secondary metabolites produced from actinomycetes|
Click here to view
|Figure 2: Antimicrobial activity of actinomycetes isolated by using humic vitamin and starch casein nitrate medium|
Click here to view
| Conclusions|| |
Of the 50 isolates obtained from six soil samples, 64.0% of their actinomycetes were isolated using HV agar medium compared to 36.0% using SCN agar medium. The HV agar medium contained soil humic acid as the sole source of carbon and nitrogen. In addition, the secondary metabolites produced from 13 isolates (26.0%) exhibited a broad spectrum of antimicrobial activity against Gram-positive bacteria and yeast. Thus, Sammuk Mountain soil from Chonburi province, Thailand, is an important source for exploration of antibiotic-producing actinomycetes.
The authors extend their appreciation to the Faculty of Science, Burapha University, for funding the work through the research group project No.: 20/2560. We thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Department of Chemistry and Department of Biotechnology, Faculty of Science, Burapha University for providing research facilities. Special thanks to Professor Dr. Ronald Beckett for comments and grammatical suggestions on the manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Devadass JB, Paulraj GM, Ignacimuthu S, Theoder AS, Al Dhabi NA. Antimicrobial activity of soil actinomycetes isolated from Western Ghats in Tamil Nadu, India. J Bacteriol Mycol 2016;3:224-32.
Brunel AS, Guery B. Multidrug resistant (or antimicrobial-resistant) pathogens – Alternatives to new antibiotics? Swiss Med Wkly 2017;147:w14553.
Liao JF, Jan YM, Huang SY, Wang HH, Yu LL, Chen CF. Evaluation with receptor binding assay on the water extracts of ten CNS-active Chinese herbal drugs. Proc Natl Sci Counc Repub China B 1995;19:151-8.
Li JX, Yu ZY. Cimicifugae
rhizoma: From origins, bioactive constituents to clinical outcomes. Curr Med Chem 2006;13:2927-51.
Okudoh VI, Wallis FM. Antimicrobial activity of rare actinomycetes isolated from natural habitats in KwaZulu-Natal, South Africa. S Afr J Sci 2007;103:216-22.
Elisha IL, Botha FS, McGaw LJ, Eloff JN. The antibacterial activity of extracts of nine plant species with good activity against Escherichia coli
against five other bacteria and cytotoxicity of extracts. BMC Complement Altern Med 2017;17:133.
Elbendary AA, Hessain AM, El-Hariri MD, Seida AA, Moussa IM, Mubarak AS, et al
. Isolation of antimicrobial producing actinobacteria from soil samples. Saudi J Biol Sci 2018;25:44-6.
Silambarasan S, Kumar EP, Murugan T, Saravanan D, Balagurunathan R. Antibacterial and antifungal activities of actinobacteria isolated from Rathnagili hills. J Appl Pharm Sci 2012;2:99-103.
Sripreechasak P, Suwanborirux K, Tanasupawat S. Characterization and antimicrobial activity of Streptomyces strains from soils in Southern Thailand. J Appl Pharm Sci 2014;4:24-31.
Supong K, Sripreechasak P, Tanasupawat S, Danwisetkanjana K, Rachtawee P, Pittayakhajonwut P. Investigation on antimicrobial agents of the terrestrial Streptomyces
sp. BCC71188. Appl Microbiol Biotechnol 2017;101:533-43.
Shirling EB, Gottlieb D. Methods for characterization of Streptomyces
species. Int J Syst Bacteriol 1996;16:313-40.
Wadetwar RN, Patil AT. Isolation and characterization of bioactive actinomycetes from soil in and around Nagpur. Inter J Pharm Sci Res 2013;4:1428-433.
Usha R, Kingsley BA. Streptomyces
Sps – A promising source of antimicrobial agent. J Anal Pharm Res 2018;7:323-8.
Sharma D, Kaur T, Chadha BS, Manhas RK. Antimicrobial activity of actinomycetes against multidrug resistant Staphylococcus aureus
, E. coli
and various other pathogens. Trop J Pharm Res 2011;10:801-8.
Charousová I, Medo J, Halenárová E, Javoreková S. Antimicrobial and enzymatic activity of actinomycetes isolated from soils of coastal islands. J Adv Pharm Technol Res 2019;8:46-51.
Takahashi Y, Matsumoto A, Seino A, Ueno J, Iwai Y, Omura S. Streptomyces avermectinius
sp. Nov. an avermectin-producing strain. Int J Syst Evol Microbiol 2002;52:2163-8.
Magaldi S, Mata-Essayag S, Hartung de Capriles C, Perez C, Colella MT, Olaizola C, et al.
Well diffusion for antifungal susceptibility testing. Int J Infect Dis 2004;8:39-45.
Valgas C, Souza SM, Smânia EF, Smânia A Jr. Screening methods to determine antibacterial activity of natural products. Braz J Microbiol 2007;38:369-80.
Hayakawa M, Nonomura H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol 1987;65:501-9.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]