Niranjan Parajuli Et Al Paper

Journal of Institute of Science and Technology, 24(2), 7-16 (2019) ISSN: 2469-9062 (print), 2467-9240 (e) © IOST, Tribhuvan University Doi: http://doi.org/10.3126/jist.v24i2.27246 Research Article ISOLATION AND CHARACTERIZATION OF SOIL MYXOBACTERIA FROM NEPAL Nabin Rana1, Saraswoti Khadka1, Bishnu Prasad Marasini1, Bishnu Joshi1, Pramod Poudel2, Santosh Khanal1, Niranjan Parajuli3 1Department of Biotechnology, National College, Tribhuvan University, Naya Bazar, Kathmandu, Nepal 2Research Division, University Grant Commission (UGC-Nepal), Sanothimi, Bhaktapur, Nepal 3Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal *Corresponding author: [email protected] (Received: May 22, 2019; Revised: October 26, 2019; Accepted: November 1, 2019) ABSTRACT Realizing myxobacteria as a potential source of antimicrobial metabolites, we pursued research to isolate myxobacteria showing antimicrobial properties. We have successfully isolated three strains (NR-1, NR-2, NR-3) using the Escherichia coli baiting technique. These isolates showed typical myxobacterial growth characteristics. Phylogenetic analysis showed that all the strains (NR-1, NR-2, NR-3) belong to the family Archangiaceae, suborder Cystobacterineae, and order Myxococcales. Furthermore, 16S rRNA gene sequence similarity searched through BLAST revealed that strain NR-1 showed the closest similarity (91.8 %) to the type strain Vitiosangium cumulatum (NR-156939), NR-2 showed (98.8 %) to the type of Cystobacter badius (NR-043940), and NR-3 showed the closest similarity (83.5 %) to the type of strain Cystobacter fuscus (KP-306730). All isolates showed better growth in 0.5-1 % NaCl and pH around 7.0, whereas no growth was observed at pH 9.0 and below 5.0. All strains showed better growth at 32° C and hydrolyzed starch, whereas casein was efficiently hydrolyzed by NR-1 and NR-2. Besides, preliminary antimicrobial tests from crude extracts showed activities against Gram-positive, Gram-negative bacteria, and fungi. Our findings suggest that the arcane soil habitats of Nepal harbor myxobacteria with the capability to produce diverse antimicrobial activities that may be explored to overcome the rapidly rising global concern about antibiotic resistance. Keywords: Antimicrobial, Cystobacterineae, Myxobacteria, Soil habitat of Nepal (Schäberle & Hack, 2014). Natural products, especially INTRODUCTION microbe-derived compounds, have been proven to be a The emergence of public health-threatening multi-drug good source for antibiotics (Leeds et al., 2006). Currently, resistance (MDR) bacteria and pathogenic fungi caused a anti-infective molecules produced from myxobacteria severe threat to human health and modern medicine have come into focus (Schäberle et al., 2014; Muller & (Butler, 2004). The heavy use of antibiotics and Wink, 2014). They are excellent producers of secondary fungicides in agriculture, food industries, farm animals as metabolites, which are known to exhibit antibacterial, well as unwanted prescriptions of drugs from health antifungal, antiviral, antitumor, immunosuppressant, anti- personnel create an environment for microorganisms, tuberculosis, and antimalarial activities (Weissman & which are nowadays termed as ―superbugs‖, to develop Muller, 2009). resistance against them, (Landers et al., 2012). According Myxobacteria are Gram-negative rod-shaped, aerobic to the US Center for Disease Control and Prevention bacteria belonging to the delta subgroup of Proteobacteria. (CDCP, 2016), superbugs kill 700,000 people annually. If They can be distinguished from other bacteria for their this trend continues, then by 2050 A.D, 10 million people unique gliding motility, swarming colonies on the agar will die each year. The dramatic increment in resistance surface, cooperative feeding behaviors, and produce towards commercially available antibiotics and fungicides fruiting bodies under starvation (Kaiser, 2004; Mauriello demands to explore new chemical entities (Weissman & et al., 2010; Shimkets, 1990; Dawid, 2000). Müller, 2009). However, the pipeline for the development of new antibiotics is limited (Fenical & Jensen, 2006). In search of myxobacteria, a soil sample from a tropical Though there has been increased research towards rain forest was considered to be the best source (Dawid, identifying novel chemical entities from both natural 2000). Furthermore, they have been isolated from products and synthetic molecules, the development of new substrate rich in cellulosic plant material (bark of trees, compounds as drugs are not satisfactory partly to re- rotten wood materials) and from the dung of herbivorous isolation of already identified compounds from natural animals such as rabbit, deer, moose, etc. Based on a products and toxic side effects from the compound biodiversity guided survey, the ecosystem of Nepal ranges developed synthetically (Amin et al., 2012; Lam, 2006; from tropical to alpine and provides a remarkable Ishida et al., 2012). Therefore, research to identify new ecological diversity. Thus, screening of myxobacteria has putative antibiotics has to be pursued and intensified immense potential for isolating new bacteria that produce Isolation and characterization of soil myxobacteria from Nepal novel bioactive metabolites that hold tremendous promise the myxobacteria according to the protocol previously for human therapy against infectious diseases. developed by Reichenbach and Dworkin (1992). The organisms were stained by Gram's stain as well as (0.01 % According to Reichenbach (1999), there is a possibility of w/v) Congo-red aqueous solution flooded on five days’ finding novel myxobacteria choosing a unique sampling culture plates (McCurdy, 1969). For cryopreservation of site, exhibiting significant biological complexity. The pure strains, pieces of agar taken from an actively isolation methods and culture media could be designed to growing swarm colony were placed in cryotube vial and mimic the natural environment during cultivation. stored at -70 C at National College, affiliated to Myxobacteria are highly specialized in bio- Tribhuvan University, Nepal. macromolecules degradation, and they require various nutrients for their growth and survival (Reichenbach, Growth response to temperature, pH, salt 1999; Thaxter, 1904). Hence baiting with E. coli and inoculation of filter paper with soil are the favorable Growth response at different temperatures; 37 C, 32 C, methods to isolate myxobacterial species (Reichenbach, 30 C, 28 C, 25 C, 18 C and 4 C, at various pH; 5.5, 2005; Garcia et al., 2009), predatory as well as cellulose- 6.0, 6.5, 7.2, 8.4 and 9.0 and at salt concentration; 0 %, decomposing genera. This research was focused on the 0.2 %, 0.5 %, 1.0 %, 2.0 %, 3.0 %, 4.0 % and 5.0 % (w/v) isolation of myxobacteria from Nepalese soil and was tested in VY/2 agar medium adjusting the pH, salt characterized them based on morphological and molecular concentration accordingly Garcia et al. (2009). The methods. relative growth was determined by measuring the diameter of swarms in successive 1, 4, 6, 10, and 12th day MATERIALS AND METHODS (McDonald, 1967). Sample collection Physiological tests From different geographical regions of Nepal, soil Casein hydrolysis was determined by the agar well samples were taken from the upper part, digging a few diffusion method on a medium containing 2 % (v/v) skim centimeters down from the soil profile. Southern, milk after incubating 24 hrs at 32 C (Isolauri et al., Western, and Eastern provinces were chosen to isolate the 1995). Starch hydrolysis was detected on the medium myxobacterial strains having diverse biological containing 0.2 % (w/v) solution of potato starch, and the characteristics. Samples were collected on a sterile plastic reaction was determined after flooding 4-week old culture zip bag and transported to the laboratory in dried form with a 2 mL (0.01 M) iodine solution (Harley, Dawid (2000). Myxobacteria were screened from those 2004). Chitin decomposition was tested using synthetic S soil samples collected from different parts of Nepal. agar with 0.7 % (w/v) chitin as the sole nutrient source (Reichenbach, 2005). Culture media CY Agar: Casitone (Difco), 0.3 %; Yeast extract (Difco), Microbial predation tests 0.1 %; CaCl2.2H2O, 0.1 %; Agar, 1.5 % (w/v); pH to 7.2. Overnight culture of E. coli ATCC 25922, Klebsiella VY/2 Agar: Bakers’ Yeast (Commercial yeast cake), 0.5 pneumoniae ATCC 700603, Staphylococcus aureus %; CaCl2.2H2O, 0.1 %; Cyanocobalamin 0.5mg/L; Agar, ATCC 25923, Salmonella typhimurium ATCC 14028, and 1.5 % (w/v) pH to 7.2. WCX: CaCl2.2H2O, 0.1 %; Agar, 36 hrs old culture of Streptomyces coelicolor A3(2) & 1.5 %; HEPES (N-2-(hydroxyl-ethyl) piperazine-N’-2- Saccharomyces cerevisiae were spotted on water buffered ethane sulfonic acid), 20 mM, 25 µg/mL of cycloheximide agar. The growth of the myxobacteria and bacterial lysis, was used for the isolation of myxobacteria. Chitin as indicated by swarm spreading on the spotted pellet of decomposition was tested using synthetic S agar (0.5 g the above-mentioned culture, was observed on a phase- CaCl2.2H2O, 0.5 g MgSO4.7H2O, 0.06 g K2HPO4, 8 mg contrast microscope. Ferric EDTA, 50 mM HEPES, 1-liter distilled water; pH DNA Isolation and PCR Amplification 7.2) with 0.7 % (w/v) chitin as the sole nutrient source. Genomic DNA was extracted from the actively growing E. coli baiting technique eight days old culture as described by Wilson (2001). The Myxobacteria were isolated by the baiting technique from ethanol precipitated DNA

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