Isolation and Screening of Antibiotics Producing Streptomyces spp from the Soil Collected around the Root of Alnus nepalensis from Godawari

©NJB, Biotechnology Society of Nepal 46 Nepjol.info/index.php/njb Isolation and Screening of Antibiotics Producing Streptomyces spp from the Soil Collected around the Root of Alnus nepalensis from Godawari Amina Baniya 1,2, Sushma Singh 1,2, Minu Singh 1,2, Pragya Nepal 1, Mahesh Adhikari 1, Sagar Aryal2, Anurag Adhikari 2* 1 Asian Institute of Management and Technology, Lalitpur, Nepal 2 Kathmandu Research Institute for Biological Sciences, Lalitpur, Nepal Abstract Actinomycetes are considered as the most invaluable prokaryotes whose genome mining show a great number of putative secondary metabolite biosynthesis pathways as well as gene clusters ranging from 20 to 50 per genome. The genus Streptomyces has been explored for its ability to produce 60% antibiotics worldwide. Alnus nepalensis (Alder) has been found to harbor diverse Eubacteria in its rhizosphere. To evaluate the antibiotic production potential from Actinomycetes, we collected soil samples from rhizosphere (5-7 cm deep) of Alder tree. Primary screening was done by cross-streak method against Multidrug Resistant (MDR) such as Methicillin resistant Staphylococcus auereus (MRSA), Vancomycin resistant Enterococcus feacalis (VRE), Imepenem resistant Acinetobacterbaumannii, Vancomycin resistant Klebsiella pneumonia and Imepenem resistant E. coli as well as Non-MDRs (E. coli, Bacillus subtilis, Klebsiella pneumoniae, S. aeureu and Enterococcus feacalis). Extraction of antibiotics was done using rota-vapour from extract obtained by solid-substrate fermentation technique followed by solvent extraction. Secondary screening was done using well diffusion assay against MDRs. Among total of 40 isolates of Actinomycetes recovered, 14 showed remarkable zone of inhibition (ZOI) to various MDRs. NASA 303 showed 26 mm of ZOI against VRE, NASA 101 had ZOI of 34 mm against MRSA, NASA 319 had 33.7 mm ZOI against Imepenem resistant E. coli, NASA 306 had 36 mm of ZOI against Vancomycin resistant Klebsiella pneumoniae, and NASA 108 showed ZOI of 29.6 mm against Imepenem resistant E. faecalis. This investigation revealed that the Actinomycetes found in Rhizosphere of Alder tree had MDR killing potent antibiotics, which needs to be further explored.


Introduction
Nepal is a narrow rectangular country in the Himalayas.It is bounded by cold, arid Tibetan plateau in north and hot humid planes to the south.Being a transition zone between the two extremes of north and south, the vegetation and the microbes in this goldilocks zone has thrived spectacularly.The alder (Alnus nepalensis) of Nepal [1] is one of the examples for the diversified ecosystem, which houses amazing diversity of microbes within its rhizosphere region.Alder is semi-deciduous tree; present throughout the Himalaya at 500-3000m elevation from Pakistan through Nepal and Bhutan to Yunnan in southwest China.Moreover, the Alder houses microbes capable of producing antimicrobial metabolites that prevents it from being infected by different pathogenic fungi and bacteria.Its rhizosphere can be screened in-search of novel antibiotic producing microbes [2].Actinomycetes are filamentous bacteria that belong to the phyla Actinobacteria and the order Actinomycetales.Actinomycetes are considered as the most invaluable prokaryotes in medicinal and biotechnology industries because of their ability to produce number of bioactive molecules, particularly of the antibiotic compounds.Streptomyces that belong to the genus Actinomycetes has been considered for the production of 60% of the antibiotics [3][4][5].The rapidly decreasing costs of genome sequencing has made genome mining, an invaluable resource for drug discovery: A great number of putative secondary metabolite biosynthesis pathways have been discovered using these genome data [6,7].In Actinomycetes species, about 20-50 gene clusters per genome encoding pathways for secondary metabolite biosynthesis are present [6,8].Intriguingly, a large number of these pathways are cryptic: they are not expressed under standard laboratory conditions, and their products are therefore unknown.Exciting proof-of-principle successes have already been achieved in awakening cryptic secondary metabolites [10].It has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media (in-vitro) and remain unexplored [9].The rRNA and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species.Accessing this "missing" microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today [10].In recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species [11].However, by and large, the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives [12].One of the oldest unresolved microbiological phenomena is why only a small fraction of the microbiological population grows on artificial media.The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty.Here we utilize this approach and develop a novel platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments.We followed the simple method of isolation of Actinomycetes by using the selective media starch casein agar.Microbial derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of existing ones are formidable challenges.Bacteria are known producers of metabolites used in pharmaceuticals and industries respectively.However the emergence of multidrug resistant pathogens has rekindled the need to discover new antimicrobials from the environment.Among many metabolites, antibiotics are one produced by Streptomyces spp.which is known to be found around the rhizosphere of Alnus nepalensis.The findings of this study will therefore shed light on isolating and screening of Streptomyces spp. that produces antibiotic from the soil around the root of Alnus nepalensis.The world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era.There is an urgent need to investigate alternative treatment options while there are still a few antibiotics left.New resistance mechanisms emerge and spread globally threatening our ability to treat common infectious diseases, resulting in death and disability of individuals who, until recently, could continue a normal course of life.Without effective anti-infective treatment, many standard medical treatments will fail or turn into very high risk procedures.Infections caused by resistant microorganisms often fail to respond to the standard treatment, resulting in prolonged illness, higher health care expenditures, and a greater risk of death.When infections become resistant to first-line drugs, more expensive therapies must be used.A longer duration of illness and treatment, often in hospitals, increases health care costs as well as the economic burden on families and societies.The achievements of modern medicine are put at risk by antimicrobial resistance.Without effective antimicrobials for prevention and treatment of infections, the success of organ transplantation, cancer chemotherapy and major surgery would be compromised.Thus, world urgently needs new and more than ever effective antimicrobials in order to treat the ever-increasing threat of antibiotics resistance.

Sample collection and growth of Actinomycetes
One gram soil sample was collected from random rhizosphere region of Alder tree, situated at an altitude of 1580 msl and brought to laboratory for optimization by dry heat (100° C for 1 hour) and wet heat (80° C for 40 minutes) methods.Starch Casein Agar (SCA) media was taken as selective media and 10-fold serial dilution technique was followed using NaCl.Spread plate technique was performed and isolated colonies of Actinomycetes were selected and revived for pure culture.

Isolation and characterization of Actinomycetes
Identification of Actinomycetes: It was done by morphological method.The morphological method consists of macroscopic examination and characterization.
Microscopically the actinomycetes isolates were differentiated by their colony characters, e.g.size, shape, color, consistency etc. Colonies showing white powdery characteristics was identified.Isolated colonies were sub-cultured to obtain pure culture.

Extraction of antibiotics
Solid substrate fermentation: 100 gm of rice grain was selected for fermentation by pouring in jam bottle and mixed with 100 ml buffer solution and autoclaved.40 such jam bottles were made.For each NASA culture inoculation of culture was done in each bottle and left for 10 days at 28°C in environment chamber.This was smashed and dried again in environmental chamber and then smashed rice was dissolved into 200 ml of methanol and ethylacetate on 1:1 ratio.Then this was kept on water bath shaker at 28°C for 24 hrs at 120 rpm.After 24 hrs the solution was filtered from Whatman paper.Extraction using Rota vapor: Filtrate was taken for extraction and crude antibiotic compounds were obtained after use of Rota vapor at 60° temperature in 100 rpm.This crude compound was in the form of liquid and obtained after color phase change.
Secondary screening: Well diffusion method was performed against similar MDR and non-MDR freshly grown in Trypton Soya broth for 3 hours.MHA media plates were made and 1-4 holes were punched; one hole contained solvent (methanol and ethylacetate) in ratio of 1:1.Other 3 holes contained different extract from Rota vapor method.All the plates with solvent and numbers of extract were left for nearly 1-2 hrs for diffusion.Then these plates were swabbed with MDR and Non-MDR microorganisms according to the requirements.Spread plate technique was used for it.One control plate with antibiotic disc placed on MHA media was also prepared.These spread plates with microorganisms were incubated at 37 ° C for 24 hrs.This plating was done for all the extract along with solvent.Then zone of inhibition was observed around extract holes.
= zone of inhibition absent; + = zone of inhibition present .Thus, Actinomycetes are found to produce drugs such as vancomycin, imepenem, etc., due to which we selected Actinomycetes in our project.Isolation of Actinomycetes from soil was done using a different method-pretreatment of soil for 1 week by air drying followed by isolation of Actinomycetes using selective media composed of starch yeast extract agar (SYE), yeast extractmalt extract agar (ISP2), oatmeal agar (ISP3), inorganic salt-starch agar (ISP4), starch-casein agar (SCA), glucose asparagine agar (GAA), Actinomycete isolation agar (AIA) and marine agar (MA)[24], whereas other authors used crowded plate method for isolation of Actinomycetes [25].Microbially derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of It was found that root soil of the analyzed trees such as pine, alder, birch contained more microorganisms than soil distal to the roots.Thus, we selected the rhizosphere of alder tree, the indigenous tree of Nepal.The presented analysis of the diversity of Actinomycetes in the isolation (different soils and rhizosphere of different trees) indicated a lack of significant differences between the microorganisms.The only sources of isolation which were relatively diverse were the root-free soil of birch and the birch rhizosphere.The lowest diversity of Actinomycetes was found in bulk soil and the action rhizal rhizoplane of alder [31].Multidrug resistant bacteria are found in context of Nepal that are entirely native to this geographical location and thus differs from those found in other parts of the world.Hence, we need to screen and isolate Actinomycetes that are native to this environmental condition as well as novel in order combat those MDRs.

Conclusion
In this study, we isolated Actinomycetes from soil around the roots (rhizosphere) of Alder tree.We demonstrated that the rhizosphere of Alder tree houses clinically important microorganisms.Further study of these isolated strains can be done to determine exact molecules responsible for anti-MDR property.Whereas the macroscopic morphology of the isolates could merely provide presumptive genus identification, 16S rRNA assay and subsequent sequencing analysis, in the future, may possibly resolve their species level identity.Since, MDRs are unique to certain geographical locations, Actinomycetes capable of combating such MDRs must be prevailed in that particular environmental condition.Thus, intensive research to isolate such clinically significant Actinomycetes must be encouraged using indigenous natural resources.

Table 1 :
Description of samples collected from different sites of Godawari and number of obtained Actinomycetes

Table 2 :
Primary screening of Actinomycetes for antibacterial activity by perpendicular streak method

Table 3 :
Secondary screening of antibacterial activity of the Actinomycetes isolated

Table 4 :
Morphological feature of obtained Actinomycetes