Screening of Secondary Metabolites Produced by Streptomyces Species from a Soil Sample that Can Produce Anti-Nematodal and Antiprotozoal Avermectins

Avermectins are a group of secondary metabolites produced by Streptomyces avermitilis, which act on invertebrates. They activate glutamate-gated chloride channels in their nerves and muscles which in turn disrupt pharyngeal function and locomotion. Avermectin ingested insects are paralyzed and starve to death. Ten isolates identified as avermectin producers were characterized by morphological, colony characters and biochemical tests. Secondary screening leads to the identification of four isolates PM2; PM4; PM7 and PM10 which produced 10, 8.4, 3.8 and 6.9mg respectively as identified by HPTLC. Bio-autography illustrated their anti-nematodal and antiprotozoal activity; the zone of clearance (turbid) was recorded 43, 24, 32 and 37 mm respectively for PM2 through PM10. Original Research Article Sharma et al.; JPRI, 33(6): 90-98, 2021; Article no.JPRI.65484 91


INTRODUCTION
Soil is the habitat for several microorganisms which include bacteria, actinomycete, fungi, and algae. Soil microbes are an outstanding source for the isolation and identification of therapeutically important products [1].
Actinomycetes produce a large number of antibiotics. These Antibiotics have been reported to possess bactericidal, fungicidal, neuritogenic, anticancer, antipyretic, analgesic, anti-protozoal and anti-nematodal activities. These antibiotics can be purified from a single genus Streptomyces [2]. The genus Streptomyces was introduced by Waksman and Henrici in 1943 [3]. This Genus belongs to the Streptomycetaceae family [4]. More than 500 species of the genus Streptomyces have been described and most if not all Streptomyces's Species are antibiotic producers [5].
Streptomyces species are chemoorganotrophic, filamentous, Gram-positive organisms that are present in a wide variety of soil in rhizospere, dead and decay litter containing soil [6]. These are nonacid-alcohol fast; since these are present in dead and decay containing soil they share habitat with fungi, and appear very similar to fungi [7].
The colonies are slow-growing, having high G+C content 69-78% in their DNA [8]. These organisms have a soil-like odor due to the production of a volatile metabolite, geosmin. Streptomyces produces Avermectin. The avermectins are a potential candidate for the anti-helminthic and insecticidal agent. The same genus is also reported producing herbicidal, insecticidal, and nematocidal activities [9].
The avermectins were discovered in the early 1980s. Their selective toxicity towards nematodes and arthropods at very dilute doses, but relatively low or no toxicity to mammals make them potential future drugs. In invertebrates, they activate glutamate-gated chloride channels in their nerves and muscles and disrupt pharyngeal function and locomotion thereby causing paralysis. Avermectin ingested insects are paralyzed and starve to death. The selective toxicity that is, they do not harm vertebrates, has led to the conclusion that avermectins affect a specific cellular target which is either missing or out of reach in the resistant organisms [10].
Avermectin B1a is an indispensable drug in mass treatment programs to eradicate two widespread serious diseases namely river blindness and lymphatic filariasis, caused by nematodes, that affect millions of people worldwide [10].

Isolation
Isolation of Streptomyces species was done from agricultural soil sample situated 18.150663 and 74.576782 with a GPS coordinates of 180 9' 2.3868" N and 740 34' 36.4152" E. The soil sample was collected at least 3-5 cm deep from thesoil.
The isolation and enumeration of Streptomyces were performed by serial dilution and spread plate method, yeast malt extract glucose agar medium (YMG agar) was used. Incubation was done at 28 C. Individual colonies growing on YMG agar were transferred on Streptomyces isolation agar medium for purification of Streptomyces [11].

Identification
The classification and identification of microorganisms are based on morphology, colony and biochemical characteristics [12]. This includes colony characters, detailed morphology, Gram's staining, motility, biochemicalcharacteristics.

Seedmedium
The cultures were maintained on the YMG agar medium. Loop full culture of the different species was streaked on agar slant and inoculated into 50mL of yeast malt glucose (YMG) medium. The fermentation broth was incubated in an orbital shaker at 150rpm for 16-18 hours at 30°C [7].

Avermectin production
Production of avermectin from the soil isolates was studied in synthetic medium 2 (SM2) growths medium. Each production medium was inoculated with 5 mL of inoculum separately. After transferring the seed medium, each growth medium was incubatedat 30°C inashaker for 10days at 150rpm [6].

Elicitation
Since avermectins are secondary metabolites and produced in very low quantity and intracellular (Periplasmic space). Avermectins are produced in response to nematode growth in the territory of Streptomyces species. The nematodes were immobilized in 0.8% agarose and these beads were introduced in fermentation broth to elicit the production of Avermectins.

Extraction of avermectin
The fermentation broths were centrifuged at 4°C for 20 minutes at 6000rpm. the cell biomass was separated. The cell biomass in the form of the pellet was mixed with an appropriate amount of methanol to completely dissolve it. The mixture was centrifuged again and the supernatant was collected for avermectin analysis by highperformance thin-layer chromatography HPTLC[11].

Determined of Avermectin Concentration by High-performance Thin-layerChromatography
The ten purified samples were analyzed for avermectin drug content. The sample Application was done by Linomat 5. Five microliters of the ten samples were spotted onto the plate of pre-coated with silica gel 60F-254 (20cm×10cm) was used as a stationary phase. The Mobile phase was n-hexane: acetone: ethyl acetate (13:7: 0.2 v/v/v). The plated were developed in Twin trough chamber (CAMAG) Densitometeric scanning was done from 190nm to 500nm in absorbance mode with Camag TLC scanner III, using deuterium and tungsten lamp operated by win CATS software (Version 1.2.0). The slit dimension of 8.00 mm × 0.60 mm and a scanning speed of 20 mm s −1 were set [13].

Bio-autography
To evaluate the anti-nematode and antiprotozoal activity of avermectins the TLC plate was overlaid by 1% agarose containing the nematodes and protozoa and incubated at 37 C. the appearance of turbid spot indicated the death of nematodes and protozoa. The zone of turbidity wasmeasured.

Isolation
Initially, colonies were relatively smoothsurfaced but later they develop a mesh of aerial mycelium and appear floccose, granular.

Identification
Physical parameters such as their tolerance to pH, temperature were evaluated. The organisms' best grow at neutral pH ranging from 7 to 7.4 and temperature 28 C to 35 C. The other physical parameters and their results are tabulated.  Similarly in an attempt to classify the organisms following Biochemical tests were performedcarbohydrate namely, glucose, lactose, maltose, potato starch. The ability to utilize citrate, liquefy gelatin. Catalase test, which evaluate the ability of organisms' respiration using oxygen, and protects them from toxic byproducts of oxygen metabolism i.e. reactive oxygen species (ROS). If the microorganism is capable of reducing the Sulphur compound to sulfides was determined by the H2S productiontest.

Image 1: Streptomyces growing on YMG agar plates (PrimaryScreening)
A positive test was denoted by + sign and negative test by -sign, ++ was strongly positive and +sign within circle indicated the organism can ferment the sugar aerobically i.e. by producing acid and gas (in form of a bubble in Durham's tube). Standard protocols were performed as mentioned in Bergey's Manual.

Fermentation Process
50 ml of synthetic medium 2 (SM2) was seeded by 5ml YGM media containing PM1 to PM10 isolates in previously sterilized 330ml glass bottles. These were incubated at 30 C in orbitek shaker incubator at 105rpm.Turbidityindictedgrowthwhichwasassess edspectrophotometericallyat600nm.
The area under the curve as obtained by densitometry for pure avermectin and thatof an obtained peak was 875.8, 736.1, 334.0 and 550.1mm squared for PM2, PM4, PM7, and PM10 respectively. Thus the concentrations of avermectins for the four isolates in the same sequence were 0.200, 0.168, 0.076 and 0.125mg/ml. Thus 50ml broth contained 10, 8.4, 3.8 and 6.9mg avermectins for PM2, PM4, PM7, and PM10 respectively. As shown in thegraph.

CONCLUSION
Ten isolates of Streptomyces were obtained after primary screening of soil, post fermentation process and HPTLC analysis revealed that isolates PM2, PM4, PM7, and, PM10 produced 10 mg, 8.4 mg, 3.8 mg and 6.9 mg of Avermectins/50 ml of fermentation medium. The anti-nematodalactivity was evaluated by Bioautography.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.