Screening Biological Activities of a Streptomyces Species Isolated from soil of Agumbe, Karnataka, India

Prashith Kekuda TR 1*, Shobha KS 1, Onkarappa R 1, Goutham SA 1, Raghavendra HL 2
  1. Department of Studies and Research in Microbiology, Sahyadri Science College (Autonomous), Shivamogga-577203, Karnataka, India.
  2. Faculty of Medical Sciences, Wollega University, Post Box No: 395, Nekemte, Ethiopia.
Corresponding Author: Prashith Kekuda. T.R Email: [email protected]
Received: 13 May 2012 Accepted: 26 June 2012
Citation: Prashith Kekuda T R*, Shobha K S,Onkarappa R, Goutham S A, Raghavendra HL “Screening Biological Activities of a StreptomycesSpecies Isolated from soil of Agumbe, Karnataka,India” Int. J. Drug Dev. & Res., July-September 2012,4(3): 104-114
Copyright: © 2012 IJDDR, Prashith Kekuda TR et al. This is an open access paper distributedunder the copyright agreement with SerialsPublication, which permits unrestricted use,distribution, and reproduction in any medium,provided the original work is properly cited.
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Abstract

The present study was undertaken to screen biological activities namely antimicrobial, insecticidal, pancreatic lipase inhibitory and cytotoxic potential of crude extract of a Streptomyces species isolated from soil of Agumbe, Karnataka, India. Primary and Secondary screening for antimicrobial activity was determined by Cross streak method and Agar well diffusion method respectively. Insecticidal efficacy of extract was tested against 3rd instar larvae of Aedes aegypti. Pancreatic lipase inhibitory activity was determined against chicken pancreatic lipase. The brine shrimp lethality test was conducted to determine cytotoxic nature of crude extract. The actinomycete isolate was identified as a species of the genus Streptomyces on the basis of microscopic, cultural and biochemical characteristics. In both primary and secondary screening, Gram positive bacteria were found most sensitive. Candida albicans has shown high sensitivity when compared to Cryptococcus neoformans. The mortality of the 3rd instar larvae of A. aegypti was found to be concentration dependent and the mortality was 100% at extract concentration 1.5mg/ml and higher. The inhibition of lipase was found to be dose dependent and an inhibition of over 50% was observed at extract concentration 50mg/ml. In cytotoxic assay, the degree of lethality of extract was directly proportional to the concentration. The LC50 of extract was found to be 21.95μg/ml and hence the extract is toxic. The soils of Western ghats are rich sources for microorganisms with potent biological activities and screening programs are to be conducted to reveal the presence of pharmacologically active actinomycete isolates. Further studies are needed to be carried out to isolate and characterize the active principles present in the crude extract and determine their biological activity.

Key words

Streptomyces, Western ghats, Agar well diffusion, Pancreatic lipase, Brine shrimp, Larvicidal activity

INTRODUCTION

Microbes produce some of the most important medicines. They are the sources of drugs for bacterial and fungal infections, transplant rejection, high cholesterol and others. Microbial natural products are notable not only for their potent therapeutic activities but also for the fact that they frequently possess the desirable pharmacokinetic properties required for clinical development [1]. Actinomycetes are the most widely distributed groups of microbes in nature. These are Gram positive bacteria frequently filamentous and sporulating organisms with DNA rich in G+C from 57-75%. They have showed marked chemical and morphological diversity but form a distinct evolutionary line of organisms. Majority of the Actinomycetes are free living, saprophytic bacteria found widely distributed in soil, water and colonizing plants. These organisms are one of the major groups of soil population. Soil Actinomycetes are prokaryotes with extremely various metabolic possibilities. These are prolific producers of various bioactive compounds, and have provided over two third of naturally occurring antibiotics discovered and continue to be major source of novel and useful compounds such as antibiotics, enzymes, pigments, immunomodulators etc [2-6]. The present study was undertaken to screen biological activities namely antimicrobial, insecticidal, pancreatic lipase inhibitory and cytotoxic potential of crude extract of a Streptomyces species isolated from soil of Agumbe, Karnataka, India.

MATERIALS AND METHODS

Isolation and Identification of Actinomycete Isolate

The actinomycete species of this study was isolated from a soil sample of Agumbe, Karnataka, India. The soil sample was collected from a depth of 5cm in sterile pouch. The soil sample was dried, serially diluted and plated on Starch casein agar (HiMedia, Mumbai). The inoculated plates were incubated at 30±2oC for a week aerobically. The isolate was subcultured on fresh medium and identified to genus level based on colony morphology (colony color, distinctive reverse color of vegetative mycelium, production of diffusible pigment, and surface colony appearance), microscopy (cover slip method), staining (Gram’s and Acid fast staining) and biochemical reactions (Starch hydrolysis, gelatin liquefaction, casein hydrolysis, sugar fermentation, hydrogen sulfide production) [7-10]. The isolate was maintained on Starch casein agar slant.

Primary Screening for Antimicrobial Activity of the Isolate

Preliminary screening for inhibitory metabolite producing ability of the isolate was tested by Crossstreak method against two Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis), two Gram negative bacteria (Proteus vulgaris and Pseudomonas aeruginosa) and two human pathogenic fungi (Candida albicans and Cryptococcus neoformans). The isolate was inoculated as a single streak in the centre of the petridish containing media and incubated at 30±2oC for 3-4 days to permit growth and antibiotic production. Later the test bacteria and fungi were inoculated by streaking perpendicular to the growth of isolate. The plates were incubated for 24-48 hours at 37oC in case of bacteria, 72 hours at room temperature in case of fungi. After incubation, inhibition of test bacteria and fungi around the growth of isolate was taken as positive for inhibitory activity [11,12].

Fermentation and Extraction

Loop-full culture of the isolate was inoculated into bottles containing sterile Starch casein broth medium aseptically. The inoculated bottles were incubated at 28oC for 12 days aerobically. After incubation, the contents of the bottles were filtered through sterile Whatmann filter paper No. 1 aseptically. The filtered broths were concentrated to pasty mass by keeping in hot air oven at 40oC [13]. The crude extract was subjected to antimicrobial, insecticidal, pancreatic lipase inhibitory and cytotoxic activity.

UV/Visible absorption of extract

The UV-Visible absorption spectra of the extract was tested by using SHIMADZU UV-2550 spectrophotometer at 200-400nm to determine the λ maximum of the band [14].

Antibacterial Activity of Extract

The antibacterial efficacy of extract was tested by Agar-well-diffusion method [15] against two Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis) and two Gram negative bacteria (Proteus vulgaris and Pseudomonas aeruginosa). Briefly, 24 hours old Nutrient broth (HiMedia, Mumbai) cultures of test bacteria were swabbed uniformly on solidified sterile Nutrient agar (HiMedia, Mumbai) plates using sterile cotton swab. Then, wells of 6mm diameter were bored in the inoculated plates with the help of sterile cork borer and the extract (20mg/ml of sterile water) and Standard (Chloramphenicol, 1mg/ml of sterile water) were added separately into respectively labeled wells. The inoculated plates were incubated at 37°C for 24 hours in upright position and the zone of inhibition formed around the well was measured. The experiment was carried in triplicates to get average reading.

Antifungal Activity of Extract

The antifungal efficacy of extract was tested by Agarwell- diffusion method against two human pathogenic fungi namely Candida albicans and Cryptococcus neoformans [15]. Briefly, 48 hours old Sabouraud dextrose broth (HiMedia, Mumbai) cultures of test fungi were swabbed uniformly on solidified sterile Sabouraud dextrose agar (HiMedia, Mumbai) plates using sterile cotton swab. Then, wells of 6mm diameter were bored in the inoculated plates with the help of sterile cork borer and the extract (20mg/ml of sterile water) and Standard (Amphotericin B, 1mg/ml of sterile water) were added separately into respectively labeled wells. The inoculated plates were incubated at 37°C for 24 hours in upright position and the zone of inhibition formed around the well was measured with a ruler. The experiment was carried in triplicates to get average reading.

Insecticidal Activity of Extract

The insecticidal efficacy of different concentrations of extract was tested against 3rd instar larvae of Aedes aegypti. Twenty larvae were placed separately into beakers containing different concentrations of extract. The larvicidal effect of the extract was determined by counting the number of dead larvae after 24 hours. Dead larvae were identified when they failed to move after probing with a needle in siphon or cervical region. The test was repeated thrice and the percentage of larval mortality for each concentration of extract was calculated [16].

Pancreatic Lipase Inhibitory Activity of Extract

The inhibitory activity of crude extract was tested against lipase extracted from the chicken pancreas. The activity of lipase was determined by incubating an emulsion containing 8ml of olive oil, 0.4ml of phosphate buffer and 1ml of chicken pancreatic lipase for an hour in rotary shaker, followed by stopping the reaction by addition of 1.5ml of a mixture solution containing acetone and 95% ethanol (1:1). The liberated fatty acids were determined by titrating the solution against 0.02M NaOH using phenolphthalein as an indicator. Lipase inhibitory activity of different concentrations of extract was tested by mixing 100μl of each concentration of extract, 8ml of oil emulsion and 1ml of chicken pancreatic lipase followed by incubation of 60 minutes. The reaction was stopped by adding 1.5 ml of a mixture solution containing acetone and 95% ethanol (1:1). The liberated fatty acids were determined by titrating the solution against 0.02M NaOH. The inhibition of lipase activity (%) was calculated using the formula: Lipase inhibition = A- B/A×100, where A is lipase activity, B is activity of lipase when incubated with the extract [17].

Cytotoxic Activity of Extract

Cytotoxic Activity of Extract The brine shrimp lethality test was conducted according to the method of Kekuda et al. [18] to determine cytotoxic nature of crude extract. Brine shrimp Artemia nauplii eggs (Nihon Animal Pharmaceutical Inc., Tokyo, Japan) were hatched in a container filled with air-bubbled artificial sea water which was prepared with 10g of a commercial salt mixture (GEX Inc., Osaka, Japan) and 500 ml of distilled water. After 36-48 hours, the phototropic shrimps were collected by pipette for bioassay. The different concentrations of extract (10-1000μg/ml) were tested in vials containing 5ml of brine and 25 shrimp in each of three replicates. The vials were incubated at 25°C and surviving shrimps were counted after 24 hours. LC50 value was calculated by regression analysis.

RESULTS

Identification of Actinomycete Isolate

The actinomycete isolate was identified as a species of the genus Streptomyces on the basis of microscopic, cultural and biochemical characteristics. The colony was brown in color with white centre. Spores were arranged in long chains (Figure 1). The isolate was Gram positive and nonacid fast. The isolate was found to produce starch hydrolysis and gelatin liquefaction and utilized glycerol. Casein was not hydrolyzed, sucrose and maltose were not utilized and hydrogen sulfide was not produced. The isolate produced brown pigment.

UV-Visible Absorption Maxima of Solvent Extract

The absorption maxima (λmax) of the extract of Streptomyces isolate was found to be 283.5nm.

Primary Screening for Antimicrobial Activity of The Isolate

The result of screening for antimicrobial activity of the isolate by cross-streak method is depicted in Table 1. Result was recorded as the absence of growth of test microbes around the growth of isolate and was taken as ‘+’. All the tested bacteria and fungi were shown to be susceptible as their growth around the isolate was inhibited.

Antibacterial Activity of Extract

The antibacterial activity of extract was investigated against four bacteria by agar well diffusion method. Results were recorded as presence or absence of zones of inhibition around the well. The inhibitory zone around the well indicated the absence of bacterial growth and is reported as positive and the absence of zone as negative. It was found that the extract caused marked inhibition of Gram positive bacteria when compared to Gram negative bacteria. Among bacteria, B. subtilis was inhibited to high extent followed by S. aureus, P. aeruginosa and P. vulgaris. Inhibition caused by standard antibiotic was higher than that of extract (Table 2)

Antifungal Activity of Extract

The antifungal activity of extract was tested by agar well diffusion method. Among fungi, C. albicans was inhibited to higher extent when compared to C. neoformans. Here also, inhibition caused by standard antibiotic was higher than that of extract (Table 3).

Insecticidal Activity of Extract

The insecticidal efficacy of different concentrations of extract was evaluated against 3rd instar larvae of A. aegypti. The mortality of the 3rd instar larvae of A. aegypti was found to be concentration dependent. The mortality was 100% at extract concentration 1.5mg/ml and higher. There was no mortality of larvae in control beakers i.e., beakers containing no extract (Table 4).

Pancreatic Lipase Inhibitory Activity of Extract

Inhibitory activity of different concentrations of extract was tested against chicken pancreatic lipase using olive oil as the substrate. It was observed that the activity of lipase was affected when incubated with the extract. The inhibition of enzyme was found to be dose dependent i.e., inhibition of enzyme was increased on increasing the concentration of extract. An inhibition of >50% was observed at extract concentration 50mg/ml whereas lipase inhibition was not observed at extract concentration 2.5mg/ml (Table 5).

Cytotoxic Activity of Extract (Brine Shrimp Lethality)

The cytotoxic activity of different concentrations of extract, in terms of mortality of brine shrimps, is presented in Table 6. The degree of lethality of extract was directly proportional to the concentration of the extract i.e., higher the concentration, mortality was higher. Highest lethal effect was observed at 1000μg/ml extract concentration at which the mortality was 100%. LC50 values greater than 1000μg/ml were considered inactive (non-toxic). The LC50 of extract was found to be 21.95μg/ml and hence the extract is toxic. Lethal effect was lesser (33%) in case of 10μg/ml extract concentration.

DISCUSSION

Actinomycetes are the abundant organisms that form thread-like filaments in the soil. They form a large group of Gram positive bacteria that grow as hyphae like fungi responsible for the characteristically “earthy” smell of soil. Actinomycetes are belonging to the order Actinomycetales, characterized by the formation of substrate and aerial mycelium on solid media, presence of spores and a high GC content of the DNA. The majority of soil actinomycetes form a very important class of bacteria since they produce numerous natural products such as antibiotics and enzymes. More than 50% of the known natural antibiotics produced are from actinomycetes. Actinomycetes involve in important ecological processes in a wide range of habitats such as degradation of plant and animal in soils, degradation of high molecular weight compounds like hydrocarbons in the polluted soil, biological control of soil environments by nitrogen fixation and production of various secondary metabolites [19-24].
Actinomycetes are very well known and successfully exploited as a source of secondary metabolites. Out of the approximately 10,000 known antibiotics, 45-55 % are produced by streptomycetes. Various antimicrobial substances from actinomycetes have been isolated and characterized including aminoglycosides, anthracyclines, glycopeptides, betalactams, macrolides, nucleosides, peptides, polyenes, polyester, polyketides, actionomycins and tetracyclines. Most of the antibiotics are extracellular metabolites which are normally secreted in culture media and have been used as herbicides, anticancer agents, drugs, immunoregulators and antiparasitic drugs [25]. A number of literatures are available on antibacterial and antifungal activity of actinomycete metabolites. A polypeptide antibiotic Chandramycin, isolated from Streptomyces lydicus, showed activity against Clostridium perfringens and Clostridium septicum [26]. A lactone group of antibiotics Okilactomycin from Streptomyces griseoflavus strain showed weak activity against S. aureus and S. pyogenes [27]. Frigocyclinone, a new angucyclinone antibiotic isolated from Streptomyces griseus NTK 97 exhibited antibacterial activities against Gram positive bacteria [28]. An octaketide antibiotic Fogacin isolated from Streptomyces strain TU-6319 showed moderate activity against B. subtilis [29]. P3-1 partially purified antibacterial substance, from Streptomyces sp. No 87 was found to possess antibacterial activity [30]. Antifungal activity of actinomycetes isolated from cultivated field soil, garden soil, compost, decaying organic matter and stored agricultural products was evaluated by Jain and Jain [31]. Out of 287 isolates, a total of 166 isolates were found antagonistic to C. albicans, while 164, 134 and 132 actinomycetes showed antagonistic properties against A. niger, M. gypseum and Trichophyton sp., repectively. Jain and Jain [32] determined antifungal activity of heptane group of antibiotics from Streptomyces sampsonii against four fungi namely Candida albicans, Aspergillus niger, Microsporium gypseum and Trichophyton sp. In our study, it was found that the extract caused marked inhibition of test bacteria and fungi.
Actinomycetes play an important role in the biological control of insects through the production of insecticidally active compounds. In a study involving 41 actinomycete soil isolates to evaluate their efficiency against third larval instar of the house fly Musca domestica, Streptomyces and Streptoverticillium were recorded as the most effective genera [33]. A macrotetrolide antibiotic recoverd from the acetone extract of Streptomyces aureus, isolated from Saitama Prefecture, Japan, exhibited significant insecticidal activity against Callosofruchus chinensis [34]. The ethyl acetate extract of Streptomyces sp. KN-0647 isolated from a forest soil sample of Dali Cangshan mountain, Yunnan Province, China displayed growth inhibition on the pathogenetic insects Spodoptera exigua, Dendrolimus punctatus, Plutella xylostella, Aphis glycines and Culex pipiens. The active compound was isolated and identified as quinomycin A [35]. An actinomycete, Streptomyces sp. 173, isolated from seawater and sediments, was found to have strong insecticidal activity against both brine shrimp and H. armigera, similar to that of avermectin B1 [36]. A new member of the tartrolone series of macrodiolides, tartrolone C, was isolated from a Streptomyces species on the basis of its insecticidal activity [37]. In a previous study, we have reported larvicidal effect of two Streptomyces species isolated from the soil of Agumbe, Karnataka against second instar larvae of Aedes aegypti [16]. In this study, the insecticidal efficacy of different concentrations of extract was evaluated against 3rd instar larvae of A. aegypti and it was found that the extract caused mortality of larvae in a dose dependent manner.
One of the most important strategies in the treatment of obesity includes the development of inhibitors of nutrient digestion and absorption, in an attempt to reduce the energy intake through gastrointestinal mechanisms, without altering any central mechanisms [38,39]. Pancreatic lipase inhibition is one of the most widely studied mechanisms used to
determine the potential efficacy of natural products as antiobesity agents. Orlistat, one of the two clinically approved drugs for obesity treatment, has been shown to act by inhibiting pancreatic lipase. Although it is one of the best-selling drugs worldwide, it has certain side effects such as oily stools, oily spotting, and flatulence, among others. The success of orlistat has prompted research for the identification of inhibitors that lack some of these side effects. At present, the potential of natural products for the treatment of obesity is still largely unexplored and might be an excellent alternative strategy for the development of safe and effective antiobesity drugs [40-42]. Naturally occurring compounds present an exciting opportunity for the discovery of newer anti-obesity agents. In a previous study, we have investigated pancreatic lipase inhibitory activity of butanol extract of a Streptomyces species isolated from soil of Agumbe Karnataka. The inhibition of enzyme was found to be dose dependent [17]. A marked dose dependent inhibition of chicken pancreatic lipase by extract was observed in this study.
The brine shrimp lethality assay was proposed by Michael et al. [43], and later developed by Vanhaecke et al. [44], and Sleet and Brendel [45]. It is based on the ability to kill laboratory-cultured Artemia nauplii brine shrimp. It is considered to be very useful in determining various biological activities such as cytotoxic, phototoxic, pesticidal, trypanocidal, enzyme inhibition, and ion regulation activities [46-51]. The assay represents a rapid, inexpensive and simple bioassay for testing bioactivity which in most cases correlates reasonably well with cytotoxic and antitumor properties. It is considered as useful tool for preliminary assessment of toxicity. This method needs no special equipment and no aseptic technique. It utilizes a large number of organisms for validation, relatively small amount of sample and does not require animal serum as needed for other methods of cytotoxicity testing [52-54]. A number of studies have been carried out on cytotoxic nature of actinomycete extracts and purified metabolites on brine shrimp. Choudury et al. [55] showed cytotoxic activity of a brown antibiotic pigment Di- (2-ethyl hexyl)-phthalate isolated from an Actinomycetes strain. The chloroform extract as well as the isolated antibiotic showed marked cytotoxic effect against brine shrimp. From ethyl acetate extract of a Streptomyces strain, Sultan et al. [56] isolated three active metabolites from and tested their toxicity against brine shrimp. The metabolites as well as extract were found to cause a marked lethal effect on brine shrimp. Safaeian et al. [57] showed marked cytotoxic effect of actinomycetes isolated from Persian gulf on two Artemia species namely A. urmiana and A. franciscana. Manivasagan et al. [58] tested lethal effect of an isolated fraction BC I from the solvent extract of Streptomyces strain PM-32 against brine shrimp. The fraction showed strong cytotoxicity against brine shrimp. Ripa et al. [59] showed potent lethal effect of ethyl acetate extract and a purified compound of Streptomyces rajshahiensis against brine shrimp. Two compounds namely sannastatin and vicenistatin, isolated from the cultures of Streptomyces sannanensis, displayed significant growth inhibitory activity against brine shrimp larvae [60]. In a previous study, we have determined cytotoxic activity of extract of a Streptomyces species recovered from the soil of Agumbe, Karnataka. It was observed that the mortality of brine shrimp larvae by the extract was concentration dependent. Mortality of larvae was 100% at extract concentration 1000μg/ml and the LC50 of solvent extract was found to be 42.11μg/ml [17]. In this study also, the extract caused dose dependent lethal effect on brine shrimp and thus the extract is toxic.

CONCLUSION

The genus Streptomyces is the largest producer of bioactive compounds and it remains important to discover new leader compounds from Streptomycetes for drug development. The soils of Western ghats are rich sources for microorganisms with potent biological activities and screening programs are to be conducted to reveal the presence of pharmacologically active actinomycete isolates. In this context, the present study highlighted the antimicrobial, insecticidal, pancreatic lipase inhibitory and cytotoxic potential of crude extract of a Streptomyces species isolated from the western ghat area of Agumbe, Karnataka. The metabolites of actinomycetes from western ghat area of Karnataka may prove to be useful. Further studies are needed to be carried out to isolate and characterize the active principles present in the crude extract and determine their biological activity.

Conflict of Interest

NIL

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