A main objective of the present study was to isolate and identify the antibacterial activity of actinomycetes strains obtained from terrestrial habitat and to utilize the antibacterial production profitably for the eradication of pathogens which cause disease to human being. The actinomycetes strains were isolated from terrestrial soil sample of King Saud University, Riyadh, Kingdom of Saudi Arabia. The isolated actinomycetes strains were identified and selected for further antibacterial activity. Streptomycetes spp. showed the best level of antibacterial effect against selected human pathogens like Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The Streptomyces may offer a potential use in development of treatments for diseases based on the normal physiological role of their secondary metabolites. Hence, it is anticipated that the isolation and identification of actinomycetes may be useful in the discovery of novel antibiotics from actinomycetes.
Key words: Terrestrial actinomycetes, Streptomycetes spp., human pathogens, antibacterial activity, soil sample.
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Actinomycetes are Gram-positive prokaryotes, which produce branching mycelium of two kinds' viz., substrate and aerial mycelium. Actinomycetes are widely distributed in natural and man-made environments, and play an important role in the degradation of organic matter. They are also well known as a rich source of antibiotics and bioactive molecules. Actinomycetes hold a prominent position for their diversity and ability to produce novel substances. The terrestrial soil actinomycetes have potential biotechnological applications, and are a new resource for structurally diverse secondary metabolites 1. For the purpose of screening of novel bioactive molecules, several factors must be considered which include choice of screening source, pretreatment, selective medium, culture conditions and recognition of colonies in primary isolation plates 2. Employing pretreatment of soil, drying and heating, stimulated the isolation of rare actinomycetes 3. An alternative approach was to make the isolation procedure more selective by adding chemical such as phenol to the soil suspension4.
Actinomycetes are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of about half of the discovered bioactive secondary metabolites, notably antibiotics 5, antitumor agents 6, immunosuppressive agents 7 and enzymes 8. Because of the excellent track record of actinomycetes in this regard, a significant amount of effort has been focused on the successful isolation of novel actinomycetes from terrestrial sources for drug screening programs in the past fifty years. Recently, the rate of discovery of new compounds from terrestrial actinomycetes has decreased, whereas the rate of re-isolation of known compounds has increased 9. Thus, it is crucial that new groups of actinomycetes from unexplored or underexploited habitats be pursued as sources of novel bioactive secondary metabolites.
Considering the practically useful compounds, today about 130 to 140 microbial products and a similar number of derivatives (including semi-synthetic antibiotics) are applied in human medicine, mostly in chemotherapy and veterinary medicine. Furthermore, some of 15 to 20 compounds are used in agriculture mainly as pesticides, plant protecting agents and food additives. The majority of these compounds, except fungal penicillins, cephalosporins and several bacterial peptides and few others, are also produced by actinomycetes. The high percentage of new compounds derived from new target-oriented screening methods is also of actinomycetal origin.
The productivity of Streptomyces strains as antibiotic producers remains unique amongst Actinomycetales strains. The relatively low occurrence of non-streptomycetes species as producers of secondary metabolites is due to difficult techniques required for the isolation of these strains from the environment. Other problem is their complicated preservation and cultivation methods, which frequently require some specific and unusual conditions. These are the main reasons for regarding these microbes as rare organisms and the difficulties for investigations and manufacturing of their products. Most of the isolates recovered on agar plates by conventional isolation techniques have been identified as 1094 Journal of Food, Agriculture & Environment, Vol.10 (2), April 2012
Streptomyces, which are the dominant actinomycetes in soil 2, 10, 11. Rare actinomycetes (non-streptomycetes) have usually been regarded as strains of actinomycetes whose frequency is lower than that of streptomycete strains. As basic knowledge of the habitat, physiology and productivity of molecules of rare actinomycetes increased, ecologically significant properties of actinomycetes assumed significance which made the screening source to expand into uncommon environments. Aim of the present study was to isolate and identify the high antibacterial activity producing actinomycetes strains from terrestrial soils, to characterize them, to determine their antibacterial efficiency and to optimize the suitable conditions for their growth and antibacterial activity.
Materials and Methods
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Sample collection and isolation of actinomycetes: The soil samples were collected from four different terrestrial locations of King Saud University, Riyadh, Kingdom of Saudi Arabia at random in sterile polythene bags, brought to the laboratory and stored for further analysis. For isolation of actinomycetes starch-casein agar medium 12 (g/l: soluble starch 10; casein 0.3; potassium nitrate 0.2; sodium chloride 2; dipotassium hydrogen phosphate 2; magnesium sulphate 0.5; calcium carbonate 0.02; ferrous sulphate 0.07; agar 18; pH 7.2) (Difco chemicals) was used. Streptomycin 40 Î¼l/ml and griseofulvin 50 Î¼l/ml (Himedia) were used to prevent bacterial and fungal contaminants, respectively. The collected soil samples were subjected to pretreatment by heating in oven at 100Â°C for 30 min. Soil samples were serially diluted up to 10-7 and 0.1 ml of aliquots were spread over the starch-casein agar plates. After incubation at 28Â°C for 7-10 days, actinomycetes colonies were purified by streak plate technique on cultivation medium.
Characterization of actinomycetes: The actinomycetes were characterized morphologically following the methods given in the International Streptomycetes Project (ISP) 13. The characters including colony morphology of the strains such as the colour of aerial mycelium, reverse side colour, size of the colony and production of diffusible pigments were observed after incubation at 28Â°C for 7-10 days on starch casein agar medium. The microscopic morphology of strains such as formation of aerial and substrate mycelium and spore arrangement, which are highly characteristic and useful in the identification of actinomycetes, were observed by cover slip technique 2, 14 with light microscopy.
Screening of antibacterial activity of actinomycetes
Preliminary screening: Antibacterial activity of actinomycetes was subjected to primary screening by cross streak plate technique 15. Straight-line inoculation of the actinomycetes isolates was made on modified nutrient agar medium (g/l: beef extract 3; yeast extract 3; NaCl22; peptone 5; glucose 20; agar 20; pH 7.0) (Difco chemicals) and incubated at 28Â°C for 4-5 days. After incubation the test organisms (The test bacteria were obtained from King Saud Medical Complex (KSMC) Riyadh, Kingdom of Saudi Arabia), Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa were streaked at right angles of actinomycetes culture and incubated at 37Â°C. Based on the inhibition zone, measured after 24-48 h the antagonistic actinomycetes were selected for further study.
Secondary screening: The selected antagonistic actinomycete isolates were inoculated into starch-casein broth, and incubated at 28Â°C in a shaker (200-250 rpm) for seven to ten days. After incubation the culture broth was filtered through filter papers such as blotting paper, Whatman No.1, and then through membrane filter to get cell free extract. To the cell-free culture filtrates equal volume of solvents, namely, acetone, butanol, chloroform, distilled water and ethyl acetate, were added and centrifuged at 5000 rpm for 10 min to extract the compounds 16. The obtained compounds of actinomycetes were tested for their antibacterial activity by disc diffusion method against four different test organisms namely, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. They were swabbed over the Muller-Hinton agar plates for bacterial organisms, respectively. The sterile discs (Himedia) were dipped in extracts of actinomycetes, placed on pathogen-seeded plates and incubated at 37Â°C for 24-48 h. After incubation the diameter of inhibition zone was measured to evaluate the antibacterial activity of the isolates.
Colony morphology on different media: The selected two antagonistic actinomycetes were cultured on four different culture media, namely nutrient agar, actinomycetes isolation agar, glycerol yeast extract agar and starch casein agar, and incubated for 7-10 days at 28Â°C. Colony morphology including colour of aerial mycelium, reverse side colour, size of the colony and production of diffusible pigments of the isolates was recorded as recommended by ISP 13.
Physiological and biochemical characterization: The physiological characteristics of the isolates such as, growth at different pH (2, 4, 6, 8 and 10), temperature (20, 25, 30, 40 and 45Â°C) and NaCl concentration (1.5, 3, 5 and 7 g/l) were recorded in starch casein broth. The antibiotic sensitivity pattern of the isolates were also recorded for various antibiotics. The biochemical characterizations of the isolates were also studied by the procedures of ISP 17-20.
Results and Discussion
In the present study, the strains of actinomycetes were isolated from the terrestrial soil samples in accordance with differences in the colony morphology. The strains were purified by visual, microscopic and cultivation methods and maintained on the same media, which was used for isolation, at 4Â°C. A strain produced white series colour of aerial mycelium on starch-casein agar medium. Similar findings were previously reported 3, 21.
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After primary screening for antibacterial activity the isolates were antagonistic against Gram-positive and Gram-negative bacteria. In a similar study 22, 50 isolates of actinomycetes from 10 farming soil samples collected in Manisa Province, Turkey, and approximately 34% of the isolates produced broad and narrow spectrum antibiotics, 16% of the isolates produced antibacterial substance that were active against only Gram-positive bacteria 6% of the isolates were active against Gram-negative bacteria and 12 % of the isolates were active against both Gram-positive and Gram-negative bacteria. On the basis of antibacterial activity and the spectrum of activity broadness, the antagonistic isolates were Streptomyces sp. Acetone extract of Streptomycetes sp. isolate had antibacterial activity against Staphylococcus aureusJournal of Food, Agriculture & Environment, Vol.10 (2), April 2012 1095
(13 mm), Bacillus subtilis (11 mm), E.coli (16 mm) and Ps. aeruginosa (10 mm). Among them, highest activity was against E.coli.
The potential antibiotic producing strain was characterized following the procedures recommended by ISP. The strain grew well on starch casein medium and produced gray colour aerial mycelium, yellow white on starch agar medium and brown colour diffusible pigment on glycerol-yeast extract agar medium. In the present study light microscopic studies of isolates showed fairly long, straight and flexuous aerial mycelium formed from the branched mycelium. Sporophores were arranged singly in pairs or occasionally in tufts along the straight aerial mycelium. Mature spore chains contained more than 20 spores per chain. The spore surface is smooth. Whirls and other special morphologies such as sporangia, zoospores or sclerotia were not observed. Aerial mycelium is generally white in colour with yellow colour reverse pigment. The cultural and physiological characteristics are summarized in Tables 1 and 2. The morphological, cultural and physiological characteristics of isolate showed that the strain could be classified with published descriptions of various Streptomycete species.
Production of antibacterial metabolites is often influenced by the components of medium and cultural conditions, such as aeration, agitations, pH, temperature, salinity, time course, nitrogen and carbon sources and suitable media, which often vary from organism to organism. Optimization of antibiotic production necessitates the complete knowledge on optimal fermentation conditions for the strain used 23-27. So in the present study the required conditions have been optimized for the productions of antibacterial compounds have been optimized justifiably, using the terrestrial actinomycetes. It has been reported that Actinopolyspora sp. (AH1) grown on tyrosine agar showed good antibacterial activity against Staphylococcus aureus compared to maltose-yeast extract agar, starch-casein agar and glucose-asparagine agar.
However, AH1 grown on glycerol glycine agar and glycerol- asparagine agar did not show any antimicrobial activity. Strain AH1 grown on glucose-asparagine agar exhibited antimicrobial activity against Trichoderma sp. AH1 grown on maltose-yeast extract agar showed good antifungal activity against Trichoderma sp. compared to SCA and tyrosine agar 28. In the present study, isolates of actinomycetes showed good antibacterial activity (Table 3), when the culture was grown on starch-casein medium against all the pathogens tested. Thus, the present study and previous findings support and conclude that the antibacterial activity depends on the medium in which the culture is grown.
Temperature is an important factor that greatly influences the diversity and distribution of biota. It has profound effect in the physiology, morphology, sporulation, biochemistry and also antimicrobial metabolite production of organisms 29, 30. In the present study the actinomycetes were grown on starch-casein medium and incubated at five different temperatures 15, 20, 25, 30 and 35Â°C (Table 4), and 25Â°C was optimum for the antibacterial activity. It is also clear from the present study that temperature plays an important role on the production of antibacterial compounds. The change in pH of the culture medium induces production of new products that adversely affect antibiotic production 31. It is a well-known factor that each particular microorganism has an optimum, minimum and maximum pH. Actinomycetes are marked by relative intolerance of acidity usually preferring neutral or slightly alkaline soil for the growth and antagonistic activity 32, 33.
In the present study, the actinomycetes were grown on starch- casein broth at five different pH such as 2, 4, 6, 8 and 10 for 7 days, and pH 6 was found to be optimum for maximum antibiotic production for the isolates. All the isolates showed minimal antibacterial effect up to pH 10 (Table 5). Thus, it is concluded that the antibacterial metabolite production is greatly influenced by pH. In the present study five different salinity levels (1, 1.5, 2, 2.5 and 3 g/l of NaCl) were tested on the antibacterial compound production by actinomycetes, and the optimal NaCl concentration for the antibacterial compound production was 1.5 g/l. All the isolates showed minimal or poor antibacterial effect at NaCl concentration up to 2.5-3 g/l of (Table 6).Thus, it is obvious from the present findings that the salts play a vital role on the production of antibacterial compounds in the actinomycetes.
The antagonistic actinomycetes were incubated in the broth culture (Table 7) for production of antibiotics at different incubation periods such as 3, 4, 5, 6, 7, 8 and 9 days. It was found that the
Table 3. Antibiotic activity of Streptomyces species. Isolate name Name of the pathogen Zone of inhibition in millimeter (mm) S.aureus 13 B.subtilis 11 E.coli 16 Streptomyces species Ps.aeruginosa 10 Table 1. Morphological characters of identified Actinomycetes (Streptomyces spp.). No. Characters Streptomyces spp 1. Isolated from Terrestrial soil 2. Color White 3. Sporophore Rectiflexibiles 4. Spore surface Smooth 5. Spore chain More than 20 spores 6. Pigments Melanoid - Reverse side Yellow Soluble - 7. Carbon source utilization Arabinose + Inositol + Mannitol + Fructose + Xylose + Sucrose + Raffinose + Rhamnose + 8. Antibacterial activity + Name of the test Result Melanin production Negative H2S Positive Starch Positive Gelatin liquefaction Positive Nitrate reduction Positive Table 2. Physiological characteristics of Streptomyces spp. 1096 Journal of Food, Agriculture & Environment, Vol.10 (2), April 2012
inhibition zone increased with the increase in the incubation period in the production medium and the maximum inhibition was found with strain which was incubated for more than 7 days. Similar type of observation, i.e. a declining trend in the development of inhibition zone due to increase in incubation period (at 168 h), was reported 34-36. Thus, the present study reports the required optimum number of days as well for the production of antibacterial compounds and the development of inhibition zone.