Study the inhibitory effect of Streptomyces spp against the growth some pathogenic bacterial

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Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 12 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.812.038 Study the Inhibitory Effect of Streptomyces spp against the Growth some Pathogenic Bacterial Bayader Abdel Mohsen, Mohsen Hashim Risan* and Asma G. Oraibi College of Biotechnology, Al-Nahrain University, Iraq *Corresponding author ABSTRACT Keywords Bacteria, actinomycetes, Streptomyces, antibacterial, Iraq Article Info Accepted: 07 November 2019 Available Online: 10 December 2019 The aimed of the study is screening of antibiotic producing Streptomyces isolates. Thirty soil samples, collected from different areas in the city of Baghdad, were screened for Streptomyces effectiveness as a source for active antibacterial, 26 (86.6%) samples were suspected to contain Streptomyces, out of them, 24 (80%) isolates were obtained with different morphological characteristics. Suspected Actinomycetes colonies were subcultured in ISP2 agar media carefully to obtain a pure isolate which was characterized as colored in aerial and substrate mycelium, dried, rough\ smooth, with an irregular/regular margin; generally convex colony. The isolates were identified as Streptomyces sp. based on their morphological, physiological and biochemical characteristics. Most Streptomyces isolates were screened for their antibacterial activity against Escherichia coli and Staphylococcus aureus on malt extract yeast extract agar medium (ISP2) using a cross-streak technique. Introduction Actinomycetes are a group of Gram-positive bacteria with high guanine and cytosine content in their DNA (Kumari et al., 2006; Khucharoenphaisan et al., 2012; Al-Rubaye et al., 2018 a, Risan et al., 2019). The major group of Actinomycetes, Streptomyces spp. can produce an array of secondary metabolites having antibacterial or antifungal properties which applied for the human pharmaceutical use (Hughes et al., 2008). It has been reported that most of the actinomycetes are widely used in industries due to their ability to produce numerous antibiotics (Raja and Prabakarana, 2011; Al-Rubaye et al., 2018b), enzymes, vitamins, growth hormones and anti-cancerous agents (Berdy, 1995). Streptomyces genus can also produce valuable metabolites, enzyme inhibitors commercially valuable enzymes like lipases, cellulases, 270 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 amylase and proteases (Ravel et al., 2000). Over 600 species of Streptomyces bacteria have been described (Euzeby, 2008). As with the other Actinomyces, Streptomyces are Gram-positive, and have genomes with high guanine and cytosine content The Streptomyces are found predominantly in soil and this results in decaying vegetation (Amin et al., 2016; Risan et al., 2016; Qasim and Risan 2017). Most Streptomyces produces spores, and are noted for their distinct "earthy" odor that results from the production of a volatile metabolite, geosmin (Madigan and Martinko 2005). Streptomyces are a unique collection of prokaryotes microorganisms having diverse morphological, biochemical, cultural and physiological characters (Chavan Dilip et al., 2013). Materials and Methods of sterile distilled water (stock suspension). The samples were shaking in a shaker at 120 rpm for 30 minutes at room temperature. Serial dilutions from 10-1 to 10-3 were made from the stock suspension and left for 10 minutes. After shaking, 0.1 ml of each dilution was pipetted and put on supplemented Yeast extract-malt extract agar (YEME) with Tetracycline 50 μg/L and Nystatin 50 μg/L, then spread by a sterile swab to make a uniform distribution of the suspension on the surface of the media. The inoculated plates were incubated at 28°C for 7 to 10 days. Based on cultural characteristics, suspected colonies of actinomycetes were selected for being characterized as small, white, and pinpoint, rough, chalky and a clear zone of inhibition around them. Soil samples collection Thirty soil samples were collected from December 2018- January 2019. Samples were collected from different areas in the city of Baghdad. The total number of soil samples and the areas for sampling selected for this study are shown in table 1. Different areas were used for the isolation of Streptomyces spp. from each area. The samples were taken up to a depth of 10-15 cm after removing approximately 3 cm of the soil surface. The samples were placed in polyethylene bags, closed tightly and stored in a refrigerator. Soil samples were incubated at 70°C for 2 hours to kill other microorganisms, followed by a screening procedure for the Streptomyces isolation (Korn and Kutzner, 1992; Risan et al., 2017). Isolation and identification of Streptomyces spp. from soil samples About one gram of dried soil samples were used to make suspension, by adding it to 99 ml The suspected colonies were subjected for their identification by types of Gram’s stain, aerial and substrate mycelium color, pigment production and pigment color. The colonies were transferred from the first screening step (mixed culture) into separate agar plates and incubated at 28±1°C for 7 days to obtain a pure growth of actinomycetes species, the last steps were repeated several times. The pure culture was kept at 4°C for a further study (Oskay et al., 2004; Risan et al., 2016). Pathogenic bacteria used for antimicrobial activities Two isolates, including Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli) were used to determine the antibacterial activity of Streptomyces isolates (both of them isolated from urine). These microorganisms were obtained from the College of Biotechnology/Al-Nahrain University, and activated by culturing in a 271 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 Nutrient Broth at 37±0.1°C for 24 hrs using 45 colonies. The inoculum was standardized by a turbidity standard (McFarland standard), for example 0.5 McFarland = 1.5 x 108 CFU/ml adjusted by the naked eye (Cockerill et al., 2012) (Table 2). Morphological characterization Morphological characterization was done according to the directions given for the International Streptomyces Project (ISP2). The morphological characterization of each isolate was first performed by: Colony characteristics Suspected Streptomyces isolates which grew on ISP 2 and GYE medium were characterized morphologically according to the colony characteristics as follows: Mass color or mature, sporulating aerial surface growth. The color of substrate mycelium as viewed from the reverse side. Diffusible melanin. soluble pigments other than Mature cultures spore mass surface was observed after 7-14 days of incubation and the color of aerial mycelium of Streptomyces was determined by a code collected by Prauser (1964) for color tabs of Baumann Farbtonkarte Atlas. Gram's stain A single colony was transferred by a loop to a clean glass slide. The smear was stained with crystal violet, treated with iodine, decolorized by the ethanol (95%), and stained with safranine, then examined by a microscope (Aghighi et al., 2004). Physiological Characterization and biochemical The physiological and biochemical tests are important in the characterization of Streptomyces spp. following the directions given for the International Streptomyces project (ISP) (Shirling and Gottlieb, 1966; Macfadden, 2000), and some the biochemical tests described by Bergey's Manual of Systematic Bacteriology 2nd Edition Volume 2 (2005) Melanin production It was investigated as follows: ISP2 or ISP4 agar slants were streaked by Streptomyces spp. and incubated at 28ºC for 7 to 10 days to detect a deep brown to black diffusible pigment (+). The absence of the color was recorded as negative (-). Carbon utilization test It was done by using Starch, Glycerol or dextrose as a carbon source. The preparation was done as described in the ISP2 and ISP4. ISP2 or ISP4 agar slants supplemented with indicator were streaked by Streptomyces spp. and incubated at 28ºC for 7 to 10 days. The positive result was detected by growing the bacteria in this media and changing the color of media to pink. Citrate utilization test Simmon's citrate agar slants were streaked by Streptomyces spp. and incubated at 28ºC for 7 to 10 days. The positive result was detected by changing the medium color from green to blue which indicated the Streptomyces ability to utilize citrate. Indole production test A loopful of Streptomyces spp. culture was inoculated in test tubes containing indole broth 272 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 and incubated at 28ºC for 7 to 10 days. The production of indole derivatives by the isolates was determined by the addition of Kovac's reagent. The formation of a red colored ring in the tubes indicates a positive reaction. Catalase test A drop of 3% hydrogen peroxide solution was added immediately on loopful with Streptomyces culture on a sterile glass slide to observe the bubbles formation which indicated the production of catalase. Antibacterial activity of Streptomyces spp. Pathogenic bacteria used for antibacterial activity The pathogenic microorganisms used as reference isolates for testing the antibacterial activity. Two isolates, including Gram positive (S. aureus) and Gram negative (E. coli) were used to determine the antibacterial activity. The routine inoculum prepared by activation of the mentioned bacteria in a Nutrient Broth (NB) at 37±0.1°C for 24 hours using 4-5 colonies. The inoculum was standardized by a turbidity standard (McFarland standard), for example 0.5 McFarland = 1.5 x 108 CFU/ml adjusted by naked eye (Cockerill et al., 2012; Risan et al., 2018). Primary screening for activities of Streptomyces antimicrobial Initial screen (primary screen) for antimicrobial activities were done by the cross-streak method according to Oskay, (2009) and Kumar et al., (2010), in which the isolated Streptomyces used against two different microbial pathogens. The Streptomyces were streaked as across lines in the middle of plates poured with MullerHinton agar and inoculated plates were incubated at 28°C for 7 days, after the Streptomyces were completely cultivated, the tested bacterial pathogens were streaked perpendicular to the Streptomyces, then plates were reincubated at 37°C for 24 hrs. The antimicrobial activities were observed by the naked eye in which the reference strains failed to grow near the Streptomyces line. Results and Discussion Isolation, purification and identification of Streptomyces isolates Thirty soil samples, collected from different areas in the city of Baghdad, were screened for Streptomyces effectiveness as a source for active antibacterial. Actinomycetes were observed in addition to other microorganisms as mixed colonies after culturing the diluted soil sample (10-1 to 10-6) for 7-10 days on ISP2 agar. Figure 1 shows white to gray small powdery colonies suspected to be Actinomycetes isolates. In this figure, a single Actinomycete colony is shown among the mixed colonies. The single colony of Actinomycetes isolate was clearly observed in figure 2. Colonies other than Actinomycetes found within the culture may be due to the presence of their spores in the soil or they were not killed by heating. The suspected colonies were grown on ISP2 agar and selected in accordance to their color (either gray or creamy or white) with colony diameter size ranged from to 10 mm) and their morphology (which have smooth surface at the beginning then became powdery, soft and granular by forming the aerial mycelium), the same results were reported by Risan et al., (2017). From 30 soil samples, 26 (86.6%) samples were suspected to contain Streptomyces, out of them, 24 (80%) isolates were obtained with different morphological characteristics. Suspected Actinomycetes colonies were sub-cultured in ISP2 agar media carefully to obtain a pure isolate which was 273 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 characterized as colored in aerial and substrate mycelium, dried, rough\ smooth, with irregular/regular margin; generally convex colony. Most colonies that were isolated possess earthy odors as described by Williams et al., (1983). Selection by streaking a plate for single colonies As observed in figure 3, a single colony was formed by the streak plate method, to purify cultures of actinomycetes. This plating technique serially dilutes the number of bacteria in each streak, the first streak probably has a very high concentration of bacteria since it comes from a concentrated stock. By dragging a new (or freshly sterilized) tool across only one small part of the initial line, we spread a small part of the initial line out over a much larger area (the second line). This second line has less bacteria, and therefore increases the chances of seeing individual colonies. The dilution was repeated many times by streaking the entire plate from the initial concentrated streak, so somewhere on the plate a single isolated colony was picked as reported by Williams et al., (1993) and Singh and Agrawal (2003). Identification and Streptomyces spp. characterization of Morphological characterization The isolates of Streptomyces were identified according to the variability in their colony morphology and microscopic characteristics like the aerial and substrate mycelium, soluble pigment, spore chain arrangement (Table 3). Some Streptomyces isolates produced diffusible pigment in the surrounding media in accordance with the aerial mycelium colour. Soluble pigment was also observed in 15 isolate. Figure 4 shows distinctive yellowish (isolate 30) series established in the Bergey’s manual of determinatives bacteriology (Buchanan and Gibbons, 1974) and in the the Bergey’s manual of systemic Bacteriology\ category 4. As shown in figure 5a, a colony morphology showed different Streptomyces isolates with regular edge and irregular edge. The mycelium surface is shown in some species with rough surface and smooth surface in others. The aerial mycelium colour either white, dark, pale gray or greenish gray. Substrate mycelium was either dark brown or light brown while one isolate showed a dark green figure 5b. Table.1 Distribution of soil samples according to the selected areas at Baghdad city No 15 10 5 Type Soil samples Soil samples Soil samples Areas of study Al- Jadria Al- Qadesia Qr Al- Aamerya Table.2 The source of pathogenic bacteria used for detection the antibacterail activity of Streptomyces isolates Source of samples Biotechnology College\ Al-Nahrain University Type Staphylococcus aureus Site of isolation Urine Escherichia coli Urine 274 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 Table.3 Morphological characteristics of Streptomyces isolates Isolate No. Isolates name Colony morphology Arial mycelium Substrate Mycelium Reverse side pigments Mycelium surface Soluble pigment Spore chain morphology 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. B3-2 B12 B1-3 B3-4 B1-4 B18 BT6 B25 BT5 BH14 B1 1-3C 4-3C B2-4 B3 B21 B4-4 B1-4 B3-3 B5 B5-5 BM3 B23 B5-2 Irregular edge-circular Regular edge-circular Irregular edge-circular Regular edge-circular Regular edge-circular Irregular edge-circular Regular edge-circular Irregular edge-circular Irregular edge-circular Regular edge-circular Regular edge-circular Regular circular Regular edge-circular Irregular edge-circular Irregular edge-circular Regular edge-circular Regular edge-circular Regular circular Irregular circular Iregular circular Irregular circular Regular Irregular circular regular Light gray Light gray Light gray Light gray gray Light gray gray White gray gray Light gray Light gray White gray Light gray gray White gray gray gray gray Light gray gray White Gray White gray gray Light brown Light brown Light brown Yellowish Darck brown Darck brown brown Light brown Darck brown Light brown Light brown Brown Light brown Light brown brown Light brown brown brown Light brown Brown Light yellow brown Light brown Light brown smooth smooth smooth Smooth Rough smooth rough smooth rough rough smooth smooth smooth smooth rough smooth smooth smooth smooth smooth rough smooth rough smooth brown Light brown Light brown Yellow No pigment Light yellow Light brown No pigment No pigment light yellow dark No pigment Dark yellow No pigment Light yellow No pigment Light yellow No pigment Dark brown Light yellow Light yellow No pigment Light yellow Light brown straight straight straight straight straight straight straight spiral straight straight straight spiral straight straight straight spiral straight rectiflexible straight Straight straight straight rectiflexible straight Table.4 Biochemical tests of Streptomyces spp No 1. 2. 3. 4. 5. Test Melanin Catalase Citrate Utilization Indole production Sugar utilization Reaction Black to brown Bubbles Deep blue color No color zone Growth 275 Result Negative Positive Positive Negative Positive Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 Fig.1 Actinomycetes first screening in ISP2 agar from soil samples dilution 10-3 at 28°C for 7-10 days Fig.2 Colorful chalky/dusty appearance of the single Actinomycete colony 276 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 Table.5 Primary screening of antibacterial activities of Streptomyces isolated from sediment soils against S. aureus and E. coli by cross streaking method Isolates B3-2 B12 B1-3 B3-4 B1-4 B18 BT6 B25 BT5c BH14 Be1 1-3C 4-3C B2-4 B3 B21 B4-4 B1-4 B3-3 B5 B4-3 BM3 B23 B5-2 S.aureus + + + + + + + + + + + + + + + + + E. coli + + + + + + + + + + + + + + - Notes ____ Selected Selected Selected ____ Selected ____ Selected Selected Selected Selected ____ ____ Selected ____ Selected ____ ____ Selected ____ Selected ____ ____ ____ Fig.3 Single colony formation of Streptomyces spp. cultured on ISP2 formed by streak plate method 277 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 Fig.4 Streptomyces spp. cultured on glycerol yeast extract media at 28°C for 7-10 days. Left isolate without pigment, Right isolate with yellow pigment Fig.5a Arial mycelium of Streptomyces grown in ISP2 media at 28°C for 7-10 days 278 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 270-283 Fig.5b Substrate mycelium of Streptomyces grown in ISP2 media at 28°C for 7-10 days Fig.6a Antimicrobial activity of 13 Streptomyces isolates against S. aureus (Staph) and E.coli (E coli), using cross streaking method with positive result 279
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