Antimicrobial Activities of Some Actinomycete Strains Isolated from the Sinai Egypt Soils

12 Arab Congress of Plant Protection ,ACPP ,4 -10 November, 2017 Hurghada Egypt Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Microbiology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the research across the whole spectrum of the subject. These including bacteriology, virology, mycology and parasitology. In addition, the journal promotes research on the impact of living organisms on their environment with emphasis on subjects such a resource, depletion, pollution, biodiversity, ecosystem.....etc www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.

Seventy three pure actinomycete colonies were isolated from 48 rhizospheric soil samples revealing different locations in Sinai.These isolates were tested for their antimicrobial activities against Bacillus subtilis and Staphylococcus aureus as gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as gram-negative bacteria, Candida albicans as an unicellular fungi and Aspergillus niger, Aspergillus flavus, Rhizoctonia solani and Fusarium oxysporum as filamentous fungi.Isolates S6, S13 and S35 were the most effective against the tested bacteria and fungi.They had the lowest Minimum Inhibitory Concentration (MIC) values and showed the highest and widest spectrum of antibacterial and antifungal activities.Factors controlling actinomycetes active metabolite(s) productivity were studied.
. Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation.MICs are considered the `gold standard' for determining the susceptibility of organisms to antimicrobials and are therefore used to judge the performance of all other methods of susceptibility testing.MICs are used in diagnostic laboratories to confirm unusual resistance, to give a definitive answer when a borderline result is obtained by other methods of testing, or when disc diffusion methods are not appropriate (Andrews, 2001).
The present study aimed to investigate the antimicrobial activity of some soil actinomycetes isolates against bacteria and fungi and spot the light on the factors affecting the productivity of their active metabolite(s)

MATERIALS AND METHODS Sample collection and preparation:
Forty eight soil samples were collected from fifteen different localities distributed through South and North Sinai Governorates, Egypt.The samples were collected aseptically from a 10-15 cm depth in clean plastic bags with a sterile spatula.The samples were immediately brought to the laboratory of the Plant Protection Department, DRC.Soil samples were airdried and were sieved through a 2 mm sieve.To reduce the vegetative bacterial cells and allow the spores of actinomycetes to survive the sieved soils were mixed before plating with Ca CO 3 in a 10:1 (w/w) ratio.

Isolation of actinomycetes colonies:
One gram of the prepared soil was suspended with 10 ml of sterile distilled water and incubated at room temperature (25 ± 2°C) for 1h on a rotary incubator shaker with vigorous shaking.Soil suspension (100 µl) was spread on starch nitrate agar (SCA) Petri-dishes and incubated at 30°C for 4 days.Colonies of actinomycetes were picked up using sterile toothpicks, placed onto SCA plates and incubated for 7 days.Pure colonies of actinomycetes were then subcultured onto SCA slants and incubated for 7 days at 30°C (Sandeepa and Menaka, 2014).

Preparation of actinomycete filtrates :
Actinomycetes filtrates were prepared by cutting 3 discs (9 mm in diameter) of pure actinomycete colony , inoculated in a 250 ml Erlenmeyer flask containing 100 ml of a liquid medium (starch nitrate broth), and incubated on a shaker ( 200 rpm) at 30 °C. for 7 days (Walker et. al., 1966).At the end of the incubation period, the culture was centrifuged at 15,000 rpm (1260 g) for 20 min.and the supernatant was stored at 4 °C.until used.

Screening of actinomycete filtrates for their antimicrobial activity. 1. Diffusion plate method:
Estimation of antimicrobial activity of actinomycete filtrates was carried out using the agar well diffusion method (Cooper, 1963) based on the observation of inhibition clearing zone of microbial growth on an agar medium.The seeded plates with target test organisms were cut by a sterile cork borer to make holes (9 mm in diameter and 2 cm apart).Only 0.1 ml of each actinomycete filtrate was transferred into a hole under aseptic conditions and allowed to diffuse at room temperature for 2 hrs.then the plates were incubated at 37°C for 24 h.Clear inhibition zone around each hole was measured in mm.

Minimal inhibition concentration method:
The most potent isolates were further investigated for their minimal inhibitory concentration (MIC).Minimal inhibitory concentration (MIC) value was defined as the lowest concentration of each natural product which completely inhibited microbial growth.Chosen filtrates were dried under reduced pressure and series of concentrations (100, 50, 25, 12.5, 6.25 and 3.125 mg/ml) were prepared and the minimal inhibitory concentration was determined by the disk diffusion method as described by Klancnik et al., (2010).

Factors affecting the productivity of actinomycete (Streptomyces lavendulae SST-32) active metabolite(s):
Several factors were tested for their role on the productivity of bioactive substance(s) by the actinomycetes Streptomyces lavendulae. 1. Suitable nutritional media (starch-nitrate, yeast extract-malt extract and Czapek's medium).

B-
Nitrogen source: KNO 3 , NaNO 3 , (NH 4 ) 2 .SO 4 , NH 4 NO 3 , urea, casein, yeast extract and peptone).The nitrogen sources were added in an equi-molecular amount of nitrogen except peptone and yeast extract which used at the concentration of 0.2% for each.

RESULTS and DISCUSSIONS Primary Screening Test:
Forty eight soil samples were collected from 15 sites in north and south Sinai.From them seventy three actinomycete isolates were obtained and purified.Soil types, associated plants and number of isolates from each site were described and tabulated in a previous study (Abdel-Rahman et.al., 2017).Actinomycete isolates were subjected to primary screening for their antimicrobial activities against soil bacteria ( Staphylococcus aurous and Escherichia coli ) and fungi (Candida albicans and Aspergillus flavus).Isolates which caused  10 mm inhibition zone on tested bacteria and fungi were considered as active isolates and selected for secondary screenings.Of these 73 isolates only 23 isolates (Table 1) were considered as active isolates.Twelve of them (S1, S2, S6, S7, S8, S13, S19, S28, S35, S36, S41and S46) had excellent antimicrobial action against the four tested organisms, while the other 11 isolates (S3, S4, S5, S14, S16, S20, S21, S22, S23, S27 and S40) came in the second category.On the contrary twenty six actinomycete isolates failed to exhibit any antimicrobial activity, while the remainder twenty four isolates were exhibited moderate to slight effects.

Antibacterial screening of selected active isolates:
Of the total 73 isolates 23 isolates were selected as active isolates and were tested for their antibacterial action against B. subtilis and S. aureus as Grampositive bacteria and E. coli and P. aeruginosa as Gram-negative bacteria.As represented in Table (2), P. aeruginosa was found to be the most resistant species in comparison to the other tested bacteria as only 6 isolates (S3, S6, S13, S27, S35 and S46) could inhibit its growth, while on the other hand B. subtilis bacteria was the most sensitive species as all the tested isolates harmed its growth.The other two species (S. aureus and E. coli) were in between.Isolates number S4, S19, S21 and S40 were found to possess strong activity against Gram negative bacteria as they completely inhibited the growth of the two tested species (E. coli and P. aeruginosa).On the basis of the broad spectrum and highly inhibition zone only five actinomycete isolates (S3, S6, S13, S35 and S46) had the most wide antibacterial spectrum as they evidently inhibited the growth of the four tested bacteria.These five most potent isolates were further investigated for their minimal inhibitory concentration (MIC).

Highly active isolates
Active isolates

Moderate and slightly active
Not active Series of concentrations (100, 50, 25, 12.5, 6.25 and 3.125 mg/ml) of the most potent isolates were prepared and tested for their MIC against four tested bacteria (B.subtilis and S. aureus as G + bacteria and E. coli and P. aeruginosa as G -bacteria).As shown in Table (3) the MIC value of isolate S6 against B. subtilis was 6.25 mg/ml as compared with 12.5 mg/ml for the other four actinomycete isolates.The same trend was observed from isolate S6 against the other tested bacteria which meant that isolate S6 was the most potent isolate as it prevent the growth of E. coli at a concentration of 3.125 mg/ml and showed a MIC value 6.25 mg/ml against the other three tested bacteria.On the same way, results in that table also indicated that the minimum concentrations needed to inhibit the visible growth of all the four tested bacteria were 12.5, 6.25, 25, 25 and 25 mg/ml from isolates S3, S6, S13, S35 and S46,respectively.Table (3): MIC (mg/ml) of selected actinomycete isolates against the 4 tested bacteria.

Antifungal screening of the selected active isolates:
Of the total 73 isolates 23 isolates were selected as active isolates and were tested for their antifungal action against C. albicans, A. niger, A. flavusm, R. solani and F. oxysporum.Results represented in Table (4) showed that isolates S6, S13 and S35 were the most potent against F. oxysporum, R. solani and C. albicans but they were less effective against A. niger.Isolates S4, S19 and S23 were the most effective against this fungus.On the same way isolates S1, S6, S13 and S23 had the greatest values of inhibition zones of A. flavus radial growth.On the other hand seven of the 23 tested isolates had no visual harm effects on the growth of A. niger fungus.
On the basis of the broad spectrum and highly inhibition zone results in Table (4) showed that only five actinomycete isolates (S1, S6, S13, S35 and S36) had the most wide antifungal spectrum as they evidently harmed the growth of all the five tested fungi.These five most potent isolates were further investigated for their minimal inhibitory concentration (MIC).

Minimum Inhibitory Concentration (MIC) of the most potent isolates against the tested fungi:
Results in Table ( 5) showed that the minimum concentration needed to inhibit the visible growth of all the five tested fungi was obtained when they were treated with isolate S6 filtrate at a concentration of 6.25 mg/ml.Filtrates of isolates S13 and S35 came in the following category with 12.5 mg/ml MIC value.On the other hand isolates S1 and S36 had the least MIC value with 25 mg/ml.These results are in harmony with Deshmukh and Vidhale, (2014) who noticed that in a primary screening out of 147 actinomycete isolates 50 isolates (34.01%) showed an activity against 2 test bacteria such as Staph.aureus and E.coli by agar overlay technique.In secondary screening, out of 50 primary isolates 19 actinomycete isolates were preceded for an antibacterial activity against Staphy.aureus (MTCC 7443), B. subtilis (MTCC 441), E.coli (MTCC 443) and P. aeruginosa (MTCC 424) by agar well diffusion method.Similar results have been reported by Attimarad et.al,. (2012) who showed that only six isolates namely ACT-A2, ACT-A3, ACT-A4, ACT-A5, ACTA7 and ACT-A15 showed significant antibacterial activity against both grampositive and gram-negative bacteria.Alpana et.al. (2010) showed that among 316 isolates, 98 (31.01%) isolates exhibited antifungal activity against one or more pathogens.Out of 98 active isolates, 19, 67, 42, 37, 18 and 25 isolates showed activity against C. albicans, Trichophyton rubrum, Microsporum canis, M. gyseum, A. flavus, A. fumigatus respectively, while 7 isolates showed activity against all the fungal pathogens.

Isolate
No.

27
The previous results with bacteria and fungi studies confirmed that isolate S6 was the most efficient with the highest antimicrobial activity.This actenomycete isolate was isolated from the rhizosphere of Tamarix nilatie plants grown in a sandy soil at El-Tor area -Sinai and taxonomically was identified as Streptomyces lavendulae SST-32 (Abdel-Rahman et.al., 2017).Factors affecting the productivity of actinomycete (Streptomyces lavendulae SST-32) active metabolite(s).Three media named starch-nitrate, yeast extract-malt extract and Czapek's medium were tested to detect the suitable nutritional media needed for the production of the maximum yield of the bioactive metabolite(s) by S. lavendulae SST-32.As shown in Table (6) starch-nitrate medium was the best medium for the production of the bioactive metabolite as compared with the two other media and that shaking cultivation technique was more suitable than the static one.Concerning to the suitable incubation period and the initial pH value of the medium, results revealed that the yield of the active metabolite(s) increased gradually with increasing the incubation period up to the seventh day and began to decrease after that.By the same way adjusting the initial pH of the medium at 7 gave the maximum inhibition zone (23.3 mm) and yielded the highest mycelium dry weight (0.36 mg/50 ml When S. lavendulae SST-32, was allowed to grow on a growth medium containing different carbon sources data represented in Table ( 6) revealed that, the highest active metabolite(s) productivity could be obtained in the presence of soluble starch followed by D-glucose as carbon source.By the same way when S. lavendulae SST-32, was allowed to grow on a growth medium containing different nitrogen sources results showed that inorganic forms of nitrogen (KNO 3 , NaNO 3 , (NH 4 ) 2 .SO 4 and NH4NO 3 ) were more suitable than the organic forms (urea, casein, yeast extract and peptone) and that sodium nitrate in the medium produced the highest active metabolites.
Similar results were obtained by Aly et. al. (2006) who reported that, maximum antibacterial recorded against B. subtilus found at 30°C.Jicheng et.al. (2007) noticed that the range of pH for the proper growth of Streptomyces is between 7 and 7.5.As well, the initial pH influences the apparent decomposition of antibiotic, and pH changes also influence culture conditions for most fermentation processes (Parente et. al. 1994).Yang et. al. (2006) found that, KNO 3 , and (NH 4 ) 2 SO 4 favored antibiotic production by Xenorhabdus spp.
As a conclusion, the most parameters affecting the productivity of the actinomycete Streptomyces lavendulae SST-32 active metabolite(s) were growing the actinomycete on starch-nitrate medium containing soluble starch and sodium nitrate as carbon and nitrogen sources, adjusting initial pH on 7 and incubation in a shaking system at 30 o C for seven days.
This study proves that the Egyptian soil is rich in microorganisms which can be a valuable source for different biological control agents.This would be a bright direction to develop new bioactive pesticides.The study also lights the scope on the antimicrobial activities of actinomycete metabolites and the ability to use them as environmentally and friendly alternatives for the extensive use of chemical pesticides in integrated pest management programs.
2. Cultivation technique (Static and Shaking)..3.Incubation period (up to 10 days) 4. pH values: The initial pH values of the media were adjusted to cover the range from 4 to 10 before sterilization. 5. Incubation temperature: 20, 25, 30, 35, 40 and 45 °C.6. Nutritional requirements: Starch nitrate medium lacking its carbon or nitrogen sources was supplemented with different types of sugars or nitrogen sources at equi-molecular amounts.