Microsoft Word - Alazar et al_Isolation and Characterization of Antibiotic Producing Actinomycetes _DOI East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 *Corresponding author: 1Department of Biology, Hawassa University, Hawassa, Ethiopia, P.O. Box-05, 2Department of Aquatic Sciences, Fisheries and Aquaculture, Hawassa University, Hawassa, KEYWORDS: Actinomycetes; Aantibacterial activity; Disk diffusion methods; Hawassa; Pathogens ABSTRACT In recent years, antimicrobial resistance has surged significantly in developing countries, becoming a major public health concern. With nearly all bacterial pathogens developing multidrug resistance to commonly used antibiotics, there is an urgent need to discover novel drugs to tackle this challenge. Actinomycetes are considered as one of the most diverse groups of filamentous bacteria capable of thriving into different types of ecological niches due to their bioactive potential. Therefore, this study was aimed at isolation and characterization of Actinomycetes from 20 soil samples that were collected from different sites of Hawassa city, Southern Ethiopia. The Actinomycetes were isolated using serial dilution followed by spread plate techniques and antimicrobial activity screening done using modified agar disc diffusion method. Actinomycete Isolation Agar (AIA) was used to isolate Actinomycetes. A total of twenty nine different Actinomycetes, identified as AB1-AB29, were isolated and differentiated based on their variations in colony morphology and mycelial structure. Using modified agar disk diffusion methods, their secondary metabolites were assessed for antibiotic activities on E.coli, Salmonella typhi, Klebsiella pneumoniae, Staphylococcus aureus and Shigella boydii. Moreover, Actinomycete isolates with broad spectrum activity were also tested against Methicillin resistant Staphylococcus aureus (MRSA) using modified agar disk diffusion methods. Out of 29 isolates, 19(65.5%) Actinomycetes showed antimicrobial activity against selected bacterial pathogens. Most of the isolates (84.2%) showed good antimicrobial activity against Salmonella typhi and yet significantly lower than the control drug Ciprofloxacin. Maximum zone of inhibition was 29.2mm observed against S.typhi. As the result indicates the Actinomycetes isolates showed higher inhibition zone against Gram negative bacteria than Gram positive bacteria. The study indicated that soils of Hawassa may have potential group of Actinomycetes with broad spectrum antimicrobial activity. It is therefore recommended that combining several molecular analysis methods, such as DNA re- association and PCR-based fingerprinting techniques, could greatly enhance our understanding of the overall genetic diversity of soil Actinomycetes obtained in this study. INTRODUCTION Antibiotics are substances normally of low molecular weight capable of inhibiting or slowing the growth of pathogenic microorganisms. They are often secondary metabolite produced by microorganisms and seem to have no definite role in the growth of the cell source. Microorganisms produce antibiotics normally during their late log phase of growth until their stationary phase. One of their key benefits to the source organism is said to be their ability to inhibit the growth and survival of other microorganisms in the same East African Journal of Biophysical and Computational Sciences Journal homepage : https://journals.hu.edu.et/hu-journals/index.php/eajbcs Isolation and Characterization of Antibiotic Producing Actinomycetes from Soils of Hawassa, Southern Ethiopia Email: keshamo78hope@gmail.com, +251- 91 6831673 https://dx.doi.org/10.4314/eajbcs.v5i1.3S Alazar Ergena Keshamo2*, Abayneh Agena1and Zufan Bedewi1 Research article East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 26 environment in nature hence providing the source with a competitive advantage. Antibiotic producing microorganisms can then compete with others and survive in nature for a long time (Onlamoon, 2008). Antimicrobial compounds are produced by various living organisms such as bacteria, fungi, and plants primarily as a defense mechanism against competing microorganisms. Out of these numerous groups of organisms that produce antimicrobial agents, the Actinomycetes are the most capable and well-studied candidate (Gebreselema et al., 2013). Actinomycetes are slow-growing, Gram-positive bacteria that are characterized by a high guanine-cytosine (G&C) content, ranging from 55% to 75% (Ningthoujam et al., 2009). They resemble fungi because of their filamentous appearance and spore production property and bacteria because of the presence of peptidoglycan in their cell wall and possession of flagella (Mythili and Das, 2011). Actinomycetes are inexhaustible producers of antimicrobial agents (Atta et al., 2011). Actinomycetes are estimated to contribute around 45% of all bioactive microbial metabolites discovered to date, totaling approximately 23,000 (Berdy, 2005). The secondary metabolites obtained from the class Actinomycetes are of special interest because of their versatile anti-bacterial, anti-fungal, anti- oxidant, anti-tumor and anti-viral. Among Actinomycetes, Streptomyces species produce around 7,600 compounds. Since most of these secondary metabolites produced by Streptomycetes are promising and powerful antibiotics, they are the primary antibiotic- producing organisms exploited by pharmaceutical industry (Sudha et al., 2015). They are responsible for the formation of more than 60 % of known antibiotics, further 15 % are made from a number of related Actinomycetes, Actinomadura, Micromonospora, Streptoverticillium, and Thermo Actinomycetes (Jensen et al., 2007; Ramesh et al., 2009). The emergence of resistance to the commercially available antibiotics and multidrug-resistant pathogenic bacteria are issues of extreme concern in present time for the whole human community. Due to these issues, there is rapid spread of infectious diseases leading to illness and death especially among the aged and immune-compromised patients (Hong et al., 2009). To overcome this situation the discovery of novel drugs with lesser side effects is need of present time. Natural materials like soils are commonly preferred in researches based on the notion that several samples collected from several geographic locations are more likely to yield novel dwelling microorganisms and therefore confidently, different secondary metabolites are exploited as a result of the associated geographical variation. Besides, the attempt of isolation and characterization of microorganisms from relatively unknown or unstudied areas is promising to identify additional and new microbes or their bioactive substances (Moncheva et al., 2002). In Ethiopia, a few studies showed the presence of antibiotic producing microorganisms from different ecosystems. Biniam (2008) isolated antimicrobial producing Actinomycetes from Hora and Chitu lakes. Based on a research conducted by Moges (2009), antibiotic producing thermophlic Actinomycete can be isolated from a mushroom compost. Atsede and Fassil (2018) also isolated and screened antibiotic producing Actinomycetes from soil East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 27 collected from the rhizosphere of plants and agricultural soils of Ethiopia. Nevertheless, there is no previous study conducted or scientific report made on antibiotic producing microorganisms from soil samples collected in Hawassa city. Therefore, the present study aimed to isolate and characterize antibiotic- producing Actinomycetes from soil samples of Hawassa, Southern Ethiopia. MATERIALS AND METHODS Description of the study area The study was conducted in Hawassa City. It is located 275 km away from Addis Ababa, the capital city of Ethiopia. It is located at 70’ 03’’ latitude and 80’29” east and lies at an altitude of 1708 m above sea level. The city has a total area of 15,720 hectares and the city Administration of Hawassa consists of 8 sub cities and 32 kebeles. The city experiences a sub humid type of climate having an average annual temperature of about 20.3oCand mean annual precipitation of about 933.4 mm (Hawassa city Administration, 2007) Collection of Soil Samples Twenty (20) samples of soil were collected from four different sites of Hawassa city namely: Main campus (5), Monopol (5), Mount Tabor (5) and Mount Alamura (5). Two hundred grams of soil samples were taken from a depth of 11- 16 cm from the soil surface using sterile spoon (Chaudhary et al., 2013). The samples were collected and placed in dry, clean, sterile polyethylene bags and transported aseptically to Microbiology Laboratory of the Department of Biology, Hawassa University where the entire research work was conducted. The collected samples were labeled with details such as: name of collection site, date of collection and pH of soil. The pH of soil was measured before collecting the soil samples. The collected soil samples were desiccated under ambient conditions for a week to reduce gram negative bacteria (Oskay et al., 2004). The soil samples from sterile plastic bag were grinded using sterile mortar and pestle and sieved aseptically using 250 µm pore size mesh to remove small pieces of stones and organic matter. The samples were then placed in polyethylene bags to avoid external contamination and kept in refrigerator at 4oC until used. Isolation and Cultivation of Actinomycetes From each sample, 1g of soil sample was added in the test tube containing 10 ml distilled sterile water and shaken well using vortex mixer for 3 minutes and serially diluted by using serial dilution method up to 10-7. From these tubes or stock cultures, different titration and dilutions were prepared. Accordingly, 1 ml of the stock culture was used to prepare the final volume of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 by serial dilution method. Thereafter 0.1 ml of the suspension from 10-3, 10-5, and 10-7 was taken and aseptically spread onto Actinomycete Isolation Agar (AIA) medium (Millipore, and Sigma, Germany) by applying spread plate technique and incubated at 30°C for 7 d. To get pure colonies, each colony were sub cultured by streaking on nutrient agar. The pure colonies were isolated and identified by the color of hyphae, colony morphology and the presence or absence of aerial and substrate mycelium (Reddy et al., 2011). After incubation, the slants containing pure Actinomycetes isolates were preserved at 4°C for the further studies. East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 28 Screening of Actinomycetes for Antimicrobial Activity Test Microorganisms Antimicrobial properties against selected microorganisms acquired from the Ethiopian Health and Nutrition Research Institute (EHNRI) and Hawassa University Referral Comprehensive Specialized Hospital were investigated in vitro. The test bacteria used for primary screening were Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Shigella boydii and Salmonella typhi. Methicillin-resistant Staphylococcus aureus (MRSA) (clinical isolate) which was provided by Hawassa University Referral Comprehensive Specialized Hospital was also used for secondary screening. Primary Screening of the Isolates by Disc Diffusion Method Antimicrobial screening was done using disc diffusion method as described by Kirby Bauer (1979). The stocked Actinomycetes isolates were resuscitated by sub-culturing on Nutrient Agar plates. The colony was then picked and inoculated into 5ml nutrient broth and incubated at 30oC for 10 days. Thereafter, the prepared culture was standardized to 0.5 McFarland turgidity standard using the spectrophotometer (optical density of 1.0 at 625 nm) by adding sterile distilled water to obtain the desired cell density of 1.5 X 108 (cell/ml) (CLSI, 2012). Paper discs (6 mm in diameter) were prepared from Whatman No 1 filter papers and sterilized by autoclaving at 121oC, 15psi for 15 minutes (Ngeny et al., 2013). The disc (6 mm in diameter) was soaked with 15μl of the 7d old culture broth and properly positioned on Mueller Hinton Agar inoculated with the test isolates. Standard antibiotic (Ciprofloxacin) was used as a positive control and filter paper disc soaked with sterile distilled water was used as a negative control. They were then incubated at 37oC for 24 hours. The isolates with antimicrobial activities were identified by measuring the inhibition zone in millimeters (mm) using a ruler. The absence of growth or a less dense growth of test bacteria near the disc was considered as positive for production and secretion of antibacterial metabolite by the isolates (Kekuda et al., 2010). Secondary Screening of the Isolates Based on the zone of inhibition in primary screening, Actinomycete isolates that have broad spectrum of inhibitory activity were further assessed against Methicillin - resistant S. aureus (clinical isolate). Antimicrobial activity of the secondary metabolites that had broad spectrum activity in primary screening was determined following Kirby Bauer disk diffusion method (Brown, 2009). Sterile paper discs, immersed for 30 minutes in one-week-old Actinomycetes broth cultures, were used to inoculate Actinomycetes on Muller Hinton Agar (MHA) media seeded with methicillin resistant Staphylococcus aureus (MRSA) (clinical isolate). The petri dishes were incubated at 37°C for 24 hours. Standard antibiotic (Ciprofloxacin) disc was used as a positive control and filter paper disc soaked with sterile distilled water was used as a negative control. The antibacterial activity was determined by measuring the diameter of the inhibitory zones with a ruler (CLSI, 2012). East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 29 Morphological Characterization A loop full of the isolates were streaked on each medium and incubated at 30oC for 7 to 10 days. The color of aerial mycelium, reverse color, and nature of the colony was observed and recorded. Microscopic Characterization The arrangement of spores and sporulating properties of the selected isolates were observed microscopically by using cover slip culture method by placing sterile cover slip at an angle of 45°C in the Actinomycete isolation agar medium. A loop full of each isolate was taken separately from 7 d old culture, inoculated at the insertion of cover slip and incubated at 30°C for 7 d. Then, the cover slip was removed by using sterile forceps and placed upward on a clean glass slide. Finally, the cover slip was observed for the morphology of isolates under the microscope at 100x magnifications (Cappuccino and Sherman, 2002). Gram Stain A thin smear of the 7 d old Actinomycetes cultures were inoculated into grease free slides. Thereafter, they were heat fixed and placed in a staining rack. The slides then flooded with crystal violet for one minute and then rinsed with distilled water gently. Gram’s iodine was then gently flooded on the smears and allowed to stand for one minute before gently rinsing with distilled water. This was then decolorized using 95% ethyl alcohol for 20 seconds and immediately rinsed with water to avoid over decolonization. Finally Safranin was gently flooded on the smears and let to stand for one minute before rinsing with distilled water. The slides were then blot dried using absorbent paper and then viewed using a light-microscope under oil-immersion (100x) (Cappuccino and Sherman, 2002). Physiological Characterization Temperature on Growth The identified isolates were streaked on Actinomycetes isolation agar and incubated at 25°C, 30°C, 37°C and 40°Cat pH of 7 and after 7 d their growth was observed. The optimum temperature for maximum growth was determined by visual examination of the growth. Biochemical Characterization Urea hydrolysis Sterile urea agar slants were inoculated with the isolate, incubated at 30°C for 7 d and inspected for color change (Betson., 1994; Collee et al., 1996). Catalase test The presence of catalase enzyme in some microorganisms causes decomposition of hydrogen peroxide to water and oxygen and this helps them in survival since hydrogen peroxide is lethal to cells. A modified version of the method described by Cappuccino and Sherman, (2002) was used in which isolates were grown on starch casein agar plates at 30oC for 7 d and thereafter a colony was picked with a sterile stick and placed on a sterile glass slide containing a drop of hydrogen peroxide. Production of bubbles was indicative of positive results hence the production of free oxygen (Collee et al., 1996). East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 30 Starch Hydrolysis The isolates were streaked on starch agar plates and incubated at 30°C. After 7 days of incubation, hydrogen hydrolysis was checked by pouring iodine solution on the agar for hydrolysis of starch. Production of clear zone around the microbial growth indicates starch hydrolysis (a positive result). Starch in the presence of iodine imparts a blue-black color to the medium indicating the absence of starch- splitting enzymes and representing a negative result (Benson, 1994; Collee et al., 1996). Citrate test Simmons citrate agar slant were prepared and a single colony of the isolates were streaked on the surface of the slant culture. Then the slants were incubated at 30ºC for 7d. After 7d of incubation the citrate utilizing bacteria were produced a blue color in slant surface of the media as a result of alkaline end products. This indicates the tested bacteria were citrate positive (Collee et al., 1996) Oxidase test A small amount of Actinomycete isolates were obtained from 7 day old culture and put on sterile filter paper. Then 1-2 drops of tetramethyl phenylenediamine dihydrochloride was added to the culture and the reaction was observed. A positive reaction was indicated by a color change to dark blue or purple and a negative test will result in the absence of color (Betson., 1994; Collee et al., 1996). Statistical Analysis The collected data were analyzed using SPSS version 20.0. Inhibition zone measurements obtained in triplicate were compared using a One-way ANOVA followed by Duncan's multiple range test to determine the effect of different isolates against different test pathogens. All results statistical results with p < 0.05 were considered to be statistically significant. RESULTS Isolation of Actinomycetes Twenty nine (29) isolates of Actinomycetes were isolated from 20 different soil samples of which 12(41.38%) from Mount Tabor, 11(37.93%) from Main campus, 4(13.79%) from Mount Alamura and 2(6.89%) from Monopol (Table 1). All the 29 isolates grown on Actinomycetes isolation agar showed morphology typical of Actinomycetes. The colonies were slow growing, aerobic, wrinkled and with aerial and substrate mycelia of variable colors. Table 1: Actinomycetes isolates from soil samples collected from different sites of Hawassa city Collection sites No. of soil samples No. of isolates Codes Mount Tabor 5 12 AB1-AB12 Main campus 5 11 AB13-AB23 Mount Alamura 5 4 AB24-AB27 Monopol 5 2 AB28-AB29 East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 31 Antimicrobial activity screening of the isolated Actinomycetes From the 29 isolated Actinomycetes, 19 (65.5%) showed antimicrobial activity against at least one of five test bacteria isolates. Sixteen (16) Actinomycetes isolates showed antimicrobial activity against S. typhi, thirteen (13) showed antimicrobial activity against S. aureus, nine (9) showed antimicrobial activity against K. pneumonia, seven (7) Actinomycetes isolates showed antimicrobial activity against S. boydii and two (2) showed activity against E. coli (Fig.1). Figure 1: Number of active Actinomycetesthat inhibited tested microorganisms (Sb-Shigella boydii, Ec-Escherichia coli, Kp-Klebsiella pneumonia, St-Salmonella typhi, Sa- Staphylococcus aureus) Antimicrobial Screening Total number of isolates which showed positive result in antibacterial activity (at least against one test bacteria) was 19 (65.5%). Salmonella typhi was susceptible to all the isolates apart from isolates AB2, AB10 and AB14. On the other hand, E. coli was resilient to most of the isolates apart from isolates AB9 and AB25. Shigella boydii was also resistant to most of the isolates apart from isolate AB1, AB2, AB6, AB13, AB19, AB22 and AB25. Among the tested isolates AB1, AB4, AB5, AB6, AB9, AB13, AB22, AB25 and AB27 proved to be a broader spectrum antibiotic as they worked good against most of the test organisms. Among broader antibiotic spectrum isolates AB13 and AB25 were proven to inhibit four of five test pathogens. Isolates AB16 and AB20 showed poor activity. They were only active for Salmonella typhi. Isolates AB2, AB3, AB7, AB10, AB14, AB19, AB24 and AB28 showed dual inhibition. Among active isolates tested against human pathogens, six (6) isolates were active against Gram negative bacteria and only East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 32 thirteen (13) isolates were active against both Gram positive and Gram negative bacteria, and no isolate was found active against only Gram positive bacteria (Table 2). Table 2: Sensitivity of selected test microorganisms for Actinomycetes isolated from different sampling sites Actinomycetes Isolates Test microorganisms Spectrum activity Sampling site E.coli K.pneumonia S.boydii S.typhi S.aureus AB1* Tabor - - + + + 3 AB2 Tabor - - + - + 2 AB3 Tabor - - - + + 2 AB4* Tabor - + - + + 3 AB5* Tabor - + - + + 3 AB6* Tabor - - + + + 3 AB7 Tabor - + - + - 2 AB8 Tabor - - - - - 0 AB9* Tabor + + - + - 3 AB10 Tabor - + - - + 2 AB11 Tabor - - - - - 0 AB12 M.C - - - - - 0 AB13* M.C - + + + + 4 AB14 M.C - + - - + 2 AB15 M.C - - - - - 0 AB16 M.C - - - + - 1 AB17 M.C - - - - - 0 AB18 M.C - - - - - 0 AB19 M.C - - + + - 2 AB20 M.C - - - + - 1 AB21 M.C - - - - - 0 AB22* M.C - + + + - 3 AB23 M.C - - - - - 0 AB24 Alamura - - - + - 1 AB25* Alamura + - + + + 4 AB26 Alamura - - - - - 0 AB27* Alamura - + - + + 3 AB28 Monopol - - - + + 2 AB29 Monopol - - - - - 0 Legend: + = active against test organism; - = inactive against test organism, *= Show broad spectrum activity, M.C=Main campus From a total of 29 isolates of Actinomycetes tested for antimicrobial activity against human pathogenic bacteria: Escherichia coli, Klebsiella pneumonia, S. boydii, Salmonella typhi and Staphylococcus aureus, 19 (65.5%) isolates showed antimicrobial activity against at least one test microorganism. All of the inhibition zones produced by isolates showed significant differences when compared with control ciprofloxacin tested against test organisms (P<0.05). The antimicrobial activity of all the isolates tested against K. pneumonia were statistically significant (P<0.05) from ciprofloxacin (25.16 mm) which was the control drug (Table 3). Escherichia coli was resistant to all of the isolates except isolates AB9 and AB25. The two isolates that showed antimicrobial activity against E. coli were East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 33 significant as compared with control drug ciprofloxacin (18.33 mm). When compared with control drug ciprofloxacin (18.33 mm), isolate AB25 showed good antimicrobial activity (15.8mm) as opposed to isolate AB9 (9.45mm). The zones of inhibition of active isolates against S. boydii were significant (P<0.05). Isolate AB6 exhibited good activity (21.90mm) when compared with control ciprofloxacin (26.25mm) as opposed to the rest which were active against S. boydii. Table 3: Primary screening of antimicrobial activity (mm) of Actinomycetes isolates ‘-’: refers inactive, ‘-ve control ’:filter paper disc soaked with sterile distilled water, ‘+ve’ control: Ciprofloxacin Salmonella typhi was sensitive to most of the Actinomycete isolates (84.2%). The highest zone of inhibition was also shown for isolate AB28 against Salmonella typhi (29.11mm). Inhibition zones produced by AB4, AB6, AB16 and AB28 against Salmonella typhi were 26.63mm, 26.21mm, 26.10mm and 29.11mm, respectively, which were strong active when compared with control ciprofloxacin (30.33mm) but all of them were statistically significant (P<0.05). Salmonella typhi showed resistant against isolates AB2, AB10 and AB14. Thirteen isolates (68.42%) exhibited antimicrobial activity against Staphylococcus aureus which was the highest next to Salmonella typhi. The isolate AB28 (18.69 mm) showed the highest inhibitory activity against Staphylococcus aureus when compared to others. Isolates that showed the Isolates Test microorganisms Gram negative Gram positive E.coli K.pneumonia S.boydii S. typhi S.aureus AB1 - - 9.25±0.97 13.75±0.91 11.88±0.41 AB2 - - 15.79±0.82 - 11.20±0.75 AB3 - - - 17.62±0.42 12.50±0.68 AB4 - 10.88±0.41 - 26.63±0.33 10.74±0.44 AB5 - 11.92±1.10 - 18.55±0.62 13.57±0.72 AB6 - - 21.90±0.40 26.21±0.55 11.04±0.37 AB7 - 17.95±0.44 - 13.30±0.69 - AB9 9.45±0.50 14.48±0.48 - 11.24±0.67 - AB10 - 10.51±0.78 - - 11.35±0.44 AB13 - 12.43±0.60 7.93±0.68 17.30±0.27 12.00±0.50 AB14 - 10.54±0.77 - - 15.08±0.54 AB16 - - - 26.10±0.38 - AB19 - - 11.68±0.85 18.99±0.58 - AB20 - - - 8.32±0.17 - AB22 - 13.58±0.66 8.00±1.00 10.80±0.38 - AB24 - - - 15.00±1.00 16.86±0.34 AB25 15.80±0.18 - 10.65±0.75 21.80±0.34 6.57±0.25 AB27 - 10.25±0.86 - 20.00±0.00 14.33±0.76 AB28 - - - 29.11±0.57 18.69±0.32 +ve control 18.33±0.57 25.16±0.76 26.25±0.66 30.33±0.57 25.00±1.00 -ve control - - - - - East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 34 second, third and fourth antimicrobial activities against Staphylococcus aureus were AB24 (16.86mm), AB14 (15.08mm) and AB27 (14.33mm), respectively. All the active isolates tested against Staphylococcus aureus also showed statistically significant result (P<0.05) as shown in Table 3. Among active isolates tested against pathogens, six (6) isolates were active against Gram negative bacteria only, thirteen (13) isolates were active against both Gram positive and Gram negative bacteria and none of the isolate was found active against Gram positive bacteria. Among the 19 active isolates from soil, the highest number was from mount Tabor (47.37%) followed by Main campus (16.58%), Mount Alamura (15.79%) and Monopol site (5.26%). According to the spectrum of 19 active Actinomycetes, it was found that most Actinomycetes inhibited two tested microorganisms (8 isolates) followed by 7, 2, and 2 isolates that inhibited 3, 4 and 1 tested microorganisms, respectively (Table 4). Table 4: Antimicrobial activity (mm) of selected isolates against Methicillin resistant Staphylococcus aureus (MRSA) in secondary screening Isolates Control MRSA AB1 AB4 AB5 AB6 AB9 AB13 AB22 AB25 AB27 Cipro - 11±0 14±1 8±1 16±1 - - 13±1 - 24±1 Cipro: ciprofloxacin, MRSA: methicillin resistant Staphylococcus aureus -: no inhibition zone Secondary Screening Based on primary screening, 9 isolates with wider spectrum activity were further tested against Methicillin resistant Staphylococcus aureus (MRSA) (clinical isolate) using modified disc diffusion method. Secondary screening of 9 isolates selected based on primary screening revealed that 5 isolates exhibited inhibition against clinical isolate of Methicillin resistant Staphylococcus aureus (MRSA), with inhibition zone size above 8mm to 16mm diameter. Isolates AB1, AB13, AB22 and AB27 did not show any antimicrobial activity during secondary screening against MRSA. The antimicrobial activities of isolates were statistically significant (P<0.05) when compared to the standard antibiotics of ciprofloxacin. Among those five isolates, 4 of them were from mount Tabor site and 1 from Mount Alamura site. Three isolates (AB5, AB9 and AB25) revealed maximum zone of inhibition (i.e. large diameter) against Methicillin resistant Staphylococcus aureus (MRSA) (clinical isolate). Two of them were isolated from Mount Tabor site and one was from Mount Alamura (Table 4). Characterization of selected Actinomycete isolates After taking the pH of all soil samples, it was found that almost all soil samples were neutral to alkaline except three samples from Monopol site which were acidic (less than pH value of 6). Based on secondary screening against Methicillin resistant Staphylococcus aureus East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 35 (MRSA) (clinical isolate), five (5) active broad spectrum Actinomycete isolates namely AB4, AB5, AB6, AB9, and AB25 were used in characterization. Morphological Characterization Macroscopic features of the selected isolates were studied by culturing the isolates on Actinomycetes Isolation Agar (AIA), Nutrient Agar and yeast extract malt agar. The isolates showed different growth patterns on each of the medium. The growth of the Actinomycete isolates were highest in Actinomycetes Isolation Agar (AIA), moderate growth was observed at yeast extract malt agar, and low growth was seen at Nutrient Agar comparatively. The nature of colonies was found rough, smooth, chalky, and powdery and it was noted that colonies had different colors ranging from white, whitish, yellow, brown, and pink colonies on Actinomycetes Isolation Agar (AIA) plates. Some colonies were very hard to pick from agar surface, which is also a characteristic of Actinomycetes. The microscopic observations showed that all the isolates were Gram positive as they retained the primary color (crystal violet) hence appeared blue and this is a characteristic of Actinomycetes (Table 5). Table 5: The morphological characteristics of the isolates on Actinomycetes isolation agar Isolates Appearance of colonies Gram stain AB4 White powdery Gram positive AB5 Brown rough Gram positive AB6 Pink Gram positive AB9 Yellow smooth Gram positive AB25 Whitish yellow Gram positive Physiological and Biochemical Characterization In biochemical tests, all the isolates were able to hydrolyze both starch and urea. Citrate was positive for all the isolates, all the isolates showed positive result for catalase test and oxidase was also positive for all the isolates. Isolates AB4 was grown at a temperature range of 25-30°C, AB9 on a temperature range of 30- 37°C while none of the isolates were able to grow at the temperature of 40°C. Optimum temperature for most of the isolates was found at 30°C (Table 6). Table 6: Physiological and biochemical characteristics of selected isolates Types of test Characteristics of isolates AB4 AB5 AB6 AB9 AB25 Starch + + + + + Citrate + + + + + Catalase + + + + + Urea + + + + + Oxidase + + + + + Opt. To 25-30°C 30°C 30°C 30-37°C 30°C Opt. To: optimum temperature; +: positive; -: negative East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 36 DISCUSSION Antibiotic resistance is one of the most pressing public health issues worldwide. Presently, antibiotic-resistant organisms are extensively emerging and causing great challenge for a number of infectious diseases and current clinical care. As a result, there has been growing curiosity in searching valuable antibiotics from marine and soil Actinomycetes in diversified ecological niches (Abo-Shadi et al., 2010). Actinomycetes are the richest sources of bioactive compounds (Suthindhiran and Kannabiran, 2009). Almost 70% of all recognized antibiotics have been isolated from Actinomycetes of which 75% and 60% are used in medicine and agriculture respectively (Kumar et al., 2012). Isolation of Actinomycetes has always been facing difficulties while comparing with other bacteria and fungi (Williams and Cross, 1971). This may be partly associated with their long incubation period. Conversely, Actinomycetes isolation ratio has increased by pretreatment of the samples by air desiccation for a week (Oskay et al., 2004). In this study, out of total 29 Actinomycete isolates, 19 (65.5%) exhibited antimicrobial activity on the test pathogens. Primary screening using the disc diffusion methods revealed 65.5% (n=19/29) of the isolates were effective inhibitors against the test pathogens. This finding is higher than the finding (26.7%) of the previous study done by Abebeet al. (2013) from soil samples of Gondar town, North West Ethiopia. However, this result is in line with the report of Atsede and Fassil (2018) that 60% of Actinomycete isolates exhibited antibiotic activity against at least one test pathogen. Sawasdee et al. (2011) reported that 80% of the isolates revealed antimicrobial activity against at least one test microorganism and this value was higher than the present findings. Khasabuli and Kibera (2014) also reported that all the isolates IS1-IS15 showed positive results against at least one tested pathogen. Test bacteria showed varied responses to metabolites of Actinomycete i.e., being susceptible to one isolate and tolerant to the other isolate. Salmonella typhi was susceptible to most of the isolates other than AB2, AB10 and AB14. This was consistent with the report of Khasabuli and Kibera (2014). Escherichia coli were resistant to all of the isolates except from AB9 and AB25 isolates. Shigella boydii was susceptible to metabolites from isolates such as AB1, AB2, AB6, AB13, AB19, AB22 and AB25 while it was resistant to the other metabolites from the rest isolates. The results of this study showed that the inhibition zones were maximum against Gram negative bacteria when compared to Gram positive bacteria. This finding differs from the previous reports of Abebe et al. (2013), Gebreselema et al. (2013) and Atsede and Fassil (2018) who reported higher inhibitory effect in Gram positive pathogens than in Gram negative. Several other studies conducted elsewhere also showed Gram positive isolates were more susceptible to the antibiotics produced by Actinomycetes than Gram negative bacteria (Sawasdee et al., 2011; Sheik et al., 2017). However, a study done in Chennai, India reported higher inhibitory effect on Gram negative bacteria than Gram positive bacteria (Fatima et al., 2017) which is in line with the present study. Kamal et al. (2018) also reported highest inhibitory effect in Gram negative pathogens than Gram positive bacteria. The reason for higher antibacterial activity of East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 37 Actinomycetes towards Gram negative bacteria in comparison to Gram positive bacteria tested might be due to the nature of the cell wall of the Gram negative bacteria which is easier to break than those of the Gram positive bacteria. However, this hypothesis did not hold good with the findings of some researchers, who observed much higher inhibitory reaction against the Gram positive bacteria than the Gram negative bacteria (Basilio et al., 2003; Oskay et al., 2004; Sacramento et al., 2004). On secondary screening using the disk diffusion methods against Methicillin resistant S. aureus (MRSA) five out of nine Actinomycete isolates showed antimicrobial activity with different inhibition zones. Based on the study conducted by Yucel and Yemac (2010), the inhibition zone of Actinomycetes isolates against MRSA ranged from 0-15 mm. In this study, however, the inhibition zone of nine isolates against MRSA ranged from 0-16 mm which was considered to be good when compared to Yucel and Yemac’s results (Yucel and Yemac, 2010). However, the present result is less when compared to the activity reported by Abebe et al. (2013) who found zone of inhibition ranged from 0-20mm. Such differences in the results of these studies might be partly associated with the variation in the strains and biotypes of Actinomycetes sourced from the different habitats. According to the present result, ciprofloxacin had 24±1 mm inhibition zone against MRSA, which had greater inhibition zone when compared to the isolates tested. The results of the current study reveal that, the type of culture medium and incubation temperature has a substantial effect on the production of antibiotics by the antibiotic producing organisms. The aerial mycelium, substrate mycelium growth and colony color exhibited distinct disparity based on the culture media in which the isolates were cultured. Among the three culture media used, the preferred medium in this experiment was Actinomycete Isolation Agar where the maximum numbers of colonies were isolated and this may be due to the inclusion of appropriate amount of nutrient in this media under 30°C. This is in agreement with the results of Atsede and Fassils (2018) who reported 30°C was optimum temperature for most of the potential Actinomycetes isolates. Kumar et al. (2012) also reported AIA as the best media for the isolation of Actinomycetes. All the potential isolates in this study have the ability to hydrolyze starch and urea. Oxidase, citrate and catalase tests were also positive for all potential isolates. Therefore, after observation of cultural, morphological, physiological and biochemical characteristics it was confirmed that these isolates obtained from soil of Hawassa belong to the species of the genus Streptomyces. Previous studies conducted by Kalyani et al. (2012); Sudha and Hemalatha (2015); Midhun and Girijasankar (2016); Ramendra et al. (2016); Sreejetha et al. (2016); Sujatha and Swethalatha (2016) showed that Streptomyces sp. being producers of useful bioactive metabolite have an antibacterial effect with a broad spectrum of activities. Another study showed that Streptomyces species produced about 7,600 compounds which have antimicrobial properties which are highest among Actinomycetes producing antibiotics in the soil (Das et al., 2010). CONCLUSION & RECOMMENDATIONS Antimicrobial resistance is a global problem which demands for novel antimicrobial structure East Afr. J. Biophys. Comput. Sci. (2024), Vol. 5, Issue 1, 25-39 38 against pathogenic microbes. Actinomycetes are famous for antibiotic production and continued to be explored in hope of getting novel antibiotics. The isolates from soil of Hawassa city might be a promising candidate for discovering novel. Actinomycetes isolates recovered from mount Tabor samples account largest number of antimicrobial bioactive compounds. It is therefore recommended that molecular analysis methods such as DNA re- association and PCR-based fingerprinting techniques (or a combination of these) may tremendously help to deliver broader insight about the entire genetic diversity of soil Actinomycetes community. Perhaps, these methods may lead to the improvement in isolating valuable strains of soil Actinomycetes obtained in this study. Acknowledgements The authors are greatly thankful to Hawassa University for providing the necessary facilities to carry out this research work. 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