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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



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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 



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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. 



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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). 

 



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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). 

 



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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 

 



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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 



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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 



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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 - - - - - 



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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 



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(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 



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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 



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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 



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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. Moreover, the 
Ethiopian Health and Nutrition Research 
Institute (EHNRI) and Hawassa University 
Referral Comprehensive Specialized Hospital 
are cordially acknowledged for provision of the 
test pathogens. 

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