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Many culture, one goal: finding solutions for better health

University of GondarFNCS

Department of BiologyApplied Microbiology

Isolation, Screening and Characterization of Antibiotic Producing Actinomycetes from Soil in Gondar, Northwest Ethiopia

By Abebe BizuyeAdvisor: Felek Moges (MSc., phD., Associate Professor)

September, 2012Gondar, Ethiopia

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1. Introduction2. Objectives3. Materials and Methods 4. Result 5. Discussion6. Conclusion and Recommendations7. Acknowledgments

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1.1 Definition of Actinomycetes “Unicellular microbes, 1µm in diameter, filamentous,

branching monopodially, seldom dichotomous, producing colonies of radiating structure’’ (Neukirch, 1902)

Most have a filamentous mycelium and produce spores, characteristics of fungi

They were originally considered to be an intermediate group between bacteria and fungi but now are recognized as prokaryotic G+ bacteria (Lacey, 1997).

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Are distinguished from other bacteria by their: Morphology, DNA rich in G+C and Nucleic acid sequencing and pairing 1.2 Occurrence of ActinomycetesUniversal, widely distributed in natural and man-made

environments In soils, manure, dust aquatic, plant bodies Influenced by geographical location such as physical and

chemical conditions (George et al., 2011) 5

1.3 Importance of Actinomycetes

In ecological system, biological control of diseases, production of secondary metabolites

Secondary metabolites are: Produced during depletion of nutrients, biosynthesis of an inducer or

decrease in growth rate Secreted during the generation of aerial hyphae from the vegetative

mycelium (Miguelez et al., 2000) Important in industry, environment and Medicine Produce above 70% antibiotics

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Streptomycetes have produced 45-55% antibiotics that have:Antibacterial, antifungal, antiviral, antiparasitic, antitumor,

and immunosuppressive actions (Berdy, 2005)But, it needs discovery and increasing productivity of

antibiotics Because:1) Many microbes have no successful antibiotics 2) Many important drugs have side effect to the host

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3) Most antibiotics are expensive and cannot easily found in the market

4) Many human pathogens are developing multidrug resistance

For example, Antibiotics like penicillin and erythromycin, which used to be one-time miracle cures, are now less effective because bacteria have become more resistant (Pearson and Carol, 2008)

MRSA, VRSA strains, and others cause an enormous threat to the treatment of serious infections (Ilic et al., 2005)

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To avoid this happening, searching and producing new and effective antibiotics from microbes is necessaryHowever, no documented research was done on antibiotic

producing actinomycetes in Gondar So, an attempt was made to isolate and characterize antibiotic

producing actinomycetes in Gondar town soil areasTo achieve this, the following objective was designed

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2.1 General ObjectiveTo isolate, screen and characterize antibiotic producing

actinomycetes from soil samples in Gondar 2.2 Specific ObjectivesTo isolate antibiotic producing soil actinomycetes To test the antimicrobial activities of actinomycete

isolates against clinical and standard strainsTo characterize and identify promising isolates

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3.1 Study Area and Period

Gondar town Is located in NW Ethiopia, North Gondar Zone of the Amhara Region,

North of Lake Tana and Southwest of the Simien Mountains Has a latitude and longitude of 12°36′N 37°28′E with an elevation of

2133m above sea level According to 2008 Ethiopian Statistical Agency report, Gondar town

has 20 Kebeles with 231, 977 total populations Kebele 16 and 18 sites were randomly selected for study The study was carried out from January 2012 to August 2012

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15 sample collection (Aghamirianand Ghiasian, 2009)

For stock culture preparation

Serial dilution (up to 10-5)1ml suspension

inhibitorsSpread plate

Incubation at 370C for 7 days(Narendra et al., 2010)

Identification (Aghamirian and Ghiasian, 2009) and isolation of pure colonies

Subculture to SC slant for preserving

Primary screening

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Preparation of NA and PDA plates

Streak actinomycete isolates

Incubate at 370C for 7 days

streak test organisms & Incubation

selection of promising isolates

Cultivate promising isolates in 200ml SC broth

Inoculate 10% of culture to SC broth and natural media

Incubate at 370C for 7 days for fermentation

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Submerged state fermentation Solid state fermentation

The inoculated sc broth in incubator shaker

Centrifuge (Nonoh et al., 2010) to obtain cell free supernatant

The inoculated Natural media in thermostat water bath

solvent extraction (Selvameenal et al., 2009)

filtration and conc. to obtain crude extracts

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Incubate at 370C for 7 days

Well diffusion assay

Well preparation on MHA and PDA

Swabbing of test organisms

Loading of 60µl of filtrate & dissolved extracts

Incubation 24hrs at370C for bacteria 48hrs at 280C for fungi

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3.4 Determination of MIC and MBC of Crude Extract for Isolate Ab18 and Ab43

The crude extract solutions (76mg/ml) from Ab18 and Ab43 were used as stock concentration for analysis of MIC and MBC

Two-fold serial dilution of the extract was made to obtain 38, 19, 9.5, 4.75mg/ml using nutrient broth

S. boydi inoculums (0.1ml) were inoculated into each dilution and incubated at 37oC for 24hrs

The lowest concentration which showed no turbidity in the test tube was recorded as the MIC

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MIC test tubes were sub-cultured on MHA and incubated at 37oC for 24hrs The MBC was the plate with the lowest concentration of

extract which can kill the test organisms3.5 Characterization of Promising Isolates Morphological, cultural, biochemical and physiological

methods were used for characterization of isolates

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3.6 Data AnalysisThe collected data were analysed using tables, % and SPSS 16

Version softwareThe different inhibition zone measurements were compared by

performing One-way ANOVA ranked with Duncan’s multiple range tests with descriptive analysis type on different isolates against different pathogensAll statistical results with p<0.05 were considered to be

statistically significant

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4.1 Sampling and Isolation of ActinomycetesFrom a total of 15 soil samples, 30 different actinomycete

isolates were obtained 2 (6.67%) were isolated from kitchen house, 10 (33.33%)

breeding area and 3 (10%) house waste disposal area K16One (3.33%) was isolated from rhizosphere soil and 14

(46.67%) industrial waste disposal area of K18 (Table1).

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Sample

sites (S)

District Kebele Specific soil sample area Soil depth (cm) Numbers of isolates and

their code

S1 Tewodros

Campus

18 Rhizosphere 11 Ab1

8 0

5 0

S2 Johannes 16 Cooking house 11 Ab4

8 Ab5

5 0

S3 Gibirina 16 Cattle breeding area 11 Ab6, Ab7, Ab8, Ab9

8 Ab11, Ab12, Ab13, Ab15

5 Ab16, Ab18

S4 Condomini

um

16 House waste disposal area 11 Ab20, Ab21

8 Ab22

5 0

S5 Taxi

Mazoria

18 Industrial waste disposal

area

11 Ab24, Ab26, Ab27, Ab28,

Ab29, Ab30, Ab31

8 Ab32, Ab33, Ab34, Ab37

5 Ab41, Ab43, Ab44

Total 30

Table 1: Description of samples collected from different sites of Gondar town and the

number of isolates obtained

4.2 Primary screeningEight (26.67%) isolates were shown antimicrobial activityFive (62.5%) from industrial waste disposal area, two (25%)

from cattle breeding area and one (12.5%) from kitchen house3(37.5%) (Ab24, Ab41 and Ab44) against G- only, 1(12.5%) (Ab18) against both G- and G+ only, and 4(50%) (Ab5, Ab13, Ab28 and Ab43) were active against G-, G+ and fungus

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5(62.5%) isolates were shown antagonistic activity against S. aureus, E. coli, S. typhi and K. pneumonia4(50%) isolates were shown antagonistic activity

against S. cerevisiaeHowever, P. aeruginosa was resistant for all isolates There was significance d/c b/n (p<0.05) antagonistic

activities of isolates against pathogens (Table 2).

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Isolate Inhibition zones against test organisms (mm)

S. aureus P.

aeruginosa

E. coli S. typhi K.

pneumonia

S.

cerevisiae

Ab5 10b+2 0 8b + 1 7b+ 1 0a+0 13c+1

Ab13 13c+1 0 0 a + 0 0 a + 0 12b+1 10b+1

Ab18 15d+1 0 15cd+1 14c+1 20d+2 0a+0

Ab24 0 a +0 0 0 a + 0 0 a + 0 15 c +1 0a+0

Ab28 20e+1 0 16d+2 25d+ 1 15c+2 15c+2

Ab41 0a+0 0 0a +0 8b+ 2 0a+0 0a+0

Ab43 25f+1 0 30e+2 32e+2 20d+1 15c+2

Ab44 0a +0 0 13 c + 1 0 a + 0 0 a +0 0a+0

Table 2. Comparison of the antagonistic activity of isolates against test organisms

Values are means + SD. The outcomes not sharing a common superscript letter in

the same column are significantly different at p<0.05. These were tested by One-

way ANOVA ranked with Duncan’s multiple range tests with descriptive analysis type.

4.3 Secondary screeningThe supernatant from isolates were shown antimicrobial

activityStatistical result showed that, there was significance d/c

(p<0.05) in antimicrobial activity among the supernatantHowever, E. coli, C. albicans and MRSA2 pathogens were

shown resistant (Table 3)Except isolate Ab28, crude extracts of isolates were also shown

strongly active (p<0.05) against test organisms (Table 4)24

Table 3. The antimicrobial activity of the supernatant from broth cultured

promising isolates against test organisms

Test organisms Zone of inhibition (mm)

Ab18 Ab28 Ab43

S. aureus 0a+0 15b+0 14b+1

E. coli 0a+0 0a+ 0 0a+ 0

S. typhi 0a+ 0 19c+2 16b+1

K. pneumonia 12b+1 0a+ 0 0a+ 0

S. boydi 35c+1 12a+1 28b+1

C. albicans 0a+ 0 0a+ 0 0a+ 0

MRSA2 0a+ 0 0a+ 0 0a+ 0

MRSA4 0a+0 14c+1 10b+1

Values are means + SD. The outcomes not sharing a common superscript letter in the same row

are significantly different at p<0.05. These were tested by One-way ANOVA ranked with

Duncan’s multiple range tests with descriptive analysis type.

Table 4: Agar well diffusion assay of ethyl acetate extract of

promising isolates against test organisms

Antibiotics Zone of inhibition (mm) against test organisms

S. aureus E. coli S. typhi K. pneumonia S. boydi C. albicans

Ab18 30c+1 35d+1 25c+2 14c+1 37c+1 15c+1

Ab28 0a+0 0a + 0 0a+ 0 0a + 0 15a+1 12b+1

Ab43 34d+1 25b+1 17b+1 0 a + 0 30b+1 0a+0

Ab18&43 25b+1 0a+ 0 15b+1 12b+1 40d+1 0a+0

AMC30 25 b+1 0a+ 0 15b+1 0a+0 45e+1 -

TE30 40e+1 33c+1 26c+1 13bc+1 46e+1 -

Cycloh - - - - - 25d+1

Values are means + SD. The outcomes not sharing a common superscript letter in

the same column are significantly different at p<0.05. These were tested by One-

way ANOVA ranked with Duncan’s multiple range tests with descriptive analysis type.

The extracts (except Ab28) were also shown promising and encouraging antimicrobial activities against two MRSA strains (Table 5)

4.4 Determination of MIC and MBC of Crude Extracts The MIC and MBC value of the crude extracts against S. boydi

was shown an encouraging result (Table 6)5.4 Characterization of Promising Isolates Isolates were shown G+ reaction, round colonies and spiral shapes

of cells White gold shine color of colonies and cylindrical spore

morphology were observed for isolates Ab18 and Ab43

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Table 5: Antimicrobial activity of ethyl acetate extract of promising isolates

against MRSAs

Antibiotics Inhibition zone(mm)

MRSA2 MRSA4

Ab18 20d+1 15b+1

Ab28 0a+0 0a+0

Ab43 16b+1 17c+1

Ab18&43 18c+1 18c+1

Penicillin 0a+0 0a+0

Methicillin 0a+0 0a+0

VA30 30e+1 20d+1

Values are means + SD. The outcomes not sharing a common superscript letter in

the same column are significantly different at p<0.05. These were tested by One-way

ANOVA ranked with Duncan’s multiple range tests with descriptive analysis type.

Table 6: MIC and MBC of ethyl acetate extracts

from isolates Ab18 and Ab43 against S. boydi

Test organism MIC and MBC activity of antibiotics (mg/ml)

Ab18 Ab43 Amoxicillin

MIC MBC MIC MBC MIC MBC

S. boydi ATCC9289 9.5 9.5 9.5 19 9.5 9.5

Red color of colonies and oval spore morphology were shown by isolate Ab28

Gray colour of aerial and substrate mycelium were shown on czapex’s and MHA by isolates Ab18, Ab28 and Ab43

White colour of aerial and red colour of substrate mycelium were shown on SC and OMA for isolate Ab28 (Table 7).

These isolates were shown for + and - result of indole and H2S production respectively

Isolates Ab18 and Ab43 were shown a + result for urea, catalase (- for Ab18) and starch test

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Table 7: Morphological and cultural characteristics of selected isolates

Properties Selected potent isolates

Ab18 Ab28 Ab43

Colony Shape Round Round Round

Color White gold shine Red White gold shine

Gram reaction Positive Positive Positive

Shapes of cells Spiral Spiral Spiral

Spore morphology Cylindrical Spherical or oval Cylindrical

SCA Growth Very good Very good Very good

CAM White White White

CSM Gray Red Gray

Pigmentation No Red No

OMA Growth Good Very good Very good

CAM White White White

CSM Gray Red Gray

Pigmentation No Red No

CA Growth Good Very good Very good

CAM Gray Gray Gray

CSM Gray Gray Gray

Pigmentation No No No

MHA Growth Very good Very good Very good

CAM Gray Gray Gray

CSM Gray Gray Gray

Pigmentation No No No

However, Ab28 were shown a - result for urea hydrolysis, catalase reaction and starch hydrolysis tests Glucose fermentation for the three isolates and gas production

for only Ab18 and Ab28 was observed (Table 8) Except isolate Ab18 at pH=10.8, the promising isolates were grown in

the range of 5-10.8 pH, 20-370C and up to 4% NaClHowever, all isolates were not grown at 8% NaCl conc. Isolates Ab28 and Ab43 were shown susceptible against C30, DO30,

T30 and VA30 antibiotics but resistant against AM30

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Properties Selected potent isolates

Ab18 Ab28 Ab43

Biochemical test

Indole production + + +

Citrate utilization - - +

H2S production - - -

Urea hydrolysis + - +

Catalase - - +

Starch hydrolysis + - +

TSI K/A & gas K/A & gas K/A

Carbon source

utilization

Starch ++ - +++

Dextrose ++ +++ +++

Key: - (Negative), + (Positive), +++ (Very good), ++ (Good), K/A (Base/Acid)

Due to some inconvenient conditions, the antibiotic sensitivity result for isolate Ab18 was not reported (Table 9)Based on the above described:Morphological Cultural Biochemical and Physiological characterization, the three promising isolates

were grouped under genus Streptomyces

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Table 9: Physiological characteristics of selected isolatesProperties Selected potent isolates

Ab18 Ab28 Ab43

pH

5 Good Good Very good

7 Good Good Very good

10.8 Very good Poor Very good

Temperature (0C)

20 Good Good Good

27 Good Good Very good

37 Very good Very good Very good

NaCl concentration

(%)

Without Very good Very good Very good

1 Very good Very good Very good

4 Good Good Very good

8 No No No

Antibiotic sensitivity

(zone of inhibition in

mm)

AMC30 Not reported Resistant Resistant

C30 Not reported 30 27

DO30 Not reported 25 30

TE30 Not reported 14 15

VA30 Not reported 16 16

Successful isolation of microbes requires an understanding of sample potential areas and growth factors

In the present study, soil samples were taken from rhizosphere like Kannan and Vincent (2011), industrial and public waste disposal areas

26.67% isolates were shown antimicrobial activity which was >21.88% (Abo-Shadi et al., 2010) and <59.09% (Thakur et al., 2007) results

Inhibition zone of crude extract against test organisms were 0-40mm which were > 0-18mm (Gurung et al., 2009) result

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In case of MRSAs were ranged from 0-20mm which were > 0-15mm (Yucel and Yemac, 2010)Combination of d/t plant extracts showed good antimicrobial

activity against food borne diarreagenic bacteria (Karmegan et al., 2008) Combined effect of extracts of Ab18 and Ab43 were shown

significant antimicrobial activity against S. boydi (40mm)The MIC (9.5mg/ml) and MBC(9.5 and19mg/ml) result of the

crude extracts against S. boydi were shown comparable to the Amoxicillin(9.5mg/ml)

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Different researchers were used different methods to identify isolated actinomycetes at genus level. For example,

Sundramoothi et al (2011) were grouped their isolates at Genus of Nokardiabased on biochemical, morphological and cultural characterization

Moncheva et al (2002) were grouped their isolates at Genus Streptomyces In the present study, the three selected isolates were characterized by some

morphological, cultural, biochemical and physiological methods to identify at least at genus level.

Based on 7th edition of Bergey’s Manual of Determinative Bacteriology (Breed et al., 1957), isolates were grouped under genus Streptomyces

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Selection and identification of potential sampling sites are useful for isolation of antibiotic producing actinomycetes Isolation and screening methods in optimum conditions are

important for searching and characterizing of new:Antibiotic producing microbesAntibioticsExploration of novel antibiotics from actinomycetes is an

important activity for potential medical, agricultural, pharmaceutical and industrial applications

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The discovery of potent antibiotics from three isolates could add a new dimension to microbial natural product research in this particular area

Isolates, capable of growing under selective nutrition and conditions of pH and salinity; possess a significant capacity to produce compounds having potential antimicrobial activity against human pathogens

Therefore, isolation, screening and characterization of new actinomycete species from potential soil sites in optimum condition can lead to the discovery of new antibiotics for the treatment of infectious diseases

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Based on the above conclusions, the following recommendations are forwarded.

There should be a great attention:-

At species and molecular level of characterization Optimization of isolates, purification and structural elucidation of

extracts For finding new antibiotic producing actinomycetes from their

diversified ecological niches For collaborative work among researchers and organizations to find

solutions for MDR pathogens

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My advisor, Dr. Feleke Moges for his inspiring guidance Staff members of Biotechnology, Chemistry, Veterinary and

MicrobiologyDepartment of Biology for sponsoring me to study the MSc

programmeDashin Beer FactoryMy family God for everything!!!

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10QU 4UR ATTENTION!