Streptomycete Isolation Of Bacteria From Soil Biology Essay

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Streptomycete produce antibiotics which are used for clinical treatments and prove beneficial for us. At the same time they also provide resistance to the antibiotics that are produced by them and therefore they transfer resistance to pathogenic organisms although they themselves are non pathogenic. This results in cases where the medical cases become untreatable as the organism causing the disease develops resistance to the antibiotic that is used in its treatment. Hence there is an urgent need to develop new antibiotics which can combat these resistant pathogens and screen the microorganisms involved. Streptomycetes have always been the method for discovering novel antibodies.

Streptomycetes are the most widely studied and well known genus of the actinomycete family. They mostly dwell in the soil and play an important role in decomposition. They are also responsible for the production of more than half of the world's antibiotics, and are hence of very high importance in the medical field.

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Streptomycetes resemble fungi in their structure. They have a branched, filamentous arrangement of cells which results in the formation of a network which is also known as mycelium. They metabolize many compounds such as sugars, alcohols, amino acids, and aromatic compounds by producing extracellular hydrolytic enzymes. Their metabolic diversity is due to their extremely large genome which has hundreds of transcription factors that control gene expression, allowing them to respond to specific needs.

Streptomycetes are most widely known for their ability to synthesize antibiotics. Over 50 different antibiotics have ben isolated from streptomycetes, providing most of the world's antibiotics. With the newly sequenced genome of Steptomyces coelicolorcomes the possibility of deriving still more antibiotics that have so far remained undiscovered.

The genes of the unusually large genome of Streptomyces coelicolor are grouped together in clusters, each cluster making a different antibiotic chemical. The genome contains around 20 clusters, of which only four were previously known. Projects to use the genome data in the synthesis of new antibiotics are already underway. Many of these projects are focusing on the possibility to using genetic engineering to to create entirely new chemicals by splicing together machinery from the numerous templates provided by Streptomyces coelicolor.

Bleomycin is an antibiotic drug with anticancer properties produced by Streptomyces verticillus. It was isolated in 1966 by Umezawa et al. and its mechanism of action is breaking the DNA double helix by the production of free radicals. It is used for malignant tumors, specifically germ cell tumors, lymphomas, head and neck and Kaposi's sarcomas.

Materials and Methods:-

Pretreated soil samples from different sources were used for the experiment. My group used soil sample from crockle park. The other soil sources used were:

1. Garden Soil. King George VI. Building. Newcastle University.

2. Leazes Park. Sycamore tree. Newcastle upon Tyne.

3. Thrunton Woods, soil from around heather. Rothbury, Northumberland.

4. Thrunton Wood, mixed woodland soil. Rothbury, Northumberland.

5. Derwent Woodlands, mixed woodland soil. Durham.

6. Newcastle University Cockle Park Experimental Farm.

Cockle Park . Plot 6. Untreated control has had hay cut annually since 1896 without manure or fertiliser applied. pH of soil 5

7. Cockle Park Plot 3. Treated with straw-based farmyard manure. pH of soil. 5.3

8. Alston river, near lead mines,(contaminated) stream sediment.

9. Jesmond Dene. Newcastle upon Tyne.

10. Cockle Park Plot. 5. Treated with straw-based farmyard manure. pH of soil 5.3

Isolation of Bacteria from soil:-

Firstly 1g of prepared soil was taken and mixed in 9ml of sterile dH2o and was vortexed for 1 min. After this the sample was allowed to sit for 30 mins and then serial dilutions upto 10-6 was done. Micropipettor was then used to plate 100µl of aliquots from dilution 10-2 to 10-5 onto the oatmeal agar (OA) plates which were amended using 100µg per ml cycloheximide. These plates were then incubated for 5 days at 280c.

Determination Of CFU's:-

Plate count method was used to determine the total number of bacteria and Streptomycete colonies and there number was noted.

Isolation and Purification of Streptomycete:-

Individual spores of Streptomycete were collected using toothpick and were then streaked onto the OA plates. Total of eight isolates were collected and streaked onto the two available OA plates these were marked as subcultures 1-4 and subcultures 5-8. These plates were then incubated for 7 days at 280c.

Purification of selected Streptomycete Subculture:-

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From the previously chosen eight subcultures best four were selected for further work. Sterile toothpick was used to streak each isolate on half a plate of OA as heavily as possible. Two OA plates were used in total and labeled as spore collection 1-2 and spore collection 3-4. These plates were then incubated for 7 days at 280c.

Spore Collection:-

Micropipettor was used to aliquot 0.75 ml of sterile 20% (v/v) glycerol into four 1.5 ml sterile microfuge tubes. These tubes were then labeled as spore suspension 1, 2, 3, and 4. Sterile cotton swab was then moistened by dipping into one of the microfuge tubes of sterile 20% (v/v) glycerol. Collect the streptomycete spores were collected by gently rolling the moist sterile swab across the surface of the spore collection plate. This step was then repeated for each of the four isolates using one fresh swab for each isolate. These swabbed spore collection plates were saved as they will be again used to collect plugs.

Antibiotic assays:-

Four OA plates were taken and labeled as streptomycete 1-4 antibiotic assay. Using a micropipettor, 100 μl of spore suspension 1 was added to the OA plate labeled streptomycete 1 antibiotic assay, same steps were repeated for streptomycete 2,3 and 4 antibiotic assay plates. A sterile bent glass rod was used to spread the spores and make good, solid lawns. These lawns were then allowed to dry for 5 mins. A sterile forceps was used to add antibiotic disc to each plate. Disc used by my group was Nalidixic acid. The disc were tapped using the forceps to make sure that they adhere properly to the plate. On each antibiotic plate 10µl spots of other streptomycete spore suspension were made using fresh tips every time. These plates were then incubated at 280c for 7 days.

Two Mueller Hinton(MH) plates were then taken and labeled as Gram positive antibiotic assay and gram negative antibiotic assay. One MH plate was divided into half and two sterile swabs were used to make two half-lawns of the Pseudomonas flourescens and Escherichia coli that were provided to us. The second MH plate was also then divided into half and two sterile swabs were used to make two half-lawns of the Micrococcus luteus and Bacillus subtilis provided. Each lawn was then labeled with the name of the swabbed bacteria. A flame sterilized cork borer was then used to punch four holes in the swabbed spore collection plate 1. The cork borer was sterilized again and the steps were repeated again for plates 2, 3 and 4. With the use of sterilized spatula these plugs were then placed into the four lawns in the MH plate. These plates were then incubated at 28oc for 7 days.

Data Collection:-

At the end of the incubation period, the antibiotic assay plates were observed for zones of inhibition. The Streptomycete antibiotic assay plates were checked for the zones of inhibition produced by the antibiotic discs and plugs of the streptomycete. Gram negative and Gram Positive assay plates were observed for the zones of inhibition produced by the plugs of streptomycete. Zone sizes were measured in mm from the edge of the antibiotic disc and the edges of plugs/spots of streptomycete spore isolates to the edge of the zones. Data was recorded in the tabular form.

Gram Staining:-

A toothpick was used to gather aerial filaments and spores from a portion of a plate that contained streptomycete isolate. A smear of the spores was made using a drop of water on a glass slide , this was then air dried and heat fixed by passing the slide over the flame of Bunsen burner. These were then flooded for 1 minute with crystal violet reagent. The slide was then washed in a gentle and indirect stream of tap water for 2 seconds. Gram's iodine was then flooded onto the slide and waited for 1 minute. Slide was then again washed in tap water for 2 minutes. Decolorizing agent was then added onto the slide for 15 seconds. The slide was then flooded with safrannine for 1 minute. The slide was then washed under the gentle and indirect stream of tap water and was dried using absorbent paper. The results were then observed under the oil immersion microscope.

Results and Discussion

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Negligible growth was observed in soil sample taken from White Moss Lake sediment, Thrunton soil (heather) and Alston Woods. Growth was observed in the other soil samples. The average number of cfu from different isolation plates was taken and the average value was used to calculate cfu/g and recorded as shown below:

Total

cfu/g

Streptomycete

Cfu/g

Crockle Park

11.9 X 104

5.2 X 104

Leezes Park

7 X 104

Too many

Hamsterley Forest

2 X 104

Colony characteristics of the four isolates chosen from the eight subcultures:-

Spore collection1: white colonies with grayish centre and fluffy spores.

Spore collection2: small white sporulating powdery colonies.

Spore collection3: large white colonies

Spore collection4: white colonies with brownish centre.

Plates A and B containing the subcultures 1-4 and 4-8

Plate observed after second step purification of selected streptomycete subculture

The four best isolates were used for antibiotic assay and the two gram negative and gram positive assays as described earlier. The results recorded are shown below:

Zone size radius (mm)

Spot, plug, or disk

Lawn

Streptomycete 1

Streptomycete 2

Streptomycete 3

Streptomycete 4

Antibiotic disk-nalidixic acid

Streptomycete 1

Resistant

Streptomycete 2

5.5

3.5

4

Resistant

Streptomycete 3

5

4.5

Resistant

Streptomycete 4

6

4

Resistant

E. coli

4.5

P. fluorescens

0

M. luteus

6.5

B. subtilis

7.5

Antisense pHN678

Zone size diameter (mm)

Spot or plug

streptomycete 1

0

streptomycete 2

0

streptomycete 3

0

streptomycete 4

0

Triclosan

19

Antisense pHN678-fabI

Zone size diameter (mm)

Spot or plug

streptomycete 1

0

streptomycete 2

0

streptomycete 3

0

streptomycete 4

0

Triclosan

25

The different zones and zone sizes produced are shown in the bar graph:

Gram staining resulted in the Gram positive test results as the Streptomycetes are Gram positive bacteria. Some isolates had a few mycelia and a numerous circular spores while as at the same time some were seen as having numerous mycelia. These results demonstrated that streptomycetes are inhibited by each other and they also resist each other which even has a effect on the microbial soil community. Depending on the streptomycete species present in a particular soil and the antibiotics produced there will be low number of bacteria communities inhibited by the antibiotic and high number that resist the bacteria. On the other hand there can be high number of different bacteria communities which have acquired resistance to the antibiotics produced by streptomycetes present in the soil. Soil bacteria generally acquire antibiotic resistant genes by horizontal gene transfer. Organisms acquire resistance to antibiotics at an increasingly alarming rate. Results from this study demonstrated resistance to the commercial antibiotics used as can be seen. Resistance to antibiotics by micro-organisms has very serious implications on the clinical use of antibiotics. Acquired antibiotics resistance among dangerous bacterial pathogens is an increasing medical problem. The source of resistance genes is from the Streptomycete that make the antibiotics and therefore need self protective mechanisms to avoid being killed by the very antibiotics that they produced thus avoiding suicide.