GENETIC VARATION IN PATHOGENIC E.coli USING RAPD : GENETIC VARATION IN PATHOGENIC E.coli USING RAPD Group : Group HARISH REMELLA
HARSHA VARDHAN REDDY
Under the guidance of
INTERNAL GUIDE EXTERNAL GUIDE
Murthy & Madhu Smitha Tamilarasan Introduction : Introduction Escherichia coli is a common inhabitant of human and animal intestines of all warm blooded animals. It is a facultative anaerobe. The temperature range is 10-400C (optimum 370C) is good for the growth of E.coli.
Colonies are large, thick, grayish white, moist, smooth opaque or partially translucent discs. : From birth, E. coli colonizes in the gastrointestinal tract where it interacts in symbiosis with its host. It is a wide indicator for fecal pollution (Whitman, 2005).
E. coli produces two kinds of toxins – hemolysens and enterotoxins. Hemolysens do not appear to be relevant in pathogenesis though they are produced more commonly by virulent strains. Thompson J S (1990). : Enterotoxins are important in pathogenesis of diarrhea. Three distinct types of E. coli enterotoxins have been indentified – heat labile toxin (LT), heat stable toxin (ST) and verotoxin (VT) (Pickett, 2004).
Normally the objective of PCR is to generate defined fragments of DNA from highly
specific primers. In the case of RAPD short oligonucleotide primers are arbitrarily selected to amplify a set of DNA fragments randomly distributed throughout the genome. OBJECTIVES OF THE STUDY : OBJECTIVES OF THE STUDY Morphological and Biochemical characterization of E.coli: microscopic and biochemical tests
Preparation of Pure culture: streak plate method
Genomic DNA isolation: using phenol chloroform method
Qualitative analysis of DNA: Agarose gel electrophoresis
5. Quantitative analysis of DNA: Nanodrop spectrophotometer
6. Determining genetic variations in E.coli strain’s using RAPD SAMPLE COLLECTION : SAMPLE COLLECTION Faeces
Intestinal sample GRAM STANING PROCEDURE : GRAM STANING PROCEDURE A] Make thin smears of samples on separate glass slides.
B] Let the smears air dry.
C] Heat fixing the smears.
D] Hold the smears using slide rack or clothes pin.
E] Cover each smear with crystal violet for 30 seconds.
F] Wash each slide with distilled water for a few seconds, using
G] Cover each smear with grams iodine for 60 seconds.
H] Wash off the iodine solution with 95% ethyl alcohol.
Add ethyl alcohol drop by drop , until no more color
flows from smear [The gram- bacteria are not
positive affected, while all gram-negative bacteria
are completely decolorized ].
I] Wash the slides with distilled water and drain.
J] Apply saffranin to smears for 30 seconds [counter-staining].
K] Wash with distilled water and blot dry with absorbent paper.
L] Let the stained slides air dry. MORPHOLOGICAL IDENTIFICATION Light microscopic view Gram negative E.coli Eosin methylene blue agar media : Eosin methylene blue agar media This medium is used for the isolation, cultivation and differentiation of gram negative bacteria based on lactose fermentation. Slide 10: Faeces sample urine sample Slide 11: especially Escherichia coli that utilize lactose and sucrose appear as colonies with a green metallic sheen or blue – black to brown colour. Bacteria that do not ferment lactose appear as colourless or transparent light purple colonies. Intestinal sample Biochemical analysis : Biochemical analysis In order to observe biochemical activities of microorganisms which were specific to individual genus and species various kinds of specially prepared media were inoculated with pure cultures of microorganisms.
Many distinctive enzyme activities can be
demonstrated by observing for the by products resulting from the action of enzymes on specific substrates within the specially prepared media.
Different mediums were used for the biochemical characterization of the isolated and selected bacteria for their identification according to Bergey’s Manual of Determinative Bacteriology. Indole production test : Indole production test Tryptophan Indole + pyruvic acid + ammonia.
Indole + p – dimethylamino benzaldehyde = Cherry red compound
Tryptophan is decomposed in to its metabolic products like indole, pyruvic acid and ammonia by the enzymes, tryptophanase. The indole is detected by calorimetric reaction by p – dimethyl amino benzaldehyde (kovac’s reagent). Slide 14: Procedure
Peptone broth was prepared
test culture was inoculated in test
incubated at 37oC for 24 hours
0.2ml of kovac’s reagent was added
cherry red colour in the alcohol layer indicates a positive reaction. Methyl red test : Methyl red test The methyl red test is employed to detect the ability of microorganisms to oxidize glucose. Procedure : Procedure peptone and phosphate wher dissolved in distilled water
At 7 ph
5ml of media was pored into each test tube
sterilized at 121oC for 15 m
solution was sterilized by filtration and 0.25ml was added to each tube MR-VP Broth preparation Test culture was inoculated in the MR broth After incubation 5-6 drops of methyl red solution was added. incubated at 37oC for 48 hours Slide 17: A bright red colour indicate a positive test. [Yellow or orange colour indicates a negative reaction] positive Voges – Proskauer (vp)Test : Voges – Proskauer (vp)Test In VP oxidation will occur under alkaline condition in the presence of a catalyst, alpha napthol Procedure : Procedure The test culture was inoculated in the VP broth
incubated at 37˚ C for 48 h
1ml of 40 % potassium hydroxide (plus creatine) and 3ml of a 5% solution of alpha – napthol was added in absolute ethano
A positive reaction is indicated by the development of a pink colour negative Citrate utilization test : : Citrate utilization test : Citrase
Citric acid → oxaloacetic acid + acetic acid
Pyruvic acid + carbon dioxide
CO2 + 2Na+ + H20 → Na2CO3(alkaline PH)
Bromothymol blue → Green to Prussian blue
During this reaction, the medium becomes alkaline as the carbon dioxide combines with sodium and water to form sodium carbonate changes the bromothymol blue fom green to deep Prussian blue. Slide 21: Simmons citrate medium dispensed in test tubes Procedure sterilized at 121oC
for 15 minutes The organisms from a saline suspension to test on the agar slopes and was inoculated 96 hours at 37oC A positive test shows a blue colour on the streak of growth Retention of original green colour and no growth on the line of streak indicates a negative reaction. negative Oxidase test : Oxidase test During aerobic respiration, oxidase enzymes play a vital role in the operation of electron transport system. Cytochrome oxidase theat catalyzes the oxidation of a reduced cytochrome by molecular oxygen, resulting in the formation of water or hydrogen peroxide. This test depends on the presence of certain oxidases in bacteria that will catalyzes the transport of electrons between electron donors in the bacteria and a redox dye (Tetramethyl p-paraphenylene diamine dihydrochloride).The dye is reduced to a deep purple colour. Procedure : Procedure The test sample is cultured on a suitable solid medium 1% solution of Tetramethyl p-phenylene diamine dihydrochloride solution poured onto a plate so as to cover the surface and then decant The colonies of oxidase positive organisms rapidly develop a purple colour. negative CATALASE TEST : CATALASE TEST Accumulation of hydrogen peroxide and super oxide leads to the death of the organisms unless they are degraded enzymatically, 4H+
2e- Superoxide dismutase
2O2 → 2O2 → 2H2O2
Superoxide radical Hydrogen peroxide
2H2O2 → 2H2O
Hydrogen peroxide Catalase Water Slide 25: Procedure A nutrient agar slopes were prepared and streaked with test cultures the test tubes were incubated
at 37˚ C for 24 hours After incubation, 1ml of 3% hydrogen peroxide was added after 5 minutes the test tubes were examined immediately for the evolution of bubbles, which indicates a positive test. positive Nitrate reduction test : Nitrate reduction test Among bacteria, there exists a variation in energy metabolism. A number of bacteria are capable of respiring under completely anaerobic conditions by utilizing nitrate, sulfate or carbonate as a terminal inorganic electron acceptor.
NO3- + 2e + H → NO2 - + H2O
Reduction of nitrate in the presence of a stable electron donor to nitrite. Procedure : Procedure Nitrate medium was prepared
sterilized at 121˚C for 15 minutes
After sterilization, inoculate the medium with culture incubated at 350 C for 96 hours.
Following incubation, add 0.1 ml of the test reagent of the test culture.
A red colour developing within a few minutes indicates the presence of nitrate and hence the ability of the organism to reduce nitrate positive Starch hydrolysis : Starch hydrolysis Starch is a linear polymer of glucose molecule linked together by glucosidic bonds. Starch as such cannot be transported in to the cell for energy production, because of its high molecular weight. To assimilate starch for energy and catabolic reactions, it must be degraded in to basic glucose units by starch hydrolyzing enzymes. These enzymes are secreted by the microorganisms in to the medium which degrade starch primarity to glucose. The resulting low molecular weight soluble glucose molecules are now able to pass in to the cell for energy production via, glycolysis. Slide 29: Procedure About 75ml of starch agar plates were prepared a single line of streak of the organisms was made across the centre of the starch agar plate. The plates were incubated at 37oC for 24 hours After incubation the plates were flooded with iodine solution Hydrolysis is indicated by clear zones around the growth and uncharged starch gives as blue colour Slide 30: negative TABLE : BIOCHEMICAL TEST ANALYSIS : TABLE : BIOCHEMICAL TEST ANALYSIS Gram Negative : Gram Negative Bacilli Medium-sized
nucleatum Coiled &
Chromobacterium Cocci Neisseria
Veillonella Slide 33: Gram Negative Rods Oxidase Test Enterobacteriaceae Aeromonas, Pseudomonas, Vibrio Slide 34: Family Enterobacteriaceae Lactose Fermentation Citrobacter diversus
Erwinia carotovora Erwinia chrysanthemi
(subsp. ozaenae and pneumoniae)
Serratia rubidaea Indole Citrobacter diversus
Klebsiella oxytoca Citrate Citrobacter diversus
(VP and H2S pos.)
(VP pos and H2S neg.) Escherichia coli
(MR pos ) Slide 35: ISOLATION OF DNA FROM BACTERIAL CELLS :phenol chloroform method. REAGENTS USED
Lysis buffer : 10 ml tris HCL , 5 ml EDTA , 0.5% SDS , 1ml Nacl .
Phenol : chloroform mixture in ratio 1:1
Chloroform : isoamyl alcohol mixture in ratio 24:1 and sodium actetate .
TE (tris , EDTA ) buffer Slide 36: Procedure
Inoculate samples in LB broth; incubate at 37°c for 24 hours.
Take 2ml of broth in eppendoff tubes.
Centrifuge at 6000rpm for 10min.
Discard the supernatant and store the pellets containing cultures.
Resuspend the pellets in 1ml lysis buffer.
Incubate at 45º C in water bath for 10min.
Add 1ml phenol: chloroform mixture and centrifuge at 10,000rpm for 10min.
Decant supernatant in a separate eppendoff tube and add equal volume of chloroform and Iso amyl alcohol mixture and 1/20th volume of 3M Sodium acetate and centrifuge at 10,000rpm for 10min
To the upper aqueous layer add double the volume of chilled ethanol and incubate at -20º C for overnight.
Centrifuge at 12,000rpm for 10min and air dry the pellets.
Dissolve the DNA pellet in 20-50µl of TE buffer.
Load on a 0.8% agarose gel and run at 50 V and 75mA for an hour. QUALITATIVE ANALYSIS OF DNA : QUALITATIVE ANALYSIS OF DNA AGAROSE GEL ELECTROPHORESIS
This procedure examines the integrity of the isolated DNA and checks the contaminations. Agarose gel electrophoresis is based on the principle that biological molecules are charged species, which can move in an electric field. Slide 38: Electrophoresis using 0.8% agarose gel
1) 0.24 gm of agarose was dissolved in 30 ml of 1X TAE (Tris Acetate EDTA) buffer.
2) The solution was heated until it became transparent and then it was allowed to attain lukewarm condition.
3) Then 3 µl of Ethidium Bromide (EtBr) was added to the gel and then poured in a gel casting tray and allowed to solidify after fixing comb.
4) The solidified gel was placed in electrophoresis tank and then the tank was filled with 1X TAE buffer to a certain level so that the gel gets submerged in the buffer.
5) 6 µl of the isolated DNA was mixed with 4 µl of Bromophenol blue dye and whole
10 µl mixture was added in the respective wells for every samples.
6) The electrophoresis was carried out at 50V, 75 mA current for an hour. Then the gel was viewed in alpha imager. S1 S2 S3 QUANTITATIVE ANALYSIS OF DNA: NANODROP SPECTROPHOTOMETRY : QUANTITATIVE ANALYSIS OF DNA: NANODROP SPECTROPHOTOMETRY Switch on the system.
Click ND -1000 software.
Select Nucleic acid.
Place 2µl of distilled water on the pedestals to initiate the instrument.
Place 2µl of TE buffer to blank.
Place the samples on the pedestals and select measure to analyse the results at
260/280nm, the concentration profile was obtained from the nanodrop readings. Protocol: Sample 3 : Sample 3 Sample 1 Sample 2 RAPD : RAPD This method based on PCR developed in 1990.
RAPD is different from conventional PCR as it needs one primer for amplification. The size of primer is normally short (10 nucleotides), and therefore, less specific.
the primers can be designed without the experimenter having any genetic information for the organism being tested.
more than 2000 different RAPD primers can be available commercially
Genomic DNA normally has complimentary sequences to RAPD primers at many locations. RAPD technology : RAPD technology A B C Genomic DNA + Taq polymerase + Arbitrary primers A + d ntp’s + Buffer PCR (under relaction conditions) Slide 43: Electrophoresis PCR 300 bp 260 bp 520 bp A B C Slide 44: PCR REACTION CONDITION:
The following conditions were set to run the PCR
Step 1: (1 repeat)
Initial denaturation at 95º C for 5 mins.
Step 2: (40 repeats)
Final denaturation at 95 ºC for 1 min.
Annealing temperature is 35 ºC for 1 min.
Extension at 72 ºC for 2 mins
Step 3: (1 repeat)
Final extension at 72 ºC for 7 mins
Storage at 4 ºC for 1 min.
PCR products were analyzed on 1.5% agarose gel and visualized by ultraviolet illumination. Slide 45: M L1 L2 L3 L4 L5 L6 L7 L8 L9 OPU 15
L2-E.Coli sample 2(urine).
L3-E.Coli sample 3 (intestinal). OPU 16
L5-E.Coli sample 2(urine).
L6-E.Coli sample 3 (intestinal). OPU 17
L8-E.Coli sample 2(urine).
L9-E.Coli sample 3 (intestinal). OPU15
5′- ACGGGCCAGT- 3′
5′ - CTGCGCTGGA - 3′
5′ - ACCTGGGGAG - 3′ Advantages: : Advantages: No prior knowledge of DNA sequences is required
Random distribution throughout the genome
The requirement for small amount of DNA
Easy and quick to assay
Commercially available primers are applicable to any species
The potential automation of the technique
RAPD bands can often be cloned and sequenced to make SCAR (sequence-characterized amplified region) markers Limitations: : Limitations: Dominant nature (heterozygous individuals can not be separated from dominant homozygous)
Sensitivity to changes in reaction conditions, which affects the reproducibility of banding patterns
The scoring of RAPD bands is open to interpretation
The results are not easily reproducible between laboratories Slide 49: Sample 1 produced 24 bands using primer OPU15 of which 6 bands where polymorphic and the percentage of polymorphism was found to be 25% .
Sample 2 produced 26 bands using primer OPU16 of which 10 bands where polymorphic and the percentage of polymorphism was found to be 38%.
Sample 3 produced 11 bands using primer OPU17 of which 2 bands where polymorphic and the percentage of polymorphism was found to be 18%.
The total polymorphism of all the three samples was obtained by calculating the mean value whish was found to be 27% .This indicates that there is genetic variation among the three E.Coli samples but the extent of variation is very small CONCLUSION Reference : Reference Cui S, Schroeder C M, Zhang D Y & Meng J, Rapid sample preparation method for PCR-based detection of Escherichia coli 0157: H7 in ground beef, J Appl Microbiol, 95 (2003)
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Cave H E, Bingen J, Elion & Denamur E, Differentiation of Escherichia coli strains using randomly amplified polymorphic DNA analysis, Res Microbiol, 145 (1994) 141-150.
Souza V, Rocha M, Valera A & Eguiarte L E, Genetic structure of natural population of Escherichia coli in wild host in different continents, Appl Environ Microbiol, 65 (1999) 3373-3365.
Yuri K, Nakata K, Katae S, Tsukamoto T & Hasegawa A, Serotype and virulence factor of Escherichia coli strains isolated from dogs and cats, J Vet Med Sci, 61 (1999) 37-40. Slide 51: Yuri K, Nakata K, Katae H, Yamamoto, S & Hasegawa A, Distribution of uropathogenic virulence factors among Escherichia coli strains isolated from dogs and cats, J Vet Sci, 60 (1998) 287-290.
Wang G, Whittam T S, Berg C M & Berg D E, RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains, Nucleic Acids Res, 21 (1993) 5930-5933.
Cave H, Bingen E, Elion J & Denamur E, Differentiation of Escherichia coli strains using randomly amplified polymorphic DNA analysis, Res Microbiol, 145 (1994) 141-150.
Bihong , Xuhua (2005) Genetic Variation in Clones of Pseudomonas pseudoalcaligenes After Ten Months of Selection in Different Thermal Environments in the Laboratory , CURRENT MICROBIOLOGY :Vol. 50 , p 238–245
Mueen Aslam, Frances Nattress, et al (2003) Origin of Contamination and Genetic Diversity of Escherichia coli in Beef Cattle, Appl Environ Microbiol. :69(5): 2794–2799. THANK YOU : THANK YOU