DNA FINGERPRINTING FOR DIFFERENTIATING E.coli ISOLATES FROM VARIOUS SOURCES USING RAPD TECHNIQUE

ABOUT AUTHOR:
Rudra Sharan Dwivedi
Regenerative Medical Services Ltd.
Assistant Manager QC
rudsd1987@gmail.com

ABSTRACT
Insightful and efficient methods for typing pathogenic microbes are significant for identification of specific strain, to trace the root of infection and to understand the evolution of virulence. A genetic characterization of six different isolates of Escherichia coli from sewage, clinical and contaminated soil samples using random amplified polymorphic DNA technique (RAPD)was carried out. Four primers of OPA series from operon technologies (OPA4, OPA5, and OPA6) were used to test out for polymorphism. Out of three primers tested OPA4 produced distinct bands in five strains with slight variation among isolates from different habitat. So we concluded that the RAPD could be used for discrimination of the E.coli isolates from different habitat.

REFERENCE ID: PHARMATUTOR-ART-1852

INTRODUCTION
DNA Finger printing or Genetic profiling is a technique employed by Biological scientists for the identification of living organisms in genetic level on the basis of their respective DNA profiles. Deoxyribo nucleic acid (DNA) is the fundamental building component of all living cells. The characteristic and physical features of all living things are determined by the specific arrangement of DNA base pair sequences in the cell. The distinct arrangements of adenine, guanine, thiamine and cytosine that regulate the production of specific proteins and enzymes control all the metabolic activities of living organism.

The chemical structure of every one’s DNA is the same. The difference between the human beings, animals, microorganisms and other living organisms lies in the order of the base pair sequence. Thus these differences in the DNA sequence of same species are called DNA Polymorphism. The DNA Polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. These are particularly useful for genetic analysis because the DNA of any organism exhibits an abundance of individual variation. various methods available for DNA fingerprinting are Restriction fragment length polymorphisms (RFLPs), Random amplified polymorphic DNA (RAPD), Amplified fragment length polymorphism (AFLP), etc. Among these methods RAPD has appreciable advantages that enable its extensive versatility and wide use. Some of its advantages are, it is simple and quick, Option not to use radioisotopes, DNA quality may be low (quick extraction possible), A large number of bands are produced per primer, Primers are readily available.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction in which the segments of DNA are randomly amplified. RAPD is performed by selecting the arbitrary, short primers-oligonucleotides (8-12 nucleotides), then proceeded with the PCR using a large template of genomic DNA, so that the fragments will be randomly amplified. Some of the applications areassessment of genetic variation in populations and species, to study the phylogenetic relationships among species and subspecies, to construct and understand genetic linkage maps, gene tagging, and identification of microbes, tracing the source of infections etc.The identification and classification of bacterial pathogens are of crucial importance in industrial, medical, agricultural, environmental and in study of microbial ecology. Accurate, rapid and cost-effective methods to identify pathogens are important for the diagnosis, surveillance and control of economically important diseases.

E. coli was discovered by German pediatrician and bacteriologist Theodor Escherich in 1885. E.coli is a gram negative, rod shaped, non-sporulating, facultative anaerobic bacterium that is common inhabitant of lower intestine of warm blooded animals. Because of its long history of laboratory culture and ease of manipulation, E. coli also plays an important role in modern biological engineering and industrial microbiology.

Scientific classification
Domain: Bacteria
Phylum: Proteobacteria
Class: Gamma Proteobacteria
Order:  Enterobacteriales
Family: Enterobacteriaceae
Genus:  Escherichia
Species: E. coli
Binomial name: Escherichia coli

Optimal growth of E. coli occurs at 37°C.E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide. Since many pathways in mixed-acid fermentation produce hydrogen gas, as in the case when E. coli lives together with hydrogen-consuming organisms such as methanogens or sulfate-reducing bacteria. E.coli can be identified as Gram negative rods by performing Gram’s staining, with no particular cell arrangement. Then, MacConkey agar inoculated with the sample will show deep red colonies, as the organism is lactose producing and fermentation of this sugar will cause the medium's pH to drop, leading to darkening of the medium. And growth on Levine EMB agar produces black colonies with greenish-black metallic sheen. Most E.coli strainsare harmless but some strains are found to be highly pathogenic and could cause Gastroenteritis, urinary tract infections and neonatal meningitis. In rare cases, virulent strains are also responsible for haemolytic-uremic syndrome (HUS), peritonitis, mastitis, septicemia and Gram negative pneumonia. The harmless strains are part of normal flora of the gut; symbiotic relation benefits their host by producing vitamin K and prevents the establishment of other pathogenic bacteria within the intestine. It can live on wide range of substrates.

A strain of E. coli is a sub-group within the species that has unique characteristics that distinguish it from other E. coli strains. These differences are often detectable only on the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. For example, a strain may gain pathogenic capacity, the ability to use a unique carbon source, the ability to inhabit a particular ecological niche or the ability to resist antimicrobial agents. Although virulent strains typically cause no more than a bout of diarrhea in healthy adult humans, particularly virulent strains, such as O157:H7 or O111:B4 can cause serious illness or death in the elderly, the very young or the immunocompromised.E. coli normally colonizes an infant's gastrointestinal tract within 40 hours of birth, arriving with food or water or with the individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. It is the primary facultative organism of the human gastrointestinal tract. As long as these bacteria do not acquire genetic elements encoding for virulence factors, they remain benign commensals. Different strains of E. coli are often host-specific, making it possible to determine the source of fecal contamination in environmental samples. Depending on which E. coli strains are present in a water sample, for example, assumptions can be made about whether the contamination originated from a human, other mammal or bird source. New strains of E. coli evolve through the natural biological process of mutation, and some strains develop traits that can be harmful to a host animal.E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population. This process led to the spread of the gene encoding shiga toxin from Shigella to E. coli O157:H7, carried by a bacteriophage. The strains of E. coli, such as O157:H7, O121 and O104:H21, produce toxins. Food poisoning caused by E. coli are usually associated with eating unwashed vegetables and meat contaminated post-slaughter. If E. coli bacteria escape the intestinal tract through a perforation (for example from an ulcer, a ruptured appendix, or a surgical error) and enter the abdomen, they usually cause peritonitis that can be fatal without prompt treatment. However, E. coli are extremely sensitive to such antibiotics as streptomycin or gentamicin. Perhaps, E. coli quickly acquires drug resistance capacity.

As sensitive and efficient methods for typing pathogenic microbes are important for tracing routes of infection, for understanding the spread of virulent genes and for the evaluation of effective therapeutic methods, deep perception of microbes in genetic level becomes necessary. And the main theme is about applying analytical method that is cost effective, less time consuming, accurate and more revealing.Thus RAPD gains it versatility in various diagnostic and analytical techniques as it is proved to be one of the best methods which generate results based on genetic level.In this study the PCR- based RAPD fingerprinting method is used as a genetic marker to detect genetic variation among E.coli isolated from different environmental samples. The scientific data presented in this study may be used as a valuable tool in the study of the molecular epidemiology of pathogenic E.coli.

MATERIALS AND METHODS
The work is concerned with differentiating the dissimilar strains belong to different habitat by genetic analysis; the samples were collected from the sources supposed to be the habitat of pathogenic and non pathogenic bacteria.

I. Sample collection:
Samples were collected from various clinical and non clinical environmental sources.
The samples were collected from natural environments and so the E.Coli has to  be separated from mixed culture. For this reason the selective as well as differential media was used to ensure the isolation of specific microbe of interest.

II. Microbial Isolation:
1. From Soil samples,
1gm of sample was taken and dissolved in 10ml of sterile distilled water and dilution was made up for the ranges from 10^-1 to 10^-7. Then the diluted samples of 10^-3 to 10^-6 were inoculated in the nutrient agar media.

2. From Sewage samples,
0.1ml of sample was serially diluted from 10^-1 to 10^-7 and then diluted samples of 10^-3 to 10^-6 were inoculated in the nutrient agar media.

3. From Clinical samples,
The plates inoculated with stool samples were obtained from clinical pathology diagnostic laboratory, from those reddish colonies were transferred to EMB media.

III. Characterization:
a. Gram’s staining
Gram staining is widely used characterising method that reveals the morphology of the cell.The bacteria which retain the primary stain (appear dark blue or violet) (i.e. not decolorized when stained with grams’ method) are called gram-positive, whereas those that lose the crystal violet and counter stained by safranin (appear red) are referred to as gram-negative. The differences in staining responses to the gram stain can be related to chemical and physical differences in their cell walls. The gram-negative bacterial cell wall is thin, complex, multi-layered structure and contains relatively a high lipid contents, in addition to protein and mucopeptides. The higher amount of lipid is readily dissolved by alcohol, resulting in the formation of large pores in the cell wall which do not close appreciably on dehydration of cell-wall proteins, thus facilitating the leakage of crystal violet-iodine (CV-I) complex and resulting in the decolorization. Bacterium that later takes the counter stain and appears red. In contrast, the gram-positive cell walls are thick and chemically simple, composed mainly of protein and cross-linked mucopeptides. When treated with alcohol, it causes dehydration and closure of cell wall pores thereby not allowing the loss of (CV-I) complex and cells remain purple. As the discrimination is based on the composition of cell wall of the bacteria. The gram positive bacterium retains the crystal violet and appears purple and the gram negative bacterium gets stained with safranin and appears pink.

After 24 hours incubation at 37?C, Bacterial colonies were screened by Gram’s staining for gram negative rods.

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Procedure:

• A thin Smear of culture broth was made over the clean glass slide.
• The slide was gently heated to enable fixing of smear over the slide.
• Crystal violet (primary stain) solution was flooded over the smear and left undisturbed for 1 minute.
• It was Gently Rinsed with water.
• Gram’s iodine (mordant) was flooded over the smear and left for 1 minute.
• The slide was washed with water.
• Ethanol was flushed over the smear (decolouriser).
• The slide was washed with water.
• Safranin was flooded and left for 2 minutes.
• The slide was again washed with water and dried.
• The smear over the slide was examined using microscope.

And the colonies identified as Gram negative rods were streak plated on EMB agar media. After 24 hours incubation in EMB agar media the streaks showed greenish metallic sheen were confirmed as E.coli.(E.coli degrades lactose and produces acids which changes the colour of media and shows characteristic metallic sheen)

IV. Genomic DNA isolation:
The micro organisms were cultured in LB (Luria Bertani) media. This special media is used before DNA isolation and in other microbial genetic manipulations as this media is believed to recover the cells from shocks and provides ready nutrients for the growth.

LB (Luria Bertani) Composition:
Tryptone  - 10.00g
NaCl  - 10.00g
Yeast extract  - 5.00g
Distilled water  - 1000ml.
PH  - 7

Add all the above mentioned components to prepare 1L.media and autoclave it for sterilization.

Phenol: Chloroform  method for DNA extraction is widely used and highly productive method for the genomic DNA isolation from bacterial cell. It involves many reagents that helps in selective isolation of DNA and purifies it from other cell debris contaminations. The lysis buffer helps in breaking and release of intercellular components. The Tris HCl changes the P^H so the ionic condition, EDTA chelates ions and destabilises and disturbs the enzymes and cell wall, NaCl alters osmolity and SDS denatures proteins. Treatment with Phenol: chloroform biphase mixture helps to separate out the denatured proteins. Where chloroform: isoamyl alcohol biphase mixture removes the trace phenol. Ethanol enhances precipitation of DNA. Storage at 4 ? C ensures the stability of DNA.

Procedure
* E.Coli is inoculated in LB broth and incubated at 37?C for 48 hours.
* 2 ml of broth was taken in microfuge tube.
* Broth in microcentrifuge tube was centrifuged at 6000 rpm for 10 minutes.
* Supernatant was discarded and the E.Coli cell in form of pellet was retained in the tube.
* Pellet was resuspended in 1 ml lysis buffer.
* Then the tubes were incubated in the boiling water bath for 10 minutes.
* 1 ml of phenol: chloroform (1:1) ratio mixture was added and then centrifuged for 10 minutes at 10,000 rpm.
* Equal volume of chloroform: isoamyl alcohol (24:1) ratio and 1/20th volume of 3M Sodium acetate was added to the supernatant and centrifuged at 10,000 rpm for 10 minutes.
* Double volume of chilled ethanol was added to the upper aqueous layer and incubated at -20?C for 20 minutes.
* Then the tube was centrifuged for 10 minutes and the pellet was air dried.
* Finally the DNA pellet was dissolved in 20-50μl of TE buffer.
* DNA was stored at 4?C to ensure its stability

Reagents composition
* Lysis buffer
10 mM Tris HCl
5 mM EDTA
0.5% SDS
1M NaCl 

* TE buffer (Tris EDTA buffer)
10mM Tris
1mm EDTA

V. Quantitative and Qualitative analysis of DNA:
a. Quantitative analysis of DNA by nanodrop spectrophotometry.
Quantitative analysis was done using  nanodrop^TM  spectrophotometer.

The sample shows 1.8 OD indicates the purest form, the negative deviation indicates the protein contamination and the positive deviation indicates the RNA contamination.

Procedure:

• TE buffer was used as Blank and the samples were analysed at 260 nm

• The optical density and concentration profile was obtained from the spectrometric readings.

b. Qualitative analysis by agarose gel electrophoresis.
Qualitative analysis was done using 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.  In this method agarose serves as the stabilizing medium and the molecules are separated based on the charge, size and shape.  Under neutral alkaline pH, the phosphate groups of DNA become ionized into polyanions, as a consequence the DNA when placed in an electric field moves towards anode.  The gel contains microscopic pores.  Smaller molecules move through these pores more easily then larger ones.Matrix of agarose gel acts as a molecular sieve through which DNA fragment move on application of electric current. Higher concentration of agarose gives firmer gels, i.e., spaces between cross-linked molecules is less and hence smaller DNA fragments easily crawl through these spaces. As the length of the DNA increases, it becomes harder for the DNA to pass through the spaces, while lower concentration of agarose helps the movements of larger DNA fragment as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye through the gel

Factors affecting the rate of DNA migration in Agarose gels:

• Molecular size of the DNA
• Agarose concentration
• Conformation of the DNA
• Applied voltage
• Direction of the electric field
• Presence of intercalating dye
• Composition of the electrophoresis buffer

Electrophoresis using 1.5% agarose gel.

• 0.45 g of agarose was dissolved in 30 ml of 1X TAE (Tris Acetate EDTA) buffer.

• The solution was heated until it became transparent and then it was allowed to attain lukewarm condition.

• Then 3μι of Ethidium bromide was added to the gel and then poured in a gel casting tray and allowed to solidify after fixing comb.

• The solidified gel was moved to 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.

• 6μι of the isolated DNA was mixed with 4μι of Bromophenol blue and whole 10μι mixture was added in the respective wells for every samples.

• The electrophoresis was done with 100 V for 40 minutes.

• Then the gel was viewed in gel documentation unit to analyse the DNA bands.

Reagent composition:
50X TAE (1ι)
242 g of Tris base
57.1 ml of glacial acetic acid
100 ml of 0.5M EDTA (P^H 8.0)

VI. RAPD:

PCR primers
Primers are oligonucleotides that flank the template DNA due to its complementarities so that the flanked region of DNA template will be amplified. Three OPA series primers (Operon technologies, USA) were used for the RAPD analysis. These primers are arbitrary primers.

PCR
PCR is invitro nucleic acid synthesising reaction it amplifies the template DNA flanked by primers. Thermocycler controls the temperature at various stages of the replication cycle.

* PCR reaction mixture:
The components were taken to match the final concentration of 1X assay buffer, 1mM/ μι MgCl₂ , 0.6mM/ μι dNTPs, Taq polymerise 0.1 unit/ μι, 50 pM/ μι of forward primer, 50 pM/μι of reverse primer.

For 25 μι of total reaction mixture,
10X Assay buffer
(Includes 25 mM of MgCl)- 2.5 μι.
dNTPs- 1.5 μι.
Taq polymerise- 0.83 μι.
Forward primer- 2.55 μι
Reverse primer- 2.55 μι.
DNA- 20 ng/ μι
Nuclease free water- rest to make up 25 μι reaction volume In PCR tube.

Procedure
All the above mentioned components were taken in a PCR tube and loaded in a Thermocycler.

The following conditions were set to run the PCR
Step 1:
Initial Denaturation at 94?C for 5 minutes.

Step 2: Repeat- 45 cycles
Denaturation at 94?C for 30 seconds.
Annealing temperature is Tm (based on primer) for 30 seconds.
Extension at 72?C for 2 minutes.

Step 3:
Final extension at 72?C for 5 minutes.
Storage at 4?C.

Calculation of Tm/ Annealing temperature for PCR reaction:
As the annealing temperature greatly depends upon the sequence of primer use the Tm has to be calculated for every primer using formula,

T_m = [4(G+C) + 2(A+T)].
Tm for OPA4 (^5’CCGCCCAAAC^3’) - 34?C.
Tm for OPA5 (^5’TCTGTCGAGG^3’) - 32?C.
Tm for OPA6 (^5’CACCTTTCCC^3’) -32?C

After the completion of PCR the PCR products were analysed using 2% agarose gel electrophoresis and the results were examined using Gel documentation system.

CONCLUSION
RAPD analysis is a sensitive method for E. coli fingerprinting. Its success depends on the selection of primers, purity of reagents. Results of this study indicate the potential of RAPD fingerprints for differentiating E. coli isolates from different habitats. It obviously describes that the information about virulence, habitats, phylogenetic relationships and other special distinct traits of different species and strains can be obtained by RAPD fingerprinting by using the right primers. It could also be used for medical diagnoses and in the identification of new strains and isolates. The identity among different isolates can also be checked by using a particular primer and its strain specific RAPD marker band for epidemiology studies.

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