MOOC4, Module 36

Isolation and Characterization of E.Coli

Main Body:

Systematic Position:

Kingdom: Eubacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria.

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Species: coli

History and discovery: The genus was with Salmonella, but the two genera got diverged around

102 million years ago.

1. Theodor Escherich (1885): discovered this organism in the feces of healthy individuals

and called it Bacterium coli commune due to the fact it is found in the colon.

Fig.1: Theodor Escherich (1857-1911)

2. W. Migula (1895): Renamed the bacteria as Bacillus coli because of its rod shaped

nature.

Fig.2:W. Migula (1863-1938)

3. Castellani and Chalmers (1919): Gave the modern name after the original discoverer.

Fig.3: Castellani (1877-1971)

4. Joshua Lederberg and Edward Tatum (1946): They first described the phenomenon

known as bacterial conjugation using E. coli as a model bacterium, and it remains the

primary model to study conjugation.

Fig.4: Joshua Lederberg (1925-2008)

Fig.5: Edward Tatum (1909-1975)

5. Seymour Benzer (1955) used E. coli and phage T4 to understand the topography of gene

structure. It proved the linear structure of gene.

Fig.6: Seymour Benzer (1921-2007)

6. S.N. Cohen and H. Boyer (1972) in E. coli, using plasmids and restriction enzymes to

create recombinant DNA, became a foundation of biotechnology.

Fig.7: S.N. Cohen (1935-)

Fig.8: H. Boyer (1936-)

Cell Structure:

1. It is a typical cocci bacillus in shape that is it is neither fully round nor rod shaped in

nature.

Fig.9: E.coli under light microscope

2. Strains that possess flagella are motile. The flagella have a peritrichous arrangement.

Fig.10: Peritrichous flagella of E.coli

3. The first complete DNA sequence of an E. coli genome (laboratory strain K-12

derivative MG1655) was published in 1997. It was found to be a circular DNA molecule

4.6 million base pairs in length, containing 4288 annotated protein-coding genes

(organized into 2584 operons), seven ribosomal RNA (r RNA) operons, and 86 transfer

RNA (tRNA) genes. The coding density was found to be very high, with a mean distance

between genes of only 118 base pairs. The genome was observed to contain a significant

number of transposable genetic elements, repeat elements, cryptic prophages,

and bacteriophage remnants.

Fig.11: Genome of E.coli

4. Many proteins previously thought difficult or impossible to be expressed in E. coli in

folded form have also been successfully expressed in E. coli. For example, proteins with

multiple disulphide bonds may be produced in the periplasmic space or in the cytoplasm

of mutants rendered sufficiently oxidizing to allow disulphide-bonds to form.

Fig.12: Protein and its processing in E.coli

Characteristic features:

1. Escherichia coli is a Gram-negative, facultatively anaerobic.

Fig.13: E.coli trapped in the vili.

2. It is commonly found in the lower intestine of warm-blooded organisms.

Fig.14; Shiga toxin producing strain of E.coli.

3. Most E. coli strains are harmless; some serotypes can cause serious food poisoning in

their hosts producing Shiga toxin.

Fig.15: Binding and release of Shigatoxin

4. They produce vitamin K and also prevent colonization of pathogenic bacteria.

Fig.15: Chorismate to dihydroxy napthanoic acid to phylloquinone conversion

5. Under favourable conditions it takes only 20 minutes to reproduce.

6. Cells are able to survive outside the body for a limited amount of time, which makes

them ideal indicator organisms to test environmental samples for fecal contamination.

7. It is non-sporulating, reproducing mostly by binary fission.

Fig.16: Binary fission of E.coli under SEM

8. 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.

Fig.17: Conjugation in E.coli

Growth of the bacterium:

1. The bacterium can be grown easily and inexpensively in a laboratory setting in nutrient

agar medium and thus.E. coli is the most widely studied prokaryotic model organism, and

an important species in the fields of biotechnology and microbiology.

Fig.18: Discrete colonies of E.coli in nutrient agar plate

Optimal growth of E. coli occurs at 37 °C (98.6 °F) but some laboratory strains can multiply at

temperatures of up to 49 °C (120 °F).

2. Growth can be driven by aerobic or anaerobic respiration, using a large variety of redox

pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino acids.

The reduction of substrates such as oxygen, nitrate, fumarate, dimethyl

sulfoxide and trimethylamine N-oxide

Fig.19: Metabolic reduction of trimethylamine oxide

Metabolism;

1. It is a facultative anaerobic organism (that makes ATP by aerobic respiration if oxygen is

present, but is capable of switching to fermentation or anaerobic respiration if oxygen is

absent).

2. E. coli uses mixed-acid fermentation in anaerobic conditions,producing a series of organic

acid like lactate, succinate, ethanol, acetate and carbon dioxide.

Fig.20: Fermentation of a series of organic acid in E.coli

The mixed-acid fermentation produce hydrogen gas, these pathways require the levels of

hydrogen to be low, as is the case when E. coli lives together with hydrogen-consuming

organisms, such as methanogens or sulphate-reducing bacteria.

Diversity:

Escherichia coli encompass an enormous population of bacteria that exhibit a very high degree

of both genetic and phenotypic diversity. Genome sequencing of a large number of isolates of E.

coli and related bacteria shows that a taxonomic reclassification would be desirable. However,

this has not been done, largely due to its medical importance and E. coli remains one of the most

diverse bacterial species: only 20% of the genome is common to all strains.

Fig.21: Comparison of non-pathogenic and pathogenic strain of E.coli

Fig.22: Twelve major sero groups of E.coli.

Serotypes

A common subdivision system of E. coli, but not based on evolutionary relatedness, is by

serotype, which is based on major surface antigens (O antigen: part of lipopolysaccharide layer;

H antigen: flagellin; K antigen: capsule), e.g. O157:H7). It is however common to cite only the

sero-group, i.e. the O-antigen.

Fig.23: X gal positive E.coli

Fig.24: Indole positive E.coli

At present about 190 sero-groups are known. The common laboratory strain has a mutation that

prevents the formation of an O-antigen and is thus non-typeable.

Neotype strain

E. coli is the type species of the genus (Escherichia) and in turn Escherichia is the type genus of

the family Enterobacteriaceae.

The original strain described by Escherich is believed to be lost, consequently a new type strain

(neotype) was chosen as a representative: the neotype strain is ATCC 11775, also known as

NCTC 9001, which is pathogenic to chickens and has an O1:K1:H7 serotype. However, in most

studies either O157:H7 or K-12 MG1655 or K-12 W3110 is used as a representative E.coli.

Fig.25: Aggregation of pathogenic O157:H7 by producing adhesin

Pathogenicity of E.coli:

1. Virulent strains of E. coli can cause gastroenteritis, urinary tract infections,

and neonatal meningitis.

Fig.26: Stages of neo-natal meningitis

2. In rare cases, virulent strains are also responsible for hemolytic-uremic

syndrome, peritonitis, mastitis, septicemia and Gram-negative pneumonia.

3. UPEC (uro-pathogenic E. coli): It is one of the main causes of urinary tract infections. It

is part of the normal flora in the gut and can be introduced in many ways. In particular for

females, the direction of wiping after defecation (wiping back to front) can lead to fecal

contamination of the urogenital orifices. Anal intercourse can also introduce this bacteria

into the male urethra, and in switching from anal to vaginal intercourse the male can also

introduce UPEC to the female urogenital system. For more information, see the databases

at the end of the article or UPEC pathogenicity.

Fig.27: Attachment and colonization of E.coli in human intestine.

4. EHEC: In 2011, one E. coli strain, Escherichia coli O104:H4, has been the subjected of

a bacterial outbreak that began in Germany. Certain strains of E. coli are a major cause of

foodborne illness. The outbreak started when several people in Germany were infected

with enterohemorrhagic E. coli (EHEC) bacteria, leading to hemolytic-uremic syndrome

(HUS), a medical emergency that requires urgent treatment. The outbreak did not only

concern Germany, but 11 other countries, including regions in North America. On 30

June 2011 the German Bundesinstitut für Risikobewertung (BfR) (Federal Institute for

Risk Assessment, a federal, fully legal entity under public law of the Federal Republic of

Germany, an institute within the German Federal Ministry of Food, Agriculture and

Consumer Protection) announced that seeds of fenugreek from Egypt were likely the

cause of the EHEC outbreak.

Fig.28: Exposure and spread of EHEC

Role in biotechnology

1. E. coli also plays an important role in modern biological engineering and industrial

microbiology.

2. E. coli is a very versatile host for the production of heterologous proteins, and various protein

expression systems have been developed which allow the production of recombinant

proteins in E. coli.

3. Researchers can introduce genes into the microbes using plasmids which permit high level

expression of protein, and such protein may be mass-produced inindustrial

fermentation processes. One of the first useful applications of recombinant DNA technology was

the manipulation of E. coli to produce human insulin.

Fig.29: Production of recombinant insulin using E.coli

4. Proteins requiring post-translational modification such as glycosylation for stability or

function have been expressed using the N-linked glycosylation system of Campylobacter jejuni

engineered into E. coli.

Fig.30: Expression of C.jejuni transglycosylase system in E.coli for stability in gut

5. Modified E. coli cells have been used in vaccine development, bioremediation, production

of biofuels; lighting, and production of immobilized enzymes.

Model organism

1. E. coli is frequently used as a model organism in microbiology studies. Cultivated strains

(e.g. E. coli K12) are well-adapted to the laboratory environment, and, unlike wild type

strains, have lost their ability to thrive in the intestine.

2. E. coli was an integral part of the first experiments to understand the genetics of

bacteriophage.

3. Production of bio fuel cell

Fig.31: Production of potential bio fuel

4. E. coli was one of the first organisms to have its genome sequenced; the complete

genome of E. coli K12 was published by Science in 1997.

Fig.32: Genetic similarity of K12 with other bacteria

5. The long-term evolution experiments using E. coli, begun by Richard Lenski in 1988,

have allowed direct observation of major evolutionary shifts in the laboratory. In this

experiment, one population of E. coli unexpectedly evolved the ability to aerobically

metabolize citrate, which is extremely rare in E. coli. As the inability to grow aerobically

is normally used as a diagnostic criterion with which to differentiate E. coli from other,

closely related bacteria, such as Salmonella, this innovation may mark a speciation event

observed in the laboratory.

Fig. 33: Composite nature of the E.coli genome from transposon, pathogenicity islands, phage

and plasmids.

6. By evaluating the possible combination of nanotechnologies with landscape ecology,

complex habitat landscapes can be generated with details at the nano-scale. On such

synthetic ecosystems, evolutionary experiments with E. coli have been performed to

study the spatial biophysics of adaptation in an island biogeography on-chip.

7. Studies are also being performed attempting to program E. coli to solve complicated

mathematics problems, such as the Hamiltonian path problem.

Fig.34: Hamiltonian Path

Isolation and Characterization of E.coli.

Introduction:

Escherichia coli is considered as the normal bowel flora of different species of mammals and

birds but some strains of E. coli possess pathogenic character due to the acquisition of virulent

factors. Microbial characteristics associated with virulent E. coli include production of

enterotoxin, verotoxin, colicins and siderophores, type-1 pili and motility, resistance to the lytic

action of the host complement and antibiotics. It can be collected from contaminating water—

where it is taken as indicator organisms.

Fig 35. Structure of E. coli

Collection of sample:

Site selection:

The study design should specify requisite site specifications for the study. For example, many

study designs use a beach area or fishing access point as the specific point of comparison

between streams. A rationale described in the Sampling and Analysis Plan (SAP) for determining

representative sampling areas for the anticipated stream types would be a site selection

specification.

Fig 36. Surface of water for collection

Protocol: surface water

The species Escherichia coli (E. coli) is the principal indicator of suitability of a Water body for

recreational use.

1. Bottles were labeled and made it in replicate number.

2. a satellite (autoclaved) 250 ml container was used to physically collect the sample from

the water body-aseptically collected

3. the satellite container is filled to 200 ml, and container is capped with vigourous shaking

Fig 37. Collection of water of sample

Sample Preservation

1. If the samples will not be processed immediately, bacteria samples should be iced or

refrigerated at a temperature of < 10°

2. E. coli samples must not be held more than six hours between collection and initiation of

analyses. Samples must be processed within 2 hours of arriving to a lab

Methods used:

Materials needed:

Mac Conkey agar

EMB agar

Gram stains

TSI agar

Hanging drop slide

Dextrose, ,maltose, lactose sugars

Durham’s tube

3% H2O2

Methyl Red

Kovac’s reagent

Barritt’s reagent

Procedure:

Culture of the samples

All the samples were cultured primarily in nutrient broth at 37ºC for 18-24 h,

Fig 38. Nutrient broth culture of E.coli

then subcultured onto the MacConkey, brilliant green and EMB agar by streak plate

method to observe the colony morphology (shape, size, surface texture, edge and

elevation, colour, opacity etc). The organisms showing characteristic colony morphology

of E. coli was repeatedly subcultured onto EMB agar until the pure culture with

homogenous colonies were obtained.

Fig 39. E.coli on EMB agar

Fig 40. E.coli on MacConkey agar

Standard culture brought from MTCC should be compared after each step

Fig 41. Standard culture of E.coli

Microscopic study by Gram’s staining method

Gram’s staining was performed as per procedures described by Merchant and Packer (1969) to

determine the size, shape and arrangement of bacteria. The organisms revealed gram negative,

pink colored with rod shaped appearance and arranged in single or in pair were suspected as E.

coli.

Fig 42. Gram staining of E.coli

Motility test by hanging drop technique

The motility test was performed by hanging drop technique to differentiate the motile bacteria

from the non-motile one. Hanging drop slide was prepared by broth culture and examined under

100X power objective. The motile organisms were suspected as E. coli.

Fig 43. Motility of E.coli on hanging drop slide

Reaction of the organism in TSI agar slant

The test organisms were cultured into TSI agar slant by stab or streak method. Yellow slant,

yellow butt, presence of gas bubbles and absence of black precipitate in the butt (due to the

production of H2S) indicative of E. coli

Fig 44.E. coli on TSI agar

Carbohydrate fermentation test

The test was performed by inoculating 0.2 ml of nutrient broth culture of the isolated organisms

into the tubes containing five basic sugars such as dextrose, maltose, lactose, sucrose and

mannitol and incubated for 24 h at 37ºC. Acid production was indicated by the color change

from red to yellow and gas production was noted by the accumulation of gas bubbles in the

inverted Durham’s tube.

Fig 45. Carbohydrate fermentation by E.coli

Catalase test

A volume of 3 ml of catalase reagent (3% H2O2) was taken in a test tube. Single colony from the

pure culture of E. coli was taken with a glass rod and merged in the reagent and observed for

bubble formation which indicated positive test. Absence of bubble formation indicated negative

result.

Fig 46. Catalase test

Methyl Red test

Single colony from the pure culture of the test organism was inoculated in 5 ml of sterile MR-VP

broth. After 5 days incubation at 37ºC, 5 drops of methyl red solution was added and observed

for color formation. Development of red or yellow color indicated positive or negative result,

respectively .

Fig 47. Methyl Red test

Voges -Proskauer (V-P) test

The test organisms were grown in 3 ml of sterile MR-VP broth at 37ºC for 48 h and then 0.6 ml

of 5% alphanapthol and 0.2 ml of 40% potassium hydroxide containing 0.3% creatine was added

per ml of broth culture. Following well shaking, the broth was allowed to stand for 5-10 minutes

to observe the color formation. Development of pink-red color indicated positive result.

Fig 48. Voges -Proskauer (V-P) test

Indole test

The test organisms were cultured in 3 ml of peptone water containing tryptophan at 37ºC for 48

h. One ml of diethyl ether was added, shaked well and allowed to stand until the ether rises to the

top. Then 0.5 ml Kovac’s reagent was gently run down the side of the test tube to form a ring in

between the medium and the ether. Development of brilliant red colored ring indicated positive

test.

Fig 49. Indole test

RESULTS AND DISCUSSION

All the E. coli isolates were able to produce bright pink colonies on MacConkey agar,

yellowish green colonies surrounded by an intense yellow green zone on BG agar and

characteristic metallic sheen colonies on the EMB agar.

In case of E. coli isolated from cattle, slight variation in colony character on EMB agar

was observed showing greenish red colonies with faint metallic sheen. Differences in

colony morphology manifested by the isolates may be due to loosing or acquiring some

properties by the transfer of host or choice of host tissue

Fig 50. Greenish red colonies with metallic sheet of E.coli in EMB agar

In Gram’s staining, the morphology of the isolated bacteria exhibited pink coloured,

small rod shaped, Gram negative bacilli

Fig 51. Gram staining of E.coli

The hanging drop technique all the isolates revealed motility as observed by several

authors .

Fig 52. Motility of E.coli

Reactions in TSI agar slant revealed yellow slant and butt with gas but no production of

hydrogen sulphide gas was observed.

E. coli fermented dextrose, maltose, lactose, sucrose and mannitol with the production of

both acid and gas but E. coli isolated from drain sewage did not ferment maltose and

isolate from pigeon showed less production of acid and gas during sucrose fermentation.

Fig 53. Lactose fermentation of E.coli

Precaution:

During sampling of surface waters, water will be collected from a depth of 15.2 cm

Surface scum should be avoided.

Inside the bottle should not be touched

While sampling the hands will be near the base of bottle

Lid should be closed tightly

Bottles should be shacked to homogenize the sample.

Standard culture should be taken—to compare after each step to assess natural sample.