Transcript E. coli
E.coli
Commensal, Pathogen,& Genetic tool
Dr.T.V.Rao MD
Dr.T.V.Rao MD
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Enterobacteriaceae
Commonly present in large intestine
Non sporing , Non Acid fast, Gram – bacilli.
A complex family of organisms,
Some are non pathogenic
A few are highly Pathogenic,
Some commensals turn out to be pathogenic.
as in UTI after catheterization.
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Characters of
Enterobacteriaceae
All Enterobacteriaceae
Gram-negative rods
Ferment glucose with acid production
Reduce nitrates into nitrites
Oxidase negative
Facultative anaerobic
Motile except Shigella and Klebsiella
Non-capsulated except Klebsiella
Non-fastidious
Grow on bile containing media (MacConkey agar)
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Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonell, Shigella
Proteus, Yersinia
There are several selective and differential media used to
isolate distinguishes between LF & LNF
The most important media are:
MacConkey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
In addition to Triple Sugar Iron (TSI) agar
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Escherichia coli
Named by Escherichia
Wide group of bacteria on basis of
Bio typing and Serotyping
Produce infections in Humans and
Animals
Detection of E.coli in water indicates
pollution and contamination.
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Classification
Domain: Bacteria
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Gamma
Proteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: Escherichia coli (E.
coli)
E.coli
Morphology Gram - ve Straight
rods,
1-3 X 0.4 -0.7 microns,
Appear in singles or in pairs,
Motile by peritrichate flagella.
Very few strains non motile
Not spore forming, Non acid fast.
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Enterobacteriaceae:
Genetic Properties
Chromosomal DNA has 39-59% guanineplus-cytosine (G+C) content
Escherichia coli is the type genus and
species of the Enterobacteriaceae
Species of Enterobacteriaceae more closely
related by evolutionary distance to
Escherichia coli than to organisms of other
families (Pseudomonadaceae,
Aeromonadaceae)
E.coli
Cultural characters
Aerobic / Facultative Anaerobic
Grows between 10 – 40 c optimal at 37 c
Grown in simple medium
Produce Large grayish ,Thick white , moist smooth
opaque colonies
May contain capsule.
On MacConkey medium Produce Bright pink
Lactose fermenters.
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E.coli
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Identification of Enterobacteriaceae
Biochemical reactions
Oxidase test
All members of Enterobacteriaceae are oxidase negative
Pseudomonas is oxidase positive
O/F test
All members of Enterobacteriaceae are O+/F+
Pseudomonas is O+/F-
Nitrate reductase
All members of Enterobacteriaceae are nitrate reductase positive
Pseudomonas is nitrate reductase negative
E.coli
Biochemical Characters,
Glucose,Lactose,Mannitol,Maltose
fermented. with A/G
I,M,Vi,C tests.
Indole +
Methyl Red +
Voges Proskauer – ve
I,M,Vi,C tests.
Citrate –ve
Urease not produced.
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Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth
on MacConkey agar
Method:
MacConkey agar is inoculated with tested organism
using streak plate technique
Incubate the plate in incubator at 37 C/24 hrs
Results:
LF organism appears as pink colonies (e.g. E. coli)
NLF organism appears as colorless colonies (e.g.
Shigella)
Flame & Cool
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1
3
4
5
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Flame & Cool
Flame & Cool
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E.coli
Antigenic Structure
Somatic 0 170
Capsular K 100
Flagella H 75
Virulence factors
Surface Antigens Toxins
O Endotoxic activity
K protects against the phagocytosis
Fimbriae promote virulence ( important in UTI )
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Toxins and E.coli
E.coli produce Exotoxins
Hemolysins, Enterotoxins causes
Diarrheas,
Important toxins produces.
Heat labile HL Heat stable HS
Vero toxins VT Like Shigella toxins
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Toxins
Enterotoxins – produced by enterotoxigenic
strains of E. coli (ETEC). Causes a
movement of water and ions from the
tissues to the bowel resulting in watery
diarrhea. There are two types of enterotoxin:
LT – is heat labile and binds to specific
Gm1 gangliosides on the epithelial cells of
the small intestine where it ADPribosylates Gs which stimulates adenylate
cyclase to increase production of cAMP
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Mechanism of action of
Toxins
Increased cAMP
alters the activity of
sodium and
chloride
transporters
producing an ion
imbalance that
results in fluid
transport into the
bowel
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Toxins in E.coli
Produce Enterotoxin L T and S T
Labile toxin 1956 De experiments in Rabbit
illeal loop causes outpouring of fluids
E.coli Labile toxin like Cholera toxin
L T contains component A and B
A = Active B= Binding
B causes Binding with Gm I Ganglioside
receptor on Intestinal epithelial cells
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E. coli toxins
• Both enterotoxins
are composed of
five beta subunits
(for binding) and
1 alpha subunit
(has the toxic
enzymatic
activity).
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Toxins E.coli
Labile toxin
Component A Activated to A1 and
A2
A1 Activates adenyl cyclase in the
enterocytes to form cyclic adenosine
5 monophosphate
Causes to increase outflow of water
and electrolytes in the gut lumen
causes Diarrhea
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Toxins of E.coli
Stable Toxin
ST A and ST B
ST A Acts by activation of Cyclic guano
sine monophosphate.( C GMP )
Causes fluid accumulation in Intestine.
E.coli ( Some ) produce Verocytotoxin
causes cytotoxicity to Vero cells.
Acts like Shigella dysentery toxin
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E.coli a Complex Microbe
More than 700
serotypes of E. coli
have been
identified. The
different E. coli
serotypes are
distinguished by
their “O” and “H”
antigens on their
bodies and flagella,
respectively.
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Classification of E.coli
1.Enteropathogenic EPEC
2.Enterotoxigenic
ETEC
3.Enteroinvasive
EIEC
4.Enterohemorrhagic EHEC
5.Enteroaggresive
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EAEC
Enteropathogenic E.coli
Causes diarrheal disease in children,
EPEC O26/O11
Produce Verocytotoxin
Infantile enteritis, Involves upper part of Intestine
Brush border of the intestine is lost
Intimacin – EPEC adhesion factor.
Frequent in summer months
Poor hygiene predisposes.
Out breaks in Institutions
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Enteropathogenic E. coli
destruction of surface microvilli
• fever
Gut lumen
• diarrhea
• vomiting
• nausea
• non-bloody stools (not generally seen as dysentery)
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Laboratory Diagnosis EPEC
Confirm with
Polyvalent sera
Test Sero groups
with polyvalent
and monovalent
sera.
HEp2 – adherence.
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Enterotoxigenic E.coli
Produce Heat stable /Heat labile toxins
Adheres to epithelium of small intestine.
Present with Nausea, Vomiting and Lose stool
H L like cholera toxin
Causes accumulation of fluids
Adhesive factors
Fimbriae specific receptor in the intestinal
epithelium CFA
Mortality in children < 5 years
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Enterotoxigenic E. coli
Heat labile toxin
like choleragen
Adenyl cyclase activated
cyclic AMP
secretion water/ions
Heat stable toxin
Guanylate cyclase activated
cyclic GMP
uptake water/ions
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Enterotoxigenic E.coli
Causes travelers diarrhea
Water contaminated with Human and Animal feces
predisposes.
Laboratory Diagnosis
Demonstration of Enterotoxin LT and ST
Tissue culture tests,
ELISA
Passive agglutination tests.
Animal experiments in Rabbit ileal loop test.
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Treatment and Prophylaxis
in Travelers diarrhea
Doxycycline,
Trimethoprim,
Norfloxacillin
Fluroquinolones
Avoid contaminated food,
Safe protected water ,prefer bottled water,
Hot foods, Hot Drinks,
Boiled milk
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Entero invasive E.coli
Some are non motile
strains,
Atypical resembles
like Shigella.
Clinically mild
diarrhea
Sereny test positive
animal Rabbit.
ELISA
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Enteroinvasive E. coli (EIEC )
• Dysentery
- resembles shigellosis
Gut lumen
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Enterohemorrhagic E.coli
Produce Verocytotoxin
Mild diarrhea - can be fatal hemorrhagic
colitis. and uremic syndrome.
Present in Human and Animal feces.
Hemorrhagic complication with O157 in
Japan and USA.
Salads vegetables, Radish Proper cooking
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EHEC ( contd )
Culture
DNA detection
methods.
Cytotoxic effects
on Vero cells.
Detection with
monovalent sera
O157/H7
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Enterohemorrhagic E.coli
can cause HUS
HUS develops when the toxin from
E. coli bacteria, known as Shiga-like
toxin (SLT) , enters the circulation
by binding to special receptors. These
Shiga-toxin receptors, known as Gb3 receptors , are
probably heterogeneously distributed in the major
body organs allowing disparate thrombotic (blood
clotting) impacts in different HUS victims, although
the greatest receptor concentration appears to be in
the kidneys, especially in children.
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Mechanism of HUS
As the inflammatory reaction process
accelerates, red blood cells are destroyed
and cellular debris aggregates within the
microvasculature while the body’s inherent
clot breaking mechanisms are disrupted. The
result is formation of micro thrombi within
particularly susceptible organs such as the
kidneys and brain. Because there exists no
way to halt the progression of HUS,
The patients are supported with medical
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care
Enteroaggresive E.coli
EAEC
Can cause Diarrhea Detect
by Culture methods
• Brick-like aggregates on
cell surfaces
•
Mucus biofilm inhibits
fluid absorption
•
Diarrhea
Detection of Enterotoxin
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E.coli resembles Shigella
spp
The E. coli serotypes
that are responsible for
the reports of
contaminated foods
and beverages are
those that produce
Shiga toxin, because
the toxin is identical to
that produced by
another bacteria
known as Shigella
dysentery
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Treatment – E.coli
Gastrointestinal disease
Fluid replacement
Antibiotics
not used usually unless
systemic infections prevails
e.g. hemolytic-uremia
syndrome
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E. Coli leading cause of
UTI
Clinical significance
Is the leading cause
of urinary tract
infections which
can lead to acute
cystitis (bladder
infection) and
pyelonephritis
(kidney infection).
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Urinary Tract Infections
E.coli produce urinary tract infection.
Majority of UTI s are produce by E.coli.
Instrumentation, Prostatic enlargement,
Urinary caliculi ,Pregnancy, increase the
predisposition
Asymptomatic Bacteriuria in pregnant women,
Pyelonephritis,
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Facts on UTI
Women suffer more than males Short urethra Pregnancy, Sexual
intercourse /Honey moon cystitis.
Other factors
Urethral obstruction,
Urinary stones
Congenital malformation's
Neurological disorders,
Catheterization , Cystoscopy
Usually cystitis is produced from fecal strains entering urethra
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Culturing for E.coli
Mid stream sample/semi quantitative
culturing (Kass et al ) >_ 1.00,000/ml of
urine. ( significant Bacteriuria )
Urine should not be kept in wards for > 2
hours and to be preserved at 4 c
Culture by standard loop method.
Fixed volume cultured on MacConkey agar
Lactose fermenters I M Vi C
Antibiotic sensitivity45 tested.
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Other infection with
E.coli
Pyogenic infections.
Intraabdominal infections
Peritonitis. Abscess.
Septicemias
Produce Drug resistant infections.
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Other Important E. coli
Infections
Neonatal meningitis – is the leading cause
of neonatal meningitis and septicemia
with a high mortality rate.
Usually caused by strains with the K1
capsular antigen.
Gastroenteritis – there are several
distinct types of E. coli that are
involved in different types of
gastroenteritis:
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E.coli
Antimicrobial therapy- E. coli is usually
susceptible to a variety of chemotherapeutic
agents, though drug resistant strains are
increasingly prevalent.
It is essential to do susceptibility testing.
Treatment of patients with EHEC infections is
not recommended because it can increase the
release of shiga-like toxins and actually
trigger HUS
Escherichia coli as a
Genetic tool.
The study of Escherichia coli and its plasmids and
bacteriophages has provided a vast body of
genetical information, much of it relevant to the
whole of biology. This was true even before the
development of the new techniques, for cloning and
analysing DNA, that have revolutionized biological
research during the past decade.. Much of the
background of knowledge necessary for the cloning
and expression of genetically engineered
information, as well as the techniques themselves,
came from work with this organism.
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Why E.coli is preferred
E. coli cells only have about 4,400 genes
whereas the human genome project
has determined that humans contain
approximately 30,000 genes. Also,
bacteria, including E. coli, live their
entire lifetime in a haploid state, with
no second allele to mask the effects of
mutations during protein engineering
experiments.
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How Does Molecular
Cloning Work?
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Cloning of any DNA sequence
involves the introduction of a
foreign piece of DNA into an
extra chromosomal element
(cloning vector) of an
organism which then produces
copies of the vector as it
replicates itself, thereby
amplifying the DNA of
interest. The whole process
can be summarized in the
following steps:
fragmentation, ligation,
transfection,
screening/selection, and
conformation of insert.
E.coli Preferred in
Genetic Engineering
It is a favorite organism for genetic
engineering as cultures of it can be made
to produce unlimited quantities of the
product of an introduced gene. Several
important drugs (insulin, for example)
are now manufactured in E. coli.
However, E. coli cannot attach sugars to
proteins so proteins requiring such
sugars have to be made in the cells of
eukaryotes such as yeast cells and
mammalian cells grown in cell culture.
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Programme Created by
Dr.T.V.Rao MD for
Microbiologists, Medical and
Paramedical Students in the
Developing World
Email.com
[email protected]
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Reference
www.slideshare.com
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