E. coli

Report
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
2
1
3
4
5
Dr.T.V.Rao MD
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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