Dr.T.V.Rao MD

E.coliCommensal, Pathogen,&

Genetic toolDr.T.V.Rao MD

Dr.T.V.Rao MD 2

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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 Enterobacteriacea

eAll 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 fermentersE. coli, Citrobacter,

Klebsiella, Enterobacter

Non-lactose fermenterSalmonell, ShigellaProteus, Yersinia

There are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are:

MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agarIn 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.

Classification Domain: Bacteria

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gamma Proteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Species: Escherichia coli (E. coli)

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E.coliMorphology 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.

Enterobacteriaceae: Genetic Properties

Chromosomal DNA has 39-59% guanine-plus-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)

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E.coliCultural 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

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

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E.coliBiochemical 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 EnterobacteriaceaeDifferentiation between LF & NLF by

Growth on MacConkey agarMethod: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

Flame & Cool

Flame & Cool

1 23

45

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E.coliAntigenic 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 ADP-ribosylates 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.coliLabile 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.coliStable 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 EAEC

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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• diarrhea• vomiting • nausea • non-bloody stools (not generally seen as dysentery)

Gut lumen

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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 choleragenAdenyl 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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• Dysentery- resembles shigellosis

Enteroinvasive E. coli (EIEC )

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 care

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Enteroaggresive E.coliEAEC

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

diseaseFluid 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 significanceIs the leading

cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection).

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 sensitivity tested.

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Other infection withE.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:

E.coliAntimicrobial 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

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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?

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.

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E.coli Preferred in Genetic

EngineeringIt 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

doctortvrao@gmail.com

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