3. 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.
3
4. 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)
4
5. 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
5
6. 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.
6
7. 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.
7
8. 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.
8
10. 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
12. 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
Flame & Cool
Flame & Cool
1 2
3
4
5
12
13. E.coli
Antigenic Structure
O antigens: heat stable.
● K antigens: surround cell be heat-inactivated.
The K antigens of some salmonellae, e.g. S. Typhi, are called Vi
antigens.
● H antigens: flagaellar protein antigens possessed
by motile enterobcteria. They are heat. Salmonella serovars often
produce two different antigenic types of H antigens, called phase 1
and phase 2.
Surface Antigens Toxins
O Endotoxic activity
K protects against the phagocytosis
Fimbriae promote virulence ( important in UTI )
13
14. Diseases
Bacteremia (most commonly isolated gram-negative rod)
Urinary tract infection (most common cause of bacterial UTIs);
limited to bladder (cystitis) or can spread to kidneys
(pyelonephritis) or prostate (prostatitis)
At least five different pathogenic groups cause gastroenteritis
enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic
(EHEC), enteroinvasive (EIEC), enteroaggregative (EAEC) E. coli); most
cause diseases in developing countries, although EHEC is an
important cause of hemorrhagic colitis (HC) and hemolytic uremic
syndrome (HUS)
Neonatal meningitis (usually with strains carrying the K1 capsular
antigen)
Intra abdominal infections (associated with intestinal perforation)
Most infections are endogenous
14
15. 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
15
16. 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. It known as traveler's diarrhea; infant
diarrhea in developing countries. watery diarrhea, vomiting,
cramps, nausea, low-grade fever. Plasmid-mediated. heat-
stable and/or heat-labile enterotoxins that stimulate
hypersecretion of fluids and electrolytes.
LT – is heat labile similar to 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.
ST A and ST B. Causes fluid accumulation in Intestine. Acts
like Shigella dysentery toxin
16
17. 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
17
18. 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.
18
19. 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
19
20. 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
Dr.T.V.Rao MD 20
21. 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.
Dr.T.V.Rao MD 21
22. 22
Enteropathogenic E. coli
destruction of surface microvilli
• fever
• diarrhea
• vomiting
• nausea
• non-bloody stools (not generally seen as dysentery)
Gut lumen
23. Laboratory Diagnosis EPEC
Confirm with
Polyvalent sera
Test Sero groups
with polyvalent
and monovalent
sera.
HEp2 – adherence.
Dr.T.V.Rao MD 23
24. Entero invasive E.coli
Causes dysentery
(similar to shigellosis),
fever and colitis, with
blood, mucus, and many
pus cells in faecal
specimens. Due to
bacteria invading and
multiplying in epithelial
cells. Many serogroups
are involved.
24
26. Enterohemorrhagic E.coli
Causes life-threatening haemorrhagic diarrhoea (colitis)
in all ages, without pus cells, and without fever. It can
progress to haemolytic uraemic syndrome with renal
failure.
EHEC is due to cytotoxins damaging vascular
endothelial cells, and is mainly associated with the
serogroup 0157:H7. It is referred to as VTEC
(verocytotoxin-producing E. coli, because it is toxic to
vero monkey cells in culture).
Infection occurs by ingesting contaminated meat
products, unpasteurized milk and dairy products.
26
27. EHEC ( contd )
Culture
DNA detection
methods.
Cytotoxic effects
on Vero cells.
Detection with
monovalent sera
O157/H7
Dr.T.V.Rao MD 27
29. 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
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.
29
30. Enteroaggresive E.coli
EAEC
Causes chronic watery
diarrhoea and vomiting,
mainly in children. Due to
the bacteria adhering to
tissue cells often in stacks
(aggregates).
30
31. 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).
31
32. 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,
Dr.T.V.Rao MD 32
33. Facts on UTI
Women suffer more than males Short urethra Pregnancy, Sexual
activity/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
33
34. Other infection with
E.coli
Pyogenic infections.
Intraabdominal infections
Peritonitis. Abscess.
Septicemias
Produce Drug resistant infections.
Dr.T.V.Rao MD 34
35. 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.
Dr.T.V.Rao MD 35
36. 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.
36
37. LABORATORY
FEATURES
Specimens: Depending on site of infection, specimens include
urine, pus, faeces, cerebrospinal fluid (infants), and blood for
culture.
Morphology: E. coli is a Gram negative usually motile rod.
Inactive strains (formerly described as Alkascens-Dispar) are
nonmotile. A minority of strains are capsulate
Culture: E. coli is an aerobe and facultative anaerobe.Optimum
temperature is 36–37 ºC
Blood agar: E. coli produces 1–4 mm diameter. It may appear
mucoid. Some strains are haemolytic.
MacConkey agar and CLED agar: E. coli ferments lactose,
producing smooth pink colonies on MacConkey agar and
yellow colonies on CLED agar. Some strains are late or non-
lactose fermenting.
37
38. Sorbitol MacConkey agar: E. coli (VTEC) 0157 is non-sorbitol
fermenting, producing colourless colonies. Most other E. coli
strains and other enterobacteria ferment sorbitol. E. coli (VTEC)
0157 can be identified by testing the colonies using 0157 latex
reagent.
Biochemical reactions:
KIA: Most strains of E. coli produce an acid butt and an acid
slope with gas production and no H2S blackening
Most strains of E. coli:
● Indole positive
● Lysine decarboxylase (LDC) positive.
● Beta-glucuronidase (PGUA) positive (E. coli 0157
is PGUA negative).
● Reduce nitrate to nitrite, giving a positive urine nitrite test.
● Citrate and H2S negative 38
39. 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
