IM Sechenov First Moscow State Medical University

Microbiology, Virology and Immunology Department

Enteric bacterial
pathogens
by Assoc. Prof. E.V. Budanova
Enteric infections are infectious diseases
caused by bacteria, bacterial toxins, viruses,
fungi or protozoa that are acquired via
alimentary tract and generally characterized
by gastrointestinal tract damage

Etiology: bacteria, bacterial toxins, viruses,

fungi or protozoa

Diarrhea is the main symptom of enteric

infection

Diarrheal pathogens can be divided into

those causing inflammatory diarrhea or
 Types of diarrhea
Inflammatory Secretory (non-
Is associated with invasive)
GIT cells invasion No GIT epithelial cells
invasion and damage
Neutrophils blood,
No fecal leukocytes
mucus may present
Develop mainly due to
in stool
Adenyl cyclase
Is caused by activation leading to loss
Shigella, EIEC, non- of electrolytes and fluids
typhoid Salmonella, Is caused by ETEC,
Yersinia sp. and V.cholerae and some
some others others
 EPIDEMIOLOGY of Enteric
Infections
Human-to-Human Transmission
Animal-to-Human Transmission may occur in
paratyphoid fever B (e.g., from cows)
Fecal-Oral Route of Transmission
Modes of Transmission waterborne, food
borne and with dirty hands or
contaminated equipment (utensils)

The rule of 4 F characterizing the

enterics transmission: fingers, flies,
food and feces are the principle factors of
enteric infections transmission
The Fecal-Oral Route of
Transmission
Morbidity and Mortality
rate
According to WHO reports:
in the world
over 500,000,000 cases annually,
about 3,000,000 deaths
In developing countries 4500 children aged
under 5 years die from diarrhea every day
S.Typhi causes 16,6 millions cases of typhoid
fever and 600,000 deaths every year
The Main Factors Contributing to Enteric
Infections Outbreaks
Poor personal hygiene practices
Improper sewage disposal
Improper purification and low levels
of chlorination of water supply
Undercooking of foods and improper
boiling of water and drinks, etc.
The Enterobacteriaceae
members: main features
 TAXONOMY of Enteric Bacteria
Family Enterobacteriaceae
Genus Salmonella (cause enteric
fevers and gastroenteritis
salmonellosis)
Genus Shigella (cause bacterial
dysentery shigellosis)
Genus Escherichia (cause enteric
and parenteral escherichiosis)
Genus Yersinia (cause enteric
yersiniosis, pseudotuberculosis
and plague*)
Morphology and Tinctorial Properties of
Enteric bacteria
Small gram-negative rods with round
ends that lie separately
Do not form capsules and endospores
May possess microcapsule
Have peritrichous flagella (except,
Shigella sp.)
 Cultural Properties of Enteric
Bacteria
Facultative anaerobes
Easily grow on simple media
Optimal conditions for growth: t=37,
18-24 h, of culture medium 7.2 7.4
On lactose-containing differential culture
media form colored colonies (lac +
E.coli) or colorless colonies (lac -
bacteria)
 Biochemical Features of
Enterobacteriaceae

Highly active:
catalase +, oxydase -,
reduce nitrates to nitrites
glucose lactos citrat LDC indol H 2S
e e e
E.coli +(AG) + - -/+ + -
Salmonella + - + + - +
sp. (A/AG)
Shigella sp. +(A) -/+ - - - -
 Biochemical Features of Enteric
Bacteria
Kligler iron agar

Citrate
+

Catalase +

Facultative Reduce
anaerobes nitrates to
nitrites
 Antigenic properties of Enteric
Bacteria
Have -, -, -antigens
S. Typhi has capsular Vi-
The bacterial species can be subdivided into serogroups and
serovars by their antigenic properties

Kauffmann-White Scheme of Salmonellae Classification:

By -antigen are divided into 5 serogroups (, , C, D, E)
By H-antigen are divided into species/serotypes (serovars)
 Taxonomy and Classification of
Salmonellae
Genus Salmonella
Species :
S.bongori (causes diseases in cold-blood animals
only)
S.enterica (includes human & animal pathogens)
and is subdivided into 6 subspecies: enterica,
salame, arizone, diarizone, houtenae, indica
Among enterica subspecies there are 2200
serotypes (serovars), eg.:
S. Typhi
S. Paratyphi A and B
S. Typhimurium
S. Enteritidis etc.
Attention: the serotypes names are
capitalized and not italicized!
 The -antigen structure
Core polysaccharide (family-specific)
O-side chains that consist of repeating units of
oligosaccharides and disaccharides(species-specific,
serogroup-specific)
 Virulence Factors of Enteric
Bacteria
Adhesins (pili, outer membrane
proteins)
Colonization factors can form
biofilms
Invasive proteins (ipa BCD)
Shigellae
Endotoxin
Exotoxins:
Enterotoxins heat-labile (LT)
activates adenylate cyclase; heat-
stable (ST) activates guanylate
cyclase
Cytotoxins Shiga toxin and
Shiga-like toxin
Antiphagocytic factors
(Salmonella sp.)
The Third Type Secretory System
(TTSS)
TTSS is a syringe-like
structure
It provides direct secretion
and translocation of a
number of Salmonella
virulence factors into the
host cell
The effector proteins
translocated into cell
mediate intracellular actin
rearrangements, resulting in
invasion of bacterium and
its internalization within
membrane-bound vacuole
 Resistance to the Unfavorable
Conditions

Not very resistant

Sensitive to disinfectants, heating, UV
Can survive freezing
Can persist in a nonculturable but
viable state in natural samples
Types of interaction of enteric bacteria with human intestine
Type I attachment and colonization of
small intestine enterocytes
(enterotoxigenic E.coli ETEC, V.cholerae)
Can attach to enterocytes
and multiply on their
surface (colonization)
without invasion and cell
damage. The enterotoxin
causes imbalance of water
and electrolytes due to
activation of adenylate
cyclase leading to
secretory diarrhea
symptoms
 Types of interaction of enteric bacteria with human intestine
Type II attachment and local damage of brush
border of enterocytes and/or colonocytes
(enteropathogenic E.coli EPEC)
Can attach to enterocytes and
colonocytes and multiply on their
surface without invasion inside
the target cells but can cause
local damage of apical part of
brush border of the epithelial
cells leading to erosions of
mucosal cells and inflammation.
If enterotoxin is formed it causes
the secretory diarrhea
symptoms
 Types of interaction of enteric bacteria with human intestine
Type III invasion and multiplication in
colonocytes of large intestine
(enteroinvasive E.coli EIEC, Shigella sp.)

Can multiply in epithelial

cells of large intestine
causing cytotoxic effect that
results in development of
erosion and ulceration of
colon and inflammation.
Symptoms of invasive
diarrhea are observed
 Types of interaction of enteric bacteria with human intestine
Type IV transcytosis without cell damage
(Salmonella sp.) or transcytosis with cell
damage of small intestine epitheliocytes
(Yersinia sp.)
Transit of bacteria trough -cells
into lamina propria
Multiplying in phagocytes
Sensitization
Damage to lymph tissue and
secondary damage to enterocytes
If enterotoxin is formed it causes
the secretory diarrhea symptoms
 PATHOGENESIS of Enteric
Infections
Portals of entry mouth, GIT
Adhesion in small or large intestine
Interaction of enteric bacteria with human intestine
Local process (gastroenteritis or colitis
symptoms) accompanied with secretory or
invasive diarrhea
Bacteriaemia occurs in enteric fevers
leading to
Fixation in RES organs (liver, spleen, kidneys,
bone marrow, lungs, etc.)
Portals of exit - feces, vomits (in typhoid
fever salmonellae may occur in urine, mother
breast milk, sweat)
 CLINICAL PICTURE of Enteric
Infections
The main sign diarrhea (in enteric
fevers rash on the trunk is seen)
Incubation period from 12-72 h. to 2-3
days (7-14 days in enteric fevers)
Abrupt onset
Intoxication
Loss of water and electrolytes
(dehydration)
CLINICAL PICTURE of Typhoid
Fever
 IMMUNITY after enteric
infections
After enteric fevers: Strong, life-long
immunity
Cell-mediated immunity is due to activated
macrophages
Antibodies are not protective, i.e. they are
witnesses of infectious process
Dynamic of antibodies development (prevalence
in appropriate periods):
-antibodies acute period;
-antibodies convalescent stage;
Vi-antibodies carrier state

No protective immunity in other enteric

bacterial infections!!!
 Microbiological Diagnosis of Enteric
Infections
Specimens:
Stool specimens, vomit specimens;
foodstuffs
Methods of Microbiological Diagnosis:
Bacteriological (the main)
Serological (in chronic cases of
shigellosis)
Bacteriological
method
 Microbiological Diagnosis of Typhoid and
Paratyphoid Fevers
rests on pathogenesis of disease
Specimens:
Blood (on the 1st week)
feces, bone marrow aspirates, urine,
bile (2nd-3d weeks)
Methods of Microbiological
Diagnosis:
Rapid Tests (IF, ELISA; PCR)
Bacteriological
Serological
Detection of Typhoid Fever Carrier State:
Serological examination of a persons serum
(the IHA with Vi-erythrocytic diagnosticum)
 TREATMENT of Enteric Infections
Antibiotics (ampicillin,
chloramphenicol are drugs of
choice)
Bacteriophages
Probiotics
Rehydration
(fluid and electrolyte replacement)
 Prophylaxis of Enteric
Infections
Vaccines against typhoid fever:
Killed alcohol treated typhoid fever vaccine
(parenteral)
Parenteral typhoid fever polysaccharide
vaccine, containing Vi-antigen (for children
above 2 years old)
The oral live attenuated vaccine (Ty21) (for
children aged above 6 years and adults)
Bacteriophages for immediate
protection:
The typhoid fever and shigellosis
bacteriophages in pills
 Prevention of Enteric
Infections
Observe the rules of personal
hygiene
Wash your hands with soap
Boil water before using for
drinking, cooking or washing dishes
Dont add ice made of tap water
to bottled drink water and
beverages
Peel fruits! Avoid seafood salads
and those made of raw fruits and
vegetables
Dont taste food on the
marketplace
Dont bathe and swim in
stagnant basin
Store food in refrigerator
Cook food properly
Escherichia coli
Theodore Escherich
(1857-1911)

German pediatrician
First isolated E. coli
from the child with
infants cholera
symptoms (1885)
He has given the
name: Bacterium coli
commune
 Taxonomy of E.coli

Family Enterobacteriaceae
Genus Escherichia
Species E.coli
 Morphology and Tinctorial
Properties of E.coli

Small gram-negative straight rods with

round ends that lie separately
Do not form capsules and endospores
May possess microcapsule
Have peritrichous flagella
Have pili (fimbriae)
 Cultural Properties of E.coli
Facultative anaerobes
Easily grow on simple media
Make liquid media uniformly turbid
As lactose-positive bacteria form
colored colonies on differential culture
media
Optimal conditions: grow best at 37,
7.2 7.4 within 18-24 h
 Biochemical Features of E.coli
Highly biochemically active:
Oxidase -
reduce Nitrates to Nitrites
Glucose + (AG)
Lactose +
2S -
Indole +
Citrate -
 Antigenic properties of E.coli
Have -, -, -antigens
- (170 variants), - (~100), - (~70)
By -antigen structure are divided into
serogroups
that are identified in slide agglutination test
Resistance of E.coli to Unfavourable
Conditions
Not resistant
Detection of coliforms
(E.coli and E.coli-like
bacteria) is used for
detection of fecal
pollution of water and
soil
 Classification of E.coli
There are two biovars:
The Biovar 1 includes opportunistic E.coli
that are gut normal microflora members and also
major cause of opportunistic infections particularly
in persons with impaired host immunity.
They can cause parenteral escherichosis

The Biovar 2 includes the diarrheagenic

E.coli that induce diarrhea by several
mechanisms.
They cause enteric escherichosis
 The Diarrheagenic E.coli
Enteropathogenic E.coli (EPEC) causes
infants diarrhea
Fecal-oral route of transmission via dirty hands
Cause local damage of brush border of
enterocytes, inducing secretory diarrhea
Typical serotype is E.coli 111
 Virulence Factors of EPEC
Type IV pili (syn.: Bundle-forming pili,
Bfp)
The outer membrane protein intimin
allows intimate adhesion to enterocytes
produces attaching and effacing (A/E)
lesion making pedestal
Translocated intimin receptor (Tir)
Shiga-like toxin (SLT)
Endotoxin
 The Diarrheagenic E.coli
Enterotoxigenic E. coli (ETEC)
produces cholera-like illness but much
milder in degree (i.e., it mimics
cholera). This infection is also known as
travelers diarrhea
Fecal-oral route of transmission, water-
borne disease
Cause damage of enterocytes, inducing
secretory diarrhea
Typical serotype is E.coli 78
 Virulence Factors of ETEC
Adhesion and colonization factors (outer
membrane proteins, pili)
Endotoxin
Heat-labile exotoxin (LT) and
Heat-stable exotoxin (ST)
LT and ST activate adenylate or guanylate
cyclase (respectively) leading to osmotic
imbalance and electrolytes and fluids loss
(i.e., secretory diarrhea)
 The Diarrheagenic E.coli
Enteroinvasive E. coli (EIEC ) causes
dysentery-like illness indistinguishable
clinically from shigellosis
Fecal-oral route of transmission, modes of
transmission with contaminated food, water
and dirty hands
Invade colonocytes like Shigella sp.
Cause invasive diarrhea
Typical serotype is E.coli 124
 Virulence Factors of EIEC
Adhesion factors (outer membrane
proteins, pili)
Invasive proteins (ipa B,C,D)
Shiga-like toxins (SLT)
Endotoxin
SLT inhibits protein synthesis in colonocytes
causing tissue destruction, necrosis and
ulceration resulting in blood and mucus in
stools
 The Diarrheagenic E.coli
Enterohemorrhagic E. coli (EHEC) causes
hemorrhagic colitis (HC) characterized by bloody
and copious diarrhea and renal failure development
(so-called haemorrhagic uraemic syndrome,
HUS), and in rare cases trombocytopenia
Fecal-oral route of transmission, food-borne disease
Invade both enterocyes and colonocytes
Cause invasive diarrhea
The common cause is E.coli 157:7
 Virulence Factors of EHEC
Adhesion factors (intimin)
Invasive proteins (ipa B,C,D)
Shiga-like toxins (SLT)
Verotoxin
Hemolysin
Endotoxin
Causes damage to ascending and
transverse colon and caecum
 The Diarrheagenic E.coli
Enteroaggregative E.coli (EAggEC, EAEC)
causes persistent diarrhea (more than 14 days)
in children in developing countries
Recent outbreaks also implicate EAEC as a cause of
food-borne illness in industrialized countries
Derived their name from characteristic attachment
pattern resembling stacked brick
Typical strain is E.coli O42
 Pathogenesis of EAggEC-
associated diarrhea
Is not well understood
Bacteria adhere to both small and large bowel
mucosa and elaborate enterotoxins and
cytotoxins, which result in secretory diarrhea
and mucosal damage

Virulence Factors:
aggregative adherence fimbriae AAF/I
and AAF/II
protein dyspersin promotes bacterial spread
across the mucosa
heat-stable enterotoxin designated EAST1
cytotoxins (Pet and Pic)
 Pathogenesis of EAggEC-
associated diarrhea
1. Adhesion
2. Enhancement of mucus production
leading to the deposit of a thick mucus-
containing biofilm. The blanket may promote
persistent colonization and nutrient
malabsorption
3. Elaboration of toxins or inflammation,
which result in damage to the mucosa and
intestinal secretion
 Clinical Manifestation of EAggEC-
associated diarrhea

watery, mucoid, secretory diarrhea

grossly bloody stools (rare)
low grade fever
no vomiting
Malnourished hosts may be unable to
repair the mucosal damage and may
thus become prone to the persistent
diarrhea syndrome
 ATTENTION:

The E.coli biovars show similar

morphology, tinctorial properties, cultural
and the most of biochemical tests. That
is why:
they can be differentiated only by
detection their antigenic
properties, virulence factors and
genome
 Clinical Picture and Immunity
after Disease
Clinical manifestations depend on E.coli
serogroup and virulence factors
Strong immunity does not develop
Colonization resistance mechanisms of
normal microflora of GIT can promote
prevention of the disease
 Microbiological Diagnosis of
E.coli -associated Diarrhea
Specimens:
Feces, vomit specimens; foods, water

Methods and Tests:

Bacteriological examination
Genetic tests (the DNA fragment probe for EAEC
infections)
 Treatment and Prophylaxis
of E.coli - associated Diarrhea
Treatment:
Antibiotics (in severe cases and for small
children)
Probiotics (colibacterinum,
bifidumbacterinum, bificol, etc.)
No vaccines are available for prophylaxis
Vibrio cholerae the
causative agent of
cholera
R.H.H. Koch
(1843-1910)

Famous German
bacteriologist
Nobel laureate (1905) for
discovering tubercle bacilli
and tuberculosis research
He first isolated and
described the causative
agent of cholera (1883)
that is also known as
Kochs vibrio
Z.V. Yermoljeva (1898-1974)
Soviet microbiologist, professor
(USSR)
Made an outstanding contribution
to microbiology in Soviet Union
Investigated V.colerae and other
vibrios
Ingested living V.colerae to study
its virulence, pathogenesis and
symptoms of disease
Discovered fluorescent vibrios
received her name
Performed experiments with
bacteriophages
Morbidity and Mortality rate
According to WHO annual reports:
Is registered in 52 countries
Endemic in India, South-East Asia and
Africa as long as recorded history
The increased number of reported
cases reaching 1990s:
more than 200,000 cases
over 6,000 deaths every year
 TAXONOMY and Classification

Family Vibrionaceae
Genus Vibrio
Species V.cholerae

Species V.colerae is subdivided into:

Serogroups (1 and 139 cause cholera)
1 serogroup splits into two biovars
classic and eltor
139 serogroup includes one biovar
Bengal
Morphology and Tinctorial Properties
of V.cholerae

Small gram-negative comma-shaped

bacteria
Do not form capsules and endospores
Motile (monotrichous)
 Cultural Properties of
V.cholerae

Facultative anaerobes but grow best

in aerobic conditions
Alkaliphiles: require 8.5-9.0
Optimal conditions: grow at 37 within
18-24 h
Cultural Properties of
V.cholerae
Grow on the surface of liquid
medium making gentle film
within 6 to 8h of incubation
(picture above: V.cholerae growth
in the 1% peptone water)

Being sucrose-positive form

yellow colonies on TCBS medium
(bottom picture)
 Biochemical Features of
V.cholerae
Highly biochemically active:
Glucose +
Lactose +
Glycogen +
Starch +
Indole +
Dissolve gelatin (the funnel-like
appearance)
Heibergs triad (belongs to group 1/chemovar1):
Mannose
+
Sucrose +
Arabinose
 V.cholerae biovars
V.cholerae O1 is divided into biovars classic
and eltor by the following biological
features:
Sensitivity to bacteriophages C or eltor
(respectively)
Agglutination of chicken erythrocytes
Hemolysis of sheep erythrocytes
Hexamine test
Polymyxin sensitivity/resistance
(respectively)
 Antigenic Properties of
V.cholerae
Have - and -antigens
by O-antigen structure are divided into O-
serogroups:
the common cause of cholera are 1 and 139 serogroups
O1-antigen consists of , , subunits
O1-serogroup is divided into three serovars:
Ogawa; Inaba; - Hikojima
are identified in slide agglutination test
Virulence Factors of V.cholerae
Adhesins (pili)
Enzymes (mucinase,
neuraminidase,
fibrinolysin,
plasmocoagulase)
Endotoxin
Exotoxin (cholerogen
toxin) that consists of
two subunits:
B (binding) and A
(active)
The Mode of Cholerogen Toxin
Action
activation
cholerogen of adenylate
cyclase

osmotic overproduction
imbalance of cAMP
 Resistance to Unfavourable
Conditions
Sensitive to acid pH
and disinfectants
Can live in sea water
and stagnant basin
Can live in
environment as free-
living bacteria
Have symbiotic
relationship with the
Plankton becoming
eaten by fish,
shellfish, shrimp and
other sea animals
 Cholera is human acute enteric
infection transmitted via fecal-oral
route that is characterized by dramatic
electrolytes and fluids loss with stools
and vomits leading to severe
dehydration and circulatory shock

Cholera is a quarantine infection

 EPIDEMIOLOGY of Cholera
Saproantroponosis
The source of infection is human
(patient and carrier of vibrios)
Fecal-oral Route of Transmission
Waterborne and food-borne
transmission (with sea foods), via
direct contact (during epidemics)
Cholera is worldwide distributed
 PATHOGENESIS of Cholera
Portals of entry mouth, GIT
The Infectious dose is 108 109 CFU/ml; many bacteria
die in gastric juice
Adhesion in small intestine, colonization of
enterocytes
Releasing of cholerogen that activates
adenylate cyclase leading to osmotic imbalance
Secretory diarrhea, vomiting
Dehydration resulting in thickening of the
blood, circulatory collapse
Bacteriaemia does not occur
 Cholera: Clinical
Manifestations
Incubation period from 2h to days (usually 2-3
days)
Abrupt onset
Watery diarrhea (10 or more times/day) and
vomiting
The rice-water appearance of stool
Low grade fever
Acidosis and circulatory shock due to
dehydration
Terminal stage body temperature lower than
35 (cholera algid)
Cholera: Clinical Manifestations
 Immunity after Cholera
Both Cell-mediated and Humoral
Not-protective immunity: not strong
and of short duration
 Microbiological Diagnosis of
Cholera
Specimens:
Stool and vomit specimens, autopsy material;
water, foods

Methods and Tests:

Rapid tests (IF, ELISA; PCR)
Bacterioscopical examination (presumptive),
a shoal of fish picture
Bacteriological examination
 Treatment of Cholera
Antibiotics (fluoroquinolones)
Carrier state eradication (nitrofurantoin)
Rehydration (fluid and electrolyte replacement
therapy)
NOTE: Glucose stimulates uptake of sodium and subsequent
osmotic absorption of water

NB: Mortality rate of untreated cholera averages

50-60%,
in effective treatment death rate is less than 1%
 Immunoprophylaxis of
Cholera
Killed parenteral V.cholerae vaccine
Combined parenteral V.cholerae vaccine
(contains V.cholerae 1-antigen and
cholerogen-toxoid) induces both
antibacterial and antitoxic immunity
Live attenuated V.cholerae vaccine for
oral administration (OChV) is recommended
for travelers since 2006. Has 85-90%
protective efficacy for 6 months