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ENTEROBACTERIACEAE
Dr.Babasaheb N. Kumbhar
M.V.Sc
[email protected]
Enterobacteriaceae
Habitat
 digestive tube(colon) of human and animals
 150 Species
 Facultative anaerobes
 Diarrheal illnesses
 3 million death/year
 4 billion infectoions/ worldwide
Enterobacteriaceae
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Classification – more than15 different genera
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Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Klebsiella
Enterobacter
Hafnia
Serratia
Enterobacteriaceae
Proteus
 Providencia
 Morganella
 Yersinia
 Erwinia
 Pectinobacterium
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Family Enterobacteriaceae
Certain E .coli strains
can be considered
true pathogens
True pathogen
(nonmotile)
(nonmotile)
True pathogen
True pathogen
Morphology and General Characteristics
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Are facultative anaerobes
Gram-negative, non-sporing, rod shaped bacteria
If motile, motility is by peritrichous flagella(Except
Shigella & Klebsiella)
Many are normal inhabitants of the intestinal tract of
man and other animals
Some are enteric pathogens and others are urinary or
respiratory tract pathogens
Differentiation is based on biochemical reactions and
differences in antigenic structure
Morphology and Physiology
Short gram-negative rods.
Facultative anaerobes.
Grow readily and rapidly on
simple media.
K. pneumoniae
Klebsiella spp. have large capsule
(form large and very mucoid colonies);
those of Enterobacter have smaller
capsule; the others produce diffusible
slime layers (form circular, convex and
smooth colonies).
ENTEROBACTERIACEAE PHYSIOLOGY
Glucose is fermented with strong acid
formation and often gas
 Reduce nitrates to nitrite
 Do not liquify alginate
 Oxidase negative
 Basis for speciation within a family
- differences in carbohydrate they ferment
- variations in end-product production
- variation in substrate utilization
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Enterobacteriaceae
Meningitis
Opportunistic pathogens
Pneumonia
Escherichia coli
Klebsiella pneumoniae
Sepsis
Enterobacter aerogenes
Serratia marcescens
Diarrhea
Proteus spp.
Providencia spp.
Citrobacter spp.
Obligate pathogens
Salmonella spp.
Shigella spp.
Yersinia spp.
Some E. coli strains
UTI
Incidence of Enterobacteriaceae
Associated with Bacteremia
CULTURAL CHARACTERISTICS
On non differential or nonselective media
blood agar or infusion agar
- no species distinction
- appear as moist, smooth, gray colonies
 Selective media -To isolate Shigella and
salmonella from fecal matter
 Differential media – selectively inhibit
gram-positive organisms and to separate
enterics in broad categories
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Modes of Infection
• Contaminated food and water (Salmonella spp.,
Shigella spp., Yersinia enterocolitica, Escherichia coli
O157:H7)
• Endogenous (urinary tract infection, primary bacterial
peritonitis, abdominal abscess)
• Abnormal host colonization (nosocomial pneumonia)
• Transfer between debilitated patients
• Insect (flea) vector (unique for Yersinia pestis)
Urinary Tract Infection, Pneumonia
• Escherichia coli, Klebsiella pneumoniae, Enterobacter
spp., and Proteus mirabilis
• Pneumonia: Enterobacter spp., Klebsiella pneumoniae,
Escherichia coli, and Proteus mirabilis
• Wound Infection: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
• Bacteremia: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
Intestinal Infection
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Shigella sonnei (serogroup D)
Salmonella serotype enteritidis
Salmonella serotype typhimurium
Shigella flexneri (serogroup B)
Escherichia coli O157:H7
Yersinia enterocolitica
MacConkey (MAC) Agar
Eosin Methylene Blue (EMB) Agar (Levine)
Antigenic Structure
– Most are motile by peritrichous flagella --H
antigens.
– Capsule – K antigen ( Vi for Salmonella).
– Cell envelope (wall)
– LPS (endotoxin) –
O antigen.
– various outer
membrane proteins.
– Pili - various antigen
types, some encoded by plasmids
Pathogenesis and Immunity
Common virulence factors
Endotoxin (Lipid A of LPS)
Capsule
Antigenic phase variation
Acquisition of growth factors (e.g. Fe)
Resistance to serum killing
Antimicrobial resistance
Type III secretion systems: possessed by some Enterobacteriaceae
pathogens, e.g., E. coli, Yersinia, Salmonella, and Shigella; facilitate
transport of bacterial virulence factors directly into host cells.
HE Agar: Growth of Enteric Pathogens and Commensals
Escherichia coli
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Toxins: two types of enterotoxin; Shiga-type toxin;
Enteroaggregative ST-like toxin; Hemolysins;
Endotoxin
 Virulence factors that protect the bacteria from host
defenses: Capsule/Iron capturing ability (enterochelin)
E. coli
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May be hemolytic on CBA – more common in pathogenic
strains
KEY tests for the normal strain:
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TSI is A/A + gas
LIA K/K
Urea –
Indole +
Citrate –
Motility +
There is an inactive biotype that is anaerogenic, lactose –,
and nonmotile.
Enteropathogenic E. coli
fever
infant diarrhea
vomiting
nausea
non-bloody stools
Destruction of surface microvilli loose attachment mediated by
bundle forming pili (Bfp);
Stimulation of intracellular calcium level;
rearrangement of intracellular actin,
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Enterotoxigenic E. coli
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A watery diarrhea, nausea, abdominal cramps and low-grade
fever for 1-5 days.
Travellers diarrhea and diarrhea in children in developing
countries
Transmission is via contaminated food or water.
E.coli-Enteroinvasive (EIEC)
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The organism attaches to the intestinal mucosa via pili
Outer membrane proteins are involved in direct penetration,
invasion of the intestinal cells, and destruction of the intestinal
mucosa.
There is lateral movement of the organism from one cell to
adjacent cells.
Symptoms- fever,severe abdominal cramps, malaise, and watery
diarrhea followed by scanty stools containing blood, mucous,& pus.
resembles shigellosis
E.coli-c. Enteropathogenic (EPEC)
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Malaise and low grade fever diarrhea, vomiting, nausea, nonbloody stools
Bundle forming pili are involved in attachment to the intestinal
mucosa.
This leads to changes in signal transduction in the cells, effacement
of the microvilli, and to intimate attachment via a non-fimbrial
adhesion called intimin.
This is a problem mainly in hospitalized infants and in day care
centers.
Enterohemorrhagic (EHEC)
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Hemorrhagic
– bloody, copious diarrhea
– few leukocytes
– afebrile
 hemolytic-uremic syndrome
– hemolytic anemia
– thrombocytopenia
(low platelets)
– kidney failure
• Usually O157:H7
Transmission electron micrograph
Enteroaggregative E. coli
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a cause of persistent, watery diarrhea with
vomiting and dehydration in infants.
 That is autoagglutination in a ‘stacked brick’
arrangement.
 the bacteria adheres to the intestinal mucosa
and
elaborates
enterotoxins
(enteroaggregative heat-stable toxin, EAST).
 The result is mucosal damage, secretion of
large amounts of mucus, and a secretory
diarrhea.
Shigella
Shigella species
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Shigella
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Contains four species that differ antigenically and, to a lesser
extent, biochemically.
S. dysenteriae (Group A)
 S. flexneri (Group B)
 S. boydii (Group C)
 S. sonnei (Group D)
bacillary dysentery, shigellosis, bloody feces, intestinal pain, pus
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Biochemistry
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TSI K/A with NO gas
LIA K/A
Urea –
Motility All ferment mannitol except S. dysenteriae
S. sonnei may show delayed lactose fermentation
Shiga toxin
1. Chromosomally encoded
2. Neurotoxic
3. Enterotoxic
4. Cytotoxic
Enterotoxicity can make the disease clinically
appear as a diarrhea.
The toxin inhibits protein synthesis (acting on the
60S ribosome and lysing 28S rRNA).
Clinical significance
man only "reservoir"
 mostly young children
– fecal to oral contact
– children to adults
 transmitted by adult food handlers
– unwashed hands
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Diagnosis of Shigella infection
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Specimen: stool.
Culture and Identification
Quick immunological methods:
Immunofluorescent “ball” test;
Coagglutination.
XLD Agar: Growth of Shigella and Proteus
Salmonella
Only a few types that are commonly associated
with characteristic human diseases 1. S. enteritidis
2. S. cholerae-suis
3. S. typhi
several syndromes including gastroenteritis, enteric
(typhoid) fever or septicemia
Salmonella
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Biochemistry
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TSI K/A + gas and H2S: S. typhi produces only a small
amount of H2S and no gas , and S. paratyphi A produces
no H2S
LIA K/K with H2S with S. paratyphi A giving K/A results
Urea –
Motility +
Citrate +/Indole -
Virulence factors
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Endotoxin – may play a role in intracellular survival
Capsule (for S. typhi and some strains of S. paratyphi)
Adhesions – both fimbrial and non-fimbrial
Salmonella typhi
• The organism is transmitted from:
1. a human reservoir
2. in the water supply (if poor sanitary conditions)
3. in contaminated food
The antigenic structures of salmonellae used in serologic typing
Virulence factors
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Endotoxin – may play a role in intracellular survival
Capsule (for S. typhi and some strains of S. paratyphi)
Adhesions – both fimbrial and non-fimbrial
Type III secretion systems and effector molecules – 2 different systems may be
found:
– One type is involved in promoting entry into intestinal epithelial cells
– The other type is involved in the ability of Salmonella to survive inside macrophages
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Outer membrane proteins - involved in the ability of Salmonella to survive inside
macrophages
Flagella – help bacteria to move through intestinal mucous
Enterotoxin - may be involved in gastroenteritis
Iron capturing ability
XLD Agar: Appearance of Salmonella
Diagnosis
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A. Specimens
a) Enteric fever: blood, bone marrow, stool, urine.
b) Food poisoning: stool, vomitus, suspected food.
c) Septicemia: blood.
B. Culture and identification
C. Widal test
Klebsiella
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NF of GI tract, but potential pathogen in other areas
TSI A/A + gas
LIA K/K
Urea +
Citrate +
MR-, VP+
Motility Has both O and
K antigens
Klebsiella
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Virulence factors
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K.Pneumoniae on BA and MAC
Capsule
Adhesions
Iron capturing ability
Clinical significance
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Causes pneumonia, mostly in immunocompromised hosts.
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Permanent lung damage is a frequent occurrence (rare in
other types of bacterial pneumonia)
A major cause of nosocomial
infections such as septicemia and
meningitis
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K.Pneumoniae On BA
Proteus
General characteristics: “swarming” phenomenon
on nonselective agar (P.vulgaris; P.mirabilis and
P.myxofaciens)
 P.vulgaris strains (OX-19, OX-K, OX-2)have
common antigen with Rickettsia (Weil-Felix test).
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urinary tract infections; food poisoning.
Some enteric bacteria are motile.
Klebsiella species are not motile,
while Proteus species move very
actively by means of peritrichous
flagella, resulting in "swarming"
on solid medium.
Some strains of E. coli produce
hemolysis on blood plates.
Proteus spp.
CITROBACTER
Resident of soil, water, stool
 • C.freundii
 • UTI and bacteremia
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Serratia
Serratia
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A free-living saprophyte
TSI A/A or K/A; +/- gas (does not ferment lactose)
LIA usually K/K
Citrate +
Motility +
Urea +/Has been found in RT and UT infections
Is resistant to many antimicrobics
Negative test
e.g. Klebsiella
Positive test
e.g. E. coli
Citrate Utilization Test
Urease Test
Positive
test
Indole test
Negative test
Special Features:
Positive
Klebsiella,
Enterobacter
Negative
E. coli
Used in the differentiation of
genera and species. e.g. E.
coli (+) from Klebsiella (-).
Results
MR/VP test
Methyl Red test
Red: Positive MR (E. coli)
Voges-Proskauer test
Pink: Positive VP (Klebsiella)
No pink: Negative VP (E. coli)
Yellow or orange: Negative MR (Klebsiella)
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Gram
stain
Oxidase
Nitrate
reductase
O/F
MacCon
key
SS
EMB
E. coli
-ve rod
-ve
+ve
O+/F+
LF
LF
Metallic
sheen
Citrobacter
-ve rods
-ve
+ve
O+/F+
LF
LF
Dark
Klebsiella
-ve rods
-ve
+ve
O+/F+
LF
LF
Dark
Enterobacter
-ve rods
-ve
+ve
O+/F+
LF
LF
Dark
Salmonella
-ve rods
-ve
+ve
O+/F+
NLF
NLF/
H2S
Colorless
Shigella
-ve rods
-ve
+ve
O+/F+
NLF
NLF
Colorless
Proteus
-ve rods
-ve
+ve
O+/F+
NLF
NLF/
H2S
Colorless
Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
TSI
Indole
MR
VP
Citrate
Urease
Motility
E. coli
A/A/-
+ve
+ve
-ve
-ve
-ve
Motile
Citrobacter
freundii
A/A/-
+ve
+ve
-ve
+ve
-ve
Motile
Klebsiella
pneumoniae
A/A/-
-ve
-ve
+ve
+ve
+ve
Non
motile
Enterobacter
cloacae
A/A/-
-ve
-ve
+ve
+ve
+ve
Motile
Salmonella
typhi
A/Alk/+ -ve
+ve
-ve
+ve
-ve
Motile
Shigella
boydii
A/Alk/-
-ve
+ve
-ve
-ve
-ve
Non
motile
Proteus
mirabilis
A/Alk/+ -ve
+ve
-ve
+ve
+ve
Motile
Swarwing
Oxidase Test
Negative
Positive
Enterobacteriaceae
Pseudomonas
MacConkey’s agar & TSI
 O/F test: O+/F-
 Nitrate test: +ve further
reduction to N2
colorless colonies on MacConkey
Pink colonies on MacConkey
& acidic butt and slant on TSI & acidic butt alkaline slant onTSI
 Growth on cetrimide agar:
Lactose non-fermenter
Pale colonies with green
pigmentation
Lactose fermenter
IMViC test
& EMB
No H2S production
(no blacking in TSI)
IMViC
IMViC
++ - - - ++
& black colonies
Motility
with metalic
shines on EMB
Shigella
H2S production
(blacking in TSI)
Urease production
+ve
SS agar
Not motile
E.coli
Klebsiella
-ve
Motile
Proteus
colorless colonies with black centers
Salmonella
References:
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