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ENTEROBACTERIACEAE
Morphology & Identification
• Gram-negative non-spore forming rods, catalase +ve, Oxidase ve.
• Primarily normal flora of gastrointestinal tract.
• transient colonizers of skin, water streams.
• Facultative anaerobes: mixed acid fermentation.
• All ferment glucose; all reduce nitrates to nitrites.
• Lactose fermentation: usually normal flora positive and
pathogens negative.
• Primary isolation media include eosin-methylene-blue (EMB)
and MacConkey agar.
• Differential selective media for specific organisms include:
Salmonella-Shigella (SS) medium, bismuth sulfite media,
Hektoen enteric agar, XLD, deoxycholate-citrate agar.
Table TSI-1: Result of TSI reactions
Genus
TSI Reaction
Butt
Slant
H2S
Gas
Enterobacter
A
A
-
+
Escherichia
A
A or K
-
+
Klebsiella
A
A
-
+
Citrobacter
A
K or A
V
+
Proteus vulgaris
A
A or K
+
+
Edwardsiella
A
K
V
+
Morganella
A
K
-
+
Serratia
A
K or A
-
V
Shigella
A
K
-
-
Salmonella typhi
A
K
+
-
Antigenic Structure
– Most are motile by peritrichous flagella --H
antigens.
– Capsule – K antigen ( Vi for Salmonella).
– LPS (endotoxin) – O antigen.
– various outer membrane proteins.
– Pili - various antigen types, some encoded by
plasmids
• O (lipopolysaccharide)
• H (flagellar)
• K (capsular)
– OKH (e.g., E. coli O55:K5:H21)
Opportunistic diseases
-Enterobacteriaceae
–
–
–
–
septicemia,
pneumonia,
meningitis
urinary tract infections
e.g.,
Citrobacter
Enterobacter
Escherichia
Hafnia
Morganella
Providencia
Serratia
Enterobacteriaceae:
Foodborne infections
–
–
–
–
Escherichia coli
Salmonellae
Shigellae
Yersinia entercolitica
Reiter's syndrome
• Associated with histocompatibility antigen (HLA) B27
•Most common in young males. most common form of arthritis
affecting men between 20-40 years old
•Symptoms
•Arthritis (pain, swelling, stiffness, redness)
•Conjunctivitis
•iritis/uveitis (rare but requires immediate treatment)
•Urinary tract infection (dysuria) may include reproductive
organs
•Skin rash
•Heart problems in 10% of cases.
•Causes:– Enterobacteriaceae
*Salmonella
*Shigella
*Yersinia
– Not Enterobacteriaceae
*Campylobacter
*Chlamydia
• community acquired
• otherwise healthy people
– Klebsiella pneumoniae
* respiratory diseases
* prominent capsule
–urinary tract infection
–fecal contamination
*E. coli
*Proteus
– urease (degrades urea)
– alkaline urine .. Cause stones
Escherichia coli
Escherichia coli
• Toxins: two types of enterotoxin; Shiga-type toxin;
ST toxin; Hemolysins; Endotoxin, LT toxin
• Adhesions: colonization factors; both pili or
fimbriae ;non-fimbrial factors involved in
attachment. There are at least 21 different types
of adhesions.
• Virulence factors that protect the bacteria from
host defenses: Capsule/Iron capturing ability
Outer membrane proteins
E.coli urinary tract infection
Is the leading cause of urinary tract infections which can lead to
acute cystitis (bladder infection) and pyelonephritis (kidney infection).
Polyuria, dysuria, hematouria, pyuria, flank pain..Can cause bacteremia.
Typically uropathogenic E. coli produce hemolysins
E.coli-Meningitis and Sepsis
• Neonatal meningitis – is a leading cause of
neonatal meningitis and septicemia with a
high mortality rate. Usually caused by strains
with the K1 capsular antigen.
Enteropathogenic E. coli
•
•
•
•
•
•
•
infant diarrhea
fever
vomiting
nausea
non-bloody stools
Destruction of surface microvilli
This is a problem mainly in hospitalized infants
and in day care centers.
Enterotoxigenic E. coli
• A watery diarrhea, nausea, abdominal
cramps and low-grade fever for 1-5 days.
• Traveller’s diarrhea
• diarrhea in children in developing
countries
• Transmission is via contaminated food or
water.
Enterotoxigenic E. coli
• Causes diarrhea like cholera but milder
• caused by LT, ST, or LT/ST and colonization
factors
Enterotoxigenic E. coli
• Heat labile toxin
– Adenyl cyclase activation
– cyclic AMP
– secretion water/ions
• Heat stable toxin
– Guanylate cyclase activation
– cyclic GMP
– uptake water/ions
E.coli-Enteroinvasive (EIEC)
• 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 include fever, severe abdominal cramps,
and watery diarrhea followed by bloody diarrhea
(+ mucous, and pus).
• resembles shigellosis
Enteroinvasive E. coli (EIEC)
• Dysentery
- resembles shigellosis
- The EIEC are like Shigella:
non-motile and non-lactose
fermenter.
Enterohemorrhagic E. coli
Also called Shiga toxin producing E.
coli (STEC)
• Usually O157:H7
Transmission electron
micrograph
E.coli Enterohemorrhagic (EHEC)
• Hemorrhagic colitis
– bloody, copious diarrhea
– few leukocytes
– afebrile
• hemolytic-uremic syndrome
– hemolytic anemia
– thrombocytopenia (low platelets)
– kidney failure
Enterohemorrhagic E. coli
O157:H7
Diagnosis
•Usually sorbitol –ve
•MUG –ve
•Can be also identified by
serology (for shiga toxin)
•Testing using Vero cells
•PCR
Enteroaggregative E. coli
• a cause of persistent, watery diarrhea with
vomiting and dehydration in infants.
• the bacteria adheres to the intestinal mucosa
and elaborates enterotoxins (heat-stable
toxin (ST)).
• The result is mucosal damage, secretion of
large amounts of mucus, and a secretory
diarrhea.
Sanitary significance
• Total bacterial
number: number of
bacteria contained per ml or gm of the
sample; the standard of drinking water is
less than 100.
• Coliform bacteria index: the number of
coliform bacteria detected out per 1000
ml sample; the standard of drinking water
is less than 3
Escherichia coli
•
•
•
•
•
•
Genetically E. coli and Shigella are genetically highly closely related. For practical reasons (primarily to avoid confusion)
they are not placed in the same genus. Not surprisingly there is a lot of overlap between diseases caused by the two
organisms.
1) Enteropathogenic E. coli (EPEC). Certain serotypes are commonly found associated with infant diarrhea. The use of
gene probes has confirmed these strains as different from other groups listed below. There is a characteristic
morphological lesion with destruction of microvilli without invasion of the organism that suggests adhesion is
important. Clinically one observes fever, diarrhea, vomiting and nausea usually with non-bloody stools.
2) Enterotoxigenic E. coli (ETEC) produce diarrhea resembling cholera but much milder in degree. Also cause "traveler’s
diarrhea". Two types of plasmid-encoded toxins are produced. a) Heat labile toxins which are similar to choleragen (see
cholera section below). Adenyl cyclase is activated with production of cyclic AMP and increased secretion of water and
ions. b) Heat stable toxins; guanylate cyclase is activated which inhibits ionic and water uptake from the gut lumen.
Watery diarrhea, fever and nausea result in both cases.
3) Enteroinvasive E. coli (EIEC) produce dysentery (indistinguishable clinically from shigellosis, see bacillary dysentery
below).
4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157: H7. These organisms can produce a hemorrhagic
colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). Outbreaks are often
caused by contaminated hamburger meat. The organisms can disseminate into the bloodstream producing systemic
hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia and kidney failure). Production of Vero toxin
(biochemically similar to shiga toxin thus also known as "shiga-like") is highly associated with this group of organisms;
encoded by a phage. Hemolysins (plasmid encoded) are also important in pathogenesis.
As noted above, there are at least 4 etiologically distinct diseases. However, in the diagnostic laboratory generally the
groups are not differentiated and treatment would be on symptomatology. Generally fluid replacement is the primary
treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic
stage (e.g.hemolytic-uremia syndrome). Two major classes of pili are produced by E. coli : mannose sensitive and
mannose resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the
host epithelium allowing attachment. This aids in colonization by E. coli.
Shigella
Shigella
• S. flexneri, S. boydii, S. sonnei, S.
dysenteriae
–bacillary dysentery
–shigellosis
• bloody feces
• intestinal pain
• pus
Genral features
• Contain pili.
• Most strains can not ferment lactose; S.
sonnei can slowly_ ferment lactose.
• According to O antigen, 4 serogroups.
• Easily acquiring drug-resistance.
Shigellosis
• within 2-3 days
–epithelial cell damage
Shiga toxin
• enterotoxic
• cytotoxic
• inhibits protein synthesis
– lysing 28S rRNA
Shigella attachment and penetration
• Within 2-3 days
• Epithelial cell
damage
• After the incubation period: fever, cramping,
abdominal pain, and watery diarrhea (due to
the toxin)for 1-3 days.
• This may be followed by frequent, scant stools
with blood, mucous, and pus (due to invasion
of intestinal mucosa).
• It is rare for the organism to disseminate.
• The severity of the disease depends upon the
species. S. dysenteria is the most pathogenic
followed by S. flexneri, S. sonnei and S. boydii.
Clinical significance
• man only "reservoir”
• Highly infectious 103 rather then 105-108 for
salmonellae and vibrios.
• Transmission via:
– fecal to oral contact
– Insects (flys..etc)
• transmitted by adult food handlers
– unwashed hands
Diagnosis of Shigella infection
•
•
•
•
Specimen: fresh stool, mucus flecks, rectal swabs / or
serum to demonstrate rise in titer of agglutination
antibodies.
Primary media: MacConkey, EMB,
Selective media: Hektoen enteric agar, SS agar, XLD,
Deoxycholate-citrate agar
Identification: TSI, Slide agglutination
Treating shigellosis
• manage dehydration
• patients respond to antibiotics ,
Problem of drug-resistance
+ dysentery vaccine.
Shigella
• Shigella (4 species; S. flexneri, S. boydii, S. sonnei, S. dysenteriae) all
cause bacillary dysentery or shigellosis, (bloody feces associated with
intestinal pain). The organism invades the epithelial lining layer, but
does not penetrate. Usually, within 2-3 days, dysentery results from
bacteria damaging the epithelium lining layers of the intestine often
with release of mucus and blood (found in the feces) and attraction of
leukocytes (also found in the feces as "pus"). Shiga toxin
(chromosomally encoded) is neurotoxic, enterotoxic and cytotoxic plays
a role. The toxin inhibits protein synthesis (acting on the 80S ribosome
and lysing 28S rRNA). This is primarily a fecal-oral disease and it can be
transmitted by infected food handlers, contaminating food. The source
in each case is unwashed hands. Man is the only "reservoir".
• Patients with severe dysentery are usually be treated with antibiotics
(e.g. ampicillin). In contrast to salmonellosis, patients respond to
antibiotic therapy and disease duration is diminished.
Salmonella
• Salmonellosis may
present as one of
several syndromes
including gastroenteritis,
enteric (typhoid) fever
or septicemia.
Virulence factors
•
•
•
•
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
• 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
Enteric or typhoid fever
• Enteric or typhoid fever occurs when the bacteria
leave the intestine and multiply within cells of the
reticuloendothelial system.
• The bacteria then re-enter the intestine, causing
gastrointestinal symptoms.
• Typhoid fever has a 10-14 day incubation period
and may last for several weeks.
• Salmonella typhi is the most common species
isolated from this type salmonellosis.
• Human reservoir: carrier state common
• Contaminated food: water supply
• Poor sanitary conditions
Typhoid
Septicemia
-occurs 10-14 days
– lasts 7 days
gall bladder
–shedding, weeks
•acute phase, gastroenteritis
gastrointenteritis
Typhoid -Therapy
• Antibiotics
– essential
• Vaccines
Vi (capsular) antigen :protective
Salmonella gastroenteritis
• Salmonella gastroenteritis is the most common
form of salmonellosis and generally requires an 848 hour incubation period and may last from 2-5
days.
• Symptoms include nausea, vomiting and diarrhea
(non-bloody stool). Salmonella enteritidis is the
most common isolate.
• Poultry, eggs. no human reservoir
• self-limiting (2 - 5 days)
Salmonella septicemia
• Salmonella septicemia (bacteremia) may be
caused by any species but S. choleraesuis is
the most common. This disease resembles
other Gram-negative septicemias and is
characterized by a high, remittent fever with
little gastrointestinal involvement.
Immunity (S. typhi)
• Vi (capsular) antigen
– protective
Diagnosis – the samples
• A. Specimens
• a) Enteric fever: blood, bone marrow, stool,
urine.
• b) gastroenteritis: stool, vomit, suspected
food.
• c) Septicemia: blood.
Culture
•
•
•
•
Differential media: MacConkey, EMB,
Selective: Hektoen enteric agar, SS agar, XLD,
Deoxycholate-citrate agar
Enrichment: Selenite F or tetrathionate.
Identifications: TSI, Bismuth sulfite media,
Slide agglutination
Table TSI-1: Result of TSI reactions
Genus
TSI Reaction
Butt
Slant
H2S
Gas
Enterobacter
A
A
-
+
Escherichia
A
A or K
-
+
Klebsiella
A
A
-
+
Citrobacter
A
K or A
V
+
Proteus vulgaris
A
A or K
+
+
Edwardsiella
A
K
V
+
Morganella
A
K
-
+
Serratia
A
K or A
-
V
Shigella
A
K
-
-
Salmonella typhi
A
K
+
-
Salmonella
•
•
•
Using appropriate antibodies more than 2000 antigenic “types” have been recognized.
There are, however, only a few types that are commonly associated with characteristic
human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi).
Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (S.
enteritidis) and is transmitted from contaminated food (such as poultry and eggs). It does
not have a human reservoir and usually presents as gastroenteritis (nausea, vomiting and
non-bloody stools). The disease is usually self-limiting (2-5 days). Like Shigella they invade
the epithelium and do not produce systemic infection. In uncomplicated cases of
salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. choleraesuis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic
therapy is required. .
The severest form of salmonella infections "typhoid" (enteric fever), caused by
Salmonella typhi. Although it is one of the historical causes of widespread epidemics and
still is in the third world. The organism is transmitted from a human reservoir or in the
water supply (if sanitary conditions are poor) or in contaminated food. It initially invades
the intestinal epithelium and during this acute phase, gastrointestinal symptoms are
noted. The organism penetrates, usually within the first week, and passes into the
bloodstream where it is disseminated in macrophages. Typical features of a systemic
bacterial infection are noted. The septicemia usually is temporary with the organism
finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks.
At this time the gastroenteritis (including diarrhea) is noted again. The Vi (capsular)
antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one
person e.g. a food handler can cause a lot of spread. Antibiotic therapy is essential.
Vaccines are not widely effective and not generally used
 Typhoid fever is still a big health-problem in many countries,
including Saudi Arabia.
 The WHO estimates that there are worldwide about 16
million of clinical cases annually, of which about 600,000
result in death.
 Foodborne infections account for 1.3 billion cases of acute
diarrhea with 3 million deaths world-wide
Klebsiella
– Normal flora of GI tract, but potential pathogen in other
areas
– Virulence factors
• Capsule
• Adhesions
• Iron capturing ability
– Clinical significance
• Causes pneumonia, mostly in immunocompromised hosts.
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
Klebsiella
• K. pneumoniae: may cause primary
pneumonia, urinary tract and wound
infections, bacteremia, meningitis, etc.
• K. rhinoscleromatis: pathogen of
granumatous destruction of nose and pharynx.
• K. ozaenae: causes chronic atrophic rhinitis.
Proteus
• General characteristics: “swarming” phenomenon,
urease positive.
• (P.vulgaris and P.mirabilis)
• P.vulgaris strains have common antigen with
Rickettsia (Weil-Felix test).
• urinary tract infections; food poisoning.
• CLED (cystine-lactose-electrolyte-deficient
medium)
End of Todays Lecture
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• Any questions
• Good luck