Gastrointestinal Pathogens Causing Diarrhea, Dysentery, Enteric Fever, Pseudomembranous Colitis and Food Poisoning
Lecture 33 - Paradigms of Gastrointestinal Infections - The Five Faces of E. coli
Reading Assignments: (1) Text Chapters 16, 17, 57, 73 (2) “Colonization I: Adherence”, pp. 189-201, In McClane, B.A. and T.A. Mietzner. 1999. Microbial Pathogenesis: A Principles-Oriented Approach, Fence Creek Publishing, Madison, CT. (3) “Infectious Diarrhea”, In Gates, R. H. 1998. Infectious Disease Secrets, Hanley and Belfus, Inc., Philadelphia. (4) Gilligan, P.H., M. L. Smiley, and D. S. Shapiro. 1997. (Case 65, pp. 297-300), Cases in Medical Microbiology and Infectious Diseases (2nd. Ed.), ASM Press, Washington, D.C.
A. Infections caused by E. coli can be confined to the intestinal tract or they can disseminate throughout
the body. Diseases associated with E. coli include diarrhea, dysentery, hemolytic uremic syndrome,
bladder and kidney infections, septicemia, pneumonia and meningitis.
B. How can one pathogen cause so many different diseases? Because, each single pathogenic E. coli
has acquired a different set of virulence genes that are encoded on a variety of mobile genetic
elements including plasmids, bacteriophages, transposons and pathogenicity islands. All 5 of the
E. coli diarrheal pathogens carry at least one virulence-related property on a plasmid. (Review
Stanley Falkow's Article – What is a Pathogen? pp. 361-362).
C. The “Five Faces of E. coli “ –the E. coli strains that cause diarrheal diseases. These strains include
several emerging pathogens of worldwide public health importance and have been the focus of a
tremendous amount of research in the last decade. Material presented taken from Reading
Assignment (2) and a recent review in the January 1998 issue of Clinical Microbiology Reviews – by
James P. Nataro and James B. Kaper entitled “Diarrheagenic Escherichia coli “.
TABLE 12-7 FIVE DISTINCT GROUPS OF E. COLI THAT CAUSE INTESTINAL DISEASE (SEE READING ASSIGNMENT (2), p. 196)
3 CATEGORIES OF VIRULENCE FACTORS: ADHERENCE, TOXINS, AND SIDEROPHORES.
Virulence factors shared in common:
A. Adherence
Type 1 pili- allow E. coli to attach to mannose receptors associated with
the host cell surface and may allow strains to co-aggglutinate through
bacteria-bacteria interactions
B. Toxins
Endotoxin- elicit inflammatory response
C. Siderophores- iron acquisition
1. enterobactin (enterochelin)- genes carried chromosomal genes
2. aerobactin – caried on plasmids – associated with extraintestinal
infections
Pathogenic strategies vary depending on the virulence factors carried by each of the five groups of E. coli.
2. CATEGORIES OF E. COLI CAUSING INFECTIOUS DIARRHEA
A. Enterotoxigenic E. coli (ETEC)
1. Diseases (associated with two major clinical syndromes)
a. Infant (weanling) diarrhea in children of the developing
world
Endemic in the developing world. ETEC contamination in the environment is
prevalent and most infants will be exposed to the pathogen upon weaning.
Sporadic infant diarrhea due to ETEC varies from 10-30% in endemic areas;
highest incidence in warm wet months, when multiplication of ETEC in food
and water is the highest. Mucosal immunity develops with increasing age and
children and adults have a very low incidence of symptomatic disease.
Immune asymptomatic individuals may shed large numbers of virulent ETEC
organisms in the stool and contaminate food and water sources. A high
infectious dose is required for infection.
b. Traveler's diarrhea (See Reading Assignment (3) pp. 241- 242)
The predominant cause of traveler's diarrhea among immunologically naïve)
adults from the developed world visting areas where ETEC is endemic.
2. Symptoms
Illness has an abrupt onset with a short incubation period (14-50h); diarrhea
watery without pus or blood. Diarrhea may be mild, brief, and self limiting, or
may result in severe purging similar to V. cholerae infection.
Most life-threatening cases occur in weanling infants in the developing world.
3. Pathogenesis (See Reading Assignment (2) and Text Fig. 16.2, p. 181)
a. Overview: ETEC strains (like V. cholerae) colonize the mucosa of the small
intestine using Type I pili and other CFA's (pili) and elaborate two enterotoxins:
labile toxin (LT) and stable toxin (ST). Strains may express LT alone, ST alone, or
LT and ST. The result of the action of the enterotoxins is watery (secretory)
diarrhea, without white or red blood cells. The mucosa is not visibly damaged
and there is no inflammatory response.
b. Virulence Factors
1. Colonization Factors: Adherence is mediated by several distinct pili called
colonization fimbrae (CFAs)
a. CFA/I- rigid rod-shaped fimbriae
b. CFA/II and CFA IV - Flexible fibrillar pili
CFA genes are encoded on plasmids which also encode LT
and/or ST.
2. Enterotoxins
a. Heat labile toxin (LT) – closely related to CT : 1) oligomeric structure
like CT (5 B subunits; 1 A subunit) (2) identical action to CT- binds to
GM1 ganglioside, internalized into a vesicle, activation of Gs
regulatory subunit of adenylate cyclase (at the basolateral membrane
of the intestinal epithelial cell), increased cAMP. Net result is
increased electrolyte and fluid secretion and inhibition of resorption
resulting in increased fluid in the lumen of the intestine -watery
diarrhea.
b. Heat stable toxins (STa and STb) – small monomeric protein toxins;
bind to membrane spanning enzyme guanylate cyclase (GC),
stimulating GC activity, leading to increased intracellular cGMP levels.
This lead to net intestinal fluid secretion.
B. Enteropathogenic E. coli (EPEC) (See Case Study “Baby D”in Text p. 177)
1. Disease
a. EPEC in the Developing World - associated with infant diarrhea (infants less
than 2 years).
b. EPEC in the Developed World
1. 1940's and 1950's – associated with frequent outbreaks of infant
diarrhea – nosocomial and community outbreaks – 50% mortality
2. Last two decades – outbreaks associated with day care centers and
on pediatric wards. Outbreaks in adults - foodborne. A recent study
where DNA probe technology was used to survey infants with
diarrhea in Seattle indicated that the frequency of EPEC exceeded
the rates of E. coli O157:H7, Campylobacter, Salmonella, and
Shigella.
2. Epidemiology – Transmission is via the fecal-oral route with contaminated hands,
contaminated weanling foods or formula, or contaminated fomites serving as vehicles.
Numerous studies demonstrate the spread of infection though hospital nurseries or in
day care from an index case. Admission of an infected infant to a pediatric ward can
result in contamination of cribs linens, toys, tabletops, hand towels, scales, carriages,
pacifiers, etc.
Reservoirs – symptomatic or asymptomatic children and asymptomatic carriers,
including mothers and persons who handle infants. Uncommon adult outbreaks linked
to food and water.
3. Pathogenesis
a. Overview- EPEC strains adhere to the mucous membrane of the small
intestine via Type 1 pili and plasmid mediated bundle forming pili (BFP),
destroy the brush border, and cause extensive rearrangement of host cell
actin (attaching-and-effacing lesions – characteristic histopathology). The
severe watery diarrhea which results is not due to an enterotoxin but to the
direct damage to brush border with the disruption of absorption by the
enterocytes. A host inflammatory response is found but without fecal
leukocytes in the stool.
b. Steps in Pathogenesis (3 stages):
1. EPEC bind loosely to the cell surface via plasmid- encoded bundle-
forming pili (BFP). The binding of the bacteria is necessary for the
next stage.
2. The binding induces several signal transduction pathways in the
eukaryotic cell, leading to increased intracellular calcium,
rearrangement of intracellular actin, and destruction of the micovilli.
The bacterial genes responsible for the signal transduction are
encoded on a 35 kb pathogenicity island called the locus of
enterocyte effacement (LEE). Included in the LEE are a type III
secretion system, multiple secreted proteins, and a bacterial adhesin
called intimin (involved in the final stage of pathogenesis).
3. The bacterium bind closely to the epithelial membrane via an
afimbrial adhesin (the outer membrane protein intimin, and bacterial
gene products cause disruption and rearrangement of the
cytoskeleton, forming the characteristic EPEC pedestal with
intimately adherent organisms (the attaching-and-effacing lesion.)
Mechanisms of diarrhea- dramatic loss of microvilli leads to malabsorption
diarrhea. Recent reports indicate that an increased intestinal permeability in
response to EPEC infection and a local inflammatory response may also
contribute to diarrhea.
C. Enteroaggregative E. coli (EAEC)
1. Disease – persistent watery diarrhea in infants in developing countries. High
prevalence on the Indian subcontinent and in South America. Causes sporadic
endemic diarrhea and epidemics in hospital nurseries. Characteristic symptoms
include watery, mucoid, persistent, secretory diarrhea with low-grade fever and
to no vomiting.
2. Pathogenesis – not well understood
a. Discovered by investigators observing that some E. coli strains isolated during
studies of diarrhea adhered to Hep-2 cells in tissue culture via aggregative
adherence – characterized by layering of the bacteria in a stacked brick
configuration.
b. Overview – EAEC strains adhere via Type I and AAF pili and enhance mucus
secretion from the mucosa; bacteria are seen trapped in a densely packed
bacteria-mucus biofilm. A cytotoxin elaborated by the bacteria damages
the enterocytes. Diarrhea may result from the interference of absorption by
the thick mucus-biofilm, damage to the enterocyte, and the host's
inflammatory response.
c. Steps in Pathogenesis (proposed 3 stage model):
1. Initial adherence to the intestinal mucosa and/or the mucus layer using Type 1
pili and flexible bundle forming pili – aggregative adherence fimbriae (AAF)
2. Enhanced mucus production, leading to deposition of a thick mucus
containing biofilm encrusted with EAEC. The blanket may promote persistent
colonization and perhaps nutrient malabsorption.
3. Elaboration of an EAEC cytotoxin (Enteroaggregative ST-like toxin-(EAST) )
results in damage to the intestinal cells. It is possible that malnourished hosts
may be particularly impaired in their ability to repair this damage leading to
perisistent-diarrhea syndrome.
Although the site of EAEC infection in the human intestine has not
been determined, it is probably the small intestine.
D. Enterohemorrhagic E. coli (EHEC)
1. History
a. Two key epidemiological observations – 1983
1. Riley, et al –linked outbreak of HC to E. coli O 157:H7
2. Karmali, et. al – sporadic cases of HUS associated with fecal cytotoxin and
cytotoxin producing E. coli strains in stools
Led do a recognition of a new class of enteric pathogens causing intestinal and
renal disease.
b. Detection of cytotoxin production in cultured cells:
Karmali – add culture filtrates of E. coli O157:H7 to cultured Vero cells produced
a unique cytopathic effect.
O'Brien – extracts of E. coli O157:H7 were cytotoxic for HeLa cells and the
cytotoxic activity could be neutralized by antitoxin against S. dysenteriae type 1
Shiga Toxin. Showed many diarrheal E. coli strains produced shiga-like
toxins (SLT) and that SLT and Vero cytotoxin were the same toxin.
Karmali – proposed the Vero cytotoxin/ SLT was the common virulence factor
between HC and HUS and that it damaged both intestinal and renal tissue.
c. E. coli O 157:H7 – a new emerging pathogen? YES!
1. Was it really new a new pathogen or just previously undiagnosed?
CDC, Public Health Laboratory in UK, and Laboratory Center for Disease
Control in Canada did retrospective studies on isolates from the 70's and
80's– could not find it.
2. Where did it come from?
HUS known prior to 1982 to be rarely associated with Stx-producing S.
dysenteriae. The genes for Stx are encoded on a bacteriophage. O 157:H7
strains closely related to O55:H7 EPEC strains, a serotype long associated
with worldwide outbreaks of infant diarrhea. Non-O157:H7 strains of E. coli
that produced Stx may have been around for decades, but it was only with the
emergence in the early 1980's of the O157:H7 clone that produce Stx that this
pathogenic class of E. coli was recognized.
2. Pathogenesis
a. Overview – O 157:H7 E. coli strains produce a bloody diarrheal syndrome known as
Hemorrhagic Colitis (HC). Disease in pediatric patients may include Hemolytic
Uremic Syndrome (HUS) which results in acute kidney failure. Although multiple
virulence factors may play a role in pathogenesis, two are well recognized: attaching
and effacing lesions, and the production of one or more Shiga toxins. Shiga toxin
is responsible for symptoms in the gastrointestinal tract and after absorption in the
bloodstream, for damage to the kidneys.
b. Attachment and Effacement (A/E)- similar to EPEC
1. Localized adherence: EHEC 60 MDa plasmid – encodes enterohemolysin and
fimbrial antigen possibly involved in the initial colonization step.
2. Signal transduction – encoded on LEE on the chromosome similar to EPEC.
3. Intimate adherence – mediated by intimin- also encoded by the LEE
c. Shiga Toxin (Stx)
1. Two major groups of Stx – the genes are encoded on bacteriophages
inserted into the chromosome.
a. Stx 1 – identical to Stx of S. dysenteriae type 1
b. Stx 2 – share sequence homology with Stx1
c. Strains can express Stx 1, 2 or both.
d. Stx 1 and 2 are A-B extotoxins:
1. 5 B subunits bind to a glycolipid receptor
globotriaosylceramide (Gb3).
2 The holotoxin is transported into the cell in an endocytic
vesicle and then to the Golgi apparatus and ER.
3. The A subunit is translocated to the cytoplasm, and the A1
subunit removes an adenine residue from the 28S rRNA,
disrupting protein synthesis and causing cell death.
2. STx is essential for development of HC and bloody diarrhea.
3. In humans, Stx produced in the intestine is assumed to translocate to the
bloodstream across epithelial cells. Human renal endothelial cells have a
high concentration of Gb3 receptors, suggesting that binding to this tissue
causes renal injury and development of HUS. Stx is believed to damage the
glomerular endothelial cells, leading to narrowing of capillary lumina and
blockage of glomerulomicrovasculature with platelets and fibrin.
4. Stx also induces macrophages to express TNF and IL-6 and these cytokines
may also play a role in the pathogenesis of renal damage.
d. Enterohemolysin – found on 60MDa plasmid- lysis of red cells to release heme and
hemoglobin to enhance growth and serve as a source of iron? (see below). Lysis of
leukocytes? Contributes to inflammation and HUS.
e. Iron transport – special system allows the bacterium to utilize heme or hemoglobin
directly as an iron source. (69dK outer membrane protein synthesized under
conditions of Fe stress)
3. Epidemiology
a. Transmission – contaminated food or water; most outbreaks by round beef. Foods
other than ground beef can serve as vehicles including roast beef, raw milk, salami,
unpasteurized apple cider and juice, sandwiches, and ranch dressing. Recent
outbreaks – fruits and vegetables – radish sprouts, lettuce and alfalfa sprouts.
Unchlorinated municipal water and swimming water have also caused outbreaks.
Person-to person spread in day care centers and chronic-care facilities.
b. Animal Reservoir – the intestinal tracts of healthy cattle. Beef is contaminated
during slaughter and the grinding process carries the pathogens from
the surface throughout the meat. If cooking is incomplete, the bacteria
survive and are ingested, reach the colon, and multiply.
c. Infectious dose – low – less than 50 organisms
d. Fecal excretion can occur for several weeks
e. Geographic and Seasonal Factors
1. Found worldwide but most commonly reported in Canada, the U.S., and the
U.K.
2. Infections most common in warmer months, peak incidence from June-Sept.
4. Clinical Manifestations
a. Incubation period – 3-4 days
b. Nonbloody diarrhea preceded by crampy abdominal pain and a short-lived fever.
c. Diarrhea becomes bloody within 1-2 days with abdominal pain that lasts 4-10 days
“all blood and no stool”- HC.
d. With most patients, the bloody diarrhea will resolve without apparent sequelae, but in
about 10% of patients younger than 10 years (and in elderly patients), the illness
progresses to HUS.
e. HUS (defined by a triad of hemolytic anemia, thrombocytopenia, and renal failure)
1. Initial clinical manifestations include oligouria or anuria, edema, pallor and
sometimes seizures.
2. Most patients recover with supportive therapy, but 3-5% of affected children
will have severe sequelae including renal impairment, hypertension, or CNS
symptoms.
f. More severe disease associated with Stx 2.
5. Treatment
a. Supportive care
Treat HUS with dialysis, hemofiltration, transfusions of packed RBC's and
platelets. 50% of patients with HUS require dialysis and 75% receive blood
transfusions. Severe disease may require kidney transplant.
b. Antibiotics?
1. Use of antibiotics does not appear to alter the course of the disease.
2. Retrospective studies indicate that antibiotics may increase the risk of HUS
possibly by (1) lysis of bacteria leading to increased release of toxin; (2)
antibiotic therapy kills the intestinal microflora of the colon leading to
increased systemic absorption of the toxin.
c. The use of antimotility agents is associated with HUS by delaying the clearance of the
organisms and thereby increasing the toxin absorption.
E. Enteroinvasive E. coli (See Lecture 34)
3. E. COLI O4:H5 – A CAUSE OF URINARY TRACT INFECTIONS (See Reading Assignment (2)
BONUS EXAM #3 – Describe the Reservoir and Tranmission, the Virulence Factors and the
Pathogenesis of uropathogenic E. coli O4:H5
Two key epidemiological observations- 1983
Riley – linked outbreaks of hemorrhagic colitis (hc) to e. Coli
o157:h7
Karmali – linked sporadic cases of hemolytic uremic syndrome
(hus) to cytotoxin producing e. Coli strains
A new class of enteric pathogens that caused intestinal and
Renal disease