Lecture 12 BIOL 533 1
Enterobacteriaceae
BIOL 533
Lecture 12
Medical Microbiology
Lecture 12 BIOL 533 2
Enterobacteriaceae
? Diversity of species
? Ecology
– Found worldwide in soil,water,vegetation,
and microbial flora of animals and humans
– Some are always associated with disease
? e.g.,Shigella,Salmonella,Yersinia pestis
– Some are normal flora that can become
opportunistic pathogens
? e.g.,E,coli,K,pneumoniae,P,mirabilis
Lecture 12 BIOL 533 3
Enterobacteriaceae
? Epidemiology
– Animal reservoir,most Salmonella infections
– Human carrier,Salmonella typhi,Shigella
– Endogenous spread in a susceptible patient
? Can involve all body sites
? 5% hospitalized patients develop nosocomial
infections,primarily caused by Enterobacteriaceae
such as Escherichia
? Sites of infection
Lecture 12 BIOL 533 4
Microbial Physiology
and Structure
? Cell morphology
– Moderate-sized Gram— rods
? Non-spore-forming
? Motile (with peritrichous flagella) or non-motile
– Physiology
? All are facultative anaerobes
? Simple nutritional requirements,
– Ferment glucose
– Reduce nitrates to nitrites
Lecture 12 BIOL 533 5
Distinguishing Characteristics
? Oxidaseˉ,
– Distinguishes among other fermentative and
non-fermentative Gram— bacilli
? Lactose fermentation (red colonies on
MacConkey agar)
– Separate Escherichia,Klebsiella,Enterobacter
from other lactose— Enterobacteriaceae
Lecture 12 BIOL 533 6
Distinguishing Characteristics
? Resistance to bile salts
– Separate Shigella and Salmonella from normal
flora in this group
? Eosin Methylene Blue (EMB)
– Lactose,eosinY,methylene blue; Lac+; grow
with green sheen
Lecture 12 BIOL 533 7
Virulence Factors
? Antigens
– Somatic ―O‖ LPS
? Major cell wall Ag; heat stable
? Specific ―O‖ antigens associated with each genus;
however,cross reactions are common
– Salmonella and Citrobacter
– Escherichia and Shigella
Lecture 12 BIOL 533 8
Virulence Factors
? Capsular K
– Either protein or polysaccharide
? Heat-labile
? May interfere with detection of ―O‖
? Removed by boiling organisms
– Capsular antigen of Salmonella typhi referred
to as Vi antigen
Lecture 12 BIOL 533 9
Virulence Factors
? Capsular K,continued
– Shared by different genera both inside and
outside of family
? Cross reactions
– E,coli K1 with N,meningitidis and Haemophilus
meningitidis
– K,pneumoniae with S,pneunomiae
– Organisms with specific antigens have been
associated with increased virulence
(e.g.,E,coli K1 with neonatal meningitis)
Lecture 12 BIOL 533 10
Virulence Factors
? Flagella H
– Heat-labile proteins
? Can be absent or undergo antigenic variation
(present in two phases)
? Specific H antigens assocated with disease
Lecture 12 BIOL 533 11
Virulence Factors
? General role in pathogenesis of ―O,‖ ―K,‖
and ―H‖ antigens
– Specific antigens associated with meningitis,
gastroenteritis,and urinary tract infections
? Role that Ag’s play in these diseases is not clear
– Some capsular Ag are poor immunogens
? Protect against antibody-mediated phagocytosis
– Flagellar Ag probably play a role in adherence
Lecture 12 BIOL 533 12
Virulence Factors
? Pili
– Attachment to host cells
Lecture 12 BIOL 533 13
Pathogenesis of Escherichia
? E,coli present in gastrointestinal tract in
normal flora
? Bacterial sepsis (multiplication in blood)
– Primary focus-infection of urinary tract or
spread from gastrointestinal tract
– Death can occur in immunocompromised
patients and infections resulting from
intestinal perforation
Lecture 12 BIOL 533 14
Pathogenesis of Escherichia
? Neonatal meningitis
– E,coli and group B streptococci most common
– 75% E,coli possess Capsular K1 antigen
– Colonization of infants with E,coli at delivery
is common; disease is not
Lecture 12 BIOL 533 15
Pathogenesis of Escherichia
? Urinary tract infections
(80% community and most nosocomial)
– Originate from gastrointestinal tract
– Important virulence factors
? Resistance to serum-killing
? Production of hemolysins
? Pili-mediated binding (not demonstrated in vivo)
? Production of slime layer that participates in cell
adhesion
Lecture 12 BIOL 533 16
Pathogenesis of Escherichia
? Gastroenteritis
(countries with poor hygiene)
– Enterotoxigenic (ETEC)
? Mediated by heat-labile (like cholera) and heat-
stable exotoxins (activates guanylate cyclase and
stimulates secretion of fluid)
– Both are coded from plasmid-borne genes
? World-wide:both adults and children
– Incubation 1-2 days; persists 3-4 days
– Mild symptoms,including cramps,nausea,vomiting,
watery diahrrea
Lecture 12 BIOL 533 17
Pathogenesis of Escherichia
? Gastroenteritis,continued
– Enteroinvasive (EIEC)
? Invade and destroy colonic epithelium
– Fever and cramps with blood and leukocytes in stool
– Uncommon; often food-borne
– Enteropathogenic (EPEC; childhood diarrhea)
? Organism adheres to enterocyte plasma
membrane and causes destruction of microvilli
producing watery diarrhea
– Adhesiveness mediated by plasmid-encoded pili
Lecture 12 BIOL 533 18
Pathogenesis of Escherichia
? Gastroenteritis,continued
– Enteropathogenic (continued)
? Infants< 1 year affected
– Enterohemorrhagic (EHEC; hemorrhagic
colitis)
? Produces cytotoxin (verotoxin)
? Severe abdominal pain,bloody diarrhea,little or no
fever
? Warm months of year; affects children < 5 years
Lecture 12 BIOL 533 19
Pathogenesis of Escherichia
? Gastroenteritis,continued
– Enteroaggregative (EaggEC; watery diarrhea)
? Infants < 6 months
? AIDS patients
Lecture 12 BIOL 533 20
Pathogenesis of Different Toxins
? Cholera and ETEC
– Colonize mucosal surface by toxin coregulated
pilus (cholera; TcpA) or colonization factor Ag
(Cfa; E,coli)
– Ctx or LT binds to receptor and taken up by
vesicles; transported from basolateral
membrane to AC complex
Lecture 12 BIOL 533 21
Pathogenesis of Different Toxins
? Cholera and ETEC (continued)
– ADP-ribosylation yields cAMP (cholera-like)
? ETEC (heat stable; ST) binds to membrane-bound
guanylate cyclase complex that produces cGMP
– Both cAMP and c-GMP reduce Na+ absorption
in vilus cells
? Increase CI— secretion in crypt cells; yields watery
diarrhea
Lecture 12 BIOL 533 22
Pathogenesis of Different Toxins
? EPEC
– Attaches to small bowel by bundle forming
pilus (BfpA)
– Binding yields signal transduction events
? Phosphorylation of major epithelial protein Hp-90
? Activation phospholipase C
? Increase inositol triPO4 (IP3) and Ca
? Damage to microvilli
Lecture 12 BIOL 533 23
Pathogenesis of Different Toxins
? EPEC (continued)
– Intimin mediates intimite association
– 39 kDa protein causes polymerization of actin
and other cytoskeletal proteins and
rearrangement of cytoskeletal structure
? Form characteristic EPEC pedestal (attaching
effacing lesion) with intimately attached organism;
not known how host gets diarrhea)
Lecture 12 BIOL 533 24
Pathogenesis of Different Toxins
? Interestingly,E,coli 0:157H:7 has
pedestal and Shiga toxin (char,Shigella)
Lecture 12 BIOL 533 25
Pathogenesis of Salmonella
? Source of most infections
– Ingestion of contaminated water,food
? Poultry,eggs,and dairy products
? Salmonella typhi spread by food or water;
contaminated by food-handlers
? Need to ingest large number of organisms (106-8)
– By fecal-oral contact in children
Lecture 12 BIOL 533 26
Pathogenesis of Salmonella
? Gastroenteritis (most common)
– Symptoms 6-48 hours after ingestion
? Nausea,vomiting,non-bloody diarrhea
? Elevated temperature,abdominal cramps,muscle
cramps,headache
– Symptoms persist for 2 days to a week before
abating
? Antibiotics are normally not employed because
carrier state can develop
Lecture 12 BIOL 533 27
Pathogenesis of Salmonella
? Gastroenteritis (continued)
– More acid-sensitive than Shigella
? Infect patients with decreased stomach acid
– Large inoculum needed
? Decreased by 10-100X in the presence of
bicarbonate
Lecture 12 BIOL 533 28
Pathogenesis of Salmonella
? Septicemia (pediatric and geriatric patients)
– 10% patients can get,
? osteomyelitis,
? endocarditis,or
? arthritis
Lecture 12 BIOL 533 29
Pathogenesis of Salmonella
? Enteric fever
(S,typhi,typhoid; S,paratyphi,paratyphoid)
– Paratyphoid is milder
– Symptoms after 10-14 day incubation period
? Gradually increasing remittant fever
? Headache,muscle aches,malaise,and decreased
appetite; gastrointestinal symptoms occur
? Symptoms persist for a few days
Lecture 12 BIOL 533 30
Pathogenesis of Salmonella
? Enteric fever (continued)
– Carriers (―Typhoid Mary‖)
? 1-5% of patients will carry after a year
? Gall bladder-primary source
Lecture 12 BIOL 533 31
Mechanism of Pathogenesis
? Sense acid environment produces ~40
proteins with importance to pathogenesis
– Organisims escape killing in,small bowel,and
distal illeum of colon
– Penetrate mucosal barrier; not clear whether
involves,
? M cells -OR-
? apical membrane of gut epithelial cells -OR-
? Tight junction between cells
Lecture 12 BIOL 533 32
Mechanism of Pathogenesis
? Sense acid environment (continued)
– Contact of organism’s cells in culture
producing ruffling of plasma membrane
(cytoskeletal rearrangements) lead to uptake
into phagocytic vesicles
– Interaction with epithelial cells activates
inflammatory response yielding damage to
intestinal mucosa
Lecture 12 BIOL 533 33
Mechanism of Pathogenesis
? Interaction with epithelial cells (continued)
– Assembly non-pili appendages (15 minutes)
? S,typhimurium
– 14 genes of inv operon
– In 30 minutes,ruffles appear
? Bacterial appendages disappear
? Assembly mutants,both assembly and
disassembly
– inv A E assemble; never disassemble
– inv C G never assemble
Lecture 12 BIOL 533 34
Mechanism of Pathogenesis
? Biochemical events activated during
invasion
– Activation,mitogen activated
? Protein kinase (MAP kinase)
? Linked to surface receptor
– Binding produces activation
? Phospholipase A2 (PLA2)
– Release arachidonic acid
– Produce prostaglandin leukotrienes
– Increase in intracellular Ca+2
Lecture 12 BIOL 533 35
Mechanism of Pathogenesis
? Biochemical events (continued)
– All these produce ruffling,but also alter
electrolyte transport leading to diarrhea
– Bacteria remain in vesicles for hours
? Resistant to lysosomal contents and antibacterial
peptides made by intestinal epithelial cells
(cryptins)
– Move from vesicles to basement membrane
leading to lamina propria
Lecture 12 BIOL 533 36
Mechanism of Diarrhea
? Exact mechanism of diarrhea unknown
? Invasion produces IL8 that leads to local
leukocyte attraction
? Ability to invade and produce inflammation
necessary,but not sufficient to produce
diarrhea; found by experiments in animals
? Other signal necessary
? Some have cholera toxin-like molecule
Lecture 12 BIOL 533 37
Pathogenesis of S,typhi
? Typhoid Fever
– Survive in macrophage; studied in mice
– Causes typhoid-like illness in mice; diarrhea in
humans
Lecture 12 BIOL 533 38
Pathogenesis of S,typhi
? Virulence regulated signal transduction
(PhoP/PhoQ)
– Mutations
? Decreased survival in macrophage
? Increased sensitivity to acid pH
? Sensitivity to mammalian antimicrobial peptides
? Attenuation of virulence
Lecture 12 BIOL 533 39
Pathogenesis of Salmonella
? Invasive non-typhoidal strains
– Virulence plasmid
? 8 kb conserved Salmonella plasmid virulence genes
(spv)
? Turned on,
– when enter eukaryotic cells
– resistance to complement
Lecture 12 BIOL 533 40
Properties of Shigella
? Species
– Shigella sonnei (industrial countries)
– Shigella flexneri (underdeveloped countries)
? Pediatric disease (1-4 years)
– Associated day-care centers,nursuries,and
custodial institutions
– Spread by fecal-oral route (hands)
– 200 bacilli can establish disease
Lecture 12 BIOL 533 41
Properties of Shigella
? Clinical syndromes (1-3 days after
ingestion)
– Abdominal cramps
– Diarrhea
– Fever
– Bloody stools
Lecture 12 BIOL 533 42
Properties of Shigella
? Pathogenesis
– Colonize small intestine and multiply during first 12
hours
– Initial sign of infection—profuse watery diarrhea
without histological evidence of mucosal invasion
? Mediated by enterotoxin
– Invasion of colonic epithelium results in lower
abdominal cramps,difficulty defecating,abundant pus
and blood in stool
– Bacteremia is uncommon
Lecture 12 BIOL 533 43
Antibiotic Therapy of Shigella
? Antibiotic treatment is recommended to
reduce spread to other contacts
? Fluoroquinolines-adults
– Under 17-damage to cartilage and joints
? Determined by animal studies
? FDA does not allow use in children
? New ?-lactam cephalosporin in use
Lecture 12 BIOL 533 44
Pathogenesis of Shigella
? Survival in stomach
– Sense acid environment
– Sigma factor RNA polymerase (formed in
stationary phase)
Lecture 12 BIOL 533 45
Pathogenesis of Shigella
? Survival in stomach (continued)
– Controls group of genes concerned with acid
resistance; acid resistance increased
? Invasion-ability less
? When reach small intestine,invasion ability returns
and acid resistance repressed
? Acid resistance enhanced by anaerobic conditions
found in large intestine
? Likely when excreted,acid-resistance is expressed;
ready for next host
Lecture 12 BIOL 533 46
Large Intestine Invasion
? Bacterial multiplication occurs inside
intestinal epithelial cell
? Invasion and survival
– multiple genes both on chromosome and
plasmid (large virulence)
Lecture 12 BIOL 533 47
Large Intestine Invasion
? Invasion steps
– Get close to mucosal surface (unknown
mechanism); no flagella; non-motile; cells
can’t be invaded on luminal surface,but can
be on basal
? First enter M cells (Ag sampling cells)
– Depends on plasmid coded outer membrane proteins
? Invasion plasmid Ag; IpaB C D
Lecture 12 BIOL 533 48
Large Intestine Invasion
? Invasion steps (continued)
– Released into lamina propria (intercellular
space)
? ingested by macrophage
– They release IL1 that produces inflammatory response;
increase subsequent invasion close to basal surface
– Entry of Shigella into mucosal epithelial cells;
rearrangement of actin cytoskeletal elements
Lecture 12 BIOL 533 49
Large Intestine Invasion
? Invasion steps (continued)
– Go from phagosome into cytoplasm and
mutiplies
– How do they infect other cells? As they
multiply,they make protein IcsA that causes
intracellular spread of 1 pole of rod; ATPase
causes polymerization of actin (host)
Lecture 12 BIOL 533 50
Large Intestine Invasion
? Deposition of actin propels bacteria
forward
– Fingerlike projection pokes adjacent cell
– Surrounded by a combination of old and new
membrane; produces lysis and entry of
organism into cytoplasm of new cell
Lecture 12 BIOL 533 51
S,dysenteriae Pathogenesis
? S,dysenterieae type 1 also possesses,
– shiga toxin
– cytotoxin-kills intestinal epithelial and
endothelial cells
Lecture 12 BIOL 533 52
S,dysenteriae Pathogenesis
? Shiga toxin
– Irreversibly inactivates mammlian 60 S
ribosomal SU; stops protein synthesis
– Mechanism,
? Targets sodium absorptive villus cell; produces
decrease in Na+ absorption; more fluid
accumulates in lumen
? Affects toxin mucosal epithelial cells yielding
bloody diarrhea
Lecture 12 BIOL 533 53
S,dysenteriae Pathogenesis
? Infections in monkeys
– Strains with inactivated Shiga toxin gene
cause disease with much less damage to
mucosa and less bleeding; therefore,both
invasion and toxin formation are important
– S,dysenteriae type 1 most severe
– S,flexneri causes severe illness with
dysentery and bloody diarrhea; has no genes
for Shiga toxin
Lecture 12 BIOL 533 54
S,dysenteriae Pathogenesis
? Interestingly,S,sonnei has same invasion
process as other two,but with no
dysentery,only watery diarrhea
? Reason for difference between type 1 and
others may be difference in intensity of
inflammatory response
Lecture 12 BIOL 533 55
Pathogenesis of
Yersinia pestis
? Clincal syndromes
– Bubonic plague (incubation period-7 days
after bite from infected flea)
? High fever and inflammation of lymph nodes in
groin or armpit
? Absence of treatment—bacteremia (75% die)
Lecture 12 BIOL 533 56
Pathogenesis of
Yersinia pestis
? Clincal syndromes
– Pneumonic plague (incubation 2-3 days)
? Have fever and malaise
? Develop pulmonary symptoms within 1 day
? Untreated >90% die
Lecture 12 BIOL 533 57
Pathogenesis of
Yersinia enterocolitica
? Associated with
– Contaminated meat or milk
– Colder climates during winter months
Lecture 12 BIOL 533 58
Pathogenesis of
Yersinia enterocolitica
? Gastroenteritis
– Diarrhea,fever,abdominal pain
? Lasting for 1-2 weeks
? Chronic form can persist for months or year
– Can mimic appendicitis,particularly in children
– Adults can have septicemia,arthritis,
intrabdominal abscess,hepatitis,and
osteomyelitis
Lecture 12 BIOL 533 59
Pathogenesis of
Klebsiella pneumoniae
? Associated pneumonia
– Frequently associated with
? Necrotic destruction of alveolar spaces
? Production of blood-tinged sputum
– Can also cause wound,soft tissue,and
urinary tract infections
Lecture 12 BIOL 533 60
Pathogenesis of
Proteus mirabilis
? Urinary tract infections
? Produce large amounts urease
– Urea into carbon dioxide and ammonia
? Changes renal pH
– Facilitates formation of stones
– Also toxic for uroepithelium
? Presence of pili may decrease virulence
– Better phagocytosis
Lecture 12 BIOL 533 61
Prevention and Control
? Difficult,because enterobacteria are
normal flora
? Prevention
– Plague
? Effective vaccines
? Prophylactic use of tetracycline for medical
workers in contact with pneumonic plague
– Vaccines for Salmonella typhi
Lecture 12 BIOL 533 62
Prevention and Control
? Treatment
– Use of antibiotic susceptibility testing
– E,coli and Proteus normally respond well to
antibiotic treatment
Lecture 12 BIOL 533 63
Lecture 12
? Questions?
? Comments?
? Assignments..,
Enterobacteriaceae
BIOL 533
Lecture 12
Medical Microbiology
Lecture 12 BIOL 533 2
Enterobacteriaceae
? Diversity of species
? Ecology
– Found worldwide in soil,water,vegetation,
and microbial flora of animals and humans
– Some are always associated with disease
? e.g.,Shigella,Salmonella,Yersinia pestis
– Some are normal flora that can become
opportunistic pathogens
? e.g.,E,coli,K,pneumoniae,P,mirabilis
Lecture 12 BIOL 533 3
Enterobacteriaceae
? Epidemiology
– Animal reservoir,most Salmonella infections
– Human carrier,Salmonella typhi,Shigella
– Endogenous spread in a susceptible patient
? Can involve all body sites
? 5% hospitalized patients develop nosocomial
infections,primarily caused by Enterobacteriaceae
such as Escherichia
? Sites of infection
Lecture 12 BIOL 533 4
Microbial Physiology
and Structure
? Cell morphology
– Moderate-sized Gram— rods
? Non-spore-forming
? Motile (with peritrichous flagella) or non-motile
– Physiology
? All are facultative anaerobes
? Simple nutritional requirements,
– Ferment glucose
– Reduce nitrates to nitrites
Lecture 12 BIOL 533 5
Distinguishing Characteristics
? Oxidaseˉ,
– Distinguishes among other fermentative and
non-fermentative Gram— bacilli
? Lactose fermentation (red colonies on
MacConkey agar)
– Separate Escherichia,Klebsiella,Enterobacter
from other lactose— Enterobacteriaceae
Lecture 12 BIOL 533 6
Distinguishing Characteristics
? Resistance to bile salts
– Separate Shigella and Salmonella from normal
flora in this group
? Eosin Methylene Blue (EMB)
– Lactose,eosinY,methylene blue; Lac+; grow
with green sheen
Lecture 12 BIOL 533 7
Virulence Factors
? Antigens
– Somatic ―O‖ LPS
? Major cell wall Ag; heat stable
? Specific ―O‖ antigens associated with each genus;
however,cross reactions are common
– Salmonella and Citrobacter
– Escherichia and Shigella
Lecture 12 BIOL 533 8
Virulence Factors
? Capsular K
– Either protein or polysaccharide
? Heat-labile
? May interfere with detection of ―O‖
? Removed by boiling organisms
– Capsular antigen of Salmonella typhi referred
to as Vi antigen
Lecture 12 BIOL 533 9
Virulence Factors
? Capsular K,continued
– Shared by different genera both inside and
outside of family
? Cross reactions
– E,coli K1 with N,meningitidis and Haemophilus
meningitidis
– K,pneumoniae with S,pneunomiae
– Organisms with specific antigens have been
associated with increased virulence
(e.g.,E,coli K1 with neonatal meningitis)
Lecture 12 BIOL 533 10
Virulence Factors
? Flagella H
– Heat-labile proteins
? Can be absent or undergo antigenic variation
(present in two phases)
? Specific H antigens assocated with disease
Lecture 12 BIOL 533 11
Virulence Factors
? General role in pathogenesis of ―O,‖ ―K,‖
and ―H‖ antigens
– Specific antigens associated with meningitis,
gastroenteritis,and urinary tract infections
? Role that Ag’s play in these diseases is not clear
– Some capsular Ag are poor immunogens
? Protect against antibody-mediated phagocytosis
– Flagellar Ag probably play a role in adherence
Lecture 12 BIOL 533 12
Virulence Factors
? Pili
– Attachment to host cells
Lecture 12 BIOL 533 13
Pathogenesis of Escherichia
? E,coli present in gastrointestinal tract in
normal flora
? Bacterial sepsis (multiplication in blood)
– Primary focus-infection of urinary tract or
spread from gastrointestinal tract
– Death can occur in immunocompromised
patients and infections resulting from
intestinal perforation
Lecture 12 BIOL 533 14
Pathogenesis of Escherichia
? Neonatal meningitis
– E,coli and group B streptococci most common
– 75% E,coli possess Capsular K1 antigen
– Colonization of infants with E,coli at delivery
is common; disease is not
Lecture 12 BIOL 533 15
Pathogenesis of Escherichia
? Urinary tract infections
(80% community and most nosocomial)
– Originate from gastrointestinal tract
– Important virulence factors
? Resistance to serum-killing
? Production of hemolysins
? Pili-mediated binding (not demonstrated in vivo)
? Production of slime layer that participates in cell
adhesion
Lecture 12 BIOL 533 16
Pathogenesis of Escherichia
? Gastroenteritis
(countries with poor hygiene)
– Enterotoxigenic (ETEC)
? Mediated by heat-labile (like cholera) and heat-
stable exotoxins (activates guanylate cyclase and
stimulates secretion of fluid)
– Both are coded from plasmid-borne genes
? World-wide:both adults and children
– Incubation 1-2 days; persists 3-4 days
– Mild symptoms,including cramps,nausea,vomiting,
watery diahrrea
Lecture 12 BIOL 533 17
Pathogenesis of Escherichia
? Gastroenteritis,continued
– Enteroinvasive (EIEC)
? Invade and destroy colonic epithelium
– Fever and cramps with blood and leukocytes in stool
– Uncommon; often food-borne
– Enteropathogenic (EPEC; childhood diarrhea)
? Organism adheres to enterocyte plasma
membrane and causes destruction of microvilli
producing watery diarrhea
– Adhesiveness mediated by plasmid-encoded pili
Lecture 12 BIOL 533 18
Pathogenesis of Escherichia
? Gastroenteritis,continued
– Enteropathogenic (continued)
? Infants< 1 year affected
– Enterohemorrhagic (EHEC; hemorrhagic
colitis)
? Produces cytotoxin (verotoxin)
? Severe abdominal pain,bloody diarrhea,little or no
fever
? Warm months of year; affects children < 5 years
Lecture 12 BIOL 533 19
Pathogenesis of Escherichia
? Gastroenteritis,continued
– Enteroaggregative (EaggEC; watery diarrhea)
? Infants < 6 months
? AIDS patients
Lecture 12 BIOL 533 20
Pathogenesis of Different Toxins
? Cholera and ETEC
– Colonize mucosal surface by toxin coregulated
pilus (cholera; TcpA) or colonization factor Ag
(Cfa; E,coli)
– Ctx or LT binds to receptor and taken up by
vesicles; transported from basolateral
membrane to AC complex
Lecture 12 BIOL 533 21
Pathogenesis of Different Toxins
? Cholera and ETEC (continued)
– ADP-ribosylation yields cAMP (cholera-like)
? ETEC (heat stable; ST) binds to membrane-bound
guanylate cyclase complex that produces cGMP
– Both cAMP and c-GMP reduce Na+ absorption
in vilus cells
? Increase CI— secretion in crypt cells; yields watery
diarrhea
Lecture 12 BIOL 533 22
Pathogenesis of Different Toxins
? EPEC
– Attaches to small bowel by bundle forming
pilus (BfpA)
– Binding yields signal transduction events
? Phosphorylation of major epithelial protein Hp-90
? Activation phospholipase C
? Increase inositol triPO4 (IP3) and Ca
? Damage to microvilli
Lecture 12 BIOL 533 23
Pathogenesis of Different Toxins
? EPEC (continued)
– Intimin mediates intimite association
– 39 kDa protein causes polymerization of actin
and other cytoskeletal proteins and
rearrangement of cytoskeletal structure
? Form characteristic EPEC pedestal (attaching
effacing lesion) with intimately attached organism;
not known how host gets diarrhea)
Lecture 12 BIOL 533 24
Pathogenesis of Different Toxins
? Interestingly,E,coli 0:157H:7 has
pedestal and Shiga toxin (char,Shigella)
Lecture 12 BIOL 533 25
Pathogenesis of Salmonella
? Source of most infections
– Ingestion of contaminated water,food
? Poultry,eggs,and dairy products
? Salmonella typhi spread by food or water;
contaminated by food-handlers
? Need to ingest large number of organisms (106-8)
– By fecal-oral contact in children
Lecture 12 BIOL 533 26
Pathogenesis of Salmonella
? Gastroenteritis (most common)
– Symptoms 6-48 hours after ingestion
? Nausea,vomiting,non-bloody diarrhea
? Elevated temperature,abdominal cramps,muscle
cramps,headache
– Symptoms persist for 2 days to a week before
abating
? Antibiotics are normally not employed because
carrier state can develop
Lecture 12 BIOL 533 27
Pathogenesis of Salmonella
? Gastroenteritis (continued)
– More acid-sensitive than Shigella
? Infect patients with decreased stomach acid
– Large inoculum needed
? Decreased by 10-100X in the presence of
bicarbonate
Lecture 12 BIOL 533 28
Pathogenesis of Salmonella
? Septicemia (pediatric and geriatric patients)
– 10% patients can get,
? osteomyelitis,
? endocarditis,or
? arthritis
Lecture 12 BIOL 533 29
Pathogenesis of Salmonella
? Enteric fever
(S,typhi,typhoid; S,paratyphi,paratyphoid)
– Paratyphoid is milder
– Symptoms after 10-14 day incubation period
? Gradually increasing remittant fever
? Headache,muscle aches,malaise,and decreased
appetite; gastrointestinal symptoms occur
? Symptoms persist for a few days
Lecture 12 BIOL 533 30
Pathogenesis of Salmonella
? Enteric fever (continued)
– Carriers (―Typhoid Mary‖)
? 1-5% of patients will carry after a year
? Gall bladder-primary source
Lecture 12 BIOL 533 31
Mechanism of Pathogenesis
? Sense acid environment produces ~40
proteins with importance to pathogenesis
– Organisims escape killing in,small bowel,and
distal illeum of colon
– Penetrate mucosal barrier; not clear whether
involves,
? M cells -OR-
? apical membrane of gut epithelial cells -OR-
? Tight junction between cells
Lecture 12 BIOL 533 32
Mechanism of Pathogenesis
? Sense acid environment (continued)
– Contact of organism’s cells in culture
producing ruffling of plasma membrane
(cytoskeletal rearrangements) lead to uptake
into phagocytic vesicles
– Interaction with epithelial cells activates
inflammatory response yielding damage to
intestinal mucosa
Lecture 12 BIOL 533 33
Mechanism of Pathogenesis
? Interaction with epithelial cells (continued)
– Assembly non-pili appendages (15 minutes)
? S,typhimurium
– 14 genes of inv operon
– In 30 minutes,ruffles appear
? Bacterial appendages disappear
? Assembly mutants,both assembly and
disassembly
– inv A E assemble; never disassemble
– inv C G never assemble
Lecture 12 BIOL 533 34
Mechanism of Pathogenesis
? Biochemical events activated during
invasion
– Activation,mitogen activated
? Protein kinase (MAP kinase)
? Linked to surface receptor
– Binding produces activation
? Phospholipase A2 (PLA2)
– Release arachidonic acid
– Produce prostaglandin leukotrienes
– Increase in intracellular Ca+2
Lecture 12 BIOL 533 35
Mechanism of Pathogenesis
? Biochemical events (continued)
– All these produce ruffling,but also alter
electrolyte transport leading to diarrhea
– Bacteria remain in vesicles for hours
? Resistant to lysosomal contents and antibacterial
peptides made by intestinal epithelial cells
(cryptins)
– Move from vesicles to basement membrane
leading to lamina propria
Lecture 12 BIOL 533 36
Mechanism of Diarrhea
? Exact mechanism of diarrhea unknown
? Invasion produces IL8 that leads to local
leukocyte attraction
? Ability to invade and produce inflammation
necessary,but not sufficient to produce
diarrhea; found by experiments in animals
? Other signal necessary
? Some have cholera toxin-like molecule
Lecture 12 BIOL 533 37
Pathogenesis of S,typhi
? Typhoid Fever
– Survive in macrophage; studied in mice
– Causes typhoid-like illness in mice; diarrhea in
humans
Lecture 12 BIOL 533 38
Pathogenesis of S,typhi
? Virulence regulated signal transduction
(PhoP/PhoQ)
– Mutations
? Decreased survival in macrophage
? Increased sensitivity to acid pH
? Sensitivity to mammalian antimicrobial peptides
? Attenuation of virulence
Lecture 12 BIOL 533 39
Pathogenesis of Salmonella
? Invasive non-typhoidal strains
– Virulence plasmid
? 8 kb conserved Salmonella plasmid virulence genes
(spv)
? Turned on,
– when enter eukaryotic cells
– resistance to complement
Lecture 12 BIOL 533 40
Properties of Shigella
? Species
– Shigella sonnei (industrial countries)
– Shigella flexneri (underdeveloped countries)
? Pediatric disease (1-4 years)
– Associated day-care centers,nursuries,and
custodial institutions
– Spread by fecal-oral route (hands)
– 200 bacilli can establish disease
Lecture 12 BIOL 533 41
Properties of Shigella
? Clinical syndromes (1-3 days after
ingestion)
– Abdominal cramps
– Diarrhea
– Fever
– Bloody stools
Lecture 12 BIOL 533 42
Properties of Shigella
? Pathogenesis
– Colonize small intestine and multiply during first 12
hours
– Initial sign of infection—profuse watery diarrhea
without histological evidence of mucosal invasion
? Mediated by enterotoxin
– Invasion of colonic epithelium results in lower
abdominal cramps,difficulty defecating,abundant pus
and blood in stool
– Bacteremia is uncommon
Lecture 12 BIOL 533 43
Antibiotic Therapy of Shigella
? Antibiotic treatment is recommended to
reduce spread to other contacts
? Fluoroquinolines-adults
– Under 17-damage to cartilage and joints
? Determined by animal studies
? FDA does not allow use in children
? New ?-lactam cephalosporin in use
Lecture 12 BIOL 533 44
Pathogenesis of Shigella
? Survival in stomach
– Sense acid environment
– Sigma factor RNA polymerase (formed in
stationary phase)
Lecture 12 BIOL 533 45
Pathogenesis of Shigella
? Survival in stomach (continued)
– Controls group of genes concerned with acid
resistance; acid resistance increased
? Invasion-ability less
? When reach small intestine,invasion ability returns
and acid resistance repressed
? Acid resistance enhanced by anaerobic conditions
found in large intestine
? Likely when excreted,acid-resistance is expressed;
ready for next host
Lecture 12 BIOL 533 46
Large Intestine Invasion
? Bacterial multiplication occurs inside
intestinal epithelial cell
? Invasion and survival
– multiple genes both on chromosome and
plasmid (large virulence)
Lecture 12 BIOL 533 47
Large Intestine Invasion
? Invasion steps
– Get close to mucosal surface (unknown
mechanism); no flagella; non-motile; cells
can’t be invaded on luminal surface,but can
be on basal
? First enter M cells (Ag sampling cells)
– Depends on plasmid coded outer membrane proteins
? Invasion plasmid Ag; IpaB C D
Lecture 12 BIOL 533 48
Large Intestine Invasion
? Invasion steps (continued)
– Released into lamina propria (intercellular
space)
? ingested by macrophage
– They release IL1 that produces inflammatory response;
increase subsequent invasion close to basal surface
– Entry of Shigella into mucosal epithelial cells;
rearrangement of actin cytoskeletal elements
Lecture 12 BIOL 533 49
Large Intestine Invasion
? Invasion steps (continued)
– Go from phagosome into cytoplasm and
mutiplies
– How do they infect other cells? As they
multiply,they make protein IcsA that causes
intracellular spread of 1 pole of rod; ATPase
causes polymerization of actin (host)
Lecture 12 BIOL 533 50
Large Intestine Invasion
? Deposition of actin propels bacteria
forward
– Fingerlike projection pokes adjacent cell
– Surrounded by a combination of old and new
membrane; produces lysis and entry of
organism into cytoplasm of new cell
Lecture 12 BIOL 533 51
S,dysenteriae Pathogenesis
? S,dysenterieae type 1 also possesses,
– shiga toxin
– cytotoxin-kills intestinal epithelial and
endothelial cells
Lecture 12 BIOL 533 52
S,dysenteriae Pathogenesis
? Shiga toxin
– Irreversibly inactivates mammlian 60 S
ribosomal SU; stops protein synthesis
– Mechanism,
? Targets sodium absorptive villus cell; produces
decrease in Na+ absorption; more fluid
accumulates in lumen
? Affects toxin mucosal epithelial cells yielding
bloody diarrhea
Lecture 12 BIOL 533 53
S,dysenteriae Pathogenesis
? Infections in monkeys
– Strains with inactivated Shiga toxin gene
cause disease with much less damage to
mucosa and less bleeding; therefore,both
invasion and toxin formation are important
– S,dysenteriae type 1 most severe
– S,flexneri causes severe illness with
dysentery and bloody diarrhea; has no genes
for Shiga toxin
Lecture 12 BIOL 533 54
S,dysenteriae Pathogenesis
? Interestingly,S,sonnei has same invasion
process as other two,but with no
dysentery,only watery diarrhea
? Reason for difference between type 1 and
others may be difference in intensity of
inflammatory response
Lecture 12 BIOL 533 55
Pathogenesis of
Yersinia pestis
? Clincal syndromes
– Bubonic plague (incubation period-7 days
after bite from infected flea)
? High fever and inflammation of lymph nodes in
groin or armpit
? Absence of treatment—bacteremia (75% die)
Lecture 12 BIOL 533 56
Pathogenesis of
Yersinia pestis
? Clincal syndromes
– Pneumonic plague (incubation 2-3 days)
? Have fever and malaise
? Develop pulmonary symptoms within 1 day
? Untreated >90% die
Lecture 12 BIOL 533 57
Pathogenesis of
Yersinia enterocolitica
? Associated with
– Contaminated meat or milk
– Colder climates during winter months
Lecture 12 BIOL 533 58
Pathogenesis of
Yersinia enterocolitica
? Gastroenteritis
– Diarrhea,fever,abdominal pain
? Lasting for 1-2 weeks
? Chronic form can persist for months or year
– Can mimic appendicitis,particularly in children
– Adults can have septicemia,arthritis,
intrabdominal abscess,hepatitis,and
osteomyelitis
Lecture 12 BIOL 533 59
Pathogenesis of
Klebsiella pneumoniae
? Associated pneumonia
– Frequently associated with
? Necrotic destruction of alveolar spaces
? Production of blood-tinged sputum
– Can also cause wound,soft tissue,and
urinary tract infections
Lecture 12 BIOL 533 60
Pathogenesis of
Proteus mirabilis
? Urinary tract infections
? Produce large amounts urease
– Urea into carbon dioxide and ammonia
? Changes renal pH
– Facilitates formation of stones
– Also toxic for uroepithelium
? Presence of pili may decrease virulence
– Better phagocytosis
Lecture 12 BIOL 533 61
Prevention and Control
? Difficult,because enterobacteria are
normal flora
? Prevention
– Plague
? Effective vaccines
? Prophylactic use of tetracycline for medical
workers in contact with pneumonic plague
– Vaccines for Salmonella typhi
Lecture 12 BIOL 533 62
Prevention and Control
? Treatment
– Use of antibiotic susceptibility testing
– E,coli and Proteus normally respond well to
antibiotic treatment
Lecture 12 BIOL 533 63
Lecture 12
? Questions?
? Comments?
? Assignments..,
