Enterobacteriaceae

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Enterobacteriaceae
Medical Microbiology

• BIOL 533
• Lecture 12

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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

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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

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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

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Distinguishing Characteristics

• Oxidase
• Distinguishes among other fermentative and
  non-fermentative Gram bacilli
• Lactose fermentation (red colonies on MacConkey
  agar)
• Separate Escherichia, Klebsiella, Enterobacter
  from other lactose Enterobacteriaceae

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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

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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

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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

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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)

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Virulence Factors

• Flagella H
• Heat-labile proteins
• Can be absent or undergo antigenic variation
  (present in two phases)
• Specific H antigens assocated with disease

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Virulence Factors

• General role in pathogenesis of O, K, and H
  antigens
• Specific antigens associated with meningitis,
  gastroenteritis, and urinary tract infections
• Role that Ags 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

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Virulence Factors

• Pili
• Attachment to host cells

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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

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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

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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

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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-wideboth adults and children
• Incubation 1-2 days persists 3-4 days
• Mild symptoms, including cramps, nausea,
  vomiting, watery diahrrea

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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

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Pathogenesis of Escherichia

• Gastroenteritis, continued
• Enteropathogenic (continued)
• Infantslt 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 lt 5 years

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Pathogenesis of Escherichia

• Gastroenteritis, continued
• Enteroaggregative (EaggEC watery diarrhea)
• Infants lt 6 months
• AIDS patients

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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

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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

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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

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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)

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Pathogenesis of Different Toxins

• Interestingly, E. coli 0157H7 has pedestal and
  Shiga toxin (char. Shigella)

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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

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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

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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

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Pathogenesis of Salmonella

• Septicemia (pediatric and geriatric patients)
• 10 patients can get
• osteomyelitis,
• endocarditis, or
• arthritis

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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

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Pathogenesis of Salmonella

• Enteric fever (continued)
• Carriers (Typhoid Mary)
• 1-5 of patients will carry after a year
• Gall bladder-primary source

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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

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Mechanism of Pathogenesis

• Sense acid environment (continued)
• Contact of organisms 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

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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

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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 Ca2

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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

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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

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Pathogenesis of S. typhi

• Typhoid Fever
• Survive in macrophage studied in mice
• Causes typhoid-like illness in mice diarrhea in
  humans

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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

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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

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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

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Properties of Shigella

• Clinical syndromes (1-3 days after ingestion)
• Abdominal cramps
• Diarrhea
• Fever
• Bloody stools

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Properties of Shigella

• Pathogenesis
• Colonize small intestine and multiply during
  first 12 hours
• Initial sign of infectionprofuse 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

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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

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Pathogenesis of Shigella

• Survival in stomach
• Sense acid environment
• Sigma factor RNA polymerase (formed in stationary
  phase)

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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

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Large Intestine Invasion

• Bacterial multiplication occurs inside intestinal
  epithelial cell
• Invasion and survival
• multiple genes both on chromosome and plasmid
  (large virulence)

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Large Intestine Invasion

• Invasion steps
• Get close to mucosal surface (unknown mechanism)
  no flagella non-motile cells cant 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

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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

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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)

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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

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S. dysenteriae Pathogenesis

• S. dysenterieae type 1 also possesses
• shiga toxin
• cytotoxin-kills intestinal epithelial and
  endothelial cells

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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

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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

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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

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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 treatmentbacteremia (75 die)

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Pathogenesis of Yersinia pestis

• Clincal syndromes
• Pneumonic plague (incubation 2-3 days)
• Have fever and malaise
• Develop pulmonary symptoms within 1 day
• Untreated gt90 die

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Pathogenesis of Yersinia enterocolitica

• Associated with
• Contaminated meat or milk
• Colder climates during winter months

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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

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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

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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

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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

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Prevention and Control

• Treatment
• Use of antibiotic susceptibility testing
• E. coli and Proteus normally respond well to
  antibiotic treatment

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Lecture 12

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