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Enterobacteriaceae

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Title: Enterobacteriaceae


1
Enterobacteriaceae
Medical Microbiology
  • BIOL 533
  • Lecture 12

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

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

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

5
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

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

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

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

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)

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

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 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

12
Virulence Factors
  • Pili
  • Attachment to host cells

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

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

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

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

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

18
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

19
Pathogenesis of Escherichia
  • Gastroenteritis, continued
  • Enteroaggregative (EaggEC watery diarrhea)
  • Infants lt 6 months
  • AIDS patients

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

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

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

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)

24
Pathogenesis of Different Toxins
  • Interestingly, E. coli 0157H7 has pedestal and
    Shiga toxin (char. Shigella)

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

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

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

28
Pathogenesis of Salmonella
  • Septicemia (pediatric and geriatric patients)
  • 10 patients can get
  • osteomyelitis,
  • endocarditis, or
  • arthritis

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

30
Pathogenesis of Salmonella
  • Enteric fever (continued)
  • Carriers (Typhoid Mary)
  • 1-5 of patients will carry after a year
  • Gall bladder-primary source

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

32
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

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

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 Ca2

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

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

37
Pathogenesis of S. typhi
  • Typhoid Fever
  • Survive in macrophage studied in mice
  • Causes typhoid-like illness in mice diarrhea in
    humans

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

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

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

41
Properties of Shigella
  • Clinical syndromes (1-3 days after ingestion)
  • Abdominal cramps
  • Diarrhea
  • Fever
  • Bloody stools

42
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

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

44
Pathogenesis of Shigella
  • Survival in stomach
  • Sense acid environment
  • Sigma factor RNA polymerase (formed in stationary
    phase)

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

46
Large Intestine Invasion
  • Bacterial multiplication occurs inside intestinal
    epithelial cell
  • Invasion and survival
  • multiple genes both on chromosome and plasmid
    (large virulence)

47
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

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

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)

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

51
S. dysenteriae Pathogenesis
  • S. dysenterieae type 1 also possesses
  • shiga toxin
  • cytotoxin-kills intestinal epithelial and
    endothelial cells

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

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

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

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 treatmentbacteremia (75 die)

56
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

57
Pathogenesis of Yersinia enterocolitica
  • Associated with
  • Contaminated meat or milk
  • Colder climates during winter months

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

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

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

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

62
Prevention and Control
  • Treatment
  • Use of antibiotic susceptibility testing
  • E. coli and Proteus normally respond well to
    antibiotic treatment

63
Lecture 12
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