Enterobacteriaceae

1. Medical Microbiology Enterobacteriaceae 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 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

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-wide:both 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) • 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

19. Pathogenesis of Escherichia • Gastroenteritis, continued • Enteroaggregative (EaggEC; watery diarrhea) • Infants < 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 0:157H:7 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 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

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 Ca+2

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 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

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 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

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