Diarrheogenic E.coli

toxins produced by E. coli and mechanism by which E.coli produce diarrhoea.

1. Toxins produced by E. coli and Mechanisms by which e.coli Produce diarrhea PRESENTED BY : DR. KUMAR VIKRAM MD (MICROBIOLOGY) IGIMS, PATNA

2. HISTORY : Escherich’s legacy to Science

3. GENUS AND SPECIES DEFINITION

4. E. Coli : The Good

5. E.coli : The Enemy within

6. Common themes in E. Coli virulence Like most mucosal pathogens, E. coli can be said to follow a requisite strategy of infection: ( i ) colonization of a mucosal site, (ii) evasion of host defenses , (iii) multiplication, and (iv) host damage. The most highly conserved feature of diarrheagenic E. coli strains is their ability to colonize the intestinal mucosal surface despite peristalsis and competition for nutrients by the indigenous flora of the gut. Diarrheagenic E. coli strains possess specific fimbrial antigens that enhance their intestinal colonizing ability and allow adherence to the small bowel mucosa.

7. Once colonization is established, E. Coli may cause diarrhea by three general paradigms: ( i ) enterotoxin production (ETEC and EAEC), (ii) invasion (EIEC), and/or (iii) intimate adherence with membrane signalling (EPEC and EHEC).

8. First recognized in the late 1960s as a cause of cholera-like diarrhoea in india. Resemble vibrio cholerae in that they induce profuse,watery diarrhea by elaboration of toxins that act on the mucosal cells. Adhere to the small intestinal mucosa, but do not invade . Enterotoxigenic E. coli

9. no apparent histological changes and little inflammation. a common cause of dehydrating diarrhea in children in developing countries , particularly when they are weaned. considered to be the leading cause of travelers’ diarrhea accounting upto 75% of these cases

10. VIRULENCE FACTORS

11. Oligomeric toxins that are closely related in structure and function to the cholera enterotoxin (CT) expressed by vibrio cholerae . Heat-labile toxin ( lt ) is inactivated by incubation at 100°c for 30 min. 2 major serogroups : LT I LT II Heat Labile Toxin

12. LT-I is expressed by E. coli strains that are pathogenic for both humans and animals. Composed of : One A subunit The A1 domain constitutes the active toxin and is linked to the A2 domain via a disulfide bond The A2 fragment is the helical portion of the molecule and anchors the A subunit to the B pentamer Five B subunits Bind to ganglioside GM 1 receptor. LT - I

13. MECHANISM OF ACTION

14. Mechanism of Action

15. OTHER MECHANISMS Prostaglandins (PGE1 and PGE2): S timulate electrolyte transport and intestinal motility. Enteric nervous system : Serotonin and vasoactive intestinal polypeptide, both of which can stimulate intestinal epithelial cell secretion via the ENS, are released into the human small bowel after treatment with CT. Intestinal inflammatory response :CT has been reported to stimulate production of the proinflammatory cytokine interleukin-6 (IL-6), thereby activating the enteric immune system and potentially generating arachidonic acid metabolites that stimulate secretion.

16. 55 to 57% identity to LT-I and CT in the A subunit. no homology to LT-I or CT in the B subunits. LT-II increases intracellular cAMP levels by similar mechanisms to those involved with LT-I toxicity. But LT-II uses GD1 as its receptor rather than GM1 There is no evidence that LT-II is associated with human or animal disease. LT - II

17. Small, monomeric toxins that contain multiple cysteine residues, whose disulfide bonds account for the heat stability of these toxins. 2 classes: ST I (ST a) – soluble in methanol ST Ip (porcine) ST Ih (human) ST II (ST b) – insoluble in methanol Heat stable Toxins

18. ST - I

19. Colonization factors A heterogenous group of proteinacious surface structures Fimbrial , non- fimbrial or fibrillar . The more recent nomenclature refer to these structures as coli surface (CS) antigen. At least 21 colonization factor antigens and coli surface antigens specific for humans have been defined. CFA/I and CFA/ II are the most commonly found adhesins in human ETEC.

20. CFAs Of Human ETEC strains

21. Vero cytotoxin -producing E.coli(VTEC)

22. Vero cytotoxin -producing E.coli (VTEC) Described first by Konowalchuk in 1977. In 1983, one particular serotype of E. Coli (O157:H7) was identified as the causative agent involved in two outbreaks of a distinctive bloody diarrheal syndrome (Riley et al. 1983). Since then, these organisms have received much attention as a cause of epidemic or sporadic bloody and non-bloody diarrhea, HUS , and thrombotic thrombocytopenic purpura .

23. Also called as shiga toxin producing E. Coli(STEC). - Because toxins are closely related to shiga toxin and that have a cytotoxic effect on vero cells, The term enterohaemorrhagic E. Coli (EHEC) is applied to those STEC serotypes that have the same clinical, epidemiological and pathogenetic features associated with the prototype strain E. Coli O157:H7.

24. Virulence factors

25. Vero cytotoxin Encoded on Stx bacteriophage Originally discovered in Shigella dysenteriae (Stx1-like) Multiple variants-VT1, VT2 (VT2c, d, e, f, g) AB-5 toxin (5 B components and one A component)

26. Vero cytotoxin

27. Enterohemolysin Found in nearly all O157:H7 strains and is widely distributed among non-O157 VT-producing E. coli strains. Two other genetically distinct phage encoded hemolysins , called Ehly1 and Ehly2 , have been reported to be produced by many VT-producing E. coli strains. There are no data to suggest in vivo expression or any role in pathogenesis for these hemolysins .

28. EPEC is an important category of diarrheagenic E. Coli which has been linked to infant diarrhea in the developing world. Most EPEC infections occur in the first 3 years of life. EPEC infections show a marked seasonality and are associated with warm season peaks. Enteropathogenic E. Coli

29. Pathogenesis Attaching-and-effacing histopathology hallmark of infections due to EPEC characterized by effacement of microvilli and intimate adherence between the bacterium and the epithelial cell membrane. Marked cytoskeletal changes, including accumulation of polymerized actin , are seen directly beneath the adherent bacteria; the bacteria sometimes sit upon a pedestal-like structure

30. Pathogenesis In 1992, Donnenberg and Kaper proposed a three-stage model. In 1998 Knutton et al. proposed a four-stage model.

31. Have the capacity to invade interstitial epithelial cells in vivo. EIEC strains are biochemically, genetically, and pathogenetically related closely to Shigella spp. Like Shigella spp., EIEC strains are generally lysine decarboxylase negative, nonmotile , and lactose negative. Except Lower acid resistant Inability to produce shiga toxin Presentation: Majority - watery diarrhea . Occasionally - Dysentery syndrome, manifested as blood, mucus, and leukocytes in the stool; tenesmus ; and fever Enteroinvasive E. coli

32. Pathogenesis Acquisition of the invasive plasmid ( pINV ) encodes the ability to invade host tissues. Comprises ( i ) epithelial cell penetration, (ii) lysis of the endocytic vacuole, (iii) intracellular multiplication, (iv) directional movement through the cytoplasm, (v) extension into adjacent epithelial cells

33. Cellular pathogenesis

34. Sereny test Instillation of a suspension of freshly isolated EIEC or Shigella into the eyes of guinea pigs Mucopurulent conjunctivitis

35. Enteroaggregative E. coli Named so because they appear aggregated in a stacked brick formation on Hep – 2 cells. second most common cause of travelers’ diarrhea after ETEC in both developed and developing countries. commonly being recognized as a cause of endemic and epidemic diarrhea worldwide. associated with persistent diarrhea .

36. Pathogenesis aggregative adherence fimbriae (AAF) Plamid encoded Enteroaggregative heat stable toxin (EAST) Fluid secretion Related to ST of ETEC

37. Diffusely Adherent E.coli heterogenous group that generates a diffuse adherence pattern on HeLa and HEp-2 cells. associated with the watery diarrhea that can become persistent in young children in both developing and developed countries as well as recurring urinary tract infections.

38. The association of the bacteria with the membrane and the formation of long finger-like projections emanating from the cell. These projections wrap around the bacterium in a phenotype termed “ embedding.” Invasion is rarely seen.

39. Conclusion A wealth of data concerning the virulence mechanisms of diarrheagenic E. Coli has been accumulated over the years even though these complicated phenomena are not yet fully understood. This versatile organism affects a wide range of eukaryotic cell processes via an array of diverse genetic elements enabling each pathotype to colonize, multiply, and disseminate and understanding each pathogenic step at molecular level may help in devising effective measures for intervention in infection.

40. References Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998; 11: 142-201. Topley and Wilson’s Microbiology and Microbial infections, 10 th Edition Jafari et al , Escherichia coli: a brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran, IRAN. J. MICROBIOL. 4 (3) : 102-117 WHO Fact Sheet (April 2013) on diarrhoeal disease

41. Thank you