E.coli Commensal , Pathogen ,& Genetic tool

1. E.coliCommensal, Pathogen,& Genetic tool Dr.T.V.Rao MD Dr.T.V.Rao MD

2. Classification of Bacteria Dr.T.V.Rao MD

3. Enterobacteriaceae • Commonly present in large intestine • Non sporing , Non Acid fast, Gram – bacilli. • A complex family of organisms, • Some are non pathogenic • A few are highly Pathogenic, • Some commensals turn out to be pathogenic. as in UTI after catheterization. Dr.T.V.Rao MD

4. Characters of Enterobacteriaceae • All Enterobacteriaceae • Gram-negative rods • Ferment glucose with acid production • Reduce nitrates into nitrites • Oxidase negative • Facultative anaerobic • Motile exceptShigella and Klebsiella • Non-capsulated except Klebsiella • Non-fastidious • Grow on bile containing media (MacConkey agar) Dr.T.V.Rao MD

5. Classification of Enterobacteriaceae • There are several selective and differential media used to isolate distinguishes between LF & LNF • The most important media are: • MacConkey agar • Eosin Methylene Blue (EMB) agar • Salmonella Shigella (SS) agar • In addition to Triple Sugar Iron (TSI) agar Dr.T.V.Rao MD

6. Escherichia coli • Named by Escherichia • Wide group of bacteria on basis of Bio typing and Serotyping Produce infections in Humans and Animals Detection of E.coli in water indicates pollution and contamination. Dr.T.V.Rao MD

7. Domain: Bacteria Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Escherichia Species: Escherichia coli (E. coli) Classification

8. E.coli • Morphology Gram - ve Straight rods, • 1-3 X 0.4 -0.7 microns, • Appear in singles or in pairs, • Motile by peritrichate flagella. • Very few strains non motile • Not spore forming, Non acid fast. Dr.T.V.Rao MD

9. Enterobacteriaceae: Genetic Properties • Chromosomal DNA has 39-59% guanine-plus-cytosine (G+C) content • Escherichia coli is the type genus and species of the Enterobacteriaceae • Species of Enterobacteriaceae more closely related by evolutionary distance to Escherichia coli than to organisms of other families (Pseudomonadaceae, Aeromonadaceae)

10. E.coliCultural characters • Aerobic / Facultative Anaerobic • Grows between 10 – 40 c optimal at 37 c • Grown in simple medium • Produce Large grayish ,Thick white , moist smooth opaque colonies • May contain capsule. • On MacConkey medium Produce Bright pink Lactose fermenters. Dr.T.V.Rao MD

11. E.coli Dr.T.V.Rao MD

12. Identification of EnterobacteriaceaeBiochemical reactions • Oxidase test • All members of Enterobacteriaceae are oxidase negative • Pseudomonas is oxidase positive • O/F test • All members of Enterobacteriaceae are O+/F+ • Pseudomonas is O+/F- • Nitrate reductase • All members of Enterobacteriaceae are nitrate reductase positive • Pseudomonas is nitrate reductase negative

13. E.coliBiochemical Characters, Glucose,Lactose,Mannitol,Maltose fermented. with A/G I,M,Vi,C tests. Indole + Methyl Red + Voges Proskauer – ve I,M,Vi,C tests. Citrate –ve Urease not produced. Dr.T.V.Rao MD

14. Flame & Cool Flame & Cool Flame & Cool Identification of EnterobacteriaceaeDifferentiation between LF & NLF byGrowth on MacConkey agar • Method: • MacConkey agar is inoculated with tested organismusing streak plate technique • Incubate the plate in incubator at 37 C/24 hrs • Results: • LF organism appears as pink colonies (e.g. E. coli) • NLF organism appears as colorless colonies (e.g. Shigella) 2 1 3 4 5 Dr.T.V.Rao MD

15. E.coliAntigenic Structure • Somatic 0 170 • Capsular K 100 • Flagella H 75 • Virulence factors Surface Antigens Toxins O Endotoxic activity K protects against the phagocytosis Fimbriae promote virulence ( important in UTI ) Dr.T.V.Rao MD

16. Toxins and E.coli • E.coli produce Exotoxins • Hemolysins, Enterotoxins causes Diarrheas, • Important toxins produces. • Heat labile HL Heat stable HS Vero toxins VT Like Shigella toxins Dr.T.V.Rao MD

17. Toxins • Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin: • LT – is heat labile and binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP Dr.T.V.Rao MD

18. Mechanism of action of Toxins • Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel Dr.T.V.Rao MD

19. Toxins in E.coli • Produce Enterotoxin L T and S T • Labile toxin 1956 De experiments in Rabbit illeal loop causes outpouring of fluids • E.coli Labile toxin like Cholera toxin • L T contains component A and B • A = Active B= Binding • B causes Binding with Gm I Ganglioside receptor on Intestinal epithelial cells Dr.T.V.Rao MD

20. E. coli toxins • Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity). Dr.T.V.Rao MD

21. Toxins E.coliLabile toxin • Component A Activated to A1 and A2 • A1 Activates adenyl cyclase in the enterocytes to form cyclic adenosine 5 monophosphate • Causes to increase outflow of water and electrolytes in the gut lumen causes Diarrhea Dr.T.V.Rao MD

22. Toxins of E.coliStable Toxin • ST A and ST B • ST A Acts by activation of Cyclic guano sine monophosphate.( C GMP ) • Causes fluid accumulation in Intestine. • E.coli ( Some ) produce Verocytotoxin causes cytotoxicity to Vero cells. • Acts like Shigella dysentery toxin Dr.T.V.Rao MD

23. E.coli a Complex Microbe • More than 700 serotypes of E. coli have been identified.  The different E. coli serotypes are distinguished by their “O” and “H” antigens on their bodies and flagella, respectively. Dr.T.V.Rao MD

24. Classification of E.coli 1.Enteropathogenic EPEC 2.Enterotoxigenic ETEC 3.Enteroinvasive EIEC 4.Enterohemorrhagic EHEC 5.Enteroaggresive EAEC Dr.T.V.Rao MD

25. Enteropathogenic E.coli • Causes diarrheal disease in children, • EPEC O26/O11 • Produce Verocytotoxin • Infantile enteritis, Involves upper part of Intestine • Brush border of the intestine is lost • Intimacin – EPEC adhesion factor. • Frequent in summer months • Poor hygiene predisposes. • Out breaks in Institutions Dr.T.V.Rao MD

26. Enteropathogenic E. coli • destruction of surface microvilli • fever • diarrhea • vomiting • nausea • non-bloody stools (not generally seen as dysentery) Gut lumen

27. Laboratory Diagnosis EPEC • Confirm with Polyvalent sera • Test Sero groups with polyvalent and monovalent sera. • HEp2 – adherence. Dr.T.V.Rao MD

28. Enterotoxigenic E.coli • Produce Heat stable /Heat labile toxins • Adheres to epithelium of small intestine. • Present with Nausea, Vomiting and Lose stool • H L like cholera toxin • Causes accumulation of fluids • Adhesive factors Fimbriae specific receptor in the intestinal epithelium CFA Mortality in children < 5 years Dr.T.V.Rao MD

29. Enterotoxigenic E. coli • Heat labile toxin • like choleragen • Adenyl cyclase activated • cyclic AMP • secretion water/ions • Heat stable toxin • Guanylate cyclase activated • cyclic GMP • uptake water/ions

30. Enterotoxigenic E.coli • Causes travelers diarrhea • Water contaminated with Human and Animal feces predisposes. • Laboratory Diagnosis Demonstration of Enterotoxin LT and ST Tissue culture tests, ELISA Passive agglutination tests. Animal experiments in Rabbit ileal loop test. Dr.T.V.Rao MD

31. Treatment and Prophylaxis in Travelers diarrhea • Doxycycline, • Trimethoprim, • Norfloxacillin • Fluroquinolones • Avoid contaminated food, • Safe protected water ,prefer bottled water, • Hot foods, Hot Drinks, • Boiled milk Dr.T.V.Rao MD

32. Entero invasive E.coli • Some are non motile strains, • Atypical resembles like Shigella. • Clinically mild diarrhea • Sereny test positive animal Rabbit. • ELISA Dr.T.V.Rao MD

33. Enteroinvasive E. coli (EIEC ) • Dysentery • - resembles shigellosis Gut lumen

34. Enterohemorrhagic E.coli • Produce Verocytotoxin • Mild diarrhea - can be fatal hemorrhagic colitis. and uremic syndrome. • Present in Human and Animal feces. • Hemorrhagic complication with O157 in Japan and USA. • Salads vegetables, Radish Proper cooking Dr.T.V.Rao MD

35. EHEC ( contd ) • Culture • DNA detection methods. • Cytotoxic effects on Vero cells. • Detection with monovalent sera O157/H7 Dr.T.V.Rao MD

36. Dr.T.V.Rao MD

37. Enterohemorrhagic E.coli can cause HUS • HUS develops when the toxin from E. coli bacteria, known as Shiga-like toxin (SLT) , enters the circulation by binding to special receptors.These Shiga-toxin receptors, known as Gb3 receptors , are probably heterogeneously distributed in the major body organs allowing disparate thrombotic (blood clotting) impacts in different HUS victims, although the greatest receptor concentration appears to be in the kidneys, especially in children. Dr.T.V.Rao MD

38. Mechanism of HUS • As the inflammatory reaction process accelerates, red blood cells are destroyed and cellular debris aggregates within the microvasculature while the body’s inherent clot breaking mechanisms are disrupted. The result is formation of micro thrombi within particularly susceptible organs such as the kidneys and brain. Because there exists no way to halt the progression of HUS, • The patients are supported with medical care Dr.T.V.Rao MD

39. Enteroaggresive E.coliEAEC • Can cause Diarrhea Detect by Culture methods • Brick-like aggregates on cell surfaces • Mucus biofilm inhibits fluid absorption • Diarrhea • Detection of Enterotoxin Dr.T.V.Rao MD

40. E.coli resembles Shigella spp • The E. coli serotypes that are responsible for the reports of contaminated foods and beverages are those that produce Shiga toxin, because the toxin is identical to that produced by another bacteria known as Shigella dysentery Dr.T.V.Rao MD

41. Treatment – E.coli Gastrointestinal disease • Fluid replacement • Antibiotics • not used usually unless systemic infections prevails • e.g. hemolytic-uremia syndrome

42. E. Coli leading cause of UTI • Clinical significance • Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection). Dr.T.V.Rao MD

43. Urinary Tract Infections E.coli produce urinary tract infection. Majority of UTI s are produce by E.coli. Instrumentation, Prostatic enlargement, Urinary caliculi ,Pregnancy, increase the predisposition Asymptomatic Bacteriuria in pregnant women, Pyelonephritis, Dr.T.V.Rao MD

44. Facts on UTI • Women suffer more than males Short urethra Pregnancy, Sexual intercourse /Honey moon cystitis. • Other factors Urethral obstruction, Urinary stones Congenital malformation's Neurological disorders, Catheterization , Cystoscopy Usually cystitis is produced from fecal strains entering urethra Dr.T.V.Rao MD

45. Culturing for E.coli • Mid stream sample/semi quantitative culturing (Kass et al ) >_ 1.00,000/ml of urine. ( significant Bacteriuria ) • Urine should not be kept in wards for > 2 hours and to be preserved at 4 c • Culture by standard loop method. • Fixed volume cultured on MacConkey agar Lactose fermenters I M Vi C • Antibiotic sensitivity tested. Dr.T.V.Rao MD

46. Other infection withE.coli • Pyogenic infections. • Intraabdominal infections • Peritonitis. Abscess. • Septicemias • Produce Drug resistant infections. Dr.T.V.Rao MD

47. Other Important E. coli Infections • Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate. • Usually caused by strains with the K1 capsular antigen. • Gastroenteritis – there are several distinct types of E. coli that are involved in different types of gastroenteritis: Dr.T.V.Rao MD

48. E.coli • Antimicrobial therapy- E. coli is usually susceptible to a variety of chemotherapeutic agents, though drug resistant strains are increasingly prevalent. • It is essential to do susceptibility testing. • Treatment of patients with EHEC infections is not recommended because it can increase the release of shiga-like toxins and actually trigger HUS

49. Escherichia coli as a Genetic tool. • The study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade.. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism. Dr.T.V.Rao MD

50. Why E.coli is preferred • E. coli cells only have about 4,400 genes whereas the human genome projecthas determined that humans contain approximately 30,000 genes. Also, bacteria, including E. coli, live their entire lifetime in a haploid state, with no second allele to mask the effects of mutations during protein engineering experiments. Dr.T.V.Rao MD