MCB 428 Lab 8

1. MCB 428Lab 8 Gastrointestinal Tract Infections

2. Helicobacter pylori Infection of the Stomach • Very acidic pH in stomach • Mucosal cells protected by thick mucin layer • Protons do not diffuse readily thru this layer – alkaline • Hypoxic area – low O2 • H. pylori is a microaerophile • Just needs to survive until it reaches mucin layer

3. Urea Helicobacter pylori Infection of the Stomach • Moves to mucin layer by means of flagella • Secretes urease: urea + H+ + 2H2O HCO3- + 2NH4+ • Neutralizes acid as it moves along • “Safe” once it reaches mucin layer

4. Colonization of H. pylori • LPS contains Lewis antigen – mimics oligosaccharide found on human cells • Does not elicit intense inflammatory response • Most of those colonized do NOT develop ulcers • Most of bacteria beyond the reach of neutrophils

5. Formation of Stomach Ulcers • Neutrophils damage gastric mucosa • Strains associated with gastric disease (ulcers, cancer) produce vacuolating toxin A (VacA) • Causes formation of vacuoles, cavities containing fluid; these rupture causing cell damage • Also inhibits T cell activation • Numbers of bacteria may be important • Some strains may be able to multiply more rapidly • Route of transmission uncertain

6. Formation of Stomach Ulcers-Other Causes- • Genetic factors may pre-dispose someone to ulcer formation • Injury of the gastric mucosal lining and weakening of mucus defenses • Excess secretion of HCl and psychological stress • Chronic use of anti-inflammatory medicines e.g., aspirin • Cigarette smoking

7. Eliminating H. pylori Infections • Difficult to culture – did not tolerate low pH in lab • Antibiotics that killed H. pylori in lab not effective in humans • Problem with delivery of antibiotic to site of infection • Growth in stomach different than growth in tube • Therapy consists of two antibiotics and compound that heals ulcer (10-14 days)

8. Heliobacter pylori (Gastric mucosal cells) Associated with gastric ulcers and gastric cancer

9. Detecting Infection • Breath tests • Detects ammonia produced by urease • Detects *CO2 after patient drinks 13-C or 14-C urea • Can give false negatives • Test for antibodies to H. pylori • Can give false positives (asymptomatic) • Biopsy urease test • Invasive, but rapid • Allows for culturing and susceptibility testing

10. Food-borne and Water-borne Infections • Many pathogens spread by this route • Statistics inadequate to determine if incidents increasing or decreasing • Probably under-reported • Size of outbreaks HAS increased (multi-state) • Safe handling and packaging of foods has improved • Centralization of food production, processing, and distribution means more people affected when lapses do occur

11. Link to Contaminated Water • Outbreaks often traced to contaminated meat in headlines • Raw fruits and vegetables also contaminated • Some of these were imported • Raspberries from Guatemala -- 1996 • Manure run-off enters groundwater • Contaminates irrigation water sprayed on vegetables • Case of exported radish seeds contaminated with E. coli O157:H7 (causes dysentery)

12. Water-borne Infections • Old sewage systems a problem • Centralized water supply • Case of contamination with Cryptosporidium parvum • Milwaukee, WI -- 1993 • Springfield lake contaminated with Leptospira interrogans from infected wild animals • CDC estimates 900,000 cases in U.S. each year – resulting in 900 deaths • Numbers probably much higher (under reported)

13. Food-Borne Infections • Estimates suggest food-borne disease results in annual costs of $5-6 billion in U.S. • CDC estimates 76 million suffer from food-borne illness in U.S. annually; 5,000+ deaths

14. Toxin Mediated Food-Borne Disease (Toxinoses) • Clostridium perfringens • 249,000 cases per year (7 deaths) • Staphylococcus aureus • 185,000 cases per year (2 deaths) • Clostridium botulinum • 58 cases per year (4 deaths)

15. Staphylococcus aureus • Gram+ cocci • Normal microbiota of the nose in 30% • Commonly colonizes skin and mucosal membranes • Responsible for a wide range of diseases • Produces a number of toxins and other virulence factors • Can infect essentially any tissue • Hospital acquired strains are highly antibiotic resistant

16. S. aureus Toxins • Produces about 8 types • Hemolysins (lyse red blood cells) • Leukocidin (kills phagocytes) • Superantigens • Toxic shock syndrome toxin • Enterotoxin

17. Staphylococcal Food Poisoning • Previously cooked food gets contaminated with someone’s nasal flora • 30% of Staphylococcus aureus isolates make enterotoxin • Organism grows and produces toxin • Toxin is heat and acid stable -- reheating food doesn’t help • Toxin is ingested

18. Staphylococcal Food Poisoning • Toxinoses -- bacterial cells are irrelevant • Symptoms have rapid onset (6-12 hours) and rapid resolution (12-24 hours) • Projectile vomiting and diarrhea • Mechanism is not clear • Directly affects “vomiting center” of brain -- emetic reflex center • Superantigen and emetic effects are genetically separable components of the toxin

19. Symptoms • Diarrhea • Ion pumps in cells of small intestine maintain flow of ions and water • Action of microbes on mucosa disrupts pumps • Dehydration due to diarrhea can kill • Rehydration therapy important • Small (80%) and large intestines hold and absorb water

20. Types of Diarrhea • Secretory diarrhea • Disruption of ion pumps • No damage to mucosal cells • Malabsorptive diarrhea • Mucosal cells damaged by microbe or by inflammatory response • Can lead to dysentery (bloody diarrhea) • Some pathogens disrupt absorption of nutrients • Vitamin deficiency and steatorrhea (malabsorption of fats)

21. Acid Tolerance Response • If bacteria such as Salmonella and E. coli are exposed to acidic pH (<5), they become able to tolerate even lower pH (1-3) for a couple of hours • Pumping of protons out of the cytoplasm • How induced • Bacteria in acid environment in foods • Bacteria ingested with large meals

22. Escherichia coli • Normal microbiota • Usually harmless unless they acquire virulence factors (e.g., plasmids and DNA from transduction) • Recurrent urinary tract infections -- UPEC • Traveler’s diarrhea and endemic diarrhea • ETEC • EPEC • New killer strains • EHEC

23. ETEC • Enterotoxigenic E.coli • 79, 000 cases/year in U.S. • 3-9 million cases worldwide • Cause of traveler’s diarrhea • Leading cause of infant deaths worldwide • Causes secretory diarrhea • Attach to mucosa cells by pili

24. ETEC • Produce toxins • Heat-labile toxin (LT) • Heat-stable toxin (ST)

25. Heat-labile Toxin (LT) of E. coli • LT toxin is an A-B toxin (Type III) • B component binds to epithelial cells and forms pore in membrane • Helps A component get inside cell

26. Mechanism of action of LT Toxin • A component locks adenylate cyclase in ON form • Levels of cAMP rise • Ion pumps malfunction • Small intestine flooded with ions and water

27. Mechanism of action of ST Toxin • ST toxin gets inside cell (mechanism unknown) • Levels of cGMP rise • Ion pumps malfunction • Small intestine flooded with ions and water

28. EPEC • Enteropathogenic E.coli • 79,000 cases/year in U.S. • Causes malabsorptive diarrhea • Most common cause of infant diarrhea • Causes ~3 million DEATHS worldwide • Binds intestinal cells via pili and damages surface • Toxic proteins injected into cell via Type III secretion system • No antibody response to toxin (direct transfer of toxin)

29. Delivery of EPEC Toxic Protein

30. EPEC Invading Host Cells EPEC induces the formation of pedestal-like structures on the host cell surface upon which they rest

31. EHEC • Enterohemorrhagic E.coli • E. coli O157:H7 (O = O antigen; H = flagellar antigen) • 20, 000 cases/year; 200 deaths • Causes malabsorptive diarrhea that can become dysentery • More deadly than ETEC or EPEC • Produce Shiga-like toxin (Shigella) that damages endothelial cells in kidney • More serious in children

32. EHEC Shiga-like Toxin • Bacteriophage encoded • A-5B Toxin • A portion: endonuclease cuts 28S rRNA • Seems to target endothelial (blood vessel) cells • Leads to Hemolytic Uremic Syndrome (HUS) -- ~8% • Kidney damage and death

33. Treatment Options • Antibiotics not used • Usually self-limiting • May increase toxin production in EHEC strains • Rehydration therapy • For severe EHEC infections, may need dialysis

34. Food-Borne Infections • Estimates suggest food-borne disease results in annual costs of $5-6 billion in U.S. • CDC estimates 76 million suffer from food-borne illness in U.S. annually; 5,000+ deaths • Caused by Salmonella, Campylobacter, E. coli, Listeria monocytogenes, other bacteria • Most commonly caused by viruses

35. Salmonella-related Infections • Salmonella typhi • Cause of typhoid fever • Very rare in U.S. – 400 cases/year • 17 million cases worldwide • Results from contaminated drinking water • Antibiotic therapy effective

36. Typhoid Fever • S. typhi multiplies in phagocytic cells • High sustained fever (103-104F) • Typhoid = Greek for “smoke” or “clouds” (fever delirium) • Weakness • Stomach pains • Headache • Diarrhea follows fever

37. Salmonella-related Infections • Non-typhoid Salmonella (mostly enteriditis) • Main reservoirs are poultry and eggs • Has been increasing in U.S. • Estimates of 1-4 million cases annually • Gastroenteritis (inflammation of stomach and intestinal lining) • Usually self-limiting

38. Salmonella • Gram negative motile rod • Facultative anaerobe (grows +/- O2) • Closely related to E. coli • Has 2 Type III secretion systems • Invasion of M cells and epithelial cells • Survival in macrophages

39. Salmonella Pathogenesis • Once inside the small intestine, they swim to mucosa and invade underlying tissue • Has affinity for M (membranous) cells • Specialized for uptake and of macromolecules and microorganisms • Not killed by ordinary macrophages, only activated ones

40. Salmonella Pathogenesis • Inflammatory response to infection causes pain and damages mucosal cells, producing malabsorptive diarrhea • Reactive arthritis • Caused by circulating bacterial antigens • No long-term damage to joints

41. Protein secreted thru Type III secretion system induces endocytosis; Once inside the vesicle, it secretes another protein to prevent fusion with a lysosome Steps in a Salmonella Infection If growth limited to intestine: - intestinal symptoms - malabsorptive diarrhea

42. Bacteria leave M cell and invade macrophages, resist digestion and multiply; macrophages are migratory and can carry the infection throughout the body Steps in a Salmonella Infection • If spreads to other tissues: • - Fever, sepsis, death • - Immunocompromised • patients

43. Salmonella

44. Salmonella-based Vaccines • Targets and stimulates GALT cells • Easily manipulated genetically • Need strain that replicates a few times but does not cause symptomatic disease

45. AING • Acute Infectious Nonbacterial Gastroenteritis • Caused by viruses • Symptoms are fever, vomiting, malaise, and diarrhea • More serious in infants

46. How Do Viruses Cause Diarrhea? • Virus preferentially affects older, fully differentiated epithelial cells • Causes stunting of villi • Younger cells are not efficient at absorption • Malabsorptive diarrhea Graphics by Jack Ikeda

47. Intestinal Villi Large surface area means better absorption

48. Norwalk Virus • Named for AING in Norwalk, OH 1972 • Exceptionally small virus (25-30 nm) with round shape and no envelope • (+) ssRNA genome • Capsid composed of single protein subunit • Self-limiting disease • Most outbreaks tied to food handlers

49. Norovirus (Norwalk–like Viruses) • 2/3 of Adults have antibodies – not protective • 12 Days of Diarrhea and/or Vomiting • Abrupt onset and is accompanied by a varying combination of signs and symptoms, including abdominal cramps, myalgias, malaise, headache, nausea, and lowgrade fever

50. Norovirus Outbreak NewsGazette Dec. 23, 1999 Article provided by Jack Ikeda