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MCB 428 Lab 8

MCB 428 Lab 8. Gastrointestinal Tract Infections. 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 O 2 H. pylori is a microaerophile

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MCB 428 Lab 8

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  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

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