1. Gastrointestinal Bacterial Infections Liliana Rodríguez, MPH, RM (AAM), M(ASCP) Liliana Rodríguez, MPH, RM (AAM), M(ASCP) UT Health Science Center at Houston UT Health Science Center at HoustonLiliana.F.Rodriguez@uth.tmc.edu

2. Learning Objectives Understand the definition of diarrhea and other gastrointestinal syndromes. Understand the definition of diarrhea and other gastrointestinal syndromes. Learn the classification of diarrhea and the microorganisms associated with each type. Learn the classification of diarrhea and the microorganisms associated with each type. Learn the mechanisms utilized by microorganisms to cause disease in the GI tract, and be able to list examples. Learn the mechanisms utilized by microorganisms to cause disease in the GI tract, and be able to list examples.

3. Learning Objectives Learn the definitions of endotoxin, exotoxin, enterotoxin and cytotoxin and be able to list examples of each. Learn the definitions of endotoxin, exotoxin, enterotoxin and cytotoxin and be able to list examples of each. Understand the mechanisms of defense in the GI tract and the role of the normal flora. Understand the mechanisms of defense in the GI tract and the role of the normal flora. Understand the global impact of diarrheal disases. Understand the global impact of diarrheal disases.

4. Learning Objectives Understand the general morphology, physiology, and virulence factors of major bacterial pathogens of the GI tract. Understand the general morphology, physiology, and virulence factors of major bacterial pathogens of the GI tract. Understand the molecular pathogenesis of the major GI bacterial diseases. Understand the molecular pathogenesis of the major GI bacterial diseases. Learn the epidemiology, clinical features, diagnosis, basic treatment and prevention of GI bacterial infections. Learn the epidemiology, clinical features, diagnosis, basic treatment and prevention of GI bacterial infections.

5. The Gastrointestinal Tract

6. GI Tract (Enteric) Infections  Most common infections seen by primary care physicians in USA.  Leading cause of infant mortality worldwide, killing 2-3 million children/year.  In some countries, a 7 years-old or younger child’s chance of dying of a diarrheal illness can be as high as 50%.  300 children die of diarrhea in the U.S. each year along with 4000 elderly, AIDS and transplant patients.

7. GI Tract (Enteric) Infections  Diarrheal disease control is a major goal of the World Health Organization.  Traveler's diarrhea has a high economic impact.  GI Tract can be the major portal of entry but not prime target (Polio, hepatitis A, botulism), OR  Major portal of entry and prime target in most cases (Shigellosis, Cholera, Traveler’s Diarrhea).

8. Clinical Features  fever  vomiting  abdominal pain  diarrhea  presence of these symptoms varies with different diseases, and the different stages of infection

9. Definition of Diarrhea  An abnormally frequent discharge of semisolid or fluid fecal matter.  Usually involving increased fluid and electrolyte loss.  Can be watery or bloody.  Method by which the host forcibly expels the infecting microorganism(s).

10. Classification of Diarrhea I.Watery diarrhea  no blood or pus  caused by enterotoxins  small bowel (proximal or distal)  self-limiting  examples: Traveler’s diarrhea, cholera

11. Classification of Diarrhea II. Bloody diarrhea  structural damage to the intestine  large bowel  invasion or damage to the mucosa by cytotoxins cytotoxins  usually requires treatment  examples: non-typhoid Salmonella, Yersinia and Campylobacter Yersinia and Campylobacter

12. Classification of Diarrhea III. Dysentery - inflammatory disorder  frequent passage of stool, blood and pus  tenesmus, fever, cramps  colon  serious, requires antibiotic treatment  typically caused by Shigella, Entamoeba histolytica (amoeba) IV. Diarrhea can also be classified as acute (<2 weeks) and chronic (>6 weeks) weeks) and chronic (>6 weeks)

13. Pathophysiology of Diarrhea  Impaired fluid absorption  Increased fluid production* toxin-mediatedtoxin-mediated mucosal injury (adhesion to enterocytes)mucosal injury (adhesion to enterocytes)  Motor disturbances

14. Important Terms Gastroenteritis – A syndrome characterized by GI symptoms including nausea, diarrhea and abdominal discomfort. Gastroenteritis – A syndrome characterized by GI symptoms including nausea, diarrhea and abdominal discomfort. Enterocolitis - Inflammation involving the mucosa of the small and large intestines Enterocolitis - Inflammation involving the mucosa of the small and large intestines Enteric fever (typhoid fever) – Disseminated infection involving multiple organs. Associated with Salmonella typhi. Enteric fever (typhoid fever) – Disseminated infection involving multiple organs. Associated with Salmonella typhi.

15. Other GI Clinical Syndromes  Gastric ulcers – H. pylori  Antibiotic-associated diarrhea – Cl. difficile  Food poisoning – Bacillus cereus, Clostridium perfringens, Cl. botulinum,S. aureus  In addition to bacteria, certain parasites and viruses are classified as enteric agents

16. GI Defenses Against Infection  Mouth- flow of fluids, saliva  Esophagus – flow of liquids, peristalsis  Stomach – acid pH  Small intestine- peristalsis, bile, IgA, Peyer’s patches, shedding, normal flora  Large intestine- NF, peristalsis, shedding, mucus

17. Normal Flora 10 11 to 10 12 viable bacteria/gm of feces 10 11 to 10 12 viable bacteria/gm of feces Bacteriocins Bacteriocins Occupation of the mucosal surface Occupation of the mucosal surface Immunogens Immunogens Vitamin K Vitamin K

18. Distribution of Intestinal Flora Predominant Concentration Organisms (per gram) Obligate anaerobes Streptococci Staphylococci Neisseria Lactobacilli Streptococci Anaerobes Bacteroides Coliforms E. coli Streptococci Candida Protozoa None >10 6 10 2 <10 4 10 6 10 9 10 11

19. Cast of Characters  Family Enterobacteriaceae Escherichia coli (many serotypes)Escherichia coli (many serotypes) Salmonella spp. (many serotypes)Salmonella spp. (many serotypes) Shigella spp. (4 species)Shigella spp. (4 species) Yersinia enterocolitica (minor cause of diarrhea)Yersinia enterocolitica (minor cause of diarrhea)  Family Vibrionaceae Vibrio choleraeVibrio cholerae Vibrio parahemolyticusVibrio parahemolyticus  Family Campylobacteriaceae Campylobacter jejuniCampylobacter jejuni

20. Acquisition of GI Pathogens  Infections are acquired by the fecal-oral route (fecally-contaminated food, fluids, fingers, fomites, flies)  Infective dose High, 10 6-8 (ETEC, Vibrio cholerae) High, 10 6-8 (ETEC, Vibrio cholerae) Intermediate 10 3-5 (Salmonella, Campylobacter) Intermediate 10 3-5 (Salmonella, Campylobacter) Low, 10-100 cells (Shigella, EHEC) Low, 10-100 cells (Shigella, EHEC)  Evasion of host defenses

21. Mechanisms of Pathogenesis A. Colonization 1. Adhesins fimbria or pili fimbria or pili colonizing factor antigen (CFA) colonizing factor antigen (CFA) 2. Receptors 2. Receptors glycoproteins glycoproteins D- mannose D- mannose

22. Pathogenesis B. Toxin Production  Endotoxin - LPS Gram-negative bacteria, extremely toxic to humans  Exotoxins –excreted Enterotoxins - proteins that affect the small intestine without demonstrable histopathology. Vibrio cholerae, ETEC Enterotoxins - proteins that affect the small intestine without demonstrable histopathology. Vibrio cholerae, ETEC Cytotoxins –produced by Shigella dysenteriae type 1 and certain E. coli serotypes; kill mammalian cells by inhibiting protein synthesis. Cytotoxins –produced by Shigella dysenteriae type 1 and certain E. coli serotypes; kill mammalian cells by inhibiting protein synthesis.

23. Pathogenesis C. Invasion of Intestinal Mucosa MucuMucu Microbial cell Epithelial cell Mucus

24. Enterobacteriaceae Largest family of GNR Largest family of GNR Found in soil, water, vegetation, humans, animals Found in soil, water, vegetation, humans, animals Large rods, non-spore forming, facultative Large rods, non-spore forming, facultative Major reservoirs for Major reservoirs for R-factors R-factors They ferment glucose They ferment glucose Most have surface pili for attachment Most have surface pili for attachment Oxidase negative Oxidase negative

25. Enterobacteriaceae (cont...)  Colon normal flora (E. coli, Klebsiella, Enterobacter).  Frank pathogens (Salmonella, Shigella, Yersinia, certain serotypes of E. coli).  Some typically encapsulated (K. pneumoniae).  Most are motile by peritrichous flagella except Shigella, Klebsiella and some E. coli.  Can cause disease in other organs, such as the urinary and respiratory tract.

26. Classification Identification to species level by biochemical characteristics Below species level by ID of surface antigens, called serotyping cell wall antigens: O (somatic) flagellar antigens: H capsular antigens: K

27. Production of fever by Lipid A

28. Escherichia coli  Most abundant facultative bacteria in GI tract  Large rods, non-spore forming, facultative  Lactose fermenter (unlike Salmonella and Shigella)  Nornal flora of colon (human and many animals)

29. Escherichia coli  Five groups cause intestinal disease. All share the property of adherence to the epithelium of the intestine. Enterotoxigenic ETECEnterotoxigenic ETEC EnteroaggregativeEAECEnteroaggregativeEAEC EnteroinvasiveEIECEnteroinvasiveEIEC EnteropathogenicEPECEnteropathogenicEPEC EnterohemorrhagicEHECEnterohemorrhagicEHEC

30. Enterotoxigenic E. coli (ETEC) The most important cause of diarrhea in children in developing countries. Main cause of traveler’s diarrhea. Affects 50% of Americans traveling to Africa or Latin America. Spread by contaminated water or food.

31. ETEC: Epidemiology   High infecting dose   Worldwide distribution   All ages affected   No seasonal incidence   Infects only humans   Affects small intestine   80,000 cases/yr in the US

33. ETEC: Virulence Factors  General type 1 pili bind to D-mannose bind to D-mannose  Colonization factor antigens (CFA), bind to glycoproteins in the host’s cells

34. ETEC: Enterotoxins  LT - Heat-labile Similar to choleragen Similar to choleragen Immunogenic Immunogenic Plasmid-mediated Plasmid-mediated LTI and LTII LTI and LTII  ST- Heat-stable Non-immunogenic Non-immunogenic Plasmid-mediated Plasmid-mediated ST-a and ST-b ST-a and ST-b

36. ETEC: Clinical features  Watery diarrhea.  No white or red cells in stool. No inflammatory process in the gut.  Incubation period 1-4 days.  Abdominal cramps, nausea, profuse diarrhea for 3-4 days.  No fever.  Immunity mediated by sIgA antibodies against LT-toxin.

38. Enteropathogenic E. coli (EPEC)  Major cause of diarrhea in poor countries  Infects primarily infants  Fever, nausea, vomiting and self-limiting watery diarrhea  Small intestine  Also called enteroadherent   12 species identified by serotype (O111:H2, O55:H6)

39. EPEC O:111 in small intestine

40. EPEC: Pathogenicity  Attachment to enterocytes through bundle-forming pili (Bfp)  Proteins inserted into the cell membrane  Effacement of microvillus  No enterotoxin  Diarrhea from malabsorption Attachment-Effacing Lesions

41. Enteroaggregative E. coli (EAggEC)  Cause of persistent diarrhea in developing countries  Affects mainly infants  Small intestine  Patients suffer of vomiting, low grade fever, watery (sometimes bloody) diarrhea and dehydration

42. EAggEC: Pathogenicity  Adherence to enterocytes  Cytotoxin (?)  Secretion of mucus and trapping of the bacteria in a biofilm  Shortening of the villi, mononuclear infiltration and sometimes hemorrhage

43. Enteroinvasive E. coli (EIEC)  Classical serotype O29:H -  Able to invade and destroy colonic epithelium  Food-borne disease  Important cause of pediatric diarrhea in developing countries

44. EIEC: Epidemiology  Infects only humans  Associated with travel  Infections restricted to children under 5 years of age living in poor conditions  One outbreak in USA traced to contaminated imported cheese

45. EIEC: Pathogenesis  Similar to Shigella  Attachment to the large intestine mucosa (plasmid-mediated)  Invasion of cells after endocytosis  Lysis of the endocytic vacuole, multiplication and spread to adjacent cells

46. EIEC: Clinical Features  Resembles shigellosis, but less severe  From mild, watery diarrhea, to severe bloody diarrhea  Fever  Cramps  Blood and leukocytes in stool

47. Enterohemorrhagic E. coli (EHEC)

48. EHEC: Historical Perspective  1982- First identified as a pathogen  1985- Associated with HUS  1990 outbreak in Missouri.  1991 outbreak in Massachusetts.  1993 - Multistate outbreak, 731 cases linked to hamburgers.

49. EHEC: General Characteristics  About 50 serotypes  Also known as Verotoxin E. coli (VTEC) (VTEC)  The most important are O157:H7 O111:NM, O26:H11, O104, O45  Acquisition of the Shiga-toxin gene?

50. EHEC: Epidemiology  Outbreaks or sporadic zoonosis  Main reservoir: GI tract of cattle  Worldwide outbreaks  Most prevalent in warmer months; greater incidence among children <5  Person-to-person infection common  Very low infective dose (~100 cells)  Sources: beef, unpasteurized milk, apple cider, untreated water

51. EHEC: Virulence Factors  Low infective dose; acid-tolerance  Two cytotoxins (verotoxins) encoded in lysogenic bacteriophages Shiga-like-toxin I (SLT-I) homologous to Shiga toxin except for one a.a. Shiga-like-toxin I (SLT-I) homologous to Shiga toxin except for one a.a. Shiga-like-toxin II (SLT-II) 60% homology Shiga-like-toxin II (SLT-II) 60% homology  Attachment (eae gene) attaching-effacing  Hemolysin

52. EHEC: Pathogenesis  Colonization of distal ileum, colon and cecum.  Confined to the gut mucosa.  Attachment to epithelial cells and local multiplication.  No systemic invasion.

53. The problem is…..  Shiga-like toxin (SLT, cytotoxin, verotoxin) promotes inflammation of the colonic mucosa resulting in purulent exudate and bleeding  Damage of the intestinal lining cells  2-7% → HUS due to systemic absorption of SLT  SLT damage to small renal arteries

55. E. coli Diarrheal Diseases Diagnosis  Stool culture in special media (EIEC difficult)  Clinical (EHEC)  Serotypes determined w/polyvalent antiserum and adhesion in cell cultures  Test for toxins (ETEC, EHEC) in tissue culture, ELISA, latex agglutination test  DNA probes EMB Agar with E. coli

58. EPEC, EAggEC, EIEC, EHEC  Fluid Replacement.  No need for antibiotics or antispasmodics except in EIEC cases, where some antibiotics reduce duration of diarrhea.  HUS  Supportive  Dialysis in renal failure  Antibiotics contraindicated

59. Prevention and Control  Pure water supply  Adequate systems for sewage  Boiling water  Promotion of breast feeding (EPEC, EAggEC)  Improved sanitation

60. E. coli typeSymptomsepidemiologypathogenesisprimary site Enterotoxigenic (ETEC) traveler ' s diarrhea infant diarrhea; watery, no fever, no WBC worldwide; all ages; humans only LT or ST toxins pili small intestine Enteropathogenic (EPEC) Fever, nausea, vomiting. Watery diarrhea, no WBC Important cause of pediatric diarrhea adherence to enterocytes, destroys villi small intestine Enteroinvasive (EIEC) bloody diarrhea, and fever (resembles Shigella) humans only associated with travel plasmid-mediated invasion. Destruction of colon cells large intestine Enterohemorrhagic (EHEC) O157:H7 Little or no fever Hemorrhagic colitis. HUS Zoonosis foodborne Cytotoxic Shiga-like verotoxin large intestine Enteroaggregative (EAEC) Vomiting, low grade fever. Watery/bloody diarrhea infant diarrhea in underdeveloped countries adherence to enterocytes heat-stable toxin small intestine Summary