病原生物学教研室 Department of pathogenic Biology of Gannan Medical University

1. 医学微生物学 Medical Microbiology 病原生物学教研室 Department of pathogenic Biology of Gannan Medical University 张文平

2. Chapter 11 Enteric Bacilli (Enterobacteriaceae)

3. Classification • Enterobacteriaceae (肠杆菌科细菌） • Vibrio cholerae(霍乱弧菌) • Vibrio parahaemolyticus (副溶血性弧菌) • Helicobacter pylori (幽门螺杆菌) • Campylobacter(弯曲菌属)

4. Characteristics • large Gram- negative rods • associated with intestinal infections (bacillary dysentery, typhoid, and food poisoning ) • oxidase negative • glucose fermenters & nitrate reducers • the pathogenicity of a particular enteric bacterium can be determined by its ability to metabolize lactose

5. Escherichia coli Coliform —enterobacteria which ferment lactose

6. Characteristics Morphology • usually motile, produce peritrichous flagella • some produce polysaccharide capsules • grow well on non-selective media, forming smooth, colourless colonies 2-3 mm in diameter in 18h on nutrient agar • temperature (15-45℃)

7. Characteristics Colonies on selective media • blue and violet colonies on EMB agar • red colonies on MacConkey agar

8. Characteristics Biochemical reaction • aerobic or anaerobic --can grow in the presence or absence of O2. • typically oxidase-negative • most strains ferment lactose & glucose with the production of acid and gas • IMViC reaction : + + - - distinguish!

9. Reactions on TSI agar slantsi

10. Characteristics Antigenic structure • Over 700 antigenic types (serotypes) are recognized based on O, H, and K antigens. • Lipopolysaccharide LPS(heat-stable somatic antigens ) -- O antigens • flagellar antigens -- H antigens • "capsular" antigens -- K antigens

11. Diseases caused by E. coli • E. coli is responsible for three types of infections in humans: urinary tract infections (UTI), neonatal meningitis, intestinal diseases (gastroenteritis).

12. Pathogenic E. coli • Five classes (virotypes) of E. coli that cause diarrheal diseases are now recognized: • enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAggEC).

13. Pathogenic E. coli

14. Pathogenic E. coli Enterotoxigenic E. coli (ETEC) • cause diarrhea in • infants and travelers

15. Pathogenic E. coli ---- ETEC pathogenesis of ETEC involves two steps: • intestinal colonization, fimbrial adhesins e.g. CFA I, CFAII, K88. K99 non invasive • diarrheagenic enterotoxin(s) -- LT and/or ST toxin • both traits are plasmid-encoded

16. Pathogenic E. coli ---- ETEC Enterotoxins • LT (heat-labile) toxin • ST (heat-stable) toxin

17. Pathogenic E. coli ---- ETEC heat-labile toxin(1) • similar to Vibrio cholerae enterotoxin. • two types of subunits. • the B subunit binds the toxin to the target cells via a specific receptor that has been identified as Gm1 ganglioside. • the A subunit is then activated by cleavage of a peptide bond and internalized

18. Pathogenic E. coli ---- ETEC heat-labile toxin(2) • The actived subunit A then catalyzes the ADP-ribosylation (transfer of ADP-ribose from nicotinamide adenine dinucleotide [NAD]) of a regulatory subunit of membrane-bound adenylate cyclase, the enzyme that converts ATP to cAMP. • This activates the adenylate cyclase, which produces excess intracellular cAMP, which leads to hypersecretion of water and electrolytes into the bowel lumen.

19. Pathogenic E. coli ---- ETEC heat-stable toxin • ST enterotoxin causes an increase in cyclic GMP in host cell cytoplasm. • Two major classes -- Sta & STb • Those termed STa stimulate intestinal guanylate cyclase, the enzyme that converts guanosine 5'-triphosphate (GTP) to cyclic guanosine 5'-monophosphate (cGMP). Increased intracellular cGMP inhibits intestinal fluid uptake. • Those termed STb do not seem to cause diarrhea by the same mechanism.

20. Pathogenic E. coli ---- ETEC Laboratory methods for isolation and identification of ETEC

21. Pathogenic E. coli ---- ETEC Control • controlled by preventing transmission and by stressing the importance of breast-feeding of infants • The best treatment is oral fluid and electrolyte replacement (intravenous in severe cases). • Antibiotics are not recommended -----antibiotic-resistant pathogenic E coli

22. Pathogenic E. coli Enteroinvasive E. coli(EIEC) • Shigella-like E coli strains • dysentery-like diarrhea (mucous, blood), severe inflammation, fever • nonfimbrial adhesins, possibly outer membrane protein • invasive (penetrate and multiply within epithelial cells) • does not produce shiga toxin

23. Pathogenic E. coli ---- EIEC Cellular pathogenesis of invasive E coli

24. Pathogenic E. coli ---- EIEC • Sereny test -- Invasive E coli, like Shigella, causes a rapid keratoconjunctivitis when placed on the conjunctiva of the guinea pig eye. • Virulent Sereny test-positive isolates carry a large (usually 140-megadalton) plasmid responsible for this property.

25. Pathogenic E. coli Enteropathogenic E. coli (EPEC) • non fimbrial adhesin (intimin) • moderately invasive (not as invasive as Shigella or EIEC) "attaching and effacing" • does not produce LT or ST; some reports of shiga-like toxin • usually infantile diarrhea; watery diarrhea similar to ETEC, some inflammation, no fever; symptoms probably result mainly from invasion rather than toxigenesis

26. Enterohemorrhagic E. coli (EHEC) represented by a single strain (serotype O157:H7) adhesins not characterized, probably fimbriae moderately invasive produces shiga toxin but not LT or ST copious bloody discharge (hemorrhagic colitis), intense inflammatory response, may be complicated by hemolytic uremia pediatric diarrhea caused by this strain can be fatal due to acute kidney failure (hemolytic uremic syndrome [HUS]). Pathogenic E. coli

27. Pathogenic E. coli Enteroaggregative E. coli(EAggEC) • adhesins not characterized • non invasive • produce ST-like heat-labile plasmid-encoded toxin (EAST) and a hemolysin • persistent diarrhea in young children without inflammation, no fever

28. treatment is based on symptomatology. fluid replacement is the primary treatmen Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome). Pathogenic E. coli Treatment

29. Shigella agents of bacillary dysentery

30. Shigella Structure • Gram-negative, • nonmotile, • facultatively anaerobic, • non-spore-forming rods • failure to ferment lactose or decarboxylate lysine • closely related with Escherichia coli

31. Shigella Classification four serogroups with multiple serotypes • A (S dysenteriae, 12 serotypes); • B (S flexneri, 6 serotypes); • C (S boydii, 18 serotypes); • D (S sonnei, 1 serotype).

32. Shigella bacillary dysentery--shigellosis

33. Shigella Virulence 1. invasin • encoded by large extra-chromosomal elements (plasmids)--Invasion Plasmid Antigens (Ipa) B and C. • induces the endocytic uptake of shigellae by M cells, epithelial cells, and macrophages. • deform the plasma membrane of contiguous cells. • IcsB plasmid-encoded protein lyses the plasma membranes, resulting in intercellular bacterial spread.

34. Shigella Histopathology of acute colitis following peroral infectionwith shigellae

35. Shigella Virulence 2.Endotoxin • cause fever, shock, bloody, mucoid stools, and abdominal pain (cramps and tenesmus) .

36. Shigella Virulence 3.exotoxin-- Shiga toxin(vero toxin) • chromosomally-encoded • neurotoxic, enterotoxic and cytotoxic • The toxin inhibits protein synthesis (acting on the 70S ribosome and lysing 28S rRNA). Its enterotoxicity can make the disease clinically appear as a diarrhea.

37. Shigella Diagnosis -- Sampling • Positive cultures are most often obtained from blood-tinged plugs of mucus in freshly passed stool specimens obtained during the acute phase of disease • Rectal swabs may also be used if the specimen is deposited in a buffered glycerol saline holding solution

38. Shigella Diagnosis -- Isolation • primary differential/selective media: Salmonella-Shigella (SS) Agar(contain bile salts& pH indicators) MacConkey, Hektoen Enteric Agar,

39. Shigella Diagnosis -- Isolation • colorless, non-lactose-fermenting colonies • Secondly tubed slants of Kligler's Iron Agar or Triple Sugar Iron Agar. • Shigella species produce an alkaline slant and an acid butt with no bubbles of gas in the agar.

40. Shigella Diagnosis -- Identification • slide agglutination tests with antisera for serogroup and serotype confirm the identification • polymerase chain reaction (PCR). • Enzyme-linked immunosorbent assay (ELISA)

41. Shigella Epidemiology • occurring by fecal-oral contact • it can be transmitted by infected adult food handlers who contaminate food. • Man is the only "reservoir"

42. Shigella Treatment and Control • Managing of dehydration is of primary concern. • Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin). • Patients respond to antibiotic therapy and disease duration is diminished

43. Salmonella salmonellosis

44. Salmonella  Structure & Antigenic Types • Gram-negative, flagellated, facultatively anaerobic bacilli • three major antigens: H or flagellar antigen; O or somatic antigen; Vi antigen (only a few serovars)

45. Salmonella  Virulence Factors (1)the ability to invade cells ---- invasin: Vi (capsular) antigen (2) a complete lipopolysaccharide coat ---- LPS (endotoxin) (3) the ability to replicate intracellularly, (4) possibly the elaboration of toxin(s)

46. Salmonella

47. Salmonella Invasion of intestinal mucosa by Salmonella.

48. Salmonella -- Clinical Manifestations 1) Gastroenteritis--food poisoning • Symptoms usually begin 6 to 48 hours after ingestion of contaminated food or water • the cardinal manifestation is diarrhea. • nausea, vomiting, abdominal cramps, myalgia, headache, fever (38oC to 39oC) and chills are common • The duration of fever and diarrhea is usually 2 to 7 days

49. Salmonella -- Clinical Manifestations 2) Septicemia • an intermediate stage of infection – no intestinal symptoms and the bacteria cannot be isolated from fecal specimens. • it may remain localized in the intestine or disseminate to the bloodstream

50. Salmonella -- Clinical Manifestations 3) Enteric fevers ---- typhoid • severe systemic form, may be fatal • The best studied enteric fever is typhoid fever, the form mainly caused by S typhi • may be preceded by gastroenteritis • an incubation period of 10 to 14 days • symptoms of enteric fevers are nonspecific:fever, anorexia, headache, myalgias, and constipation