C hair of Medical Biology, M icrobiology, V irology, and I mmunology

1. Chair of Medical Biology,Microbiology, Virology, and Immunology PATHOGENIC ENTEROBACTERIACEAE

2. Classification of the Enterobacteriaceae Genera

3. Shigella a. Slender, gram-negative rod; non lactose-fermenting (except for S. sonnei) b.In contrast to E. coli: no H2S production, no lysine decarboxylation, no acetate utilization c.Invasive (key to pathogenesis) d.In contrast to Salmonella: non-motile; no gas from glucose fermentation; no H2S production e.Toxin production limited to a few strains

4. f.All have O antigens-four groups (A-D) g.Differentiating species ( S. dysenteriae - no mannitol fermentation; S. boydii - C antigen group; S. flexneri - B antigen group; S. sonnei-orniltine decarbexylase production)h. Specimens i. Rectal swab from colonic ulcer is best for culture. j. Fecal specimen - must be immediately innoculated onto transport media or culture media.k. Sensitive to acids present in feces.

5. Morphology. Morphologically dysentery bacilli correspond to the organisms of the family Enterobacteriaceae. Dysentery bacilli have no flagella and this is one of the differential characters between these organisms and bacteria of the coli-typhoid-paratyphoid group.

6. Dysentery bacilli Intracellular Shigella

7. Cultivation. Colonies of dysentery bacilli on Ploskirev's medium

8. Colonies of Salmonella on Mac-Conkey medium

9. Fermentative properties. None of the species of dysentery bacilli liquefy gelatin nor produce hydrogen sulphide. They ferment glucose, with acid formation, with the exception of the Newcastle subspecies which sometimes produce both acid and gas during this reaction. With the exception of the Sonne bacilli, none of them ferment lactose.

10. Shigellae Biochemical Properties

11. Test for determination of motility and producing hydrogen sulphide • S. flexneri – nonmotile, no produce hydrogen sulphide; • Enterobacter cloacea – motile, no produce hydrogen sulphide; • 3. Proteus mirabilis – motile, produce hydrogen sulphide.

12. Toxin production. S. dysenteriae produce thermolabile exotoxin which displays marked tropism to the nervous system and intestinal mucous membrane. This toxin may be found in old meat broth cultures, lysates of a 24-hour-old agar culture, and in desiccated bacterial cells. An intravenous injection of small doses of the exotoxin is fatal to rabbits and white mice. Such an injection produces diarrhoea, paralysis of the hind limbs, and collapse.

13. The dysentery exotoxin causes the production of a corresponding antitoxin. The remaining types of dysentery bacilli produce no soluble toxins. They contain endotoxins, which are of a gluco-lipo-protein nature, and occur in the smooth but not in the rough variants. Thermolabile substances exerting a neurotropic effect were revealed in some S. sonnei strains. They were extracted from old cultures by treating the latter with trichloracetic acid.

14. Antigenic structure. Dysentery bacilli are subdivided into 4 subgroups within which serovars may be distinguished. The antigenic structure of shigellae is associated with somatic O-antigens and surface K-antigens.

15. Antigens

16. Classification. Dysentery bacilli are differentiated on the basis of the whole complex of antigenic and biochemical properties. S. sonnei have four fermentative types which differ in the activity of ramnose and xylose and in sensitivity to phages and colicins.

17. Epidemiology and Pathogenesis of Shigellosis. Humans seem to be the only natural hosts for the shigellae, becoming infected after the ingestion of contaminated food or water. Unlike Salmonella, the shigellae remain localized in the intestinal epithelial cells, and the debilitating effects of shigellosis are mostly attributed to the loss of fluids, electrolytes, and nutrients and to the ulceration that occurs in the colon wall.

18. Pathogenesis of shigellosis in humans

19. Shigella dysenteriae type 1 secreted one or more exotoxins (called Shiga toxins), which would cause death when injected into experimental animals and fluid accumulation when placed in ligated segments of rabbit ileum. The mechanism whereby Shiga toxin causes fluid secretion is thought to occur by blocking fluid absorption in the intestine. In this model, Shiga toxin kills absorptive epithelial cells, and the diarrhea results from an inhibition of absorption rather than from active secretion.

20. To cause intestinal disease, shigellae must invade the epithelial cells lining of the intestine. After escaping from the phagocytic vacuole, they multiply within the epithelial cells. Thus, Shigella virulence requires that the organisms invade epithelial cells, multiply intracellularly, and spread from cell to cell by way of finger-like projections to expand the focus of infection, leading to ulceration and destruction of the epithelial layer of the colon.

21. Gross pathology of shigellosis

22. Histopathology of acute colitis following peroral infection with shigellae.

23. Immunity. Immunity acquired after dysentery is specific and type-specific but relatively weak and of a short duration. For this reason the disease may recur many times and, in some cases, may become chronic. This is probably explained by the fact that Shigella organisms share an antigen with human tissues.

24. Laboratory diagnosis. Reliable results of laboratory examination depend, to a large extent, on correct sampling of stool specimens and its immediate inoculation onto a selective differential medium. The procedure should be carried out at the patient's bedside, and the plate sent to the laboratory.

25. Rules the correct procedure of material collection : • carry out bacteriological examination of faeces before aetiotropic therapy has been initiated; • collect faecal samples (mucus, mucosal admixtures) from the bedpan and with swabs (loops) directly from the rectum (the presence in the bedpan of even the traces of disin­fectants affects the results of examination); • inoculate without delay the collected material onto enrichment media, place them into an incubator or store them in preserving medium in the cold; • send the material to the laboratory as soon as possible.

26. Bacteriological examination. Faecal samples are streaked onto plates with Ploskirev's medium and onto a selenite medium containing phenol derivatives, beta-galactosides, which retard the growth of the attendant flora, in particular E. coli. The inoculated cultures are placed into a 37 °C incubator for 1S-24 hrs. The nature of tile colonies is examined on the second day. Colourless lactose-negative colonies are subcultured to Olkenitsky's medium or to an agar slant to enrich for pure cultures. On the third day, examine the nature of the growth on Olkenitsky's medium for changes in the colour of the medium column without gas formation. Subculture the material toHiss' media with malonate, arabinose, rhamnose, xylose, dulcite, salicine, and phenylalanine. Read the re­sults indicative of biochemical activity on the following day. Shigellae ferment carbohydrates with the formation of acid

27. To determine the species of Shigellae, one can employ the following tests: 1.Theagglutination test is performed first with a mixture of sera containing those species, and variants of Shigellae that are prevalent in a given area, and then the slide agglutination test with monoreceptor species sera. 2. The coagglutination test which allows to determine the specificity of the causative agent by a positive reaction with protein A of staphylococci coated with specific antibodies. On a suspected colony put a drop of specific sensitized protein A of Staphylococcus aureus, then rock the dish and 15 min later examine it microscopically for the appearance of the agglutinate (these tests may also be carried out on the second day of the investigation with the material from lactose-negative colonies). 3. Direct and indirect immunofluorescence test.

28. IFT: Salmonella enterica serovar Typhimurium inside (green) and on the surface (blue) of human intestinal epithelial cells. Actin is labelled in red.

29. 4.The indirect haemagglutination (IHA) test with erythrocyte diagnosticums with the titre of 1:160 and higher is performed. The test. is repeated after at least seven days. Diagnostically important is a four-fold rise in the anti­body litre, which can be elicited from the 10th-12th day of the disease. To distinguish between patients with subclinical forms of the disease and Shigella carriers, identify immunoglobulins of the G class. 5. ELISA. For the epidemiological purpose the phagovar and colicinovar of Shigellae are also identified. 6.To determine whether the isolated cultures belong to the genus Shigella, perform the keratoconjunctival test on guinea pigs. In contrast to causal organisms of other intestinal infections, the dysentery Shigellae cause marked keratitis.

30. 7. An allergic test consisting in intracutaneous injection of 0.1 ml of dysenterin is applied in the diagnosis of dysentery in adults and children. Hyperaemia and a papule 2 to 3.5 cm in diameter develop at the site of the injection in 24 hours in a person who has dysentery. The test is strictly specific. 8.An allergy intracutaneous test with Tsuverkalov's dysenterine is of supplementary significance. It becomes positive in dysentery patients beginning with the fourth day of the disease. The result is read in 24 hrs by the size of the formed papula. The test is consid­ered markedly positive in the presence of oedema and skin hyperaemia 35 mm or more in diameter, moderately positive if this diameter is 20-34 mm, doubtful if there is no papula and the diameter of skin hyperaemia measures 10-15 mm, and negative if the hyperaemic area is less than 10 mm. 9.The nature of the isolated culture may be determined in some cases by its lysis by a polyvalent dysentery phage

31. Treatment of Shigellosis • Intravenous replacement of fluids and electrolytes; • antibiotic therapy (ampicillin frequently is not effective, and alternativetherapies includesulfamethoxazole/trimethoprim and, the quinolone antibiotics such as nalidixic acid and ciprofloxacin)

32. Dysentery control is ensured by a complex of general and specific measures; (1) early and a completely effective clinical, epidemiological, and laboratory diagnosis; (2) hospitalization of patients or their isolation at home with observance of the required regimen; (3) thorough disinfection of sources of the disease; (4) adequate treatment of patients with highly effective antibiotics and use of chemotherapy and immunotherapy; (5) control of disease centres with employment of prophylaxis measures; (6) surveillance over foci and the application of prophylactic measures there; (7) treatment with a phage of all persons who were in contact with the sick individuals; (8) observance of sanitary and hygienic regimens in children's institutions, at home and at places of work, in food industry establishments, at catering establishments, in food stores.

33. Vibrio Cholerae Morphology.Cholera vibrios are shaped like a comma or a curved rod measuring 1-5 mcm in length and 0.3 mcm in breadth They are very actively motile, monotrichous, nonsporeforming, noncapsulated, and Gram-negative.

34. Gram’s stain Scanning electron micrograph V. cholerae

36. Cultivation. Colonies of V. cholerae on bismuth-sulphit-agar

37. Colonies of V. cholerae on blood agar

38. Fermentative properties. The cholera vibrio liquefies coagulated serum and gelatin; it forms indole and ammonia, reduces nitrates to nitrites, breaks down urea, ferments glucose, levulose, galactose, maltose, saccharose, mannose, mannite, starch, and glycerine (slowly) with acid formation but does not ferment lactose in the first 48 hours, and always coagulates milk. The cholera vibrio possesses lysin and ornithine decarboxylases and oxidase activity. B. Heiberg differentiated vibrios into biochemical types according to their property of fermenting mannose, arabinose, and saccharose.

40. Toxin production. • an exotoxin (cholerogen) which is marked by an enterotoxic effect • the endotoxin also exerts a powerful toxic effect fibrinolysin • hyaluronidase • collagenase • mucinase • lecithinase • neuraminidase • proteinases

41. Mechanism of action of cholera enterotoxin according to Finkelstein. Cholera toxin approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside. Conformational alteration of holotoxin occurs, allowing the presentation of the A subunit to cell surface. The A subunit enters the cell. The disulfide bond of the A subunit is reduced by intracellular glutathione, freeing A1 and A2. NAD is hydrolyzed by A1, yielding ADP-ribose and nicotinamide. One of the G proteins of adenylate cyclase is ADP-ribosylated, inhibiting the action of GTPase and locking adenylate cyclase in the "on" mode.

42. Cholera toxin activates the adenylate cyclase enzyme in cells of the intestinal mucosa leading to increased levels of intracellular cAMP, and the secretion of H20, Na+, K+, Cl-, and HCO3- into the lumen of the small intestine.

43. Antigenic Determinants of Vibrio cholerae

44. Pathogenesis and diseases in man. Cholera is undoubtedly the most dramatic of the water-borne diseases. The cholera vibrios are transmitted from sick persons and carriers by food, water, flies, and contaminated hands.

46. Cholera is characterized by a short incubation period of several hours to up to 6 days (in a disease caused by the El Tor vibrio it lasts three to five days), and the disease symptomsincludegeneral weakness, vomiting, and a frequent loose stool. The stools resemble rice-water and contain enormous numbers of torn-off intestinal epithelial cells and cholera vibrios. The major symptom of cholera is a severe diarrhea in which a patient may lose as much as 10 to 20 L or more of liquid per day. Death, which may occur in as many as 60% of untreated patients, results from severe dehydration and loss of electrolytes.

47. Phases in the development of the disease: 1. Cholera enteritis (choleric diarrhoea) which lasts 1 or 2 days. In some cases the infectious process terminates in this period and the patient recovers. 2. Cholera gastroenteritis is the second phase of the disease. Profuse diarrhoea and continuous vomiting lead to dehydration of the patient's body and this results in lowering of body temperature, decrease in the amount of urine excreted, drastic decrease in the number of mineral and protein substance, and the appearance of convulsions. The presence of cholera vibrios is revealed guite frequently in the vomit and particularly in the stools which have the appearance of rice water.

48. 3. Cholera algid which is characterized by severe symptoms. The skin becomes wrinkled due to the loss of water, cyanosis appears, and the voice becomes husky and is sometimes lost completely. The body temperature falls to 35.5-34° C. As a result of blood concentration cardiac activity is drastically weakened and urination is suppressed.

50. Immunity acquired after cholera is high-grade but of short duration and is of an anti-infectious (antibacterial and antitoxic) character. It is associated mainly with the presence of antibodies (lysins, agglutinins, and opsonins). The cholera vibrios rapidly undergo lysis under the influence of immune sera which contain bacteriolysins.