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

1. Chair of Medical Biology,Microbiology, Virology, and Immunology PATHOGENIC ENTEROBACTERIACEAE Lecturer As. Prof. O.V. Pokryshko

2. Classification of the Enterobacteriaceae Genera

5. The organism was isolated from feces in 1885 by T. Escherich. E. coli is a common inhabitant of the large intestine of humans and mammals. It is also found in the guts of birds, reptiles, amphibians, and insects. The bacteria are excreted in great numbers with the feces and are always present in the external environment (soil, water, foodstuffs, and other objects).

6. Escherichia coli. Morphology. E coli are straight rods measuring 0.4-0.7 in breadth and 1-3 in length. There are motile and non-motile types.

7. Scanning electron micrograph

9. Cultivation. Colonies of E. coli on meat-peptone agar

10. Colonies of E. coli on Endo's medium

11. Colonies of E. colion Ploskirev's medium

12. Colonies of E. coli on blood agar

13. Escherichia coli is highly motile and will show turbidity throughout the tube.

14. Fermentative properties. Positive (left) reactions of isolates E. coli in glucose fermentation broth. Note the formation of acid (yellow color) and gas. Observe the bubble in the Durham tube. “+” -vetest “—” -vetest

15. Indole reaction A.Salmonella B. E. coli is the positive microbe. A B

16. Note the bubble formation. Catalase positive E. coli can reduce nitrate to nitrite.

17. Toxin production. • a gluco-lipo-protein complex with which their toxic, antigenic, and immunogenic properties • endotoxins • thermolabile neurotropic exotoxins • haemotoxins • pyrogenic substances, • proteinases, • deoxyribonucleases, urease, • phosphatase • hyaluronidase • aminoacid decarboxylases

18. Antigenic structure. The antigenic structure of E. coli is characterized by variability and marked individuality. Along with the H- and O-antigens, the presence of other antigens has been shown in some strains, i.e. the surface somatic (membranous, capsular) K-antigens which contain the thermolabile L- and B-antigens and the thermostable A- and M-antigens. On the basis of antigenic structure an antigenic formula is derived which fully reflects the antigenic properties of the strain For example, one of the most widely spread serotypes is designated 0111 : K58 : H2.

19. Pathogenesis of E. coli diarrheal disease

20. Pathogenesis and diseases in man. • Definite E. coli serogroups are capable of causing various acute intestinal diseases in humans: • the causative agents of colienteritis in children are O-groups-25, -26, -44, -55, -86, -91, -111, -114, -119, -125, -126, -127, -128, -141, -146, and others (they cause diseases in infants of the first months of life and in older infants); • the causative agents of dysentery-like diseases are E. coli of the O-groups-23, -32, -115, -124, -136, -143, -144, -151, and others; • the causative agents of cholera-like diarrhoea are the O-groups-6, -15, -78, -148, and others, they produce thermolabile and thermoresistant enterotoxins.

23. Escherichia coli Virulence Factors

24. Enterotoxigenic Escherichia coli. Enterotoxin-producing E coli, called enterotoxigenic E.coli (ETEC), produce one or both of two different toxins – a heat labile toxin called LT and a heat-stable toxin called ST.

25. Enterohemorrhagic Escherichia coli(EHEC) is the etiologic agent of hemorrhagic colitis, a disease characterized by severe abdominal cramps and a copious, bloody diarrhea. These organisms are also known to cause a condition termed hemolytic-uremic syndrome (HUS), which is manifested by a hemolytic anemia, thrombocytopenia (decrease in the number of blood platelets), and acute renal failure.

26. Enteroinvasive Escherichia coli. The disease produced by the enteroinvasive E. coli (EIEC) is indistinguishable from the dysentery produced by members of the genus Shigella, although the shigellae seem to be more virulent because considerably fewer shigellae are required than EIEC to cause diarrhea. The key virulence factor required by the EIEC is the ability to invade the epithelial

27. Enteropathogenic Escherichia coli(EPEC). The ability of the EPEC to cause diarrhea. EPEC strains routinely have been considered noninvasive, but data have indicated that such strains can invade epithelial cells in culture. However, EPEC strains do not typically cause a bloody diarrhea, and the significance of cell invasion during infection remains uncertain.

31. Immunity. In individuals who had suffered from diseases caused by pathogenic E. coli serovars, cross immunity is not produced owing to which re-infection may occur.

32. Laboratory diagnosis. Tested material:the patients' faeces, throat and nasal discharges, material obtained at autopsy (blood, bile, liver, spleen, lungs, contents of the small and large intestine, pus), water, foodstuffs, and samples of washings from objects and hands of staff of maternity hospitals, hospitals, and dairy kitchens

33. The tested material is inoculated onto solid nutrient media (Endo's, Levin's) and, simultaneously, onto Ploskirev's media. Blood is first inoculated into broth and then subcultured on solid media when development of a septic process is suspected. The pure culture isolate is identified by its morphological, cultural, biochemical, serological, and biological properties.

34. The corresponding O-group to which an enteropathogenic-serovars belong is determined by means of the agglutination reaction. Besides, the immunofluorescence method employing type specific labelled sera is also used. It yields a preliminary answer in one to two hours. In serological diagnosis of colienteritis beginning with the third to fifth day of the disease the indirect haemagglutination reaction is used which excels the agglutination reaction in sensitivity.

35. E. coli by immunofluorescence method

36. Treatment. • Patients with colienteritis are prescribed • antibiotics (tetracycline with vitamins C, B1 and B2) • biopreparations (coli autovaccine, coli bacteriophage, colicin, bacterin, lactobacterin, bificol, bifidumbacterin). • Physiological solutions with glucose are injected for controlling toxicosis.

37. Prophylaxis. To prevent diseases caused by pathogenic serovars of E. coli, special attention is given to early identification of individuals suffering from colienteritis, and also to their hospitalization and effective treatment. Regular examination of personnel is necessary in children's institutions as well as of mothers whose children are suffering from dyspepsia. Considerable importance is assigned to observation of sanitary regulations in children's institutions, infant-feeding centres, maternity hospitals, and children's nurseries. Protection of water and foodstuff's from contamination with faeces, the control of flies, and gradual improvement of standards of hygiene of the population are also particularly important.

38. Sanitary significance of E. coli. This organism is widely spread in nature. It occurs in soil, water, foodstuff's, and on various objects. For this reason E. coli serves as an indicator of faecal contamination of the external environment. Detection of E. coli is of great importance in estimating the sanitary index of faecal contamination of water, foodstuff's, soil, beverages, objects, and hand-washings. The degree of contamination of water, soil and foodstuff's is determined by the coli titre or coli index (these terms have been discussed in the chapter concerning the spread of microbes in nature).

39. Salmonella Enteric Fever and Paratyphoid Salmonellae: Salmonella typhi Salmonella paratyphiA Salmonella schottmuelleri (S. paratyphi B)

40. Salmonella Morphology. The morphology of the typhoid and paratyphoid salmonella corresponds with the general characteristics of the Enterobacteriaceae family. Most of the strains are motile and possess flagella, from 8 to 20 in number.

41. Salmonella typhi Scanning electron micrograph Gram’s staining

42. Cultivation. Colonies of S. paratyphi on Ploskirev's medium

43. Colonies of S. typhus on Ploskirev's medium

44. Colonies of Salmonella on Mac-Conkey medium

45. Colonies of Salmonella on CLED medium

46. Colonies of S. typhus on on bismuth-sulphite agar

47. Fermentative properties.

48. Toxin production. S. typhi contains gluco-lipo-protein complexes. The endotoxin is obtained by extracting the bacterial emulsion with trichloracetic acid. This endotoxin is thermostable, surviving a temperature of 120° C for 30 minutes, and is characterized by a highly specific precipitin reaction and pronounced toxic and antigenic properties. Investigations have shown the presence of exotoxic substances in S. typhi which are inactivated by light, air, and heat (80° C), as well as enterotropic toxin phosphatase, and pyrogenic substances.

49. Antigenic structure. S. typhi possesses a flagella H-antigen and thermostable somatic O- and Vi-antigens. All three antigens give rise to the production of specific antibodies in the body, i. e. H-, O-, and Vi-agglutinins. H-agglutinins bring about a large-flocculent agglutination, while 0- and Vi-agglutinins produce fine-granular agglutination.