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

1. Chair of Microbiology, Virology, and Immunology Escherichia. Klebsiella.

2. The Enterobacteriaceae contain gram negative rods, which, if motile, are peritrichously flagellated. Because members of this family are morphologically and metabolically similar, much effort has been expended to develop techniques for their rapid identification. In general, biochemical properties are used to define a genus, and further subdivision frequently is based on sugar fermentation and antigenic differences.

3. Classification of the Enterobacteriaceae Genera

5. O ANTIGENS. All gram-negative bacteria possess a lipopolysaccharide (LPS) as a component of their outer membrane. This toxic LPS (also called endotoxin) is composed of three regions, lipid A, core, and are peating sequence of carbohydrates called the O antigen. Based on different sugars, alpha- or beta-glycosidic linkages, and the presence or absence of substituted acetyl groups, Escherichia coil can be shown to possess at least 173 different O antigens, and 64 have been described in the genus Salmonella.

6. K ANTIGENS. K antigens exist as capsule or envelope polysaccharides and cover the O antigens when present, inhibiting agglutination by specific O antiserum.Most K antigens can be removed by boiling the organisms in water.

7. H ANTIGENS. Only organisms that are motile possess H antigens because these determinants are in the proteins that makeup the flagella. However, to complicate matters, members of the genus Salmonella alternate back and forth to form different H antigens. The more specific antigens are called phase 1 antigens and are designated by lower-case letters (a, b, c, and so on), whereas the less-specific phase 2 H antigens are given numbers.

8. Escherichia coli. Morphology. E coli are straight rods measuring 0.4-0.7 in breadth and 1-3 in length. They occur as individual organisms or in pairs and are marked by polymorphism. There are motile and non-motile types. The G+C content in DNA is 50-51 per cent. The cell surface has pili on which certain phages are adsorbed. The microcapsule is not always clearly defined.

13. Fermentative properties. E. coli does not liquefy gelatin. It produces indole and hydrogen sulphide, and reduces nitrates to nitrites; ferments glucose, levulose, lactose, maltose, mannitol, arabinose, galactose, xylose, rhamnose, and occasionally saccharose, raffinose, dulcitol, salycin, and glycerin, with acid and gas formation. It also coagulates milk. There are varieties of the bacteria which ferment saccharose, do not produce indole, have no flagella, and do not ferment lactose.

14. Toxin production. - a gluco-lipo-protein complex with which their toxic, antigenic, and immunogenic properties are associated; - haemolytic properties (O124 and others) - endotoxins and thermolabile neurotropic exotoxins; - haemotoxins and pyrogenic substances, proteinases, deoxyribonucleases, urease, phosphatase, hyaluronidase, amino acid decarboxylases

16. Escherichia coif Virulence Factors

17. Pathogenesis and diseases in man. Definite E. coli serogroups are capable of causing various acute intestinal diseases in humans: (1) 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); (2) the causative agents of dysentery-like diseases are E. coli of the O-groups-23, -32, -115, -124, -136, -143, -144, -151, and others; (3) the causative agents of cholera-like diarrhoea are the O-groups-6, -15, -78, -148, and others, they produce thermolabile and thermoresistant enterotoxins.

22. Laboratory diagnosis. 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 are all used for laboratory examination during colienteritis. If possible, faecal material should be seeded immediately after it has been collected. The throat and nasal discharges are collected with a sterile swab. Specimens of organs obtained at autopsy are placed in separate sterile jars.

23. Enteric Fever, Paratyphoid Salmonellae

25. Morphology. The morphology of the typhoid 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. It is possible that the flagella form various numbers of bunches.

31. Toxin production. S. typhi contains gluco-lipoprotein 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.

32. Antigenic structure. S. typhi possesses a flagellar H-antigen and thermostable somatic 0- 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.

34. Classification. At present, about 2000 species and types of this genus are known. F. Kauffmann and P. White classified the typhoid-paratyphoid salmonellae into a number of groups according to antigenic structure and determined 65 somatic O-antigens. For instance, S. typhi (group D) contains three different O-antigens — 9, 12, and Vi. S. paratyphi A alone constitutes group A, and S. schottmuelleri belongs to group B. It has been proved by F. Andrewes that the flagellar H-antigen is not homogeneous but is composed of two phases: phase 1 is specific and agglutinable by specific serum, phase 2 is non-specific and agglutinable not only by specific, but also by group sera. Salmonellae, which possess two-phase H-antigens, are known as diphasic, while those which possess only the specific H-antigen are monophasic.

35. Pathogenesis and diseases in man. The causative agent is primarily located in the intestinal tract. Infection takes place through the mouth (digestive stage). Cyclic recurrences and development of certain pathophysiological changes characterize the pathogenesis of typhoid fever and paratyphoids. There is a certain time interval after the salmonellae penetrate into the intestine, during which inflammatory processes develop in the isolated follicles and Peyer's patches of the lower region of the small intestine (invasive stage).

36. As a result of deterioration of the defence mechanism of the lymphatic apparatus in the small intestine the organisms enter the blood (bacteriemia stage). Here they are partially destroyed by the bactericidal substances contained in the blood, with endotoxin formation. During bacteraemia typhoid salmonellae invade the patient's body, penetrating into the lymph nodes, spleen, bone marrow, liver, and other organs (parenchymal diffusion stage). This period coincides with the early symptoms of the disease and lasts for a week.

37. On the third week of the disease a large number of typhoid bacteria enter the intestine from the bile ducts and Lieberkuhn's glands. Some of these bacteria are excreted in the faeces, while others reenter the Peyer's patches and solitary follicles, which had been previously sensitized by the salmonellae in the initial stage. This results in the development of hyperergia and ulcerative processes. Lesions are most pronounced in Peyer's patches and solitary follicles and may be followed by perforation of the intestine and peritonitis (excretory and allergic stage). The typhoid-paratyphoid salmonellae together with products of their metabolism induce antibody production and promote phagocytosis. These processes reach their peak on the fifth-sixth week of the disease and eventually lead to recovery from the disease.

38. Clinical recovery (recovery stage) does not coincide with the elimination of the pathogenic bacteria from the body. The majority of convalescents become carriers during the first weeks following recovery, and 3-5 per cent of the cases continue to excrete the organisms for many months and years after the attack and, sometimes, for life. Inflammatory processes in the gall bladder (cholecystitis) and liver are the main causes of a carrier state since these organs serve as favourable media for the bacteria, where the latter multiply and live for long periods. Besides this, typhoid-paratyphoid salmonellae may affect the kidneys and urinary bladder, giving rise to pyelitis and cystitis. In such lesions the organisms are excreted in the urine.

39. Immunity. Immunity acquired after typhoid fever and paratyphoids is relatively stable but relapses and reinfections sometimes occur. Antibiotics, used as therapeutic agents, inhibit the immunogenic activity of the pathogens, which change rapidly and lose their O- and Vi-antigens.

40. Laboratory diagnosis. The present laboratory diagnosis of typhoid fever and paratyphoids is based on the pathogenesis of these diseases. 1. Isolation of haemoculture. Bacteraemia appears during the first days of the infection.

41. 2. Serological method. Sufficient number of agglutinins accumulate in the blood on the second week of the disease, and they are detected by the Widal reaction. Diagnostic typhoid and paratyphoid A and B suspensions are employed in this reaction. The fact that individuals treated with antibiotics may yield a low titre reaction must be taken into consideration. The reaction is valued positive in patient's serum in dilution 1 : 200 and higher.

42. 3. A pure culture is isolated from faeces and urine during the first, second, and third weeks of the disease. The test material is inoculated into bile broth, Muller's medium, Ploskirev's medium, or bismuth sulphite agar.

43. Treatment. Patients with typhoid fever and paratyphoids are prescribed chloramphenicol, oxytetracycline, and nitrofuran preparations. These drugs markedly decrease the severity of the disease and diminish its duration. Great importance is assigned to general non-specific treatment (dietetic and symptomatic). The eradication of the organisms from salmonellae carriers is a very difficult problem.

44. Prophylaxis. General measures amount to rendering harmless the sources of infection. This is achieved by timely diagnosis, hospitalization of patients, disinfection of the sources, and identification and treatment of carriers. Of great importance in prevention of typhoid fever and paratyphoids are such measures as disinfection of water, safeguarding water supplies from pollution, systematic and thorough cleaning of inhabited areas, fly control, and protection of foodstuff's and water from flies. Washing of hands before meals and after using the toilet is necessary. Regular examination of personnel in food-processing factories for identification of carriers is also extremely important.

45. In the presence of epidemiological indications specific prophylaxis of typhoid infections is accomplished by vaccination. Several varieties of vaccines are prepared: typhoid vaccine (monovaccine), typhoid and paratyphoid B vaccine (divaccine).