Bohomolets Microbiology Lesson #7


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Bohomolets Microbiology Lesson #7

  1. 1. LessonN7: LABORATORY DIAGNOSIS OF SHIGELLOSIS (BACTERIAL DYSENETERY)1.Scientifically methodical ground of theme Shigellosis is a disease caused by bacteria of genus Shigella, is distributed worldwide and is an important diarrhealillness wherever sanitary practices are lacking. Reported cases in the United States have averaged about 21,500 peryear.2.Educational purposeSTUDENTS MUST KNOW: 1. Structure, staining properties and cultivation of Shigellae 2. Antigenic structure and classification of Shigellae. 3. Fermentative properties and toxin production of Shigellae. 4. Epidemiology and pathogenesis shigellosis. 5. Methods of laboratory diagnosis of shigellosis, main methods of prophylaxis and treatment.STUDENTS SHOULD BE ABLE TO: – prepare the smears from tested material; – stain the smears by Gram’s technique; – make microscopical examination of the smears; – value the growth of Shigellae on different media; – value the biochemical properties of Shigellae according to Hiss’media. – carry out agglutination test for identification of Shigellae;3.Chart of topic content. The causative agent of dysentery was described in 1888 by A. Chantemesse and in 1891 by A. Grigoryevand F. Widal. In 1898 this organism was studied in detail by K. Shiga in Japan and in 1900-1901 by V. Kruse inGermany (Shiga bacillus). Morphology. Morphologically dysentery bacilli correspond to the organisms of the familyEnterobacteriaceae. Dysentery bacilli have no flagella and this is one of the differential characters between theseorganisms and bacteria of the coli-typhoid-paratyphoid group. Cultivation. Dysentery bacilli are facultatively aerobic and grow readily on common media at pH 6.7-7.2,the optimum temperature for growth being 37° C, they do not grow at 45° C. On solid media they form small (1-1.5mm in diameter), fragile, semitransparent colonies which are similar to those of the typhoid bacteria. In meat brothdysentery bacilli produce a diffuse turbidity. Fermentative properties. None of the species of dysentery bacilli liquefy gelatin nor produce hydrogensulphide. They ferment glucose, with acid formation, with the exception of the Newcastle subspecies whichsometimes produce both acid and gas during this reaction. With the exception of the Sonne bacilli, none of themferment lactose. Toxin production. S. dysenteriae produce thermolabile exotoxin which displays marked tropism to thenervous system and intestinal mucous membrane. This toxin may be found in old meat broth cultures, lysates of a24-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 injectionproduces diarrhoea, paralysis of the hind limbs, and collapse. The dysentery exotoxin causes the production of a corresponding antitoxin. The remaining types ofdysentery bacilli produce no soluble toxins. They contain endotoxins, which are of a gluco-lipo-protein nature, andoccur in the smooth but not in the rough variants. Antigenic structure. Dysentery bacilli are subdivided into 4 subgroups within which serovars may bedistinguished. The antigenic structure of shigellae is associated with somatic 0-antigens and surface K-antigens. Classification. Dysentery bacilli are differentiated on the basis of the whole complex of antigenic (Table 1)and biochemical (Table 2) properties. S. sonnei have four fermentative types which differ in the activity of ramnoseand xylose and in sensitivity to phages and colicins. Resistance. Dysentery bacilli live in the external environment for a period of 5-10 days (in soil, foodstuffsand water, and on objects, plates and dishes). Direct sunlight and a 1 per cent phenol solution destroy the organismsin 30 minutes and at a temperature of 60° C the organisms perish in 10 minutes. The bacilli are easily killed bytreatment with chloramine and calcium chloride solutions. The Shiga bacilli are most sensitive to physical andchemical factors, while the Sonne bacilli are relatively resistant to them. Dysentery bacilli may acquire resistanceto drugs (sulphonamides, antibiotics) and to ionizing radiation. 1
  2. 2. Pathogenicity for animals. Monkeys are susceptible to dysentery bacilli. They contract the infection fromsick people or carriers in the nurseries. In some cases they become sources of contamination of personnel innurseries and zoological gardens. Table 1 International Classification of Shigellae Subgroup Species and Subserotype Antigenic formula serotype s Type Group antigen antigens A. Does S. not ferment dysenteriae, mannite 1-12 B. S. flexneri 1a I 2,4 Ferments 1 1b I:S 6:2,4 mannite as 2 2a II 3,4 a rule 2b II 7,8 3 3a III 6:7,8 3b III 6:3,4 3c III 6 4 4a IV B:3,4 4b IV B:6:3,4 5 V 7,8 V (3,4) 6 Some strains VI 2,4 ferment glucose with acid and gas – formation – X variant 7,8 Y variant 3,4 C. S. boydii, Ferments 1-18 mannite as a rule D. S. sonnei Ferments mannite, slowly lactose and saccharose Parenteral infection causes fatal toxicosis in rabbits. An intravenous injection of a S. dysenteriae cultureexerts a highly toxic effect. The resulting infection constitutes diarrhoea, paresis or paralysis of thelimbs, followed by collapse and death. Autopsy reveals hyperaemia of the intestinal mucous membrane,haemorrhages, necrosis, and ulcerations. Infected white mice die within the first four days. When cultures of virulent dysentery bacilli are introduced into the respiratory tract of white mice, theorganisms multiply. However, attempts to reproduce dysentery in white mice are of no success. Kittens andpuppies are more susceptible. Guinea pigs display low susceptibility to dysentery bacilli, but infection through theeye conjunctiva results in keratoconjunctivitis which is assumed to be a specific lesion. Table 2 2
  3. 3. Shigellae Biochemical Properties Indole production Ornithine de-carboxylation Catalase Fermentation of carbohydrates glucose mannite succrose lactose dulcite Subgroup S. dysenteriae – + – – – – – – –A S. fiexneri – B – + + + – – – + S. boydii – C – + + + – + – – S. sonnei – D + + + – + – + + slowly slowly Note: “+”, fermentation of carbohydrates, formation of undol and catalase; “–”, the absence of carbohydratefermentation and indol and ornithine formation; “±”, weak formation of indol and ornithine and weak carbohydratefermentation. 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 remainlocalized in the intestinal epithelial cells, and the debilitating effects of shigellosis are mostly attributed to the lossof fluids, electrolytes, and nutrients and to the ulceration that occurs in the colon wall. It has been known for many years that Shigella dysenteriae type 1 secreted one or more exotoxins (calledShiga toxins), which would cause death when injected into experimental animals and fluid accumulation whenplaced in ligated segments of rabbit ileum. These toxins are essentially identical to the Shiga-like toxins producedby the EIEC and the EHEC. Thus, Shiga toxin consists of one A subunit and five B subunits and seems to kill anintestinal epithelial cell by inactivating the 60S ribosomal subunit, halting all protein synthesis. Moreover, althoughall virulent species of Shigella produce Shiga toxins, there seems to be a wide variation in the amount of toxinformed. The mechanism whereby Shiga toxin causes fluid secretion is thought to occur by blocking fluid absorptionin the intestine. In this model, Shiga toxin kills absorptive epithelial cells, and the diarrhea results from aninhibition of absorption rather than from active secretion. Of note is that, like the EHEC, Shigella species can cause HUS. Moreover, Shiga-like toxins have beendetected in certain strains of Vibrio cholerae and Vibrio parahaemolyticus that were associated with HUS,indicating an important role of Shiga toxin in this malady. There has also been a report indicating that tumornecrosisfactor-alpha acts synergistically with Shiga toxin to induce HUS. To cause intestinal disease, shigellae must invade the epithelial cells lining of the intestine. After escapingfrom the phagocytic vacuole, they multiply within the epithelial cells in a manner similar to that described for EIECstrains. Thus, Shigella virulence requires that the organisms invade epithelial cells, multiply intracellularly, andspread from cell to cell by way of finger-like projections to expand the focus of infection, leading to ulceration anddestruction of the epithelial layer of the colon. Interestingly, for Shigella to be fully invasive, both plasmid andchromosomally encoded products seem to be required. The invasion plasmids is identical for the Shigella and theEIEC and contains at least four genes, IpaA, IpaB, IpaC, and IpaD that encode for a series of proteins termedinvasion-plasmid antigens, which arc involved in the virulence of these organisms. Interestingly, IpaB acts both asan invasin that triggers phagocytosis of the bacterium and as a cytolysin that allows escape from the phagocyticvacuole. The elaboration of toxic products causes a severe local inflammatory response involving bothpolymorphonuclear leukocytes and macrophages, resulting in a bloody, mucopurulent diarrhea. During 1990, over 27,000 cases of shigellosis were reported to the Centres for Disease Control (CDC) and,of these, the most prevalent species in the United States was S sonnei. The disease produced by this species istransmitted by a fecal-oral route, and most of patients are preschool-age children, particularly those in day-carecentres. Immunity. Immunity acquired after dysentery is specific and type-specific but relatively weak and of a shortduration. For this reason the disease may recur many times and, in some cases, may become chronic. This isprobably explained by the fact that Shigella organisms share an antigen with human tissues. 3
  4. 4. Laboratory diagnosis. Reliable results of laboratory examination depend, to a large extent, on correctsampling of stool specimens and its immediate inoculation onto a selective differential medium. The procedureshould be carried out at the patients bedside, and the plate sent to the laboratory. In hospital conditions the stool is collected on a paper plate or napkin, placed into a bedpan. The latter shouldbe washed previously with running water or, better still, with boiling water, be dry, and should contain nodisinfectants. It is best to collect the faeces directly from the rectum by means of a rectal tube or rectal swab. Thespecimen should be sown in the isolation department immediately after collection. Portions of the stool, containingpus and mucus, are picked out with a swab and plated on Ploskirevs medium. The plates are incubated at 37°C for24 hours The isolated pure culture is identified by its biochemical and serological properties. An accelerated method of dysentery diagnosis is employed to shorten the examination period. In some casesan agglutination reaction, similar to the Widal reaction, is used. This test is relevant to retrospective diagnosis. The nature of the isolated culture may be determined in some cases by its lysis by a polyvalent dysenteryphage and by the reaction of passive haemagglutmation as well as by the method of immunofluorescence. Thismethod is used for demonstrating antigens of Shigella organisms in smears from faeces or in colonies by means ofspecific sera treated with fluorochromes. An allergic test consisting in intracutaneous injection of 0.1 ml of dysenterin is applied in the diagnosis ofdysentery in adults and children. Hyperaemia and a papule 2 to 3.5 cm in diameter develop at the site of theinjection in 24 hours in a person who has dysentery. The test is strictly specific. Treatment and Control of Shigellosis. Intravenous replacement of fluids and electrolytes plus antibiotictherapy are used for severe cases of shigellosis. Ampicillin frequently is not effective, and alternative therapiesinclude sulfamethoxazole/trimethoprim and, with increasing sulfamethoxazole/trimethoprim resistance, thequinolone antibiotics such as nalidixic acid and ciprofloxacin. In the Far East, India, and Brazil where shigellosis ismore common than in the United States, multiple antibiotic resistance because of the acquisition of plasmids hasbecome common. Shigellosis also is common in Latin America. Efforts to control the disease usually are directed toward sanitary measures designed to prevent the spread oforganisms. This is particularly difficult in view of the fact that many persons remain asymptomatic carriers afterrecovery from an overt infection. Such individuals provide a major reservoir for the spread of the shigellae. The injection of killed vaccines is worthless, because humoral IgG does not seem to be involved inimmunity to the localized intestinal infection. Live vaccines that cannot grow in the absence of streptomycin (ie,streptomycin-dependent vaccines) have been developed and used in clinical trials, but success has been equivocal.It seems that the organisms must invade and colonize the intestine to induce a local immunity. An engineeredvaccine designed to induce this type of immunity used an avirulent E coli K12 into which was transferred a 140-megadalton plasmid obtained from a virulent strain of Shigella flexneri. The transfected plasmid endowed the E.coli K12 strain with the ability to invade intestinal epithelial cells, and its use as an oral vaccine in monkeysconferred significant protection against oral challenge with virulent S flexneri. Acquired immunity seems to resultfrom both a cell-mediated immune response and an IgA antibody production. Interestingly, human milk contains a globotriaosylceramide that binds to Shiga and Shiga-like toxins. Thissuggests that human milk could contribute to a protective effect by preventing these toxins from binding to theirintestinal target receptors. Thus, dysentery control is ensured by a complex of general and specific measures; (1) early and a completelyeffective clinical, epidemiological, and laboratory diagnosis; (2) hospitalization of patients or their isolation athome with observance of the required regimen; (3) thorough disinfection of sources of the disease; (4) adequatetreatment of patients with highly effective antibiotics and use of chemotherapy and immunotherapy; (5) control ofdisease centres with employment of prophylaxis measures; (6) surveillance over foci and the application ofprophylactic measures there; (7) treatment with a phage of all persons who were in contact with the sickindividuals; (8) observance of sanitary and hygienic regimens in childrens institutions, at home and at places ofwork, in food industry establishments, at catering establishments, in food stores. Dysentery is an infectious disease with the predominant involvement of the large intestine and generalintoxication caused by bacteria of the genus Shigella: S. dysenteriae, S. sonnei, S. flexneri, S. boydii. Material used for isolating the causal organism of dysentery includes faeces of patients, convalescents, andcarriers, less frequently, vomited matter and waters from stomach and intestine lavage. Shigellae may be recoveredin washings off hands, cutlery and crockery, and various other objects (toys, door handles, etc.) as well as in milkand other foodstuffs. The results of laboratory examination depend to a large degree on the correct procedure ofmaterial collection. The following rules should be strictly adhered to: (1) carry out bacteriological examination offaeces before aetiotropic therapy has been initiated; (2) collect faecal samples (mucus, mucosal admixtures) fromthe bedpan and with swabs (loops) directly from the rectum (the presence in the bedpan of even the traces of disin- 4
  5. 5. fectants affects the results of examination); (3) inoculate without delay the collected material onto enrichmentmedia, place them into an incubator or store them in preserving medium in the cold; (4) send the material to thelaboratory as soon as possible. Bacteriological examination. Faecal samples are streaked onto plates with Ploskirevs medium and onto aselenite medium containing phenol derivatives, beta-galactosides, which retard the growth of the attendant flora, inparticular E. coli. The inoculated cultures are placed into a 37 °C incubator for 1S-24 hrs. The nature of tilecolonies is examined on the second day. Colourless lactose-negative colonies are subcultured to Olkenitskys medium or to an agar slant to enrich forpure cultures. On the third day, examine the nature of the growth on Olkenitskys medium for changes in the colourof the medium column without gas formation. Subculture the material to Hiss media with malonate, arabinose,rhamnose, xylose, dulcite, salicine, and phenylalanine. Read the results indicative of biochemical activity on thefollowing day. Shigellae ferment carbohydrates with the formation of acid (Table 2). For serological identification the agglutination test is performed first with a mixture of sera containing thosespecies, and variants of Shigellae that are prevalent in a given area, and then the slide agglutination test withmonoreceptor species sera. To determine the species of Shigellae, one can employ the following tests: 1. Direct and indirect immunofluorescence test. 2. The coagglutination test which allows to determine the specificity of the causative agent by a positivereaction with protein A of staphylococci coated with specific antibodies. On a suspected colony put a drop ofspecific sensitized protein A of Staphylococcus aureus, then rock the dish and 15 min later examine itmicroscopically for the appearance of the agglutinate (these tests may also be carried out on the second day of theinvestigation with the material from lactose-negative colonies). 3. Another test, which is highly specific for dysentery, is ELISA. For the epidemiological purpose thephagovar and colicinovar of Shigellae are also identified. To determine whether the isolated cultures belong to the genus Shigella, perform the keratoconjunctival teston guinea pigs. In contrast to causal organisms of other intestinal infections, the dysentery Shigellae cause markedkeratitis. Depending on the findings obtained, the presence of Shigella bacteria in the test material is either confirmedor ruled out. For the serological diagnosis of dysentery the indirect haemagglutination (IHA) test with erythrocytediagnosticums 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 thedisease. To distinguish between patients with subclinical forms of the disease and Shigella carriers, identifyimmunoglobulins of the G class. An allergy intracutaneous test with Tsuverkalovs dysenterine is of supplementary significance. It becomespositive in dysentery patients beginning with the fourth day of the disease. The result is read in 24 hrs by the size ofthe formed papula. The test is considered markedly positive in the presence of oedema and skin hyperaemia 35 mmor more in diameter, moderately positive if this diameter is 20-34 mm, doubtful if there is no papula and thediameter of skin hyperaemia measures 10-15 mm, and negative if the hyperaemic area is less than 10 mm. Another technique that can be employed is determination of the indicator of neutrophil damage in thepresence of dysenterine. Examination of water, milk, and washings off various objects for Shigellae is conducted utilizing the abovementioned techniques. Of especial importance for examination of these objects is the test aimed at determining theincrease in the phage litre, which is also employed for demonstration of Shigella bacteria in the patients faeces. To carry out this test, the indicator phages and reference strains of Flexners and Sonnes Shigella bacteria areused. A rise in the phage titre by 3-5 orders (4-) is considered as weak positive reaction, by 5-7 orders (++) and7-10 orders (+++), positive, and by over 10 orders (++++), markedly positive. The immunofluorescence test for Shigella recovery is employed in examining objects containing minoramounts of the causative agents and for rapid laboratory diagnosis of dysentery.4. Student’s independent study program 1. Structure, staining properties of causative agents of shigellosis. Cultivation. 2. Antigenic structure, classification, biochemical properties of shigellae. 3. What virulence factors provide Shigellae pathogenicity? 4. Resistance of Shigellae. 5. Epidemiology of shigellosis. 6. Pathogenesis of disease in man. Main clinical findings. 5
  6. 6. 7. Laboratory diagnosis of dysentery: a – rules of material collection and features of inoculation of tested material onto nutrient media; b – bacteriological method of diagnosis; c – phagotyping and colicinotyping of Shigellae, their value; d – serological method of diagnosis (agglutination test, IHAT); e – allergic method; 8. Specific and nonspecific prophylaxis of shigellosis. Treatment. 6. Students’ practical activities: 1. To study under microscope morphology of various Shigellae species. 2. To study peculiarities of Shigellae growth on MPA, Levin’s and Ploskirev’s media, and in MPB. 3. To study biochemical properties of Shigellae in Hiss’media. 4. To study growth of Shigellae on Ploskirev’s medium, subcultivate colonies onto slant MPAExamine colonies on plate’s media. Select suspected colonies, check purity and inoculate the agar slope for enrichof culture. Shigellae do not ferment lactose and form pale (colorless) small, semitransparent,colonies. 6. To determine species and serovar of Shigellae in agglutination test with group and subtypes sera. 7. Control questions and tests III. Tests and Assignments for self– assessment Select the correct answers: 1. Shigellae are: a – gram-negative; b – peritrichates; c – nonmotile; d – gram-positive; e – without capsule. 2. Growth of Shigellae on differential diagnostic media: a – on Endo’s medium the colonies are semitransparent colorless; b – on Levin’s medium the colonies areviolet; c – on Ploskirev’s medium the colonies are red; d – on Ploskirev’s medium the colonies are colourless; e –on Endo’s medium the colonies are red. 3. Biochemical activities of Shigellae: a – S. flexneri ferments mannite; b – S. dysenteriae ferments mannite; c – S. boydii ferments mannite; d –S. dysenteriae ferments a glucose with gas; e – S. flexneri ferments lactose. 4. Resistance of Shigellae: a – S. sonnei is resistance; b – S. flexneri can survive in drinking tap water about 2 months; c – S.dysenteriae is most sensitive; d – S. sonnei survives in water about 2 months; e – S. flexneri in drinking watersurvives about 2 weeks. 5. Pathogenicity of Shigellae is stipulated for: a – adhesins; b – production of cytotoxins; c – production of leukotoxin; d – formation of endotoxin; e –intracellular reproduction. 6. Rules of material collection for bacteriological examination: a – to collect feces on fasting stomach; b – to collect feces before ethiotropic therapy; c – to take feces(mucus, mucosal admixtures, “saga boluses of mucus”); d – it is necessary to wash a bedpan with disinfectantsolution; e – it is necessary to streak the feces immediately onto nutrient media. 8. For serological diagnosis of shigellosis such tests are used: a – IHAT; b – agglutination test; c – flocculation test; d – determination of antibodies in a spit; e –precipitation test. 9. Stool culture reveled S.zonne. What additional researches of isolated culture should be carried out todetermine the source of infection? A. Drugs susceptibility tests B. Phagotyping C. Agglutination testing D. Complement fixation reaction. E. Neutralization testing 10. Stool culture from a patient with typical clinical picture of shigelosis didn’t reveal shigella due to earlytreatment with antibiotics. But there is four times increase of antishigella antibody titer between the first and thesecond test serum samples, which is obtained in PHAT. What does it mean? A. Vaccinal reaction B. Excludes diagnosis of shigelosis C. A patient was ill with dysentery before 6
  7. 7. D Nonspecific reaction E Confirms diagnosis of shigelosis 11. Stool culture from a patient with enteric infection revealed Shigella sonnei. Choose one of the followingtests, which could be used for identification of obtained pure culture? A. Complement fixation test B. Precipitation. C. Agglutination. D. Neutralization. E. Lysis test 12. A patient complained of nausea, liquid stool mixed with bloody fiber and mucus, fever, weakness. He washospitalized to the infectious department of the hospital. A doctor suspected shigellosis .What diagnostic testshould be administered to confirm this diagnosis? A. Culture . B..Serologic C..Microscopy D..Allergic E.Test on laboratory animals Real-life situation to be solved: 1. A patient N., was hospitalized to the hospital with complaints of a pain in the left iliac region, diarrheawith bloodstained and mucus feces, excruciating, pulling rectal pains, painful desire to defecate, tenesmus. A. What enteric disease may be in this patient? B. What tested material is it necessary to collect and what roles of this collection? C. Make scheme of laboratory examination. 2. After inoculation of the feces of the patient with acute dysentery the small, semitransparent,colourlesscolonies were grown on the Ploskirev’s medium. Some of them were inoculated on Hiss’s medium andagglutination test with mixture of Flexner’s and Sonnei’s sera were performed. Continue the laboratory diagnosis for determination of S. flexneri.7. List of literature:1. I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,chapter25, p.183-188. 7