1. Lesson N11: LABORATORY DIAGNOSIS of clostridiosis 1.Scientifically methodical ground of theme Clostridium is genus of gram-positive, spore â€“forming rods, that is widely distributed in nature. The large genus (over 120 species) is extremely varied in its habitats. Saprobic members reside in soil, sewage, vegetation and organic debris and commensals inhabit the bodies of humans and other animals. Infection caused by pathogenic speciesare not normally communicable but occur when spores are introduced into injured skin. Clostridial diseases can be divided into (1) wound and tissue infections, including myonecroses (gas gangrene), antibiotic-associated colitis, and tetanus and food intoxication of perfringens and botulism varieties. Most of these diseases are caused when exotoxins some of which are highly potent, act on specific cellular targets 2.Educational purpose STUDENTS MUST KNOW: 1. Structure, staining properties and cultivation of gas gangrene Clostridia ,C. tetani and C. botulinum. 2. Antigenic structure of Clostridia. 3. Fermentative properties and toxin production of Clostridia. 4. Ecology of gas gangrene Clostridia,C. tetani and C. botulinum. Resistance. Pathogenicity for animals. 5. Epidemiology and pathogenesis of gas gangrene, tetanus and botulism. 6. Methods of laboratory diagnosis of gas gangrene tetanus and botulism, main methods of prophylaxis and treatment STUDENTS SHOULD BE ABLE TO:• stain the smears by Gramâ€™s technique;• examine the smears under the microscope;• value the growth of Clostridia on different nutrient media;• inoculate spesimen into Kittâ€“Tarozzi medium or Vrublevsky medium• draw the scheme of laboratory diagnosis of tetanus and boptulism. 3.Chart of topic content. FigN1. Clostridia as human pathogens A. The gas gangrene group: 1.Established pathogens Cl. perfringens Cl.novyi Cl.septicum
2. 2. Less pathogenic Cl. hystolyticum Cl.fallax3. Doubtful pathogens Cl. bifermentans Cl. sporogenesB. Tetanus Cl. tetaniC. Food poisoning1. Gastroenteritis Cl. perfringens(typeA)2.Botulism Cl.botulinumD. Acute colitis Cl.difficileFigN2:Important species of clostridiumspecies Chief important to humans Description of roleCl. perfringens pathogen Principal cause of gas gangrene and myonecrosis; common agent in enterotoxigenic food poisoningCl.novyi pathogen Second most cause of gas gangreneCl.septicum pathogen Third most cause of gas gangreneCl. tetani pathogen Cause of tetanusCl.botulinum pathogen Cause of botulismCl.difficile opportunist Involved in antibiotic- associated colitisCl. iodofilum industrial uses Produces organic acids and alcohols for commertial useCl.acetobutylicum industrial uses Produces organic acids and alcohols and benzeneCl.butyricum industrial uses Produces butyric acid in butter and cheeseCl. cellobiovarum industrial uses Digest celluloseFigN3: Circumstances and events that may lead to gas gangreneTissue damage and contamination with clostridial spores and pyogenic microorganismsPresence of foreign bodies, including soil with damaging saltsInflammatory reaction
3. Extravasation of fluid, oedemaImpaired tissue perfusion; stasisPoor oxygenation, with reduction in local EhImpaired phagocytosis and intraleukocytic bactericidal mechanismsMultiplication of facultative bacteriaFurther reduction of Eh and pHGermination and out growth with production of toxinMultiplication of obligate anaerobes with production of toxins, aggressins and gas in affectedtissuesFurther impairment of local blood supply and extension of area of tissue damageProfound toxaemia and shock; severe hypotension; multiple organ failure; coma; deathFigN4: Mode of action Ð¡l. perfringens exotoxin (enzymatic lysis)Cl. perfringensAlpha toxin(phospholipase C)hydrolyses phosphoryichlorine in cell membraneCell deathCell membraneLysisFigN5: Mode of action Ð¡l.tetani exotoxin (effects on nerve-muscletransmission)Continuous stimulation by excitatory transmitterCl. tetani
4. ExcitatorytransmitterTetanospasminInhibitory transmitterrelease blockedInhibitory transmitterFigN6: Mode of action Ð¡l. botulinum exotoxin (effects on nerve-muscle transmission)Ð¡l. botulinumACH- containing vesiclesMotorend plateStimulation blockedToxin blocks release of ACHfrom vesiclesMuscle fibre4. Studentâ€™s independent study program 1. Structure, staining , biochemical properties of causative agent of tetanus, gas gangrene andbotulism. Cultivation. 2. Toxin production and virulence factors of these causative agents. 3. Ecology and resistance. 4. Epidemiology of tetanus, gas gangrene and botulism. Pathogenesis. Clinical pictures. 5. Main methods of laboratory diagnosis of clostridiosis.
5. 6. Active and passive prophylaxis of clostridiosis. Preparation, which are used for this purpose.Methods of their obtaining and determination of activity.5. Studentsâ€™ practical activities:1To study morphology of gas gangrene Clostridia, C. botulinum and C. tetani in smears from theculture and impression preparations (touch smears) from the tissues.Draw and record youobservation.Note forms and localization of spores of different Clostridia species, staining reaction by Gram. C.tetani appears by microscopy as a straight, slender rod with rounded ends.The spores aresperical, terminal and bulging, giving the bacteria characteristic â€œdrum stickâ€a appearance.Cl. botulinum with oval, subterminal and swelling the sporangium spore resembles tennis racquet.Ð¡l.tetani.Cl. botulinum2.To inoculate a specimen (wound exudate) into Kitt-Tarozzi (Vrublevskyi) medium, Wilson-Blair medium and milk.Tested material (wound exudate) introduce into into Kitt-Tarozzi (Vrublevskyi) medium, Wilson-Blair which has been cooled to 450 C and milk by bacterial loop (stab culture) 3. To familiarize with changing of media ( Kitt-Tarozzi (Vrublevskyi) medium, Wilson-Blair medium and milk) after cultivation Cl. perfringens. Into the Kitt-Tarozzi medium Cl. perfringens grow with intensive gas formation. On litmus milkone can observe characteristic fermentation with lightening of the serum and formation of asponge clot of brick colour (â€œstormy clotâ€i reaction). In the Wilson-Blair medium occursblack growth with ruptures. These changing may be observed after 3-6 hours of growth Cl. perfringens. Therefore this israpid method of determination of Cl. perfringens in the tested material. 4. To familiarize with biological preparations which are used for laboratory diagnosis, activeand passive prophylaxis and treatment of gas gangrene, tetanus and botulism. 7. Control questions and tests Select the correct answers. 1. A patient injured in an accident has been suspected of probable development of anaerobicwound infection (gas gangrene). What is the most appropriate treatment before making a specificlaboratory diagnosis?
6. A. Plasma. B. ToxoidC. Typo specific immune serumD. Polyvalent specific serumE. Placenta gamma globulin. 2. Patient developed symptoms of bulbar paralysis: ptosis, diplopia, aphonia, dysphagia aftereating of tinned mushrooms. Botulism was diagnosed previously. What test should be used todetermine toxin type? A. Neutralization B. Agglutination. C. Precipitation. D. Complement fixation test E. Immunofluorescence testing . 3. The fish of domestic preparation was a reason of food poisoning. The fish was examined atbacteriological laboratory. After cultivation in anaerobe condition there were revealed rods withsubterminal situated spores. What diagnosis is possible? A. Botulism. B. Salmonellosis. C. Cholera. D. Shigellosis. E. Enteric fever.4. The causative agents of gas gangrene have such properties: ÐT â€“ C. perfringens is motile; b â€“ C. perfringens has a capsule; c â€“ C. novyÑ– formsspores; d â€“ C. novyÑ– has a capsule; e â€“ C. septicum is motile.
7. 5. Features of growth of gas gangrene Clostridia: ÐF â€“ grow on simple media; b â€“ on blood agar form hemolysis zones; c â€“ on sugar brothform pellicle; d â€“ grow on Kitt-Tarozzi medium; e â€“colonies form in agar stab cultures.6. Methods of laboratory diagnosis of gas gangrene:ÐM â€“ allergic test; b â€“ bacteriological; c â€“ neutralization test; d â€“ CFT; e â€“ biological.7. The causative agent of tetanus has such morphological and staining properties: ÐT â€“ rod; b â€“ produces capsule; c â€“ has flagella; d â€“ produces spores; e â€“ Gram-negative; f â€“ Gram-positive.8. Cultivation of C. tetani: ÐC â€“ aerobe; b â€“ anaerobe; c â€“ grows on simple nutrient media; d â€“ grows on Kitt-Tarozzi medium; e â€“ zones of hemolysis are produced around the colonies on blood agar.9. Toxin production of C tetani: ÐT â€“ produces enterotoxins; b â€“ produces tetanospasmin; c â€“ has necrotic factor; d â€“produces tetanolysin; e â€“ produces virulence enzymes.10. Bacteria preparations used for passive and active immunization: ÐB â€“ Pertussis-diphtheria-tetanus vaccine; b â€“ chemical associated adsorbed vaccinewhich contains O- and Vi-antigens of typhoid, paratyphoid B, and a concentrated purified andsorbed tetanus anatoxin; c â€“ antitoxic antitetanus serum; d â€“ BCG vaccine; e â€“ associateddiphtheria-tetanus toxoid.11. The causative agent of botulism has such morphological and tinctorial properties: ÐT â€“ is motile; b â€“ has a capsule; c â€“ produces spores; d â€“ in stained preparationoccurs in chains; e â€“ Gram-negative.12. Cultivation of C. botulinum: ÐCit produces zones of hemolysis around colonies; d â€“ it grows on Kitt-Tarozzi medium; e â€“ itgrows on Ploskirevâ€™s medium.13. Toxin production of C. botulinum:
8. Ð â€“ produces exotoxins; b â€“ does not produce hemolysin; c â€“ produces neurotoxin; d â€“ produces enterotoxin; e â€“ does not produce virulence enzymes. 14. Methods of laboratory diagnosis of botulism: ÐM 15. What specific preparations are used for treatment and prophylaxis of botulism:• anatoxin (toxoid); b â€“ botulinuss vaccine; c â€“ botulinum antitoxin; d â€“ bacteriophage; e â€“ antibotulinum immunoglobulin. 16.The most important protective function of the antibody stimulated by tetanus immunization is: A. to opsonize the pathogen (C.tetani) B. to prevent growth the pathogen C. to prevent adherence the pathogen D. to neutralize the toxin of pathogen 17. Each of following statements concerning Clostridium perfringens is correct except: A. It is an important cause of gas gangrene B. It is an important cause of food poisoning C. It produces an exotoxin that degrades lecithin and causes necrosis and hemolysis D. It produces a toxin that inhibits the release of acetylcholine at the synapse 18. Each of following statements concerning Clostridium tetani is correct except: A. It is a gram-positive, spore-forming rod B. Pathogenesis is due to production of an exotoxin that blocs inhibitory neurotrasmitters C. It is a facultative organism; it will grow on a blood agar plate of the presence of room air D. Its natural habitat is primary the soil 19. Which one of following statements concerning immunization against diseases cased by clostridia is correct? A. Antitoxin against tetanus protects against botulism as well, because the two toxins share antigenic sites B. Vaccines containing alpha toxin (lecithinase) are effective in protecting against gas gangrene
9. C. The toxoid vaccine against Clostridium difficile should be administered to immunocompromised patients D. Immunization with tetanus toxoid induces effective protection against tetanus toxin20. Each of following statements concerning wound infection caused by Clostridium perfringens iscorrect except: A. An exotoxin plays a role in pathogenesis B. Gram â€“positive rods are foud in the exudate C. The organism grows only in human cell culture D. Anaerobic culture of the wound site should be ordered21. Each of following statements regarding tetanus is correct except: A. The causative agent is gram-positive anaerobic rods B. The antigen in the vaccine is tetanus toxoid, which is chemically modified toxin C. The natural habitat of the organism is the soil.Infection typically occurs when spores enter the body in contaminated wounds D. The toxin lyses red blood cells, causing severe anemia and kidney failure Real-life situation to be solved: 1. A patient injured the femur during transport accident. In the deep wound were left the piecesof clothes and soil. In 2 hours after the accident the primary surgical treatment of the wound wasmade and surgeon placed the sutures. In a day patientâ€™s condition became grave. The femurincreased in its size, the skin became pale and lucid. Muscles look like boiled meat. There arefoamy discharge from the wound with unpleasant smell. During palpation of the tissues near thewound the crepitaion was determined. A. What disease may be in this patient? B. What bacteria cause the disease? C. What laboratory examinations can confirm the diagnosis? D. What specific preparation is used for treatment? 2. A patient was injured during transport accident. After the primary surgical treatment of thewound 3000 IU of antitetanic serum was injected to the patient. A. How is it necessary to inject antitoxic serum to prevent complications. B. How much time does this immunity last? 3. A patient with diagnosis of tetanus was admitted to the infectious diseases hospital. Twomonths ago after the trauma he was injected with antitetanic serum. There were not any traumasduring last months. A. Could tetanus develop in this patient?
10. B. What clinical findings testify about tetanus? C. Why tetanus was developed in patient? D. What mistakes did the doctor make? E. What immune response does antitetanic serum create? 4. One family (3 persons) was hospitalized to the infectious disease department withcomplaints of aphonia, disturbance of swallowing, double vision, ptosis of the upper lips, difficultbreathing, weakness in arms and legs. In a day they died. To determine the cause of intoxicationthe examination of canned fish was perfumed. A. What microbes can cause this disease? B. What methods of diagnosis must be used for examination of food remains? C. How canned fish was infected with these causative agents?7. List of literature:1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,chapter18, p. 44-71.