Bohomolets Microbiology Lecture #20


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Bohomolets Microbiology Lecture #20

  1. 1. Diphtheria Tuberculosis
  2. 2. Diphtheria is acute human infectious disease caused by toxigenic Corynebacterium diphtheriae, transmitted by airborne droplets, manifesting in local fibrinous inflammation of mucous membrane of nasopharynx and trachea, and common intoxication, cardiovascular, nervous, urogenital system injury
  3. 3. Species of Corinebacterium genus <ul><li>C. diphtheriae </li></ul><ul><li>C. xerosis, species that normally lives in the eye, skin, and mucous membranes and is an occasional opportunist is eye and postoperative infections. </li></ul><ul><li>C. pseudodiphtheriticum (hofmannii) , a normal inhabitant of the human nasopharynx, can colonize natural and artificial heart valves. </li></ul>
  4. 4. Features of Corynebacterium diphtheriae <ul><li>Gram-positive rods of variable size </li></ul><ul><li>Club-shaped/slender rod (with swell on the one end) </li></ul><ul><li>Pleomorphic, can develop filamentous forms </li></ul><ul><li>Nonmotile </li></ul><ul><li>Arranged in palisades or in V-, X- or L-shaped formation </li></ul><ul><li>Older cells are filled with volutin granules that consist of polyphosphate. The granules stain metachoromatically </li></ul><ul><li>Aerobic and facultative anaerobic </li></ul><ul><li>Require special media for cultivation </li></ul><ul><li>(encriched with blood,serum or egg). Scanty </li></ul><ul><li>growth in ordinary media </li></ul>
  5. 5. Morphology of С. diphtheriae Methylene blue stain Neisser’s stain Metachromatically stained volutin inclusions
  6. 6. Cultivation of C. diphtheriae <ul><li>Loeffler’s media with solidified serum </li></ul><ul><li>Blood agar </li></ul><ul><li>Tellurite agar. Contains a potassium tellurite that is reduced to elemental tellurium within C.diphtheriae. The typical gray-black color of the colony is diagnostic criterion </li></ul>
  7. 7. Biochemical activity of C.diphtheriae + Nitrate reduc-tion Gelatin hydrosysis Catalase - - + - + + Urease Maltose Sucrose Glucose
  8. 8. Biotypes of C.diphtheriae <ul><li>Gravis </li></ul><ul><li>Mitis </li></ul><ul><li>Intermedius </li></ul><ul><li>Belphanti </li></ul>
  9. 9. Differentiation criterions for C.diphtheriae biotypes Less strains are toxigenic More strains are toxigenic Toxigenicity Yes No Hemolysis No Yes Fermentation of starch No Yes Fermentation of glycogen Diffuse turbidity Surface pellicle and granular deposit Growth in broth S colonies R colonies Growth on solid media Mitis Gravis Feature
  10. 10. С. diphtheriae biotypes cultural properties Gravis forms large colonies R-form (rough surface, with uneven edge) Mitis forms small colonies S-form (smooth surface, with flat edge)
  11. 11. Virulence factors of C. d iphtheriae <ul><li>Exotoxin (diphtheritoxin). It consist of 2 fragments: </li></ul><ul><ul><ul><li>B - for binding to the target cells </li></ul></ul></ul><ul><ul><ul><li>A – active part that inactivates the elongation factor in the ribosomes and thereby stops protein synthesis </li></ul></ul></ul><ul><li>Enzymes : catalase , dehydroginase , DNAses , neuraminidase , hyaluronidase </li></ul><ul><li>Cord-factor ( in microcapsule ) – toxic effect on mitochondria </li></ul><ul><li>Surface lipids – antiphagocytic factor </li></ul>
  12. 12. C. d iphtheriae exotoxin <ul><li>Diphtheritoxin affects the body on two levels: </li></ul><ul><li>Local. The toxin causes inflammation, fever, sore throat, nausea, severe swelling in the neck. </li></ul><ul><li>It necrotizes cells in the site of bacteria attachment and causes formation of pseudomembrane . This film consist of solidified fibrous exudate cells, fluid that develops in the pharynx, and bacilli. </li></ul><ul><li>If membrane forms into the larynx and trachea, it can cause airway abstruction (mechanical asphyxiation). </li></ul><ul><li>Systemic (toxemia). Toxin is carried by the blood to certain target organs, primarily the heart, nerves and kidneys. </li></ul><ul><li>It causes myocarditis accompanied by arrhythmias and circulatory collapse, recurrent laryngeal nerve palsy. </li></ul>
  13. 13. The clinical appearance in diphtheria infection Inflammation of the pharynx and tonsils marked by a pleudomembrane and swelling over the entire area.
  14. 14. Toxigenisity of C.diphtheria The toxigenisity of C.diphtheria is connected with bacteriophage. The DNA that codes for diphtheria toxin is part of the genetic material of a temperate bacteriophage. During the lysogenic phase of viral growth, the DNA if this virus integrates into the bacterial chromosome and the toxin is synthesized. C.Diphtheria cells that are not lysogenized by this phage do not produce exotoxin and are nonpathogenic.
  15. 15. Laboratory diagnosis <ul><li>Bacteriological method . Isolating of pure culture and demonstrating toxin production </li></ul><ul><li>Microscopy of smears of the throat swab that are stained by Gram method and methylene blue (only presumptive diagnosis because possible presence of nontoxigenic corynebacteria and other pleomorphic gram-positive rods) </li></ul><ul><li>Gen diagnosis – PCR analysis </li></ul>
  16. 16. Tests for toxicity of C.diphtheriae <ul><li>In vitro: </li></ul><ul><ul><ul><li>Immunoprecipitation test in the agar </li></ul></ul></ul><ul><ul><ul><li>Immunoenzyme reaction </li></ul></ul></ul><ul><ul><ul><li>PCR test </li></ul></ul></ul><ul><li>In vivo: </li></ul><ul><ul><ul><li>Biological probe on the guinea pigs and rabbits </li></ul></ul></ul>
  17. 17. Immunoprecipitation test for diphtheria toxin 1 2 3 4 5
  18. 18. Biologic test for toxin detection
  19. 19. Treatment <ul><li>Antitoxin </li></ul><ul><li>Antimicrobial drugs – antibiotics from the penicillin or erythromycin family </li></ul>
  20. 20. Prevention <ul><li>Immunization with diphtheria toxoid. </li></ul><ul><li>Usually given as a combination of diphtheria toxoid, tetanus toxoid, and inactivated pertussis bacilli) </li></ul><ul><li>Toxoid is prepared by treating the exotoxin with formaldehyde during 4 to 6 weeks at temperature 37 0 C. </li></ul><ul><li>Immunization does not prevent nasopharyngeal carriage of the microorganisms </li></ul>
  21. 21. The incidence and fatality rates for diphtheria in the USA
  22. 22. <ul><li>Tuberculosis is a chronic bacterial infection of the lungs (and often other body organs) characterized by slight fever, weight loss, sweating at night, and chronic cough productive of blood-streaked sputum </li></ul>
  23. 23. Classification of mycobacteria that are agents of tuberculosis <ul><li>Порядок Actinomycetales </li></ul><ul><ul><li>Family Mycobacteriaceae </li></ul></ul><ul><ul><ul><li>Genus Mycobacterium </li></ul></ul></ul><ul><ul><ul><ul><li>Species that are human pathogens : </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Mycobacterium tuberculosis (human , 92 %) </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Mycobacterium bovis ( cow , 5 %) </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Mycobacterium africanum (3 %) </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>M. avium ( causes tuberculosis in immunocompromised humans, in AIDS patients - 15-14 %); </li></ul></ul></ul></ul></ul>
  24. 24. Properties of Mycobacterium tuberculosis <ul><li>Pleomorphic long, thin rods with a tendency to be filamentous or branching. </li></ul><ul><li>Do not form capsules, flagella, or spores </li></ul><ul><li>They are stained poorly by the dyes used in Gram’s stain </li></ul><ul><li>Obligate aerobe </li></ul><ul><li>Resistant to acids and alkalia, to various germicides, to dehydration </li></ul><ul><li>Slowly growing bacilli </li></ul><ul><li>Cell wall consists of 60% lipids and contains several complex lipids: </li></ul><ul><ul><li>Mycolic acids (long-chain fatty acids), which contribute to the organism’s acid-fastness </li></ul></ul><ul><ul><li>Wax D </li></ul></ul><ul><ul><li>Phosphatides </li></ul></ul>
  25. 25. M.Tuberculosis in sputum Red acid-fast bacilli against acid-susceptible blue cells. M.Tuberculosis bacilli are frequently localized intracellularly Ziehl-Neelsen stained specimen
  26. 26. Cultivation of M.tuberculosis <ul><li>M.tuberculosis forms non-pigmented R-colonies </li></ul><ul><li>Laboratory media for M.tuberculosis cultivation (Lowenstein-Jensen media) contains complex nutrients (eg, egg yolk, potato) and dyes (eg, malachite green). The dyes inhibit the unwanted rapidly growing normal flora that interfere with the isolation of M.tuberculosis </li></ul>
  27. 27. Virulence factors of M.tuberculosis <ul><li>Cord factor ( trehalose dimycolate ) – destruction of cell mitochondria , inhibition of leukocyte migration </li></ul><ul><li>Lipids and phosphatides – toxic effect for cells </li></ul><ul><li>Resistance to digestion in macrophages and intracellular multiplication </li></ul><ul><li>Protein, combined with waxes, (tuberculin) elicits delayed hypersensitivity </li></ul><ul><li>Do not produce exotoxins and have no endotoxin </li></ul>
  28. 28. Cord formation in infected tissue Red-stained M.tuberculosis and blue macrophages
  29. 29. Pathogenesis <ul><li>Transmission </li></ul><ul><li>By respiratory aerosol </li></ul><ul><li>By food (nonpasterised milk) </li></ul><ul><li>By contact </li></ul><ul><li>Vertical - from the mother to the fetus ( rarely ) </li></ul><ul><li>Infectious dose is around 10 cells </li></ul><ul><li>Natural reservoir : </li></ul><ul><li>M.tuberculosis – humans </li></ul><ul><li>M.bovis – animals (cow) </li></ul><ul><li>Initial site of infection is the lung (commonly) </li></ul>
  30. 30. Tubercle formation Primary tubercolosis. The bacilli in lung are phagocytosed by macrophages and multiply intracellulary. After 2 weeks, delayed hypersensitivity to the TB develop. Macrophages fuse together to form large multinucleated cells. Lymphocytes and macrophages then wall off the infected area from the surrounding tissue. The localized collection of inflamatory cells is called a granuloma (tubercle). In most cases, growth of the TB is halted by granuloma formation. Serondary reactivation tuberculosis. Live bacilli can remain dormant and become reactivated weeks, months, or years later. They multiplies, spreads into the bronchial tube and upper respiratory tract.
  31. 31. Forms of extrapulmonary tuberculosis <ul><li>Tuberculosis of kidney and urinary tract </li></ul><ul><li>Tuberculosis of genital organs </li></ul><ul><li>Tuberculosis of bones and joints </li></ul><ul><li>Tuberculosis of eyes </li></ul><ul><li>Tuberculosis of intestine </li></ul><ul><li>Tuberculosis of regional lymph nodes </li></ul><ul><li>Tuberculosis of skin </li></ul><ul><li>Tubercular meningitis </li></ul>
  32. 32. Immunity and prevention <ul><li>BCG vaccine contains a strain of alive attenuated M.bovis (bacilli Calmette-Guerin or BCG) </li></ul><ul><li>Success rate of vaccination is around 80% in children and to 50% in adults. </li></ul><ul><li>The length of protection is 5 to 15 years. </li></ul>Resistance is mediated by cellular immunity, ie, by CD4-positive T cells
  33. 33. Laboratory diagnosis <ul><li>Bacterioscopy of sputum. Acid-fast staining by Zuehl-Neelsen method and fluorescent acid-fast staining. Sensitivity of the method is 5-10 thousands of bacilli in a milliliter of sputum </li></ul><ul><li>Bacteriologic method . Isolation of bacilli on Lowenstein-Jensen or Middlebrook media) and its identification. Sputum is pretreated with acid or alkali for contaminents inhibition. Sensitivity of the method is 20-100 bacilli in a ml of sputum, duration is to 6-8 weeks. </li></ul><ul><li>Gen diagnosis – PCR analysis. Sensitivity – 10 and more bacilli in a ml, duration – 2 hours </li></ul><ul><li>Biological method . Infecting guinea pegs, rabbits. The most sensitive – 1-5 bacilli in a ml of sputum </li></ul><ul><li>Serological diagnosis (immuno-enzyme test, indirect agglutination test, complement-fixation test) </li></ul><ul><li>Allergic probe – Mantoux test </li></ul>
  34. 34. A fluorescent acid-fast stain of M.tuberculosis from sputum
  35. 35. Mantoux test – tuberculin skin test Hypersensitivity to the tubercle bacilli is detected by injecting small amount of tuberculin or PPD (purified protein derivative) into the skin. <ul><li>Results - appearance of induration and erythema in the site of injection after 48-72 hours: </li></ul><ul><li>Negative - less than 5 mm </li></ul><ul><li>Intermediate – 5 to 10 mm </li></ul><ul><li>Positive – more than 10-15 mm </li></ul><ul><li>Interpretation : </li></ul><ul><li>5-10 mm – neither person is vaccinated with BCG or probably infected (if not vaccinated) </li></ul><ul><li>Reaction of more than 10 mm are assumed to be infected with M.tuberculosis </li></ul><ul><li>If negative test person is not infected and not immune and should be vaccinated with BCG </li></ul>
  36. 36. Treatment <ul><li>Multiple-drug therapy is used to prevent the drug-resistant mutants circulation. </li></ul><ul><li>Duration of treatment is 4 to 24 months </li></ul><ul><li>Treatment for most patients with pulmonary tuberculosis is with 3 drugs: </li></ul><ul><li>Isoniazid </li></ul><ul><li>Rifampin </li></ul><ul><li>Pyrazinamide </li></ul><ul><li>For immunocompromiced patients and humans with disseminated form ethambutol or streptomycin is added </li></ul><ul><li>Ciprofloxacin, amikacin, cycloserine. </li></ul>
  37. 37. Atypical mycobacteria <ul><li>The atypical mycobacteria are classified onto 4 groups according to their rate of growth and whether they produce pigment under certain conditions </li></ul><ul><li>Photochromogens (M.kansasii) produce a eyllow-orange-pigmented colony only when exposed to light </li></ul><ul><li>Scotochromogens (M.scrofulaceum) produce the pigment chiefly in the dark </li></ul><ul><li>Nonchromogens (M.avium) produce little or no yellow-orange pigment in presence or absence of light </li></ul><ul><li>Rapidly growing mycobacteria (M.fortuitum) produce colonies in less than 7 days. </li></ul>
  38. 38. Cultural properties of mycobacteria M. tuberculosis M. kansasii (photochromogenic) M. gordonae (scotochromogenic)
  39. 39. Agent of leprosy Mycobacterium leprae is agent of leprosy. M.leprae has not been grown in the laboratory media or in cell culture. It can be cultivated in the mouse footpad on in the armadillo. The bacilli replicates intracellularly, typically within skin hystiocytes, endothelial cells, and nerve cells. Red acid-fast bacilli Ziehl-Neelsen stain
  40. 40. Leprosy The incubation period averages several years (due to very slow multiplication of bacilli) There are 2 distinct forms of leprosy – tuberculiod and lepromatous When lepromatous form deformation of hands, nose, lips, chin. Leprosy is a chronic, progressive disease of the skin and nerves