Bohomolets Microbiology Lecture #21


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By Ms. Kostiuk from Microbiology department

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  • Bacteria can be classified into some divisions based upon their requirments for gaseous oxygen or air, which contain 20% oxygen.
  • Why are some anaerobes killed in the presence of O 2 , but others can tolerate in even though they cannot use it? Oxygen can be converted into a number of forms that are highly toxic. Some of these toxic forms, such as hydrogen peroxide (H 2 O 2 ), are formed by metabolic processes involving O 2 . Other toxic compounds, such as superoxide O 2 -), are produced as a chemical reaction on light. Cells that are not killed in the presence of oxygen contain enzymes that can convert these toxic compounds to nontoxic forms.
  • Therefore clostridia are anaerobes, they need special condition of growing in host orgamism (without oxygen) and this feature influence to pathogenesis – as usual, bacterium must infect wound
  • Spores are not sensitive to Gram-staining and are shown as uncolored space in the vegetative cell or separately situated uncolored oval cells.
  • Theta toxin
  • The enzymatic activity of  toxin can be demonstrated by growing the organism on a media containing egg yolk. It hydrolyses lecithin in the egg yolk and changes its color.
  • Almost every sample of soil and dusty surface has spores of the gas gangrene bacillus. However, only rarely does contamination of a wound result in gas gangrene. The low oxygen condition results from the presence of aerobic bacteria that deplete oxygen. Such condition stimulate spore germination, rapid vegetative growth in the dead tissue, and release of exotoxins.
  • Surgical removal of infected tissue is of primary importance. If treatment is not indicated early, the disease if invariably fatal.
  • Diagnosis depends mostly on patient’s symptoms
  • Diplopia – double vision Dyphagia – cannot swallow
  • Cl.Botulinum species has 8 distinctly different types (A, B, C1, C2, D, E, F, G), which vary in distribution among animals, regions of the world, and the type of exotoxin. Human disease is usually associated with types A, B, E, and F, and animal disease with types A, B, C, D, E. In C, D, F strains, toxin production results from lysogenic conversion
  • The toxin is moderately inactivated by boiling contaminated food for 15 minutes. The antitoxin only neutralizes toxin circulating in the blood stream, and the nerves already affected by it recover slowly, requiring weeks or months.
  • The most available method is identification of toxin in patient’s blood and food.
  • Tetanus is a neuromuscular disease whose alternate name, lockjaw, refers to an early effect of the disease on the jaw muscle.
  • Cl.tetani shows 2 striking features. The first feature is the spherical endospore that forms at the end of the bacillus, in contrast to the oval endospore that develops near the center the center of the rods in other pathogenic species of Cl.
  • The second feature is its swarming growth that spreads to cover the entire surface of solid media.
  • Laboratory diagnosis has limited value. The main position – clinical symptoms of tetanus is so striking and high specific than do not require laboratory conformation. Isolation of Cl.tetani from wounds does not prove that a person has tetanus. Conversely, failure to find the organism in a patient’s wound cultures does not eliminate the possibility of tetanus. False negative results occur in up to 2 thirds of patients. The reasons for these observations are that tetanus exdopsores may contaminate sounds that are not sufficiently anaerobic to allow germination and toxin production, or the person may simply be immune to the toxin by prior vaccination.
  • Paralysis develops since lower extremities and tail to upper part of body. In human - on the contrary, tetanus develop since back of the head and go down to extremities.
  • Tetanus has a high mortality but is easily prevented by immunization.
  • Cl.defficile is part of the flora of the gastrointestinal tract approximately 3% of the general population.
  • Bohomolets Microbiology Lecture #21

    1. 1. Agents of anaerobe infections Clostridia
    2. 2. General gropes of bacteria with respect to oxygen requirements <ul><li>Obligate (strict) aerobes </li></ul><ul><li>Obligate (strict) anaerobes </li></ul><ul><li>Facultative anaerobes </li></ul><ul><li>Microaerophiles </li></ul><ul><li>Aerotolerant microorganisms </li></ul><ul><li>Capnophiles </li></ul>
    3. 3. Obligate anaerobes There are microorganisms that cannot multiply is any oxygen is present. Some members are actually killed by traces of oxygen because they cannot modify the toxic forms of oxygen produced in metabolism. Among the more important anaerobic pathogens are some species of Clostridium, Bacteroides
    4. 4. Enzyme content of bacteria with different requirement for oxygen Neither catalase nor superoxide dismutase Strict anaerobe Superoxide dismutase Aerotolerant Small amount of catalase and superoxide dismutase Microaerophile Catalase Superoxide dismutase Facultative anaerobe Catalase – H 2 O 2  H 2 O + O 2 Superoxide dismutase O 2 - +2H+  O 2 + H 2 O 2  H 2 O + O 2 Strict aerobe Enzyme content for O 2 detoxification Name
    5. 5. Cultivation of anaerobes
    6. 6. Anaerobe microorganisms that are medically important <ul><li>Spore-forming : </li></ul><ul><ul><li>Gram-positive bacteria </li></ul></ul><ul><ul><ul><li>Clostridium </li></ul></ul></ul><ul><li>Nonspore-forming : </li></ul><ul><li>Gram-positive bacteria : </li></ul><ul><ul><li>Actinomyces </li></ul></ul><ul><ul><li>Bifidobacterium </li></ul></ul><ul><ul><li>Lactobacillus </li></ul></ul><ul><ul><li>Propionobacterium </li></ul></ul><ul><li>Gram-positive cocci : </li></ul><ul><ul><li>Peptococcus </li></ul></ul><ul><ul><li>Peptostreptococcus </li></ul></ul><ul><ul><li>Gram-negative bacteria and curved forms </li></ul></ul><ul><ul><ul><li>Anaerovibrio </li></ul></ul></ul><ul><ul><ul><li>Bacteroides (>40 species) </li></ul></ul></ul><ul><ul><ul><li>Fusobacterium </li></ul></ul></ul><ul><ul><ul><li>Prevatella </li></ul></ul></ul><ul><ul><ul><li>Campylobacter </li></ul></ul></ul><ul><ul><ul><li>Leptotrichia </li></ul></ul></ul><ul><ul><ul><li>Porphyromonas </li></ul></ul></ul><ul><ul><ul><li>Treponema </li></ul></ul></ul><ul><ul><ul><li>Borellia </li></ul></ul></ul><ul><ul><li>Gram-negative cocci </li></ul></ul><ul><ul><ul><li>Veilonella </li></ul></ul></ul>
    7. 7. General properties of Clostridia <ul><li>Gram-positive spore-forming rods </li></ul><ul><li>Oval or spherical spores often have diameter more than the vegetative cell (therefore is named Clostridium) </li></ul><ul><li>Anaerobes </li></ul><ul><li>Catalase-negative </li></ul><ul><li>Widely distributed in soil, vegetation, and commensals inhabit the bodies of humans and other animals </li></ul><ul><li>There are over than 120 species </li></ul><ul><li>General virulence factor of pathogenic clostridia is very powerful exotoxin </li></ul>
    8. 8. Clostridia that are implicated in serious human disease <ul><li>Cl.tetani causes tetanus </li></ul><ul><li>Cl.botulinum causes botulism </li></ul><ul><li>Cl.perfringens </li></ul><ul><li>Cl.novyi </li></ul><ul><li>Cl.septicum </li></ul><ul><li>Cl.histolyticum </li></ul><ul><li>Cl.sporogenes </li></ul><ul><li>Cl.difficile cause causes pseudomembranous colitis (or antibiotic-associated colitis) </li></ul>cause gas gangrene first three – in monoculture last two – only in association with one of first
    9. 9. Clostridium perfringens <ul><li>The main causative agent of gas gangrene (or anaerobe infection of wound). </li></ul><ul><li>Gram-positive rod with central or terminal endospore </li></ul><ul><li>Encapsulated </li></ul><ul><li>Nonmotile </li></ul><ul><li>Anaerobes </li></ul><ul><li>Commonly founded in human and animal intestines as well in soil </li></ul><ul><li>Besides gas gangrene causes food poisoning with diarrhea, abdominal cramps, nausea and vomiting ( symptoms are connected with spore germination in the intestine and enterotoxin production) </li></ul>
    10. 10. Smear of Cl.perfringens
    11. 11. Cl.perfringens in the smear from wound exudate
    12. 12. Sporulation of Cl. perfringens
    13. 13. <ul><li>Five different types of Cl.perfringens are recognized, depend on which toxins they produce – A, B, C, D, E </li></ul>
    14. 14. Cl.perfringens virulence factors <ul><li> exotoxin - lecithinase (the most potent one) kills leukocytes, causes red blood cell rupture, edema and tissue destruction by degrading the lecithin component of their membranes. On blood agar causes  -hemolysis </li></ul><ul><li> exotoxin causes  -hemolysis on the blood agar </li></ul><ul><li>Enzymes: </li></ul><ul><ul><li>Collagenase </li></ul></ul><ul><ul><li>Hyaluronidase </li></ul></ul><ul><ul><li>DNase </li></ul></ul>
    15. 15. Lecithinase and hemolytic activities of Cl. perfringens Absence of lecithinase effect when antitoxin is used Hemolysis Lecithinase effect Absence of hemolysis when antitoxin is used
    16. 16. Pathogenesis of gas gangrene <ul><li>Agent – Cl.perfringens: other Clostridia less frequently </li></ul><ul><li>Sours of infection. The natural habitat of C.perfringens included both the soil and the human intestine. </li></ul><ul><li>Transmission. Ednospores commonly contaminate wounds (especially puncture and gunshot), crushing injuries (if anaerobic condition) </li></ul><ul><li>Incubation period – 1 to 5 days </li></ul><ul><li>Spores germinate, vegetative bacilli multiply and release enzymes for invasion and exotoxin </li></ul>
    17. 17. Gas gangrene therefore the gas forms in tissues due to fermentation of muscle carbohydrates, amino acids and glycogen.
    18. 18. Changes in the infected tissue The gas formed in the tissue can destroy muscle structure
    19. 19. Pathogenesis of gas gangrene Symptoms: pain, edema, and a bloody exudate in the lesion, fever, tachycardia, and blackened necrotic tissue filled with bubbles of gas.
    20. 20. Treatment <ul><li>Surgical removal of all dead and infected tissues </li></ul><ul><li>Hyperbaric oxygen treatment (it inhibits growth of the clostridia, thereby stopping release of toxin, and it also improves oxygenation of injured tissue) </li></ul><ul><li>Antibiotics administration (penicillin) </li></ul>
    21. 21. Laboratory diagnosis <ul><li>Bacterioscopy. Digested muscle, Gram-positive rods, and few of no leukocytes </li></ul><ul><li>Bacteriological method . Cultivation bacilli in anaerobic condition. Identification according morphology, biochemical properties; toxin production. </li></ul>
    22. 22. Botulism is the most feared type of food poisoning because it can result in paralysis and death <ul><li>Gram-positive anaerobe rod with subterminal spore </li></ul><ul><li>Widely distributed in soils around the world </li></ul><ul><li>Has very powerful exotoxin </li></ul>
    23. 23. Cl. botulinum morphology
    24. 24. Cl.botulinum sporulation process
    25. 25. Virulence factor <ul><li>Exotoxin. It is neurotoxin, meaning that it acts against the nervous system, and is one of the most powerful poisons known. </li></ul><ul><li>The toxin acts by blocking the transmission of nerve signals to the muscles, producing paralysis. </li></ul>100 milligrams of the toxin would be sufficient to kill all population of the Earth. 1 gram of purified crystallized botulinum toxin contains 10 12 fatal doses for a human
    26. 26. <ul><li>There are clinical forms of botulism: </li></ul><ul><li>Botulinum food poisoning is intoxication associated with eating poorly preserved food, contaminated by Cl.botulinum spores. These spores later germinate (in anaerobic condition) and growth of the bacteria results in the release of exotoxin into the food. </li></ul><ul><li>The neurotoxin survives stomach acid and pepsin, is absorbed into the blood stream, and is carried to nerves. Incubation period 12-36 hours. </li></ul><ul><li>Wound botulism. The spores enter a wound or puncture, germinate, and produce toxin </li></ul><ul><li>Infant botulism, in which the bacilli grow in the gut and produce toxin (in contrast the infant, the adult intestinal tract normally inhibits this sort of infection) </li></ul>Wound and infant botulism are generally milder than first form
    27. 27. Symptoms <ul><li>Diplopia, dysphagia, weakness, nausea, vomiting and diarrhea. </li></ul><ul><li>Nerve involvement leads to generalized paralysis and respiratory insufficiency </li></ul><ul><li>Respiratory paralysis is the most common cause of death. </li></ul><ul><li>Despite treatment, about one fourth of the victims of botulism die. </li></ul>
    28. 28. Serotypes of Cl.botulinum and its connection with toxigenicity Bacterial chromosome - G Bacterial chromosome + F Temperate bacteriophage + E Temperate bacteriophage - D Temperate bacteriophage - С2 Temperate bacteriophage - С1 Bacterial chromosome + В Bacterial chromosome + А Location of tox-gen Human sensitivity Serotype
    29. 29. Treatment and prevention <ul><li>Botulism is treated by administering the antitoxin specific for the causative serotype of Cl.botulinum. </li></ul><ul><li>Infectious botulism is treated with antibiotics – penicillin (addition to antitoxin) </li></ul><ul><li>Immunization with toxoid is not generally available </li></ul>
    30. 30. Laboratory diagnosis <ul><li>Biological method. Botulinum toxin is demonstrable in uneaten food and the patient’s serum by neutralization test in mouse (protection test) – mice are inoculated with a sample of the clinical specimen and will die unless protected by antitoxin. </li></ul>
    31. 31. Susceptibility of animals to botulinum toxin <ul><li>Susceptible : </li></ul><ul><ul><li>Mouse </li></ul></ul><ul><ul><li>Guinea pig </li></ul></ul><ul><ul><li>Cat </li></ul></ul><ul><ul><li>Hors </li></ul></ul><ul><ul><li>Duck </li></ul></ul><ul><li>Unsusceptible : </li></ul><ul><ul><li>Rat </li></ul></ul><ul><ul><li>Dog </li></ul></ul><ul><ul><li>Chicken </li></ul></ul>
    32. 32. Guinea pig, effected by botulinum toxin
    33. 33. Clostridium tetani <ul><li>Cause tetanus or lockjaw </li></ul><ul><li>Gram-positive motile anaerobic rod with spherical terminal spore </li></ul><ul><li>Does not ferment carbohydrates but ferment proteins </li></ul><ul><li>Is a common resident of cultivated soil and the gastrointestinal tract of animals </li></ul><ul><li>Has potent exotoxin </li></ul>Ziehl-Neelsen staining Red acid-fast spore at the end of bacillus
    34. 34. Cl.tetani morphology Spherical terminal spores
    35. 35. Cultural properties of Cl.tetani Swarming growth on the solid media
    36. 36. Cl.tetani sporulation process
    37. 37. Virulence factor <ul><li>Extremely potent exotoxin with neurotoxic activity </li></ul><ul><li>It consist of 2 components: tetanospasmin and tetanolysin (causes lysis of erythrocytes) </li></ul><ul><li>Tetanospasmin attaches to motor nerves where they join the muscles. The toxin is taken into the nerve by endocytosis and is carried intra-axonally to the central nervous system, where it binds to ganglioside receptors and blocks release of inhibitory madiators at spinal synapses. </li></ul><ul><li>So tetanospasmin alters the usual regulation mechanisms for muscle contraction. As result, the muscle are released from normal inhibition and begin to contract uncontrollably. </li></ul>
    38. 38. Clinical symptoms The first symptoms are clenching of the jaw, followed in succession by extreme arching of the back, flexion of the arms, and extension of the legs.
    39. 39. Death is most often due to paralysis of the respiratory muscles and respiratory collapse. The fatality rate ranging from 10% to 70%.
    40. 40. Pathogenesis <ul><li>Spores usually enter the body through accidental puncture wounds, burns, frostbite, and crushed body parts. Spores germinate and release exotoxin. </li></ul><ul><li>Neonatal tetanus occurs when spores enter the newborn through umbilical wound after delivery. </li></ul>Baby with neonatal tetanus
    41. 41. Laboratory diagnosis has limited value <ul><li>Bacteriological method. </li></ul><ul><li>Biological method. Infecting of mouse causes tetanus and death. Neutralization test in mice can be used – mice protected by antitoxin (antibodies to botulinum exotoxin) remain live. </li></ul>
    42. 42. Tetanus in animals
    43. 43. Treatment and prevention <ul><li>Tetanus is treated by administering tetanus antitoxin to neutralize any toxin not yet attached to motor nerve cells </li></ul><ul><li>An antibiotics such as penicillin </li></ul>For prevention use toxoid (formaldehyde-treated exotoxin)
    44. 44. C lostridium difficile Cl.defficile causes pseudomembranous or antibiotic-associated colitis. <ul><li>Antibiotics suppress drug-sensitive normal flora, allowing Cl.defficile to multiply and produce exotoxins A and B: </li></ul><ul><li>toxin A is an enterotoxin that causes watery diarrhea </li></ul><ul><li>toxin B is a cytotoxin that causes damage to the colonic mucosa, leading to pseudomembrane formation </li></ul>Clindamycin and ampicillin are 2 of many antibiotics that cause this colitis
    45. 45. Action of B cytotoxin to colonic mucosa Yellow-white plaques formation on the colonic mucosa.
    46. 46. Diagnosis, treatment, prevention <ul><li>Diagnosis. </li></ul><ul><ul><li>Exotoxin B is detected in filtrates of stool samples by its cytotoxin effect on cultured cells. It is identified by inhibition of cytotoxicity by specific antibody. </li></ul></ul><ul><ul><li>An ELISA (immuno-enzyme test) is available for detection both exotoxins A and B. </li></ul></ul><ul><li>Treatment. The causative antibiotics should be withdrawn. Oral metronidazole should be given and fluids replaced. </li></ul><ul><li>Prevention. There are no vaccines </li></ul>