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Bacillus anthracis


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Bacillus anthracis

  1. 1. Bacillus anthracis Dr Kamran Afzal Asst Prof Microbiology
  2. 2. Clinically Important GPRs <ul><li>Spore forming </li></ul><ul><ul><li>Bacillus </li></ul></ul><ul><ul><li>Clostridium </li></ul></ul><ul><li>Non-spore forming </li></ul><ul><ul><li>Corynebacterium </li></ul></ul><ul><ul><li>Listeria </li></ul></ul><ul><ul><li>Lactobacillus </li></ul></ul><ul><li>Bacilli with branching filaments </li></ul><ul><ul><li>Actinomyces </li></ul></ul><ul><ul><li>Nocardia </li></ul></ul>
  3. 3. Identity of Genus Bacillus <ul><li>Growth – large, rough/ irregular colonies </li></ul><ul><li>GPR with spores (sporangium) </li></ul><ul><li>Spores –resistant to heat, drying, chemical disinfectants, UV radiation, staining, may lie freely </li></ul><ul><li>Inclusions – lipid, volutin or crystalline proteins </li></ul>
  4. 4. Species <ul><li>34 species of Bacillus </li></ul><ul><li>B. anthracis – only true infectious pathogen </li></ul><ul><li>B. cereus – saprophyte with pathogenic potential </li></ul><ul><li>Most species – saprophytes, resident in soil, some opportunistic infections </li></ul>
  5. 5. Bacillus anthracis <ul><li>Aerobic, large Gram positive bacillus </li></ul><ul><li>Rods – square ends, filamentous chains in culture, single/ pairs </li></ul><ul><li>Non-motile and non-flagellate </li></ul><ul><li>Spores – central and oval </li></ul><ul><li>Capsule – sheep/ horse blood agar, nutrient agar with10% serum </li></ul>
  6. 6. Virulence Factors <ul><li>Capsule: Antiphagocytic </li></ul><ul><li>Exotoxins: </li></ul><ul><li>AB model </li></ul><ul><ul><li>Binding </li></ul></ul><ul><ul><li>Activating </li></ul></ul><ul><li>Protective antigen (PA), edema factor (EF) and lethal factor (LF) </li></ul><ul><ul><li>Make up 50% of proteins in the organism </li></ul></ul><ul><li>Individually non-toxic </li></ul><ul><ul><li>PA+LF  lethal activity </li></ul></ul><ul><ul><li>EF+PA  edema </li></ul></ul><ul><ul><li>EF+LF  inactive </li></ul></ul><ul><ul><li>PA+LF+EF  edema and necrosis; lethal activity </li></ul></ul>
  7. 7. Staining <ul><li>Gram stain - GPR </li></ul><ul><li>Polychrome Methylene blue by McFadyean method –Blue bacilli with irregular purple capsule </li></ul><ul><li>Giemsa stain – Purple bacilli with red capsule </li></ul><ul><li>Unstained spaces in bacilli – Spores </li></ul>
  8. 8. Culture <ul><li>Aerobe/ facultative anaerobe </li></ul><ul><li>Temp range – 12 to 45 0 C </li></ul><ul><li>Nutrient agar, blood agar, selective media </li></ul><ul><li>Colonies – grey granular discs 2-3 mm after 24 hrs on nutrient agar (Medusa head), sticky membranous consistency </li></ul><ul><li>Non-hemolytic – BA </li></ul>
  9. 9. <ul><li>Growth </li></ul><ul><ul><li>In broth silky strands, surface pellicle and floccular deposit </li></ul></ul><ul><ul><li>In gelatin stab inverted fir tree appearance, slow liquefaction 7 days 20 0 C </li></ul></ul><ul><li>Boichemical </li></ul><ul><ul><li>Acid from glucose, maltose, sucrose, no gas </li></ul></ul><ul><li>Catalase + </li></ul><ul><li>Nitrate to nitrite </li></ul><ul><li>Egg-yolk agar </li></ul><ul><ul><li>Weak lecithinase </li></ul></ul>
  10. 10. Endospore <ul><li>Dehydrated cells </li></ul><ul><ul><li>Highly resistant to heat, cold, chemical disinfectants, dry periods </li></ul></ul><ul><li>Protoplast carries the material for future vegetative cell </li></ul><ul><li>Cortex provides heat and radiation resistance </li></ul><ul><li>Spore wall provides protection from chemicals and enzymes </li></ul><ul><li>Oxygen required for sporulation </li></ul>
  11. 11. Anthrax <ul><li>Bacillus anthracis was the first disease-causing bacterium discovered in 1877 by Robert Koch </li></ul><ul><li>Koch also discovered tuberculosis bacillus and cholera vibrio </li></ul><ul><li>Anthracis from the Greek word “anthrakos” meaning “coal”, which refers to the black lesions formed by the cutaneous form of the disease </li></ul>
  12. 12. <ul><li>Caused by spores </li></ul><ul><li>Primarily a disease of domesticated and wild animals </li></ul><ul><ul><li>Herbivores such as sheep, cows, horses, goats </li></ul></ul><ul><li>Natural reservoir is soil </li></ul><ul><li>Anthrax zones </li></ul><ul><ul><li>Soil rich in organic matter (pH < 6.0) </li></ul></ul>
  13. 14. Who gets it? <ul><li>People who work with imported animal hides or furs in areas where standards are insufficient to prevent exposure to anthrax spores </li></ul><ul><li>People who handle potentially infected animal products in high-incidence areas </li></ul><ul><li>People who work directly with it in the lab </li></ul><ul><li>Military personnel deployed to sensitive areas are at high risk for exposure to the organism </li></ul>
  14. 15. Epidemiology <ul><li>Forms of Anthrax </li></ul><ul><ul><ul><li>Inhalational (<5% cases; 45-89% mortality) </li></ul></ul></ul><ul><ul><ul><li>Cutaneous (95%; <1-20% mortality) </li></ul></ul></ul><ul><ul><ul><li>Gastrointestinal (<5%; >50% mortality) </li></ul></ul></ul>
  15. 16. Pathogenesis <ul><li>Inhalation Anthrax </li></ul><ul><li>The infection begins with the inhalation of the anthrax spore </li></ul><ul><li>Macrophages lyse and destroy some of the spores </li></ul><ul><li>Survived spores are transported to lymph nodes </li></ul>
  16. 17. <ul><li>Disease immediately follows germination </li></ul><ul><li>Spores replicate in the lymph nodes </li></ul><ul><li>Bacterial toxins released during replication result in mediastinal widening and pleural effusions </li></ul>
  17. 18. <ul><li>Inhalation Anthrax is the most lethal type of Anthrax </li></ul><ul><li>Incubation period </li></ul><ul><ul><ul><li>1–7 days </li></ul></ul></ul><ul><ul><ul><li>Possibly ranging up to 42 days </li></ul></ul></ul><ul><ul><ul><li>(depending on how many spores were inhaled) </li></ul></ul></ul><ul><li>Case fatality </li></ul><ul><ul><ul><li>Untreated (97%) </li></ul></ul></ul><ul><ul><ul><li>With antimicrobial therapy (75%) </li></ul></ul></ul>
  18. 19. <ul><li>Cutaneous Anthrax </li></ul><ul><li>95% of anthrax infections </li></ul><ul><li>When the bacterium enters a cut or scratch on the skin </li></ul><ul><li>May also be spread by biting insects </li></ul><ul><li>Spore germinates, toxin production results in itchy bump, a vesicle and then painless black ulcer </li></ul>
  19. 20. <ul><li>Gastrointestinal Anthrax </li></ul><ul><li>GI anthrax may follow after the consumption of contaminated, poorly cooked meat </li></ul><ul><li>There are 2 different forms of GI anthrax </li></ul><ul><ul><li>Oral-pharyngeal </li></ul></ul><ul><ul><li>Abdominal </li></ul></ul>
  20. 22. Lab Diagnosis <ul><li>Specimens - Exudates, blood, CSF, aspirated fluids, tissues </li></ul><ul><li>Pulmonary anthrax – sputum </li></ul><ul><li>Blood – septicemic stage </li></ul><ul><li>Malignant pustule : </li></ul><ul><ul><li>Specimen fluid from vesicle </li></ul></ul><ul><ul><li>Microscopy by Gram/ Giemsa/ McFadyean stains </li></ul></ul><ul><ul><li>Culture on nutrient/ blood agar and nutrient broth at 37 0 C </li></ul></ul><ul><ul><li>Stain from culture </li></ul></ul>
  21. 23. Presumptive Identification Clinical specimen (blood, CSF, etc.) Isolate on BA Colony morphology No hemolysis Non motile Spores Gram stain Malachite green Gram stain Capsule production
  22. 24. Confirmatory Identification Isolate Phage lysis Capsule Bicarbonate media Horse blood (McFadyean Stain) (McFadyean stain India ink stain) DFA Capsule antigen Cell wall
  23. 25. Biochemical Tests
  24. 26. Rapid Detection Methods <ul><li>- ELISA assay for antigen / antibody detection </li></ul><ul><li>- Immunofluorescence examinations </li></ul><ul><li>- PCR for detection of nucleic acid </li></ul>
  25. 27. Radiological Diagnosis <ul><li>Useful for inhalational anthrax </li></ul><ul><li>Find a widened mediastinum and pleural effusion </li></ul><ul><ul><li>Resulting less available space in lungs </li></ul></ul><ul><li>Computed Tomography (CT) scan </li></ul>
  26. 28. Treatment <ul><li>Hospitalization </li></ul><ul><li>IV antibiotics </li></ul><ul><ul><ul><li>Empirical until sensitivities known </li></ul></ul></ul><ul><li>Intensive supportive care </li></ul><ul><ul><ul><li>Electrolyte and acid-base imbalances </li></ul></ul></ul><ul><ul><ul><li>Mechanical ventilation </li></ul></ul></ul><ul><ul><ul><li>Hemodynamic support </li></ul></ul></ul>
  27. 29. <ul><li>Before 2001, 1st line of treatment was penicillin G </li></ul><ul><ul><ul><li>Penicillin resistance, ß-lactamase </li></ul></ul></ul><ul><ul><ul><li>60 days course of antibiotics </li></ul></ul></ul><ul><li>Ciprofloxacin </li></ul><ul><ul><ul><li>500 mg tablet every 12h or 400 mg IV every 12h </li></ul></ul></ul><ul><li>Doxycycline </li></ul><ul><ul><ul><li>100 mg tablet every 12h or 100 mg IV every 12h </li></ul></ul></ul><ul><li>For inhalational, need another antimicrobial agent </li></ul><ul><ul><ul><li>clindamycin </li></ul></ul></ul><ul><ul><ul><li>rifampin </li></ul></ul></ul><ul><ul><ul><li>chloramphenicol </li></ul></ul></ul><ul><ul><ul><li>aminoglycosides </li></ul></ul></ul><ul><ul><ul><li>cephalosporins </li></ul></ul></ul>
  28. 30. Post-Exposure Prophylaxis <ul><li>Indications </li></ul><ul><ul><li>Exposure to anthrax spores </li></ul></ul><ul><ul><li>Not for contacts of cases </li></ul></ul><ul><li>Oral antibiotics </li></ul><ul><ul><li>Ciprofloxacin 500 mg po bid OR </li></ul></ul><ul><ul><li>Doxycycline 100 mg po bid </li></ul></ul><ul><ul><li>Duration 60-100 days </li></ul></ul><ul><li>Vaccination </li></ul><ul><ul><li>May reduce PEP duration to 30 days </li></ul></ul>
  29. 31. Case Study <ul><li>63 year-old man was admitted to the emergency department because of fever, vomiting and confusion </li></ul><ul><li>On physical examination, the patient was lethargic and disoriented. His temperature was 39 o C, blood pressure 150/80 mm Hg, pulse 110/min and respiration 18/min </li></ul><ul><li>Rhonchi on auscultation of the chest </li></ul><ul><li>No neck rigidity </li></ul>
  30. 32. <ul><li>Patient admitted to hospital </li></ul><ul><li>Blood cultures ordered </li></ul>
  31. 33. <ul><li>Blood culture incubator signals that there is growth in one of the bottles </li></ul><ul><li>It is removed and a Gram stain is performed </li></ul>
  32. 34. <ul><li>Lab Diagnosis </li></ul><ul><li>In vitro tests – to differentiate from B. cereus , capsule demonstration, McFadyean stain, motility, pencillin sensitivity, egg-yolk lecithinase activity, virulence in mice </li></ul><ul><li>In vivo test – intra-peritoneal inoculation in mice </li></ul>
  33. 35. As Biological Warfare Agent <ul><li>Why is this agent considered to be the number-one biological threat? </li></ul>
  34. 36. Benefits of Using Anthrax as a BW <ul><li>Highly lethal (Inhalational Anthrax) </li></ul><ul><li>Long shelf-life </li></ul><ul><li>Relatively easy availability </li></ul><ul><li>Short incubation period (Relative to most other BW) </li></ul><ul><li>Anthrax is stable in various types of weapons systems </li></ul><ul><li>Dangerous as both powder or aerosolized forms </li></ul><ul><li>Only need a small amount for a ‘Mass Effect’ </li></ul>
  35. 37. Pakistan’s Experience of Bioterrorism <ul><li>A total of 230 suspected samples of Anthrax from 194 sources were analyzed for anthrax spores at NIH from Nov 2001 to March 2002 </li></ul><ul><ul><li>71 samples were from clinical specimens </li></ul></ul><ul><li>(anterior nares, skin, blood) </li></ul><ul><ul><li>159 were from non-clinical environmental samples (powders, swabs from inanimate objects, papers, envelopes, packages, plastics etc) </li></ul></ul><ul><li>The samples were received from Foreign mission, media organizations, banks, government institutions, universities, hospitals and individuals </li></ul>
  36. 38. <ul><li>Out of these, 141 samples yielded growth suggestive of Bacillus species </li></ul><ul><li>On the basis of colony morphology, Gram’s stain and other preliminary laboratory tests 62 isolates were found suspicious for B. anthracis , however all the samples were negative by animal inoculation </li></ul><ul><li>The suspected anthrax parcel/letter bombs in Pakistan during the investigation period were hoaxes </li></ul>