1. II MBBS Dr Ekta Chourasia Microbiology
Bacillus
II MBBS
Dr Ekta Chourasia
Microbiology

2. Dr Ekta Chourasia, Microbiology
Introduction
Sporing rod shaped bacteria: 2 groups
Aerobic – Bacillus
Anaerobic – Clostridia
Important Bacillus species:
Bacillus anthracis
Bacillus cereus
Bacillus stearothermophilus
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Dr Ekta Chourasia, Microbiology

3. Dr Ekta Chourasia, Microbiology
Bacillus: key words
Sporing Gram+ve bacilli
Bacillus anthracis
Anthrax
Hide porter’s disease
Wool sorter’s disease
Malignant pustule
Eschar
M’fadyean’s reaction
Bamboo stick appearance
Medusa head colony
String of pearl’s reaction
PLET medium
Ascoli’s test
Duckering
Anthrax vaccine
Bioterrorism
Bacillus cereus
Gastroenteritis
Bacillus stearothermophilus
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Dr Ekta Chourasia, Microbiology

4. History of Bacillus anthracis
1st pathogenic bacterium to be seen under microscope – Pollender, 1849
1st communicable disease shown to be transmitted by inoculation of infected blood – Davaine, 1850
1st bacillus to be isolated in pure culture & shown to possess spores – Koch, 1876
1st bacterium used for the preparation of an attenuated vaccine – Pasteur, 1881
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Dr Ekta Chourasia, Microbiology

5. Dr Ekta Chourasia, Microbiology
Pathogenicity
Anthrax – zoonotic disease primarily involves cattle & sheep.
Animals – infected by ingestion of spores present in the soil
Large no of bacilli are shed in discharges from the mouth, nose & rectum - sporulate in the soil.
Human anthrax – contracted from animals, directly or indirectly.
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Dr Ekta Chourasia, Microbiology

6. Pathogenicity: virulence factors
Two virulence factors –
Capsular polysaccharide – inhibits phagocytosis, encoded by a plasmid
Anthrax toxin : made up of 3 fractions
Edema factor (EF or Factor I)
Protective antigen factor (PA or Factor II)
Lethal factor (LF or Factor III)
* They are not toxic individually, the whole complex produces local edema & generalised shock. Toxin production is plasmid mediated
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Dr Ekta Chourasia, Microbiology

8. Dr Ekta Chourasia, Microbiology
Human Anthrax
The disease may be
Cutaneous
Pulmonary, or
Intestinal
* All types lead to fatal septicemia
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Dr Ekta Chourasia, Microbiology

9. Dr Ekta Chourasia, Microbiology
1. Cutaneous Anthrax
95 % of human cases of anthrax
Route of entry: Skin
Sites involved – face, neck, hands, arms & back
Papule Vesicles containing colorless or blood stained fluid Malignant Pustule
‘Malignant pustule’ – satellite lesions filled with serum or yellow fluid arranged around a central necrotic lesion which is covered by a black eschar
Also known as ‘Hide Porter’s disease’
Resolves spontaneously, 10-20% of untreaed may develop fatal septicemia or meningitis
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Dr Ekta Chourasia, Microbiology

10. Dr Ekta Chourasia, Microbiology
2. Pulmonary Anthrax
Also called ‘Wool Sorter’s disease’ – common in workers in wool factories
A life- threatening hemorrhagic pneumonia caused by Inhalation of spores
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Dr Ekta Chourasia, Microbiology

11. 3. Gastrointestinal Anthrax
Rare
By ingestion of inadequately cooked meat
containing B. anthracis spores
* Human anthrax can be
Industrial – in meat packing or wool factories
Nonindustrial – frequent association with animals like butchers, veterinarians, farmers
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Dr Ekta Chourasia, Microbiology

12. Dr Ekta Chourasia, Microbiology
Laboratory Diagnosis
Specimen
Fluid or pus from local lesion, blood, sputum
Microscopy
Culture
In septicemic anthrax, blood culture should be
done
Serological test
Animal inoculation
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Dr Ekta Chourasia, Microbiology

13. Dr Ekta Chourasia, Microbiology
Microscopy
Large aerobic, non motile,
Gm+ve bacilli
Arranged singly, in pairs or in short chains, the entire chain is surrounded by a capsule
Capsules are produced in the presence of bicarbonates or 10-25% CO2
Spores are oval and centrally located, non bulging
Spores are stained by special stains – Sudan black B.
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Dr Ekta Chourasia, Microbiology

14. Dr Ekta Chourasia, Microbiology
Microscopic features
Staining blood films with polychrome methylene blue:
- Pink amorphous material around blue bacillus
(M’ Fadyean’s reaction): represents
capsular material – used for the
presumptive diagnosis of anthrax in
animals.
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Dr Ekta Chourasia, Microbiology

15. Cultural Characteristics
Grow on blood or nutrient agar, at 37°C
Irregular, round, raised, dull, opaque, greyish white colonies with a frosted glass appearance.
Low power – edge of the colony is composed of long, interlacing chains of bacilli, resembling locks of matted hair – “Medusa Head Appearance”
Gelatin stab culture – “inverted fir tree” appearance, with slow liquefaction starting from top.
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Dr Ekta Chourasia, Microbiology

16. Medusa Head Appearance
Inverted fir tree
Medusa Head Appearance
wavy colonies with small
projections

17. Cultural Characteristics
“String of Pearls reaction” – solid medium containing units of Pn/ ml, in 3-6 hrs the cells become large, spherical and occur in chains on agar surface, resembling a string of pearls.
- differentiates B. anthracis from B. cereus
Selective medium – PLET medium – contains polymyxin, lysozyme, EDTA & thallous acetate : to isolate it from mixtures containing other spore bearing bacilli.
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Dr Ekta Chourasia, Microbiology

18. Dr Ekta Chourasia, Microbiology
Smear from colony
Morphology in stained
smears from cultures
“Bamboo stick appearance” : bacilli arranged end to end in long chains.
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Dr Ekta Chourasia, Microbiology

19. Dr Ekta Chourasia, Microbiology
Laboratory Diagnosis
Animal inoculation
By rubbing contaminated tissues over shaven skin of a guinea pig
Serology
Ascoli’s thermoprecipitation test – to demonstrate anthrax Ag in tissue extracts
EIA (using purified anthrax toxin Ag)
PCR to detect anthrax contamination of animal & agricultural products
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Dr Ekta Chourasia, Microbiology

20. Dr Ekta Chourasia, Microbiology
Resistance
Bacilli destroyed at 60°C in 30 mins.
Animal carcasses – bacilli remain viable in BM for a wk & in skin for 2 wks.
Spores – highly resistant, survive in soil for 60 yrs
Spores can be destroyed by
4% KMnO4 in 15 mins
‘Duckering’ – using formaldehyde solution for animal products imported into non endemic countries
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Dr Ekta Chourasia, Microbiology

21. Dr Ekta Chourasia, Microbiology
Duckering
For disinfection of wool – 2% soln of formaldehyde at °C for 20 mins
Animal hair & bristles – 0.25% at 60°C for 6 hrs
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Dr Ekta Chourasia, Microbiology

22. Dr Ekta Chourasia, Microbiology
Prophylaxis
General methods of prevention
Improvement of factory hygiene
Proper sterilisation of animal products
Animal carcasses to be buried deep in quicklime or cremated
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Dr Ekta Chourasia, Microbiology

23. Dr Ekta Chourasia, Microbiology
Prophylaxis
Active immunisation of
Domestic animals with live attenuated spore vaccines
Persons with occupational risk (butchers, farmers, veterinarians) with a cell- free vaccine containing purified protective antigen as immunogen. 3 doses IM with annual booster injections.
* Anthrax infection in humans give life long permanent immunity & secondary infections are very rare.
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Dr Ekta Chourasia, Microbiology

24. Dr Ekta Chourasia, Microbiology
Anthrax vaccines
Original anthrax vaccine – developed by Pasteur – live attenuated bacilli vaccine – strain rendered avirulent by the loss of plasmids which encodes anthrax toxin
Live attenuated anthrax spore vaccine
Sterne vaccine – contains spores of a noncapsulated avirulent mutant strain - loss of plasmid which controls capsule production
Mazucchi vaccine – contains spores of stable attenuated Carbazoo strain
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Dr Ekta Chourasia, Microbiology

25. Dr Ekta Chourasia, Microbiology
Biological warfare
Large epidemics (occasionally)
In 1979 – former Soviet Union: due to accidental release of spores from a military facility engaged in biological research
In 1980s – Zimbabwe: affected 10,000 persons.
* Hence the need to develop better human vaccine.
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Dr Ekta Chourasia, Microbiology

26. Dr Ekta Chourasia, Microbiology
Treatment
Bacillus anthracis is sensitive to:
- Penicillin
- Doxycycline
- Ciprofloxacin
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Dr Ekta Chourasia, Microbiology

27. Dr Ekta Chourasia, Microbiology
Anthracoid bacilli
Belongs to the genus Bacillus
Occasionally cause human infections
Includes B. cereus, B. subtilis, B. licheniformis & other species.
These and a variety of non pathogenic aerobic spore bearing bacilli appear as laboratory contaminants & resemble anthrax bacilli – Pseudoanthrax or Anthracoid bacilli.
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Dr Ekta Chourasia, Microbiology

28. Differences b/n Anthrax & Anthracoid bacilli
Anthrax bacilli
Nonmotile
Capsulated
Grow in long chains
Medusa head colony
No growth in Pn agar (10units/ml)
Weak or no hemolysis
Inverted fir tree growth & slow gelatin liquefaction
No growth at 45C
Anthracoid bacilli
Generally motile
Noncapsulated
Grow in short chains
Not present
Grow usually
Hemolysis well marked
Rapid liquefaction
Usually grows
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Dr Ekta Chourasia, Microbiology

29. Dr Ekta Chourasia, Microbiology
Bacillus cereus
Readily isolated from soil, vegetables and a wide variety of foods including milk, cereals, spices, poultry & meat.
Causes foodborne gastroenteritis – 2 patterns of disease (diarrhoeal & emetic); both types are mild & self limited, requiring no specific therapy.
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Dr Ekta Chourasia, Microbiology

30. Bacillus cereus clinical presentation Gastroenteritis DIARRHOEAL FORM
EMETIC FORM
Incubation period > 6 hours
Diarrhoea
Lasts hours
Incubation period < 6 hours
Severe vomiting
Lasts 8-10 hours

31. Types of Gastroenteritis
Type I
Wide range of foods including cooked meat & vegetables
Diarrhoea & abdominal pain develops 8 –16 hrs after consumption
Few bacilli seen in fecal specimens
Caused by serotypes 2,6,8,9,10 or 12.
Enterotoxin resembles LT of E.coli
Type II
Chinese fried rice exclusively.
Acute nausea & vomiting 1-5 hrs after meals, diarrhoea rare
Large no of bacilli in cooked rice & fecal samples.
Caused by serotypes 1,3 or 5
Toxin resembles staphylococcal enterotoxin
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Dr Ekta Chourasia, Microbiology

32. Dr Ekta Chourasia, Microbiology
Diagnosis
Primarily depends on clinical diagnosis & food sources
Laboratory Diagnosis
Specimen – stool, vomitus, food, blood
Microscopy – not of much help
Culture
Test for toxin – to differentiate from staphylococcal food poisoning.
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Dr Ekta Chourasia, Microbiology

33. Dr Ekta Chourasia, Microbiology
Culture
Blood agar
Special MYPA medium: Mannitol - egg yolk - phenol red – polymyxin agar : to isolate B.cereus from feces & other sources.
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Dr Ekta Chourasia, Microbiology

34. Dr Ekta Chourasia, Microbiology
Treatment
Rehydration
Antibiotics – in systemic infections
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Dr Ekta Chourasia, Microbiology