Gram positive aerobic spore forming organisms, primarily a zoonotic disease responsible to cause deadliest infections in humans due to inhalation, ingestion of spores of these organisms present in dust, animal wool, or in dead animals. Causes Cutaneous, Pulmonary and Intestinal Anthrax. Grow well on ordinary media. Detected by M'Fadyean's Reaction.

1. BACILLUS
Mr. Gunjal Prasad N.
Assistant Prof.
M.Sc. Medical Microbiology,
PGDCR&RA

2. INTRODUCTION
 Sporogenous OR Spore forming rod shaped
bacteria: 2 groups
1. Aerobic – Bacillus – Non Bulging spores.
2. Anaerobic – Clostridia- Bulging spores.
 Important Bacillus species:
 Bacillus anthracis
 Bacillus cereus
 Bacillus stearothermophilus

3. INTRODUCTION
 Ubiquitous, present in Soil, Air, Dust, &Water.
 Frequently isolated as “ LAB CONTAMINANTS”.
 B. anthracis, the causative agent of an important Zoonotic
disease called “ANTHRX”.
 B. cereus can cause “FOOD POISOINING”.
 All members are generally “MOTILE” except B. anthracis,
which is “NON-MOTILE”.
 Temp. range for growth 25 -750C.
 Salt conc. 2% - 25%.
 Also gained importance recently because of its ability to
be used as “Biological Weapon”.

4. Historical Interest
 First organism observed under microscope,
Pollender, 1849.
 First communicable disease shown to be
transmitted by inoculation of infected blood –
Davaine, 1850.
 First bacillus to be isolated in pure culture and
shown to possess spores – Koch, 1876.
 First bacteria to be used for preparation of
attenuated vaccine by Pasteur, 1881.

5. BACILLUS ANTHRACIS
 Causative agent of “ANTHRX”.
 A disease primarily of animals, & man gets
infected secondarily (Zoonosis).

6. MORPHOLOGY
 Gram positive, Non-Acid fast, Non-Motile.
 Large (3-10 µm X 1-1.6 µm), rectangular
 Capsule is made up of polypeptide, polymer of d-glutamic
acid.
 This capsule is Plasmid coded (pX02).
 It inhibits complement mediated phagocytosis.
 Capsules not formed under ordinary conditions only if media
containing bicarbonate or are incubated at 10 to 25 % CO2.
 If media contains serum, albumin, charcoal or starch – Capsule
formation may occur in absence of CO2.

7.  The bacilli are arranged end to end in long
chains.
 The ends are often concave & somewhat
swollen so that a chain of bacilli present a
“bamboo-stick” appearance.
 Spore are oval & central in position & are of
the same width as the bacillary body so that
they do not cause bulging of vegetative cell.
 Spores are formed in culture and in soil but
never in host body.
MORPHOLOGY

8. Spores
 Spores are stain by special methods – Sudan black – B – Fat
globules maybe made out within bacilli. “Hot malachite
green (Ashby’s method) OR 0.25% Sulphuric acid as spores
are Acid fast”.
 When blood films are stained with polychrome methylene blue
for a few seconds and observed – an amorphous purplish
material is noticed around the bacilli.
 This represents the capsular material and is characteristic of
anthrax bacillus.
 This is called as “Mc Fadyean’s reaction” & used
as presumptive diagnosis ofAnthrax in animals.

9. CULTURE
 B. anthracis is an aerobe & facultative anaerobe, with a
temp. range for growth being 12-450C (optimum 35-
370C).
 NUTRIENT AGAR MEDIA
 Colonies are irregular, round, 2-3 mm in diameter, dull,
raised, opaque & grayish white with frosted glass
appearance.
 Under low power microscope, the edge of the colony is
found to be composed of long, interlacing chains of
bacilli, resembling locks of matted hairs, the so called
“Medusa head” appearance.

10. CULTURE
 BLOOD AGAR MEDIA
 Colonies are Non - haemolytic,
 Though occasional strains produce a narrow zone of
haemolysis.
 SELECTIVE MEDIUM
 A selective medium (PLET) consisting of Heart infusion
agar with Polymyxin, Lysozyme, Ethylene diamine
tetracetic acid (EDTA) &Thallous acetate has been devised
for isolation of B. anthracis from mixtures containing other
spore-bearing bacilli.

11. PATHOGENICITY
o Naturally anthrax is disease of cattle and sheep, less or more
other animals are also susceptible (Zoonosis).
Subcutaneous Inoculation of G.P. with B. anthracis
culture filtrate.
Animal dies within 24-72 hrs.
Autopsy – Site of inoculation – shows local gelatinous
hemorrhagic edema.
Spleen – Extensive subcutaneous congestion, enlarged, dark
red, friable - easily broken into pieces or become powder.
Blood – Dark red & coagulates less firmly than normally.
Bacilli are found in large number in local lesions, heart, blood,
spleen (more than 108 bacilli / ml)

12. PATHOGENESIS
 Bacilli remain attached to interior capillaries,
number is more – so obstruction occur in
blood flow.

13. VIRULENCE FACTORS
oTwo major virulence factor –
o Capsular polypeptide
o Anthrax toxin
o Each produced by separate plasmid.
o Capsular polypeptide –
oComposed of poly peptide of a high molecular weight
consisting of D-glutamic acid.
o Inhibits phagocytosis.
o Loss of plasmid (px02) controls production of capsule,
leads to loss of virulence.
o Attenuated anthrax spore vaccine is prepared by this
method (Sterne strain).

14. Anthrax Toxin
 The toxin is complex of 3 fractions:
 1. Edema Factor (OF or Factor I)
 2. ProtectiveAntigen Factor (PA or Factor II)
 3. Lethal Factor (LF or Factor III)
 These are nontoxic if act individually.
 But the complex causes local edema and
generalised shock.

15. Anthrax Toxin
 These three factors have been characterised and
cloned.
 Protective Antigen Factor or PA - It binds the target
cell surface receptors and in turns provides
attachment sites for Edema factor or OF or Lethal
Factor or LF, facilitating their entry inside the target
cell.
 The antibody to PA is protective because it blocks the
attachment of PA to target cell surface receptor and
inhibit the further action of OF or LF.

16.  Edema Factor or OF - It is an Adenyl cyclase which is
activated only inside the target cells, leading to
intracellular accumulation of cyclic AMP.
 This is believed to be responsible for the edema and
other biological effects of the toxin.
 Lethal Factor - Entry of LF into the cell causes cell
death but the mechanism of action is not known.
Anthrax Toxin

17.  Loss of the plasmid (px01) which encodes the
toxin renders the strain avirulent.
 This is believed to have been the basis for the
original anthrax vaccine developed by Pasteur.
 The avirulent Sterne vaccine strain is devoid of
the plasmid coding for the capsular
polysaccharide.
Anthrax Toxin

18. PATHOGENESIS – Animals
 Anthrax is primarily a disease of animals like cattle
& sheep, less often of horses & swine.
 Anthrax in animals presented as a fetal septicemia;
however, localized cutaneous lesions may be
produced rarely.
 Infected animals discharge large number of bacilli
from the mouth, nose & rectum.
 The bacilli sporulate in soil & remains as the source
of infection.

19. HUMAN ANTHRAX
 Humans are occasionally secondarily infected
from diseased animals.
 There are three clinical types of disease based
on route of infection.
 CUTANEOUS
 PULMONARY
 INTESTINAL ANTHRAX.
 ALLTYPES LEADTO SEPTICAEMIA.

20. HUMAN ANTHRX – Cutaneous
 95 % of human cases are of cutaneous type.
 Route of entry: Skin by inoculation.
 Involves 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 untreated may
develop fatal septicemia or meningitis.

21. HUMAN ANTHRAX – Pulmonary
 Pulmonary anthrax occurs due to inhalation
of the dust or filaments of wool from infected
animals, particularly in wool factories .
 This is also called “Wool – sorter’s Disease”
 A life- threatening hemorrhagic
pneumonia caused by Inhalation of spores.

22.  Intestinal anthrax is a rare in man and is found in
those who consume improperly cooked food/
infected meat for e.g.-AfricanTribes living in
Jungle.
Human anthrax can be
 Industrial – in meat packing or wool factories
 Nonindustrial – frequent association with
animal handlers like butchers, veterinarians,
farmers
HUMAN ANTHRAX –
Gastrointestinal

23. Difference between Cutaneous and Pulmonary Anthrax
Features Cutaneous Anthrax Pulmonary Anthrax
Other Name Hide porter’s disease (As
commonly occurs in dock
workers carrying loads of hides
and skin on their bare backs)
Wool sorter’s disease (As it is
seen in workers of wool factories
acquire infection by inhalation of
dust from infected wool.
Transmission Cutaneous exposure to spores
(enter through abraded ski)
Inhalation of spores
Characterized
by
Malignant pustule – the lesion
begins as a papule that evolves
into a painless vesicle followed by
the development of a coal-black,
necrotic eschar surrounded by
non-pitting indurated edema.
 The name anthrax, which
means coal, comes from the
black colour of the eschar.
 However, it is a non-malignant
condition
Haemorrhagic pneumonia
Bacillus spread by lymphatics or
blood, leading to –
Bacteremia
Haemorrhagic meningitis

24. Features Cutaneous Anthrax Pulmonary Anthrax
Occupational
exposure
Dock worker, butcher,
abattoir and farmer
Workers of the wool factory
Occurrence Most common (95%) Rare
Prognosis Self- limiting, rarely becomes
fatal if untreated
Fatal
Bioterrorism Rarely causes bioterrorism Most common form to cause
bioterrorism
Difference between Cutaneous and Pulmonary Anthrax

25. LABORATORY DIAGNOSIS
 A. SPECIMENS –
 Swabs
 Fluids or Pus from pustules
 Sputum &
 Blood from pulmonary & septicaemic anthrax
are generally collected.

26. MICROSCOPY
 Gram stained smear from the specimen shows
often chain of large Gram Positive Bacilli.
 Capsule appears as a clear halo around the
bacterium by India-Ink preparation/ staining.
 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.

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

28. CULTURE
 Specimen is inoculated on Nutrient Agar medium &
incubated at 370C for overnight.
 Irregular, round, raised, dull, opaque, grayish 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 HeadAppearance”.
 Gelatin stab culture – “inverted fir tree” appearance,
with slow liquefaction starting from top.

29. Medusa HeadAppearance
-wavy colonies with small
projections
Inverted fir tree

30.  “String of Pearls reaction” – solid medium
containing 0.05-0.5 units of Penicillin/ml, in 3-6
hrs. the cells become large, spherical and occur
in chains on agar surface, resembling a string of
pearls.
 Selective medium – PLET medium – contains
Polymyxin, Lysozyme, EDTA &Thallous acetate :
to isolate it from mixtures containing other spore
bearing bacilli.

31. ANIMAL INOCULATION
 White mouse or Guinea – pigs are inoculated/ injected with
exudate or culture.
 By rubbing contaminated tissue over shaven skin of a
guinea pig.
 Animal dies in 24----------------------------------------------------
-------- 48 hrs.
 Serology
1. Ascoli’s thermoprecipitation test – to demonstrate
anthrax Ag in tissue extracts
2. EIA (using purified anthrax toxin Ag)
3. PCR to detect anthrax contamination of animal &
agricultural products

32. TREATMENT
Bacillus anthracis treatment :
- Ciprofloxacin or Doxycycline, plus Clindamycin,
and/or Rifampin for 60 days.
Antibiotics for post exposure prophylaxis :
Ciprofloxacin for 60 days.
Doxycycline for 60 days or Amoxicillin for 60days (given
if strain is penicillin sensitive).
Raxibacumab: It is a monoclonal antibody that
neutralizes anthrax toxin (protective antigen). It is
intended for the prophylaxis and treatment of
inhalation anthrax.

33. PROPHYLAXIS
 General methods of prevention
1. Improvement of factory hygiene
2. Proper sterilization of animal products
3. Animal carcasses to be buried deep in
quicklime or cremated

34. PROPHYLAXIS
 Active immunisation of
1. Domestic animals with live attenuated spore
vaccines
2. Persons with occupational risk (butchers, farmers,
veterinarians) with a cell- free vaccine containing
purified protective antigen as immunogen.
3. 3 doses IM at an interval of 6 weeks with annual
booster injections.
* Anthrax infection in humans give life long
permanent immunity & secondary infections are
very rare.

35. 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 vaccines -
1. Sterne vaccine – contains spores of a non -
capsulated avirulent mutant strain - loss of
plasmid which controls capsule production.
2. Mazucchi vaccine – contains spores of stable
attenuated Carbazoo strain.

36.  Biological warfare
 Anthrax was a long feared as a potential tool in
biological warfare.
 Large epidemics (occasionally)
1. In 1979 – former Soviet Union: due to accidental
release of spores from a military facility engaged
in biological research.
2. In 1980s – Zimbabwe: affected 10,000 persons.
3. In 2001 – USA several died due to mails with
spores of B. anthracis having enhanced virulence.
* Hence the need to develop better human vaccine.

37. ANTHRACOID BACILLI
 Aerobic spore bearing bacilli resembling B.
anthracis are called “ANTHRACOID” or
“PSEUDOANTHRAX BACILLI.”
 Some of them are frequent laboratory
contaminants & have to be differentiated from B.
anthracis.
 The main differentiating features between
anthracoid bacilli & B. anthracis are shown in
table.

38. DIFFERENTIATING FEATURES BETWEEN B. ANTHRCIS & ANTHRACOID BACILLI
FEATURES B. anthracis Anthracoid bacilli
Motility Non- motile Generally motile
Capsule Capsulated Non – capsulated
Chain formation Long chains Short chains
Colony on Nutrient Agar Medusa Head Colony No such morphology
Growth in Broth No turbidity Uniform turbidity
Gelatin Stab culture Inverted Fir tree appearance
& show gelatin liquefaction
Rapid gelatin liquefaction
Haemolysis on BA Absent Usually well marked
Growth in PenicillinAgar (10
units/ml)
No growth Grow usually
Growth at 450C No growth Grow usually
Susceptibility to Gamma
phage
Susceptible Not susceptible
Pathogenicity test in animals Pathogenic Non-pathogenic
Ascoli’s precipitin test Positive Negative
Fluorescent Antibody test
with anthrax antiserum
Positive Negative

39. BACILLUS CEREUS
 Cause of FOOD POISOINING.
 Ubiquitous in nature.
 Vegetables, milk, cereals, spices, meat & poultry.
 Some spores survive cooking & germinate into vegetative bacilli
which produce ENTEROTOXIN that causes food poisoining.
 Ocular disease :
 It causes severe keratitis and panophthalmitis following trauma to
the eye that may lead to loss of vision.
 Other conditions: Rarely cause systemic infections including –
endocarditis, meningitis, osteomyelitis and pneumonia.
 The presence of medical device or intravenous drug use predispose
to these infections.

40. TYPES OF FOOD POISOINING
 1. SHORT INCUBATION PERIODTYPE (1-5 HRS).
 Characterized by acute Nausea & vomiting, 1-5 hrs
after the meal.
 Diarrhoea is not common.
 It is usually associated with consumption of cooked
rice, usually fried rice from Chinese restaurants.

41. 2. Long incubation period type (8-16)
 Characterised by
 Acute abdominal pain & diarrhoea, 8-16 hrs.
after consumption of contaminated food.
 Vomiting is rare symptom in this type.

42. GastroenteritisBacillus cereus
clinical presentation
Incubation period < 6 hours
Severe vomiting
Lasts 8-10 hours
Incubation period > 6 hours
Diarrhoea
Lasts 20-36 hours
EMETIC FORMDIARRHOEAL FORM

43. Types of Gastroenteritis
Bacillus cereus DiarrhealType EmeticType
Incubation period 8-16 hours 1-5hours
Toxin Secreted in intestine (Cl.
perfringens enterotoxin)
Performed toxin (formed in
diet, similar to Staph. aureus
enterotoxin)
Heat Heat labile Heat stable
Food items contaminated Meat, vegetables, dried
beans, cereals
Rice (Chinese fried rice)
Clinical feature Diarrhoea, fever, abdominal
cramps
Vomiting, abdominal
cramps
Serotypes 2,6,8,9,10,12 1,3,5

44. DIAGNOSIS
 Suspected food, faeces & vomitus are cultured on ordinary
media or a special such as –
 MYPA - Mannitol, Egg yolk, Phenol red, Polymyxin agar.
 PEMBA – Polymyxin B, Egg yolk, mannitol, bromothymol blue
agar.
 Spore bearing Gram Positive Bacilli may be seen on smear from
colonies.
 B. cereus is a motile bacilli, non-capsulated, not susceptible to
gamma phage & does not react with fluorescent antibody
conjugate.

45. TREATMENT
 Disease is mild and self limiting, requiring no specific
treatment.
 Rehydration
 Antibiotics – in systemic infections
 Bacillus cereus susceptible to Clindamycin,
erythromycin vancomycin, aminoglycosides and
tetracycline.
 It is resistant to penicillin (by producing β-lactamase)
and trimethoprim.

46. Bacillus thuringinesis
 It is closely related with B. cereus and may
occasionally produce food poisoning.
 It is also used as Larvicidal agent for mosquito
control.

47. Bacillus used as sterilization
control
 B. stearothermophilus & B. subtilis both are
used as biological controls for autoclave and
plasma sterilization.
 B. pumilus is used as biological control for
ethylene oxide.

48. Industrial use of Bacillus
subtilis
 B. subtilis is used in industries for various purposes –
As cleaning agent (Detergent).
 In paper and textile industries – produces amylase that
breaks down starch.
 For pollution treatment- by breaking down pollutants
(Bioremediation).
 In pesticide industries – for protecting crops against
fungi.
 In food industries – helps in fermentation.

49. Case study
 Alisha, a 30 yr. old women, was admitted to hospital after a
prolonged fever, with chills and night sweats, chest discomfort
with blood stained sputum, hospitalized.
 The doctors performed a Gram’s stain for sputum specimen and
found Gram positive rod shaped bacteria arranged in chains.
 Other tests results showed that bacterium was aerobic and spore
bearing.
 1.What is the clinical diagnosis and causative agent?
 2. Describe the pathogenesis and various forms of clinical
presentation of this infection?
 3. Mention the laboratory investigation to confirm the diagnosis?

50. Short Notes
 Malignant pustule.
 B. cereus food poisoning.
 Wool sorter’s disease.
 Hide porter’s disease.
 Mc Fadyean’s Reaction.

51. MCQ
 Gram-stain morphology of Bacillus anthrax is : a.Tennis racket
 b. Drum stick appearance
 c. Bamboo stick appearance
 d. Spectacle glass appearance
 A “Malignant pustule” is a term for:
 a. An infected malignant melanoma
 b. A carbuncle
 c. A rapid spreading rodent ulcer
 d. Anthrax of the skin.
 Incubation period for B. cereus food poisoning following consumption of
contaminated fried rice?
 a. 1-6 hrs.
 b. 8-16 hrs.
 c. 24 hrs.
 D. > 24 hrs.