Bacillus

Dr. Neha Sharma

Introduction 

  • Bacillus is a genus of Gram-positive, rod-shaped bacteria

  • Belongs to the family Bacillaceae

  • Widely distributed in soil, water, air, and vegetation

  • Organisms are aerobic or facultative anaerobic

  • Characteristically form endospores

  • Endospores are highly resistant to heat, drying, disinfectants, and chemicals

  • Spores help in survival under adverse environmental conditions

  • Includes both pathogenic and non-pathogenic species

  • Medically important species include:

    • Bacillus anthracis – causes anthrax

    • Bacillus cereus – causes food poisoning and opportunistic infections

    • Bacillus subtilis – usually non-pathogenic, used in research and industry

  • Pathogenicity is mainly due to toxin production, capsule formation, and enzymes

  • Important genus in medical microbiology, public health, and laboratory diagnosis

 

General Character

Genus: Bacillus

Family

  • Bacillaceae

Key Species

  • Bacillus anthracis – Causes anthrax

  • Bacillus cereus – Causes food poisoning (emetic & diarrheal types), eye infections, sepsis

  • Bacillus subtilis – Non-pathogenic; widely used in research, probiotics, and industry

Morphology

  • Gram reaction: Gram-positive (purple)

  • Shape: Large rod-shaped bacilli

  • Arrangement: Single cells, pairs, or chains

  • Size: Large bacteria (except B. anthracis which is relatively uniform)

Spore Formation

  • Endospore-forming

  • Spores are:

    • Oval or cylindrical

    • Central, subterminal, or terminal (species-dependent)

    • Do not bulge the cell (important differentiation point)

Motility

  • Motile: Most species (peritrichous flagella)

  • Non-motile: Bacillus anthracis (important diagnostic clue)

Oxygen Requirement

  • Aerobic or facultative anaerobic

  • Majority are obligate aerobes

  • Some species can grow anaerobically by fermentation

 

Morphology

  • Gram reaction: Gram-positive (appear purple on Gram staining due to thick peptidoglycan layer)

  • Shape: Large, straight rod-shaped bacilli

  • Size: Relatively large bacteria (≈ 1–1.5 µm × 3–10 µm)

  • Arrangement:

    • Single cells

    • Pairs

    • Chains (especially Bacillus anthracis)

  • Ends of cells:

    • Square or blunt ends (classically seen in B. anthracis – “box-car” appearance)

  • Spore formation:

    • Endospore-forming

    • Spores may be central, subterminal, or terminal

    • Spores are oval/cylindrical

    • Do not cause bulging of the bacterial cell

  • Motility:

    • Motile in most species (peritrichous flagella)

    • Non-motile: Bacillus anthracis (important diagnostic feature)

  • Capsule:

    • Usually absent

    • Present in Bacillus anthracis (polypeptide capsule made of poly-D-glutamic acid)

 

Cultural Characteristics

Cultural Characteristics of Genus: Bacillus

  • Growth requirements:

    • Non-fastidious organisms

    • Grow well on ordinary culture media

    • Optimal temperature: 35–37°C

    • Aerobic conditions preferred

On Nutrient Agar

  • Growth is rapid (18–24 hours)

  • Colonies are:

    • Large

    • Opaque

    • Rough or smooth (species dependent)

    • Irregular margins

  • Often produce a dry, matte or ground-glass appearance

On Blood Agar

  • Large, spreading colonies

  • Hemolysis pattern:

    • β-hemolysis: Bacillus cereus

    • Non-hemolytic: Bacillus anthracis (important differentiating feature)

Bacillus anthracis colonies:

  • Gray-white

  • Non-hemolytic

  • “Medusa head” appearance (curled, filamentous edges)

  • Tenacious colonies – stand up like beaten egg white when touched with loop

On Selective / Differential Media

  • Polymyxin–pyruvate–egg yolk–mannitol agar (PEMBA):

    • Used for B. cereus

    • Blue-green colonies with lecithinase activity (opaque halo)

  • Egg yolk agar:

    • B. cereus → Lecithinase positive

    • B. anthracis → Lecithinase negative

In Liquid Media (Broth)

  • Uniform turbidity

  • Surface pellicle formation may be seen

  • Sediment may be present at the bottom

Special Cultural Features

  • Spores are formed in old cultures

  • Growth often has a musty or earthy odor (especially B. subtilis)

  • Resistant spores survive adverse environmental conditions

 

Biochemical Reactions

Biochemical Reactions of Genus: Bacillus

  • Catalase test: Positive

  • Oxidase test: Variable (usually positive in many species)

  • Indole test: Negative

  • Methyl Red (MR): Variable

  • Voges–Proskauer (VP): Variable (often positive in B. cereus)

  • Citrate utilization: Variable

Carbohydrate Fermentation

  • Ferment carbohydrates aerobically

  • Acid production common; gas usually absent

  • Glucose fermented by most species

  • Lactose fermentation: variable

Enzyme Production

  • Amylase: Positive (B. subtilis strongly positive)

  • Protease: Positive

  • Lipase: Positive in B. cereus

  • Lecithinase:

    • Positive: Bacillus cereus

    • Negative: Bacillus anthracis

Nitrate Reduction

  • Most species reduce nitrate to nitrite

  • B. anthracis → Positive

Urease Test

  • Negative

Gelatin Hydrolysis

  • Positive (liquefaction of gelatin)

Special Biochemical Tests 

Test B. anthracis B. cereus B. subtilis
Catalase Positive Positive Positive
Motility Non-motile Motile Motile
Hemolysis Negative β-hemolytic Variable
Lecithinase Negative Positive Negative
Starch hydrolysis Negative Variable Positive
Nitrate reduction Positive Positive Positive

 

Pathogenicity

Bacillus anthracis 

Virulence Factors

  1. Capsule

    • Made of poly-D-glutamic acid (polypeptide, not polysaccharide)

    • Anti-phagocytic

  2. Anthrax Toxin (Tripartite Exotoxin)

    • Protective Antigen (PA): Binds to host cell receptors and facilitates entry of EF and LF

    • Edema Factor (EF): Adenylate cyclase → ↑ cAMP → edema

    • Lethal Factor (LF): Zinc-dependent metalloprotease → macrophage death, cytokine release, shock

 Toxin combinations

  • PA + EF → Edema toxin

  • PA + LF → Lethal toxin

Types of Anthrax

  1. Cutaneous anthrax (most common)

    • Entry through skin abrasion

    • Painless papule → vesicle → black eschar

    • Surrounding edema

  2. Inhalational anthrax (wool-sorter’s disease)

    • Inhalation of spores

    • Hemorrhagic mediastinitis

    • High mortality

  3. Gastrointestinal anthrax

    • Ingestion of contaminated meat

    • Ulcers, bleeding, diarrhea

Bacillus cereus

Virulence Factors

  • Emetic toxin (Cereulide)

    • Heat-stable

    • Acts on vomiting center

  • Enterotoxins

    • Heat-labile

    • Cause increased intestinal secretion

  • Tissue-destructive enzymes

    • Lecithinase, protease, hemolysin

Clinical Syndromes

  1. Food poisoning

    • Emetic type:

      • Rice, starchy food

      • Incubation: 1–6 hours

      • Nausea, vomiting

    • Diarrheal type:

      • Meat, vegetables

      • Incubation: 8–16 hours

      • Watery diarrhea, abdominal cramps

  2. Extra-intestinal infections

    • Endophthalmitis

    • Wound infections

    • Septicemia (immunocompromised)

3. Other Bacillus Species

  • B. subtilis

    • Usually non-pathogenic

    • Rare opportunistic infections

 

Laboratory Diagnosis

1. Specimen Collection

Bacillus anthracis

  • Cutaneous anthrax → Vesicular fluid / exudate, blood

  • Inhalational anthrax → Blood, sputum

  • GI anthrax → Stool, blood

 Do not open eschar (risk of aerosolization)

Bacillus cereus

  • Food poisoning → Food sample, vomitus, stool

  • Systemic infection → Blood, eye discharge, wound swab

2. Direct Microscopy

  • Gram stain

    • Large Gram-positive bacilli

    • Square ends

    • Arranged in chains (B. anthracis)

  • Spore stain

    • Central/subterminal spores

  • Capsule demonstration (B. anthracis)

    • Polychrome methylene blue → McFadyean reaction

    • Capsule appears pinkish-purple

3. Culture

Media Used

  • Blood agar

  • Nutrient agar

  • Selective media (for B. cereus)

Colony Characteristics

Feature B. anthracis B. cereus
Blood agar Non-hemolytic β-hemolytic
Colony edge Medusa head Irregular
Motility Non-motile Motile
Lecithinase Negative Positive (egg yolk agar)

4. Biochemical Tests

  • Catalase → Positive

  • Nitrate reduction → Positive

  • Motility test → Key differentiator

  • Lecithinase test

    • Positive in B. cereus

    • Negative in B. anthracis

5. Special Tests (Important for B. anthracis)

String of Pearls Test

  • Growth on penicillin-containing media

  • B. anthracis forms spherical cells in chains

  • Highly suggestive

Gamma Phage Lysis Test

  • Positive in B. anthracis

  • Specific and confirmatory

6. Molecular & Immunological Tests

  • PCR for toxin genes (PA, EF, LF)

  • ELISA for anthrax toxins or antibodies

  • Used in reference laboratories

7. Animal Inoculation Test (Historical)

  • Inoculation in mice/guinea pigs

  • Death with septicemia

  • Rarely used now

8. Diagnosis of B. cereus Food Poisoning

  • Isolation from:

    • Food sample

    • Stool/vomit

  • Demonstration of enterotoxin / emetic toxin

  • Clinical correlation essential

 

Antibiotic Resistance

General Features

  • Bacillus species show variable antibiotic susceptibility

  • Resistance is mainly due to:

    • β-lactamase production

    • Altered penicillin-binding proteins

    • Plasmid-mediated resistance genes

  • Spores are highly resistant to antibiotics, disinfectants, heat, and drying
    (important: spores survive treatment, vegetative cells are killed)

1. Bacillus anthracis

Resistance Pattern

  • Naturally resistant to:

    • Penicillin (many strains produce β-lactamase)

    • Cephalosporins (poor activity)

  • Usually sensitive to:

    • Ciprofloxacin

    • Doxycycline

    • Tetracycline

    • Erythromycin

    • Chloramphenicol

Acquired Resistance

  • Rare but reported (plasmid-mediated)

  • Bioterrorism-related strains may show multidrug resistance

 Exam pearl:

Penicillin is not the drug of choice for anthrax due to possible β-lactamase production.

2. Bacillus cereus

Resistance Pattern

  • Inherently resistant to:

    • Penicillins

    • Cephalosporins

    • β-lactam antibiotics (strong β-lactamase producer)

  • Usually sensitive to:

    • Vancomycin

    • Clindamycin

    • Ciprofloxacin

    • Gentamicin

    • Linezolid

 Very important point:

B. cereus is always resistant to penicillin.

3. Other Bacillus Species

  • B. subtilis:

    • Generally sensitive to many antibiotics

    • Resistance uncommon and clinically insignificant

Clinical Correlation

Species Key Resistance Preferred Drugs
B. anthracis Penicillin resistance (β-lactamase) Ciprofloxacin, Doxycycline
B. cereus Penicillins & cephalosporins Vancomycin, Clindamycin
B. subtilis Minimal resistance Rarely needed

 

Prevention

1. General Preventive Measures

  • Proper hygiene and sanitation

  • Safe handling and disposal of animal products (wool, hides, bones)

  • Thorough cooking and proper storage of food

  • Avoid consumption of leftover rice / improperly stored food (B. cereus)

  • Hospital infection control:

    • Sterilization of instruments

    • Proper wound care

2. Prevention of Anthrax (Bacillus anthracis)

A. Control in Animals (Most Important)

  • Vaccination of livestock (live attenuated Sterne strain)

  • Quarantine of infected animals

  • Proper disposal of carcasses:

    • Burning or deep burial with lime

  • Avoid opening carcasses of animals that died suddenly

B. Human Vaccination

  • Anthrax vaccine (AVA – Adsorbed vaccine)

    • Cell-free filtrate containing Protective Antigen

    • Given to:

      • Veterinarians

      • Laboratory workers

      • Wool, hide, and leather industry workers

      • Military personnel (high-risk groups)

C. Post-Exposure Prophylaxis (Anthrax)

  • Ciprofloxacin or Doxycycline

  • Duration: 60 days

  • Combined with vaccination in high-risk exposure

3. Prevention of Bacillus cereus Food Poisoning

  • Cook food thoroughly

  • Do not leave cooked rice or starchy food at room temperature

  • Refrigerate food promptly (<4°C)

  • Reheat food adequately before consumption

  • Maintain food hygiene in hotels, hostels, and canteens

4. Laboratory Safety Measures

  • Biosafety level precautions while handling suspected anthrax samples

  • Avoid aerosol generation

  • Proper labeling and transport of specimens

 

MCQs

1. Bacillus species are classified under which family?

A. Enterobacteriaceae
B. Bacillaceae
C. Clostridiaceae
D. Micrococcaceae
✅ Answer: B

2. Which Bacillus species causes anthrax?

A. B. cereus
B. B. subtilis
C. B. anthracis
D. B. licheniformis
✅ Answer: C

3. Gram reaction of Bacillus species is:

A. Gram-negative
B. Gram-variable
C. Gram-positive
D. Acid-fast
✅ Answer: C

4. Shape of Bacillus organisms is:

A. Cocci
B. Curved rods
C. Spiral
D. Straight rods
✅ Answer: D

5. Arrangement of Bacillus anthracis is typically:

A. Clusters
B. Chains
C. Diplococci
D. Palisades
✅ Answer: B

6. Which Bacillus is non-motile?

A. B. cereus
B. B. subtilis
C. B. anthracis
D. B. megaterium
✅ Answer: C

7. Endospores of Bacillus species are:

A. Heat sensitive
B. Acid fast
C. Highly resistant
D. Bulging the cell
✅ Answer: C

8. Spores of Bacillus usually do NOT:

A. Bulge the cell
B. Survive harsh conditions
C. Resist heat
D. Remain dormant
✅ Answer: A

9. Oxygen requirement of Bacillus species is mainly:

A. Obligate anaerobe
B. Microaerophile
C. Aerobic or facultative anaerobe
D. Capnophile
✅ Answer: C

10. Capsule of Bacillus anthracis is composed of:

A. Polysaccharide
B. Lipopolysaccharide
C. Poly-D-glutamic acid
D. Peptidoglycan
✅ Answer: C

11. Bacillus anthracis colony on blood agar is:

A. α-hemolytic
B. β-hemolytic
C. Non-hemolytic
D. Double zone hemolysis
✅ Answer: C

12. “Medusa head” colony is characteristic of:

A. B. cereus
B. B. anthracis
C. B. subtilis
D. B. megaterium
✅ Answer: B

13. Which Bacillus shows β-hemolysis on blood agar?

A. B. anthracis
B. B. cereus
C. B. subtilis
D. All
✅ Answer: B

14. Which test demonstrates capsule of B. anthracis?

A. Ziehl–Neelsen stain
B. India ink
C. McFadyean reaction
D. Albert stain
✅ Answer: C

15. McFadyean reaction uses:

A. Gram stain
B. Polychrome methylene blue
C. Carbol fuchsin
D. Malachite green
✅ Answer: B

16. Major virulence factor of B. anthracis is:

A. Endotoxin
B. Capsule only
C. Tripartite exotoxin
D. Hemolysin
✅ Answer: C

17. Protective antigen is responsible for:

A. Edema
B. Cell binding and entry
C. Cell death
D. Capsule formation
✅ Answer: B

18. Edema factor acts by increasing:

A. cGMP
B. Calcium
C. cAMP
D. ATP
✅ Answer: C

19. Lethal factor is a:

A. Lipase
B. Protease
C. Metalloprotease
D. Kinase
✅ Answer: C

20. Most common form of anthrax is:

A. Inhalational
B. Gastrointestinal
C. Cutaneous
D. Septicemic
✅ Answer: C

21. Black eschar is seen in:

A. Plague
B. Cutaneous anthrax
C. Diphtheria
D. Gas gangrene
✅ Answer: B

22. Food poisoning due to B. cereus is commonly associated with:

A. Milk
B. Rice
C. Eggs
D. Meat only
✅ Answer: B

23. Emetic toxin of B. cereus is:

A. Heat labile
B. Heat stable
C. Protein antigen
D. Endotoxin
✅ Answer: B

24. Incubation period of emetic type food poisoning is:

A. 12–24 h
B. 8–16 h
C. 1–6 h
D. 2–3 days
✅ Answer: C

25. Lecithinase test is positive in:

A. B. anthracis
B. B. subtilis
C. B. cereus
D. All
✅ Answer: C

26. String of pearls test is positive in:

A. B. cereus
B. B. anthracis
C. B. subtilis
D. Clostridium
✅ Answer: B

27. Gamma phage lysis test is specific for:

A. B. cereus
B. B. subtilis
C. B. anthracis
D. C. tetani
✅ Answer: C

28. Catalase test in Bacillus species is:

A. Negative
B. Variable
C. Positive
D. Weakly positive
✅ Answer: C

29. Which Bacillus is always resistant to penicillin?

A. B. anthracis
B. B. cereus
C. B. subtilis
D. None
✅ Answer: B

30. Drug of choice for anthrax is:

A. Penicillin
B. Amoxicillin
C. Ciprofloxacin
D. Erythromycin
✅ Answer: C

31. Spores of Bacillus are resistant to antibiotics because:

A. They multiply rapidly
B. They are metabolically inactive
C. They produce enzymes
D. They form capsule
✅ Answer: B

32. Anthrax vaccine contains:

A. Live bacilli
B. Killed spores
C. Protective antigen
D. Capsule antigen
✅ Answer: C

33. Anthrax vaccine is recommended for:

A. General population
B. Children
C. High-risk occupational groups
D. Pregnant women
✅ Answer: C

34. Best prevention of anthrax is:

A. Human vaccination
B. Antibiotics
C. Animal vaccination
D. Quarantine only
✅ Answer: C

35. Which Bacillus is commonly non-pathogenic?

A. B. anthracis
B. B. cereus
C. B. subtilis
D. B. thuringiensis
✅ Answer: C

36. B. cereus diarrheal toxin is:

A. Preformed
B. Heat stable
C. Heat labile
D. Endotoxin
✅ Answer: C

37. Blood culture in anthrax usually shows:

A. No growth
B. Gram-negative rods
C. Gram-positive bacilli in chains
D. Cocci
✅ Answer: C

38. Bacillus spores germinate when:

A. Conditions are unfavorable
B. Antibiotics present
C. Favorable conditions return
D. Oxygen absent
✅ Answer: C

39. Which medium is used for lecithinase detection?

A. Blood agar
B. MacConkey agar
C. Egg yolk agar
D. TCBS
✅ Answer: C

40. In broth culture, Bacillus typically produces:

A. Clear broth
B. Pellicle and turbidity
C. Only sediment
D. Gas bubbles
✅ Answer: B

41. Bacillus species are commonly found in:

A. Human intestine only
B. Soil
C. Water only
D. Animals only
✅ Answer: B

42. Which Bacillus is motile?

A. B. anthracis
B. B. cereus
C. Both A and B
D. None
✅ Answer: B

43. The capsule of B. anthracis helps in:

A. Toxin production
B. Sporulation
C. Anti-phagocytosis
D. Motility
✅ Answer: C

44. Bacillus cereus food poisoning is usually:

A. Fatal
B. Mild and self-limiting
C. Chronic
D. Septicemic
✅ Answer: B

45. Which Bacillus produces β-lactamase?

A. B. anthracis
B. B. cereus
C. B. subtilis
D. All
✅ Answer: D

46. A non-hemolytic, non-motile Gram-positive rod suggests:

A. B. cereus
B. B. subtilis
C. B. anthracis
D. Clostridium perfringens
✅ Answer: C

47. Post-exposure prophylaxis for anthrax is given for:

A. 7 days
B. 14 days
C. 30 days
D. 60 days
✅ Answer: D

48. Bacillus spores are best destroyed by:

A. Alcohol
B. Boiling
C. Autoclaving
D. Phenol
✅ Answer: C

49. Which toxin causes vomiting in B. cereus infection?

A. Enterotoxin
B. Cereulide
C. Endotoxin
D. Lethal toxin
✅ Answer: B

50. Bacillus belongs to which Gram-positive group?

A. Non-sporing cocci
B. Spore-forming rods
C. Branching filaments
D. Acid-fast bacilli
✅ Answer: B