Bordetella

Dr. Neha Sharma

Introduction 

  • Bordetella is a genus of small, Gram-negative, aerobic coccobacilli.

  • Bordetella pertussis is the principal causative agent of whooping cough (pertussis).

  • Whooping cough is a highly contagious respiratory disease transmitted via respiratory droplets.

  • The organism primarily infects the ciliated epithelium of the respiratory tract.

  • Bordetella produces several potent toxins and adhesins that contribute to disease severity.

  • The infection is characterized clinically by paroxysmal coughing, inspiratory whoop, and post-tussive vomiting.

  • Laboratory diagnosis involves culture, PCR, and serological methods.

  • Whole-cell and acellular pertussis vaccines play a vital role in disease prevention.

  • Re-emergence of pertussis highlights the importance of booster immunization and surveillance.

  • Understanding Bordetella biology is essential for clinical microbiology, epidemiology, and public health control.

General Character

Family

  • Bordetellaceae

Species and Clinical Significance

  1. Bordetella pertussis

    • Primary causative agent of whooping cough (pertussis) in humans

    • Causes severe disease, especially in infants and young children

  2. Bordetella parapertussis

    • Causes a milder, pertussis-like illness

    • Clinical features resemble whooping cough but are generally less severe

    • Does not produce pertussis toxin

  3. Bordetella bronchiseptica

    • Primarily causes respiratory infections in animals (e.g., kennel cough in dogs)

    • Can cause opportunistic respiratory infections in humans, especially immunocompromised individuals

Morphology and Staining Characteristics

Gram Staining

  • Gram-negative bacteria

  • Appear pink on Gram staining due to:

    • Thin peptidoglycan layer

    • Presence of an outer membrane containing lipopolysaccharide (LPS)

Shape

  • Small coccobacilli

  • Short, plump rod-shaped organisms

Arrangement

  • Usually seen as:

    • Single cells

    • Occasionally in small clusters

  • Do not form chains or spores

Oxygen Requirements

  • Obligate aerobes

  • Require oxygen for growth

  • Growth occurs best under aerobic laboratory conditions

 

Morphology

  • Size

    • Very small bacteria

    • Measure approximately 0.2–0.5 µm × 0.5–1.0 µm

  • Shape

    • Coccobacilli (short, plump rods)

    • Appear intermediate between cocci and bacilli

  • Gram Reaction

    • Gram-negative

    • Stain pink on Gram staining due to:

      • Thin peptidoglycan layer

      • Presence of outer membrane

  • Arrangement

    • Occur singly

    • Sometimes seen in small clusters

    • Do not form chains or palisades

  • Motility

    • Non-motile

    • Lack flagella

  • Capsule

    • True capsule absent

    • However, surface adhesins (e.g., filamentous hemagglutinin) provide capsule-like protective function

  • Spores

    • Non-sporing

  • Pleomorphism

    • Minimal pleomorphism

    • Morphology remains fairly uniform

  • Special Staining Features

    • Poorly stained with routine stains

    • Best visualized in:

      • Gram stain

      • Immunofluorescence staining (direct fluorescent antibody test)

 

Cultural Characteristics

1. Growth Requirements

  • Fastidious organisms, especially Bordetella pertussis

  • Require enriched media

  • Strictly aerobic

  • Optimal growth temperature: 35–37 °C

  • Optimal pH: 7.2–7.6

  • Growth is slow, visible after 3–7 days

2. Primary Culture Media

A. Bordet–Gengou Agar

  • Medium of choice for B. pertussis

  • Composition:

    • Potato infusion

    • Glycerol

    • Blood (15–20%)

  • Blood neutralizes toxic fatty acids

  • Glycerol enhances growth

Colony Morphology

  • Small, smooth, convex

  • Glistening, pearly, dome-shaped colonies

  • Described as “mercury drop” appearance

  • Non-hemolytic

B. Regan–Lowe Medium

  • Charcoal agar with:

    • 10% horse blood

    • Cephalexin (selective agent)

  • Preferred in modern laboratories

  • Advantages:

    • Less toxic than Bordet–Gengou

    • Better recovery from clinical specimens

    • Supports transport and prolonged survival

3. Growth on Other Media

  • Blood agar:

    • B. pertussis: poor or no growth

    • B. bronchiseptica: may grow

  • MacConkey agar:

    • B. pertussis → no growth

    • B. bronchiseptica → may grow (non-lactose fermenter)

4. Colony Variations (Phase Variation – B. pertussis)

Phase Characteristics Virulence
Phase I Smooth, dome-shaped, hemagglutinating Fully virulent
Phase II–IV Rough, irregular colonies Reduced or avirulent

5. Cultural Differences Among Bordetella Species

Feature B. pertussis B. parapertussis B. bronchiseptica
Growth rate Slow Faster Faster
Bordet–Gengou Good growth Good growth Good growth
Regan–Lowe Excellent Excellent Excellent
Blood agar Poor Moderate Good
MacConkey agar No growth No growth Growth present

6. Atmospheric Requirements

  • Strict aerobes

  • No growth under anaerobic conditions

  • Increased CO₂ may enhance early growth

7. Transport of Specimens

  • Organism is fragile

  • Best specimens:

    • Nasopharyngeal swab/aspirate

  • Transport media:

    • Regan–Lowe transport medium

    • Avoid cotton swabs (toxic fatty acids)

 

Biochemical Reactions

A. Oxidase Test

  • B. pertussis → Positive

  • B. parapertussis → Positive

  • B. bronchiseptica → Positive

Useful screening test

B. Catalase Test

  • All Bordetella species → Positive

C. Urease Test 

Species Urease Activity
B. pertussis Negative
B. parapertussis Positive
B. bronchiseptica Strongly Positive (Rapid)

Most important test to differentiate Bordetella species

D. Nitrate Reduction Test

Species Nitrate Reduction
B. pertussis Negative
B. parapertussis Negative
B. bronchiseptica Positive

E. Citrate Utilization

  • All Bordetella species → Negative

F. Indole Test

  • All Bordetella species → Negative

G. Carbohydrate Fermentation

  • No carbohydrate fermentation

  • Glucose, lactose, sucrose → Not fermented

  • Hence non-acid producing

Summary Table

Test B. pertussis B. parapertussis B. bronchiseptica
Oxidase + + +
Catalase + + +
Urease − + ++
Nitrate reduction − − +
Indole − − −
Citrate − − −
Sugar fermentation − − −

 

Pathogenicity

Source and Transmission

  • Source of infection: Infected human (symptomatic or asymptomatic carrier)

  • Mode of transmission: Respiratory droplets (coughing, sneezing)

  • Highly infectious; secondary attack rate is high

Portal of Entry and Site of Infection

  • Entry via upper respiratory tract

  • Organism attaches to ciliated epithelial cells of:

    • Nasopharynx

    • Trachea

    • Bronchi

  • No bloodstream invasion

Virulence Factors and Their Role

A. Adhesins (Colonization Factors)

  1. Filamentous Hemagglutinin (FHA)

    • Major adhesin

    • Mediates attachment to ciliated epithelium and macrophages

  2. Pertactin

    • Outer membrane protein

    • Facilitates tight adhesion to respiratory epithelium

  3. Fimbriae (Agglutinogens 2 & 3)

    • Help in adherence

    • Important antigens in acellular vaccines

B. Toxins (Major Determinants of Disease)

1. Pertussis Toxin (PT) 

  • AB₅ exotoxin

  • ADP-ribosylates Gi protein → ↑ cAMP

  • Effects:

    • Lymphocytosis

    • Inhibition of phagocytosis

    • Increased insulin secretion → hypoglycemia

  • Responsible for systemic manifestations

2. Adenylate Cyclase Toxin (ACT)

  • Enters phagocytes

  • Converts ATP → cAMP

  • Inhibits:

    • Neutrophil function

    • Macrophage killing

3. Tracheal Cytotoxin (TCT)

  • Peptidoglycan fragment

  • Causes:

    • Destruction of ciliated epithelial cells

    • Loss of mucociliary clearance

  • Responsible for persistent cough

4. Dermonecrotic Toxin

  • Causes local tissue damage

  • Vasoconstrictive effects

C. Endotoxin (LPS)

  • Contributes to:

    • Inflammation

    • Fever

    • Local tissue injury

 

Laboratory Diagnosis

Specimen Collection

A. Type of Specimen

  • Nasopharyngeal swab (preferred)

  • Nasopharyngeal aspirate (highest yield)

B. Technique

  • Use:

    • Dacron / calcium alginate swab

  • Avoid:

    • Cotton swabs (contain fatty acids toxic to Bordetella)

C. Transport

  • Organism is fragile

  • Transport immediately in:

    • Regan–Lowe transport medium

Direct Microscopy

  • Gram staining

    • Small Gram-negative coccobacilli

  • Sensitivity is low

  • Mainly supportive, not confirmatory

Culture

Media Used

  1. Bordet–Gengou agar

  2. Regan–Lowe medium (preferred)

Incubation

  • 35–37°C

  • Strictly aerobic

  • Observe for 3–7 days

Colony Morphology

  • Small, smooth, convex

  • Glistening “mercury-drop” colonies

  • Non-hemolytic

Culture positivity highest in catarrhal stage

Biochemical Identification

  • Oxidase: Positive

  • Catalase: Positive

  • Urease: Negative (key feature of B. pertussis)

  • Nitrate reduction: Negative

Molecular Methods 

PCR (Polymerase Chain Reaction)

  • Target genes:

    • IS481

    • Pertussis toxin gene

  • Advantages:

    • Rapid

    • Highly sensitive and specific

    • Useful even after antibiotic therapy

  • Preferred diagnostic test in modern labs

Serological Tests

ELISA

  • Detects antibodies against:

    • Pertussis toxin

    • FHA

  • Useful in:

    • Late stages

    • Adolescents and adults

  • Not useful in early disease or vaccinated children

Direct Fluorescent Antibody (DFA) Test

  • Detects organisms directly from specimen

  • Rapid but:

    • Low specificity

    • False positives common

  • Largely obsolete

Hematological Findings

  • Marked lymphocytosis

  • Total leukocyte count may exceed 20,000–50,000 /mm³

  • Characteristic of pertussis toxin effect

 

Antibiotic Resistance

Drugs of Choice and Susceptibility

Macrolides 

  • Erythromycin

  • Azithromycin

  • Clarithromycin

Alternative Drugs

  • Trimethoprim–Sulfamethoxazole (Co-trimoxazole)

    • Used in macrolide intolerance or resistance

    • Effective in patients ≥2 months of age

Resistance Patterns

A. Macrolide Resistance

  • Historically rare

  • Increasing reports worldwide

  • Mechanisms:

    • 23S rRNA gene mutations

    • Reduced macrolide binding to ribosomal target

  • Clinically significant in:

    • Prolonged illness

    • Treatment failure

B. β-lactam Antibiotics

  • B. pertussis is intrinsically resistant to:

    • Penicillins

    • Cephalosporins

  • Reason:

    • Poor penetration

    • Lack of target efficacy

  • Not recommended for treatment

C. Fluoroquinolones

  • In vitro activity present

  • Not routinely used

  • Reserved for special circumstances in adults

D. Aminoglycosides

  • Poor intracellular penetration

  • Limited clinical role

Species-wise Resistance 

Bordetella pertussis

  • Usually susceptible to macrolides

  • Emerging macrolide resistance (regional variation)

Bordetella parapertussis

  • Less sensitive to macrolides than B. pertussis

  • Generally responsive to macrolides and TMP-SMX

Bordetella bronchiseptica

  • More resistant

  • Often resistant to:

    • Macrolides

    • First-generation cephalosporins

  • Susceptible to:

    • Fluoroquinolones

    • Aminoglycosides

    • TMP-SMX

Antibiotic Susceptibility Testing

  • Not routinely done for B. pertussis

  • Performed in:

    • Outbreaks

    • Treatment failure

    • Surveillance studies

  • Methods:

    • MIC determination

    • PCR detection of resistance mutations

 

Prevention

Active Immunization 

A. Types of Pertussis Vaccines

1. Whole-Cell Pertussis Vaccine (wP)

      • Contains killed whole B. pertussis

      • Strong immunogenicity

      • More adverse effects:

        • Fever

        • Local reactions

      • Used widely in developing countries

2. Acellular Pertussis Vaccine (aP)

      • Contains purified antigens:

        • Pertussis toxin (PT)

        • Filamentous hemagglutinin (FHA)

        • Pertactin

        • Fimbriae

      • Fewer side effects

      • Used mainly in developed countries

B. Vaccines in Use

      • Given as DPT / DTaP / Tdap

        • DPT (wP)

        • DTaP (aP for children)

        • Tdap (booster for adolescents & adults)

C. National Immunization Schedule 

Age Vaccine
6 weeks DPT-1
10 weeks DPT-2
14 weeks DPT-3
16–24 months DPT booster
5 years DPT booster
Adolescents / Adults Tdap (recommended)
Pregnancy Tdap in 3rd trimester

Passive Immunization

      • No role for routine passive immunization

      • Maternal antibodies provide partial protection to neonates

Chemoprophylaxis

      • Recommended for:

        • Household contacts

        • Healthcare workers

        • High-risk individuals (infants, pregnant women)

Drugs Used

      • Macrolides (Azithromycin / Erythromycin)

      • TMP-SMX (alternative)

Isolation and Infection Control

      • Isolation of confirmed cases:

        • For 5 days after starting antibiotics

      • Use of:

        • Droplet precautions

        • Masks and hand hygiene

Health Education & Public Health Measures

      • Early reporting of cases

      • Completion of immunization schedule

      • Booster doses in adolescents and adults

      • Surveillance for:

        • Vaccine failures

        • Resurgence of disease

Reasons for Re-emergence of Pertussis

    • Waning immunity (especially with acellular vaccines)

    • Incomplete vaccination

    • Antigenic variation

    • Increased awareness and improved diagnostics

 

MCQs

1. Bordetella pertussis belongs to which family?

A. Enterobacteriaceae
B. Neisseriaceae
C. Bordetellaceae
D. Pasteurellaceae

✅ Answer: C

2. Morphology of Bordetella pertussis is best described as:

A. Gram-positive cocci
B. Gram-negative bacilli
C. Gram-negative coccobacilli
D. Acid-fast bacilli

✅ Answer: C

3. Bordetella species are:

A. Facultative anaerobes
B. Obligate anaerobes
C. Microaerophilic
D. Obligate aerobes

✅ Answer: D

4. Best specimen for laboratory diagnosis of pertussis is:

A. Throat swab
B. Sputum
C. Nasopharyngeal swab
D. Blood

✅ Answer: C

5. Culture medium of choice for Bordetella pertussis is:

A. Chocolate agar
B. Blood agar
C. MacConkey agar
D. Bordet–Gengou agar

✅ Answer: D

6. Colony appearance of Bordetella pertussis on Bordet–Gengou agar is:

A. Beta-hemolytic colonies
B. Mucoid colonies
C. Mercury drop colonies
D. Dry wrinkled colonies

✅ Answer: C

7. Regan–Lowe medium contains which selective agent?

A. Vancomycin
B. Cephalexin
C. Colistin
D. Amphotericin B

✅ Answer: B

8. Bordetella pertussis is biochemically characterized by:

A. Fermentation of glucose
B. Lactose fermentation
C. Urease positivity
D. Urease negativity

✅ Answer: D

9. Which Bordetella species is strongly urease positive?

A. B. pertussis
B. B. parapertussis
C. B. bronchiseptica
D. All of the above

✅ Answer: C

10. Oxidase reaction of Bordetella species is:

A. Negative
B. Variable
C. Weakly positive
D. Positive

✅ Answer: D

11. Bordetella pertussis causes disease mainly by:

A. Tissue invasion
B. Endotoxin only
C. Toxin-mediated damage
D. Septicemia

✅ Answer: C

12. Primary site of Bordetella pertussis infection is:

A. Alveoli
B. Bloodstream
C. Ciliated respiratory epithelium
D. Lymph nodes

✅ Answer: C

13. The most important adhesin of Bordetella pertussis is:

A. Lipopolysaccharide
B. Filamentous hemagglutinin
C. Capsule
D. Flagella

✅ Answer: B

14. Pertussis toxin acts by:

A. Inhibiting protein synthesis
B. ADP-ribosylating Gi protein
C. Blocking Na⁺ channels
D. Activating complement

✅ Answer: B

15. Pertussis toxin leads to which hematological finding?

A. Neutrophilia
B. Pancytopenia
C. Lymphocytosis
D. Eosinophilia

✅ Answer: C

16. Tracheal cytotoxin causes:

A. Alveolar edema
B. Ciliary destruction
C. Hemolysis
D. Bronchospasm

✅ Answer: B

17. Persistent cough in pertussis is mainly due to:

A. Bronchoconstriction
B. Mucus plugging
C. Loss of mucociliary clearance
D. Alveolar collapse

✅ Answer: C

18. Which toxin increases intracellular cAMP in phagocytes?

A. Endotoxin
B. Pertactin
C. Adenylate cyclase toxin
D. Tracheal cytotoxin

✅ Answer: C

19. Bordetella parapertussis differs from B. pertussis by:

A. Being urease negative
B. Producing pertussis toxin
C. Causing severe disease
D. Lacking pertussis toxin

✅ Answer: D

20. Most infectious stage of pertussis is:

A. Incubation stage
B. Catarrhal stage
C. Paroxysmal stage
D. Convalescent stage

✅ Answer: B

21. Best diagnostic test during catarrhal stage is:

A. Serology
B. Culture
C. Chest X-ray
D. Weil–Felix test

✅ Answer: B

22. Most sensitive test for diagnosis of pertussis is:

A. Culture
B. DFA
C. PCR
D. Gram stain

✅ Answer: C

23. Target gene commonly used in PCR diagnosis is:

A. toxA
B. IS481
C. mecA
D. spa

✅ Answer: B

24. Direct fluorescent antibody test is now avoided because of:

A. Low sensitivity
B. Low specificity
C. High cost
D. Time consumption

✅ Answer: B

25. Marked leukocytosis in pertussis is due to:

A. Endotoxin
B. Adenylate cyclase toxin
C. Pertussis toxin
D. Tracheal cytotoxin

✅ Answer: C

26. Drug of choice for treatment of pertussis is:

A. Penicillin
B. Cephalosporin
C. Macrolide
D. Aminoglycoside

✅ Answer: C

27. Antibiotics in pertussis mainly help by:

A. Shortening cough duration
B. Neutralizing toxins
C. Preventing transmission
D. Preventing complications

✅ Answer: C

28. Bordetella pertussis is intrinsically resistant to:

A. Macrolides
B. Fluoroquinolones
C. β-lactam antibiotics
D. TMP-SMX

✅ Answer: C

29. Mechanism of macrolide resistance in Bordetella involves:

A. Beta-lactamase production
B. Efflux pump
C. 23S rRNA mutation
D. Plasmid transfer

✅ Answer: C

30. Chemoprophylaxis for close contacts includes:

A. Penicillin
B. Azithromycin
C. Doxycycline
D. Rifampicin

✅ Answer: B

31. Most effective method of pertussis prevention is:

A. Antibiotic prophylaxis
B. Isolation
C. Vaccination
D. Passive immunization

✅ Answer: C

32. Whole-cell pertussis vaccine contains:

A. Live bacteria
B. Killed whole organisms
C. Purified toxins only
D. Recombinant antigens

✅ Answer: B

33. Acellular pertussis vaccine contains all EXCEPT:

A. Pertussis toxin
B. FHA
C. Lipopolysaccharide
D. Pertactin

✅ Answer: C

34. Advantage of acellular vaccine is:

A. Stronger immunity
B. Fewer adverse effects
C. Lifelong immunity
D. Cheaper

✅ Answer: B

35. Reason for re-emergence of pertussis is:

A. Complete vaccine failure
B. Waning immunity
C. Poor diagnosis
D. Antibiotic misuse

✅ Answer: B

36. Immunity after natural infection is:

A. Lifelong
B. Permanent
C. Short-lived
D. Partial and temporary

✅ Answer: D

37. Bordetella bronchiseptica mainly infects:

A. Humans only
B. Birds
C. Animals
D. Insects

✅ Answer: C

38. Growth of Bordetella pertussis on MacConkey agar is:

A. Lactose fermenter
B. Non-lactose fermenter
C. Poor growth
D. No growth

✅ Answer: D

39. Best transport medium for Bordetella is:

A. Cary–Blair
B. Alkaline peptone water
C. Regan–Lowe
D. Venkatraman medium

✅ Answer: C

40. Cotton swabs are avoided because they contain:

A. Alkali
B. Fatty acids
C. Alcohol
D. Enzymes

✅ Answer: B

41. Bordetella pertussis is non-motile because it lacks:

A. Pili
B. Capsule
C. Flagella
D. Fimbriae

✅ Answer: C

42. Pertussis is also known as:

A. Croup
B. Whooping cough
C. Bronchiolitis
D. Diphtheria

✅ Answer: B

43. Inspiratory “whoop” occurs due to:

A. Bronchospasm
B. Laryngeal edema
C. Forceful inspiration after coughing
D. Vocal cord paralysis

✅ Answer: C

44. Most common complication of pertussis in infants is:

A. Pneumonia
B. Otitis media
C. Sinusitis
D. Pharyngitis

✅ Answer: A

45. Pertussis toxin is best described as:

A. Heat stable toxin
B. AB5 exotoxin
C. Neurotoxin
D. Cytolysin

✅ Answer: B

46. Bordetella pertussis does NOT invade:

A. Respiratory epithelium
B. Bloodstream
C. Nasopharynx
D. Trachea

✅ Answer: B

47. Lymphocytosis in pertussis is due to:

A. Bone marrow stimulation
B. Reduced lymphocyte extravasation
C. Increased lymphocyte production
D. Viral co-infection

✅ Answer: B

48. Best stage for serological diagnosis is:

A. Catarrhal
B. Paroxysmal
C. Convalescent
D. Incubation

✅ Answer: C

49. Which antigen is common to acellular vaccines?

A. Capsule
B. Lipid A
C. FHA
D. Peptidoglycan

✅ Answer: C

50. Bordetella pertussis is best described as:

A. Invasive intracellular pathogen
B. Opportunistic pathogen
C. Toxin-mediated extracellular pathogen
D. Zoonotic pathogen

✅ Answer: C