Haemophilus - Allied Guru

Table of contents

Introduction

Haemophilus is a genus of small, pleomorphic, Gram-negative bacteria commonly found in the human upper respiratory tract.
Members of this genus may exist as normal commensals or act as pathogenic organisms under suitable conditions.
The most important pathogenic species is Haemophilus influenzae, which is responsible for a variety of respiratory and invasive infections.
Haemophilus species are fastidious organisms, requiring special growth factors for cultivation in the laboratory.
Infections caused by Haemophilus are more common in infants, young children, elderly individuals, and immunocompromised patients.
Capsulated strains, especially type b, are associated with severe systemic infections such as meningitis and septicemia.
Haemophilus infections spread mainly through respiratory droplets.
Due to its clinical importance, Haemophilus is a significant organism in medical microbiology, particularly for MLT and blood bank students.
Early diagnosis, appropriate antibiotic therapy, and effective vaccination are essential for prevention and control of Haemophilus infections.

General Character

Family: Pasteurellaceae
Gram reaction: Gram-negative
Morphology:
- Small, pleomorphic bacilli
- Often appear as coccobacilli
Arrangement:
- Single cells
- Pairs or small groups
Motility: Non-motile
Spore formation: Non-spore forming
Oxygen requirement:
- Facultative anaerobes
Growth nature:
- Fastidious organisms
- Require enriched media
Special growth factors:
- X factor (Hemin)
- V factor (NAD / NADP)
Capsule:
- Present in some strains (H. influenzae type b)
- Important virulence factor
Biochemical nature:
- Oxidase positive
- Catalase positive
Habitat:
- Normal flora of upper respiratory tract (some species)

Morphology

Gram reaction:
- Gram-negative
- Appear pink on Gram staining
Shape:
- Small pleomorphic bacilli
- Often seen as coccobacilli
Size:
- Very small bacteria (tiny rods)
Arrangement:
- Occur as single cells
- May be seen in pairs or small groups
Capsule:
- Present in some species (e.g., Haemophilus influenzae type b)
- Capsule is an important virulence factor
Motility:
- Non-motile
Spore formation:
- Non-spore forming
Pleomorphism:
- Show variation in shape and size, especially in clinical specimens

Cultural Characteristics

Growth requirement:
- Fastidious organism (requires enriched media)
Essential growth factors:
- X factor (Hemin) – required for heme synthesis
- V factor (NAD / NADP) – required for growth and metabolism
Ordinary media:
- Does not grow on simple media like nutrient agar
Chocolate agar:
- Best medium for growth
- Heating releases X and V factors
- Produces small, smooth, moist, translucent colonies
Blood agar:
- H. influenzae does not grow alone
- Shows satellite phenomenon around Staphylococcus aureus
  - Staphylococcus provides V factor
Selective media:
- Chocolate agar with antibiotics (e.g., bacitracin) used for throat specimens
Colony morphology:
- Small, convex, grayish, dew-drop like colonies
- Capsulated strains form larger, mucoid colonies
Atmosphere:
- Grows best in 5–10% CO₂ (capnophilic)
Temperature:
- Optimal growth at 35–37°C

Biochemical Reactions

General Biochemical Characteristics

Oxidase test: Positive
Catalase test: Positive (most species)
Indole test: Variable (species dependent)
Urease test: Negative
Nitrate reduction: Positive

Carbohydrate Fermentation

Fermentation occurs without gas production

Species	Glucose	Sucrose	Lactose	Indole
H. influenzae	+	–	–	Variable
H. parainfluenzae	+	+	–	+
H. ducreyi	–	–	–	–
H. aegyptius	+	–	–	–

Other Important Tests

Porphyrin test (ALA test):
- H. influenzae → Negative (requires X factor)
- H. parainfluenzae → Positive (does not require X factor)
ONPG test: Negative

Pathogenicity

Virulence Factors

Capsule
- Present in capsulated strains (especially H. influenzae type b – Hib)
- Prevents phagocytosis
- Major virulence factor
Lipooligosaccharide (LOS)
- Acts as endotoxin
- Causes inflammation and tissue damage
IgA protease
- Destroys secretory IgA
- Helps in colonization of respiratory mucosa
Fimbriae (Pili)
- Help in adherence to epithelial cells
Outer membrane proteins (OMP)
- Aid in attachment and immune evasion

Pathogenesis

Entry through respiratory or genital mucosa
Adherence to epithelial cells using pili
Local multiplication
Invasion of bloodstream (mainly capsulated strains)
Spread to distant organs → meninges, lungs, joints

Diseases Caused

Haemophilus influenzae

Upper respiratory tract infections
- Otitis media
- Sinusitis
- Epiglottitis
Lower respiratory tract infections
- Pneumonia
Invasive diseases
- Meningitis (Hib)
- Septicemia
- Septic arthritis

Haemophilus ducreyi

Chancroid
- Painful genital ulcer
- Soft, non-indurated edges
- Painful inguinal lymphadenitis (bubo)

Haemophilus aegyptius

Acute conjunctivitis (pink eye)

Host Factors

More common in:
- Children
- Immunocompromised individuals
- Unvaccinated persons

Laboratory Diagnosis

1. Specimen Collection

Haemophilus influenzae
- Sputum
- Throat swab
- Blood
- Cerebrospinal fluid (CSF)
Haemophilus ducreyi
- Pus or exudate from genital ulcer
Haemophilus aegyptius
- Conjunctival swab

2. Direct Microscopy

Gram staining
- Small Gram-negative pleomorphic bacilli / coccobacilli
CSF examination
- Shows Gram-negative bacilli in meningitis cases

3. Culture

Chocolate agar
- Medium of choice
- Incubation at 35–37°C, 5–10% CO₂
Blood agar
- Shows satellite phenomenon around Staphylococcus aureus
Selective media
- Chocolate agar with bacitracin (respiratory specimens)

4. Identification Tests

X and V factor requirement
- H. influenzae → requires both X & V
Porphyrin (ALA) test
- Negative in H. influenzae
Biochemical tests
- Oxidase: Positive
- Catalase: Positive
- Sugar fermentation pattern for species identification

5. Serological Tests

Capsular antigen detection
- Latex agglutination test (CSF)
Useful for Hib meningitis

6. Molecular Methods

PCR
- Rapid and sensitive detection
- Useful in culture-negative cases

7. Antimicrobial Sensitivity Testing

Performed due to:
- β-lactamase producing strains
Helps in guiding therapy

Antibiotic Resistance

Mechanisms of Resistance

β-lactamase production
- Most common mechanism
- Causes resistance to ampicillin and penicillin
- Enzyme breaks β-lactam ring
Altered Penicillin-Binding Proteins (PBPs)
- Seen in β-lactamase–negative ampicillin-resistant (BLNAR) strains
- Reduced affinity for β-lactam antibiotics
Efflux pumps
- Contribute to resistance against macrolides and tetracyclines
Target site modification
- Causes resistance to fluoroquinolones (rare but reported)

Commonly Resistant Antibiotics

Ampicillin
Penicillin
First-generation cephalosporins
Macrolides (in some strains)

Usually Effective Antibiotics

Amoxicillin + clavulanic acid
Second and third-generation cephalosporins
- Cefuroxime
- Ceftriaxone
Fluoroquinolones (adults)
Azithromycin (variable sensitivity)

Detection of Resistance

β-lactamase test
- Nitrocefin test (rapid)
Antibiotic sensitivity testing
- Disk diffusion / MIC methods

Prevention

1. Vaccination

Hib vaccine (Haemophilus influenzae type b)
- Most effective preventive measure
- Prevents meningitis, pneumonia, epiglottitis, septicemia
- Given in infancy as part of routine immunization
- Available as pentavalent / hexavalent vaccines

2. Chemoprophylaxis

Rifampicin
- Given to close contacts of Hib meningitis cases
- Prevents secondary spread
- Indicated for:
  - Household contacts
  - Daycare contacts

3. Infection Control Measures

Proper hand hygiene
Respiratory etiquette (covering cough/sneeze)
Isolation of infected patients when required

4. Early Diagnosis and Treatment

Prompt identification and appropriate antibiotic therapy
Reduces transmission and complications

5. Public Health Measures

Routine childhood immunization programs
Surveillance of antibiotic resistance
Health education and awareness

6. Prevention of Sexually Transmitted Infection (H. ducreyi)

Safe sexual practices
Early diagnosis and treatment of chancroid
Partner notification and treatment

MCQs

1. Haemophilus belongs to which family?

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

2. Haemophilus influenzae is a:

A. Gram-positive coccus
B. Gram-negative bacillus
C. Gram-positive bacillus
D. Acid-fast bacillus

3. Morphology of Haemophilus is best described as:

A. Cocci in chains
B. Long rods
C. Pleomorphic coccobacilli
D. Spiral shaped

4. Haemophilus species are:

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

5. Which factor is required by H. influenzae for growth?

A. Only X factor
B. Only V factor
C. Both X and V factors
D. Neither X nor V factor

6. X factor is chemically:

A. NAD
B. Hemin
C. Riboflavin
D. Biotin

7. V factor is:

A. Hemin
B. Iron
C. NAD / NADP
D. Vitamin K

8. Best culture medium for Haemophilus is:

A. Nutrient agar
B. MacConkey agar
C. Chocolate agar
D. TCBS agar

9. Satellite phenomenon is seen on:

A. Chocolate agar
B. Blood agar
C. MacConkey agar
D. Lowenstein-Jensen medium

10. Satellite phenomenon occurs due to:

A. X factor release
B. V factor release by Staphylococcus
C. Capsule production
D. Hemolysis

11. Haemophilus influenzae is oxidase:

A. Negative
B. Positive
C. Variable
D. Not done

12. Catalase reaction of Haemophilus is usually:

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

13. Which sugar is fermented by H. influenzae?

A. Lactose
B. Sucrose
C. Glucose
D. Mannitol

14. Gas production from sugars by Haemophilus is:

A. Present
B. Absent
C. Variable
D. Depends on media

15. Porphyrin (ALA) test in H. influenzae is:

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

16. Capsule of H. influenzae is made of:

A. Protein
B. Lipid
C. Polysaccharide
D. Lipopolysaccharide

17. Most virulent strain of H. influenzae is:

A. Non-typeable
B. Type a
C. Type b
D. Type f

18. Major virulence factor of H. influenzae is:

A. Flagella
B. Capsule
C. Exotoxin
D. Enzyme

19. Hib commonly causes meningitis in:

A. Elderly
B. Neonates
C. Children
D. Pregnant women

20. Haemophilus ducreyi causes:

A. Syphilis
B. Gonorrhea
C. Chancroid
D. LGV

21. Chancroid ulcer is characteristically:

A. Painless and hard
B. Painful and soft
C. Painless and soft
D. Hard with induration

22. Haemophilus aegyptius is associated with:

A. Pneumonia
B. Meningitis
C. Conjunctivitis
D. Otitis media

23. Habitat of Haemophilus influenzae is:

A. Skin
B. Intestine
C. Upper respiratory tract
D. Genital tract

24. Gram stain of Haemophilus shows:

A. Purple cocci
B. Pink bacilli
C. Blue spiral forms
D. Red acid-fast rods

25. Motility of Haemophilus is:

A. Motile
B. Non-motile
C. Peritrichous
D. Polar

26. Spore formation in Haemophilus:

A. Present
B. Absent
C. Seen in old cultures
D. Variable

27. Endotoxin of Haemophilus is:

A. LPS
B. LOS
C. Exotoxin A
D. Protein toxin

28. IgA protease helps in:

A. Capsule formation
B. Antibiotic resistance
C. Mucosal colonization
D. Spore formation

29. Drug of choice for Hib meningitis is:

A. Ampicillin
B. Penicillin
C. Ceftriaxone
D. Erythromycin

30. Most common resistance mechanism in Haemophilus is:

A. Efflux pump
B. Capsule change
C. β-lactamase production
D. Spore formation

31. BLNAR strains are:

A. β-lactamase positive
B. Ampicillin sensitive
C. β-lactamase negative but resistant
D. Fully sensitive

32. Rapid test to detect β-lactamase is:

A. Coagulase test
B. Oxidase test
C. Nitrocefin test
D. Indole test

33. Chemoprophylaxis drug for Hib contacts is:

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

34. Hib vaccine is composed of:

A. Live bacteria
B. Killed bacteria
C. Capsular polysaccharide
D. Protein toxoid

35. Hib vaccine is mainly protective against:

A. All strains
B. Type b strains
C. Non-typeable strains
D. H. ducreyi

36. Haemophilus grows best in presence of:

A. Oxygen only
B. CO₂ (5–10%)
C. Nitrogen
D. Anaerobic jar

37. Colony morphology on chocolate agar is:

A. Large dry colonies
B. Small moist translucent colonies
C. Swarming growth
D. Pigmented colonies

38. Haemophilus does NOT grow on:

A. Chocolate agar
B. Blood agar alone
C. Enriched media
D. CO₂ incubator

39. Latex agglutination test detects:

A. Antibody
B. Capsule antigen
C. Endotoxin
D. Enzyme

40. Best specimen for suspected Hib meningitis is:

A. Urine
B. Stool
C. CSF
D. Throat swab

41. Haemophilus is normal flora of:

A. Intestine
B. Skin
C. Upper respiratory tract
D. Blood

42. ONPG test in Haemophilus is usually:

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

43. Haemophilus parainfluenzae requires:

A. X factor only
B. V factor only
C. Both X and V
D. Neither X nor V

44. Gas gangrene is caused by Haemophilus:

A. True
B. False

45. Haemophilus is fastidious because:

A. Slow growing
B. Needs enriched media
C. Produces spores
D. Anaerobic

46. Which test differentiates X-factor dependency?

A. Oxidase test
B. Indole test
C. Porphyrin test
D. Catalase test

47. Hib meningitis is common in:

A. Adults only
B. Children <5 years
C. Neonates only
D. Elderly only

48. Painful inguinal bubo is seen in:

A. Syphilis
B. Gonorrhea
C. Chancroid
D. LGV

49. Haemophilus is best described as:

A. Acid-fast bacillus
B. Gram-negative fastidious bacillus
C. Gram-positive cocci
D. Spore-forming rod

50. Most effective prevention against Hib is:

A. Antibiotics
B. Hygiene
C. Vaccination
D. Isolation

Answer Key