Haemophillus Species

1. HAEMOPHILUS SPECIES Chapter 18

2. OBJECTIVES
 Discuss the microscopic characteristics of Haemophilus species.
 State the normal habitat (if any) and discuss the disease caused
by each Haemophilus species.
 Select the media and incubation condition appropriate for
culture Haemophilus species.
 Describe the colony morphology of Haemophilus influenzae.
 Explain the term satellitism as it relates to Haemophilus species.
 Explain the principle of the X and V factor and ALA test.
 Identify Haemophilus species based on X and V factor
requirements.
 Evaluate Haemophilus identification tests when given a
description of the test results or a description of the technique
used to perform the test.

3. GENERAL CHARACTERISTICS
 Most are normal inhabitants of the upper respiratory tract in
humans
 H. ducreyi is not!!!
 Infections with pathogenic Haemophilus are usually systemic
 All species require pre -formed growth factors present in blood
 X (Hemin)
 V (NAD)

4. GENERAL CHARACTERISTICS
 Optimum growth temperature is 35 -37°C
 Aerobes or facultative anaerobes
 Non-motile
 Reduce nitrates to nitrites
 Obligate parasites of the mucous membranes of humans and
animals
 Oxidase + (except H. ducreyi)
 Catalase + (except H. ducreyi)

5. SPECIMEN MANAGEMENT
 Very susceptible to drying and temperature extremes
 Specimens must be plated immediately if not submitted in a
suitable transport medium

6. MICROSCOPIC EVALUATION
 Gram negative
 Spherical, oval or rod -shaped cells
 Variable in length
 Pleomorphic
 May form filaments

7. GROWTH REQUIREMENTS
 Humid atmosphere
 Most require 5-10% CO 2
 Haemophilus will only grow on media supplemented with X
and V factors
 X factor – hemin; hematin
 V factor – nicotine adenine dinucleotide (NAD)
 Satellitism can be seen on BAP

8. SATELLITISM
 Some bacteria, such as S. aureus, produce V factor and tiny
Haemophilus can be seen growing on sheep blood agar very
close to the bacteria producing the V factor

9. MEDIA FOR ISOLATION
 Chocolate agar provides both X and V factors
 Blood agar contains the X factor, but not the V factor
(is contained inside the unlysed RBCs)

10. COLONY MORPHOLOGY
 Small, round, convex
 May be iridescent (capsules)

11. METHODS OF DIFFERENTIATION - 1
 Use media devoid of factors
 Add growth factors
 Strips
 Quad plates

12. METHODS OF DIFFERENTIATION - 2
 ALA or Prophryin test
 ALA (Delta-Aminolevulinic Acid)
 Purpose:
 To test whether non-hemin requiring species have the
enzyme necessary to synthesize heme precursors, such as
porphyrin from ALA
 Principle:
 Strains that lack the enzymes necessary to convert ALA to
protoporphyrin are dependent on the X factor to grow
 X factor dependent organisms = ALA negative
 H. parainfluenzae = ALA positive

13. DELTA-AMINOLEVULINIC ACID (ALA)
Positive Negative

14. SPECIES OF CLINICAL
INTEREST

15. HAEMOPHILUS
INFLUENZAE

16. CLINICAL SIGNIFICANCE
 Lower respiratory infections
 Pneumonia
 Meningitis
 Epiglottitis
 Otitis media
 Septic arthritis
 Bacteremia
 Pericarditis
 Cellulitis

17. VIRULENCE FACTORS
 Capsule
 Other factors that mediate bacterial attachment to human
epithelial cells

18. MICROSCOPIC MORPHOLOGY

19. COLONY MORPHOLOGY - 1
 Small
 Smooth
 Gray colonies
 “Mousy” or “Bleach-like” odor
 Chocolate
 Translucent
 Moist
 Smooth
 Convex

20. COLONY MORPHOLOGY - 2

21. BIOCHEMICAL IDENTIFICATION
 Requires X and V factors
 ALA negative
 Non-hemolytic on rabbit blood
 Positive Nitrate
 Acid production from glucose
ALA Negative

22. ANTIMICROBIAL SUSCEPTIBILITY
 Most sensitive to penicillin
 20% positive for beta -lactamase production

23. SERO-TYPING
 Serotypes a-f
 Based on capsule composition
 Different methods of sero-typing:
 Slide agglutination
 Co-agglutination
 PCR

24. HAEMOPHILUS DUCREYI

25. CLINICAL SIGNIFICANCE
 Causative agent of the sexually transmitted disease chancroid
 Painful
 Genital ulcers
 Soft chancre

26. MICROSCOPIC MORPHOLOGY
 Gram negative coccobacilli
 Clustered groups "school
of fish" or loosely coiled
parallel chains "railroad
tracks"

27. COLONY MORPHOLOGY
 Small
 Flat
 Smooth
 Yellow to gray
 Translucent

28. BIOCHEMICAL IDENTIFICATION
 Requires X factor
 ALA negative

29. HAEMOPHILUS Biotype
aegypticus
INFLUENZAE

30. CLINICAL SIGNIFICANCE
 Pink eye
 Brazilian purpuric fever (BPF)
 A life-threatening pediatric infection
 Preceded by conjunctivitis

31. BIOCHEMICAL IDENTIFICATION
 Requires both X and V factors
 ALA negative

32. HAEMOPHILUS
PARAINFLUENZAE

33. CLINICAL SIGNIFICANCE
 Causative agent of endocarditis
 Rare cause of upper respiratory tract infection

34. CHARACTERISTICS
 Requires V factor only for growth
 Colonies similar to H. influenzae, except larger
 Non-hemolytic on rabbit blood
 ALA test positive
 Acid production from glucose
 Treat with Penicillin

36. HAEMOPHILUS
HAEMOLYTICUS

37. CLINICAL SIGNIFICANCE
 Rare infections
 Endocarditis
 Cellulitis

38. COLONY MORPHOLOGY
 Colonies are identical to those of Haemophilus influenzae
 Small and Smooth
 Gray
 On chocolate agars
 Translucent
 Moist
 Smooth
 Convex
 Hemolysis on rabbit blood

39. BIOCHEMICAL IDENTIFICATION
 Requires X and V factors
 ALA negative

40. HAEMOPHILUS DIFFERENTIATION
Hemolysis
YES NO
H. influenzae
H. aegypticus
H. ducreyi
Haemophilus haemolyticus
H. parainfluenzae
H. aphrophilus
H. paraaphrophilus

41. HAEMOPHILUS DIFFERENTIATION
Growth Factors
X Factor V Factor X & V Factors
H. parainfluenzae H. influenzae
Catalase + H. aegypticus
ALA + Urease +
H. ducreyi
H. Paraaphrophilus H. haemolyticus
Catalase = Hemolysis +

42. WHAT’S NEXT?
 Review, Mahon Chapter 18
 Complete AUMoodle Review