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Chapter 18 HAEMOPHILUS SPECIES
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.
OBJECTIVES
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)
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)
Very susceptible to drying and temperature extremes
Specimens must be plated immediately if not submitted in a suitable transport medium
SPECIMEN MANAGEMENT
Gram negative
Spherical, oval or rod-shaped cells
Variable in length
Pleomorphic
May form filaments
MICROSCOPIC EVALUATION
Humid atmosphere
Most require 5-10% CO2
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
GROWTH REQUIREMENTS
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
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)
Small, round, convex
May be iridescent (capsules)
COLONY MORPHOLOGY
METHODS OF DIFFERENTIATION - 1
Use media devoid of factors
Add growth factors
Strips
Quad plates
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
DELTA-AMINOLEVULINIC ACID (ALA)
Positive Negative
SPECIES OF CLINICAL INTEREST
HAEMOPHILUS INFLUENZAE
Lower respiratory infections
Pneumonia
Meningitis
Epiglottitis
Otitis media
Septic arthritis
Bacteremia
Pericarditis
Cellulitis
CLINICAL SIGNIFICANCE
VIRULENCE FACTORS
Capsule
Other factors that mediate bacterial attachment to human epithelial cells
MICROSCOPIC MORPHOLOGY
COLONY MORPHOLOGY - 1
Small
Smooth
Gray colonies
“Mousy” or “Bleach-like” odor
Chocolate
Translucent
Moist
Smooth
Convex
COLONY MORPHOLOGY - 2
BIOCHEMICAL IDENTIFICATION
Requires X and V factors
ALA negative
Non-hemolytic on rabbit blood
Positive Nitrate
Acid production from glucose
ALA Negative
ANTIMICROBIAL SUSCEPTIBILITY
Most sensitive to penicillin
20% positive for beta-lactamase production
SERO-TYPING
Serotypes a-f
Based on capsule composition
Different methods of sero-typing:
Slide agglutination
Co-agglutination
PCR
HAEMOPHILUS DUCREYI
Causative agent of the sexually transmitted disease chancroid
Painful
Genital ulcers
Soft chancre
CLINICAL SIGNIFICANCE
Gram negative coccobacilli
Clustered groups "school of fish" or loosely coiled parallel chains "railroad tracks"
MICROSCOPIC MORPHOLOGY
COLONY MORPHOLOGY
Small
Flat
Smooth
Yellow to gray
Translucent
BIOCHEMICAL IDENTIFICATION
Requires X factor
ALA negative
Biotype aegypticus
HAEMOPHILUS INFLUENZAE
CLINICAL SIGNIFICANCE
Pink eye
Brazilian purpuric fever (BPF)
A life-threatening pediatric infection
Preceded by conjunctivitis
BIOCHEMICAL IDENTIFICATION
Requires both X and V factors
ALA negative
HAEMOPHILUS PARAINFLUENZAE
CLINICAL SIGNIFICANCE
Causative agent of endocarditis
Rare cause of upper respiratory tract infection
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
HAEMOPHILUS HAEMOLYTICUS
CLINICAL SIGNIFICANCE
Rare infections
Endocarditis
Cellulitis
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
Requires X and V factors
ALA negative
BIOCHEMICAL IDENTIFICATION
HAEMOPHILUS DIFFERENTIATION
Hemolysis
YES
Haemophilus haemolyticus
NO
H. influenzae H. aegypticus
H. ducreyi H. parainfluenzae
H. aphrophilus H. paraaphrophilus
HAEMOPHILUS DIFFERENTIATION
Growth Factors
X Factor
H. ducreyi
V Factor
H. parainfluenzae Catalase +
ALA +
H. Paraaphrophilus Catalase =
X & V Factors
H. influenzae H. aegypticus
Urease +
H. haemolyticus Hemolysis +
Review, Mahon Chapter 18
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