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 +

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