Uk standards for Microbiology Investigations Acknowledgments



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HACEK group reported to have caused human infection


Haemophilus influenza, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus paraphrohaemolyticus, Haemophilus aegyptius, Haemophilus pittmaniae, Haemophilus ducreyi, Haemophilus sputorum, Aggregatibacter aphrophilus (includes Haemophilus aphrophilus and Haemophilus paraphrophilus), Aggregatibacter segnis (formerly Haemophilus segnis), Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans), Cardiobacterium hominis, Cardiobacterium valvarum, Eikenella corrodens, Kingella kingae, Kingella denitrificans, Kingella oralis, Kingella potus

3 Identification

3.1 Microscopic Appearance

Gram stain (TP 39 - Staining Procedures)


Haemophilus species are small coccobacilli or longer rod-shaped Gram negative cells, variable in length with marked pleomorphism and sometimes forming filaments.

Other HACEK organisms produce spherical, oval or rod-shaped Gram negative cells which may be variable in length with marked pleomorphism or filament formation.



3.2 Primary Isolation Media

Chocolate agar incubated in 5-10% CO2 at 35-37°C for 24-48hr.

Blood agar incubated in 5-10% CO2 at 35-37°C for 24-48hr.

3.3 Colonial Appearance

Haemophilus species are small, round, convex colonies, which may be iridescent and develop after 24hr incubation on chocolate agar. Satellitism of H. influenzae may be seen around colonies of S. aureus on blood agar.

Colonial morphology of other HACEK organisms varies with species and isolation medium (see subheading “Characteristics” and table below).


Aerobic growth Characteristics of HACEK group organisms


HACEK group organisms

Characteristics of growth on blood agar after aerobic incubation at 35-37°C for 16-48hr

A. actinomycetemcomitans

Will not grow in air but grows in air + CO2. Minute colonies at 24hr, 1mm at 48hr. Firm, adherent, star-shaped colonies with rough surface and which may produce pitting of the agar. Some strains may be sticky. Non-haemolytic.

A. aphrophilus

Requires added CO2 for primary isolation. Opaque, yellowish colonies 1.0-1.5mm at 24hr. Haemin (X-factor) enhances growth but there is not an absolute requirement for X-factor. Some isolates require V factor (formerly H. paraphrophilus) whereas others are V-factor-independent (formerly H. aphrophilus).

Non-haemolytic.



A. segnis

Growth on chocolate agar is slow and the colonies are smooth or granular, convex, greyish-white or opaque and 0.5mm in diameter after 48hrs incubation.

C. hominis

Some strains will not grow without added CO2. May require X-factor on primary isolation. Colonies smooth, convex and opaque.

1-2mm at 48hr. Slight -haemolysis.



C. valvarum

Grows best in air +5% CO2. Slow growing, colonies smooth, round, opaque and glistening, 0.6-0.8mm after 48hr. Some strains show slight -haemolysis, others are non-haemolytic.

E. corrodens

Colonies very small, moist, clear centres surrounded by flat growth. Pitting may occur. Spreading is rare and usually confined to a very small area around the colony. Non-haemolytic. Colonies 0.5-1mm after 48hr. Requires 5-10% CO2.

K. kingae

2 types of colony: a spreading, corroding type and a smooth, convex type. Small zone of -haemolysis. Cells are often capsulate, producing mucoid colonies. Does not require 5-10% CO2.

K. denitrificans

Non-haemolytic. 2 types of colony: a spreading, corroding type and a smooth, convex type.

K. oralis

Colonies are round with slightly irregular borders and flat to umbonate, and each colony has a granular periphery. Colonies appear to corrode the agar surface.

K. potus

Colonies are circular, low convex, yellow- pigmented, smooth, entire, approximately 1.5 to 2mm in diameter, and friable on Columbia blood agar after 48hr of incubation at 37°C. Colonies are non-haemolytic.

Note 1: For descriptions of Haemophilus species, see subheading “Characteristics”

3.4 Test Procedures

Catalase Test (TP 8 - Catalase Test)

Oxidase Test (TP 26 - Oxidase Test)

Urease Test (TP36 – Urease Test)


Summary of the biochemical tests:

Organism

Catalase

Oxidase

Urease

H. influenzae

+

+

(+)

H. aegyptius

+

+

+

H. ducreyi

-

+

Unknown

H. haemolyticus

+

+

+

H. parainfluenzae

d

+

d

H. pittmaniae

d

d

-

H. parahaemolyticus

d

+

+

H. paraphrohaemolyticus

+

+

+

H. sputorum

V

+

+

A. actinomycetemcomitans

+

+

-

A. aphrophilus

-

-

-

C. hominis

-

+

-

C. valvarum

-

+

-

E. corrodens

-

+

-

K. kingae

-

+

-

K. denitrificans

-

+

-

K. oralis

-

+

-

K. potus

-

+

-
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