CHAPTER 18: Genus Haemophilus and

(HACEK group + -ella)

 Haemophilus and Similar Organisms
General Characteristics
- Normal upper respiratory tract flora
- H- aemophilus
- A- actinobacillus
- C- ardiobacterium hominis
- E- ikenella corrodens
- K- ingella
Fastidious / causes endocarditis
 Haemophilus spp.
● Pleomorphic gram-negative bacilli , very small to filamentous
:Encapsulated form are coccobacilli (CSF sample)
:Non-Capsulated are pleomorphic, long, thread, filamentous
● FASTIDIOUS
● Growth requirements: Hemin (X factor)- released from
hemoglobin
● NAD (V factor) heat labile compound
● Do not grow on SBA - bec. NADase
● Horse or Rabbit blood agar
● CAP - +
● Horse CAP = Medium of choice
Laboratory diagnosis
• Cultural characteristic
• Microscopic characteristic
• X and V factor requirement
• Porphyrin test
• Biochemical Test
X factor (+) V factor (+)
“X factor (+)” means GROWTH on X-factor
= org. needs X factor
“X factor (-)” means NO GROWTH on X-
factor
X factor (-) V factor (+)
= Organism does not require X factor
to grow. It needs only the V factor for
growth. Because the organism already
has X factor.
Porphyrin test = ability of hemophilus to convert d-ala to
porphyrin (this means the organism can synthesize heme in
its system).

Some spp. cant convert d-ala. This means that they need an
external source of hemin. They will grow in the X factor disk.

Confirmatory reagent: kovacs reagent (pdab). or Wood’s lamp

(red-orange fluorescence)

D-ala → porphyrin (add kovacs) = red color (+)

D-ala → porphyrin (wood’s lamp: source of UV light) = red-
orange fluorescence (+)
BOTH Factor X, V, Porphyrin -
: H. influenzae
: H. haemolyticus

ONLY Factor V, Porphyrin +

: H. parainfluenzae
: H. parahaemolyticus
: H. paraphrohaemolyticus
: A. segnis
ONLY Factor X, Porphyrin −
: H. ducreyi

NEITHER Factor X, V, Porphyrin +

: H. aphrophilus
• 35-37C with 5-10% CO2
• Normally inhabit the URT of humans except
H. ducreyi - found in genitals
• Susceptible to drying and temp. changes
• Colony: smooth round, flat, opaque tan on Cap
• Satellitism- can grow around S. aureus (H.
influenzae)
• Nonmotile, catalase, oxidase +
 a. Haemophilus influenzae
• Capsular serotypes – A to F
• biotype I-VIII
• H. influenzae type b- MOST COMMON CAUSE of
MENINGITIS in CHILDREN
• Major infection: Epiglotitis
• Other than b- causes respi. Infection, sinusitis, bronchitis,
pneumonia., otitis media.
Isolate should be tested for B-lactamase
Specimens: CSF, blood, sputum, swabs
• Culture: Grayish; Dew-drop colonies. Mousy odor.
 b. Haemophilus aegypticus
• Koch’s week Bacillus
• Causes pink eye conjunctivitis
• Very contagious conjuctivitis
• Similar to H. influenzae except Sucrose (+)
• causes Brazillian Purpuric Fever
 c. Haemophilus ducreyi
• Genital ulcers
• STD
• Chancroid (aka. SOFT CHANCRE)= very painful
and buboes (swollen lymph node)
• CAP with vancomycin- agar of choice
• Microscopic appearance = School of Red Fish
 HACEK group
- H- aemophilus aphrophilus
- A- ggregatibacter actinomycetemcomitans,
(formerly Actinobacillus
actinomycetemcomitans)
- C- ardiobacterium hominis
- E- ikenella corrodens
- K- ingella kingae
HACEK group
- Gram-negative bacilli
- Capnophilic
- ACEK = slow and poor growing
- likes to attach to heart valves = causing
bacterial endocarditis
- Normal flora of the oral cavity
 Aggregatibacter aphrophilus
● Old name: Haemophilus aphrophilus
(aphro=foam philia=loving)
● Does not need X factor and V factor (equal
growth all over the X/V factor test agar)
● Colonies are convex, granular, and yellow
with an opaque zone near the center on
CHOC agar.
● Catalase (-) Oxidase (+) MAC (-)
Aggregatibacter actinomycetemcomitans
• Old name: Actinobacillus actinomycetemcomitans
• Nonmotile small gram-neg coccobacilli
• Six serotypes (A to F): A, B, C are most common
• Causative agent= Sub-acute bacterial endocarditis
(insidious and protracted); periodontitis
• Collagenase and leukotoxins
• Colonies= Star-like colonies (best observed after 48
hours)
• Microscopy= Morse code appearance (Dots and dashes)
• Catalase (+) Oxidase (-) MAC (-)
A single colony of A. actinomycetemcomitans
 Cardiobacterium hominis
: Pleomorphic, nonmotile, fastidious, gram-
negative bacillus
- Endocarditis (infects the aortic valve)
- Meningitis (rarely)
- Indole (+)
- Pitting of the agar may be produced
- Microscopy: Rosettes (rare to occasional
teardrop-shaped)
- Catalase (-) Oxidase (+) MAC (-)
 Eikenella corrodens
Fastidious, gram-negative coccobacilli that grow best under
conditions of increased CO2 (with hemin)
- Nonmotile
- Asaccharolytic
- Catalase (-) Oxidase (+) MAC (-)
Associated with human bites or fights (i.e., “clenched fist
wounds,” or after the skin has been broken by human teeth).

Least common isolate of the HACEK group in adult infectious

endocarditis.

Corrodes the agar. Bleach-like odor.

 Kingella kingae
Gram neg coccobacillus to short bacillus with squared ends that
occur in pairs or short chains

Kingella species may grow on Neisseria selective agar (e.g., Thayer-

Martin medium)

Catalase (-) Oxidase (-) MAC (+)

Can also corrode the agar.

Major gram-neg bacterium isolated from degenerative joint and

bone infections (osteoarthritis) in children younger than 3 years old
 Miscellaneous
- Capnocytophaga
- Pasteurella
- Brucella
- Francisella
- Legionella
- Bordetella
 Capnocytophaga
⚫- capnophilic, fusiform or filamentous bacilli
⚫Characteristic motility: Gliding motility (without
flagella)
⚫Causative agent: Periodontitis (neutropenic px)
⚫Colonies: Large spreading colonies
⚫Oxidase, Catalase, Indole (-)
⚫Esculin HOH, NO3 reduction (+)

Capnocytophaga resemble the HACEK in their

requirements for CO2 for enhanced growth and their
isolation
 Pasteurella
Disease: Pasteurellosis (zoonosis)
Causative agent of Shipping fever (in cattles)
“Multo”- many
:Cida”- killings
- HAS CAPSULE
- Bipolar staining: safety-pin appearance is frequently
observed.
-carried in the oral cavity and respiratory and gastrointestinal
tracts of cats and dogs
Acquired via contact with domestic animals
Route: bite or scratch usually of cat
-wound infections are most common type of pasteurella infection.
 Brucella
Small gram-negative coccobacilli/bacilli, aerobic, nonmotile,
unencapsulated bacteria

- found in milk products

Causative agent of: Brucellosis, Undulant fever, Malta
fever, abortion in cattles, endocarditis
- Facultatively intracellular (phagocytes)
- Erythritol: enhances growth

Category B agent: Easy to disseminate and cause

moderate morbidity but low mortality (Center for Disease
Control anf Prevention)
Zoonotic
: B. melitensis - sheep/goat
: B. abortus - cattles
: B. suis - pigs
: B. canis - dogs

Humans acquired infections through the ingestion of

contaminated animal products, including meats and
milk, farmers can directly acquire infections through
direct contact. Inhalation of infected aerosols.
 
⚫Brucella abortus- Bangs bacillus
 CO2 (5-10%) H2S Thionine Basic fuchsin

B.abortus + + inhibited growth

B. melitensis - - growth growth

B. suis - - growth inhibited

B. canis - - growth inhibited
Specimen: Blood/ Bone marrow
 
Culture for Brucella: Castañeda bottles
3 to 4 weeks
Biphasic medium

- Castañeda medium
- TSB
- W (Wisconsin) medium: selective
 Francisella
⚫Gram negative coccobacilli; nonmotile; CAPSULE (+)
⚫Obligate aerobe
⚫Facultatively intracellular
⚫BSL LEVEL 2
⚫Requires: cysteine
⚫Tularemia- disease of the rodents, primarily rabbits
(Water rat trapper’s disease); lymphadenopathy
⚫Zoonotic infection- direct contact with blood or through
animal bite or scratch.
⚫Can also be acquired through insect vectors- primarily
ticks

⚫Culture medium: Blood Glucose cysteine medium

 Legionella
- Isolated from air conditions, humidifiers, and water towers
(Transport: 4’C or frozen)
- Aerobic
Causative agent: Legionellosis (pneumonia)
L. pneumophila (Legionnaire’s disease, Pontiac fever - mild form,
flu-like symptoms)
-poor staining capacity (facultatively intracellular)
Requires: Cysteine
Stain: Dieterle’s silver stain (dark brown –black color)
- Autofluoresce when exposed to UVL
 
L.micdadei- Pittsburg pneumonia (weakly acid-fast)
L. bozemanni- Wiga’s agent of pneumonia
Naturally found in both natural and artificial water sources
E.g. Ponds, creeks and streams, wet soil, water cooling towers,
shower heads, plumbing system hearing system.
Specimen of choice: Bronchial wash/expectorated sputum,
biopsy
Urine antigen test- common lab assay for diagnosis

Culture medium: BCYE (Blue green cut glass colonies)

Feely German medium
Identifying characteristics
• G/S: thin, poorly stain gram negative bacilli.
• Use 0.1%basic fuschin as counter stain instead of safranin
Requires: Cysteine for growth
BCYE agar (+) but not on SBA (does not contain cysteine)
Tiny colonies on CAP
- assacharolytic- most biochem- negative

L. pneumophila autofluoresce when exposed to UVL

Laboratory diagnosis:
• Isolation using special media
• Urine antigen detection
• Direct fluorescent antibody
• Serology
 Bordetella
⚫ Gram negative (poorly stained)
⚫obligate aerobe
⚫non motile except Bordetella bronchiseptica
⚫Requires: Cysteine and methionine
⚫Toluidine blue: Bipolar granules

Bordetella pertussis – agent of whooping cough

(Whooping cough bacillus)
3 stages of pertusssis

1. catarrhal: general flulike symptoms (highly infectious)

2. paroxysmal- Hallmark: repetitive coughing episodes
(whooping cough)
3. Convalescent- recovery phase
Ideal specimen: nasopharyngeal swab
 
Culture medium
1. Bordet-Gengou medium
Potato, blood, glycerol
2. Regan Lowe medium/charcoal cephalexin medium
Ampotericin, Cephalexin, horse blood, charcoal
- Preferred medium
 
Colonies- Mercury droplet colonies/pearl-like (Bordet-
Gengou)
B. parapertussis- pertussis-like syndrome

B.bronchiseptica-inhabits respiratory tract of canines

(kennel’s cough), infrequently responsible for
chronic respiratory tract infections in humans.
 Identifying characteristics

1. Small and smooth , mercury droplet and are

beta hemolytic
2. G/S: minute, poorly stained coccobacilli,
single or in pairs
3. Growth on MAC + except B. pertussis
4. B. pertussis= urease negative others are
urease +
 Motility Urease Nitrate Oxidase

B. pertussis +

B. +
parapertus
sis

B. + + + +
bronchisep
tica
 Treatment
Erythromycin

Oxygen and sedation- to prevent anoxia and

subsequent brain damage
 References:
Mahon’s 5th edition
Dr. Emerson Bernardino Lecture Notes