Haemophilus, Bordetella, Brucella,: and Francisella

Haemophilus, Bordetella, Brucella,
and Francisella
Suzan Matar (PhD Medical Microbiology and Immunology)

Department of Clinical Laboratory Sciences
Gram-Negative Rods Associated with the Respiratory Tract.
Species Major Diseases Laboratory Diagnosis Factors X and Vaccine Prophylaxis for
V Avail Contacts
Required able
for
Growth
H. influenzae Meningitis1; otitis media, Culture; capsular + + Rifampin

sinusitis, pneumonia, polysaccharide in
epiglottitis serum or spinal
fluid
B. pertussis Whooping cough (pertussis) Fluorescent antibody + Erythromycin

on secretions;
culture
L. pneumophila Pneumonia Serology; urinary None

antigen; culture
HAEMOPHILUS INFLUENZAE
Haemophilus influenzae is found on the
mucous membranes of the upper
respiratory tract in humans.
It is an important cause of meningitis in

children and causes upper and lower
respiratory tract infections in children
and adults.
Morphology and Identification
A. Typical
Organisms
In specimens from
acute infections,
the organisms are
short (1.5 m)
coccoid bacilli,
sometimes
occurring in pairs
or short chains.
Culture
- On chocolate agar
- H influenzae does not grow on sheep blood

agar except around colonies of
staphylococci (satellite phenomenon).
- Factor X acts physiologically as hemin;

factor V can be replaced by nicotinamide
adenine nucleotide (NAD)
Antigenic Structure
Encapsulated H influenzae contains capsular

polysaccharides one of six types (af).
A capsule swelling test with specific antiserum is

analogous to the quellung test for pneumococci.
Typing can also be done by immunofluorescence.
Pathogenesis
H influenzae produces no exotoxin
The capsule is antiphagocytic in the absence of
specific anticapsular antibodies.
Type b H influenzae causes meningitis, pneumonia

and epiglottitis, cellulitis, septic arthritis, and
occasionally other forms of invasive infection.
Blood of many persons older than age 35 years is

bactericidal for H influenzae
Clinical Findings
Encapsulated organisms may reach the
bloodstream and be carried to the
meninges or may establish themselves in the
joints to produce septic arthritis.
Pneumonitis and epiglottitis caused by H

influenzae may follow upper respiratory tract
infections in small children and old or
debilitated people
Adults may have bronchitis or pneumonia

caused by H influenzae.
Epidemiology, Prevention, and
Control
Encapsulated H influenzae type b is
transmitted from person to person by the
respiratory route.
H influenzae type b disease can be

prevented by administration of Haemophilus
b conjugate vaccine to children.
HAEMOPHILUS DUCREYI
H ducreyi causes chancroid (soft chancre), a

sexually transmitted disease. Chancroid consists of
a ragged ulcer on the genitalia, with marked
swelling and tenderness.
The small gram-negative rods occur in strands in the

lesions, usually in association with other pyogenic
microorganisms.
H ducreyi requires X factor but not V factor.
It is grown best from scrapings of the ulcer base that

are inoculated onto chocolate agar containing 1%
IsoVitaleX and vancomycin, 3 g/mL; the agar is
incubated in 10% CO2 at 33C.
BORDETELLA PERTUSSIS
- The organisms are minute gram-negative coccobacilli

- Bipolar metachromatic granules can be demonstrated
- A capsule is present.
Culture
Primary isolation of B pertussis requires enriched media.
Bordet-Gengou medium (potato-blood-glycerol agar)
that contains penicillin.
The plates are incubated at 3537C for 37 days

aerobically in a moist environment.
Antigenic Structure, Pathogenesis,
and Pathology
Filamentous hemagglutinin and fimbriae mediate
adhesion to ciliated epithelial cells and are essential for
tracheal colonization.
Pertussis toxin promotes lymphocytosis, sensitization to

histamine.
Adenylate cyclase toxin, dermonecrotic toxin, and

hemolysin
The tracheal cytotoxin inhibits DNA synthesis in ciliated

cells
The lipopolysaccharide in the cell wall may also be

important in causing damage to the epithelial cells of the
upper respiratory tract.
Transmission is largely by the respiratory route from early
cases and possibly via carriers.
The blood is not invaded. The bacteria liberate the toxins

and substances that irritate surface cells, causing
coughing and marked lymphocytosis.
Secondary invaders such as staphylococci or H

influenzae may give rise to bacterial pneumonia.
Obstruction of the smaller bronchioles by mucous plugs

results in diminished oxygenation of the blood.
Convulsions in infants with whooping cough.

Clinical Findings
After an incubation period of about 2 weeks, the
catarrhal stage develops, with mild coughing and
sneezing. During this stage, large numbers of organisms
are sprayed in droplets, and the patient is highly
infectious but not very ill.
During the paroxysmal stage, the cough develops its

explosive character and the characteristic whoop
upon inhalation. This leads to rapid exhaustion and may
be associated with vomiting, cyanosis, and convulsions.
The white blood count is high (16,00030,000/L), with an

absolute lymphocytosis. Convalescence is slow.
Diagnostic Laboratory Tests
Specimens
A saline nasal wash is the preferred specimen.
Direct Fluorescent Antibody Test

The fluorescent antibody (FA) reagent can be used to
examine nasopharyngeal swab specimens.
Prevention
Every infant should receive three

injections of pertussis vaccine during
the first year of life followed by a
booster series for a total of five doses.
THE BRUCELLAE
The brucellae are obligate parasites of animals and
humans and are characteristically located
intracellularly. They are relatively inactive
metabolically.
Brucella melitensis typically infects goats;

Brucella suis, swine;
Brucella abortus, cattle;
and Brucella canis, dogs.
The disease in humans, brucellosis (undulant fever,

Malta fever), is characterized by an acute
bacteremic phase followed by a chronic stage that
may extend over many years and may involve many
tissues.
Pathogenesis and Pathology
The common routes of infection in
humans are the intestinal tract (ingestion
of infected milk), mucous membranes
(droplets), and skin (contact with infected
tissues of animals). lymphatic channels
bloodstream organs (granuloma).
Clinical Findings
The incubation period ranges from 14 weeks.
Malaise, fever, weakness, aches, and sweats. The
fever usually rises in the afternoon; its fall during the
night
After the initial infection, a chronic stage may

develop, characterized by weakness, aches and
pains, low-grade fever, nervousness,
Brucellae cannot be isolated from the patient at this

stage, but the agglutinin titer may be high.
Specimens
Blood should be taken for culture, biopsy material for culture
(lymph nodes, bone, and so on), and serum for serologic tests.
B. Culture
Brucella agar was specifically designed to culture Brucella
species bacteria.
Blood culture media readily grow Brucella species bacteria.

All cultures should be incubated in 810% CO2 at 3537C and
should be observed for 3 weeks
Serology
Immunoglobulin M (IgM) antibody levels rise during

the first week of acute illness, peak at 3 months, and
may persist during chronic disease.
IgG antibody levels rise about 3 weeks after onset of

acute disease, peak at 68 weeks, and remain high
during chronic disease.
IgA levels parallel the IgG levels.
Screened by ELISA
FRANCISELLA TULARENSIS
and TULAREMIA
Francisella species are widely found in animal
reservoirs and aquatic environments.
Subspecies tularensis (type A) is the most

virulent among this group and the most
pathogenic for humans. It is associated with
wild rabbits, ticks, and flies.
Morphology and Identification
Specimens
Blood is taken for serologic
tests. The organism may be
recovered in culture from
lymph node aspirates, bone
marrow, peripheral blood,
deep tissue, and ulcer
biopsies.
Culture
Buffered charcoal yeast extract (BCYE) agar used

to grow Legionella species.
Media should be incubated in CO2 at 3537C for

25 days.
Caution: To avoid laboratory acquired infections,

biosafety level three (BSL III) practices are required
when working with live cultures suspected of
containing F tularensis.
Pathogenesis and Clinical Findings
F tularensis is highly infectious: Penetration of the skin or
mucous membranes or inhalation of 50 organisms can
result in infection.
Most commonly, organisms enter through skin abrasions.

In 26 days, an inflammatory, ulcerating papule
develops Regional lymph (necrosis, (ulceroglandular
tularemia)
Inhalation of an infective aerosol results in peribronchial
inflammation and localized pneumonitis (pneumonic
tularemia).
Oculoglandular tularemia
Oropharyngeal tularemia,
Typhoidal tularemia (septicemia)
All affected individuals have fever, malaise, headache,
and pain in the involved region and regional lymph
nodes.
Because of the highly infectious nature
of F tularensis, this organism is a
potential agent of bioterrorism and is
currently classified on the select agent
list as a category A agent.
Paired serum samples collected 2 weeks

apart can show a rise in agglutination titer.
A single serum titer of 1:160 is highly
suggestive
Prevention and Control
- Humans acquire tularemia from handling
infected rabbits or muskrats or from bites
by an infected tick or deer-fly.
- Less often, the source is contaminated

water or food or contact with a dog or cat
that has caught an infected wild animal.

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Haemophilus, Bordetella, Brucella,

Suzan Matar (PhD Medical Microbiology and Immunology)

H. influenzae Meningitis1; otitis media, Culture; capsular + + Rifampin

B. pertussis Whooping cough (pertussis) Fluorescent antibody + Erythromycin

L. pneumophila Pneumonia Serology; urinary None

It is an important cause of meningitis in

- H influenzae does not grow on sheep blood

- Factor X acts physiologically as hemin;

Encapsulated H influenzae contains capsular

A capsule swelling test with specific antiserum is

Type b H influenzae causes meningitis, pneumonia

Blood of many persons older than age 35 years is

Pneumonitis and epiglottitis caused by H

Adults may have bronchitis or pneumonia

H influenzae type b disease can be

H ducreyi causes chancroid (soft chancre), a

The small gram-negative rods occur in strands in the

H ducreyi requires X factor but not V factor.

It is grown best from scrapings of the ulcer base that

- The organisms are minute gram-negative coccobacilli

The plates are incubated at 3537C for 37 days

Pertussis toxin promotes lymphocytosis, sensitization to

Adenylate cyclase toxin, dermonecrotic toxin, and

The tracheal cytotoxin inhibits DNA synthesis in ciliated

The lipopolysaccharide in the cell wall may also be

The blood is not invaded. The bacteria liberate the toxins

Secondary invaders such as staphylococci or H

Obstruction of the smaller bronchioles by mucous plugs

Convulsions in infants with whooping cough.

During the paroxysmal stage, the cough develops its

The white blood count is high (16,00030,000/L), with an

Direct Fluorescent Antibody Test

Every infant should receive three

Brucella melitensis typically infects goats;

The disease in humans, brucellosis (undulant fever,

After the initial infection, a chronic stage may

Brucellae cannot be isolated from the patient at this

Blood culture media readily grow Brucella species bacteria.

Immunoglobulin M (IgM) antibody levels rise during

IgG antibody levels rise about 3 weeks after onset of

IgA levels parallel the IgG levels.

Subspecies tularensis (type A) is the most

Buffered charcoal yeast extract (BCYE) agar used

Media should be incubated in CO2 at 3537C for

Caution: To avoid laboratory acquired infections,

Most commonly, organisms enter through skin abrasions.

Paired serum samples collected 2 weeks

- Less often, the source is contaminated

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