HAEMOPHILUS,

BORDETELLA, BRUCELLA
DIVAE SANDRAINY
HAEMOPHILUS
• Haemophilae are small

• Sometimes pleomorphic,

• Gram negative Rods Present On The Mucous Membranes Of Humans

Haemophilus influenzae (most commonly)

Haemophilus aegyptius (acute purulent conjunctivitis)

Haemophilus ducreyi (etiologic agent of the sexually transmitted disease soft chancre or
chancroid)

Haemophilus parainfluenzae (most common species in the mouth)

Physiology and Structure
The growth of most species of Haemophilus requires supplementation of media
with one or both of the following growth-stimulating factors:

(1) hemin (also called X factor for “unknown factor”)

(2) nicotinamide adenine dinucleotide (NAD; also called V Factor for

“vitamin”).

Although both factors are present in blood-enriched media, sheep blood agar must
be gently heated to destroy the inhibitors of V factor.

For this reason, heated blood (“chocolate”) agar is used for the isolation of
Haemophilus in culture.
Cell wall of Haemophilus is typical of other gram negative rods.
Lipopolysaccharide with endotoxin activity is present in the cell wall and strain-
specific and species-specific proteins are found in the outer membrane.

The surface of many but not all, strain of H. influenzae is covered with a
polysaccharide capsule, and six antigenic serotypes (a though f) have been
identified.
Pathogenesis and Immunity
H. Parainfluenzae & H. influenza (non capsulated) colonize the upper respiratory
tract in virtually all people within the first few months of life.

They are spread locally and cause disease in the ears (otitis media), sinuses
(sinusitis), and lower respiratory tract (bronchitis, pneumonia)

Encapsulated H. influenzae (serotype b (biotype I) common cause of disease in

unvaccinated children (meningitis, epiglottitis, cellulitis)

The major virulence factor in H. influenzae type b is the antiphagocytic

polysaccharide capsule, which contains ribose, ribitol and phosphate (commonly
polyribitool phosphate (PRP))
Epidemiology
Haemophilus sp. are present in almost all individuals, primarly colonizing the
mucosal membranes of the respiratory tract.

H. parainfluenza predominant Haemophilus sp in the mouth

H. Influenzae commonly found in the upper respiratory tract

Most of the H. influenzae type b infections now occur in children who are not
immune (because of incomplete vaccination or a poor response to the vaccine)
and in elderly adults with waning immunity.
Clinical Disease
Laboratory Diagnosis
1. Specimen Collection and Transport
Direct needle aspiration should be used for sinusitis or otitis media
Sputum for lower airways is used for pneumonia
CSF for meningitis
Blood culture for epiglottitis, cellulitis, and arthritis
2. Microscopic examination of Gram stained specimens is also useful for the
rapid diagnosis of the organism in arthritis and lower espiratory tract
3. Antigen detection : Th eimmunologic detection of H. influenza antigen (PRP
capsular antigen) isa rapid and sensitive way, because PRP can be detected
with the particle agglutination test, which can detect less than 1ng/ml of PRP
in clinical specimen.
4. Culture

Used Chocolate Agar

Treatment
1. Serious infection are treated with broad-spectrum cephalosporins

2. Less severe infection (otitis and sinusitis) treated with amoxicillin

3. The primary approach to preventing H. influenzae type b disease is through

active immunization with purified capsular PRP

• The use of conjugated vaccines has been remarkably successful in reducing the
incidences of H. influenzae

• Children receive two or three doses of vaccine against H. influenzae type b

disease before the age of 6 months, followed by booster dose at age 12 to 15
months
BORDETELLA
• Small (0,2 to 0,5x1 mikro meter)
• Striclty aerobic
• Gram negative coccobacillus
• Bordet Gengou Glycerine Potato Blood Agar

Responsible for human disease :

1. Bordetella pertussis : Agent responsible for pertussis or whooping cough
2. Bordetella parapertussis : Responsible for a milder form of pertussis
3. Bordetella bronchiseptica : Responsible for respiratory disease in dogs, swine,
laboratory animals, and occasionally humans
4. Bordetella holmesii : an uncommon cause of sepsis
Infection (B. pertussis) > development of whooping cough require exposure to the
organism > bacterial attachment ciliated epithelial cells (respiratory tract) >
proliferation of the bacteria > localized tissue damage and systemic toxicity

 Attachment of the organism to ciliated epithelial cells is mediated by protein

adhesins : pertactin, filamentous hemagglutinin, and fimbria

(Similar protein are also found in B. parapertussis and B. bronchiseptica

Localized tissues damage is mediated by dermonecrotic toxin (produce

localized ischemia in mouse model) and tracheal cytotoxin (inhibits cilia
movement, disrupting mormal clearance mechanisms in the respiratory tree
leading to the characteristic pertussis cough)

Systemic toxicity is produced primarily by pertussis toxin

Pertussis toxin > inactivates the protein that control adenylate cyclase activity >
leading to an increase in cAMP levels > increase in respiratory secretions and
mucus production (characteristic of the paroxysmal stage of pertussis)

Infection is initiated when infectious aerosols are inhaled and the bacteria
become attached to and proliferate on ciliated epithelial cells. After 7-10 day
incubation period, the classical presentation of pertussis proceeds through three
stage.
Laboratory Diagnosis
Bordetella species have simple nutritional requirements, some species are highly
susceptible to toxic substance and metabolites present in common laboratory
media. These species (particularly B. pertussis) require media supplemented with
charcoal, starch, blood or albumin to absorb these toxin substances.

Enzyme-linked immunosorbent assay (ELISA) test have been developed to

detect antibodies against pertussis toxin, filamentous hemagglutinin, pertactin,
and fimbriae
Treatment
 Macrolides (Erythromycin, azithromycin, clarithromycin) are effective in
eradicating the organism
!This effect has limited value because the illness is ussualy unrecognized during the
peak of contagiousness.
!Azithromycin and clarithromycin are generally better tolerated and the preferred
macrolides
 Trimethoprim-sulfomethoxazole or fluoroquinolones can be used in patients
unable to tolerate macrolides
 Two acellular vaccines (one for children and one for adults) administered
combination with vaccines for tetanus and diphtheria
! Pediatric vaccine (ages 2, 4, 6, and 15 to 18 months, with fifth dose between the ages
of 4 and 6 years)
! Adults vaccine ( at 11 or 12 y.o and again between the ages of 19 and 65), because
pertussis is highly contagious in a susceptible population, and unrecognized
BRUCELLA
 Small (0,5 x 0,6 to 1,5 mikro meter)
 Non motile, non encapsulated
 Gram negative coccobacilli
 Grow slowly in culture (taking a week or more) and generally requires complex
growth media
 Aerobic and doen’t ferment carbohydrates
 Colonies can assume both smooth (translucent, homogenous) and rough (opaque,
granular or sticky) forms
 Determined by the O antigen of the cell wall lipopolysaccharide (LPS)
 O chain of the smooth LPS is an important maker for virulence
 Doesn’t produce a detectable exotoxin, and endotoxin is less toxic than that
produced by other gram negative rods
There are 4 species most commonly associated with human disease :

1. Brucella abortus : infects cattle and American bison

2. Brucella melitensis : Animal reservoir are goats and sheep

3. Brucella suis : swine, reindeer and caribou

4. Brucella canis : dogs, foxes, and coyotes

The organism has a predilection for infecting organs rich in erythritol, a sugar
metabolized by many Brucella strains in preference to glucose.

The disease caused by members of this genus are characterized by a number of

names based on the original microbiologists who isolated and described the
organism (Sir David Bruce) called brucellosis
Initial exposure > the organism are phagocytosed by macrophages and monocyte,
where the bacteria survive and replicate.

Phagocytosed bacteria are carried to the spleen, liver, bone marrow, lymph nodes,
and kidneys. The bacteria secrete proteins that induce granuloma formation in
these organs, and destructive changes in these and other tissues occur in patients
with advances disease.

Brucellosis in human can be acquired by direct contact with the organism

(laboratory exposure), ingestion (consumption of contaminated food products), or
inhalation. Of particular concern is the potential use of Brucella as a biological
weapon, in which exposure would most likely be by inhalation.
Clinical Disease
Acute disease develops in approximately half of he patients infected with Brucella,
with symptoms forst appearing typically 1 to 3 weeks after exposure. Initial
symptoms are nonspecific and consist of malaise, chills, sweats, fatigue,
weakness, myalgias,weight loss, anthralgias, and nonproductive cough.
Almost all patient have fever and this can be intermittent to untreated patients,
hence the name undulant fever.

Chronic infections can also develop in inadequately treated patients, with

symptoms developing within 3 to 6 months after discontinuing antibiotic therapy.
Relapses are associated with a persistent focus on infections ( in bone, spleen,
liver) and not with the development of antibiotic resistance.
Laboratory Diagnosis
 Several blood samples should be collected for culture and serologic testing
 Bone marrow cultures and cultures of infected tissues may also be useful
 Brucella organisms are readily stained using conventional techniques, but their
intracellular location and small size make them difficult to detect in clinical
specimen
 The organism grow slowly in culture, requiring enriched blood agars and extended
incubation (3 days or more)
 Blood cultures should be incubated for 2 weeks before they are considered negative
 Preliminary identification of Brucella is based on the isolates microscopic and
colonial morphology, positive oxidase and urease reactios, and reactivity with
specific antibodies
 A presumptive diagnosis can be made if there is a fourfold increase in the titer or a
single titer is 1 : 160 or greater
Treatment
Tetracyclines + doxycycline the preferred agent, are generally active against
most strains of Brucella

 However, because this is a bacteriostatic drug, relapse is common after an

initially successful response

WHO recommends the combination of doxycycline with rifampin. Because the

tetracyclines are toxic to young children and fetuses, doxycycline should be
replaced with trimethoprim-sulfamethoxazole for pregnant woman and for
children younger than 8 years.

 Treatment must be continued for 6 weeks or longer for it to be successful

 Relapse of disease is caused by inadequate therapy and not the development of

antibiotic resistance
 The avoidance of unpasteurized dairy products, the observance of appropriate
safety procedures in the clinical laboratory, and the wearing of protective
clothing by abattoir workers are further ways to prevent brucellosis.

B. abortus and B. melitensis vaccines have been used successfully to prevent
infection in animal herds

Vaccines have not been developed against B. suis or B. canis, and the existing
vaccines cannot be used in humans because they produce symptomatic disease

Lack of an effective human vaccine is of concern because Brucella could be used

as an agent of bioterorism