Miscellaneous
Small Gram-Negative Bacilli
Chapter 34 Haemophilus
Pasteruella
Chapter 35 Bordetella
Chapter 36 Francisella Brucella
Yu Chun-Keung DVM, PhD
Department of Microbiology and Immunology
College of Medicine
National Cheng Kung University
May 5, 2010
Chapter 34 Haemophilus
Family Pasteurellaceae
Genera Haemophilus
Actinobacillus
Aggregatibacter
Pasteurella
Small, G(-), non-spore-forming bacilli
Fastidious growth needs
Genus Haemophilus
“Blood-lover”
Growth require hemin (x factor) and nicrotinamide
adenine dinucleotide, NAD (v factor)
Heated blood (chocolate) agar for isolation
Important Haemophilius species

H. influenzae (an important pathogen)

H. aegyptius (acute, purulent conjunctivitis)

H. ducreyi (STD – soft chancre)

H. parainfluenzae (rarely pathogenic)
Classification H. influenzae (Hi)
Serological differentiation - polysaccharide
capsular antigens: serotype a to f
Biochemical properties - indole production,
urease activity, ornithine decarboxylase
activity : biotype I to VIII
Pathogenesis
Non-encapsulated Hi & H. parainfluenzae
(non-typeable)
Colonize URT in all people
10% of the flora of saliva: H. parainfluenzae
Opportunistic pathogens: spread locally and
cause acute and chronic otitis, sinusitis,
bronchitis, and pneumonia.
Pathogenesis - encapsulated Hi type b
Uncommon in the URT
Common cause of disease in unvaccinated
children
Adhesins  colonization of oropharynx 
release cell wall components  damage and
impair ciliary function across epithelial and
endothelial cells  blood (invasion)
Produce IgA1 proteases, facilitate colonization
LPS lipid A induces meningeal inflammation
Major virulence factor: antiphagocytic
polysaccharide capsule – polyribitol
phosphate (PRP) : ribose, ribitol,
phosphate
Phagocytic engulfment of H.
influenzae bacterium opsonized
by antibodies specific for the
capsule and somatic (cell wall)
antigen.
2004 Kenneth Todar University of
Wisconsin-Madison Department of
Bacteriology
Natural infection, vaccination, passive
transfer of material antibody - antiPRP antibody is protective (enhance
phagocytosis and complementmediated bacteriocidal activity)
Absence of anti-PRP antibody
(complement depletion, splenectomy )
leads to invasion, bacteremia and
dissemination
Clinical diseases (Hib infection)
Meningitis: Hi type b was the most common cause
of pediatric meningitis (3 -18 m) results from
bacterimic spread from nasopharynx; cannot be
differentiated from other causes of bacterial
meningitis (S. pneumoniae, N. meningitidis, E. coli).
Age-specific incidence of
bacterial meningitis caused by
Haemophilus influenzae,
Neisseria meningitidis and
Streptococcus pneumoniae
prior to 1985
2004 Kenneth Todar University of WisconsinMadison Department of Bacteriology
Clinical diseases (Hib infection)
Epiglotitis: 2-4 yrs; swelling of the
supraglottic tissue, pharyngitis, fever,
rapidly progress to complete obstruction of
the airways, life-threatening emergency.
Cellulitis: fever, reddish-blue patches on
the cheeks or periorbital area.
Arthritis: the most common form of arthritis
(single large joint) in children <2 yrs.
• Before the introduction of conjugated vaccine, Hib was a
common pediatric disease in children < 5 yrs.
• Polysaccharide vaccine for Hib were not effective for children
< 18m (the high risk population).
• Hib conjugate vaccine, which can work for infant >2m, was
introduced in 1987 which greatly reduced the incidence of
disease (>90%).
Epidemiology
Now Hib infections only occur in nonimmune
children or adults with waning immunity,
Other serotypes of encapsulated strains and
non-encapsulated strains become more
common.
Hib remains a significant pediatric pathogen
worldwide, especially in developing
countries.
Transmission
Person-to-person transmission in nonimmune population
Increased disease frequency in households
where there is a primary case or an
asymptomatic carrier.
Primary risk factor for invasive disease =
absence of anti-PRP antibody.
Close contacts should be given
chemoprophylaxis.
Diagnosis
Clinical diagnosis

Sign, Symptom, Syndrome
Laboratory diagnosis


detection / identification of antigens
(whole cell, protein, nucleic acid)
detection of immune responses (i.e.
antibodies)
Signs are those findings that a
physician can objectively detect or
measure.
Symptoms are those problems that
a patient notices or feels.
Diagnosis
Specimens:

Oral swab: avoid contamination with oral
secretions

Direct needle aspiration

Sputum from LRT

Cerebrospinal fluid and blood (>107 bacteria/ml)
Microscopy: both sensitive & specific;
G(-) bacilli in CSF in >80% cases before
antibiotics treatment
Diagnosis
Particle agglutination test (for Hib only):


Detect PRP antigen, rapid and sensitive (1
ng/ml)
Anti-PRP Ab-coated latex particles + specimen,
if PRP present, “positive” agglutination
Culture:



Hib : chocolate agar, colony 1-2 mm.
Satellite phenomenon: Hib grows around
colonies of Staph. aureus on unheated blood
agar.
H. aegyptius / H. ducreyi : fastidious
Treatment
Prompt antimicrobial therapy for systemic
Hib infections, otherwise mortality 100%
Serious infections: cephalosporins
Less severe infections (otitis, sinusitis):
ampicillin
Antibiotic chemoprophylaxis (rifampin) for
high risk group (children < 2ys with
patients around)
Prevention Hi type b conjugate vaccine
Purified capsular PRP + Carrier proteins:
Meningococcal outer membrane protein
Diphtheria toxoid
Tetanus toxoid
Three doses of vaccine (the same type)
before age of 6 months followed by booster
doses.
Haemophilus ducreyi
Cause of genital ulcers - soft chancre, 軟
性下疳
A sexually transmitted disease; most
common in Africa and Asia
Indurate and painful ulcer on genitalia with
regional lymphadenopathy
Differential: syphilis, herpes simplex,
lymphogranuloma venereum (caused by
Chlamydia trachomatis)
Haemophilus aegyptius
Koch-weeks bacillus
Acute, purulent conjunctivitis
Genus Aggregatibacter
A. actinomycetemcomitans and A.
aphrophilus
Mouth flora  blood  damaged heart
valve / artificial valve  subacute
endocarditis
Difficult to diagnosis : develop slowly and
the bacteria grow slowly in blood agar (> 5
days)
Genus Pasteurella
P. multocida and P. canis
Primarily animal pathogen.
Commensals in oropharynx of health
animals.
Human infections result from animal contact
(bites, scratches, shared food).
The most common organism in human
wounds inflicted by bites from cats and dogs.
Three general forms of disease
Localized cellulitis and regional
lymphadenopathy after animal bite or
scratch.
Exacerbation of chronic respiratory tract
disease in patients with underlying
pulmonary dysfunction.
Systemic infection in immunocompromised
patients.
Lab diagnosis
Grows well on blood and chocolate
agar
Large, buttery colonies with a musty
odor
Treatment
Susceptible to a variety of antibiotics
Penicillin, macrolides, tetracycline …
Chapter 35 Bordetella
Extremely small (0.2 x 1 μm ), G(-),
coccobacilli
Have simple nutritional requirement
Some species (i.e., B. pertussis) are
highly susceptible to toxic substances
and metabolites in media (need charcoal,
starch, blood, or albumin to absorb toxic
substances)
Important Bordetella species
B. pertussis: whooping cough / pertussis
(severe cough)
B. parapertussis: mild form of pertussis
B. bronchiseptica: respiratory disease of
animals (pigs and dogs)
B. holmesii: uncommon cause of sepsis
The four species are closely related,
differing only in the expression of virulence
genes
Pathogenesis
• Exposure (aerosol)
• Bacterial attachment to
ciliated epithelial cells of
the respiratory tract by
means of adhesins
(黏附因子)
• Proliferation
• Production of toxins
• Localized tissue damage
and systemic toxicity
Colonization of tracheal epithelial cells by
Bordetella pertussis
2004 Kenneth Todar University of WisconsinMadison Department of Bacteriology
Bacterial adhesins
Filamentous hemagglutinin: contain RGD motif:
bind (1) sulfated glycoprotein integrins on ciliated
respiratory cells; (2) CR3 on macrophages, and
trigger phagocytosis without initiating oxidative
burst (intracellular survival, escape from Ab)
Pertactin : contain RGD motif
Pertussis toxin： A classic A-B toxin with a toxic
subunit (S1) and binding subunits (S2 to S5); S2
binds lactosylceramide on ciliated respiratory cells,
S3 binds phagocytic cells
Fimbria : mediate binding in vitro; in vivo function
unknown
Toxins
S1 subunit of pertussis toxin
Adenylate cyclase toxin / hemolysin
Dermonecrotic toxin
Tracheal cytotoxin
LPS
S1 subunit of pertussis toxin
S2-S5
binding
subunit
S1 toxic
subunit
Adenosine diphosphateribosylating activity for G protein,
which regulates adenylate cyclase
activity (convert ATP to cAMP).
Increase respiratory secretion and
mucus production.
Tracheal cytotoxin: (1) target ciliated epithelial cell;
ciliostasis, extrusion of ciliated cells, impair regeneration of
damaged cells by interfering DNA synthesis (disrupt
clearance mechanism, lead to cough); (2) IL-1 production
(lead to fever)
A tracheal organ culture 72 h after
infection with B. pertussis.
Large arrow: Bordetella
Small arrow: cilia
Extruded epithelial cell with
attached bacteria
Denuded epithelium
Normal ciliated epithelial cell
Toxins
Adenylate cyclase toxin / hemolysin:
(1) activated by calmodulin and converse
ATP to cAMP, increase respiratory secretion;
(2) inhibit leukocyte functions
Dermonecrotic toxin: vasoconstriction and
tissue destruction
LPS: unknown (activate complement and
stimulate cytokine release)
Clinical disease
Infect ciliated epithelial cells of the airways,
produce disease locally, no invasion.
Catarrhal phase : resemble common cold,
sneezing, serous rhinorrhea, malaise, low-grade
fever, 1-2 wk, infectious (disease not recognized
with high number of bacteria produced)
Paroxysmal phase : a series of repetitive coughs
followed by inspiratory whoop, vomiting, and
exhaustion, 40-50 paroxysms daily, lymphoctyosis,
2-4 wk.
Convalescent phase : paroxysms diminish with
secondary complications, lasts for above 3 wk.
Clinical disease
Classic presentation may not be seen in patients
with partial immunity.

Only chronic persistent cough without whooping or
vomiting
Differential diagnosis

Mycoplasma pneumoniae

Chlamydophilia pneumoniae

Legionella pneumophila
Epidemiology
Pertussis was considered a pediatric
disease (< 1 year)
Incidence (morbidity and mortality) has
been reduced considerably after the
introduction of vaccine in 1949.
Still endemic worldwide with a dramatic
increase in recent year in US
Majority of infections are found in
adolescents and adults (reason not known).
2009.4.23
Lab diagnosis – specimen collection
and transport
Extremely sensitive to drying, do not
survive outside the host or traditional
transport medium.
Inoculate (nasopharyngeal aspirate) to
freshly prepared medium or transport
medium at bedside.
Use synthetic fiber swabs not cotton swabs
(fatty acid are toxic to Bp).
Lab diagnosis - microscopy
Fluoresceinlabeled rabbit
anti-Bp Ab
Aspirated
specimen
Direct
Fluorescein-labeled
anti-rabbit Ig Ab
Rabbit anti-Bp Ab
Indirect
Direct or indirect
fluorescent antibody
tests for antigen
detection
Aspirated specimen 
microscopic slide airdried heat fixed 
fluorescent Ab
Sensitivity 50%
.Nucleic acid amplification
Polymerase chain reaction
sensitivity 80-100%
No FDA approved test, inhouse assay
Serology
No FDA approved test
ELISA for antibodies against
filamentous hemagglutinin or
pertussis toxin
Lab diagnosis - culture
Regan-Lowe charcoal medium (horse blood,
glycerol, peptones).
35°C, humidified, 7 days,
50% sensitivity, affected by

Patient factors (stage of illness, use of
antibiotics)

Quality of specimen

Transport conditions

Culture methods
Treatment
Primarily supportive. Recovery depends on
regeneration of ciliated epithelial cells.
Antibiotics (erythromycin) are effective and can
reduce duration of clinical course.
However, the illness is usually unrecognized
during catarrhal phase (the peak of
contagiousness)
Pertussis is highly contagious; prophylaxis for
family members of a symptomatic patient.
Vaccination
DTP vaccine (diphtheria + toxoid of tetanus +
inactivated whole cell of Bp), 80-85% effective.
DTP vaccine has not been widely accepted
because of vaccine-related complications.
DTaP (acellular vaccine) : D + T + inactivated
pertussis toxin, filamentous hemagglutinin, and
pertactin or fimbriae.
Pediatic DTaP vaccination: 2, 4, 6, 15-18 m, and
4-6 yrs; Adult DTaP: 11-12 yrs and 19-65 yrs,
both high level of protection
Chapter 36 Francisella and Brucella
Zoonotic pathogens and potential
agents of bioterrorism
Very small G(-) coccobacilli, 0.5  1.5
m,
Fastidious, slow growth on culture
(>1 week)
Taxonomically unrelated
α-Proteobacteria
 Brucella
 Rickettsia
 Ehrlichia
γ-Proteobacteria
 Francisella
 Legionella
 Pasteruella
 Pseudomonas
Genus Francisella
Francisella tularensis (Tularemia)

F. tularensis subsp. tularensis (type A)

F. tularensis subsp. holarctica (type B)

F. tularensis subsp. Mediasiatica (rare as pathogen)

F. tularensis subsp. Novicida (rare as
pathogen)
Francisella philomiragia (uncommon opportunistic
pathogen)
Pathogenesis
Pathogenic strains possess antiphagocytic capsule;
protect bacteria from complement-mediated killing
Intracellular parasite Can survive for prolonged
periods in macrophages; inhibit phagosomelysosome fusion.
IFN-γ- and TNF-α-mediated activation of
macrophage is essential for controlling bacterial
replication and killing in early stage.
Specific T cell-mediated activation of macrophage
is essential for controlling bacterial replication and
killing in late stage.
Epidemiology
F. tularensis subsp. tularensis
Natural reservoirs and vectors: >200 species of mammals,
birds, blood-sucking arthropods
Type A:
North America: lagomorphs (rabbits, hares), cats, biting
arthropod
Type B:
Northern hemisphere: rodents, cats
Hunters, lab personnel and those exposed to ticks are high
risk for infection in endemic areas


Infections occur in summer and winter; warm winter +
wet summer (increase in tick population)
Actual numbers of infections > reported cases
Clinical disease - Tularemia
(Rabbit fever / Tick fever)
Clinical symptoms and prognosis
determined by route of infection
Ulcer
Cutaneous tularemia
infection
microbes.historique.net
Ulceroglandular form: cutaneous ulcer +
swollen LN, most common
 Oculoglandular form: painful conjunctivitis +
swollen cervical LN.
 Typhoidal form: sepsis
 Pneumonic form: pulmonary symptoms
 Gastrointestinal form:

Lab diagnosis
Specimen collection: highly contagious: able to penetrate
through unbroken skin and mucous membrane + aerosols.
extremely hazardous for physician and lab workers; wear
gloves and perform work in biohazard hood
Microscopy: Grain stain – not practical; direct staining with
fluorescent antibody, more sensitive and specific
Culture: not grow in common medium without cysteine (eg.
blood agar); use chocolate agar or buffered charcoal yeast
extract (BCYE) agar, take a week or longer
Serology: a  4-fold increase in Ab titer during illness or a
single titer of  1:160; antibody persist for many years (past
or current infection?); cross-reactivity between Brucella and
Francisella
T/P/C
Penicillin and cephalosporin are ineffective
(produce β-lactamase)
Streptomycin and gentamicin are effective
(high toxicity)
Prompt treatment < 1% mortality rate
Wear protective clothes and use insect
repellents, avoid reservoirs and vectors
Prophylactic antibiotics for high risk groups
Live-attenuated vaccine : partly protective
Genus Brucella
Six species with four species associated
with human diseases
B. melitensis : goat and sheep (natural host)
B. suis : swine, reindeer, caribou
B. abortus : cattle, bison
B. canis : dog, fox, coyotes
Pathogenesis
No exotoxin, endotoxin
low toxicity
Smooth colonies
associated with virulence
Granuloma
Accumulation of activated macrophages
Obligate intracellular parasites
Infect monocytes/macrophages, inhibit
phagolysosome fusion.
Spread to spleen, liver, lymph node, bone marrow,
kidneys (bacteria secrete proteins that induce
granuloma)
Epidemiology
Worldwide distribution
Animal reservoirs; natural hosts develop mild
or asymptomatic disease.
Sterility, abortion, and asymptomatic carriage.
Animal tissues (breast, uterus, epididymis,
placenta) contain erythritol (紅鮮醇) which is
required for the growth of the organism.
Milk, urine and birth products contain high
number of bacteria.
Epidemiology
Human infections
Direct contact: a lab or
occupational exposure
Ingestion: consume
contaminated food
products
Inhalation
Sources of Brucella infection.
G.G. Alton & J.R.L. Forsyth
Clinical disease (Brucellosis, Bang’s disease,
undulant fever, Malta fever)
Disease spectrum depends on the infecting
organism
B. melitensis : severe disease
B. suis : severe and chronic
B. abortus : mild
B. canis : mild
Clinical disease
Acute disease: incubation period 1-3 wks, fever
rises in afternoon, fall during night with drenching
sweat (undulant fever), weakness, malaise, chill,
weight loss, nonproductive cough, aches, pain.
Advanced disease: involve many tissues,
granulomas and abscesses. 70% GI symptoms,
20-60% bone lesions, 25% respiratory tract
symptoms
Chronic infection occur in inadequately treated
patients.
Lab diagnosis
Difficult
Multiple sampling (blood, bone marrow,
infected tissues)
Microscopy: insensitive (small size and
intracellular location)
Culture: blood agar, > 3 days to 2 wks,
sensitive and specific
Lab diagnosis - serology
A significant increase in Ab titer = evidence of
current disease; antibodies detected in all patients
(IgM, then IgA and IgG) and persist for months
and years.
5-10% of population in endemic area have high Ab
titer (>1:160)
Serologic test: use to confirm clinical diagnosis,
not as a basis of diagnosis.
Serum agglutination test (SAT): a fourfold increase
in titer or a single titer >1:160.
B. abortus cross-react with B. melitensis and B.
suis, but not with B. canis.
T/P/C
Tetracycline (doxycycline): bacteriostatic drugs,
relapse is common (due to inadequate therapy, not
antibiotic resistance); use doxycycline + rifampin for
> 6 weeks.
No vaccine for humans
Control of disease in livestock
Identification (serologic testing)
Elimination of infected herds
Vaccination
Avoidance of unpasturized dairy products
Observance of safety procedures in clinical lab