Download B. pertussis

Miscellaneous
Small Gram-Negative Bacilli
Yu Chun-Keung DVM, PhD
Department of Microbiology and Immunology
College of Medicine
National Cheng Kung University
April 30, 2008
Chapter 35 Haemophilus
Pasteruella
Chapter 36 Bordetella
Chapter 37 Brucella Francisella
Most of the G(-) bacilli are commonly
found in the environment or as normal
members of the human microbial flora.
The isolation of Bordetella, Brucella
and Francisella is always associated
with disease.
Bordetella
Extremely small (0.2 x 1 μm ), G(-),
coccobacilli
Have simple nutritional requirement
Some species (B. pertussis) are highly
susceptible to toxic substances (need
charcoal, starch, blood or albumin to
absorb toxic substance in medium, eg.
agar)
Important Bordetella species
B. pertussis: whooping/pertussis (severe
cough)
B. parapertussis: mild form of pertussis
B. bronchiseptica: respiratory disease of
animals (pigs and dogs)
The three species are closely related,
differing only in the expression of virulence
genes
Pathogenesis
• Exposure (aerosol)
• Bacterial attachment to ciliated epithelial
cells of the RT
• Proliferation
• Production of toxins
• Localized tissue damage and systemic
toxicity
Colonization of tracheal epithelial cells by Bordetella pertussis
2004 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology
Virulence Factors (Table 36-1)
Adhesins (黏附因子)
Toxins
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)
Pertactin (P69 protein) : contain RGD motif
Pertussis toxin： A classic A-B toxin. Toxic
subunit (S1) and binding subunits (S2 to S5); S2
binds ciliated respiratory cells, S3 binds
phagocytic cells and increase CR3 expression
Fimbria : mediate binding in vitro; function
unknown
Toxins
S1 subunit of pertussis toxin: target membrance
surface G protein, increase cAMP and then increase
respiratory secretion and mucus secretion
Adenylate cyclase toxin/hemolysin: converse ATP to
cAMP, increase respiratory secretion, inhibit leukocyte
function
Dermonecrotic toxin: vasoconstriction and tissue
destruction
Tracheal cytotoxin: ciliostasis, extrusion of ciliated cells,
impair regeneration of damaged cells by interfering DNA
synthesis (disrupt clearance mechanism, lead to cough);
IL-1 production (lead to fever)
LPS: unknown (activate complement and stimulate
cytokine release)
S2-S5
binding
subunit
S1 toxic
subunit
S1 subunit of pertussis toxin
Adenosine diphosphateribosylating activity for G protein,
which regulates adenylate cyclase
activity (convert ATP to cAMP).
Increase respiratory secretion and
mucus production.
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 of
non-ciliated cells
Clinical disease
Infect ciliated epithelial cells of the airways,
produce disease locally, no invasion.
Catarrhal phase (卡他期): resemble common cold,
sneezing, serous rhinorrhea, malaise, low-grade
fever, 1-2wk, infectious (disease not recognized
with high number of bacteria produced)
Paroxysmal phase (突發期): repetitive coughs and
inspiratory whoop, vomiting and exhaustion, 40-50
paroxysms daily, bronchopneumonia, 2-4wk.
Recovery phase (恢復期): lasts for above 3 wks.
Classic presentation may not be seen in patients
with partial immunity.
Epidemiology
Pertussis has been considered a pediatric
disease (< 1 year)
Incidence (morbidity and mortality) has
been reduced considerably after the
introduction of vaccine in 1949. Still
endemic worldwide.
Immunity is not lifelong.
Infection is seen in nonimmune infants,
young children and adults with waning
immunity.
Diagnosis
Clinical diagnosis

Sign, Symptom, Syndrome
Laboratory diagnosis


Detection / identification of antigens
(whole cell, protein, nucleic acid)
Detection of immune responses (i.e.
antibodies)
Lab diagnosis – specimen collection
and transport
Extremely sensitive to drying, cannot
survive outside the host or traditional
transport medium.
Inoculate (nasopharyngeal aspirate) to
freshly prepared medium (charcoal-horse
blood agar) or Regan-Lowe transport
medium at bedside.
Use synthetic fiber swabs not cotton swabs
(fatty acid are toxic to Bp).
Lab diagnosis - culture
Regan-Lowe medium (charcoal medium
with horse blood, glycerol, peptones).
35°C, humidified, 7 days, sensitivity 50%,
depend on stage of illness, use of
antibiotics etc.
Lab diagnosis - microscopy
Fluoresceinlabeled rabbit
anti-Bp Ab
Aspirated
specimen
Direct or indirect
fluorescent antibody
tests for antigen
detection
Sensitivity 50%
Fluorescein-labeled
anti-rabbit Ig Ab
Rabbit anti-Bp Ab
檢體玻片風乾熱固定
螢光抗體染色
Serology
No FDA approved test
ELISA for antibodies against filamentous
hemagglutinin and pertussis toxin.
Nucleic acid amplification
Polymerase chain reaction
sensitivity 80-100%
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)
Prophylaxis for unimmunized infants (Pertussis
is highly contagious, family members will
become carrier).
Vaccination
DTP vaccine (diphtheria, toxoid of tetanus, and
inactivated whole cell of Bp,), 80-85% effective.
Vaccine has not been widely accepted because
of vaccine-related complications.
DTaP (acellular vaccine) with inactivated
pertussis toxin and one or more bacterial
components (eg. filamentous hemagglutinin,
pertactin or fimbriae.
DTaP vaccination: 2, 4, 6, 15-8 m, and 4-6 y,
high level of protection
Chapter 37
Brucella 布魯氏桿菌屬
Francisella 法蘭西氏菌屬
Brucella and Francisella
Zoonotic pathogens and potential
agents of bioterrorists
Very small coccobacilli, 0.5  1.5 M,
G(-)
Fastidious, slow growth on culture
(>1 week)
Taxonomically unrelated
α-Proteobacteria
 Brucella
 Rickettsia
 Ehrlichia
γ-Proteobacteria
 Francisella
 Legionella
 Pasteruella
 Pseudomonas
Brucella
Six species with four species associated
with human diseases
B. melitensis : goat and sheep
B. suis : swine
B. abortus : cattle
B. canis : dog, fox
Sources of Brucella infection.
G.G. Alton & J.R.L. Forsyth
Pathogenesis
No exotoxin, endotoxin low toxicity
Obligate intracellular parasites of
animals and humans
Infect monocytes/macrophages,
replicate in phagolysome under the
control of virulence genes in virB operon
Spread to spleen, liver, lymph node,
bone marrow, kidneys (bacteria secrete
proteins that induce granuloma)
Activated macrophages
Clinical disease (Brucellosis, Bang’s disease,
undulant fever, Malta fever)
Disease spectrum depends on the infecting
organism
B. melitensis : severe disease in humans
B. suis : severe and chronic
B. abortus : mild
B. canis : mild
Clinical disease
Acute stage: incubation period >2 months, fever
rises in afternoon, fall during night with drenching
sweat (undulant fever), weakness, malaise, chill,
weight loss, nonproductive cough, aches, pain.
Chronic stage: involve many tissues for many
years, granulomas and abscesses. 70% GI
symptoms, 20-60% bone lesions, 25% respiratory
tract symptoms
Epidemiology
Worldwide distribution
Animal reservoirs; natural hosts develop
mild or asymptomatic disease.
Animal tissues (breast, uterus, epididymis,
placenta) contain erythritol (紅鮮醇)which
is required for the growth of the organism.
Epidemiology
In animals: sterility, abortion, and
asymptomatic carriage. Milk, urine and
birth products contain high number of
bacteria.
Human infections:



Direct contact (a lab or occupational
exposure)
Ingestion: consume unpasteurized milk,
milk products or cheese
Inhalation
Lab diagnosis
Multiple sampling (blood, bone
marrow, infected tissues)
Microscopy: insensitive (small size
and intracellular location)
Culture: blood agar, > 3 days
Lab diagnosis - serology
Antibodies detected in all patients (IgM, then
IgA and IgG) and persist for months and years.
A significant increase in Ab titer = evidence of
current disease
Serum agglutination test: use for confirming
clinical diagnosis; a fourfold increase in titer or
a single titer >1:160
B. abortus Ag cross reacts with those of B.
melitensis and B. suis but not B. canis
T/P/C
Tetracycline (doxycycline): bacteriostatic
drugs, relapse is common; use doxycycline +
rifampin for > 6 weeks.
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
Genus Francisella
F. tularensis spp. tularensis (type A)
F. tularensis spp. holarctica (type B)
F. tularensis spp. mediaasiatica
F. tularensis spp. philomiragia
Francisella tularensis
G(-), very small size (0.2  0.7 m) bacilli,
able to penetrate through skin and
mucous membrane + aerosols
Highly contagious: extremely hazardous
for physician and lab workers.
Fastidious growth requirement (iron and
cysteine)
Epidemiology
Northern hemisphere (20°N to Arctic circle, not
in southern regions
Animal reservoirs: wild mammals (rabbits, hares,
voles), domestic animals, bird, fish, bloodsucking arthropods (ticks)
Human infections result from:

Arthropod biting (10 organisms)

Direct contact with infected animals or pets (10)

Inhalation of infectious aerosol (mostly in lab ) (50)

Ingestion of contaminated meat or water (108)
Hunters, lab personnel and those exposed to
ticks are high risk for infection in endemic areas
Pathogenesis
Intracellular parasite
Can survive for prolonged periods in
macrophages; inhibit phagosomelysosome fusion.
Pathogenic strains possess
antiphagocytic capsule; protect bacteria
from complement-mediated killing
Tularemia
(Rabbit fever / Tick fever)
Fever, chills, malaise, fatigue
Clinical symptoms and prognosis
determined by route of infection





Ulcer
Cutaneous tularemia
infection
microbes.historique.net
Ulceroglandular form: skin, most common
Oculoglandular form: eyes, painful
conjunctivitis.
Typhoidal form: blood, sepsis with multi-organ
involvement
Pneumonic form: respiratory tract
Gastrointestinal form: oral
Lab diagnosis
Specimen collection: great
hazardous for physicians and
lab workers; wear gloves and
perform work in biohazard hood
Microscopy: Grain stain – not practical; direct
staining with fluorescein antibody
Culture: not grow in common medium without
cysteine (eg. blood agar); 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; cross-reactivity
between Brucella and Francisella
T/P/C
Streptomycin (high toxicity) and
gentamicin are effective
Penicillin and cephalosporin are
ineffective (β-lactamase)
Wear protective clothes and use insect
repellents
Prophylactic antibioties
Live-attenuated vaccine : partly protective
Chapter 35 Haemophilus
Family Pasteurellaceae (巴斯德桿菌科)
Genera Haemophilus (嗜血桿菌屬)
Actinobacillus (放線桿菌屬)
Pasteurella (巴斯德桿菌屬)
Small, G(-), non-spore-forming bacilli
Fastidious growth needs
Haemophilus
“Blood-loving”, obligate parasite of
mucus membrane.
Growth require x factor (hemin) and v
factor (nicrotinamide adenine dinucleotide,
NAD)
Heated-blood (chocolate) agar for
isolation
Haemophilus
H. influenzae (an important pathogen)
H. parainfluenzae (rarely pathogenic)
H. ducreyi (STD – soft chancre)
H. aegyptius (acute, purulent conjunctivitis)
H. influenzae biogroup aegyptius
(Brazilian purpuric fever)
Classification
Serological differentiation : polysaccharide
capsular antigens: type a to f
Biochemical properties : biotypes I to VIII;
indole production, urease activity, ornithine
decarboxylase activity
Clinical presentation : two biogroups;
biogroup aegypticus causes Brazilian
purpuric fever
Pathogenesis
Nonencapsulated Hi / H. parainfluenzae
Colonize URT in all people
10% of the flora of saliva: H. parainfluenzae
Opportunistic pathogens: spread locally
and cause acute and chronic otitis and
sinusitis, exacerbation of chronic bronchitis
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
Produce IgA1 proteases, facilitate
colonization
Major virulence factor:
antiphagocytic polysaccharide
capsule – (PRP: polyribitol
phosphate)
Anti-PRP antibody is
protective (enhance
phagocytosis and
complement-mediated
bacteriocidal activity)
Phagocytic engulfment of H. influenzae
bacterium opsonized by antibodies specific for
the capsule and somatic (cell wall) antigen.
2004 Kenneth Todar University of WisconsinMadison Department of Bacteriology
Absence of anti-PRP antibody
(complement depletion,
splenectomy ) leads to
invasion, bacteremia and
dissemination
Clinical diseases
Meningitis: Hi type b was the most common cause
of pediatric meningitis results from bacterimic
spread from nasopharynx; cannot be differentiated
from other causes of bacterial meningitis (S.
pneumoniae, N. meningitidis, E. coli).
Epiglotitis: swelling of the supraglottic tissue,
rapidly progress to complete obstruction of the
airways, life-threatening emergency.
Cellulitis: reddish-blue patches on the cheeks or
periorbital area.
Arthritis: the most common form of arthritis (single
large joint) in children <2 years old.
Age-specific incidence of bacterial meningitis caused by Haemophilus
influenzae, Neisseria meningitidis and Streptococcus pneumoniae
prior to 1985
2004 Kenneth Todar University of Wisconsin-Madison Department of
Bacteriology
Epidemiology
Before the introduction of vaccine, Hib was
responsible for >95% invasive diseases,
epiglottitis, orbital cellulitis, meningitis in children
5 m to 5 y (<3 m protected by maternal antibody).
Hi type b conjugated vaccine was introduced in
1987 which greatly reduced the incidence of
disease (>90%). Now infections occur in
nonimmune children or adults with waning
immunity, especially in many developing counties.
Hi type c and f and nonencapsulated strains
become more common.
The decline of Hib meningitis associated with the introduction of new
vaccines
• Polysaccharide vaccine for Hib were not protective for
children < 18m.
• Hib conjugated vaccine, which can work for infant >2m, was
introduced in 1987 which greatly reduced the incidence of
disease (>90%).
Transmission
Person-to-person transmission
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
Samples: Cerebrospinal fluid and blood (>107
bacteria/ml)
Microscopy: both sensitive & specific; G(-) bacilli
in CSF in >80% cases before antibiotics
treatment
Culture: chocolate agar
Satellite phenomenon: grow around colonies of
Staph. aureus on unheated blood agar.
Particle agglutination: detect PRP antigen, rapid
and sensitive (1 ng/ml)
Anti-PRP Ab-coated latex particles + specimen,
if PRP present, “positive” agglutination
Treatment
Prompt antimicrobial therapy for
systemic Hi infections, otherwise
mortality 100%
Serious infections: cephalosporins
Less severe infections: ampicillin
Antibiotic chemoprophylaxis (rifampin)
for high risk group (children < 2ys with
patients around)
Prevetion - Hi type b conjugate vaccines
Hib polysaccharide vaccine: not effective
for children < 18 months
Hib conjugate vaccine: purified capsular
PRP with different carrier proteins:
Neisseria meningitidis outer membrane protein
Diphtheria toxoid
Three doses of vaccine (the same type)
before age of 6 months followed by booster
doses.
Haemophilus ducreyi
Cause chancroid (soft chancre,軟性下疳),
a sexually transmitted disease; most
common in Africa and Asia
Indurated ulcer on genitalia with regional
lymphadenopathy
Differential: syphilis, herpes simplex,
lymphogranuloma venereum (Chlamydia
trachomatis)
H. aegyptius
Acute, purulent conjunctivitis
H. influenzae biogroup aegyptius
Brazilian purpuric fever
(conjunctivitis  fever, vomiting and
abdominal pain  petechia, purpura
and shock)
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 …