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Miscellaneous
Small Gram-Negative Bacilli
Yu Chun-Keung DVM, PhD
Department of Microbiology and Immunology
College of Medicine
National Cheng Kung University
Chapter 36 Bordetella
Chapter 37 Brucella
Francisella
Chapter 35 Haemophilus
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 博德氏菌屬
Extremely small (0.2 x 1 μm ), G(-),
coccobacilli
Fastidious growth requirement
(charcoal, starch, blood or albumin
to absorb toxic substance in
medium, eg. agar)
Bordetella
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 respiratory tract
• 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
Adhesins (黏附因子)
Toxins
Bacterial adhesins
Filamentous hemagglutinin (絲狀紅血球凝集素):
contain RGD motif: (1) sulfated glycoprotein
integrins on ciliated respiratory cells; (2) CR3
on macrophages.
Pertactin (P69 protein) : contain RGD motif
Pertussis toxin： A classic A-B toxin. Toxic
subunit (S1) and binding subunit (S2 to S5); S2
binds ciliated respiratory cells, S3 binds
phagocytic cells
Fimbria : mediate binding in vitro; function
unknown
Toxins
S1 subunit of pertussis toxin: increase respiratory
secretion
Adenylate cyclase/hemolysin toxin: increase
respiratory secretion, inhibit leukocyte function
Dermonecrotic toxin: vasoconstriction and tissue
destruction
Tracheal cytotoxin: ciliostasis, extrusion of ciliated
cells, impair regeneration of damaged cells
(disrupt clearance mechanism, lead to cough), IL1 production (lead to fever)
LPS: unknown (stimulate cytokine release)
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): sneezing, serous
rhinorrhea, malaise, low-grade fever, 1-2wk,
infectious.
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.
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.
Incidence vs. Prevalence
Epidemic vs. Endemic
Lab diagnosis - culture
Extremely sensitive to drying, cannot survive
outside the host or traditional transport medium.
Inoculate to freshly prepared medium at bedside.
Charcoal-horse blood agar or Regan-Lowe
charcoal medium with horse blood, glycerol,
peptones.
Nasopharyngeal aspirate (use synthetic fiber
swabs not cotton swabs, fatty acid are toxic to
Bp).
35°C, humidified, 7 days, sensitivity 50%.
Diagnosis - microscopy
Aspirated
specimen
Direct and indirect
fluorescent antibody
tests for antigen
detection
Sensitivity 50%
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
Erythromycin during catarrhal phase (在卡
他期無法做正確診斷，失去治療先機).
Primarily supportive, antibiotics do not
ameliorate clinical course. Recovery
depends on regeneration of ciliated
epithelial cells.
Prophylaxis for unimmunized infants
(Pertussis is highly contagious, family
members will become carrier).
Vaccination
DTP vaccine (inactivated whole cell of Bp,
toxoid of tetanus and diphtheria), 80-85%
effective.
Vaccine has not been widely accepted
because of vaccine-related complications.
Acellular vaccine with inactivated pertusis
toxin and filamentous hemagglutinin,
pertactin or fimbriae.
Chapter 37
Brucella 布魯氏桿菌屬
Francisella 法蘭西氏菌屬
α-Proteobacteria
 Brucella
 Rickettsia
 Ehrlichia
γ-Proteobacteria
 Francisella
 Legionella
 Pasteruella
 Pseudomonas
Brucella (布魯氏桿菌)
Small, 0.5  1.5 M, G(-)
Fastidious, grow slowly on culture
(>1 week)
Zoonotic pathogens and potential
agent of bioterrorists
Brucella
B. melitensis : goats and sheep
B. suis : swine
B. abortus : cattle
B. canis : dogs, fox
Sources of Brucella infection.
G.G. Alton & J.R.L. Forsyth
Brucellosis (Bang’s disease, undulant
fever, Malta fever)
B. melitensis : severe disease in
humans
B. suis : severe and chronic
B. abortus : mild disease
B. canis : mild disease
Pathogenesis
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 (granuloma)
Granuloma
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 abcesses.
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 exposure)
Ingestion: consume unpasteurized milk,
milk products or cheese
Inhalation
Lab diagnosis
Multiple sampling (blood, bone
marrow, infected tissues)
Microscopy: insensitive
Culture: blood agar, > 3 days
Serology (serum agglutination test):
use for confirming clinical diagnosis;
fourfold increase in titer or a single
titer >1:160
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
Francisella tularensis (法蘭西氏土倫桿菌)
Highly contagious: extremely hazardous
for physician and lab workers.
G(-), very small size (0.2  0.7 m), able
to penetrate through skin and mucous
membrane + aerosols
Fastidious growth requirement (iron and
cysteine)
Distribute 20° North
Pathogenesis
Animal reservoirs: wild mammals (rabbits, hares,
voles), domestic animals, bird, fish, bloodsucking arthropods (ticks)
Human infections result from arthropod biting (10
organisms), direct contact (10), inhalation (50), or
ingestion (108).
Intracellular parasite, can survive for prolonged
periods in macrophages; inhibit phagosomelysosome fusion.
Pathogenic strains possess antiphagocytic
capsule.
Tularemia (土倫病)
(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
Clinical sign
Clinical symptom
Syndrome (eg. AIDS)
Chapter 35
Haemophilus
Pasteurellaceae (巴斯德桿菌科)
Haemophilus (嗜血桿菌屬)
Actinobacillus (放線桿菌屬)
Pasteurella (巴斯德桿菌屬)
Haemophilus
“Blood-loving”, small G(-) bacilli,
obligate parasite of mucus
membrane.
growth require factor x (hemin) and
factor v (NAD); heated-blood
(chocolate) agar for isolation.
Haemophilus
H. influenzae (流行性感冒嗜血桿菌)
H. parainfluenzae
H. ducreyi (杜克氏嗜血桿菌)
H. aegyptius
H. influenzae biogroup aegyptius
Classification
Polysaccharide capsular antigens:
Type a – f
Biotypes I to VIII (biochemical properties):
indole production, urease activity, ornithine
decarboxylase activity
Two biogroups (clinical presentation):
Hi 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: 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 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. Cannot be differentiated
from other causes of bacterial meningitis (S.
pneumoniae, N. meningitidis, E. coli).
Epiglottitis: 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.
Hi type c and f and nonencapsulated strains
become more common.
The decline of Hib meningitis associated with the
introduction of new vaccines
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, “+” agglutination
Treatment
Prompt antimicrobial therapy,
otherwise mortality 100%
Serious infections: cephalosporins
Less severe infections: ampicillin
Antibiotic chemoprophylaxis
(rifampin) for high risk group
Hi type b conjugate vaccines
Hib polysaccharide vaccine: not effective
for < 18 months (CHO: no/low immunogenicity)
Hib conjugate vaccine: purified capsular
PRP + 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 (杜克氏嗜血桿菌)
Cause chancroid (soft chancre,軟性下疳),
a sexually transmitted disease.
Indurated ulcer on genitalia with regional
lymphadenopathy.
Differential: syphilis, herpes simplex,
lymphogranuloma venereum (Chlamydia
trachomatis)
H. aegyptius
Purulent conjunctivitis
H. influenzae biogroup aegyptius
Brazilian purpuric fever