Bordetella

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PAMB 650 Medical Microbiology Lecture
41 Legionella, Bordetella and Haemophilus Gram
Negative Rods of the Respiratory Tract
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Organization of Lecture

1. Overview
2. Organisms of Clinical Importance
3. Microbiology
4. Public Health
5. Pathogenic Mechanisms
6. Clinical Presentation

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LEGIONELLA
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LEGIONELLAEOverview

• Facultative intracellular pathogen
• Gram negative rod
• Requires specialized media to grow
• Stains poorly with gram stain
• Transmitted via contaminated aerosols
• No person to person transmission

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2 Species of Clinical Importance

• Legionella
• One genus
• 50 species
• ½ of species implicated in human disease
• Legionella pneumophila
• Causes 90 of all cases of legionellosis
• Majority of all confirmed cases are caused by
  serogroups 1-6
• Legionella micdade
• Most common after L. pneumophila

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Legionella micdadei

• Caution
• This strain can stain weakly acid fast on primary
  isolation, but loses this property when grown in
  vitro.
• NO RELATIONSHIP TO MYCOBACTERIA

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Microbiology

• Will not grow on standard Sheep Blood Agar
• Buffered Charcoal Yeast Extract Agar (BCYE)
• 1. Cysteine is essential for growth
• Iron is essential for growth
• Growth conditions
• 350 C
• 3-7 days

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Colony Appearance

• Ground glass
• Small 1-3 mm

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Laboratory Diagnosis of Legionella

• Culture of Legionella organism from normally
  sterile tissue
• Detection of L. pneumophila antigen in urine
• Seroconversion 4 fold or greater rise in
  specific serum antibody titer L. pneumophila
• Direct fluorescent antibody (DFA) staining

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Legionnaires disease- Public Health

• Disease - Worldwide
• Sporadic
• Epidemic community-acquired pneumonia
• Nosocomial infections
• Exposure - Water-based aerosols
• Air conditioning cooling towers
• Whirlpool spas
• Sauna or mister
• Survival Environment
• Amoebae
• Biofilms

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2 Clinical Presentations

• Legionnaire's disease
• Incubation period 2-10 days
• pneumonia
• 15-75 mortality
• erythromycin
• Pontiac fever
• Incubation period 1-2 days
• flu-like
• milder (no mortality)
• self-limiting

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Pathogenesis of Legionella

• Phagocytosis into the monocytes
• binding to complement receptors
• Inhibition of phagolysosome fusion
• Replication within the phagosome
• Lysis of the phagosome leads to apoptosis and
  release of the organism
• TH1 cells and IFN-?

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Bordetella
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Bordetella pertussis

• Strict aerobe
• Gram negative
• Small Coccobacillus -singly or in pairs
• Transmission by aerosolized droplets
• Non-invasive
• Strictly human pathogen

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DIFFERENTIATION OF BORDETELLA SPECIES DIFFERENTIATION OF BORDETELLA SPECIES DIFFERENTIATION OF BORDETELLA SPECIES DIFFERENTIATION OF BORDETELLA SPECIES DIFFERENTIATION OF BORDETELLA SPECIES DIFFERENTIATION OF BORDETELLA SPECIES
Growth on common lab media (SBA, MacConkey) Growth on Bordet-Gengou agar Urease Oxidase Motility
B. pertussis - - -
B. parapertussis - -
B. bronchiseptica
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• B. pertussis
• Small, transparent hemolytic colonies on
• Bordet-Gengou medium

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Diagnosis

• Based on symptoms
• Culture of respiratory secretions on
  Bordet-Gengou medium
• Direct fluorescent antibody testing
• PCR
• Slide agglutination

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Public Health Aspects of B. pertussis (Whooping
Cough)
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August 2, 2010
WHOOPING COUGH EPIDEMIC GROWS HEALTH OFFICIALS
URGE VACCINATION AND TIMELY DIAGNOSIS
As of July 27, the number of illnesses from the
disease this year had climbed to 2,174, a
six-fold increase from the 349 illnesses reported
for the same period last year. In addition, a
San Diego County infant has become the seventh to
die from pertussis this year. The pertussis
epidemic is a sobering and tragic reminder that
diseases long thought controlled can return with
a vengeance, Horton said. We can protect
ourselves and the most vulnerable in our
community by getting vaccinated today.
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Morbidity and Mortality Weekly Report
(MMWR) July 9, 2010 / 59(26)817

• During January--June in California
• 89 of cases were among infants aged lt6 months
• too young to be fully immunized
• Children aged 7 to 9 years and 10 to 8 years
• 10.1 cases and 9.3 cases per 100,000,
  respectively.
• Of 634 case reports
• 105 (16.6) patients were hospitalized,
• 66 (62.9) were lt3 months.
• Incidence among Hispanic infants (49.8 cases per
  100,000) was higher than among other
  racial/ethnic populations.
• 5 deaths were reported, all in previously healthy
  Hispanic infants aged lt2 months at disease onset
  none had received any pertussis-containing
  vaccines.
• The incidence of pertussis is cyclical, with
  peaks occurring every 3--5 years in the United
  States (2). The last peak was in 2005, when
  approximately 25,000 cases were reported
  nationally.

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Pertussis Among Adolescents and Adults

• Disease often milder than in infants and children
• Infection may be asymptomatic, or may present as
  classic pertussis
• Persons with mild disease may transmit the
  infection
• Older persons often source of infection for
  children

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Pertussis- Disease

• Primarily a toxin-mediated disease
• Exotoxins are controlled by central locus
• BvgAS two-component signal transduction system
  to sense the environment and regulate gene
  expression

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Pertussis- Disease

• Inflammation interferes with clearance of
  pulmonary secretions
• Cough progresses from mild (catarrhal stage) to
  sever (paroxysmal stage)
• Resolves slowly
• Evasion of host defenses
• Pertussis antigens allow evasion of host defenses
  
• Lymphocytosis promoted but impaired chemotaxis

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Pertussis Pathogenesis

• Two-stage process of disease
• Respiratory colonization
• 7-10 days
• NO symptoms
• Positive cultures toward the end of this stage
• Toxin-mediated disease

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Colonization

• Attachment requires multiple factors
• Pertussis Toxin
• Filamentous hemagglutinin
• Fimbriae

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Adhesins

• Filamentous hemagglutinin
• Dominant adhesin
• Required for tracheal colonization
• Highly immunogenic
• Primary component of acellular pertussis
  vaccines
• Fimbriae
• Required for persistent tracheal colonization
• Component of some acellular pertussis vaccines
• Required for protective immunity to infection

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Bordetella pertussis Toxins

• SIX
• DIFFERENT TOXINS

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Systemic effects of Pertussis Toxin

1. T cell Lymphocytosis with ? mitogenicity
2. ? insulin secretion
3. Histamine sensitization
4. ? IgE production
5. Impaired phagocyte function
6. ADP-ribosylates G proteins
7. Strong adjuvant
8. Primary component of pertussis vaccines

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2. Adenylate cyclase Toxin

• Calmodulin-activated with adenylate cyclase and
  hemolysin activity
• Acts as anti-inflammatory and antiphagocytic
  factor
• Secreted invasive toxin

Adenylate cyclase toxin
B. pertussis
calmodulin
?cAMP
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Other Toxins

• Dermonecrotic toxin (DNT)
• Heat-labile secreted toxin
• Transglutaminase activity that acts on small
  GTPases of the Rho family
• Induces localized necrosis

• Phenotypic Modifications seen
• Reorganization of
• actin cytoskeletal
• focal adhesions
• stress fibers
• Alterations in cell morphology

Fukui, A. et al. J Biochem 2004 136415-419
doi10.1093/jb/mvh155
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Other Toxins

• Tracheal cytotoxin
• A. Disaccharide-tetrapeptide monomeric
  by-product of peptidoglycan synthesis
• B. Causes damage to cilia, and loss of
  ciliated cells
• C. Increased IL-1 and nitric oxide production
• Type III secretion system
• Allows Bordetella to translocate effector
  proteins directly into host cells
• Required for persistent tracheal colonization
• Inhibits host immune response
• Induces necrotic cell death
• Lipopolysaccharide (LPS)
• Pyrogenic
• Mitogenic
• Activate and induce tumor necrosis factor
  production in macrophages
• LPS lacks a repetitive O-antigenic structure

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Treatment

• Erythromycin
• Vaccine
• killed bacterial cell suspension -DTP vaccine
• Vaccine- induced immunity wanes after five to ten
  years
• acellular vaccines (DTaP)
• Multicomponent acellular vaccines

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Haemophilus
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Overview- Haemophilus

• Small
• Non-motile
• Gram-negative rods
• Transmitted via respiratory droplets, or direct
  contact with contaminated secretions
• Normal flora of the human respiratory tract and
  oral cavity.

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Haemophilus species of clinical importance

• 1. H. influenzae
• -type b is an important human pathogen
• 2. H. ducreyi
• -sexually transmitted pathogen (chancroid)
• 3. Other Haemophilus are normal flora
• - H. parainfluenzae pneumonia endocarditis
• - H. aphrophilus pneumonia endocarditis
• - H. aegyptius pink eye (purulent
  conjunctivitis)

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Differentiation of Species
Growth Factor
Hemolysis
X Y
H. influenzae -
H. aegyptius -
H. ducreyi - -
H. parainfluenzae -
H. aphrophilus - - -
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Public Health Aspects-H. influenzae

• Typing based on capsule polysaccharide a ? f
• Polyribose-ribitol phosphate (PRP) capsule (type
  b)
• Nonencapsulated (nontypeable) organisms are part
  of normal flora of the respiratory tract
• 95 of invasive disease caused by type b

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Public Health Aspects

• H. influenzae type b incidence has fallen 99
  post-vaccine
• Pre-immunization
• Serotype b was the most common invasive species

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• Post-immunization
• Most cases in unvaccinated or incompletely
  vaccinated children.
• Non-encapsulated and serotype f are the most
  common
• Children - Pneumonia and meningitis less common
• Most infections (2/3) are currently attributed
  to nontypeable strains.

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Disease caused by H. influenzaeSerotype b
Clinical Microbiology Reviews, April 2000, p.
302-317, Vol. 13, No. 2
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Invasive Diseases post-immunization

• Septic arthritis
• Osteomyelitis
• Cellulitis
• Pericarditis
• Pneumonia - most frequent is serotype f
• Otitis media
• Streptococcus pneumoniae and then non-typeable Hi
  

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Pathogenic Mechanisms

• H. influenzae
• Antiphagocytic polysaccharide capsule is the
  major pathogenesis factor
• Lipopolysaccharide lipid A component from the
  cell wall (major role in non capsule strains)
• All virulent strains produce neuraminidase and an
  IgA protease
• No exotoxins

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Pathogenesis Host Factors

• Hib conjugate vaccine (PRP capsule)
• The Hib conjugate vaccine does not protect
  against nontypeable strains.
• Persons at risk for invasive H influenzae disease
  
• Asplenia
• Immunocompromised

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Public Health Aspect of other Haemophilus strains

• H. ducreyi
• Sexually transmitted disease - chancroid
• H. influenzae biogroup aegyptius
• Brazilian Purpuric Fever
• H. aegyptius
• pink eye (purulent conjunctivitis)
• H. aphrophilus
• pneumonia
• Infective endocarditis

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Haemophilus influenzae biogroup aegyptius

• Brazilian purpuric fever in children
• High fever
• Death within 48 hours

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Case Study

• History
• 13 year old white male
• fully vaccinated
• cold-like symptoms and persistent cough- 10 days
  duration
• 2 weeks later
• progressive coughing spells with inspiratory
  whoop
• posttussive vomiting

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Case Study

• Tests
• Nasopharyngeal swabs
• Bordet-Gengou medium
• Blood samples for serology
• positive IgM and IgA antibodies
• Treatment
• azithromycin

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Case Study

• History
• 4 month old white female
• 1 day history
• 1030 fever, lethargy, irritability, stiff neck
• Tests
• Cerebral spinal fluid culture
• IsoVitaleX-enriched chocolate agar