More fastidious Gram negative coccobacilli

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More fastidious Gram negative coccobacilli

• Bordetella and Legionella

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Bordetella introduction

• The genus Bordetella contains 6 species. All but
  B. avium are involved in human pathology.
• The most significant of these are B. pertussis,
  B. parapertussis, and B. bronchiseptica
• Bordetella present as small, Gram negative
  coccobacilli on primary isolation media, and tend
  to be pleomorphic upon subculture, often
  appearing as slightly longer rods
• They are obligately aerobic
• They are asaccharolytic and prove inactive
  (negative) on most biochemical testing similar to
  some of the GNNF rods. As a matter of fact, the
  genetics and siderophores of the genus is so
  similar to Alcaligenes that some microbiologists
  support combination of these two genera.

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Bordetella
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more introduction

• B. bronchiseptica, B. hinzii B. avium are
  motile where the other 3 are not.
• None of the species require hematin or NAD
  supplements. However, B. pertussis (the most
  important pathogen in the genus) requires the
  addition of charcoal, blood, etc, to neutralize
  the effects fatty acids, peroxides, and other
  substances that are toxic either by acting as
  oxidizing agents or by other means.
• B. parapertussis is also fastidious, requiring
  the same culture media as B. pertussis.
• The other species are less fastidious, and are
  routinely cultured on SBA, CBA and MacConkeys

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Clinical significance of B. pertussis

• B. pertussis causes the respiratory condition
  pertussis, also known as whooping cough.
  Worldwide cases are estimated at lt 4 x 107 with 4
  x 105 deaths annually
• The condition was described in the 16th century,
  and Bordet isolated the pathogen in 1906.
• Transmission is by respiratory aerosols. It is
  highly contagious with an infectivity exceeding
  90 of unprotected individuals!
• The condition usually runs in 3-5 year cycles,
  and is most prevalent in the northern hemisphere
  between the months of July and October
• The condition is most significant for children
  around 1 year of age, especially prior to the DPT
  vaccine.
• A recent recurrence of pertussis in older adults
  is believed to be due to waning memory from
  childhood vaccination. This extends to newborns
  of these adults no inheritance of passive
  immunity

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continued

• B. pertussis produces a toxin (at least 1) which
  causes paralysis of ciliated columnar cells in
  the trachea. This results in an extended series
  of coughs followed by a prolonged inspiratory
  whoop.
• This is exacerbated by swollen and narrowed
  glottis and vomiting, both contributing to
  blockage of the airway.
• Asphyxia and suffocation of infants is the
  primary means of mortality
• Complications such as otitis media,
  encephalopathy, inguinal hernia and rectal
  prolapse can result from coughing attrition and
  secondary infection, but these are rare

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Virulence factors of B. pertussis

• Pertussis toxin (known by several names) is the
  most significant VF. Its functions include..
• 1) lymphocytosis (hyperactivity), histamine
  hypersensitivity and other means of immune
  stimulation
• 2) pancreatic islet cell stimulation which
  destabilizes homeostasis (link DPT to childhood
  type 1 diabetes?)
• 3) tracheal cilia paralysis - mechanism unknown
  but it is known that PT works with a separate
  tracheal cytotoxin (TCT) to damage tracheal
  epithelial cells
• Filamentous hemaglutinin antigen FHA
  hemaglutination and attachment to ciliated
  tracheal cells
• Adenylate cyclase hemolysin - vs neutrophil
  activity, plus
• IgA protease
• others pertactin, LPS, fimbriae, etc

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Pertussis vaccines

• Whole agent killed vaccine (wP) introduced in the
  1920s have decreased worldwide mortality from
  1.15x106 to lt 4x105. The wP vaccine is still the
  most used due to cost. Current global coverage
  is 80.
• Possible suspected side effects, especially with
  the wP vaccine, include neurological disfunction
  or damage (seizures, hyporesponsiveness,
  encephalopathy) and contribution to onset of
  childhood type-1 diabetes (???)
• In response to health concerns with the wP
  vaccine, the first acellular vaccine was
  developed in Japan in 1981.
• It (DTaP) was approved for use in the US in 1991.
  Prior regimens in the US involved use of DTwP).
  
• The aP vaccine is a toxoid (inactivated PT) as
  well as containing pertussis antigens such as
  FHA, fimbriae, etc. it is a conjugated
  vaccine
• Ist at 6-8 weeks with boosters at 6-12 months
  4-6 yrs

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Clinical significance of other species

• B. parapertussis causes near identical
  pertussis-like symptoms in humans although more
  mild. Rarely it can be severe in the very young
  even causing death.
• B. bronchiseptica causes RT symptoms in other
  animals, most notably kennel cough in dogs. It
  is commensal in the human RT, but can be
  opportunistic causing various systemic ailments
  in immunocompromised individuals
• Both B. hinzii B. homesii have been isolated
  from immunocompromised humans, though neither
  have proven to function as a significant
  pathogen.

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Clinical specimens culture

• Specimen of choice is nasopharyngeal swab or
  aspirate to access the URT ciliated epithelium.
  Samples should be plates ASAP as B. pertussis
  dies quickly out of the body.
• Bordetella, especially B. pertussis are sensitive
  to toxic substances in growth media. B.
  pertussis will not grow on SBA or other media
  containing peptones. They are also very
  sensitive to oxidation, so reducing agents (RA)
  are necessary additives to media.
• Bordet-Gengou is commonly used for primary
  isolation. It is peptone-less and contains
  sheep blood, potato (starch as RA), glycerol and
  methicillin or cephalexin
• Regan Lowe contains horse blood, charcoal and
  antibiotics
• Grows in 3 to 5 days in high humidity plus CO2
  Colony morphology 1-mm, smooth, round, domed,
  opaque, shiny, beta hemolytic on BG medium
  resembling drops of mercury (the way the colonies
  refract light)

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B. Pertussison Bordet-Gengou
on Regan-Lowe
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Identification

• Presumptive ID Gram-negative coccobacilli with
  characteristic colony morphology, no growth on
  SBA, slow growth on Bordet-Gengou and Regan Lowe
  agar
• Definitive ID serological methods such as
  fluorescent antibody or agglutination - usually
  performed at reference labs

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Legionella introduction

• After the Philadelphia American Legion convention
  in early spring of 1976, 182 legionaires
  developed multiple system pathology (1
  pneumonia) uncommon etiology.
• CDC scientists soon described the organism as
  small, non-enteric, fastidious, thin/pleomorphic
  Gram negative rod. In 1979 it was named
  Legionella pneumophila. Since then 41 species
  have been identified.
• Legionella are obligate aerobes do not Gram
  stain well
• They are nutritionally fastidious requiring
  cysteine and iron supplements. Like B.
  pertussis, they are also sensitive to oxidizing
  agents and need charcoal as a RA. They only (?)
  grow well on BCYE with cysteine and iron, which
  is a good presumptive ID tool for respiratory
  pathogens (along with morphology symptoms).
• Legionella are motile by polar flagella

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Legionella
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Pathology L. pneumophila

• L. pneumophila primarily causes legionaires
  disease, but also a less severe condition called
  Pontiac fever
• Legionaires disease (legionellosis) causes
  typical pneumonia (consolidation, abcess) with
  pleural effusion, watery diarhea nausea,
  delirium, liver function anomalies, and renal
  dysfunction with occasional renal failure.
• It is most prevalent in the elderly and
  immunosuppressed. COPD is a common predisposing
  condition, and smoking is considered significant
  in predisposition. The condition comprises 1-5
  of all pneumonias. Mortality 10-30
• Pontiac fever is a non-pneumonic, epidemic,
  influenza-like illness. It is milder,
  self-limiting, and has short duration.
• The organism can cause pathology of virtually any
  organ system without pneumonic symptoms.
• The other species are fairly insignificant

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continued

• When alveolar macrophages phagocytize Legionella
  cells in the lungs the cells often survive - the
  organism is an intra-macrophage parasite so it
  multiplies within the macrophages.
• Much of the pulmonary pathology results from the
  hypersensitive macrophage behavior that follows
  much like happens in tuberculosis
• By an unknown mechanism, phagocytized Legionella
  cells block fusion of lysosomes with the
  macrophage

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Reservoirs of the organism

• Natural
• Natural fresh waters such as creeks and rivers,
  and soil along the bank of these waters
• Infected individuals
• Man-made
• Water cooling towers
• Condensate pans of AC units, refrigerators, etc
• Tap water, water heaters, shower heads
• Grocery store produce misters CDC reported 33
  cases from store in Bogalusa, LA in 1990
• Humidifiers

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Clinical specimens culture

• Collect 2 LRT specimens (remember sputum vs
  saliva), one for culture and another for
  serology.
• Transtracheal aspirates or bronchial washings are
  superior to sputum for culture and diagnosis
• BCYE supplemented with cysteine and iron is the
  best primary isolation medium
• Incubation humid, 3-5 days, 35oC
• Colony morphology 3 to 4 mm, circular, gray,
  convex, ground glass appearance

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L. pneumophila
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Identification

• Presumptive ID of Legionella pneumophila patient
  with symptoms indicative of legionaires disease,
  thin Gram negative rods with characteristic
  colony morphology that grow almost exclusively on
  supplemented BCYE (no growth on SBA)
• Definitive ID serological ID probably by direct
  fluorescent antibody (DFA). Can confirm with DNA
  probe.
• Biochemical tests very difficult to perform on
  this organism