14_Pertussis, Pseudomonas

Question 1

Corynebacterium diphtheria

1. carrier
2. transmission
3. symptoms

Answer 1

1. Carried in NASOPHARYNX of HUMANS
2. Transmission by droplet spread thru coughing and sneezing from one human to another; organisms do not disseminate from the throat
3. Sxs of Diphtheria:
   
   • grayish pseudomembrane can form in the throat/tonsills due to accumulated dead cells & inflammatory cells;
   • pharyngitis or tonsillitis

Question 2

Corynebacterium diphtheriae:

1. structure
2. arrangement
3. staining
4. cultures

Answer 2

1. Gram positive, rod-like
2. Frequently arrange themselves in palisades, and have club-shaped (koryne=club in Greek) swellings at their poles.
3. Stain irregularly, exhibit deeply staining bands and beads.
4. Grows on a special medium with potassium tellurite = Tinsdale agar

Question 3

C. diphtheriae:

mechanism of disease

Answer 3

1. Bacteria stay localized in the throat, but systemic symptoms appear.
2. Pathological changes incl. heart, liver, kidneys, lungs, and adrenals.
3. Bacteria cannot be cultured from these organs.
4. Organ dysfunction is due to elaboration of diphtheria toxin.

Question 4

Relationship and extraction of exotoxins in culture filtrates?

Answer 4

• Exotoxins are secreted from bacterial cells
• Exotoxins can be purified from the supernatants of liquid bacterial cultures.
• E.g. C. diphtheria produces Diphtheria toxin, which can be filtered from the supernatant fluid of the liquid culture

Question 5

Which strains of C. diphtheria produce toxins?

Answer 5

• Only strains of C. diphtheriae that are lysogenized with a beta-phage, make toxin.
• Low iron conc. induces phage to its lytic stage –> toxin is produced
• Toxins is a single polypeptide molecule of 72,000 MW

Question 6

What is the structure of the Diphtheria toxin?

Answer 6

• A single polypeptide chain that can be proteolytically nicked to produce A (Active) and B (Binding) fragments.
  
  • B fragment attaches to cell membrane –> complete molecule enters the cell
  • W/in the cell, A subunit dissociates and catalyzes a reaction that ADP-ribosylates/ inactivates elongation-factor 2 (EF-2)
  • ADP ribosylation of EF-2 by diphtheria toxin INHIBITS PRTOEIN SYNTHESIS –> KILLING THE CELL
• The A fragment has the toxic enzymatic activity, and enters the cytoplasm

Question 7

Diphtheria toxin:

1. receptor
2. inhibits what?
3. receptors are abundant where?

Answer 7

1. Toxin receptor is Heparin Binding Epidermal Growth Factor- Like Growth Factor Precursor.
2. Toxin inhibits protein synthesis in organs whose cells express the receptor and take up the toxin
3. Receptors are abundant in the heart.

Question 8

Diphtheria toxin: Immunity

1. vaccine? what is it?
2. vaccine function
3. treatment

Answer 8

1. Vaccine is a TOXOID.
   
   • A toxoid is a formalin treated toxin.
2. Vaccine induces antibodies that bind up the toxin before it reaches its receptor.
3. If disease develops, can use antitoxin for therapy.

Question 9

Formalin: define

Answer 9

Formalin (dilute formldehyde) denatures the toxin slightly so it cannot bind to its receptor, but it retains enough of its 3-D configuration so it can induce antibodies that will bind to the native toxin.

Question 10

pseudomonas aeruginosa:

1. structure
2. location
3. type of pathogen
4. metabolic needs
5. transmission

Answer 10

1. gram negative rod w/ polar flagella
2. ubiquitous in water and soil; colonizes in GI tract as normal flora (10% of people)
3. OPPORTUNISTIC Pathogen
4. prefers aerobic environment
5. NO HUMAN-HUMAN TRANSMISSION

Question 11

Pseudomonas aeruginosa: clinical symptoms

1. colonizes in which pts?
2. mortality?

Answer 11

1. chronically colonizes lungs in children with cystic fibrosis
   
   • causes respiratory infections and pneumonia
   • colonizes burns and wounds; UTIs, osteomyelitis, folliculitis, keratitis, swimmer’s ear
2. bacteremias & sepsis can result in high mortality

Question 12

pseudomonas & disease:

1. when/what capsule is produced?
2. what acts in burn pts to cause disease?

Answer 12

1. CF pts become permanently colonized in the lung with Ps. aeruginosa. These strains produce an alginate capsule that blocks phagocytosis. (Alginate is composed of mannuronic and glucuronic acids).
2. In burn patients– elastase can help the organism penetrate deeper into the tissues and become systemic.

Question 13

pseudomonas virulence factors:

3 major virulence factors

Answer 13

1. pseudomonas exotoxin A: ADP-ribosylates elongation factor 2
2. alginate capsule blocks phagocytosis in some strains
3. multiple antibiotic resistance
4. elastase activity damages lung tissues and blood vessels
5. exoenzyme S: ADP ribosylates vimentin and Ras G proteins
6. lipopolysaccharide in outer membrane

Question 14

describe quorum-sensing and biofilm formation as it relates to

pseudomonas virulence factors

Answer 14

1. Quorum-sensing
   
   • When Pseudomonas reach a threshold concentration they send out signals that turn on a panel of virulence genes in all members in the culture.
2. Biofilm formation
   
   • Pseudomonas have a strong tendency to form biofilms.
   • This process is believed to happen in the lungs of cystic fibrosis patients.

Question 15

Pseudomonas exotoxin A:

1. structure
2. function;
3. mechanism is similar to…?

Answer 15

1. a single polypeptide chain
2. ADP ribosylates Elongation Factor-2
3. Same mechanism of action as Diphtheria toxin (both toxins inhibit protein synthesis in organs whose cells express the respective receptor and take up the toxin)
   
   • but no structural homology b/w the two
   • Also the 2 toxins have different receptors

Question 16

Receptor for Pseudomonoas Exotoxin A versus Receptor for Diphtheria toxin

Answer 16

• Receptor for Pseudomonas Exo A: alpha-macroglobulin receptor/ low density lipoprotein receptor-related receptor
• Receptor for Diphtheria toxin: heparin binding epidermal growth factor- like growth factor precursor
• Both toxins inhibit protein synthesis in organs whose cells express the respective receptor and take up the toxin

Question 17

Pseudomonas diagnosis and treatment:

1. growth
2. odor
3. color
4. resistance

Answer 17

1. growth - can grow on a variety of media
2. odor - has characteristic “fruity” odor
3. color -
   
   • produces a blue-green pigment called pyocyanin
   • wounds/ burns can turn blue-green from the pigments
4. resistance to multiple antibiotics

Question 18

All are true about Pseudomonas aeruginosa EXCEPT:

1. Is fragile and difficult to grow.
2. Is resistant to most antibiotics.
3. Colonizes burns.
4. Infects cystic fibrosis patients.

Answer 18

It is not fragile or difficult to grow

Question 19

Hx of Bordetella pertussis (whooping cough)

1. # cases/yr prio to vaccine
2. 1976 - # cases
3. 2017 - # cases vs/ 2012

Answer 19

1. Prior to vaccine: 200,000-250,000 cases/yr in US
   
   • 1st vaccine was whole killed cells of the organism
2. 1976 - 1,000 cases in US
3. 2012 - 48,000 cases in US
   
   • 2017 - 18,975 cases in US;
   • either unvaccinated population, new vaccine less efficacious than old vaccine, or organism is mutating away from vaccine strain

Question 20

Bordtadella pertussis:

1. organism structure
2. host/ transmission
3. colonization
4. & clinical symptoms

Answer 20

1. structure : small, gram-negative coccobacillus
2. host/ transmission:
   
   • human host
   • spread by aerosol (droplet nuclei) from cough/sneeze
3. colonization: in ciliated epithelium of trachea and bronchi
4. & clinical symptoms
   
   • initially, mild coughing and sneezing (catarrhal)
   • after 10-14 days: organism is in lower resp tract/ inspirational whooping: Paroxysms of coughing, vomiting
   • 3-4 wk: convalescence (recovering)

Question 21

paroxysm:

define

Answer 21

a sudden attack or violent expression of a particular emotion or activity

(e.g. paroxysms of coughing)

Question 22

B. pertussis:

relevant clinical aspects and epidemiology

incidence

Answer 22

• previously considered exclusively a pediatric disease
• many adult cases currently
• highly contagious
• in US, 2017 - 13 deaths, with 9 pts being less than 1 year of age
• INCIDENCE is most common in <1 year

Question 23

Bordetella pertussis:

virulence factors (6)

Answer 23

A - F - F - P - P - T

1. Adenyl cyclase - increases cAMP
2. Fimbriae - mediates adherence to ciliated epithelial cells
3. Filamentous hemagglutinin (FHA) - adhesion to ciliated epithelial cells and clumping of red blood cells
4. Pertactin - a cell surface protein that is an adhesin
5. Pertussis toxin - raises cAMP by ADP ribosylation of a Gi protein
6. Tracheal cytotoxin - fragments of peptidoglycan that are toxic for ciliated epithelial cells

Question 24

Bordetella toxins:

1. summary,
2. and the 2 kinds

Answer 24

1. Summary: Toxins increase cAMP –> dysregulating ion balance in the cells
2. Two toxins
   
   • Pertussis toxin:
     
     • An “A” subunit and 5 “B” subunits
     • ADP ribosylates Gi protein (that normally deactivates adenylate cyclase) –> the ADP ribosylated Gi protein can’t work well –> adenylate cyclase continues to produce cAMP –> altering ions and water in cell
   • Adenylate cyclase: the bacteria makes it own adenylate cyclase, makes more cAMP

Question 25

Describe the structure/function of Pertussis vaccine?

Answer 25

• antibodies to the toxin and adherence factors are protective
• former vaccine was whole killed cells (wP). had significant toxicity
• new vaccine is “aceulluar pertussis” (aP); recommended since 1997
  
  • Given i.m. in combination with diphtheria toxoid and tetanus toxoid (DTaP) to infants at 2, 4, 6, and 18 months.
  • Boost with Tdap at: 11 to 12 years, every pregnancy, and adults every 10 years.

Question 26

Pertussis Epidemiology:

outbreaks and tentative conclusions

Answer 26

1. Tentative conclusion is that the new aP vaccine is not providing long lasting immunity.
   
   • (after 2012 outbreak in Washington state: 2,520 cases
   • & 2014 outbreak in California: 9,935 cases)
2. Conclude that vaccine encoded genes evolving faster –> new isolates are appearing that lack pertactin
   • Esp. after outbreak in UK in 2012, w/ nearly 10,000 cases and 14 infnt deaths

Question 27

Pertussis:

1. diagnosis and
2. treatment

Answer 27

• Diagnosis:
  
  1. obtain sample w/ nasopharyngeal swab or aspirate
  2. culture on special agar:
     
     • bordet-gengou medium (10-15% blood + starch base), or
     • regan-lowe medium (10% blood + charcoal base)
  3. direct immunofluorescent Ab can be used on nasopharyngeal smear
  4. USE PCR: used routinely in labs and in outbreaks
• Treatment: recommended Abx include: macrolides (e.g. erythromycin, clarithromycin, and azithromycin)

Question 28

In regard to Bordetella pertussis, which is the best answer?

• 1. Frequently causes sepsis.
• 2. Adults are resistant to infection.
• 3. Grows profusely on blood agar.
• 4. Has an ADP-ribosylating toxin that raises cAMP.

Answer 28

Has an ADP-ribosylating toxin that raises cAMP.

Question 29

Note similarities and differences of: C. diphtheriae, P. aeruginosa, B. pertussis, and V. cholerae w/ regard to:

1. stain
2. toxin structure
3. toxin features
4. toxin target
5. effect

Answer 29

Question 30

List the less common gram negative Pyogens

(pyogen: pus-producing microorganism)

Answer 30

• 1. Haemophilus ducreyi - chancroid, gential ulcers
• 2. Burkholderia cenocepacia – cystic fibrosis patients
• 3. Burkholderia pseudomallei – melioidosis. Acute or chronic pneumonia. In the tropics.
• 4. Acinetobacter baumannii – Pneumonia in ICU, sepsis
• 5. Moraxella catarrhalis (Branhamella catarrhalis) – Otitis media and lower respiratory tract infection
• 6. Aeromonas – wound infection, infection from leeches