WHOOPING COUGH: PERTUSSIS (Bordetella pertussis)
Reading Assignment: (1) Text Chapter 19 (2) Gilligan, P.H., M.L. Smiley, and D.S. Shapiro. 1997. Cases in Medical Microbiology and Infectious Diseases (2nd ed.), pp. 45-47. American Society for Microbiology, Washington, D.C. – on the WEBSITE, (3) Optional – Other articles posted on the WEBSITE
1. INTRODUCTION
Case study # 20 – Cases in Medical Microbiology and Immunology
2. MICROBIOLOGY (The Organism)
a. Bordetella species
Bordetella are extremely small (0.2-0.5 X 1um), aerobic, Gram-negative
coccobacilli. Three species have been associated with human disease:
1. B. pertussis (Latin, severe cough) – pertussis or whooping cough
2. B. parapertussis (Latin, “like pertussis”) – a mild form of pertussis in
humans
3. B. bronchiseptica – respiratory disease in dogs, swine, laboratory
animals, and occasionally humans
b. Bordetella pertussis
1. Morphology - very tiny Gram negative coccobacilli
2. Growth of B. pertussis on Laboratory Media
a. Will not grow on ordinary laboratory media. Requires
media supplemented with charcoal, blood (20%), potato (starch) to absorb toxic substances in agar.
b. Oxidizes amino acids (does not ferment sugars)
c. Grows slowly. After aerobic incubation for 3-5 days - forms tiny glistening, compact colonies (like bisected pearls, or mercury drops).
b. Media
Bordet-Gengou medium– potato agar + 20% blood
Charcoal blood agar (used today - shown in case study photo)
3. EPIDEMIOLOGY
a. Reservoir – Exclusively a human pathogen; no animal or environmental reservoir. Adult carriers with chronic bronchitis, or adolescents with mild URI's are believed to be the reservoir for the disease. Rarely recovered from the upper respiratory tract of healthy people.
b. Transmission
1. Transmission from person to person via aerosols produced by coughing and sneezing.
2. Highly contagious – infecting 90% of the members of a nonimmune family exposed to the organism. 3. Very fragile organism – susceptible to environmental changes
survives only briefly outside the human respiratory tract.
c. A Reemerging Disease Around the World – it is increasing in incidence
around the world because of new susceptible populations
1. Nonvaccinated or Inadequately Vaccinated Children
(Recent epidemic in the Netherlands suggests that epidemics
can occur in vaccinated populations as well.)
2. Adolescents and Adults with waning immunity, who are exposed
to symptomatic patients
Prior to 1950, pertussis was most common in children from ages 2-10, and virtually everyone had this illness as a child. Adults had immunity from their childhood which was maintained by repeated exposure to children with pertussis.
Currently, the highest incidence is in infants less than 6 months of age who have not yet received the full course of primary immunization (you are not protected without all three DPT shots), in unvaccinated children, and in those over 10 years old whose vaccine immunity is fading.
4. CLINICAL FEATURES
CLASSICAL WHOOPING COUGH
a. Infection initiated when aerosols are inhaled and the bacteria become
attached to and proliferate on the ciliated epithelial cells.
b. After an incubation period of 7-10 days, the patient experiences the
first of three stages of disease..
1. Catarrhal – resembles the common cold; primary feature profuse and mucoid rhinorrhea, malaise, anorexia, and low-grade fever. The peak number of bacteria are produced at this stage and because the disease is not recognized, patients in this stage are highly infectious to their contacts.
2. Paroxysmal coughing – after 1-2 weeks, the appearance of a persistent During this time, ciliated epithelial cells are extruded from the respiratory tract, and the clearance of mucus is impaired. Mucus production increases and is partially responsible for blocking the airways.
Episodes of paroxysmal coughing occur up to 50 times a day for 2 to 4 weeks. The classic whooping cough paroxysms are a series of repetitive coughs followed by an inspiratory whoop as air is drawn through the narrowed glottis. Paroxysmx - frequently followed by vomiting and exhaustion. Apnea (cessation of breathing) may follow such episodes, particulary in infants. A marked lymphocytosis is also predominant during this stage.
3. Convalescent Stage –3-4 week - the frequency and severity of paroxysmal coughing and other features of the disease gradually fade, but secondary complications can occur.
Complications include: secondary bacterial pneumonia with
superinfecting bacteria such as S. pneumoniae; complications from the paroxysmal coughing - convulsions; hemorrhage, hernias, seizures and brain damage (due mainly to pressure effects and anoxia), and death (most often in infants).
OTHER PRESENTATIONS:
The clinical features vary with age, general health and immune status. Adults and older children may show mild or atypical symptoms. There is commonly a prolonged cough which is often misdiagnosed as something else (however, adults may also experience the full blown symptoms of whooping cough). Those without the whoop are often misdiagnosed. Reports suggest that 1/3 to _ of adolescent or adult patients with cough of greater than 1 week may have pertussis
5. PATHOGENESIS
Pathogenesis is complex and multifactorial. This is a local infection of the ciliated epithelial cells without invasion. There are two broad categories of virulence factors – adhesins and toxins. The virulence factors are coordinately and sequentially regulated.
STEPS IN PATHOGENESIS
a. Entry into the trachea and bronchi by inhalation of an aerosol from
another infected individual.
b. Attachment to the ciliated epithelial cells of the respiratory tract (a good
example of tissue tropism). Infection is localized to the respiratory tract.
VIRULENCE FACTORS THAT MEDIATE ADHESION:
1. Filamentous Hemagglutin (FHA) – a cell surface protein that binds to sulfated glycolipids on ciliated epithelial cells; unusual in that this adhesin mediates binding to neutrophils and macrophages – initiating the phagocytosis of pertussis by these cells. (Pertussis is phagocytosed but does not elicit the respiratory burst, and can therefore survive intracellularly). Recently, FHA has been found to also mediate binding to the human complement regulatory protein C4BP.
2. Pertussis Toxin (PT) – again unusual –subunits of this A-B exotoxin help bind B. pertussis to the surface of ciliated epithelial cells and to phagocytic cells.
3. Pili/Fimbriae – mediate adhesin
4. Pertactin (PRN) – cell surface protein that mediates adhesion
Note the multiple mechanisms of adherence. These adhesins have been targeted in the development of acellular vaccines in order to stop this 1st step in pathogenesis.
c. Growth on the cell surface (without invasion) and release of toxins,
that cause local damage to tissues, systemic symptoms, and interfere with the phagocytosis by neutrophils and macrophages.
TOXINS
1. Pertussis Toxin (PT)
a. Classic A-B toxin with an active subunit (S1) and 5 subunits for binding that are assembled into a complete toxin. S2- S5 mediate binding of the toxin to the cell membrane, and the S1 subunit is released into the cell.
b. The active S1 subunit ADP ribosylates a membrane G protein (a guanosine triphosphate-hydrolyzing protein) that regulates adenylate cyclase activity. ADP-ribosylation of this protein results in increased levels of intracellular cAMP. Known effects in vitro include inhibition of neutrophil chemotaxis, inhibition of the respiratory burst of neutrophils., Systemic effects include promotion of lymphocytosis, and insulin secretion.
2. Adenylate Cyclase Toxin (ACT)- converts ATP to cAMP; inhibits
leukocyte chemotaxis, phagocytosis, and killing by phagocytes, induction of apoptosis of phagocytic cells.
NOTE: Both PT and ACT increase cAMP levels inside host cells, but
by different mechanisms.
3. Tracheal Cytotoxin – (a peptidoglycan fragment) – kills ciliated
epithelial cells respiratory cells leading to their extrusion from
membrane.
4. Lipopolysaccharide – pyrogenicity, activates alternate
complement pathway, stimulates cytokine release.
Toxins cause local damage to the mucosal surface, produce systemic symptoms, and fight against the neutrophils and macrophages that are being called into the area by inflammation.
.
REGULATION OF THE B. PERTUSSIS VIRULENCE FACTORS
These toxins, like diptheria toxin, are not produced constitutively, but are coordinately regulated by the bacterium, in response to environmental changes. The environmental signal for expression of diptheria toxin by C. diptheriae was iron, while expression of the virulence factors of B. pertussis are controlled by temperature, sulfate, and nicotinic acid.
Both diptheria toxin and the virulence factors for B. pertussis are examples of regulation at the level of transcription, but they have different mechanisms.
C. diptheriae and diptheria toxin:
Production of diptheria toxin is regulated by a transcriptional repressor protein DtxR. When iron concentrations are high, the repressor protein is in the iron-bound form, and it binds DNA at the promoter site, preventing RNA polymerase from transcribing the gene. When iron levels are low, the iron-free form of the protein does not bind DNA, thus allowing the toxin gene to be transcribed.
Virulence factors of B. pertussis????
B. pertussis coordinately regulates several of its virulence factors,
at the level of transcription, using a transcriptional activator rather than a transcriptional repressor. A transcriptional activator is a protein that facilitates binding of RNA polymerase to the promoter and/or initiates transcription.
This activation in the case of B. pertussis and many pathogens occurs in a two step process called a TWO COMPONENT REGULATORY SYSTEM. That is, there are two activators not one which are used to turn on the gene. (See Text p. 208-209)
In these systems, there is a sensor protein with a kinase activity (called BvgS in Bordetella pertussis for Bordetella virulence genes), and a response regulator protein (BvgA in B. pertussis) that activates transcription. The sensor protein (located in the cytoplasmic membrane) responds to an external signal and autophosphorylates. It then activates the cytoplasmic response regulator protein by phosphorylation. The phosphorylated (and active form of the response regulator protein) activates transcription of the B. pertussis virulence genes.
Sequential transcription of virulence genes:
Not all of the virulence genes are transcribed at once. First the attachment proteins - filamentous hemagglutinin and pertactin are produced, then several hours later the toxins are produced (which cause local cell damage and help protect the bacterium against the host's leukocytes). Thus this pathogen has a finely tuned response to change in environmental conditions that allow it to adapt in a stepwise fashion to the conditions within the human respiratory tract.
B. PERTUSSIS AS AN INTRACELLULAR PATHOGEN??
B. pertussis can enter and survive in vitro within eukaryotic cells such as HeLa cells, macrophages, and neutrophils. In vivo, these organisms have been detected within alveolar macrophages of HIV-infected patients, who may experience chronic infection with B. pertussis.
ANTIGENIC MODULATION IN B. PERTUSSIS????
Multiple serotypes of pertussis fimbriae exist, and these must be included in the vaccines. Antigenic variation (a variation in serotypes of the fimbriae produced) has been demonstrated in response to vaccination. This phenomenon has been observed in the Netherlands and in other European countries where a high percentage of the population is immunized. Whether these shifts are affecting vaccine efficacy and are one of the reasons contributing to the upsurge in pertussis is not known at this time.
6. DIAGNOSIS
a. A clinical diagnosis is made if the “whoop” is heard, but must be confirmed in
the microbiology laboratory.
b. Laboratory Diagnosis
(1) Specimen – nasopharyngeal swab–requires SPECIAL HANDLING!
(2) Direct identification of B. pertussis organisms present on
the swab by:
a. PCR (compares favorably with culture)
b. Direct Fluorescent Antibody
(3) Isolation on:
a. Charcoal Blood agar (has replaced the classic
Bordet-Gengou agar). Contains low
concentrations of cephalosporins to inhibit
members of the normal flora and allow the slow-growing B. pertusssis to be isolated. Colonies are identified serologically, because B. pertussis shows few metabolic activities.
NOTE: It's still important to isolate these organisms in order to track serotypes in the population and to monitor antibiotic susceptibility (at least one erythromycin R organsim has been reported.)
(4) Increasingly, serology is being used to identify patients with pertussis
- important during epidemics – used retrospectively - not to diagnose individual patients with acute symptoms.
7. TREATMENT AND RECOVERY
Once the paroxysmal stage has been reached, treatment is primarily supportive. Infants are at the greatest risk for complications, and children less than one year of age are frequently hospitalized. They are given oxygent to prevent anoxic damage to the brain. Erthromycin is is given to stop the spread to susceptible individuals and may help with the earlier stages of infection. Treatment after onset of the paroxysmal stage does not alter the outcome.
8. IMMUNITY (Natural or Vaccine-Induced)
a. Recovery from whooping cough or vaccination confers immunity.
Second infections may occur, but they are usually mild. If immunity totally wanes, then reinfections years later in adults may be severe.
b. Antibody is the first defense against B. pertussis (How does it work???)
c. There is recent evidence that cell mediated immunity may also play a
role in immunity (TH1 cells when transferred to immunodeficient mice
can protect them against a challenge with B. pertussis)
9. PREVENTION
a. For 50 years a whole cell vaccine of killed B. pertussis was included as
part of the DPT immunization series.
b. There have been major concerns about the side effects of this vaccine:
(1) 20% of infants – fever, malaise and pain at injection site
(2) 0.5% of infants – persistent screaming and/or convulsions
(3) 0.001% (1/ 100,000) –encephalopathy and permanent neurologic sequelae
Concern about these side effects have led to a marked fall in
vaccination and an increase in the incidence of whooping cough!
Adults are given DT not DPT because of concern about these side
effects.
c. Nonwhole cell pertussis vaccines “acellular vaccines”(containing only
the protective antigens) have been field tested, and licensed for use in the last two years. They consist of one, two, three, or four components of B. pertussis virulence factors that have been shown to be protective in animal studies. These vaccines are less reactive than whole cell products and provide protection against pertussis. Whereas the last dose of the cellular vaccines is given at 4-6 years of age, these vaccines may be useful to control pertussis in adolescents and adult populations. There is a field trial now being carried out to test their efficacy in subjects age 18-65 yrs. The outcome may influence adult immunization protocols in the future.