Bordetella

HISTORY:

In 1900 Bordet described a Gram-negative bacilli such as whooping cough agent.

In 1906 Bordet and Gengou have managed to grow the bacillus on an agar medium containing peptone potato and mixed with blood.

Pertussis has long been a major cause of infant mortality. Vaccination has helped to disappear France this killer.Nevertheless epidemics could recur if vaccination was not sufficiently applied.

I – DEFINITION AND CLASSIFICATION:

Bordetella are coccobacilli (0.5 to 1 | Jm long and 0.2 to 0.4 um wide) Gram negative, arranged singly or in pairs but rarely short chains;

– Aerobic strict in respiratory metabolism;

– Not fermenting any carbohydrates which do not produce gas;

– Demanding nicotinamide and sulfur derivatives (cysteine) but not factor X or V;

– Their G + C% is between 61 and 70;

– Having a tropism for the respiratory mucous membranes of mammals. Three main species, the homology DNA / DNA is very similar, are encountered in humans: B. pertussis, B. parapertussis and B. bronchiseptica. B. aviwn is an isolated case in turkeys and turkey poults.

II – PATHOGENICITY:

Whooping cough, infectious disease of the upper respiratory tract, is usually due to B. pertussis. After a silent incubation of about a week, the period of invasion is marked by a catarrh, nose and tracheobronchial which is the most contagious phase. It is followed by the period of fifths where the clinical diagnosis is obvious because of the paroxysmal cough (whoop). Reaching especially young children, pertussis in infants less than 3 months is a life-threatening severe illness, death occurs from asphyxiation. Secondary and neurological complications infections are to be feared.

B. parapertussis can also be responsible for pertussis syndromes.

B. bronchiseptica is a disease of the upper airways pets, head of atrophic rhinitis in pigs. Rarely isolated in humans, this bacterium causes pseudocoqueluches.

III – HABITAT – EPIDEMIOLOGY:

B. pertussis as a single tank man. It is believed that immune adults may be carriers of bacillus in their oropharyngeal mucosa and thus to transmit the non-immunized children. Contamination is by air.

Pertussis is evolving in a endemic epidemic mode. In countries where vaccine protection is good, epidemics are rare. By cons in countries where child immunization rate is low (below 50%), large outbreaks can occur. More than 100,000 cases of pertussis have been reported in Britain in 1977. In 1982 more than 1,000 cases a week were reported there.

In France, an estimated 1,000 per year the number of hospitalized cases of whooping cough. It is children under one year.

There has been a resurgence of pertussis in Australia from 1987.

The incidence of pertussis in European countries has declined largely due to vaccination when the disease is increasing in the US.

B. pertussis was also isolated fortuitously in patients suffering from AIDS with bronchial secretions were plated on BCYE medium for research of Legionella.

IV – PATHOPHYSIOLOGY:

Pertussis, poisoning B. pertussis is essentially a bronchopulmonary disease, the bacillus has a tropism for the ciliated cells of the respiratory epithelium. Bacteria adhere to the cilia of the cells, cause paralysis eyelashes and activation of mucous secretions. Injured cells and secretions are removed through coughing.

At the state phase of the disease, there is more than B. pertussis viable in secretions. Clinical signs are then due to virulence factors released by the bacteria.

B. pertussis has several antigens, capsular polysaccharide antigen, somatic antigen thermostable corresponding to the endotoxin of gram-negative bacteria.

Several virulence factors are produced:

– Pertussis toxin (PT), is a protein toxin released in part by the germ (various synonyms have been used to describe this toxin, reflecting its various activities: HSF, histamine-sensitizing factor, LPF, lymphocytosis-promoting factor and IPA , islet-activating protein). The oligomeric protein of 117 kDa is on different eukaryotic cells by increasing the intracellular concentration of cAMP (in particular in the epithelial cells of the respiratory tract). It acts on the site controller negative (G-protein) to adenylate cyclase membrane raising inhibition of this enzyme by a ADP-ribosylation reaction. It causes hyperlymphocytosis which is an important clinical sign in this disease.

– Adenylate cyclase exocellular B. pertussis is excreted in the form of a 200 kDa protein with a 43 kDa fragment corresponds to adenylate cyclase, the other parts having binding functions and internalization, it has a hemolytic activity, it is activated by intracellular calmodulin. Its primary role is to increase the pool of adenylate cyclase intracellular and reduce the phagocytic function of neutrophils and macrophages.

– Hemagglutins are involved in attachment B. pertussis to epithelial cells. Filamentous hemagglutinin or FHA, is carried by pili.

– Other toxins have been described: a dermonecrotic toxin (HLT = heat labile toxin), a tracheal cytotoxin (TCT = tracheal cytotoxin) acting on the ciliated cells.

V – LABORATORY DIAGNOSIS OF PERTUSSIS:

A – Isolation and identification of B. pertussis:

1. The levy:

It must be early, at the onset of clinical signs. Indeed, bacteria are scarce at the onset of coughing.

The collection of mucus is either using a soft swab calcium alginate or Dacron, introduced through the nostril to the posterior nasal cavity, or using a flexible tube attached to a syringe . Other collection methods are not recommended as they give much lower results.

2. Culture medium:

The culture media should be inoculated immediately at the bedside or default using a transport medium (Regan and Lowe Stainer and Scholte or).

Coming out of the body, B. pertussis does not grow on ordinary agar.

– The Bordet-Gengou

It is an empirical agar medium consisting of a potato infusion, glycerine and NaCl which is added 15% sterile fresh blood fibrin dice horse, sheep or rabbit.

The role of the blood is to neutralize some growth inhibitors of B. pertussis, such as fatty acids. In the middle of Regan is also added activated charcoal.

The medium can be made selective by addition of methicillin at the final concentration of 2.5 mcg / ml or more of cefalexin to the final concentration of 40 (Ag / ml.

There is also a liquid synthetic medium, the medium Stainer and Scholte.

– Incubation

She gets to 35-36 ° C for 7 days. It is important to use a system to prevent desiccation of the agar (tape, aluminum foil or jar) to perform a humid atmosphere conducive to growth.

3. Observation of the colonies:

B. pertussis grows slowly. The colonies rarely appear before the 3rd day of incubation.

– At the end of the body colonies are tiny, smooth, curved, have a look into “drops of mercury.” They gradually surround a hemolysis zone. This corresponds to Phase I. The colonies are smooth and coccoid bacterial bodies.

– After transplanting, the settlements are a rough appearance and grow on ordinary agar. This corresponds to phase IV, or rough forms. Bacterial bodies may be filamentous.

4. Identification of B. pertussis:

Identification is difficult because of the many negative characters for this bacillus. Research oxidase is positive. The various characters are indicated in the table. Identification can be supplemented or confirmed by a slide agglutination test using anti B. Serum pertussis.

Similarly, one can identify B. pertussis by direct immunofluorescence from the colonies. It has been described an aid to identification by determining cellular fatty acids by gas chromatography.

B – Other diagnostic methods:

-. Direct immunofluorescence Made on mucus is an exam that has the advantage of speed of response, but his request for interpretation of habit and it is less sensitive than culture. The interpretation is delicate and prone to errors, it is a function of the experimenter. There may be false positives (cross-react with Legionella) and false negatives. Can be found 70% positivity in the forms back to less than three weeks and only 10% after taking erythromycin.

-The Identification of serum antibodies is a test whose results late has little interest for diagnosis. ELISA was developed but can not replace culture and it should be noted that this technique is very effective in children younger than 4 months. A search for IgA anti B. pertussis was proposed from nasopharyngeal secretions.

– Research of adenylate cyclase produced by the germ (AÇ)

It is performed from nasopharyngeal swabs, immediately immersed for a few seconds in the middle Stainer and Scholte which are added ATP and calmodulin. The medium is incubated 18 hours at 37 ° C: cAMP formed was measured by a method of radiocompétition. This method recently introduced, is sensitive, reliable and fast. It is very useful for the diagnosis of pertussis in infants less than 3 months and immunocompromised children.

Diagnostic outlook improves with the detection in samples of PT and / or FHA using monoclonal antibodies and the contribution of synthetic oligonucleotide probes (PT or FHA gene) and gene amplification in vitro (PCR).

VI – OTHER BORDETELLA:

A – Bordetella parapertussis:

It is characterized by:

– Its fastest growth (36-48 hours) agar Bordet-Gengou with net hemolysis halo

– Urease (24 h)

– A subculture easier on nutrient agar or broth

– A browning Bordet-Gengou agar due to the presence of tyrosinase.

B – Bordetella bronchiseptica:

Is recognized by:

– Its easy and fast growth (24 h) on plain agar

– Growth on MacConkey agar

– Culture on medium Simmons citrate

– Mobility due to ciliature peritrichous

– Reduction of nitrate to nitrite (medium supplemented with NAD)

– The presence of a very active urease (1-4 h), which distinguishes it from Alcaligenes.

Turkey rhinotracheitis responsible strains were designated “B. bronchiseptica-like. “ They are characterized by the absence of nitrate reductase in a conventional medium (but this reaction is positive if one adds NAD) and urease.

They are now designated as B. avium.

VII – PERTUSSIS VACCINE:

A – Preparation and administration methods:

It is a suspension containing 5 billion B. pertussis per ml, inactivated by heating at 56 ° C.

It can be administered to children as young as 3 months (between 3rd and 6th months) in deep sub-cutaneous injections at 3 one month apart and a booster after one year. It can be combined with other vaccines.

B – Efficiency:

Vaccination has virtually eliminated in France outbreaks of whooping cough and mortality from this disease.

The vaccine confers immunity from 3 to 4 years, the quality may vary from one batch of vaccine in the other. It increases agglutinins, but there is no parallelism with the degree of protection. Immunization coverage is 80 to 95% after 3 injections.

C – Activity criteria:

This activity is considered conventionally by a test of survival of mice immunized after intracerebral injection of B.pertussis.

The activity of a lot of vaccine is compared to a reference vaccine by measuring survival in immunized mice. It is expressed in International Units and must be greater than or equal to 4 IU.

A mouse model for protection against infection by respiratory aerosol has been developed more recently. It allows to judge the effectiveness of protective antigens.

D – Disadvantages and cons-indications:

Pertussis vaccine may cause local or systemic reactions (low grade fever). But it is the neurological events (encephalopathies), whose risk is approximately 1 in 300,000 vaccinated, which are the cause of disaffection for the vaccine in some countries. The risk of serious accident vaccine is significantly lower risk of death from pertussis. The neurological history, even “simple hyperpyretic convulsions” are against-indication for immunization.

E – Outlook:

The current vaccine consists of inactivated whole bacterial bodies is effective but empirical. The use of more immunogenic bacterial fractions and better tolerated as pertussis toxin and filamentous hemagglutinin is still under study. Two acellular vaccines are developed, one in Japan and one in France, based on two FHA antigens and detoxified toxin, their HIV efficiency is certain but the protective value of antibodies is difficult to assess due to the difficulty of comparing two populations and the decline in the number of pertussis cases.

VIII – ANTIBIOTIC SENSITIVITY:

Antibiotic therapy has a significant influence on the development of a declared darling whose clinical signs are due to more toxic factors as the presence of bacteria.

B. pertussis is resistant to penicillin and bacitracin.

In vitro active antibiotics: erythromycin, rifampin, tetracycline, cephalosporins 3rd generation, chloramphenicol, fluoroquinolones, co-trimoxazole and gentamicin. It seems that erythromycin is the most active antibiotic to prevent infection in people who have been in contact with pertussis.