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 Bacterial identification 
  in the diagnostic 
  laboratory versus 
  taxonomy


BORDETELLA AND 
  HAEMOPHILUS  

 
BORDETELLA
    
Bordetella pertussis
 
HAEMOPHILUS
    
Haemophilus influenzae
      Haemophilus ducreyi
      H.influenzae aegyptius

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   BORDETELLA AND HAEMOPHILUS        

Dr Abdul Ghaffar 
MBIM 650/720 Medical Microbiology 

 

TEACHING OBJECTIVES

To know the general morphology and physiology the organisms

To know epidemiology and clinical symptoms

To understand the mechanisms pathogenesis

 To know the diagnostic, therapeutic and preventive procedures

 

READING: Murray, et al.: Medical Microbiology, 3rd ed., Chapter 33, pp. 265-269 and chapter 34, pp. 276-281

 

BORDETELLA

Bordetella pertussis is the only organism of major clinical significance, causing whooping cough in infants and young children. However, a closely related organism, B. parapertussis can also cause a milder form of bronchitis. B. bronchosepticus, another member of the genus Bordetella, is the causative agent of respiratory diseases in cats and swine, but can cause broncho-pulmonary symptoms in severely immunosupressed individuals.

 

Bordetella pertussis

CDC Pertussis information (requires Acrobat)

pertussis-lympho.jpg (9160 bytes) BLOOD LYMPHOCYTOSIS IN A PATIENT WITH PERTUSSIS. The lymphocytes in this blood smear from an 18-month-old  child with a Bordetella pertussis infection have lobulated nuclei.  Lymphocytosis is characteristic of this disorder and the lymphocyte morphology is often atypical. The cytology of the cells could be mistaken  for neoplastic lymphocytes. (Wright-Giemsa stain) © The Johns Hopkins Autopsy Resource (JHAR). Image Archive. 

 

Morphology and physiology

B. pertussis is an extremely small, strictly aerobic, Gram negative, non-motile cocobacillus (short rod). Compared to other Bortdetella species, B. pertussis does not grow on common laboratory media and can be distinguished from B. parapertussis in that B. pertussis is oxidase positive but urease negative, while B. parapertussis is oxidase negative and urease positive. B. bronchosepticus is positive for both enzymes.

Epidemiology and symptoms 

pertuss-stat.jpg (160868 bytes) Pertussis in the US, 1940-1999 CDC

pertuss-child.jpg (30056 bytes) This child has pertussis. It is difficult for him to stop coughing and to get air. Coughing spasms with a "whooping" sound that follows the cough are typical. The sound means child is trying to catch his breath before the next round of coughing  WHO

Most of the patients with whooping cough are less than a year old although older children may also get the disease. The severity of disease is also age-related. The organism, contained in aerosol droplets, gains access via inhalation and colonizes the bronchial ciliary epithelial cells. After a week to 10 days of incubation period, mild symptoms of rhinitis, mild cough and sneezing occur (catarrhal stage) which last 1-2 weeks. Further proliferation of the organism compromise ciliary function and is accompanied by increased frequency and intensity of symptoms. This leads to the paroxysmal stage, characterized by paroxysms of cough followed by a prolonged and distressing inspiratory gasp (whoop). The cough, which recurs at variable intervals and often every few minutes, may last for 2-3 weeks. The cough interferes with oral intake, and the swallowed mucus may induce vomiting, resulting in severe dehydration and weight loss. Hypoxia during prolonged attacks may lead to seizure, hypoxic encephalopathy or coma. The cough episodes slowly decrease and there is gradual recovery over 3-16 weeks (convalescent stage). Pneumonia (due to B. pertussis or other bacterial pathogens), otitis media, rectal prolapse and meningo-encephalitis are among the secondary complications.

Pathogenesis 

The symptoms following the infection are due to many factors. In addition to the attachment to and growth on ciliated cells, the organism produces a number of exotoxins which contribute to these symptoms.

Pertussis toxin (pertussigen)

Pertussis toxin is an oligopeptide AB-type exotoxin that is the major cause of pertussis (abnormal cough). It causes T cell lymphocytosis and has adjuvant properties. It also causes hypoglycemia, increased IgE synthesis, and increased histamine and endotoxin sensitivity. The organism inhibits many leukocyte functions, including chemotaxis, phagocytosis and respiratory burst and impairs NK cell killing. It also contributes to bacterial binding to ciliated epithelial cells. It exerts many of its effects by covalent addition of ADP-ribose to the GTP binding Gi protein and thereby preventing the deactivation of adenylate cyclase. This results in the accumulation of large amounts of cAMP which leads to increased mucus secretion and interferes with many cellular functions.

pertussis.jpg (102899 bytes) Binding of pertussis toxin to cell membrane

Adenylate cyclase toxin

This exotoxin penetrates the host cells, is activated by calmodulin and catalyzes the conversion of ATP to cAMP. Like pertussigen, it also inhibits phagocyte and NK cell functions. However, in contrast with pertussigen, the cAMP increase caused by this toxin is short-lived.

Tracheal cytotoxin

This is a peptidoglycan-like molecule (monomer) which binds to ciliated epithelial cells, thus interfering with ciliary movement. In higher concentrations, it causes ciliated epithelial cell extrusion and destruction. The destruction of these cells contributes to pertussis.

Dermonecrotic (heat-labile) toxin

Dermonecrotic toxin is a very strong vaso-constrictor and causes ischemia and extravasation of leukocytes and, in association with tracheal cytotoxin, causes necrosis of the tracheal tissue.

Filamentous haemagglutinins (agglutinogens)

These are not exotoxins but are filament-associated lipo-oligo-saccharides which are implicated in the binding of the organism to ciliated epithelial cells. Antibodies against these molecules are protective, probably by preventing bacterial attachment.

Lipopolysaccharide (LPS)

Like LPS of other gram negative bacteria, these endotoxins cause a number of patho-physiolocigal effects. When released in relatively large quantities following bacterial cell lysis, they cause irreversible shock and cardiovascular collapse. In smaller quantities, they activate a variety of inflammatory mediators ( TNF, IL1, IL6, prostaglandins, etc.) and generate complement activation products.

Diagnosis

Symptoms are characteristic. Laboratory diagnosis is made by obtaining a nasopharyngeal aspirate and primary culture on Bordet-Gengou medium (potato-glycerol-blood agar). Growth is inhibited by peptones, unsaturated fatty acids, sulphides, etc. found in ordinary media. The organism grows as small transparent hemolytic colonies. It can be serologically distinguished from B. parapertussis and B. bronchosepticus.

Prevention and treatment

A killed whole bacterial vaccine is normally administered as DPT combination. An acellular vaccine consisting of filamentous hemagglutinins and detoxified pertussigen is also available and is recommended for booster shots. Erythromycin is the current drug of choice.

 

 

HAEMOPHILUS

CDC Haemophilus information (requires Acrobat)
WHO Haemophilus information

The genus Haemophilus  contains many species but H. influenzae is the most common pathogen. Other species of Haemophilus that are of clinical importance to immuno-competent humans are H. ducreyi (causes chancroid: an STD), H. influenzae aegyptius (associated with conjunctivitis and Brazilian purpuric fever) and H. parainfluenzae (a rare cause of pneumonia and endocarditis). There are several species of Hemophilus that are normal flora, but may be pathogenic in immuno-compromised hosts. The capsulated strain of H. influenzae (type b) is most virulent, although some non-encapsulated (non typable) strains are also pathogenic.

hemo-incid.jpg (94465 bytes) Incidence of H. influenzae non-type b invasive disease among children <5 years of age, 1996. CDC/Barbara Rice ber2@cdc.gov 

Haemophilus influenzae

Morphology and physiology

hemo-dk.jpg (13406 bytes) Haemophilus influenzae - coccobacillus prokaryote (dividing); causes meningitis in children, pneumonia, epiglottitis, laryngitis, conjunctivitis, neonatal infection, otitis media (middle ear infection) and sinusitis in adults (SEM x 64,000) © Dr Dennis Kunkel, University of Hawaii. Used with permission

H. influenzae is a small Gram negative bacillus which can be grown on chocolate agar (heated blood) and requires hemin (factor X) and nicotinamide adenine dinucleotide (NAD+:factor V) for growth which is enhanced by high CO2 concentration (5%). It does not grow on normal blood agar. The factor V and factor X requirement can be used to distinguish between H. influenzae which requires both, H. parainfluenzae which requires factor V only and H. ducreyi which requires factor X only. H. influenzae are divided into several strains on the basis of capsular polysaccharides (a-f) or the absence of a capsule (non-typable).

Epidemiology and symptoms

h-influ.jpg (153740 bytes) Clinical symptoms of infection by Haemophilus hib patient.jpg (24392 bytes) This child has swollen face due to Hib infection. The tissue under the skin covering the jaw and cheek is infected. Infection is spreading into her face. She is probably very sick  Courtesy of Children's Immunization Project, St. Paul, MN  hemo-card.jpg (113445 bytes) Gross pathology of subacute bacterial endocarditis involving mitral valve. Left ventricle of heart has been opened to show mitral valve fibrin vegetations due to infection with Haemophilus parainfluenzae. Autopsy.  CDC/Dr. Edwin P. Ewing, Jr. epe1@cdc.gov

H. influenzae causes a variety of clinical symptoms some of which may depend on the presence of the bacterial capsule. Until the availability of the Hib vaccine, the type-b H. influenzae was the main cause of meningitis in children between 6 months and 5 years, although older children, adolescents and adults can also be infected. The infection initially causes a runny nose, low grade fever and headache (1-3 days). Due to its invasive nature the organism enters the circulation and crosses the blood-brain barrier, resulting in a rapidly progressing meningitis (stiff neck), convulsions, coma and death. Timely treatment may prevent coma and death, but the patient may still suffer from deafness and mental retardation. Type-b H. influenzae may also cause septic arthritis conjunctivitis, cellulitis, and epiglottitis, the latter results in the obstruction of the upper airway and suffocation. H. influenzae of other types may rarely cause some of the symptoms listed above. Non-typable strains of H. influenzae are the second commonest cause of otitis media in young children (second to Streptococcus pneumoniae). In adults, these organisms cause pneumonia, particularly in individuals with other underlying pulmonary infections. These organisms also cause acute or chronic sinusitis in individuals of all ages.

Pathogenesis

The exact mechanism of pathogenesis is not known but the presence of capsule, which is anti-phagocytic, is a major factor in virulence. Type-b H. influenzae are more invasive and pathogenic than other strains. The lipopolysaccharide is responsible for the inflammatory process. The organisms also produce IgA1-specific protease which may aid their mucosal colonization.

Diagnosis

Presumptive diagnosis is based on history, physical examination and symptoms. Blood cultures are positive in more than 50% of symptomatic patients, except those with conjunctivitis. Polyribitol phosphate (PRP), a component of the capsular polysaccharide is present in the serum, cerebrospinal fluid (CSF) and concentrated urine of more than 95% of H. influenzae-b meningitis cases. Gram-negative cocobacilli can be found in the CSF in more than 80% of meningitis cases. Some Gram-stained preparations may be useful in rapid diagnosis of septic arthritis and lower respiratory diseases.

Treatment and prevention

Unless prompt treatment is initiated, H. influenzae-b meningitis and epiglotitis are almost 100% fatal. Due to common resistance to ampicillin and some resistance to chloramphenicol, cephalosporin, which penetrates the blood brain barrier, is the antibiotic of choice in these cases. Other diseases caused by this organism can be treated with ampicillin (if susceptible) or choice of trimethoprim-sulphamethoxazol, tetracyclin and cefaclor.

hibimp.gif (29660 bytes) Countries implementing routine childhood Hib immunization  WHO

Hib-C vaccine which consists of capsular PRP conjugated to tetanus toxoid has been used successfully to provide protection and is a part of the recommended routine vaccination schedule.

 

H. ducreyi

This is a significant cause of genital ulcers (chancroid) in Asia and Africa but, is seen less commonly in the United States. The incidence is approximately 4000-5000 per year with clusters found in California, Florida, Georgia and New York. The infection is asymptomatic in women but about a week following sexual transmission to a man, it causes appearance of a tender papule with erythematous base on the genitalia or the peripheral area. The lesion progresses to become a painful ulcer with inguinal lymphadenopathy. The H. ducreyi lesion (chancroid) is distinguished from a syphilitic lesion (chancre) in that it is a comparatively soft lesion. The organism is more fastidious than H. influenzae but can be grown on chocolate agar, supplemented with IsovitaleX in 5%-10% CO2 atmosphere and the growth can be detected in 2-4 days.

 

H.influenzae aegyptius

This bacterium, previously known as H. aegyptius, causes an opportunistic organism which can result in a fulminant pediatric disease (Brazilian purpuric fever) characterized by an initial conjunctivitis, followed by an acute onset of fever, accompanied by vomiting and abdominal pain. Subsequently, the patient develops petechiae, purpura, shock and may face death. The pathogenesis of this infection is poorly understood. The growth conditions for this organism are the same as those for H. influenzae.

 

Both H. ducreyi and H. influenzae aegyptius can be treated with erythromycin. 


 

 


 






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