1. Asst. Prof. Dr. Dalya Basil Hanna
Medical Microbiology
Asst. Prof. Dr. Dalya Basil Hanna

2. The Bordetellae
There are several species of Bordetella. Bordetella pertussis, a highly communicable and important pathogen of humans, causes whooping cough (pertussis).
Bordetella parapertussis can cause a similar disease.
Bordetella bronchiseptica occasionally causes respiratory disease and bacteremia in humans, primarily in immunocompromised hosts. Newer species and their disease associations include B hinzii (bacteremia and respiratory illness), B holmseii (bacteremia among immunosuppressed patients), and B trematum (wounds and otitis media).
B pertussis, B parapertussis, and B bronchiseptica are closely related, with 72–94% DNA homology and very limited differences in multilocus enzyme analysis; the three species might be considered three subspecies within a species.

3. Bordetella pertussis Morphology & Identification
Typical Organisms: The organisms are small gram-negative coccobacilli. With toluidine blue stain, bipolar metachromatic granules can be demonstrated. A capsule is present.
Culture: Primary isolation of B pertussis requires enriched media. Bordet-Gengou medium (potato-blood-glycerol agar) that contains penicillin G 0.5µg/ml, can be used. The plates are incubated at 35–37°C for 3–7 days in a moist environment (eg, a sealed plastic bag). B pertussis is nonmotile.

4. Bordetella pertussis Growth Characteristics & variation
The organism is a strict aerobe and forms acid but not gas from glucose and lactose. Hemolysis of blood-containing medium is associated with virulent B pertussis.
There are two mechanisms for B pertussis to shift to nonhemolytic, nontoxin-producing avirulent forms. Reversible phenotypic modulation occurs when B pertussis is grown under certain environmental conditions (eg, 28°C versus 37°C, the presence of MgSO4 , etc). Reversible phase variation follows a low-frequency mutation in the genetic locus that controls the expression of the virulence factors.

5. Antigenic Structure, Pathogenesis, & Pathology
1- B pertussis produces a number of factors that are involved in the pathogenesis of disease. One locus on the B pertussis chromosome acts as a central regulator of virulence genes. This locus has two Bordetella virulence genes, bvgA and bvgS. bvgS responds to environmental signals while bvgA is a transcriptional activator of the virulence genes.
2- The filamentous hemagglutinin mediates adhesion to ciliated epithelial cells.
3- Pertussis toxin promotes lymphocytosis, sensitization to histamine, and enhanced insulin secretion.

6. Antigenic Structure, Pathogenesis, & Pathology
4- Adenylate cyclase toxin, dermonecrotic toxin, and hemolysin also are regulated by the bvg system. The tracheal cytotoxin inhibits DNA synthesis in ciliated cells and is not regulated by bvg.
5- Pili probably play a role in adherence of the bacteria to the ciliated epithelial cells of the upper respiratory tract.
6- The lipopolysaccharide in the cell wall may also be important in causing damage to the epithelial cells of the upper respiratory tract.

7. Antigenic Structure, Pathogenesis, & Pathology
B pertussis survives for only brief periods outside the human host. There are no vectors. Transmission is largely by the respiratory route from early cases and possibly via carriers. The organism adheres to and multiplies rapidly on the epithelial surface of the trachea and bronchi and interferes with ciliary action. The blood is not invaded. The bacteria liberate the toxins and substances that irritate surface cells, causing coughing and marked lymphocytosis.

8. Antigenic Structure, Pathogenesis, & Pathology
Later, there may be necrosis of parts of the epithelium and polymorphonuclear infiltration, with peribronchial inflammation and interstitial pneumonia.
Secondary invaders like staphylococci or H influenzae may give rise to bacterial pneumonia. Obstruction of the smaller bronchioles by mucous plugs results in atelectasis and diminished oxygenation of the blood. This probably contributes to the frequency of convulsions in infants with whooping cough.

9. Clinical Findings
After an incubation period of about 2 weeks, the "catarrhal stage" develops, with mild coughing and sneezing. During this stage, large numbers of organisms are sprayed in droplets, and the patient is highly infectious but not very ill.
During the "paroxysmal stage", the cough develops its explosive character and the characteristic "whoop" upon inhalation. This leads to rapid exhaustion and may be associated with vomiting, cyanosis, and convulsions. Lymphoctosis is markedly elevated. B pertussis is a common cause of prolonged (4–6 weeks) cough in adults. Rarely, whooping cough is followed by the serious and potentially fatal complication of encephalitis.

10. Diagnostic Laboratory Tests
Specimens: A saline nasal wash is the preferred specimen. Nasopharyngeal swabs or cough droplets expelled onto a "cough plate" held in front of the patient's mouth during a paroxysm are sometimes used but are not as good as the saline nasal wash.
Direct Fluorescent Antibody Test: The fluorescent antibody (FA) reagent can be used to examine nasopharyngeal swab specimens.
Culture: The saline nasal wash fluid is cultured on solid medium agar. The antibiotics in the media tend to inhibit other respiratory flora but permit growth of B pertussis.
Polymerase Chain Reaction: PCR is the most sensitive method to diagnosis pertussis. Primers for both B pertussis and B parapertussis should be included.
Diagnostic Laboratory Tests

11. Immunity & Treatment Immunity Treatment
Recovery from whooping cough or immunization is followed by immunity. Second infections may occur but are mild; reinfections occurring years later in adults may be severe. It is probable that the first defense against B pertussis infection is the antibody that prevents attachment of the bacteria to the cilia of the respiratory epithelium.
Treatment
B pertussis is susceptible to several antimicrobial drugs in vitro. Administration of erythromycin during the catarrhal stage of disease promotes elimination of the organisms and may have prophylactic value. Treatment after onset of the paroxysmal phase rarely alters the clinical course. Oxygen inhalation and sedation may prevent anoxic damage to the brain.

12. Prevention
Every infant should receive three injections of pertussis vaccine during the first year of life followed by a booster series for a total of five doses recommended prior to school entry.
Pertussis vaccine is usually administered in combination with toxoids of diphtheria and tetanus (DPT). The usual schedule is administration of doses at 2, 4, 6, and 15–18 months of age and a booster dose at 4–6 years of age.

13. Legionella
Several dozen species of Legionella exist, some with multiple serotypes.
L pneumophila is the major cause of disease in humans.
Legionella micdadei and a few other species sometimes cause pneumonia. The other legionellae are rarely isolated from patients or have been isolated only from the environment.

14. Morphology & Identification
Legionellae are fastidious, aerobic gram-negative bacteria that are 0.5– 1µm long, they often stain poorly by Gram's method. Basic fuchsin (0.1%) should be used as the counterstain, because safranin stains the bacteria very poorly.

15. Morphology & Identification
Culture
Legionellae can be grown on complex media such as buffered charcoal yeast extract agar with alpha-ketoglutarate and iron (BCYẸ, at pH 6.9, temperature 35°C, and 90% humidity. Antibiotics can be added to make the medium selective for Legionella.

16. Morphology & Identification
Growth Characteristics
The legionellae are catalase-positive. L pneumophila is oxidase- positive; the other legionellae are variable in oxidase activity.
L pneumophila hydrolyzes hippurate; the other legionellae do not. Most legionellae produce gelatinase and β-lactamase; L micdadei produces neither gelatinase nor β-lactamase.

17. Pathology & Pathogenesis
Legionellae are found in lakes, streams, and other bodies of water. Infection of debilitated or immunocompromised humans commonly follows inhalation of the bacteria from aerosols generated from contaminated air- conditioning systems, shower heads, and similar sources. L pneumophila usually produces a lobar, segmental, or patchy pulmonary infiltration. Histologically, the appearance is similar to that produced by many other bacterial pathogens. Acute purulent pneumonia involving the alveoli is present with a dense intra alveolar exudate of macrophages, polymorphonuclear leukocytes, red blood cells, and proteinaceous material. Most of the legionellae in the lesions are within phagocytic cells. There is little interstitial infiltration and little or no inflammation of the bronchioles and upper airways.

18. Pathology & Pathogenesis
The phagocyte oxidative metabolic burst is reduced. Ribosomes, mitochondria, and small vesicles accumulate around vacuoles containing the L pneumophila. The bacteria multiply within the vacuoles until they are numerous, the cells are destroyed, the bacteria are released, and infection of other macrophages then occurs. The presence of iron (transferrin-iron) is essential for the process of intracellular growth of the bacteria, but other factors important to the processes of growth, cell destruction, and tissue damage are not well understood.

19. Clinical Findings
Asymptomatic infection is common in all age groups, the incidence of the disease is highest in men over age 55 years. Factors associated with high risk include smoking, chronic bronchitis and emphysema, steroid and other immunosuppressive treatment (as in renal transplantation), cancer chemotherapy, and diabetes mellitus. When pneumonia occurs in patients with these risk factors, Legionella should be investigated as the cause.
Infection may result in nondescript febrile illness of short duration or in a severe, rapidly progressive illness with high fever, chills, malaise, nonproductive cough, hypoxia, diarrhea, and delirium. Chest x-rays reveal patchy, often multilobar consolidation.

20. Clinical Findings
During some outbreaks, the mortality rate has reached 10%. The diagnosis is based on the clinical picture and exclusion of other causes of pneumonia by laboratory tests. Demonstration of Legionella in clinical specimens can rapidly yield a specific diagnosis. The diagnosis can also be made by culture for Legionella or by serologic tests.
L pneumophila also produces a disease called "Pontiac fever," after the clinical syndrome that occurred in an outbreak in Michigan. The syndrome is characterized by fever and chills, myalgia, malaise, and headache that develop over 6–12 hours. Dizziness, photophobia, neck stiffness, and confusion also occur. Respiratory symptoms are much less prominent in Pontiac fever than in Legionnaires' disease and include mild cough and sore throat.

21. Diagnostic Laboratory Tests
Specimens: bronchial washings, pleural fluid, lung biopsy specimens, or blood.
Smears: Legionellae are not demonstrable in Gram-stained smears of clinical specimens. Silver stains are sometimes used on tissue specimens.
Culture: Specimens are cultured on BCYẸ agar .
Specific Tests: Sometimes Legionella antigens can be demonstrated in the patient's urine by immunologic methods. The urine antigen test is specific for L pneumophila serotype 1.
Serologic Tests: Levels of antibodies to legionellae rise slowly during the illness. Serologic tests have a sensitivity of 60–80% and a specificity of 95–99%.

22. Treatment
L pneumophila are intracellular parasites of macrophages, other phagocytic cells, and probably of other human cells as well. Thus, antimicrobials useable to treat Legionella infections must enter the phagocytes and have biological activity there. Macrolides (erythromycin, azithromycin, and clarithromycin), quinolones (ciprofloxacin and levofloxacin), and tetracyclines (doxycycline) are effective.