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Bacteriology Dr. Bara ׳Hamid Hadi Haemophilus and Legionella Haemophilus is a genus of Gram-negative, pleomorphic, coccobacilli bacteria belonging to the Pasteurellaceae family. These organisms inhabit the mucous membranes of the upper respiratory tract, mouth, vagina, and intestinal tract. The genus Haemophilus includes a number of species that cause a wide variety of infections but share a common morphology and a requirement for blood-derived factors during growth that has given the genus its name.The impotant one and the major pathogen is Haemophilus influenza. Other Haemophilus species cause disease less frequently. Haemophilus parainfluenzae sometimes causes pneumonia or bacterial endocarditis. Haemophilus ducreyi causes chancroid. Haemophilus aphrophilus is a member of the normal flora of the mouth and occasionally causes bacterial endocarditis. Haemophilus aegyptius, which causes conjunctivitis and Brazilian purpuric fever, and Haemophilus haemolyticus Haemophilus influenzae is a small, nonmotile Gram-negative bacterium this bacteria is classified as type-able (encapsulated) and non-type-able (non encapsulated) according to the presence or absence of capsule .Encapsulated strains of Haemophilus influenzae are coccobacilli, similar in morphology to Bordetella pertussis, the agent of whooping cough. ,and are further classified into 6 serotypes(a-f) , with the Haemophilus influenzae serotype b being the most pathogenic for humans, responsible for respiratory infections, ocular infection, sepsis and meningitis. While non encapsulated strains are pleomorphic and often exhibit long threads and filaments. The organism may appear Gram-positive unless the Gram stain procedure is very carefully carried out. Furthermore, elongated forms may exhibit bipolar staining, leading to an erroneous diagnosis of Streptococcus pneumoniae Haemophilus "loves heme", more specifically it requires a precursor of heme in order to grow. Nutritionally, Haemophilus influenzae prefers a complex medium and requires preformed growth factors that are present in blood, specifically X factor (i.e., hemin) and V factor (NAD or NADP). In the laboratory, it is usually grown on chocolate blood agar which is prepared by adding blood to an agar base at 80oC. The heat releases X and V factors from the RBCs and turns the medium a chocolate brown color. The bacterium grows best at 35-37oC and has an optimal pH of 7.6. Haemophilus influenzae is generally grown in the laboratory under aerobic conditions or under slight CO2 tension (5% CO2), although it is capable of glycolytic growth and of respiratory growth using nitrate as a final electron accept. Pathogenesis: The pathogenesis of H. influenzae infections is not completely understood, but the important Virulence factors of this bacteria are: 1. Capsule: all of the 6 encapsulated strains of H. influenzae have capsule as a virulence factor. The capsule is composed of polysaccharide containing two hexose sugers as subunits carbohydrates. But type b polysaccharide is the only capsular type which has two pentose monosaccharide instead of the two hexose sugars in other serotypes called polyribosyl-ribitol-phosphate (PRP) 2. outer membrane proteins: designated as P1 and P2. 3. lipooligosaccharide 4. neuraminidase . 5. IgA protease. 6. Fimbriae So the presence of the capsule is known to be the major factor in virulence. Encapsulated organisms can penetrate the epithelium of the nasopharynx and invade the blood capillaries directly. Their capsule allows them to resist phagocytosis and complement-mediated lysis in the non immune host. Non encapsulated strains are less invasive, but they are apparently able to induce an inflammatory response that causes disease. Clinical features: Naturally-acquired disease caused by H. influenzae seems to occur in humans only. In infants and young children (under 5 years of age), H. influenzae type b causes bacteremia and acute bacterial meningitis. Occasionally, it causes epiglottitis (obstructive laryngitis), cellulitis, osteomyelitis, and joint infections. Nontypable H. influenzae causes ear infections (otitis media) and sinusitis in children, and is associated with respiratory tract infections (pneumonia) in infants,children and adults. Diagnosis:1- Isolation of the organism from clinical material (such as CSF, blood, throat swab, sputum) on chocolate agar enriched with two growth factor X and V. 2- A capsule swelling test (Quelling test) with specific antiserum is analogous to quelling test of pneumonia 3- Capsular antigen may be detected CSF or other body fluid using immunologic tests such as latex agglutination. 4- PCR to detect bacteria and to differentiate into capsulated and non capsulated one Treatment: The recommended treatment for H. influenzae meningitis is ampicillin for strains of the bacterium that do not produce ß-lactamase, and a third-generation cephalosporin or chloramphenicol for strains that do. Amoxicillin, together with a substance such as clavulanic acid, that blocks the activity of ß-lactamase, has been unreliable in treatment of meningitis, although it is effective in treatment of sinusitis, otitis media and respiratory infections.Vaccine conjugated to diphtheria toxin or to carrier protein. The vaccine is more effective in children. Haemophilus ducreyi This is a significant cause of genital ulcers (chancroid). 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. Legionella Legionella cells are thin, somewhat pleomorphic Gram-negative bacilli that measure 2 to 20 μm. Long, filamentous forms may develop, particularly after growth on the surface of agar. Ultrastructurally, Legionella has the inner and outer membranes typical of Gram-negative bacteria. It possesses pili (fimbriae), and most species are motile by means of a single polar flagellum. It include the speciesL. pneumophila,which is the primary human pathogenic bacterium in this group and is the causative agent of Legionnaires' disease, also known as legionellosis. Although more than 70 Legionella serogroups have been identified among 50 species, L pneumophila causes most legionellosis. Transmission occurs by means of aerosolization or aspiration of water contaminated with Legionella organisms. Wounds may become infected after contact with contaminated water. Pathogenesis The pathogenesis of Legionella infections begins with a supply of water containing virulent bacteria and with a means for dissemination to humans. Person-to-person transmission has never been demonstrated, and Legionella is not a member of the bacterial flora of humans. Infection begins in the lower respiratory tract. Alveolar macrophages, which are the primary defense against bacterial infection of the lungs, engulf the bacteria; however, Legionella is a facultative intracellular parasite and multiplies freely in macrophages. The bacteria bind to alveolar macrophages via the complement receptors and are engulfed into a phagosomal vacuole. However, by an unknown mechanism, the bacteria block the fusion of lysosomes with the phagosome, preventing the normal acidification of the phagolysosome and keeping the toxic myeloperoxidase system segregated from the susceptible bacteria. The bacilli multiply within the phagosome. Thus, a cellular compartment that should be a death trap instead becomes a nursery. Eventually, the cell is destroyed, releasing a new generation of microbes to infect other cells. The symptoms of Legionella infection undoubtedly result from a combination of physical interference with oxygenation of blood, ventilation-perfusion imbalance in the remaining lung tissue, and release of toxic products from bacteria and inflammatory cells. Bacterial factors include a protease that may be responsible for tissue damage. Cellular factors include interleukin-1, which produces fever after it is released from monocytes, and tumor necrosis factor, which may be responsible for some of the systemic symptoms. Virulence appears to be multifactorial. An outer membrane protein that functions as a metalloprotease and a cytoplasmic membrane heat-shock protein elicit protective immune responses, but are not essential for expression of virulence. Clinical Features: The most common presentation of Legionella pneumophila is acute pneumonia (legionellosis). The clinical manifestations of Legionella infections are primarily respiratory. Two very different kinds of respiratory illness may result from infection; the reasons for this dichotomy are not understood. The most common presentation is acute pneumonia, which varies in severity from mild illness that does not require hospitalization (walking pneumonia) to fatal multilobar pneumonia. Typically, patients have high, unremitting fever and cough but do not produce much sputum. Extrapulmonary symptoms, such as headache, confusion, muscle aches, and gastrointestinal disturbances, are common. Most patients respond promptly to appropriate antimicrobial therapy, but convalescence is often prolonged (lasting many weeks or even months). The second form of respiratory illness is called Pontiac fever after the city in Michigan where the first epidemic was recognized. This uncommon manifestation of infection resembles acute influenza, including fever, headache, and severe muscle aches. It is self-limited, and convalescence is uneventful. Bacteremia occurs during Legionella pneumonia, and symptomatic infection outside the lungs occasionally develops. Under special conditions, bacteria introduced through portals other than the lungs, such as surgical wounds, may cause disease. Diagnosis: Legionellosis can be suspected clinically. The preferred method is culturing on special charcoal-containing agar. Direct detection of bacterial antigen in clinical specimens is potentially much faster than culturing. Control: Decontamination of identified environmental sources is of primary importance for prevention. The drug of choice is erythromycin.