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

2. Haemophilus
The Haemophilus Species is a group of small, gram-negative, pleomorphic bacteria that require enriched media, usually containing blood or its derivatives, for isolation.
Haemophilus influenzae type b is an important human pathogen.
Haemophilus ducreyi, a sexually transmitted pathogen, causes chancroid.
Other Haemophilus species are among the normal flora of mucous membranes and only occasionally cause disease.

3. Haemophilus influenzae
Haemophilus influenzae is found on the mucous membranes of the upper respiratory tract in humans. It is an important cause of meningitis in children and occasionally causes respiratory tract infections in children and adults.

4. Morphology and Identification
Typical Organisms
In specimens from acute infections, the organisms are short (1.5 µm) coccoid bacilli, sometimes occurring in pairs or short chains. In cultures, the morphology depends on both on age and on the medium. At 6–8 hours in rich medium, the small coccobacillary forms predominate. Later there are longer rods, lysed bacteria, and very pleomorphic forms. Organisms in young cultures (6–18 hours) on enriched medium have a definite capsule. The capsule is the antigen used for "typing" H influenza.

5. Morphology and Identification
Culture
On chocolate agar, flat, grayish brown colonies with diameters of 1–2 mm are present after 24 hours of incubation. IsoVitaleX in media enhances growth. H influenzae does not grow on sheep blood agar except around colonies of staphylococci ("satellite phenomenon"). H haemolyticus and H parahaemolyticus are hemolytic variants of H influenzae and H parainfluenzae, respectively.

6. Morphology and Identification
Growth Characteristics
Identification of organisms of the Haemophilus group depends in part upon demonstrating the need for certain growth factors called X and V. Factor V can be replaced by nicotinamide adenine nucleotide (NAD) or other coenzymes. Colonies of staphylococci on sheep blood agar cause the release of NAD, yielding the satellite growth phenomenon. Carbohydrates are fermented poorly and irregularly.
Nicotinamide adenine nucleotide

7. Characteristics and Growth Requirements of the Haemophilus Species Important to Humans
Requires
Hemolysis v x
Species - +
Haemophilus influenzae  
Haemophilus parainfluenzae 
Haemophilus ducreyi 
Haemophilus haemolyticus 
Aggregatibacter aphrophilus
Haemophilus paraphrophaemolyticus 
Haemophilus segnis 

8. Morphology & Identification
Variation
In addition to morphologic variation, H influenzae has a marked tendency to lose its capsule and the associated type specificity.
Transformation
Under proper experimental circumstances, the DNA
extracted from a given type of H influenzae is capable of
transferring that type specificity to other cells (transformation).
Resistance to ampicillin and chloramphenicol is controlled
by genes on transmissible plasmids.

9. Antigenic Structure
Encapsulated H influenzae contains capsular polysaccharides (MW >150,000) of one of six types (a–f). The capsular antigen of type b is a polyribose-ribitol phosphate (PRP). Encapsulated H influenzae can be typed by slide agglutination or agglutination of latex particles coated with type-specific antibodies.
A capsule swelling test with specific antiserum is analogous to the quellung test for pneumococci. Typing can also be done by immunofluorescence. Most H influenzae organisms in the normal flora of the upper respiratory tract are not encapsulated.
The somatic antigens of H influenzae consist of outer membrane proteins. Lipooligosaccharides (endotoxins) share many structures with those of neisseriae.

10. Pathogenesis
H influenzae produces no exotoxin. The non encapsulated organism is a regular member of the normal respiratory microbiota of humans. The capsule is antiphagocytic in the absence of specific anticapsular antibodies. The polyribose phosphate capsule of type b H influenzae is the major virulence factor.
The carrier rate in the upper respiratory tract for H influenzae type b is 2–4%. The carrier rate for non typeable H influenzae is 50–80% or higher. Type b H influenzae causes meningitis, pneumonia and empyema, epiglottitis, cellulitis, septic arthritis, and occasionally other forms of invasive infection. Nontypeable H influenzae tends to cause chronic bronchitis, otitis media, sinusitis, and conjunctivitis following breakdown of normal host defense mechanisms.

11. Pathogenesis
The carrier rate for the encapsulated types a and c–f is low (1–2%), and these capsular types rarely cause disease. Although type b can cause chronic bronchitis, otitis media, sinusitis, and conjunctivitis, it does so much less commonly than non typeable H influenzae. Similarly, non typeable H influenzae only occasionally causes invasive disease (about 5% of cases).
The blood of many persons over age 3–5 years is bactericidal for H influenzae, and clinical infections are less frequent in such individuals. However, bactericidal antibodies have been absent from 25% of adults in the United States, and clinical infections have occurred in adults.

12. Clinical Findings
H influenzae type b enters by way of the respiratory tract. There may be local extension with involvement of the sinuses or the middle ear. H influenzae type b and pneumococci are two of the most common etiologic agents of bacterial otitis media and acute sinusitis. The organisms may reach the bloodstream and be carried to the meninges or, less frequently, may establish themselves in the joints to produce septic arthritis. Prior to the use of the conjugate vaccine, H influenzae was the most common cause of bacterial meningitis in children age 5 months to 5 years in the United States. Clinically, it resembles other forms of childhood meningitis, and diagnosis rests on bacteriologic demonstration of the organism.

13. Clinical Findings
Occasionally, a fulminating obstructive laryngotracheitis with swollen, cherry- red epiglottis develops in infants and requires prompt tracheostomy or intubation as a lifesaving procedure. Pneumonitis and epiglottitis due to H influenzae may follow upper respiratory tract infections in small children and old or debilitated people. Adults may have bronchitis or pneumonia due to H influenzae.

14. Diagnostic Laboratory Tests
Specimens
Specimens consist of nasopharyngeal swabs, pus, blood, and spinal fluid for smears and cultures.
Direct Identification
Commercial kits are available for immunologic detection of H influenzae antigens in spinal fluid. A positive test indicates that the fluid contains high concentrations of specific polysaccharide from H influenzae type b. These antigen detection tests generally are not more sensitive than a Gram stain and therefore are not widely used.

15. Diagnostic Laboratory Tests
Culture
Specimens are grown on IsoVitaleX-enriched chocolate agar until typical colonies appear. H influenzae is differentiated from related gram-negative bacilli by its requirements for X and V factors and by its lack of hemolysis on blood agar.
Tests for X and V (nicotinamide-adenine dinucleotide) factor requirements can be done in several ways.
The Haemophilus species that require V factor grow around paper strips or disks containing V factor placed on the surface of agar that has been autoclaved before the blood was added (V factor is heat-labile). Alternatively, a strip containing X factor can be placed in parallel with one containing V factor on agar deficient in these nutrients. Growth of Haemophilus in the area between the strips or on the discs indicates requirement for both factors.

16. X and V factor test

17. Immunity
Infants under age 3 months may have serum antibodies transmitted from the mother. During this time H influenzae infection is rare, but subsequently the antibodies are lost. Children often acquire H influenzae infections, which are usually asymptomatic but may be in the form of respiratory disease or meningitis. H influenzae has been the most common cause of bacterial meningitis in children from 5 months to 5 years of age. By age 3–5 years, many unimmunized children have naturally acquired anti-PRP antibodies that promote complement-dependent bactericidal killing and phagocytosis. Immunization of children with H influenzae type b conjugate vaccine induces the same antibodies.
There is a correlation between the presence of bactericidal antibodies and resistance to major H influenzae type b infections. However, it is not known whether these antibodies alone account for immunity. Pneumonia or arthritis due to H influenzae can develop in adults with such antibodies.

18. Treatment
The mortality rate of untreated H influenzae meningitis may be up to 90%. Many strains of H influenzae type b are susceptible to ampicillin, but up to 25% produce lactamase under control of a transmissible plasmid and are resistant. Essentially all strains are susceptible to the third-generation cephalosporins.

19. Epidemiology, Prevention, & Control
Encapsulated H influenzae type b is transmitted from person to person by the respiratory route. H influenzae type b disease can be prevented by administration of Haemophilus b conjugate vaccine to children. Beginning at age 2 months, all children should be immunized with one of the conjugate vaccines. Depending upon which vaccine product is chosen, the series consists of three doses at 2, 4, and 6 months of age or two doses given at 2 and 4 months. An additional booster dose is given sometime between 12 and 15 months of age. Contact with patients suffering from H influenzae clinical infection poses little risk for adults but presents a definite risk for non immune siblings and other non immune children under age 4 years who are close contacts.

20. Haemophilus aegyptius
This organism was formerly called the Koch-Weeks bacillus; it is sometimes called H influenzae biotype III, but the current designation is H influenzae biogroup aegyptius. It resembles H influenzae closely and has been associated with a highly communicable form of conjunctivitis. H aegyptius is the cause of Brazilian purpuric fever, a disease of children characterized by fever, purpura, shock, and death.

21. Haemophilus ducreyi
Haemophilus ducreyi causes chancroid (soft chancre), a sexually transmitted disease. Chancroid consists of a ragged ulcer on the genitalia, with marked swelling and tenderness. The regional lymph nodes are enlarged and painful. The disease must be differentiated from syphilis, herpes simplex infection, and lymphogranuloma venereum.
The small gram-negative rods occur in strands in the lesions, usually in association with other pyogenic microorganisms. H ducreyi requires X factor but not V factor. It is grown best from scrapings of the ulcer base on chocolate agar containing 1% IsoVitaleX and vancomycin, and incubated in 10% CO2 at 33°C. There is no permanent immunity following chancroid infection. Treatment with intramuscular ceftriaxone, oral trimethoprim-sulfamethoxazole, or oral erythromycin often results in healing in 2 weeks.

22. Thank you