1. Pasteurellaceae

2. Classification – includes three medically important genera Haemophilus Pasteurella Actinobacillus (we won’t be discussing this rare clinical isolate) Haemophilus species – these are fastidious “blood loving” organisms that require one or both of two factors present in blood:

3. Haemophilus X=hemin - is necessary for the synthesis of iron containing respiratory enzymes such as cytochrome, cytochrome oxidase, catalase, and peroxidase. V=NAD - is a coenzyme required for oxidation- reduction reactions X is heat stable while V is heat labile Morphology and cultural characteristics G-coccobacilli to g- filamentous rods (seen on Gram stains of rough colonies) Optimal growth is at 35-37 0 C, with an atmosphere of 5- 10% CO 2. H. ducreyi grows best at 33-35 0 C

4. Microscopic appearance of Haemophilus

5. Gram stain of Haemophilus

6. Haemophilus With the exceptions of H. ducreyi (requires 4 days) and H. aegyptius (requires 2-3 days), most grow in 18-24 hours. On conventional sheep blood agar, X factor diffuses from the RBCs into the medium, but the small amount of V factor that diffuses out is destroyed by the NADase activity that is present in the blood agar. Therefore, most will grow poorly or not at all on CBA plates. Many will produce small colonies on CBA plates surrounding colonies of Staph. aureus or Pseudomonas which produce and secrete large amounts of NAD into the medium. This phenomena is known as satelliting.

7. Satelliting

8. Haemophilus Haemophilus sp. grow best on chocolate agar. Heating (80 0 C for 15 min.) destroys the NADase activity and releases the NAD from the RBCs. Why would heating for too long be detrimental for the growth of some Haemophilus sp.? If a specimen is likely to be contaminated with large amounts of NF, chocolate agar can be made selective for H. influenzae by adding bacitracin, vancomycin, and clindamycin. Colonies on chocolate agar are grayish. The hemolytic species are beta hemolytic Colonies with capsules are smooth and non-encapsulated organisms produce rough colonies

9. Haemophilus influenzae on Choc agar

10. Haemophilus The organisms are very susceptible to drying and chilling (are autolytic) so specimens should not be refrigerated and they should be processed quickly to avoid killing the organisms There are 10 different species that may be found in clinical specimens: Influenzae, parainfluenzae, haemolyticus, parahaemolyticus, aphrophilus, paraphrophilus, paraphrohaemolyticus, aegyptius, ducreyi, and segnis Biochemistry Are all oxidase +

11. Haemophilus Are speciated on the basis of hemolysis and X and V factor requirements. Fermentation and catalase production are also used. Which species require both X and V, and which require only V? H. ducreyi is the only species that requires only X, though H. aphrophilus may require it on initial isolation

12. V factor requirement

13. X factor requirement

14. X and V factor requirements

15. Speciation of Haemophilus

16. Haemophilus H. influenzae can be differentiated from H. aegyptius by its fermentation of xylose (H. aegyptius is -) H. influenzae is further subdivided into 7 biotypes, with type I being associated with more invasive disease. Immunoserologic ID H. influenzae is divided into 6 serotypes based on capsular polysaccharide. Type b is the most virulent.

17. Haemophilus Mechanisms of pathogenicity Capsule – the type b serotype, in particular, is poorly immunogenic and antiphagocytic Adhesions – both pili and outer membrane proteins may act as adhesions IgA protease Endotoxin Clinical significance – H. influenzae Nonencapsulated H. influenzae is part of the NF of the nasopharynx in 75% of children and a lower % of adults. H. influenzae type b (Hib) is a major pathogen, particularly in children where the initial focus of infection is the nasopharynx followed by invasion of local tissue and sometimes the bloodstream.

18. Haemophilus Meningitis – occurs in individuals between 2 months and three years of age. Before 2 months the infant is protected by residual antibodies from mom and after 3 years active immunity has occurred. Following infection, long term sequelae such as deafness, speech impairment, and behavior abnormalities may occur. Epiglottitis – occurs in 2-4 year olds with mostly boys being affected. Starts as a sore throat and progresses to cough and fever and then to respiratory distress with blockage of the air passage and death from suffocation. Pneumonia - frequently associated with otitis media, meningitis, and septicemia

19. Haemophilus Septic arthritis, cellulitis, and pericarditis in children under 2 years of age In adults and children, nonencapsulated varieties may cause secondary infections of acute sinusitis or bronchitis. Clinical significance-H. aegyptius (Koch- Weeks bacillus) Causes acute and contagious conjunctivitis (commonly called pink eye)

20. Haemophilus Clinical significance-H. ducreyi Causes a venereal disease called chancroid or soft chancre which is transmitted by direct contact and is more commonly seen in hot, tropical countries. After an incubation of 4-7 days, lesions appear on genitals or adjacent areas. The lesions are small, tender, and red, but they rapidly become pustular, eroded, and ulcerated. They may spread to neighboring lymphatics causing buboes, but they do not spread further. The chancre is autoinoculable, resulting in multiple lesions.

21. Chancroid

22. Haemophilus Clinical significance – other species May be part of the NF of the oropharynx and may occasionally cause endocarditis, meningitis, sinusitis, or pneumonia. Can cause invasive disease following a human bite. Antimicrobial therapy and treatment Many strains of H. influenzae now produce beta lactamase so testing is necessary. Chloramphenicl or tetracycline are usually effective There is a Hib vaccine given to infants For chancroid - sxt or erythromycin is effective

23. Pasteurellaceae Pasteurella Is a small Gram-negative bacillus Is fermentative, but anaerogenic, non- motile, and oxidase +. P. multocida is the most commonly isolated species. The organisms are usually associated with animals other than man

24. Pasteurella P. multocida won’t grow on a Mac plate and may exhibit bipolar staining

25. Pasteurella TSI results may be confusing because of weak acid production Virulence factors Capsule Endotoxin Clinical significance P. multocida is NF in the respiratory tract of a wide variety of organisms including dogs and cats. Clinical isolates in humans usually from a localized infection produced after a dog or cat bite or scratch. It may occasionally progress to osteomyelitis or arthritis.

26. Pasteurella Antimicrobial sensitivity Is exquisitely sensitive to penicillin (Sensitivity to 2U of penicillin may be used for presumptive ID) Tetracycline and chloramphenicol may also be used