1. Pasteurellaceae: Haemophilus, Pasteurella
Fastidious Gram-Negative Rods

2. Pasteurellaceae: Genera
Small, gram(-) bacilli; pleomorphic (cocci, filamentous rod)
Medically important genera:
Haemophilus - human oropharynx NF; infection of unimmunized children
Pasteurella - animal oropharynx NF; bite wound infection, RTI, bacteremia, meningitis
Aggregatibacter (former Actinobacillus) - human oral NF; periodontitis, endocarditis, bite wound

3. Haemophilus: Characteristics
Aerobic, facultative anaerobe
Optimal growth C
Atmosphere of 5-10% CO2
Catalase(+)
Oxidase(+)
Nonmotile
Most fastidious, require enriched media
Susceptible to drying and chilling; autolytic
Clinical specimens should not be refrigerated, processed immediately

4. Haemophilus: Characteristics
Obligate parasites
Mucous membranes of human, animals
“blood-lover”
Most require growth factors
X (hemin) factor - synthesis of iron containing respiratory enzymes (cytochrome, cytochrome oxidase, catalase, peroxidase)
V (NAD) factor - coenzyme for oxidation-reduction

5. Haemophilus: CBA Grow 1-4 days Some β-hemolytic
Most grow poorly, or not at all on CBA
Satellitism - grow as small colonies on CBA around Staphylococcus aureus (hemolysis secrete NAD into medium, hemin diffuses from RBC)

6. Haemophilus: Chocolate Agar
Grow best on highly enriched CHOC
Heat RBC 80°C, 15 min. - destroys NADase, releases NAD into medium
Can make selective - add bacitracin, vancomycin, clindamycin to eliminate NF
Colonies grayish:
Capsulated strains = smooth
Non-encapsulated strains = rough

7. ID Haemophilus: X & V Factor Requirement
Only V (NAD) = H. parainfluenzae
Both X (Hemin) and V (NAD) = H. influenzae

8. Haemophilus influenzae: Lab ID
Six serotypes (a-f)
by capsular polysaccharide
Type b most virulent (>95% of all invasive infections)
Hib ~20,000 pediatric infections/year prior to vaccine
Capsule contains polyribitol phosphate (PRP); used for vaccine, elicits host protective antibody response
Eight biotypes (I-VIII)
Encapsulated type I – pathogens, more invasive disease
Nonencapsulated type II, III - opportunists

9. Haemophilus influenzae: Virulence Factors
PRP capsule – protects against phagocytosis
LPS – endotoxin; induces inflammation
Protease – IgA specific; facilitates colonization of mucosal surfaces
Pili and outer membrane proteins – adhesion, attachment

10. Nonencapsulated Haemophilus influenzae
Colonize URT within first few months of newborn
Opportunistic pathogen - spread locally and cause disease:
Otitis media
Sinusitis
Bronchitis, pneumonia – elderly, patients with chronic pulmonary disease
Disseminated disease uncommon

11. Encapsulated Haemophilus influenzae
Infrequently found in URT as NF
Infect, able to penetrate nasopharynx (NP) submucosal, into bloodstream
Common infection of infants and young children

12. Encapsulated H. influenzae: Infection and Disease
Meningitis:
Follow bacteremic spread from NP
Usually infants 3-18 months age
Generally preceded by 1-3 days URT disease
High mortality
Epiglottitis:
Occurs in 2-4 year olds, mostly boys
Cellulitis, tissue swelling, life-threatening emergency
Pharyngitis, fever, breathing difficulties
Progress to complete obstruction of airway leading to death

13. Encapsulated H. influenzae Infection and Disease
Cellulitis:
In very young children
Fever, reddish-blue color on cheek, periorbital area
Arthritis:
Usually children <2 years age
Infection of large joints, secondary to bacteremia
Conjunctivitis:
H. aegyptius
Acute, contagious conjunctivitis
Commonly called “pink eye”

14. Haemophilus ducreyi
DNA studies – not Haemophilus; but related to Pasteurellaceae
STD – genital ulcers, soft chancre or chancroid
Common in hot, tropical countries (Africa, Asia); less common in Europe, North America
Diagnosed in men, usually AS in women
Following 5-7 days exposure, tender papule, progress to painful ulcer, inguinal lymphadenopathy
Does not spread further
Autoinoculable, resulting in multiple lesions

15. Haemophilus: Treatment and Prevention
Prompt treatment or causes high mortality
Antibiotic resistance:
~30% penicillin (β-lactamase)
<1% chloroamphenicol (chloramphenicol acetyltransferase)
Drug of choice – ceftriaxone (β-lactamase-resistant cephalosporin), good CSF penetration
For chancroid – treat with erythromycin
Purified HIB vaccine:
3 doses purified PRP-conjugate
Given 2-6 months of age
In USA, dramatically reduced disease in infants

16. Pasturella multocida: Characteristics
Bipolar staining
Grows well on CBA, Chocolate agar
Poor growth on Mac and other media for Gram(-) rods
Fermentative (glucose, sucrose), little or no gas
TSI confusing because of weak acid production = “sick” appearance

17. P. multocida: Infection and Disease
Three forms of disease
Animal bite wound infection - localized cellulitis and lymphadenitis
Exacerbation of chronic respiratory disease of patients with underlying pulmonary dysfunction
Systemic infection in immunocompromised patient (bacteremia, meningitis)
Exquisitely sensitive to penicillin
2 units of penicillin
may be used for presumptive ID)
Tetracycline, chloramphenicol also used

18. Class Assignment
Textbook Reading: Chapter 18 Haemophilus and Other Fastdious Gram-Negative Rods - Haemophilus, Pasteurella (Omit HACEK Group, Capnocytophaga)
Key Terms
Learning Assessment Questions

19. Case Study – H. influenzae
A 78-year-old man confined to a nursing home awoke with a severe headache and stiff neck.
Because he had a high fever and signs of meningitis, the nursing home staff took him to a local emergency department.
The CSF specimen was cloudy.

20. Case Study – H. influenzae
Analysis revealed 400 white blood cells per mm3 (95% polymorphonuclear neutrophils), a protein concentration of 75 mg/dl, and a glucose concentration of 20 mg/dl.
Small gram-negative rods were seen on Gram stain of the CSF, and cultures of CSF and blood were positive for Haemophilus influenzae.

21. Case Study - Questions
1. Discuss the epidemiology of H. influenzae meningitis,and compare it with the epidemiology of meningitis caused by Streptococcus pneumoniae and by Neiserria meningitidis.
2. Compare the biology of H. influenzae strain that is likely to be the cause of this patient’s disease with the strains that historically caused pediatric diseases (prior to vaccination).

22. Case Study - Questions
3. What other diseases does this organism cause? What other Haemophilus species cause disease, and what are the diseases?
4. What diseases are caused by Pasteurella multocida? What is the source of this organism?