Pasteurellaceae: Haemophilus, Pasteurella Fastidious Gram-Negative Rods

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

Haemophilus: Characteristics Aerobic, facultative anaerobe Optimal growth 35-370 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

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

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)

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

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

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

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

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

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

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

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”

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

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

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

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

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

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.

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.

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).

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?