Overview of Haemophilus and Fastidious Gram-Negative Bacilli Infections

1. Haemophilus

2. Haemophilus and Other Fastidious GNB • The fastidious group of gram-negative bacilli: • Haemophilus • HACEK( Haemophilus, Actinobacillus, Cardiobacteria, Eikenella & Kingella) • Legionella • Bordetella • Pasteurella • Brucella • Francisella • Bartonella

3. Haemophilus Species • Haemophilus = “blood loving” • Require either heme (X factor) or NAD (V factor) • Small pleomorphic GNB, oxidase positive

5. Haemophilusinfluenzae(Pfeiffer’s bacillus)

6. Haemophilusinfluenzae • Polysaccharide capsule • Six different serotypes (a-f) of polysaccharide capsule • 95% of invasive disease caused by type b (Hib) • Non-capsulated strains: non pathogenic (normal flora of respiratory tract)

7. Clinical Infections: • Acute epiglottitis/ laryngotracheal infection in small children • Can cause airway obstruction needing immediate tracheostomy • Cellulitis/arthritis: cheek and upper extremities • Meningitis :Children under 6 years • Contagious, vaccine has decreased incidence • Pneumonia/septicemia : In children • Conjunctivitis “pink eye” very contagious

8. Clinical Infections: nontypable strains • Otitis media • Children 6 months- 2 years • Sinusitis • Pneumonia, bronchitis • In adults • These sites are all in proximity to respiratory tract

9. Haemophilus influenzae type b Clinical Manifestations* *prevaccination era

10. Haemophilusinfluenzae type b Epidemiology • Reservoir Human Asymptomatic carriers • Transmission Respiratory droplets • Temporal pattern Peaks in Sept-Dec and March-May • Communicability Generally limited but higher in some circumstances

11. Risk factors for invasive disease • Exposure factors • household crowding • day care attendance • low socioeconomic status • In schools, hostels

13. pathogenesis…………………….. • Penetrate nasopharynx • and invades blood stream • Causes meningitis, pneumonia, bacteremia • 95% of invasive disease caused by type b 

15. HaemophilusInfluenzae: Virulence Factors • Capsule: Antiphagocytic • IgA Protease: Cleaves IgA on mucosal surfaces • Lipid A: Effects ciliated respiratory epithelium • Pili: Attachment

16. Laboratory diagnosis

17. Sample collection: • CSF, blood, sputum. Diagnosis is confirmed when the organism is isolated from a sterile body site (Blood & CSF)  • Sample should never be refrigerated • Gram Stain: Usually very small pleomorphic gram negative coccobacilli

18. Direct smear of H. influenzae in CSF in a case of meningitis. Note the TINY intracellular and extracellular pleomorphic gram-negative bacilli. Remember to look for capsules surrounding the rod.

19. Capsule detection: Quellung reaction (capsular swelling) when CSF is mixed with type b antiserum. • Methylene blue is added • Observed under microscope

20. Antigen detection: type b capsular antigen detected in CSF, by LATEX AGGLUTINATION TEST

21. CULTURE • On Chocolate Agar: semi-opaque, gray-white, convex, mucoid. (5-10% CO2)

22. Growth factors • Haemophilus species require growth factors: • X-factor ( hemin) • Heat-stable substance • Present in RBC and released with degradation of hemoglobin • V-factor (NAD): Nicotinamide adenine dinucleotide) • Heat- labile • Found in blood or secreted by certain organisms

27. Fildes agar: • Levinthals agar:

28. BIOCHEMICAL IDENTIFICATION: • Catalase + • Oxidase + • X and v factor strips or disks • Reduces nitrate to nitrite • Ferments glucose & xylose • BIOTYPING: (indole, ornithine & urease) • SEROTYPING: (antisera)

29. Antibiotic sensitivity testing:

31. Vaccine: • PRP capsular Ag of Hib is used as vaccine candidate • Conjugated to adjuvants: toxoids, OMP Chemoprophylaxis: • Oral rifampin ( household contacts & HCW) Treatment: • Cefotaxime, ceftriaxone, azithromycin levofloxacin

32. Haemophilusducreyi • Causative agent of chancroid or soft chancre (STD), highly contagious, painful • Specimens should be collected from base of lesion, inoculated directly to enriched media and held for 5 days • Gram stain appears as groups of coccbacilli that resemble a ‘school of fish” or “railroad tracks” • Requires only X factor to grow

36. Haemophilus Species Identification V=variable

37. ANTIBIOTIC THERAPY • Historically ampicillin was drug of choice. • However, resistance has developed due to production of beta-lactamase or altered penicillin binding proteins and cell wall permeability • Primary antibiotics include cefotaxime or ceftriaxone

38. Take 5!

39. HACEK Group • HACEK is an acronym of the first initial of each genus that belong in the group: • Haemophilusaphrophilus: • NAME ALERT: Now called Aggregatibacteraphrophilus • Not a true Haemophilus because does not need X nor V • Actinobacillusactinomycetemcomitans • Cardiobacteriumhominis • Eikenellacorrodens • Kingella species • Habitat • Commensals of oral cavity • Clinical Significance • Infective endocarditis • Peridontal disease • Dental caries • Infections following dental procedures

40. HACEK Group: General Characteristics • Gram-negative bacilli • Require an increased CO2 (5%-10%) environment • Slow/poor growers • Usual flora of the oralpharyngeal cavity • Opportunists in immunocompromised hosts

41. Capnocytophaga sp. • Capnophilic • Facultative anaerobe • Part of the normal oralpharygeal flora • Cause periodontal disease, sepsis

42. Pasteurella species • General characteristics • Colonizes mucous membranes of the upper respiratory tract and gastrointestinal tracts of mammals and birds • Human infections occur from bites and scratches inflicted by animals, primarily felines • Results in a localized, pus- producing infection • Can cause life-threatening systemic disease • Most common isolated species is Pasteurella multocida

43. Pasteurella multocida

44. Pasteurella multocida • Culture characteristics • Growth on 5% blood or chocolate shows small, smooth, grayish,convex colonies • Non-hemolytic • “Musty” or earthy odor • No growth on MacConkey agar

45. Pasteurella multocida • Microscopic examination • Very small gram-negative rods • Bipolar staining with Giemsa or methylene blue • “Safety-pin” appearance

46. Pasteurella multocida: Identification • Oxidase positive • Indole positive • Nonmotile • Catalase positive • Glucose fermenter

47. Brucella species • Causes infection in cattle (zoonosis) • Acquired through aerosol, percutaneous and oral routes of exposure • Brucellosis • Primarily seen with animal handlers and those who handle animal products • Also known as Malta or undulant fever • Type 3 biohazard – can be transmitted through unbroken skin • Category B Biological agent- easy to disseminate and cause moderate morbidity, but low mortality.

48. Brucella species: Identification • Colony Morphology • Small, smooth, convex, nonhemolytic • May require holding culture for 21 days • Gram Stain Morphology • Small gram-negative coccobaccilli • Nonmotile • Aerobic • Oxidase positive • Catalase positive • Urease positive

49. Francisella tularensis • Highly infectious Type 3 biohazard – can be transmitted through unbroken skin, bite from an insect, direct contact with infected animals or inhalation of aerosols • Category A Biological agent-it can be spread from person to person or disseminated, high mortality rates • Infection in rabbits, sheep, squirrels and ticks • Zoonotic infection in humans • Tularemia

50. Francisella tularensis: Identification • Colony Morphology • BAP = No growth • MAC = No growth • Choc = Small, smooth, gray gncb at 2-5 days • Requires special media (BCYE or MTM) • Oxidase: negative • Catalase: negative- weak positive • Ferments glucose • X and V negative • NOTE: Usually identified by DFA or direct agglutination tests due to risk of lab acquired infection