1. Haemophilus and Other Fastidious Gram-Negative Rods

2. Case Study
A 2-year-old unvaccinated child was seen in the emergency room because of complaints of headache and fever
A cerebrospinal fluid(CSF) sample was obtained and sent to the laboratory for culture
The Gram stain showed many white blood cells and many gram-negative, small bacilli

3. Points to Consider
What clinical findings led the physician to request a CSF culture?
Based on the patient’s age, which organism would be suspected?
What growth requirements must be met to recover the suspected agent?
Which other fastidious organisms would be considered?
Other points to consider

4. Haemophilus and Other Fastidious Gram-negative Rods
The fastidious group of gram-negative bacilli include:
Haemophilus
HACEK
Legionella
Bordetella
Pasteurella
Brucella
Francisella

5. Haemophilus Influenzae: Clinical Infections
Infections caused by typable (encapsulated) strains
Acute epiglottis or laryngotracheal infection in small children
Cellulitis/arthritis
Meningitis
Pneumonia/septicemia (in children)
Conjunctivitis

6. Haemophilus Influenzae: Clinical Infections
Infections caused by Nontypable strains
Otitis media
Sinusitis
Pneumonia, bronchitis (in adults)

7. Haemophilus Species
H. influenzae satellitism around and between the large, white, hemolytic staphylococci
Haemophilus species require hemoglobin for growth:
X-factor ( hemin): Heat-stable substance
V-factor (NAD): Heat- labile, coenzyme I, nicotinamide adenine dinucleotide, found in blood or secreted by certain organisms

8. Haemophilus Species
Direct smear of H. influenzae in CSF in a case of meningitis. Note the intracellular and extracellular pleomorphic gram-negative bacilli.

9. Haemophilus species
H.influenzae growing on chocolate agar. Notice the semi-opaque, gray-white, mucoid colonies characteristic of encapsulated strains.
Gram stain of H. influenzae from colony

10. Haemophilus Species: Identification
This organism would be identified as H. influenzae because it is using both X and V factors.

11. Haemophilus Species: Identification
This organism would be identified as H. parainfluenzae because it is using V factor only.

12. Haemophilus Species: Identification
This organism would be identified as H. aphrophilus because it is using X factor only.

13. Haemophilus Species: Identification
Under ultraviolet light, the organism on the bottom is showing a positive porphyrin reaction, whereas the organism on the top is demonstrating a negative porphyrin reaction.

14. HACEK Group and Capnocytophaga
HACEK is an acronym of the first initial of each genus that belong in the group:
Haemophilus aphrophilus
Actinobacillus actinomycetemcomitans
Cardiobacterium hominis
Eikenella corrodens
Kingella species
Capnocytophaga sp.
Has similar requirements as the HACEK group

15. HACEK Group and Capnocytophaga: General Characteristics
Gram-negative bacilli
Require an increased CO2 (5%-10%) environment
Significant cause of endocarditis
Usual flora of the oral cavity
Opportunists in immunocompromised hosts

16. HACEK Group H. aphrophilus growing on blood agar
Gram stain morphology of H. aphrophilus

17. HACEK Group
A. actinomycetemcomitans on blood agar. Note the star-shaped centers of the colonies.
Microscopic morphology of A. actinomycetemcomitans (Gram stain).

18. HACEK Group The 48-hour growth of colonies of C. hominis
Gram stain of C. hominis showing typical “rosettes”

19. HACEK Group E. corrodens on chocolate agar
Gram stain morphology of E. corrodens

20. HACEK Group and Capnocytophaga
Growth of Capnocytophaga colonies on chocolate agar. Compare this growth with Eikenella
Gram stain of Capnocytophaga species

21. HACEK group
Gram stain of Kingella kingae showing plump rods in chains. Compare this morphology with other members of the HACEK group.

22. 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
Most common isolated species is Pasteurella multocida

23. Pasteurella species P. multocida: Clinical manifestations
Localized infection after a bite or scratch
Respiratory tract infection
Life-threatening systemic diseases (e.g., meningitis, bacteremia)

24. Pasteurella Multocida
Culture characteristics
Growth on 5% blood or chocolate shows small, smooth, convex colonies
“Musty” odor
No growth on MacConkey agar; oxidase positive
Microscopic examination
Very small gram-negative rods
Bipolar staining with Giemsa or methylene blue
“Safety-pin” appearance

25. Pasteurella Multocida
P. multocida growing on blood agar and chocolate agar. No growth on MacConkey agar plate.
Gram stain morphology of P. multocida showing bipolar staining

26. Case Study
Ten members of a group of 2 dozen retirees from the tobacco industry became acutely ill with pneumonia during a 2-week cruise
The group was staying in a block of rooms together and spent time socializing over drinks and cigarettes in the cabins and saunas and at the poolside

27. Case Study
On the fifth day of the cruise, several members went to see the ship’s doctor because of a worsening cough
Chest x-rays revealed patchy lobar pneumonia in all affected individuals
The condition improved with erythromycin therapy

28. Legionella Species General characteristics Clinical infections
Ubiquitous gram-negative rods
Acquired by humans primarily through inhalation of aerosols
Clinical infections
Febrile disease with pneumonia (Legionnaire’s disease) and extrapulmonary involvement
Pontiac fever (without pulmonary involvement)
Asymptomatic infection

29. Legionella pneumophila
Gram stain of specimen showing intracellular and extracellular Legionella pneumophila
L. pneumophila in specimen stained by direct fluorescent antibody technique.

30. Legionella pneumophilaB
(A) Nonselective buffered charcoal yeast extract (BCYE) plate inoculated with sputum specimen.
(B) Selective BCYE inoculated with the same specimen but treated before inoculation. Legionella colonies are the smallest visible colonies.

31. Legionella pneumophila
L. pneumophila colony on BCYE agar after 3, 4, 5, and 7 days of incubation

32. Legionella pneumophila: Identification
Schema for identification of L. species

33. Points to Remember Clinical manifestations presented by the patient
The types of infections these organisms produce
The risk factors that predispose susceptible individuals

34. Points to Remember
What special growth supplements are required for isolation
Where these groups of organisms are usually found
Characteristic features of the organisms for identification and differentiation among closely-related species