Haemophilus and Other Fastidious Gram-Negative Rods

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Legionella pneumophila B (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.

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

Legionella pneumophila: Identification Schema for identification of L. species

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

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