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Questions and Answers
What does Haemophilus mean in Greek?
What does Haemophilus mean in Greek?
Blood lover
Which of these Haemophilus species is NOT a part of the normal flora?
Which of these Haemophilus species is NOT a part of the normal flora?
What growth factors do Haemophilus species require? (Select all that apply)
What growth factors do Haemophilus species require? (Select all that apply)
Haemophilus influenzae is the only species in the genus that can cause disease.
Haemophilus influenzae is the only species in the genus that can cause disease.
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What is the KEY identifying feature of Haemophilus influenzae in culture?
What is the KEY identifying feature of Haemophilus influenzae in culture?
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Haemophilus influenzae is always encapsulated.
Haemophilus influenzae is always encapsulated.
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Haemophilus influenzae was originally named for its role in causing the influenza pandemic.
Haemophilus influenzae was originally named for its role in causing the influenza pandemic.
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What is the most common Haemophilus species before vaccination?
What is the most common Haemophilus species before vaccination?
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What is the name commonly used for H. influenzae type B?
What is the name commonly used for H. influenzae type B?
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Haemophilus influenzae is resistant to penicillin due to its production of beta-lactamase.
Haemophilus influenzae is resistant to penicillin due to its production of beta-lactamase.
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Haemophilus influenzae biogroup aegyptus is primarily known for causing what infection?
Haemophilus influenzae biogroup aegyptus is primarily known for causing what infection?
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Which of these is a more serious consequence of Haemophilus influenzae biogroup aegyptus infection?
Which of these is a more serious consequence of Haemophilus influenzae biogroup aegyptus infection?
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Haemophilus ducreyi is known for causing which infection?
Haemophilus ducreyi is known for causing which infection?
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Haemophilus ducreyi is a common member of the normal flora.
Haemophilus ducreyi is a common member of the normal flora.
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Which of these organisms is NOT a member of the HACEK group?
Which of these organisms is NOT a member of the HACEK group?
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What does HACEK stand for?
What does HACEK stand for?
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Aggregatibacter aphrophilus requires both X and V factor for growth.
Aggregatibacter aphrophilus requires both X and V factor for growth.
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Aggregatibacter actinomycetemcomitans is a major contributor to periodontitis.
Aggregatibacter actinomycetemcomitans is a major contributor to periodontitis.
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Cardiobacterium hominis is most commonly isolated from what site?
Cardiobacterium hominis is most commonly isolated from what site?
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Besides endocarditis, what other infection is Cardiobacterium hominis associated with?
Besides endocarditis, what other infection is Cardiobacterium hominis associated with?
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What is a characteristic feature of Eikenella corrodens on agar plates?
What is a characteristic feature of Eikenella corrodens on agar plates?
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Eikenella corrodens is a common cause of bite wounds.
Eikenella corrodens is a common cause of bite wounds.
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Kingella species are commonly found in the respiratory tract of children.
Kingella species are commonly found in the respiratory tract of children.
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What is the most common Kingella species?
What is the most common Kingella species?
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Capnocytophaga species are often isolated from which site?
Capnocytophaga species are often isolated from which site?
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Pasteurella multocida is a major cause of infections in humans.
Pasteurella multocida is a major cause of infections in humans.
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Pasteurella multocida is a fastidious organism.
Pasteurella multocida is a fastidious organism.
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Which of these is NOT a characteristic feature of Pasteurella multocida?
Which of these is NOT a characteristic feature of Pasteurella multocida?
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Pasteurella multocida infections are most commonly respiratory in nature.
Pasteurella multocida infections are most commonly respiratory in nature.
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Study Notes
Gram-Negative Coccobacilli
- Gram-negative bacteria have a thin peptidoglycan layer in their cell wall
- The organisms, including Haemophilus influenzae and other related organisms, are frequently isolated from the body system.
- Haemophilus influenzae is commonly encountered, clinically significant microorganisms by that system
- Most of these organisms do not grow on MAC (Modified American Culture)
- Haemophilus influenzae species are isolated in humans including H. influenzae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, H. aphrophilus, H. paraaphrophilus, H. segnis, H. ducreyi, H. aegyptius (part of H. influenzae biogroup)
- Gram-negative coccobacilli are facultative anaerobes.
Haemophilus - General Characteristics
- Haemophilus are Greek words for "blood lover"
- Most species are part of the normal flora of the upper respiratory tract.
- Pathogenic species include Haemophilus influenzae, H. influenzae biogroup aegyptius, and H. ducreyi (genital tract pathogen)
- Optimal growth for most species involves 5-10% CO2 at 35 +/- 2 degrees Celsius
- H. aphrophilus requires CO2.
Haemophilus – Nutritional Requirements
- Fastidious bacteria do not grow on routine media like BAP, TSA, and MAC
- Require accessory growth factors found inside red blood cells (RBCs)
- X factor (hemin/hematin)
- V factor (nicotine adenine dinucleotide, or NAD)
- Some species (prefix "Para") only require the V factor
- Media with horse or rabbit blood provides necessary growth factors for differentiation
- Sheep blood contains X factor, but V factor must be supplied
- Sheep RBCs contain X and V factors with enzymes (NADase) for hydrolysis of the V factor. This needs a source for V factor
- Fastidious organisms can grow as satellites with a staph streak
- Staphylococcus aureus (Staph Streak), S. pneumoniae, Neisseria spp. naturally produce the V factor.
- Organisms requiring V factor grow near the organism supplying the V factor.
Haemophilus influenzae – Nutritional Requirements
- CHOC agar supplies both the X and V factors
- CHOC (Chocolate) media is an enriched, non-selective media that includes a nutritional base, heated RBCs, or other supplements, meant for fastidious microorganisms
- The RBCs are lysed during the heating, releasing factors while deactivating NADases
- Haemophilus species grow happily in CHOC agar.
Haemophilus influenzae
- Erroneously named during 1889–1890 influenza pandemic
- Frequently isolated from infected patients with influenza and postmortem lung cultures.
- H. influenzae is a secondary opportunistic invader, not the causative agent of influenza
- Multiple strains characterized by, or without, capsule production (typeable or non-typeable strain)
Haemophilus influenzae – Virulence Factors
- Capsule is only found in typeable strains
- Six polysaccharide capsular types (a-f), with type B being most common prior to the Hib vaccination
- Commonly implicated in meningitis in children
- IgA protease destroys secretory IgA
- Adherence by fimbriae and other structures is mostly seen in non-typeable strains and allows the organism to adhere and cause localized damage
- Lack of HiB adhesion helps prevent systemic infections
- Outer membrane proteins and LPS
Haemophilus influenzae – Infection
- Infections are most common in children
- Mode of infection: Organism enters via nasopharynx and may remain transiently (asymptomatic carriers)
- Can enter bloodstream for an invasive infection
- Two patterns:
- Invasive disease caused by encapsulated strains
- Localized infection by non-encapsulated strains
- Invasive diseases include bacteremia, septicemia, meningitis, arthritis, Epiglottitis, Tracheitis, and pneumonia
- Localized infections include in respiratory tract, conjunctiva, sinus, otitis media
Haemophilus influenzae – Specimens
- Specimens of choice include blood, CSF, sputum, and conjunctival swabs
- Specimens should be transported at room temperature without delay to prevent organism death.
Haemophilus influenzae – Colonial Morphology
- On CHOC agar: colonies are large, translucent, tannish, moist, smooth, and convex with a distinct pungent/chemical odor (mousey or bleach-like)
- Encapsulated strains appear more mucoid
- On BAP:
- No staph streak - no growth
- With staph streak - Satellite colonies
- Colonial morphology is a key factor in identifying the species.
Haemophilus influenzae – Cellular Morphology
- Organisms have pleomorphic filaments, singles, and varied appearance as tiny gram-negative coccobacilli (coccobacilli)
- Clear nonstaining areas (halos) are visible, indicative of capsules
- Staining can sometimes be faint, requiring microscope cleanliness to ensure visibility.
Haemophilus influenzae – ID (Identification)
- Identification requires recognizing tiny gram-negative and pleomorphic coccobacilli on CHOC agar
- Growth will only happen on CHOC agar; not on MAC or BAP, unless staph streak is applied to BAP, resulting in satellite colonies
- Oxidase is positive
- X and V factor test shows growth around X+V discs only
- Porphyrin test (ALA) is negative
- Catalase is positive
- Latex agglutination/coagglutination kits are useful to detect Hib antigens.
- Chromogenic tests (API NH) can also be used.
- Staph streak tests are critical to identify if organisms are V factor dependent
Haemophilus influenzae – ID (X and V Factor)
- Purpose: Identify the growth requirements of Haemophilus species
- Principle: A clear agar plate missing X and V factors is inoculated with the target organism. 3 nutrients(X factor, V factor) are added separately (as discs or filter paper)
- Results: Observations if growth appears around each factor is informative of the needs of the organism.
Haemophilus influenzae – ID (ALA Test)
- Purpose: Differentiation of Haemophilus spp.
- Principle: Determining if an isolate needs X factor; organisms that do not require X factor convert aminolevulinic acid (ALA) to hemin (producing hemin)
- Procedure: A tube of ALA or an impregnated disk, or broth is inoculated with the organism
- Results:
- Positive: Fluorescence observed under ultraviolet light
- Negative: No fluorescence
- Quality Control:
- Positive: Haemophilus parainfluenzae
- Negative: Haemophilus influenzae
Haemophilus influenzae – Treatment
- Most strains (over 35%) produce beta-lactamase, leading to resistance to ampicillin (drug of choice for meningitis and bacteremia)
- Beta-lactamase testing is crucial
- Antimicrobial treatment for beta-lactamase-producing strains include: cefotaxime, ceftriaxone, cefuroxime
- Alternate treatments include: trimethoprim/sulfamethoxazole (SXT), imipenem, ciprofloxacin (in conjunction with ampicillin).
Haemophilus influenzae – Vaccination
- Routine Hib vaccination is recommended for children under 5 years old. Needs multiple shots
- Before vaccination, Hib was a leading cause of invasive disease and acute bacterial meningitis in children under 2 years old).
Haemophilus influenzae biogroup aegyptus
- Subgroup of H. influenzae, closely related to each other. Difficult to distinguish.
- Infections include acute, contagious conjunctivitis ("pink eye")
- Brazilian purpuric fever, with recurring conjunctivitis and sudden onset of fever, rash, septicemia, shock, vascular damage
- High mortality rate (70%) within 48 hours and localized to South America
Haemophilus ducreyi – Infection
- Not part of the normal flora
- Infection: Genital ulcer disease (GUD), chancroid ("soft chancre"), highly contagious sexually transmitted disease.
- Characterized by painful, suppurative ulcers or buboes (swollen lymph nodes, especially in groin).
- Men show inguinal tenderness and genital lesions, whereas most women remain asymptomatic.
- More common in Latin America, Africa, and Asia.
- Specimens of choice include material from the ulcer and aspirates of infected lymph nodes (pus)
Haemophilus ducreyi – Cellular Morphology
- Tiny gram-negative coccobacilli (GNCB) arranged as "school of fish".
Haemophilus ducreyi – Culture and ID
- Requires enriched CHOC media
- Specimens are inoculated immediately
- Incubation in high humidity with CO2 at 35° C for up to 7 days
- X and V Factors result in growth around X factor only
- Catalase is negative
- Oxidase is negative
- Porphyrin (ALA) test is negative.
Other Haemophilus species
- H. parainfluenzae, H. haemolyticus, and H. parahaemolyticus are normal members of the upper respiratory tract flora.
- H. aphrophilus and H. paraphrophilus are typically found in the oral cavity.
- These nonpathogenic species are sometimes involved in infections such as endocarditis, respiratory tract infections, abscesses (in brain), bacteremia, urethritis, and pneumonia.
HACEK Group
- Gram-negative bacilli that do not grow on MAC.
- Fastidious, requiring increased CO2 (5-10%) for growth
- Significant cause of endocarditis
- Normal flora of the oral cavity, sometimes associated with human bites. This leads to septicemia and subacute endocarditis
- H. spp. (Other than H. influenzae and H. ducreyi) are typically nonpathogenic, but can be involved in infections
- Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans), Cardiobacterium hominis, Eikenella corrodens, and Kingella spp.
Aggregatibacter aphrophilus
- Recall from the Greek words aphros and philia for "foam loving"
- Found in dental plaque and gingivals scrapings.
- Colonial morphology: yellow, granular, convex colonies with an opaque zone near the centers
- Nonhemolytic on BAP (Blood Agar Plate).
Aggregatisbacter actinomycetemcomitans
- Primarily an animal pathogen found in oral cavities.
- Causes human tissue infections from animal bites/contact with infected animals.
- Six serotypes (a–f), with a, b, and c being most common.
- Major contributor to periodontitis.
Aggregatibacter actinomycetemcomitans – Cultural Characteristics
- Requires 24 to 48 hours to grow at 35°C with increased CO2.
- Characteristic star formation in the center of colonies; best viewed under ×100 magnification.
Aggregatibacter actinomycetemcomitans – ID and treatment
- Ferment carbohydrates in serum
- Not lactose or sucrose
- Catalase positive
- Variable Oxidase
- Negative for X and V growth factors.
- Negative for urease, indole, esculin, and citrate
- Treatments include aminoglycosides, third-generation cephalosporins, quinolones, chloramphenicol, and tetracycline.
Cardiobacterium hominis – General Characteristics
- Normal flora of the nose, mouth, and throat
- Commonly present in the GI tract
- Infection: Endocarditis(Can be found on the heart valve and is resistant to antibiotics).
Cardiobacterium hominis – Cellular Morphology
- Gram negative, pleomorphic and nonmotile bacilli
- Might show rosettes, swellings, or long filaments.
Cardiobacterium hominis – ID and Treatment
- Slow growth on BAP and CHOC
-Biochemical reactions:
-Oxidase is positive
-Catalase is negative
- Indole is positive -Negative for urease, nitrate, gelatin, and esculin -Ferments sucrose -Usual therapy: Penicillin and aminoglycosides.
Eikenella corrodens – Infections
- Normal flora of the oral and bowel cavities
- Bite wounds
- Infections include meningitis, pneumonia, osteomyelitis,arthritis, postoperative infections from bacteremia in wounds (cellulitis) and inoculation from needles/sharp instruments ("licked clean").
Eikenella corrodens – Cellular Morphology
- Gram-negative coccobacilli.
Eikenella corrodens – Colonial Morphology
- Flat, pitted colonies on BAP and CHOC
- Nonhemeolytic on BAP
- Bleach-like odor is associated with yellow colonies that pit the agar.
- Yellow pigment is often present.
Eikenella corrodens – Growth and ID
- Grow in capnophilic environment
- Nonmotile
- Nonfermenter
- Oxidase positive; catalase negative
- Lysine decarboxylase positive
- Ornithine decarboxylase positive
- Arginine dihydrolase negative
- Requires hemin (X factor) and CO2.
Kingella spp.
- Four species in this genus
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K. kingae
- Part of HACEK; associated with endocarditis in immunocompromised patients
- Isolates were found in blood, bone, joint fluid, wounds.
- Associated with endocarditis, poor dental hygiene, and oral surgery
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K. oralis K. potus K. denitrificans
- Important in pediatric cases with a predilection for bones and joints
- Causes osteoarthritis infections in children under 4 years old.
Kingella – General Characteristics
- Gram stain: Coccobacilli (or short bacilli with square ends) in pairs or chains
- Resist decolorization
- Nonmotile
- Oxidase positive; catalase negative
- Sugar fermenters (without gas production)
- Usually susceptible to most agents.
Kingella kingae – Colonial Morphology
- Grows on BAP, CHOC, + Neisseria media
- Can be confusing with Neisseria, but Gram staining helps differentiation
- Colony types
- Spreading, corroding colony
- Smooth, convex, and β-hemolytic
- Hemolysis may occur beneath colony or in proximity to it 24/48 Hours after incubation
Kingella kingae – ID requirements (according to CLSI)
- Gram-negative short coccobacilli, forming large white to beige, β-hemolytic colonies on SBA
- No growth on MAC
- Catalase is negative
- Oxidase is positive
Capnocytophaga – General Characteristics
- Group of fastidious, facultatively anaerobic, gram-negative bacilli.
- Normal flora in the oral cavity
- Infections: Septicemia;
- Neutropenic patients (oral ulcers, soft tissue infections, peritonitis, endocarditis)
Capnocytophaga – Cultural Morphology
-Thin, pointed ends GNB
Pasteurella - General Characteristics
- Many species, most relevant is Pasteurella multocida.
- Has similar characteristics to Haemophilus
- Infections (Pasteurellosis): Acquired from contact with infected animals or their products; zoonotic disease
- Primarily caused by animal bites (often cats), including respiratory tract and oral cavity of birds and mammals.
Pasteurella – Infection
- Most common presentation: soft tissue infection
- Injuries can be aggressive, with skin manifestations typically appearing within 24 hours of a bite
- Wounded can rapidly develop soft tissue inflammation
- Soft tissue infections can progress into deep tissue infections
- Rarer presentations: systemic infections, like septic arthritis, osteomyelitis, tenosynovitis, endocarditis, septicemia, meningitis, pneumonia.
Pasteurella – Cellular Morphology
- Can be easily confused with Haemophilus
- Gram-negative coccobacilli (ovoid, filamentous, or bacilli).
- Bipolar staining (resembles safety pins)
- End of bacilli retains more stain.
Pasteurella – Colonial Morphology
- Not fastidious; grows on both BAP and CHOC.
- Growth on BAP distinguishes it from Haemophilus exhibiting a "musty" odor similar to wet dog.
- Colonies are non hemolytic, grey, and have mucoid and green/brown halos on older colonies.
Pasteurella – ID
- Nonmotile
- Catalase and Oxidase positive (most isolates)
- Indole positive
- Ornithine positive
- Urease negative
- Glucose fermentation with weak acid production.
Haemophilus vs. Pasteurella
| Feature | Haemophilus | Pasteurella |
|---|---|---|
| Growth on MAC | Negative | Negative |
| Growth on BAP | Negative | Positive-mostly with a mousy odor |
| Growth on CHOC | Positive | Positive |
| Colonial Morphology | Small GNCB | Small GNB- possible bipolar staining, |
| Odor | Pungent/chemical | Musty odor |
| Source/infections | Respiratory tract (can become systemic) | Wounds(rarely respiratory) |
Knowledge Check Questions and Answers
-
(These questions are not specific and are simply grouped.)*
-
Q: Which characteristics are NOT defining for Haemophilus influenzae?
-
A: Growth around staph streak on blood agar, growth around XV disc (without being X or V), Colonies on CHOC agar. Gram negative coccobacilli is correct.
-
Q: What Haemophilus species causes soft chancres, showing a school of fish cellular morphology?
- A: H. ducreyi.
-
Q: Which Haemophilus species causes Brazilian purpuric fever?
- A: H. aegyptius.
-
Q: Which of the options is NOT a HACEK organism?
- A: Haemophilus influenzae.
-
Q: Which organism should be suspected in soft tissue infections following animal bites?
- A: Pasteurella multocida.
-
Q: Which of these is NOT a HACEK organism?
- A: Haemophilus influenzae.
-
Q: What is a differentiating characteristic between Haemophilus influenzae and Pasteurella multocida?
- A: Musty odor.
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Description
This quiz explores the characteristics and significance of gram-negative coccobacilli, particularly focusing on the Haemophilus genus. Understand their role as part of normal flora, pathogenic species, and laboratory isolation challenges. Dive into the unique traits of these bacteria and their clinical relevance.
