Haemophilus and fastidious gram-negative bacilli

10 DOH Abu Dhabi (HAAD) lab licensing practice questions on Haemophilus and fastidious gram-negative bacilli, part of Microbiology. The first 10 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Hard

   Blood cultures from an unvaccinated 18-month-old with meningitis grow small gram-negative coccobacilli. They grow on chocolate agar but not on sheep blood agar, except as tiny colonies beside a Staphylococcus aureus streak. For the X and V factor test, a heavy suspension is made from the chocolate agar growth and spread on a nutrient agar plate with X, V and XV strips. After overnight incubation in CO2, there is growth around the V and XV strips but not around the X strip. The porphyrin (ALA) test shows no fluorescence and no red color. The isolate is non-hemolytic on horse blood agar. Which identification is correct?

   1. AHaemophilus influenzae
   2. BHaemophilus haemolyticus
   3. CHaemophilus ducreyi
   4. DHaemophilus parainfluenzae

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   Answer: A · Haemophilus influenzae

   The negative porphyrin test shows the isolate cannot make heme from delta-aminolevulinic acid, so it needs X factor, and the satellitism shows it needs V factor. Its apparent growth with V alone is a known pitfall: a heavy inoculum taken from chocolate agar carries enough hemin to support growth without the X strip. The porphyrin test is not affected by this carryover and is the more reliable test. An X- and V-dependent, non-hemolytic isolate is H. influenzae. A light suspension, with no chocolate agar picked up, avoids the error.

   • B: H. haemolyticus is beta-hemolytic on horse blood agar.
   • C: H. ducreyi needs X factor but not V factor, and grows only on special enriched media.
   • D: H. parainfluenzae needs only V factor and is porphyrin positive.

2. Question 2 · Medium

   Why does chocolate agar support Haemophilus influenzae when sheep blood agar does not?

   1. AHeating concentrates the peptones in the base
   2. BHeating frees NAD and destroys the NADases
   3. CHeating raises the pH and frees the haemin
   4. DAdded starch takes up inhibitory substances in the agar

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   Answer: B · Heating frees NAD and destroys the NADases

   Gentle heating of blood in the agar base lyses the red cells and releases both haemin and nicotinamide adenine dinucleotide into the medium, and it inactivates the NADases that otherwise destroy the coenzyme. Intact sheep red cells keep these factors inside and the NADases active, so Haemophilus influenzae does not grow on plain blood agar. The same heating gives the medium its brown colour; no chocolate is involved.

   • A: The peptone content of the base does not change with heating.
   • C: Chocolate agar is not made alkaline, and pH is not the mechanism.
   • D: Starch in Thayer-Martin-type media absorbs inhibitors, which is a separate point.

3. Question 3 · Hard

   Which conditions follow CLSI for disk diffusion on a Haemophilus influenzae isolate from CSF?

   1. AHTM agar, 35 °C in 5% CO2, 16 to 18 hours
   2. BChocolate agar, 35 °C in 5% CO2, 24 hours
   3. CMueller-Hinton agar with 5% sheep blood, 35 °C in air, 24 hours
   4. DMueller-Hinton agar, 30 °C in air, 20 to 24 hours

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   Answer: A · HTM agar, 35 °C in 5% CO2, 16 to 18 hours

   Haemophilus test medium is Mueller-Hinton base with yeast extract, haematin and NAD, which supports growth without clouding the zone edges, and it is the only agar with validated Haemophilus zone criteria. Plates are read after 16 to 18 hours at 35 °C in 5% carbon dioxide. Chocolate agar is excellent for isolation but has no interpretive criteria, and quality control uses H. influenzae ATCC 49247.

   • B: Chocolate agar grows the organism but is not a susceptibility medium.
   • C: Blood-supplemented Mueller-Hinton agar has no Haemophilus zone criteria.
   • D: Plain Mueller-Hinton agar does not support growth, and 30 °C is the wrong temperature.

4. Question 4 · Easy

   The V factor that some Haemophilus species need for growth is:

   1. ACysteine hydrochloride
   2. BPyridoxal hydrochloride
   3. CHaemin (protoporphyrin IX)
   4. DNicotinamide adenine dinucleotide

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   Answer: D · Nicotinamide adenine dinucleotide

   V factor is nicotinamide adenine dinucleotide, a coenzyme needed for respiration that these organisms cannot make. X factor is haemin, needed for the cytochromes. Haemophilus influenzae needs both, while H. parainfluenzae needs V factor only, and the pattern of requirement is the basis of routine speciation.

   • A: Cysteine is a supplement for Francisella media.
   • B: Pyridoxal is needed by the nutritionally variant streptococci, not by Haemophilus.
   • C: Haemin is the X factor, not the V factor.

5. Question 5 · Hard

   A Haemophilus influenzae isolate from sputum is nitrocefin negative, but the ampicillin MIC is 4 mg/L (CLSI: susceptible 1 or less, resistant 4 or more). The best interpretation is:

   1. AAltered PBP3: a BLNAR strain
   2. BCarbapenemase production
   3. CAn inoculum heavier than the standard
   4. DA false negative nitrocefin test

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   Answer: A · Altered PBP3: a BLNAR strain

   Beta-lactamase negative ampicillin-resistant strains carry mutations in the ftsI gene that alter penicillin-binding protein 3, so they resist ampicillin without making an enzyme. The negative nitrocefin test is correct, and the MIC is what decides the report. Such strains must not be reported as susceptible to amoxicillin-clavulanate, because the inhibitor adds nothing when no beta-lactamase is present; a third-generation cephalosporin or a carbapenem is used instead.

   • B: Carbapenemases are not a recognised mechanism in this species.
   • C: A heavy inoculum would raise several MICs, and the pattern here is specific.
   • D: A nitrocefin test can be falsely negative, but the resistant MIC is explained by the PBP change.

6. Question 6 · Medium

   Haemophilus influenzae grows as tiny satellite colonies beside a Staphylococcus aureus streak on sheep blood agar because the staphylococcus:

   1. AProduces the carbon dioxide that Haemophilus needs
   2. BSecretes an iron-binding siderophore
   3. CReleases NAD and lyses red cells, freeing haemin
   4. DSuppresses the flora around its streak

   Answer and explanation in LearnFree account

7. Question 7 · Hard

   Blood cultures from a patient with suspected endocarditis who had antibiotics before admission are still negative at 5 days. Which test is most likely to identify the cause?

   1. AHolding the bottles for a further 21 days
   2. BAcridine orange staining of the bottles
   3. CRepeat blood cultures on chocolate agar
   4. DBroad-range 16S rRNA PCR on the excised valve

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8. Question 8 · Medium

   A negative porphyrin (delta-aminolevulinic acid) test shows that the isolate:

   1. ACannot make haem, so it needs X factor
   2. BNeeds 5% carbon dioxide to grow
   3. CProduces a beta-lactamase
   4. DCannot use NAD, so it needs V factor

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9. Question 9 · Medium

   Which step most improves recovery of Kingella kingae from the joint fluid of a toddler with septic arthritis?

   1. APlating the fluid on MacConkey agar
   2. BAdding colistin to the primary plate
   3. CInoculating the fluid into a blood culture bottle
   4. DIncubating the plates anaerobically

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10. Question 10 · Hard

    Blood cultures from a man with a prosthetic aortic valve flag on day 4. The smear shows pleomorphic gram-negative rods with swollen ends in rosettes; the isolate is oxidase positive, catalase negative and indole positive and grows on blood and chocolate agar in CO2. Which organism fits best?

    1. ACardiobacterium hominis
    2. BEikenella corrodens
    3. CAggregatibacter actinomycetemcomitans
    4. DHaemophilus parainfluenzae

    Answer and explanation in LearnFree account