No Organisms Were Recovered On Buffered Charcoal Yeast Extract Medium After 2 Days of Incubation

Serum samples collected from a patient with pneumonia demonstrate a rising antibody titer to Legionella. A bronchoalveolar lavage (BAL) specimen from this patient had a positive antigen test for Legionella but no organisms were recovered on buffered charcoal yeast extract medium after 2 days of incubation. What is the best explanation?

Legionella BCYE

First of all, Legionella is a pathogenic group of gram-negative bacteria with the chief species being Legionella pneumophilia which is the causative agent of legionnaires disease. Legionella lives within amoeba in the natural environment. Transmission occurs via inhalation of water droplets from a contaminated source. Upon inhalation the bacteria infects alveolar macrophages where it begins to replicate. Legionella pneumophilia is a non-encapsulated aerobic bacillus with a single flagellum. It is unable to hydrolase gelatin or produce urease. Legionella is a non-pigment producer and does not auto fluoresce. It requires cysteine and iron to survive, thus media used to inoculate legionella must contain those two nutrients to grow.

The pathogenesis of legionella begins after an incubation period of up to two weeks. The initial prodromal symptoms are flu-like that include fever, chills and dry-cough. Through advancement of the disease the patient will experience gastrointestinal tract and the nervous system involvement. Pneumonia is also a common occurrence. The most at risk for infection with legionella are those that are immunocompromised, and the elderly. Legionella is also a prevalent nosocomial infection that has a fatality rate of 28%.

The medium of choice for when legionella is suspected is a buffered charcoal yeast extract agar (BCYE). The BCYE is a modified medium from the pre-existing F-G agar. Yeast serves as the protein source instead of casein. Beef extractives and starch are not added in the BCYE like they are in the F-G agar. Macroscopic colonies of legionella pneumophilia are visible on the BCYE after 3 days, but media should be incubated at 35-37 degrees Celsius for at least 7 days for a definitive answer. This is compared to the F-G agar where visible colonies were present after 4 days of incubation.

To answer the original question, the best explanation as to why colonies were present on the BCYE after 2 days is because the medium was simply not incubated long enough. It should be incubated for 7 days to make a definitive diagnosis even though colonies should start to appear after 3 days. The colonies on the BCYE should appear gray-white with a textured, cut glass appearance or opal.

-Caleb

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