Optimization of the Amoeba Plate Test for the Isolation of Legionella and Other Opportunistic Pathogens from Environmental Water Samples

Free-living amoebae represent a reservoir for some environmental bacteria that are opportunistic human pathogens. Legionella pneumophila is a Gram-negative, amoeba-resistant bacterium, which upon inhalation can cause a life-threatening atypical pneumonia termed Legionnaires’ disease. The capacity to grow within amoebae and other protozoa is a prerequisite for L. pneumophila virulence and requires the bacterial Icm/Dot type IV secretion system (T4SS). The amoebae plate test (APT) exploits the capacity of virulent rather than Icm/Dot-deficient L. pneumophila to grow on BCYE agar plates in the presence of amoeba. Here, we apply the APT to different Legionella species and optimize the test by varying the amoeba concentration, growth temperature, agar plate culture media, and additives such as fungicides. The optimized APT enriches amoeba-resistant environmental bacteria and suppresses other microbial flora. Accordingly, the APT allows the identification in complex environmental water samples of Legionella species and further opportunistic pathogenic bacteria such as Delftia acidovorans, a rare causative agent of sepsis and pneumonia.