Optimization\nof the Amoeba Plate Test for the Isolation\nof <i>Legionella</i> and Other Opportunistic Pathogens from\nEnvironmental Water Samples

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