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Screening-level assays for potentially human-infectious environmental Legionella spp.
LAU, H. Y., A. BREHM, J. W. SANTO-DOMINGO, AND N. ASHBOLT. Screening-level assays for potentially human-infectious environmental Legionella spp. The Journal of Microbiology . Springer, New York, NY, 49(2):200-207, (2011).
The primary objective of the biofilm Legionella study is to determine if there is any correlation between the ability of Legionella isolates to invade and replicate within amoebal hosts and their in vivo virulence utilizing a murine model of acute bacterial pneumonia (6-8 week old A/j mice). If such a correlation is demonstrated, then a second objective will be development of an assay for the rapid identification of potentially pathogenic environmental isolates of Legionella (and latter, other intracellular bacterial pathogens) via co-culturing with an “indicator” amoebal host rather than infecting mice. Subsequent research will then focus on better understanding the stability and ecology of Legionella spp. in biofilms and the effect(s) of key stressors to select for virulent biotypes. For the development of a biofilm-like sampling device, the initial objective is to evaluation representative mineral iron oxyhydroxides (alpha- and gamma-FeOOH) and an iron oxide (Fe3O4) for their ability to sorb pathogen surrogates (MS2, E. coli and Saccharomyces cerevisiae representing viral, bacterial and parasitic protozoan pathogens respectively). Then, to evaluate these mineral phases on coated glass beads and glass wool by pilot plant distribution system challenges (1 h intrusion events for the same three surrogates & followed over one month) and at full-scale under normal operation of Cincinnati’s distribution system.
In spite of the fact that Legionella species can be isolated from nonclinical settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. In this study, an in vivo murine model of pneumonia and three in vitro proliferation assays using Acanthamoeba polyphaga cells, along with THP-1 human macrophage and J774 murine macrophage cell lines were evaluated to screen for the pathogenicity of various Legionella strains. Two clinical (L. pneumophila and L. longbeacheae) and two environmental (L. dumoffii and L. maceachernii) isolates were tested in this study. For the in vivo assay, A/J mice were intranasally infected independently with the different isolates. At 12 h post infection (p.i.), there were high titers of bacteria in the lungs of the Legionella infected mice. L. pneumophila and L. dumoffii densities were higher than L. longbeacheae and L. maceachernii levels in the lungs at 24 h p.i. However, only L. pneumophila persisted in the lungs of mice after 48 h, indicating that the other isolates were rapidly cleared from the pulmonary airspace. Results from the in vitro assays showed that L. pneumophila multiplied within A. polyphaga, THP-1 and J774 cells after 72 h. Although to a lesser degree than L. pneumophila, L. dumoffii also multiplied within A. polyphaga and THP-1 cells suggesting that it may be pathogenic. The results of this study demonstrate the value of using a multi-tier approach to identify potentially pathogenic Legionella strains. The results also suggest that A. polyphaga parasitism and macrophage cell infectivity may be useful screening tools for potentially human-infectious Legionella spp., as a result decreasing the amount of animals that need to be sacrificed in virulence screening studies.