Plaque assay for virulent Legionella pneumophila

Abstract

Methods of assessing virulence of Legionella pneumophila, the etiologic agent of Legionnaires disease, include the infection of guinea pigs, fertile chicken eggs, and mammalian and protozoan cell cultures. Guinea pig assays, in particular, are expensive, laborious, or unsuitable for routine screening of Legionella isolates. We have developed a virulence assay that requires the enumeration of viruslike plaques which are the result of virulent L. pneumophila infecting mouse L929 cells. Each plaque is the consequence of the initial infection of an L cell with a single bacterium. A nonvirulent mutant derived from the serial passage of virulent L. pneumophila on Mueller-Hinton agar fails to survive within L cells and consequently fails to produce plaques.

Multiplication of Legionella pneumophila in HeLa Cells in the Presence of Cytoskeleton and Metabolic Inhibitors

“…The bacterium also multiplies intracellularly within protozoa, including ciliates and amoebae (2,9,30). Various studies have described the growth of L. pneumophila in several types of non-professional phagocytic cells (MRC-5, Hep-2, McCoy, Vero, L929, HeLa) in tissue cultures (5,6,8). Virulent strains of L. pneumophila invade cells by receptor-mediated endocytosis and survive intracellularly due to failure of lysosomal migration and fusion with phagosomes (17).…”

Demonstration of spread by Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayers

“…In addition, detection of GFP provides for enhanced assay sensitivity, allowing detection of a single bacterium or few clustered bacteria in locations throughout the monolayer. This model can also be used to select clones with decreased abilities to attach, internalize or multiply intracellularly as exem-pli¢ed with Legionella pneumophila and L. monocytogenes when mutants produced plaques either smaller or larger than those produced by infections with wild-type bacilli [17,20].…”

Passage through Tetrahymena tropicalis enhances the resistance to stress and the infectivity of Legionella pneumophila

“…To determine whether the MIFs produced in Tetrahymena have similar phenotypes as those emerging after replication in amoeba, we tested sensitivity to gentamicin, an antibiotic extensively used to eliminate extracellular growth of a variety of intracellular bacterial pathogens in cell culture systems (Kihlstrom, 1977;Isberg & Falkow, 1985;Fernandez et al, 1989). Bouyer et al (2007) have studied Legionella-containing vesicles released from amoebae.…”