Intracellular replication of Legionella pneumophila

Legionella pneumophila was inoculated onto tissue culture monolayers of MRC-5, HeLa, Hep 2, and McCoy cells. Fresh and conditioned medium without cells served as controls. Cultures were harvested at various times after inoculation, serial log10 dilutions were performed, and growth of L. pneumophila was quantitated either by direct immunofluorescence or by counting colony-forming units on Feeley-Gorman agar. Growth in monolayers was demonstrated using both techniques. Within 72--96 hr after inoculation, supernatants of infected cells became cloudy, and the cells demonstrated cytopathic changes. In the medium controls, titers of L. pneumophila remained constant by immunofluorescence and declined by counting colonies on agar. When L. pneumophila was inoculated onto monolayers and gentamicin was added 3 hr later, organisms were recovered from the cells but not from supernatants. Transmission electron microscopy revealed intracytoplasmic organisms. These studies indicate that L. pneumophila acts as an intracellular pathogen in tissue cultures, and this characteristic may offer a simple method for studying growth and pathogenicity of this unusual bacillus in humans.