Plaque formation by virulent Shigella flexneri

Abstract

An in vitro tissue culture plaque assay was developed to investigate the intracellular replication and intercellular spread of virulent shigellae. Shigella plaques were formed in HeLa cell monolayers in the presence of an agarose overlay containing tissue culture medium and gentamicin, which eliminated extracellular bacterial growth. Microscopically, the plaques were characterized by a central area of dead host cells surrounded by cells infected with shigellae. Cells further away from the plaque center were uninfected. Inclusion of chloramphenicol or nalidixic acid in the overlay completely abolished plaque formation. Plaque formation was completely inhibited when infected monolayers were shifted from 37 to 30°C. Shifting infected monolayers from 30°C, where plaques do not form, to 37°C resulted in the formation of plaques. Cultures of Shigella boydii, Shigella sonnei (form I), and all six serotypes of Shigella flexneri produced plaques. Shigellae isolated from plaques were Sereny test positive, contained a 140-megadalton plasmid, and were gentamicin sensitive. Noninvasive shigellae did not form plaques.