Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 56, Issue 1, Pages 83-91Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2003.09.004
Keywords
invasion; intracellular proliferation; competitive index; mixed infections; intracellular pathogens; epithelial cells; macrophages; fluorescent proteins
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Epithelial cell lines are widely used as an in vitro model to study cell invasion by Salmonella. In turn, phagocytic cell lines are used to study Salmonella intracellular survival and proliferation. We describe a novel method, derived from the classical mixed infection procedure, to quantify invasion and proliferation defects in Salmonella enterica serovar Typhimurium. A eukaryotic cell culture is infected with two strains (e.g., a mutant and the wild-type). After infection, bacterial cells that remain extracellular are eliminated with gentamicin. At the end of the trial, intracellular bacteria are recovered and plated. Colonies from each strain are then counted for the calculation of a competitive index. Strain discrimination can be achieved either with antibiotic resistance markers or using plasmids encoding color markers (e.g., fluorescent proteins). Because both strains are exposed to the same conditions throughout the process, the procedure decreases the variability between independent trials and allows a direct measurement of the impairment of the mutant in invasion or intracellular proliferation. (C) 2003 Elsevier B.V. All rights reserved.
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