title: A novel flow cytometry-based assay for the quantification of Staphylococcus aureus adhesion to and invasion of eukaryotic cells.
abstract: Flow cytometry is a powerful tool for analyzing the adhesion to and invasion of Staphylococcus aureus (S. aureus) to eukaryotic cells. Established techniques have used bacteria that have been genetically modified to express fluorescent proteins or directly labeled with fluorochromes prior to infection. Such approaches are appropriate in most cases; however, the use of genetically or chemically altered bacteria could introduce a bias when measuring fine differences in adhesion and invasiveness. Here, we describe a combined flow cytometry-based invasion and adhesion assay that does not require the processing of bacteria prior to internalization. This method was performed on osteoblastic MG-63 cells infected with S. aureus reference strain 8325-4 and its invasion-deficient isogenic mutant, which carries deletions in the genes encoding fibronectin-binding proteins A and B. The data from this assay were compared to those obtained using the standard gentamicin protection assay. The results obtained by the two methods were consistent. Moreover, quantification of internalized bacteria was more reproducible using the flow cytometry-based assay than the gentamicin protection assay, which allowed for the simultaneous quantification of host cell adhesion and invasion.
authors.affiliation: INSERM U851, National Reference Center for Staphylococci, University of Lyon, 8 rue Guillaume Paradin, 69008 Lyon, France. trouilletsophie@aol.com
authors.forename: Sophie
authors.initials: S
authors.lastname: Trouillet
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Rasigade
authors.forename: Yannick
authors.initials: Y
authors.lastname: Lhoste
authors.forename: Tristan
authors.initials: T
authors.lastname: Ferry
authors.forename: François
authors.initials: F
authors.lastname: Vandenesch
authors.forename: Jerome
authors.initials: J
authors.lastname: Etienne
authors.forename: Frederic
authors.initials: F
authors.lastname: Laurent
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics; D001422:Bacterial Adhesion; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D017353:Gene Deletion; D006801:Humans; D010006:Osteoblasts / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000235:genetics / Q000472:pathogenicity*; D037521:Virulence Factors / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; D037521:Virulence Factors; C078635:fibronectin-binding proteins, bacterial