Epithelial cell invasion by Staphylococcus aureus

Previous work has shown Porphyromonas gingivalis contains two subpopulations, one able to invade epithelial cells to a high level and a second showing poor invasion. However it is unknown whether this applies to other invasive bacteria. Objectives: The aim of this study was to investigate the invasion of oral keratinocytes by a facultative aerobe, Staphylococcus aureus. Methods: S.aureus Oxford (107 cfu/ml) was incubated with a monolayer of H357 oral keratinocytes (105 cells/well). Non-invading bacteria were killed by gentamicin treatment (100µg/ml). Intracellular bacteria were recovered by cell lysis and the percentage invasion calculated by viable counting. This is termed “first round invasion”. Invasive and non-invasive bacteria recovered following first round were directly used in a second assay termed second round invasion. Preliminary invasion assays testing the effects of growth phase and the stability of changes in phenotype were also conducted. Results: S.aureus Oxford showed a low level of invasion in the first round (1.24% ± 0.92). In a second round of infection, invasive bacteria showed an average 3.5 fold increase in invasion to 4.43% ± 0.37. Non-invasive bacteria when used in a second round assay demonstrated only 0.18% ± 0.002 invasion. Subculture of bacteria from the “hyper-invasive” subpopulation followed by an antibiotic protection assay showed a mean invasion of 2.33% ± 2.8 indicating that this phenotype is unstable. Little variation in invasion was seen between bacteria in log or stationary growth phase. Conclusions: These data indicate that in the presence of epithelial cells an S.aureus culture contains invasive and non-invasive subpopulations. However the phenotype these subpopulations appears to be transient. The growth phase does not dictate the invasive capability or the proportion of each subpopulation within a culture. These data suggest that the existence of an invasive subpopulation within a bacterial culture may be relatively general rather than species specific.