Polymicrobial Intracellular Infections of Buccal Epithelial Cells from Human Subjects

Methods: Three mixtures were prepared. Each contained a red fluorescent (AlexaFluor 594™) universal probe (Eub338), paired with green fluorescent (AlexaFluor 488™) versions of either the Aa-, Pg-, or Bf-specific probes. Each mixture was hybridized with BEC from each of 37 healthy human subjects. Red and green versions of the complement to Eub338 were used as negative controls. Confocal microscopy was used to determine whether labeled bacteria were intracellular. Z-sections imaged at the red and the green wavelengths were superimposed to determine co-localization of labeled bacteria.

Results: The universal probe detected intracellular bacteria in every subject. BEC with intracellular Aa, Pg, and Bf were detected in 30, 28, and 27 subjects. Each BEC that was labeled with a species-specific probe also contained additional bacteria recognized only by the universal probe. In superimposed images, bacteria labeled with the specific probe often occupied smaller defined regions within larger masses of bacteria.

Conclusions: In these subjects, intracellular infections of BEC with periodontal pathogens were uniformly polymicrobial. This suggests that future studies of oral mechanisms of bacterial invasion, intracellular growth, and host cell response may need to be done with microbial consortia.

Supported by NIH grant R01 DE 14214.