Gram-Negative Bacteria Are Internalized Into Osteocyte-Like Cells.

While Gram-positive organisms are the most common causative agent of initial bone infections, the percentage of Gram-negative species increases in reoccurring bone infections. As bacterial internalization has been suggested as one cause of reoccurring bone infection, we tested the hypothesis that Gram-negative species of bacteria can be internalized into bone cells. Using the MLO-A5 and the MLO-Y4 cell lines as our cell models, we demonstrated that the Gram-negative species, Proteus mirabilis and Serratia marcescens, can be internalized in these cells using an internalization assay. This rate at which these two species were internalized was both time- and initial concentration-dependent. Confocal analysis demonstrated the presence of internalized bacteria within both cell types. Inhibition of the cellular uptake with methyl-β-cyclodextrin and chloroquine both reduced internalized bacteria, indicating that this process is, at least in part, cell mediated. Finally, we demonstrated that the presence of internalized P. mirabilis did not impact cell viability, measured either by lactate dehydrogenase (LDH) release or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity, while the presence of S. marcescens, on the other hand, both increased LDH release and reduced MTT activity, indicating a loss of cell viability in response to the organism. These results indicated that both species of Gram-negative bacteria can be internalized by bone cells and that these internalized bacteria could potentially result in reoccurring bone infections.