Introduction: While the most common microbe responsible for bone infections is Staphylococcus aureus, gram-negative bacteria have also been shown to be involved in bone infections, with their contributions increasing in chronic bone infections. Bone cells have been shown to die following exposure to Staphylococcus aureus biofilm-conditioned media. It is suggested that gram-negative bacteria may also produce a similar effect. To test this hypothesis, we exposed a pair of immortalized osteocyte-like cells, MLO-Y4 and MLO-A5 cells to media conditioned by biofilms of two strains of gram-negative bacteria.

Objective: To measure and analyze the amount of MLO-Y4 and MLO-A5 cell death in response to incubation with biofilm-conditioned media (BCM) produced by two gram-negative bacterial species, Proteus mirabilis or Serratia marcescens.

Methods: BCM was generated by taking a 1 McFarland dilution of P. mirabilis or S. marcescens, centrifuging it and combining it with filtered Serum-Free Alpha Minimal Essential Media. This biofilm solution was incubated overnight and subsequently filtered to remove all bacteria. MLO-Y4 and MLO-A5 cells were plated in 24-well plates and exposed to 100%, 50%, 25%, 10%, or 5% BCM for 1 or 4 hours. Following this incubation, the BCM was removed and cell death assessed with an MTT assay.

Results: P. mirabilis and S. marcescens BCM, both, reduced cellular viability for both cell types. 100% BCM produced similar cell death percentages for both cell types, while lower concentrations showed evidence of a slightly greater effect on the viability of S. marcescens in both cell types. Interestingly, MLO-Y4 cells showed a greater sensitivity to cell death at the earlier time point. There was little change in sensitivity after 4 hours for the MLO-Y4 cells, while the MLO-A5 cells showed significantly increased cell death at 4 hours.

Conclusion: BCM, from both bacteria, cause a reduction in cell viability. This reduction is accentuated in MLO-A5 cells with time, while it remains static in the MLO-Y4 cells. To our knowledge, this is the first study that demonstrates the effect of gram-negative bacteria biofilms on osteocyte viability. Further research should include these osteocytes exposed to planktonic conditioned media (PCM) of P. mirabilis and S. marcescens.