Interplay between in vitro multispecies biofilms and human polymorphonuclear neutrophils

Method: Calgary Biofilm Devices (CBD) were seeded with 10⁶ cells of A.naeslundii, and incubated anaerobically at 37°C.  After 48 hours, pegs were transferred to plates containing fresh media and 10⁶ cells of S.gordonii.  The next day pegs were transferred into an inoculum of 10⁶ cells of V.parvula and F.nucleatum ss. polymorphum and by the 5^th day, early biofilms were exposed to 10⁶ cells of P.gingivalis.  With this method, layered 5-species biofilms formed on the pegs of the CBD.  Human PMNs were isolated using Ficoll-Hypaque density gradient centrifugation, counted and exposed to biofilms at 3 different multiplicity of infection (MOI) ratios for 6 and 24 hours.  The experiment was repeated 3 times with each condition in triplicate.  The levels of the 5 species were assessed using checkerboard DNA-DNA hybridization.  Levels of IL-1β, IL-8, and MMP-8 were measured using Luminex; myeloperoxidase (MPO) using ELISA; and Elastase activity using an enzymatic assay.  Statistical analyzes were conducted using the Kruskal-Wallis and the Dunn’s Multiple Comparison tests.

Result: All species were present in the biofilms.  Levels of A.naeslundii (p<0.05), F.nucleatum ss. polymorphum (p<0.001), S.gordonii (p<0.001), and P.gingivalis (p<0.01) were significantly reduced after 6 hours of exposure to PMNs at the lowest MOI, compared to control biofilms not exposed to PMNs.  The impact of PMNs on levels of biofilm species after 24 hours of incubation was much smaller, and further growth of the biofilms could be observed.  The level of all PMN biomarkers increased significantly with decreased MOI ratios at both time points (p<0.01).

Conclusion: PMNs were capable of killing subgingival species grown in biofilms but could not fully eliminate all microorganisms and additional biofilm growth could be detected after 24 hours of exposure to phagocytes.