Influence of methylene blue-mediated photodynamic therapy on the resistance to detachment of streptococcus mutans biofilms from titanium substrata
Sharab, Lina Y.
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In dental settings, as well as in other natural systems, plaque-forming microorganisms develop biofilms in which the microbes become protected via their own phenotypic changes and their polymeric exudates from disinfection by washes and antibiotics. Photodynamic Therapy (PDT) is variably effective against these microorganisms, depending on such factors as whether the bacteria are Gram positive or Gram negative, plaque age and thickness, and internal biofilm oxygen concentration. This investigation applied a novel combination of PDT and water-jet impingement techniques to Streptococcus mutans (ATCC strain 27351)-formed biofilms on commercially pure titanium (cpTi) starting with three different phases (ages) of the bacteria, to examine whether the detachment shear stress –as a signature for the work required for removal of the biofilms- would be affected by prior PDT treatment independently from microbial viability. Biofilms were grown with sucrose addition to Brain Heart Infusion media, producing visible thick films and nearly invisible thin films (within the same piece) having the same numbers of culturable microorganisms, the thicker films having greater susceptibility to detachment by water–jet impingement. Colony-forming-unit (CFU) counts routinely correlated well with results from a spectrophotometric Alamar Blue (AB) assay. Use of Methylene Blue (MB) as a photosensitizer (PS) for PDT of biofilms did not interfere with the AB assay, but did mask AB reduction spectral changes when employed with planktonic organisms. It was discovered in this work that PD-treated microbial biofilms, independently from starting or PS-influenced microorganism viability, were significantly (p<0.05) and differentially more easily delaminated and ultimately removed from their substrata biomaterials by the hydrodynamic forces of water-jet impingement. Control biofilms of varying thickness, not receiving PDT treatment, required between 144 and 228 dynes/cm 2 of shear stress to delaminate from titanium while PDT-treated companion biofilms were removed at 90 to 140 dynes/cm 2 , depending on water flow rate. In comparison, it required only between 57 and 68 dynes/cm 2 shear stress to separate microbial layers from within the exopolymer matrix of control biofilms, and between 39 and 51 dynes/cm 2 to delaminate PDT-treated matrix sections of varying thickness biofilms, again depending on water flow rate. Multiple Attenuated Internal Reflection InfraRed spectra of identical biofilms, grown on germanium prisms having surface properties similar to those of cpTi, confirmed these differences in film-removal susceptibility for shear stresses as low as 10 dynes/cm 2 , and illustrated the PDT-induced preferential removal of predominantly the polysaccharide biofilm components. Scanning Electron Microscopy of Control and PDT-treated biofilms before and after water-jet impingement also confirmed these findings. These results are consistent with proposals that PDT induces oxidative embrittlement and fragmentation of plaque/biofilm matrix biopolymers, allowing easier release by hydrodynamic (rinsing) forces.