Characterisation and susceptibility to lethal photosensitisation of in vitro-grown sub-gingival biofilms
Oral biofilms are the aetiological agents of periodontal diseases. Biofilms are inherently resistant to traditional antimicrobials, hence novel treatment modalities, like photodynamic therapy (PDT), might be an efficacious alternative to traditional antimicrobials. Preliminary PDT studies involved the screening of a number of photosensitisers, Toluidine blue O (TBO) was chosen for further investigation. TBO in conjunction with a 35mW Helium Neon (HeNe) laser was then tested against a range of planktonic oral bacteria as well as both monospecies biofilms grown on membrane filters and multi- species biofilms grown on membrane filters from a salivary inoculum. TBO achieved appreciable kills against many of the planktonic organisms, whilst a 2 logio reduction was evident in both the monospecies biofilms and the saliva- derived biofilms following irradiation with 31.5 J of laser light in the presence of 81.7 pM TBO. In order to evaluate the likely efficacy of PDT in vivo it was necessary to develop a laboratory model that was capable of producing biofilms that would resemble (in terms of composition and structure) the subgingival plaques found in periodontitis patients in vivo. Consequently, biofilms were grown from subgingival plaques in a Constant Depth Film Fermentor (CDFF) under conditions mimicking those present within a periodontal pocket. The resultant biofilms were quantified using viable counts on selective agars and the microbiota was characterised using 16S rRNA gene sequencing. The community structure was analysed by denaturing gradient gel electrophoresis and community level physiological profiling. The structure of the biofilms was studied in real time using confocal scanning laser microscopy. The susceptibility to PDT (using toluidine blue and 632 nm light) of these subgingivally-derived biofilms was then determined. Reductions in viability of up to 1 logio were observed and when compared to chlorhexidine digluconate (an antimicrobial commonly used for the treatment of oral infections and often considered to be the "gold standard"), there was little difference in efficacy. In this study, a laboratory model able to produce biofilms with a composition and structure resembling those of subgingival dental plaques was developed. The biofilms produced were found to be susceptible to lethal photosensitisation using toluidine blue and light (632 nm).