Cell viability within oral biofilms

Bacteria are not distributed homogenously throughout an oral biofilm. This is primarily due to regions of differing environmental conditions, themselves a manifestation of nutrient, gaseous and ionic concentration gradients within the biofilm. Confocal laser scanning microscopy in conjunction with fluorescent staining techniques and image analysis can both visualise and quantify the distribution of viable and nonviable bacteria within the three-dimensional volume of the biofilm

Susceptibility of microcosm subgingival dental plaques to lethal photosensitisation

Photodynamic therapy (PDT) offers potential as a non-invasive treatment of periodontal 15 disease. In this study, microcosm biofilms were grown in vitro under conditions designed 16 to mimic subgingival plaques typically found in patients with periodontitis. To 17 investigate potential PDT modalities, biofilms were exposed to light from a helium/neon 18 laser in conjunction with a photosensitiser, toluidine blue (TBO), at varying output and 19 concentration, respectively. To determine cytotoxic effects, viability profiling was 20 undertaken on whole biofilms using standard plating methods, and on horizontal cross-21 sections of biofilms using confocal laser-scanning microscopy (CLSM) in conjunction 22 with a differential viability stain. A light energy dose of 94.5 J in combination with 81.7 23 .M TBO was found to be optimal, achieving significant kills of over 97%. CLSM 24 enabled visualisation of the effects of PDT in three dimensions. Viability profiling of the 25 CLSM images revealed that lethal photosensitisation was most effective in the upper 26 layers of biofilm. PDT was found to reduce the viability of subgingivally-modelled 27 plaques in vitro by a similar magnitude to chlorhexidine digluconate (CHL), which is 28 commonly used to treat periodontal disease. The findings of this study indicate that PDT 29 may be an effective alternative to conventional modalities in the treatment of periodontal 30 disease. 3

The inability of a bacteriophage to infect Staphylococcus aureus does not prevent it from specifically delivering a photosensitizer to the bacterium enabling its lethal photosensitization

Objectives: It has been demonstrated that the efficiency of lethal photosensitization can be improved by covalently binding photosensitizing agents to bacteriophage. In this study we have investigated whether a bacteriophage requires the capacity to infect the bacterium to enhance lethal photosensitization when linked to a photosensitizer.Methods: Tin (IV) chlorin e6 (SnCe6) was conjugated to bacteriophage Phi 11, a transducing phage that can infect Staphylococcus aureus NCTC 8325-4, but not epidemic methicillin-resistant S. aureus (EMRSA)-16. The conjugate and appropriate controls were incubated with these bacteria and either exposed to laser light at 632.8 nm or kept in the dark.Results: The SnCe6/Phi 11 conjugate achieved a statistically significant reduction in the number of viable bacteria of both 8325-4 and EMRSA-16 strains by 2.31 log(10) and 2.63 log(10), respectively. The conjugate could not however instigate lethal photosensitization of Escherichia coli. None of the other combinations of controls, such as an equivalent concentration of SnCe6 only, an equivalent titre of bacteriophage only or experiments conducted without laser light, yielded significant reductions in the number of viable bacteria recovered.Conclusions: The inability of a bacteriophage to infect S. aureus does not prevent it from specifically delivering a photosensitizer to a bacterium enabling its lethal photosensitization

A preliminary study of the effects of pH upon fluorescence in suspensions of prevotella intermedia

© 2016 Hope et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The quantification of fluorescence in dental plaque is currently being developed as a diagnostic tool to help inform and improve oral health. The oral anaerobe Prevotella intermedia exhibits red fluorescence due to the accumulation of porphyrins. pH affects the fluorescence of abiotic preparations of porphyrins caused by changes in speciation between monomers, higher aggregates and dimers, but this phenomenon has not been demonstrated in bacteria. Fluorescence spectra were obtained from suspensions of P. intermedia that were adjusted to pHs commensurate with the range found within dental plaque. Two fluorescent motifs were identified; 410 nm excitation / 634 nm emission (peak A) and 398 nm excitation / 622 nm emission (peak B). A transition in the fluorescence spectra was observed from peak A to peak B with increasing pH which was also evident as culture age increased from 24 hours to 96 hours. In addition to these 'blue-shifts', the intensity of peak A increased with pH whilst decreasing with culture age from 24 to 96 hours. A bacterium's relationship with the local physiochemical environment at the time of image capture may therefore affect the quantification of dental plaque fluorescence