Viable Compositional Analysis of an Eleven Species Oral Polymicrobial Biofilm

Abstract

Purpose: Polymicrobial biofilms are abundant in clinical disease, particularly within the oral cavity. Creating complex biofilm models that recapitulate the polymicrobiality of oral disease are important in the development of new chemotherapeutic agents. In order to do this accurately we require the ability to undertake compositional analysis, in addition to determine individual cell viability, which is difficult using conventional microbiology. The aim of this study was to develop a defined multispecies denture biofilm model in vitro, and to assess viable compositional analysis following defined oral hygiene regimens.Methods: An in vitro multispecies denture biofilm containing various oral commensal and pathogenic bacteria and yeast was created on poly (methyl methacrylate) (PMMA). Denture hygiene regimens tested against the biofilm model included brushing only, denture cleansing only and combinational brushing and denture cleansing. Biofilm composition and viability were assessed by culture (CFU) and molecular (qPCR) methodologies. Scanning electron microscopy and confocal laser scanning microscopy were also employed to visualize changes in denture biofilms following treatment.Results: Combinational treatment of brushing and denture cleansing had the greatest impact on multispecies denture biofilms, reducing the number of live cells by more than 2 logs, and altering the overall composition in favor of streptococci. This was even more evident during the sequential testing, whereby daily sequential treatment reduced the total and live number of bacteria and yeast more than those treated intermittently. Bacteria and yeast remaining following treatment tended to aggregate in the pores of the PMMA, proving more difficult to fully eradicate the biofilm.Conclusions: Overall, we are the first to develop a method to enable viable compositional analysis of an 11 species denture biofilm following chemotherapeutic challenge. We were able to demonstrate viable cell reduction and changes in population dynamics following evaluation of various denture cleansing regimens. Specifically, it was demonstrated that daily combinational treatment of brushing and cleansing proved to be the most advantageous denture hygiene regimen, however, residual organisms still remained within the pores of PMMA surface, which could act as a reservoir for further biofilm regrowth. We have identified an industry need for denture cleansing agents with the capacity to penetrate these pores and disaggregate these complex biofilm consortia.

Artificial dry surface biofilm models for testing the efficacy of cleaning and disinfection

“…; Sherry et al . ). There are, however, no standard protocols to measure the efficacy of biocidal formulations against dry surface biofilms (DSB).…”

Impact of frequency of denture cleaning on microbial and clinical parameters – a bench to chairside approach

“…In vitro denture cleansing study A denture plaque cleansing study and quantitative analysis of remaining viable cells was performed as previously described [26]. It was the aim to investigate whether a sequential denture cleansing technique was more advantageous than one treatment over the course of a 5-day treatment regimen.…”

“…PMMA discs were then sonicated in 1 mL phosphate-buffered saline (Sigma-Aldrich, Gillingham, UK) at 35 kHz for 10 min to remove the biomass, as previously described [21]. For quantitative analysis, both colonyforming unit (CFU) and quantitative live/dead PCR were performed, as described previously [26].…”

“…Viability of the treated biofilms was also assessed using live/dead PCR to enumerate the definitive and relative composition of the biofilms, a technique that has been shown to differentiate viable and dead cells from multispecies oral bacteria biofilm models. Samples were prepared as previously described, with some modifications [26]. In brief, 50 μM propidium monoazide (PMA) was added to each sonicated sample and incubated in the dark for 10 min to allow uptake of the dye.…”

“…Available data conservatively estimates that at least 10-fold more bacteria than yeasts colonise the surface of dentures [10], clearly indicating that denture plaque has a polymicrobial and interkingdom composition [11]. Denture plaque biofilm models, such as a recently described 11 species interkingdom model, are likely to be more representative of the polymicrobial nature of the clinical situation [26]. In this study, we sought to adopt a bench-to-chairside approach to test the appropriateness of routine daily denture cleansing methods compared to intermittent methodologies.…”