Viable Compositional Analysis of an Eleven Species Oral Polymicrobial Biofilm.

Leighann Sherry,Craig Williams,Owain R. Millington,Gordon Ramage,Christopher J. Nile,David J. Bradshaw,Alyson S. Axe,Emma Millhouse,Lindsay E. O'Donnell,Gillian Lappin

doi:10.3389/fmicb.2016.00912

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.

Highlights

Denture stomatitis (DS) is characterized as the erythema and inflammation of the oral mucosa, localized under dentures
Multi-species biofilms treated with various denture-cleansing regimens were initially quantified by Colony Forming Units (CFU) for total aerobes (Figure 2A), anaerobes (Figure 2B), and yeast (Figure 2C)
When the combinational treatment of DC and brushing (DC + B) was used, total aerobes and anaerobes were reduced to 3.3 × and 2.3 × CFU/mL (P < 0.0001), respectively

Summary

Introduction

Denture stomatitis (DS) is characterized as the erythema and inflammation of the oral mucosa, localized under dentures. There has been growing interest surrounding how fungalbacterial interactions in the oral cavity influence disease (Sumi et al, 2002; Ealla et al, 2013; O’Donnell et al, 2015a) To this end, denture biofilm systems have been developed to model and test polymicrobial infections, these tend to be limited to 2-3 organisms, or rely on undefined inocula from clinical samples. Denture biofilm systems have been developed to model and test polymicrobial infections, these tend to be limited to 2-3 organisms, or rely on undefined inocula from clinical samples This makes it difficult to reproduce and fully understand the impact of multi-species biofilm consortia in denture patients (Coulthwaite and Verran, 2008; Li et al, 2010; Urushibara et al, 2014). There is the need for the development of a defined in vitro multi-species denture biofilm, as this would provide a greater understanding to clinically relevant polymicrobial oral diseases and the treatment of these using various denture regimens

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