A Multispecies Biofilm In Vitro Screening Model of Dental Caries for High-Throughput Susceptibility Testing.

Lara A Heersema,Hugh D C Smyth

doi:10.3390/ht8020014

Abstract

There is a current need to develop and optimize new therapeutics for the treatment of dental caries, but these efforts are limited by the relatively low throughput of relevant in vitro models. The aim of this work was to bridge the 96-well microtiter plate system with a relevant multispecies dental caries model that could be reproducibly grown to allow for the high-throughput screening of anti-biofilm therapies. Various media and inoculum concentrations were assessed using metabolic activity, biomass, viability, and acidity assays to determine the optimal laboratory-controlled conditions for a multispecies biofilm composed of Streptococcus gordonii, Streptococcus mutans, and Candida albicans. The selected model encompasses several of the known fundamental characteristics of dental caries-associated biofilms. The 1:1 RPMI:TSBYE 0.6% media supported the viability and biomass production of mono- and multispecies biofilms best. Kinetic studies over 48 h in 1:1 RPMI:TSBYE 0.6% demonstrated a stable biofilm phase between 10 and 48 h for all mono- and multispecies biofilms. The 1:1:0.1 S. gordonii: S. mutans: C. albicans multispecies biofilm in 1:1 RPMI:TSBYE 0.6% is an excellent choice for a high-throughput multispecies model of dental caries. This high-throughput multispecies model can be used for screening novel therapies and for better understanding the treatment effects on biofilm interactions and stability.

Highlights

Dental caries, or tooth decay, is characterized by the acidic breakdown of dental hard tissues from the fermentation of dietary carbohydrates by oral microorganisms within biofilms on the tooth surface [1,2]
We tested nine microbiological media including bacteria promoting (Brain Heart Infusion, Todd Hewitt Broth with 0.3% yeast extract, and Trypticase Soy Broth with 0.6% yeast extract), fungal promoting media (Yeast Nitrogen Base and Sabouraud Dextrose Broth), and non-specific media (RPMI 1640, 1:1 v/v RPMI 1640:THBYE 0.3%, 1:1 v/v RPMI 1640:TSBYE 0.6%, and M9 minimal salts)
The mono-species biofilm growths were best supported by Brain Heart Infusion (BHI), 1:1 v/v RPMI 1640:TSBYE 0.6%, and Sabouraud Dextrose Broth (SDB) for S. gordonii, S. mutans, and C. albicans, respectively. 1:1 v/v RPMI 1640:THBYE 0.3% supported the 1:1:0.01 multispecies biofilm and 1:1 v/v RPMI 1640:TSBYE 0.6% supported both the 1:1:0.1 and 1:1:1 multispecies biofilms best

Summary

Introduction

Tooth decay, is characterized by the acidic breakdown of dental hard tissues from the fermentation of dietary carbohydrates by oral microorganisms within biofilms on the tooth surface [1,2]. Of the over 600 species of bacteria, fungi, viruses, and archaea that have been identified in the human oral cavity using 16sRNA, pyrosequencing (ITS), and next-generation sequencing [3,4,5,6,7], an average of 100 microorganisms are harbored at any one time in an individual’s mouth [5] and 20–30 microorganisms are predominately found in dental caries-associated biofilms, commonly referred to as dental plaque [8]. The community of microorganisms that form on biotic and abiotic substrates, including the tooth surface, occur naturally in the oral cavity and can even provide benefits to the host [8,9]. Patient risk is a factor of age, socio-economic status, and access to dental care, but is often highly dependent on the patient and dental health regimen, which can make providing proper dental treatment options challenging and which emphasizes the need for new treatment options [2,14]

Objectives

Methods

Results

Discussion

Conclusion