Development of In Vitro Denture Biofilm Models for Halitosis Related Bacteria and their Application in Testing the Efficacy of Antimicrobial Agents

Abstract

Since dentures can serve as a reservoir for halitosis-causing oral bacteria, halitosis development is a concern for denture wearers. In this study, we surveyed the prevalence of four selected halitosis-related species (Fusobacterium nucleatum, Tannerella forsythia, Veillonella atypica and Klebsiella pneumoniae) in clinical denture plaque samples, and developed denture biofilm models for these species in vitro to facilitate assessment of antimicrobial treatment efficacy. Design : Denture plaque from ten healthy and ten denture stomatitis patients was screened for the presence of aforementioned four species by PCR. Biofilm formation by these halitosis-associated species on the surfaces of denture base resin (DBR) discs was evaluated by crystal violet staining and confocal laser scanning microscopy. The efficacy of denture cleanser treatment on these mono-species biofilms was evaluated by colony counting. Results : 80% of the subjects in the denture stomatitis group and 60% in the healthy group contained at least one of the targeted halitosis-related species in their denture plaque. All halitosis species tested were able to form biofilms on DBR disc surfaces to varying degrees. These in vitro mono-species resin biofilm models were used to evaluate the efficacy of denture cleansers, which exhibited differential efficacies. When forming biofilms on resin surfaces, the halitosis-related species displayed enhanced resistance to denture cleansers compared with their planktonic counterparts. Conclusion : The four selected halitosis-related bacterial species examined in this study are present on the majority of dentures. The mono-species biofilm models established on DBR discs for these species are an efficient screening tool for dental product evaluation.

Keywords: Antimicrobial treatment; biofilms; denture; halitosis; model.

Fig. (1)

Biofilm formation on DBR disc surfaces. Representative images after crystal violet staining of biofilms formed on DBR discs by F. nucleatum ATCC 23726 (A) T. forsythia ATCC 43037 (B) V. atypica PK 1910 (C) K. pneumoniae IA 565 (D) For each set, the image on the left was a control DBR disc without biofilms growing on the surface, the image on the right was a DBR disc with biofilms growing on the surface. The experiment was performed in triplicate.

Fig. (2)

CLSM images of biofilms on DBR disc surfaces. Biofilm formation of F. nucleatum ATCC 23726 (A) T. forsythia ATCC 43037 (B) V. atypica PK 1910 (C) K. pneumoniae IA 565 (D) on DBR discs. For each set, the image on the left was taken through a 20x objective (Scale bar, 50 μm); while the image on the right was taken through a 63x objective (Scale bar, 20 μm). Four random fields of view were examined for each sample and representative images are shown.

Fig. (3)

CLSM images of F. nucleatum (A, E) T. forsythia (B, F) V. atypica (C, G) K. pneumoniae (D, H) biofilms formed on DBR disc surfaces after treatment with Polident® experimental formulation M138-12 (upper panel) and Efferdent® (lower panel), respectively. Denture biofilms were stained with SYTO59 and SYTOX Green as described in Materials and Methods, and examined by CLSM to reveal the live (green) and dead (red) cell population following treatment. Four random fields of view were examined for each sample and representative images taken through a 20x objective are shown (Scale bar, 50 μm).

Fig. (4)

Antimicrobial treatment efficacy against biofilms formed on DBR disc surfaces and planktonic cells. The biofilms and planktonic cells of F. nucleatum (A) and K. pneumoniae (B) were treated with denture cleansers as described in the Materials and Methods. The viability of biofilms after treatment was assessed by colony-forming units (CFU) on agar plates. Standard error of three replicates is presented.