A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials

Abstract

Objectives: Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva.

Methods: Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 37°C. The bacterial diversity of the biofilms was evaluated by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%-0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software.

Results: The analysis of biofilms by bTEFAP demonstrated that they contained genera typically found in human dental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5% and 0.001% w/v. At 0.5% (w/v) CPC, 90% of the total signal was from dead/damaged cells. Below this concentration range, less killing was observed. In the 0.5%-0.05% (w/v) range CPC penetration/killing was greatest and biofilm thickness was significantly reduced.

Conclusions: This work demonstrates the utility of a high-throughput microfluidic-CLSM system to grow multi-species oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials.

Keywords: Live/Dead staining; confocal scanning laser microscopy; microfluidics; multi-species biofilm; pyrosequencing.

Figure 1.

The microfluidic confocal scanning laser microscope biofilm system. (a) Diagram depicting a vertical cross-section of the BioFlux microfluidic system mounted on a Leica SPE CLSM. (b) Annotated photograph showing the channels for two microfluidic channels and accompanying reservoir and waste wells. Black annotations highlight positions of flow channels. (c) An example of a Live/Dead-stained dental plaque biofilm rendered in 2D and 3D using Imaris. Figure 1(b) is used with permission from Fluxion (Fluxionbio.com). Figure 1(a) is modified with kind permission from the American Society for Microbiology (Benoit et al. Appl Environ Microbiol 2010; 76: 4136–42).

Figure 2.

Epifluorescence images of observed cell shapes and arrangements demonstrating the multi-species composition of the biofilm in the microfluidic system. (a) Chains of cocci. (b) Aggregated rod-shaped cells with outlying diplococci. (c) Fusiforms co-localized with mixtures of other cell types (including diplococci and streptococci). (d) Simple multi-species coaggregated clusters of cells. Bars represent 10 μm.

Figure 3.

Average percentage abundance of each phylum (a) and genus (b) based on bacterial tag-encoded FLX amplicon pyrosequencing of dental plaque biofilms grown in three randomly harvested BioFlux microfluidic channels.

Figure 4.

Average percentage signal from biofilms accounted for by ‘live’/viable signal (light grey bars) and ‘dead’/damaged signal (dark grey bars) in relation to total signal captured for both. A sigmoidal relationship was observed between CPC concentration and cell viability, with greatest replicate variability for CPC concentrations falling within the linear range. Significant differences from the PBS control are highlighted (*P < 0.05, **P < 0.01).

Figure 5.

Representative biofilm renderings following each treatment. Green signal indicates viable live cells (Syto 9), red signal indicates damaged/dead cells (propidium iodide). Upper images (A₁–H₁) are of the x–y plane. Middle images (A₂–H₂) are of the x–z plane. Lower images (A₃–H₃) are an angled view of each plane (x–y–z). PBS is the negative control, 70% EtOH (ethanol) is the positive control, and all CPC treatments shown in decreasing concentration from left to right. Both controls indicate that the assay functioned correctly and decreasing biofilm damage corresponds to decreasing CPC concentration. Bars represent 30 μm.