Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses

ABSTRACT Biofilms are complex polymicrobial communities which are often associated with human infections such as the oral disease periodontitis. Studying these complex communities under controlled conditions requires in vitro biofilm model systems that mimic the natural environment as close as possible. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic the continuous flow of nutrients at the air-liquid interface in the oral cavity. The design is engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a heterogeneous, spatially uniform, dense, and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features, and bacterial composition of the biofilm remain stable from 3 to 6 days. As a proof of concept for our periodontal model, the 3 days developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and incorporation of the pathogenic species and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine and ammonium in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community. Importance Periodontitis is a multifactorial chronic inflammatory disease in the oral cavity associated with the accumulation of microorganisms in a biofilm. Not the presence of the biofilm as such, but changes in the microbiota (i.e., dysbiosis) drive the development of periodontitis, resulting in the destruction of tooth-supporting tissues. In this respect, novel treatment approaches focus on maintaining the health-associated homeostasis of the resident oral microbiota. To get insight in dynamic biofilm responses, our research presents the establishment of a periodontal biofilm model including Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis. The added value of the model setup is the combination of simulating continuously changing natural mouth conditions with spatiotemporal biofilm profiling using non-destructive characterization tools. These applications are limited for periodontal biofilm research and would contribute in understanding treatment mechanisms, short- or long-term exposure effects, the adaptation potential of the biofilm and thus treatment strategies.

flow cytometry (Figure A2).The signal was hardly captured in the green detector channel for an early exponential culture of F. nucleatum, but when reaching the stationary phase (more related to biofilm maturation), microscopy and flow cytometry data show increased fluorescent intensities (Figures A3 and A2).After performing the image thresholding and filtering steps on the separate channels in BiofilmQ (9), the quantified fluorescence intensities in the GFPmut3*, mCherry, and TMR-Star channels are presented in Figures A1b, A1c, and A1d, respectively.They align very well with the bacterial cells observed in bright field view and show hardly overlap between the channels.Flow cytometry Flow cytometry was used to investigate the autofluorescence of F. nucleatum during its growth. 1 mL of a liquid culture was centrifuged at 5000 ×g for 10 minutes and the pellet was resuspended in 0.22 µm-filtered PBS to obtain a concentration in the range between 10 7 − 10 8 cells/mL.Samples were analyzed with the Amnis ® CellStream ® (Luminex) at a flow rate of 3.66 µL/min.Side scatter is measured with a 785 nm laser (40 %).F. nucleatum was excited by the 488 nm laser (100 %) and autofluorescence was detected with a charge-coupled device (CCD) camera using the filter stack 528 nm with a bandwidth of 46 nm, referred to as the green channel.Density plots of side scatter versus the green channel are presented.Data is gated on the aspect ratio, calculated as the ratio of the length of the minor cell axis over the length of the major cell axis.A1.

Supplementary file S2 Additional information to Results
5 mL of the culture was sampled and centrifuged (4 • C, 5000 ×g, 10 min).The supernatant was filter sterilized, snap frozen in liquid nitrogen, and stored at -20 • C for further analysis.Hydrogen peroxide concentrations were finally determined with the Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) according to the manuals instructions.The fluorescence signal was measured in a Tecan Infinite 200 PRO, with 530 nm excitation and 590 nm emission detection.Using this complex protocol, the intrinsic capacity of the streptococcal strains to produce hydrogen peroxide could be quantified.
Results Monoculture experiments are performed with and without the addition of 1.5 % Larginine.Growth curves are shown in Figure A7a and A7b. Figure A7d depicts the metabolic interaction network, derived from metabolite concentration profiles (Figures A8 and A9) and coculture experiments (data not shown).Amino acids and peptides are present in the medium and contribute to growth.Total amino acid (i.e., free and protein-bound) concentrations are measured because the anaerobic strains prefer the usage of peptides to amino acids (12).
The streptococcal strains grow relatively fast (maximum OD in less than a day) while F. nucleatum and P. gingivalis-SNAP26 are slow growers.The streptococcal strains consume glucose and pyruvic acid as substrates for growth while producing organic acids, i.e., acetic acid, lactic acid and formic acid.F. nucleatum first converts pyruvic acid into biomass, acetic acid, lactic acid and formic acid.
Upon depletion of pyruvic acid, glucose, peptides, and formic acid are converted into biomass, lactic acid and butyric acid.P. gingivalis-SNAP26 primarily uses peptides for growth and subsequently, pyruvic acid is consumed to produce organic acids including propionic acid and butyric acid.
Hydrogen peroxide acts as an antimicrobial, limiting the growth of pathobionts (13), and was therefore measured (10).Quantitative concentration profiles of the hydrogen peroxide production potential during growth of the streptococcal strains are determined (Figure A7c).These have not been reported in any study before.All streptococcal strains start producing hydrogen peroxide in the exponential growth phase and continue production in the stationary phase with concentrations up to 0.28 mM, 1.28 mM, and 0.32 mM for S. gordonii -GFPmut3*, S. oralis-GFPmut3*, and S. sanguinis-pVMCherry, respectively, at a similar average final OD of 1.25.Under aerobic conditions, pyruvate oxidase (SpxB) catalyzes the generation of hydrogen peroxide, carbon dioxide, and acetyl phosphate.
The latter is further metabolized into acetate (12,14,15).With this in mind, the very high hydrogen peroxide production observed by S. oralis-GFPmut3* might be directly related to the observed higher pyruvate uptake rate and acetate production rate compared to S. gordonii -GFPmut3* and S. sanguinis-pVMCherry.
Growth and metabolism of the community members are impacted by the addition of L-arginine, as shown in Figure A7a and A7b. S. gordonii GFPmut3* and S. sanguinis-pVMCherry reaches higher optical density values (A7a).L-arginine is metabolized into biomass and converted into alkaline products, including ornithine (Figure A9).Ornithine results from the degradation of citrulline in the arginine deiminase system (12).S. oralis-GFPmut3* does not metabolize L-arginine and consequently has no growth benefit or ornithine production.Hydrogen peroxide production is not altered as a result of the addition of L-arginine.Contrarily to the streptococcal strains, growth and metabolic activity are partly impaired for F. nucleatum, and P. gingivalis-SNAP26 is even completely inhibited by the presence of L-arginine.

Figure A1 :
Figure A1: Fluorescence specificity and compatibility of the fluorochromes GFPmut3* (green), mCherry (magenta), and SNAP-Cell TMR Star (red) in a liquid culture containing S. gordonii -GFPmut3*, S. oralis-GFPmut3*, S. sanguinis-pVMCherry, F. nucleatum, and P. gingivalis-SNAP26.Fluorescence signals for all laser-detector settings are shown.Images are thresholded with the Otsu method.a. Overlap of all fluorescent channels and the bright field channel.b, c, d.Fluorescent intensities in the GFPmut3*, mCherry, and SNAP-Cell TMR Star detector channels, respectively, after image analysis with the BiofilmQ software.

Figure A2 :
Figure A2: Flow cytometry graphs of a liquid culture of F. nucleatum over time.Density plots of side scatter versus the green channel (528 nm, 46 nm bandwidth) are shown.Graphs represent 2 (a), 8 (b), and 24 hours (c) of growth, respectively.

Figure A3 :
Figure A3: Autofluorescence signal of a liquid, mature grown culture of F. nucleatum.a. Bright field channel.b. Green fluorescent channel.

Figure A4 :FigureFigure A6 :
Figure A4: Amino acid (free and protein-bound) concentrations in the effluent of the drip flow reactor reflecting metabolic activity of the biofilm.Averages and standard deviations of two biological replicates are shown.Relevant amino acids are selected to be shown.a b c d

Figure A7 :
Figure A7: Growth and metabolic characterization of the community members.a, b.Optical density over time of monoculture experiments.c.Hydrogen peroxide production potential over time for the streptococcal strains.Dots '•' represent experiments in BHI medium supplemented with sodium pyruvate.Crosses '×' represent experiments where L-arginine is supplemented.d.Metabolic interaction network of the community.Arrows pointing at metabolites represent production while arrows pointing at the bacteria represent consumption.Blocked lines represent inhibition of the bacteria.Red dashed lines show L-arginine metabolization by S. gordonii -GFPmut3* and S. oralis-GFPmut3* resulting in higher cell numbers and the inhibition of P. gingivalis-SNAP26 in the presence of 1.5% L-arginine.
Figure A8 (cont.):Glucose and organic acid concentration profiles of the community members during growth (continued).Dots '•' represent experiments in BHI medium supplemented with sodium pyruvate.Crosses '×' represent experiments where L-arginine is supplemented.

Figure A9 :
Figure A9: Amino acid (free and protein-bound) profiles of the community members during growth (continued).Dots '•' represent experiments in BHI medium supplemented with sodium pyruvate.Crosses '×' represent experiments where L-arginine is supplemented.Relevant amino acids are selected to be shown.

Figure A10 :
Figure A10: Dimensional drawings of the drip flow biofilm reactor.

Figure A11 :
Figure A11: Locations on the biofilm substratum for confocal image analysis.In each of the seven white boxes, four different z-stacks are imaged.
Glucose and organic acid concentration profiles of the community members during growth.Dots '•' represent experiments in BHI medium supplemented with sodium pyruvate.Crosses '×' represent experiments where L-arginine is supplemented.
Amino acid (free and protein-bound) profiles of the community members during growth.Dots '•' represent experiments in BHI medium supplemented with sodium pyruvate.Crosses '×' represent experiments where L-arginine is supplemented.Relevant amino acids are selected to be shown.

Table A1 :
Statistics of the depth profiles which are shown in Figure8.Differences between the negative control and treatment at each time point are compared to differences at time point 72 (i.e., reference point).No significant interactions implies analysis over all depth data.Data for all depth layers is available upon request.