An oral multispecies biofilm model for high content screening applications.

Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript

Maximum intensity projection of biofilm image stacks upon species-specific staining of a 24h four-species biofilm by FISH.

<p>(a)–(d) Separate color channels showing the staining of individual bacterial species within the four-species biofilm. (a) <i>S</i>. <i>oralis</i> (MIT-588-Alexa-405; blue), (b) <i>A</i>. <i>naeslundii</i> (ANA-103-Alexa-488; green), (c) <i>V</i>. <i>dispar</i> (VEI-217-Alexa-568; yellow) and (d) <i>P</i>. <i>gingivalis</i> (POGI-Alexa-647; red), (e) Overlay of individual images of the four-species biofilm. Image stacks of 11 single images with a z-step size of 1 μm.</p

qRT-PCR analysis representing the relative species distribution within the biofilms.

<p>These consisted of the bacterial species <i>S</i>. <i>oralis</i> (blue), <i>A</i>. <i>naeslundii</i> (green), <i>V</i>. <i>dispar</i> (yellow) and <i>P</i>. <i>gingivalis</i> (red) and were incubated anaerobically for 24 and 48 hours. Each independent biofilm approach (1–3) included three technical replicates (three wells); qRT-PCR was run in triplicate for each biofilm sample. (a) Percentage distribution based on the total cell numbers after 24 hours, (b) percentage distribution based on the viable cell numbers after 24 hours, (c) percentage distribution based on the total cell numbers after 48 hours, (d) percentage distribution based on the viable cell numbers after 48 hours.</p

Maximum intensity projection of biofilm image stacks upon species-specific staining of a 48h four-species biofilm by FISH.

<p>(a)–(d) Separate color channels showing the staining of individual bacterial species within the four-species biofilm. (a) <i>S</i>. <i>oralis</i> (MIT-588-Alexa-405; blue), (b) <i>A</i>. <i>naeslundii</i> (ANA-103-Alexa-488; green), (c) <i>V</i>. <i>dispar</i> (VEI-217-Alexa-568; yellow) and (d) <i>P</i>. <i>gingivalis</i> (POGI-Alexa-647; red), (e) Overlay of individual images of the four-species biofilm. Image stacks of 11 single images with a z-step size of 1 μm.</p

Scanning Electron Micrograph (SEM) of 24 hours old biofilms.

<p>(a) <i>S</i>. <i>oralis</i>, (b) <i>A</i>. <i>naeslundii</i>, (c) <i>V</i>. <i>dispar</i>, (d) <i>P</i>. <i>gingivalis</i> and (e) a four-species biofilm. In the mixed community, the individual species are exemplarily highlighted by arrows: <i>S</i>. <i>oralis</i> (a), <i>A</i>. <i>naeslundii</i> (b), <i>V</i>. <i>dispar</i> (c), and <i>P</i>. <i>gingivalis</i> (d).</p

Biovolume quantification and maximum intensity projections of four-species biofilms grown on (coated) glass discs.

<p>(a) Relative proportion of the total biovolume of biofilms grown on different surfaces as indicated in the figure. (b-e) Images of 24 hour-old four-species biofilms established in glassbottom wells (b), on glass discs (c), on mesoporous silica coated glass discs (d) and on mesoporous silica coated glass discs containing ciprofloxacin (e). Bacteria were stained live/dead (viable cells: green; dead cells: red).</p

CLSM of the four-species biofilms.

<p>Images of the four-species biofilms comprising the bacterial species <i>S</i>. <i>oralis</i>, <i>A</i>. <i>naeslundii</i>, <i>V</i>. <i>dispar</i> and <i>P</i>. <i>gingivalis</i> after (a) 24 hours (bottom of the biofilm), (b) 24 hours (top of the biofilm), (c) 48 hours (bottom of the biofilm) and (d) 48 hours (top of the biofilm) of biofilm growth. Bacteria were live/dead stained with viable cells visualized in green and dead cells appearing red.</p

pH value curve.

<p>pH measurement of the biofilm medium over 45 hours of biofilm growth.</p

Quantification of biovolume.

<p>(a) Total biovolume and (b) relative biovolume of the four-species biofilms, including the bacterial species <i>S</i>. <i>oralis</i>, <i>A</i>. <i>naeslundii</i>, <i>V</i>. <i>dispar</i> and <i>P</i>. <i>gingivalis</i>. Each bar shows the mean ± standard error from three different wells of one biofilm growth experiment of the three biological replicates measured after 24 and 48 hours.</p

Biovolume quantification and maximum intensity projections of grown four-species biofilms exposed to medium supplemented with antibiotics.

<p>(a) Determination of total biovolumes of 24 hour-old four-species biofilms grown under conditions as indicated in the figure. (b-h) Images of biofilms grown in glassbottom wells without supplementation of antibiotic (b), with 14 μg/mL amoxicillin (c), with 140 μg/mL amoxicillin (d), with 14 μg/mL metronidazole (e), with 140 μg/mL metronidazole (f), with 14 μg/mL amoxicillin and 14 μg/mL metronidazole (g) and with 140 μg/mL amoxicillin and 140 μg/mL metronidazole (h). Antibiotics were added to BHI / vitamin K medium. Bacteria were stained live/dead (viable cells: green; dead cells: red).</p