Fig 1.
CLSM of the four-species biofilms.
Images of the four-species biofilms comprising the bacterial species S. oralis, A. naeslundii, V. dispar and P. gingivalis 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.
Fig 2.
(a) Total biovolume and (b) relative biovolume of the four-species biofilms, including the bacterial species S. oralis, A. naeslundii, V. dispar and P. gingivalis. 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.
Fig 3.
Maximum intensity projection of biofilm image stacks upon species-specific staining of a 24h four-species biofilm by FISH.
(a)–(d) Separate color channels showing the staining of individual bacterial species within the four-species biofilm. (a) S. oralis (MIT-588-Alexa-405; blue), (b) A. naeslundii (ANA-103-Alexa-488; green), (c) V. dispar (VEI-217-Alexa-568; yellow) and (d) P. gingivalis (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.
Fig 4.
Maximum intensity projection of biofilm image stacks upon species-specific staining of a 48h four-species biofilm by FISH.
(a)–(d) Separate color channels showing the staining of individual bacterial species within the four-species biofilm. (a) S. oralis (MIT-588-Alexa-405; blue), (b) A. naeslundii (ANA-103-Alexa-488; green), (c) V. dispar (VEI-217-Alexa-568; yellow) and (d) P. gingivalis (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.
Fig 5.
Spatial-temporal distribution of the individual bacterial species within the 24 hours biofilm.
Fluorescence in situ hybridisation of the 24 hour four-species biofilm consisting of the bacterial species S. oralis (MIT-588-Alexa-405; blue), A. naeslundii (ANA-103-Alexa-488; green), V. dispar (VEI-217-Alexa-568; yellow) and P. gingivalis (POGI-Alexa-647; red). (a)–(k) show the 11 images of the biofilm with a z-step size of 1 μm. (l) shows the 3D-reconstruction of the complete biofilm.
Fig 6.
Spatial-temporal distribution of the individual bacterial species within the 48 hours biofilm.
Fluorescence in situ hybridisation of the 48 hour four-species biofilm consisting of the bacterial species S. oralis (MIT-588-Alexa-405; blue), A. naeslundii (ANA-103-Alexa-488; green), V. dispar (VEI-217-Alexa-568; yellow) and P. gingivalis (POGI Alexa-647; red). (a)–(k) show the 11 images of the biofilm with a z-step size of 1 μm. (l) shows the 3D-reconstruction of the complete biofilm.
Fig 7.
Scanning Electron Micrograph (SEM) of 24 hours old biofilms.
(a) S. oralis, (b) A. naeslundii, (c) V. dispar, (d) P. gingivalis and (e) a four-species biofilm. In the mixed community, the individual species are exemplarily highlighted by arrows: S. oralis (a), A. naeslundii (b), V. dispar (c), and P. gingivalis (d).
Fig 8.
qRT-PCR analysis representing the relative species distribution within the biofilms.
These consisted of the bacterial species S. oralis (blue), A. naeslundii (green), V. dispar (yellow) and P. gingivalis (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.
Fig 9.
pH measurement of the biofilm medium over 45 hours of biofilm growth.
Fig 10.
Biovolume quantification and maximum intensity projections of four-species biofilms grown on (coated) glass discs.
(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).
Fig 11.
Biovolume quantification and maximum intensity projections of grown four-species biofilms exposed to medium supplemented with antibiotics.
(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).