Structured Review

Thermo Fisher biofilms
Fatty acids in <t>biofilms/planktonic</t> cultures. Strain 630Δ erm was grown either as biofilms in microfermentors (like in Figure 1 ) or as planktonic cultures in Falcon tubes. Supernatants of biofilms (in gray) and planktonic cultures (in white) were recovered and their composition in volatile (A) and non-volatile fatty acids (B) was determined. Relevant fatty acids, as inferred from our transcriptomic study, are shown. ∗ indicates a statistically significant difference ( p -value
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1) Product Images from "Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture"

Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02084

Fatty acids in biofilms/planktonic cultures. Strain 630Δ erm was grown either as biofilms in microfermentors (like in Figure 1 ) or as planktonic cultures in Falcon tubes. Supernatants of biofilms (in gray) and planktonic cultures (in white) were recovered and their composition in volatile (A) and non-volatile fatty acids (B) was determined. Relevant fatty acids, as inferred from our transcriptomic study, are shown. ∗ indicates a statistically significant difference ( p -value
Figure Legend Snippet: Fatty acids in biofilms/planktonic cultures. Strain 630Δ erm was grown either as biofilms in microfermentors (like in Figure 1 ) or as planktonic cultures in Falcon tubes. Supernatants of biofilms (in gray) and planktonic cultures (in white) were recovered and their composition in volatile (A) and non-volatile fatty acids (B) was determined. Relevant fatty acids, as inferred from our transcriptomic study, are shown. ∗ indicates a statistically significant difference ( p -value

Techniques Used:

Intact biofilm architecture of the parental strain and CD2214–CD2215 mutant. The biofilms of the parental strain 630Δ erm (A–D) and its CD2214–CD2215 mutant (E–H) are shown. They were grown for 48h in 96-well polystyrene micro-titer plates, in TYt medium freshly added onto adhesive starter cells ( Supplementary Figure S5 ). After live dead staining of intact biofilms directly in the micro-titer plates, their microscopic architecture was observed in situ by CLSM. Images representative of three independent experiments (each using three clones) are shown. Raw confocal z-stacks were treated using IMARIS software. This allowed obtaining both a 3D projection upside view, with its shadow on the right (A,E) , and a section view close to the surface (B,F) , in which the white bar indicates the scale (50 μm). For the section view of each strain, magnifications of micro-aggregated forms (C,G) and rods (D,H) are also provided, with the corresponding scale bar in white (5, 10, or 15 μm as indicated). After data recovery, biovolume, maximum coverage, mean thickness and biovolume/surface ratio were quantified and a statistical analysis was performed (see Supplementary Figure S6 ).
Figure Legend Snippet: Intact biofilm architecture of the parental strain and CD2214–CD2215 mutant. The biofilms of the parental strain 630Δ erm (A–D) and its CD2214–CD2215 mutant (E–H) are shown. They were grown for 48h in 96-well polystyrene micro-titer plates, in TYt medium freshly added onto adhesive starter cells ( Supplementary Figure S5 ). After live dead staining of intact biofilms directly in the micro-titer plates, their microscopic architecture was observed in situ by CLSM. Images representative of three independent experiments (each using three clones) are shown. Raw confocal z-stacks were treated using IMARIS software. This allowed obtaining both a 3D projection upside view, with its shadow on the right (A,E) , and a section view close to the surface (B,F) , in which the white bar indicates the scale (50 μm). For the section view of each strain, magnifications of micro-aggregated forms (C,G) and rods (D,H) are also provided, with the corresponding scale bar in white (5, 10, or 15 μm as indicated). After data recovery, biovolume, maximum coverage, mean thickness and biovolume/surface ratio were quantified and a statistical analysis was performed (see Supplementary Figure S6 ).

Techniques Used: Mutagenesis, Staining, In Situ, Confocal Laser Scanning Microscopy, Software

Intact biofilm architecture of the parental strain over-expressing or not dccA . Biofilms were grown in TYt medium freshly added onto adhesive starter cells in 96-well polystyrene micro-titer plates. The procedure was essentially as described in Figure 8 , except that growth was for only 24 h and that anhydro-tetracycline was added to induce P tet promoter and dccA expression. At the end of growth, intact biofilms were stained and observed by CLMS as described in Figure 8 . Representative images are shown. For each strain, a 3D projection upside view, with its shadow on the right (A,C) , and a section view close to the surface (B,D) are shown, with the white bar indicating the scale (50 μm). The biofilm of the parental strain over-expressing dccA (630Δ erm p dccA in C,D ) and that of the control strain (630Δ erm p in A,B ) are shown. After data recovery, the same four parameters as in Figure 8 were quantified and a statistical analysis was performed (see Supplementary Figure S9 ).
Figure Legend Snippet: Intact biofilm architecture of the parental strain over-expressing or not dccA . Biofilms were grown in TYt medium freshly added onto adhesive starter cells in 96-well polystyrene micro-titer plates. The procedure was essentially as described in Figure 8 , except that growth was for only 24 h and that anhydro-tetracycline was added to induce P tet promoter and dccA expression. At the end of growth, intact biofilms were stained and observed by CLMS as described in Figure 8 . Representative images are shown. For each strain, a 3D projection upside view, with its shadow on the right (A,C) , and a section view close to the surface (B,D) are shown, with the white bar indicating the scale (50 μm). The biofilm of the parental strain over-expressing dccA (630Δ erm p dccA in C,D ) and that of the control strain (630Δ erm p in A,B ) are shown. After data recovery, the same four parameters as in Figure 8 were quantified and a statistical analysis was performed (see Supplementary Figure S9 ).

Techniques Used: Expressing, Staining

Sugar transport and metabolism in biofilms compared to planktonic cultures. A model for sugar transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in sugar transport and metabolism, and their variation between micro-fermentor biofilms ( Figure 1 ) and planktonic cultures ( Supplementary Table S2 ). The name/short identification number of proteins is indicated in green or red when their gene is down- or up-regulated during biofilm/planktonic growth, respectively (see Supplementary Table S2 for protein activity/function). For simplicity, the number of molecules is not indicated in metabolic reactions (e.g., for glycolysis, each molecule of glucose leads to two molecules of glyceraldehyde 3-phosphate and all subsequent products). PEP, Phospho-Enol-Pyruvate; THF, TetraHydroFolate; CoA, Co-enzyme A; P, phosphate.
Figure Legend Snippet: Sugar transport and metabolism in biofilms compared to planktonic cultures. A model for sugar transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in sugar transport and metabolism, and their variation between micro-fermentor biofilms ( Figure 1 ) and planktonic cultures ( Supplementary Table S2 ). The name/short identification number of proteins is indicated in green or red when their gene is down- or up-regulated during biofilm/planktonic growth, respectively (see Supplementary Table S2 for protein activity/function). For simplicity, the number of molecules is not indicated in metabolic reactions (e.g., for glycolysis, each molecule of glucose leads to two molecules of glyceraldehyde 3-phosphate and all subsequent products). PEP, Phospho-Enol-Pyruvate; THF, TetraHydroFolate; CoA, Co-enzyme A; P, phosphate.

Techniques Used: Expressing, Activity Assay

Protein export in biofilms compared to planktonic cultures. A model for protein export pathways and exported protein production in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in protein export or encoding exported proteins, and drawn as in Figure 2 . PSII, surface polysaccharide II and atypical teichoic acid.
Figure Legend Snippet: Protein export in biofilms compared to planktonic cultures. A model for protein export pathways and exported protein production in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in protein export or encoding exported proteins, and drawn as in Figure 2 . PSII, surface polysaccharide II and atypical teichoic acid.

Techniques Used: Expressing

Amino acid transport and metabolism in biofilms compared to planktonic cultures. A model for amino acid transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in amino acid transport and metabolism, and drawn as in Figure 2 . BCAA, Branched Chain Amino Acids; Fe-S, Iron-Sulfur cluster.
Figure Legend Snippet: Amino acid transport and metabolism in biofilms compared to planktonic cultures. A model for amino acid transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in amino acid transport and metabolism, and drawn as in Figure 2 . BCAA, Branched Chain Amino Acids; Fe-S, Iron-Sulfur cluster.

Techniques Used: Expressing

Comparison between CD2214–CD2215 regulon and the set of genes differentially expressed in biofilm/planktonic growth. (A) Overlap. Transcriptomes are drawn as elipses: the set of genes differentially expressed during biofilm/planktonic growth ( Supplementary Table S2 ) is on the left and the set of genes differentially expressed in strain 630Δ erm / CD2214–CD2215 mutant ( Supplementary Table S4 ) is on the right. The number of shared genes (whose expression varies in both transcriptomes) is indicated above the overlap region. As shared genes can vary in the same direction in the two transcriptomes or in opposite directions, the overlap region is divided into two parts. The number of shared genes whose expression varies in opposite directions is indicated in the dark gray part, while the number of genes whose expression varies in the same direction is in the light gray part. (B) Functions. Genes common to the two transcriptomes are shown as a pie chart. The main functional categories of genes regulated in the same direction in the two transcriptomes appear in capital letters in light gray slices. A unique dark gray slice is shown for all genes regulated in opposite directions in the two transcriptomes, whatever the functional category they belong to. The function and the product of main shared genes (designated by their name or short identification number) are indicated beside the pie chart. Protein names/short identification numbers are in green or red depending on whether their genes are, respectively, down- or up-regulated in both transcriptomes. Proteins whose genes are controlled by a c-di-GMP riboswitch ( Soutourina et al., 2013 ) are underlined.
Figure Legend Snippet: Comparison between CD2214–CD2215 regulon and the set of genes differentially expressed in biofilm/planktonic growth. (A) Overlap. Transcriptomes are drawn as elipses: the set of genes differentially expressed during biofilm/planktonic growth ( Supplementary Table S2 ) is on the left and the set of genes differentially expressed in strain 630Δ erm / CD2214–CD2215 mutant ( Supplementary Table S4 ) is on the right. The number of shared genes (whose expression varies in both transcriptomes) is indicated above the overlap region. As shared genes can vary in the same direction in the two transcriptomes or in opposite directions, the overlap region is divided into two parts. The number of shared genes whose expression varies in opposite directions is indicated in the dark gray part, while the number of genes whose expression varies in the same direction is in the light gray part. (B) Functions. Genes common to the two transcriptomes are shown as a pie chart. The main functional categories of genes regulated in the same direction in the two transcriptomes appear in capital letters in light gray slices. A unique dark gray slice is shown for all genes regulated in opposite directions in the two transcriptomes, whatever the functional category they belong to. The function and the product of main shared genes (designated by their name or short identification number) are indicated beside the pie chart. Protein names/short identification numbers are in green or red depending on whether their genes are, respectively, down- or up-regulated in both transcriptomes. Proteins whose genes are controlled by a c-di-GMP riboswitch ( Soutourina et al., 2013 ) are underlined.

Techniques Used: Mutagenesis, Expressing, Functional Assay

Biofilm of strain 630Δ erm after growth in a continuous-flow micro-fermentor. After anaerobic growth for 72 h in TYt medium, macro-colonies and biofilms can be observed on micro-fermentor walls. The medium is clear, as expected in the absence of planktonic growth. A representative picture of independent experiments is shown (A) . Magnifications (B,C) allow observing macro-colonies (arrows).
Figure Legend Snippet: Biofilm of strain 630Δ erm after growth in a continuous-flow micro-fermentor. After anaerobic growth for 72 h in TYt medium, macro-colonies and biofilms can be observed on micro-fermentor walls. The medium is clear, as expected in the absence of planktonic growth. A representative picture of independent experiments is shown (A) . Magnifications (B,C) allow observing macro-colonies (arrows).

Techniques Used: Flow Cytometry

Biofilm and planktonic cells of strain 630Δ erm . Biofilms (A–D) and planktonic cultures (E,F) of strain 630Δ erm were grown in parallel in TYt medium, respectively, for 48 h in 24-well polystyrene micro-titer plates and for 24 h in Falcon tubes. After having been recovered, fixed and washed, biofilm and planktonic cells were observed by Transmitted Light Microscopy. Representative images are shown, with a white bar indicating the scale (10 μm). In biofilm (A–D) , (i) elongated rods (∼20–30 μm) are shown by white horizontal arrows, and (ii) cells aligned side by side along the width and tightly packed into micro-aggregates are indicated by black vertical arrows. In a planktonic culture (F) , a refracting spore is indicated by a gray oblique arrow.
Figure Legend Snippet: Biofilm and planktonic cells of strain 630Δ erm . Biofilms (A–D) and planktonic cultures (E,F) of strain 630Δ erm were grown in parallel in TYt medium, respectively, for 48 h in 24-well polystyrene micro-titer plates and for 24 h in Falcon tubes. After having been recovered, fixed and washed, biofilm and planktonic cells were observed by Transmitted Light Microscopy. Representative images are shown, with a white bar indicating the scale (10 μm). In biofilm (A–D) , (i) elongated rods (∼20–30 μm) are shown by white horizontal arrows, and (ii) cells aligned side by side along the width and tightly packed into micro-aggregates are indicated by black vertical arrows. In a planktonic culture (F) , a refracting spore is indicated by a gray oblique arrow.

Techniques Used: Light Microscopy

2) Product Images from "Aggregatibacter actinomycetemcomitans mediates protection of Porphyromonas gingivalis from Streptococcus sanguinis hydrogen peroxide production in multi-species biofilms"

Article Title: Aggregatibacter actinomycetemcomitans mediates protection of Porphyromonas gingivalis from Streptococcus sanguinis hydrogen peroxide production in multi-species biofilms

Journal: Scientific Reports

doi: 10.1038/s41598-019-41467-9

The effect of the spxB gene deletion on Pg biomass in Pg-Aa-Ss tri-species biofilms under micro-aerobic conditions. ( A ) Ss WT and Ss Δ spxB biofilms with/without the supplement of Aa were cultured. The H 2 O 2 concentration in the supernatant of these biofilms was measured by the Hydrogen Peroxide Assay as described in Materials and methods. ( B ) Pg - Aa - Ss WT (left) and Pg - Aa-Ss Δ spxB tri-species (right) ( Pg = green, Ss = blue, Aa = red) biofilms were shown. ( C ) The biomass of Pg , Aa and Ss in B was quantified by COMSTAT and shown as a bar chart. Scale bars were indicated on the corresponding images. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.
Figure Legend Snippet: The effect of the spxB gene deletion on Pg biomass in Pg-Aa-Ss tri-species biofilms under micro-aerobic conditions. ( A ) Ss WT and Ss Δ spxB biofilms with/without the supplement of Aa were cultured. The H 2 O 2 concentration in the supernatant of these biofilms was measured by the Hydrogen Peroxide Assay as described in Materials and methods. ( B ) Pg - Aa - Ss WT (left) and Pg - Aa-Ss Δ spxB tri-species (right) ( Pg = green, Ss = blue, Aa = red) biofilms were shown. ( C ) The biomass of Pg , Aa and Ss in B was quantified by COMSTAT and shown as a bar chart. Scale bars were indicated on the corresponding images. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.

Techniques Used: Cell Culture, Concentration Assay, H2O2 Assay

Confocal microscopical analysis of Pg biomass in 4-day old mixed-species biofilms under micro-aerobic conditions (6% O 2 ). ( A ) Pg was incubated under static conditions for biofilm growth (left bar) and under shaking conditions (100 rpm) to prevent biofilm formation and to maintain a planktonic state (right bar is absent due to lack of growth). After 4 days, the CFUs from both samples were tested. ( B ) Single and multiplex staining of Pg (green), Aa (red) and Ss (blue) with FISH probes specific to the conserved 16s ribosomal (rRNA) genes of these bacteria (scale bar shown in top left panel = 20 µm). ( C ) Pg biomass in ( B ) was calculated for single and multiplex biofilms. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.
Figure Legend Snippet: Confocal microscopical analysis of Pg biomass in 4-day old mixed-species biofilms under micro-aerobic conditions (6% O 2 ). ( A ) Pg was incubated under static conditions for biofilm growth (left bar) and under shaking conditions (100 rpm) to prevent biofilm formation and to maintain a planktonic state (right bar is absent due to lack of growth). After 4 days, the CFUs from both samples were tested. ( B ) Single and multiplex staining of Pg (green), Aa (red) and Ss (blue) with FISH probes specific to the conserved 16s ribosomal (rRNA) genes of these bacteria (scale bar shown in top left panel = 20 µm). ( C ) Pg biomass in ( B ) was calculated for single and multiplex biofilms. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.

Techniques Used: Incubation, Multiplex Assay, Staining, Fluorescence In Situ Hybridization

The influence of the katA gene of Aa VT1169 on Pg - Ss - Aa VT1169 tri-species biofilms. ( A ) The H 2 O 2 concentrations of Pg - Ss - Aa VT1169 and Pg - Ss - Aa Δ katA were recorded at 10-minute intervals using the Hydrogen Peroxide Assay. ( B ) The biofilms of Pg - Ss - Aa VT1169 and Pg - Ss - Aa Δ katA were stained by FISH and observed by CLSM. Pg , Aa VT1169 and Ss were marked as green, red and blue, respectively. ( C ) The biomass of Pg , Aa VT1169 and Ss in ( B ) were quantified by COMSTAT. Scale bars were indicated on the corresponding images. *P ≤ 0.05, ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.
Figure Legend Snippet: The influence of the katA gene of Aa VT1169 on Pg - Ss - Aa VT1169 tri-species biofilms. ( A ) The H 2 O 2 concentrations of Pg - Ss - Aa VT1169 and Pg - Ss - Aa Δ katA were recorded at 10-minute intervals using the Hydrogen Peroxide Assay. ( B ) The biofilms of Pg - Ss - Aa VT1169 and Pg - Ss - Aa Δ katA were stained by FISH and observed by CLSM. Pg , Aa VT1169 and Ss were marked as green, red and blue, respectively. ( C ) The biomass of Pg , Aa VT1169 and Ss in ( B ) were quantified by COMSTAT. Scale bars were indicated on the corresponding images. *P ≤ 0.05, ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.

Techniques Used: H2O2 Assay, Staining, Fluorescence In Situ Hybridization, Confocal Laser Scanning Microscopy

Confocal microscopical images of 4-day old Pg-Ss dual-species biofilms to analyze the effect of H 2 O 2 on Pg biomass under micro-aerobic conditions. ( A ) Pg-Ss dual-species biofilms ( Pg = green, Ss = blue) with/without the treatment of 10,000 U/mL of catalase. ( B ) Pg-Ss wild type (WT) and Pg-Ss Δ spxB dual-species biofilms. Samples were stained by FISH probes. Orthogonal CLSM images were shown in the left panel. Scale bars were indicated on the corresponding images. In the right panel, the biomass of Pg from images in the left panel was quantified by COMSTAT analysis. ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.
Figure Legend Snippet: Confocal microscopical images of 4-day old Pg-Ss dual-species biofilms to analyze the effect of H 2 O 2 on Pg biomass under micro-aerobic conditions. ( A ) Pg-Ss dual-species biofilms ( Pg = green, Ss = blue) with/without the treatment of 10,000 U/mL of catalase. ( B ) Pg-Ss wild type (WT) and Pg-Ss Δ spxB dual-species biofilms. Samples were stained by FISH probes. Orthogonal CLSM images were shown in the left panel. Scale bars were indicated on the corresponding images. In the right panel, the biomass of Pg from images in the left panel was quantified by COMSTAT analysis. ***P ≤ 0.001, Student’s t- test. Means and standard deviations from triplicate experiments are shown.

Techniques Used: Staining, Fluorescence In Situ Hybridization, Confocal Laser Scanning Microscopy

3) Product Images from "Bundle-Forming Pili and EspA Are Involved in Biofilm Formation by Enteropathogenic Escherichia coli"

Article Title: Bundle-Forming Pili and EspA Are Involved in Biofilm Formation by Enteropathogenic Escherichia coli

Journal: Journal of Bacteriology

doi: 10.1128/JB.00177-06

Proposed model of biofilm formation by EPEC.
Figure Legend Snippet: Proposed model of biofilm formation by EPEC.

Techniques Used:

Transcription expression of EPEC virulence genes ( LEE1 , LEE2 , LEE3 , LEE4 , LEE5 , per , bfp , fliA , fliC , and flhDC ) during biofilm formation on plastic in DMEM using transcription fusions with a reporter gfp gene (plasmid pFPV25). We used as a negative control the bla :: gfp (β-lactamase promoter) fusion, which is constitutive. Transcription of all promoters was normalized by the number of bacterial cells and subtracted from basal gfp expression levels expressed by the promoterless gfp vector (pFPV25).
Figure Legend Snippet: Transcription expression of EPEC virulence genes ( LEE1 , LEE2 , LEE3 , LEE4 , LEE5 , per , bfp , fliA , fliC , and flhDC ) during biofilm formation on plastic in DMEM using transcription fusions with a reporter gfp gene (plasmid pFPV25). We used as a negative control the bla :: gfp (β-lactamase promoter) fusion, which is constitutive. Transcription of all promoters was normalized by the number of bacterial cells and subtracted from basal gfp expression levels expressed by the promoterless gfp vector (pFPV25).

Techniques Used: Expressing, Plasmid Preparation, Negative Control

Measurement of biofilm biomass during different incubation time points (3, 6, 9, 12, and 24 h) by WT EPEC (filled diamonds) and isogenic mutants in espA (filled squares), bfp (open triangles), qseA (×'s), fimA (filled circles), and flu (open circles) in DMEM on an abiotic surface under static conditions. Biomass is expressed as CFU/cm 2 . Error bars indicate standard errors of the means.
Figure Legend Snippet: Measurement of biofilm biomass during different incubation time points (3, 6, 9, 12, and 24 h) by WT EPEC (filled diamonds) and isogenic mutants in espA (filled squares), bfp (open triangles), qseA (×'s), fimA (filled circles), and flu (open circles) in DMEM on an abiotic surface under static conditions. Biomass is expressed as CFU/cm 2 . Error bars indicate standard errors of the means.

Techniques Used: Incubation

Light microscopy of biofilm formation by WT EPEC and isogenic mutants (of espA , bfpA , and qseA ) during different incubation time points (6, 12, and 24 h) in DMEM on an abiotic surface under static conditions. Magnification, ×100.
Figure Legend Snippet: Light microscopy of biofilm formation by WT EPEC and isogenic mutants (of espA , bfpA , and qseA ) during different incubation time points (6, 12, and 24 h) in DMEM on an abiotic surface under static conditions. Magnification, ×100.

Techniques Used: Light Microscopy, Incubation

4) Product Images from "Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids"

Article Title: Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids

Journal: Journal of periodontal research

doi: 10.1111/jre.12372

Inhibitory effect of prenylated flavonoids on growth and biofilm formation of P. gingivalis (A–C) P. gingivalis growth in the presence of prenylated flavonoids at 12.5 µM is shown by measuring absorbance at 620 nm different time points. Results were similar in each of three independent experiments and representative data are shown as means ± SD in a triplicate assay. (D) Biofilm formation (absorbance at 492 nm after staining with safranin) was assessed after a 24 h-culture in the presence of a compound at 1.25 µM. All the compounds at 1.25 M had no effect on growth of P. gingivalis . Results were similar in three independent experiments and representative data are shown. Results are expressed as means ± SD in a triplicate assay. Data were statistically analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. Asterisks indicate significant difference ( P
Figure Legend Snippet: Inhibitory effect of prenylated flavonoids on growth and biofilm formation of P. gingivalis (A–C) P. gingivalis growth in the presence of prenylated flavonoids at 12.5 µM is shown by measuring absorbance at 620 nm different time points. Results were similar in each of three independent experiments and representative data are shown as means ± SD in a triplicate assay. (D) Biofilm formation (absorbance at 492 nm after staining with safranin) was assessed after a 24 h-culture in the presence of a compound at 1.25 µM. All the compounds at 1.25 M had no effect on growth of P. gingivalis . Results were similar in three independent experiments and representative data are shown. Results are expressed as means ± SD in a triplicate assay. Data were statistically analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. Asterisks indicate significant difference ( P

Techniques Used: Staining

5) Product Images from "Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis"

Article Title: Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0051241

Loss of outer membrane integrity in strain RN102. Bacterial samples were collected from 48-hour-cultured biofilms for TEM analysis. TEM images of the bacterial cells and the cell appendages are shown for strains: (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). The outer membranes are indicated by arrows. Representative electron-microphotographs of each strain are shown. A 500-nm-long bar is shown in the lower left corner of each eclectron-micrograph. (F) Western blot analysis of supernatants from BW25113 and RN102. Supernatants were harvested by centrifugation from bacterial liquid culture grown for 48 hours under static conditions. Results of Western blot using anti-Crp, anti-DsbA, anti-OmpC, and anti-OmpA antisera are shown. (G) Supernatants from bacterial liquid cultures of BW25113 or RN102 grown for 48 hours under static conditions were serially diluted with TE. The diluted samples were used as template DNA for PCR using E. coli atoS gene-specific primer pairs. Lanes: 1, without dilution; 2, 10 −1 dilution; 3, 10 −2 dilution; 4, 10 −3 dilution; 5, 10 −4 dilution; 6, 10 −5 dilution; 7, 10 −6 dilution.
Figure Legend Snippet: Loss of outer membrane integrity in strain RN102. Bacterial samples were collected from 48-hour-cultured biofilms for TEM analysis. TEM images of the bacterial cells and the cell appendages are shown for strains: (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). The outer membranes are indicated by arrows. Representative electron-microphotographs of each strain are shown. A 500-nm-long bar is shown in the lower left corner of each eclectron-micrograph. (F) Western blot analysis of supernatants from BW25113 and RN102. Supernatants were harvested by centrifugation from bacterial liquid culture grown for 48 hours under static conditions. Results of Western blot using anti-Crp, anti-DsbA, anti-OmpC, and anti-OmpA antisera are shown. (G) Supernatants from bacterial liquid cultures of BW25113 or RN102 grown for 48 hours under static conditions were serially diluted with TE. The diluted samples were used as template DNA for PCR using E. coli atoS gene-specific primer pairs. Lanes: 1, without dilution; 2, 10 −1 dilution; 3, 10 −2 dilution; 4, 10 −3 dilution; 5, 10 −4 dilution; 6, 10 −5 dilution; 7, 10 −6 dilution.

Techniques Used: Cell Culture, Transmission Electron Microscopy, Western Blot, Centrifugation, Polymerase Chain Reaction

Contribution of eDNA to biofilm structure formed by RN102. (A–E) CLSM images of biofilms formed by strains: (A) BW25113 (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (D) RN102/pNT3( hldE ). Images of biofilms stained with acrydine orange are shown as digital CLSM images. In each strain, a section which has the largest sum of signals in the defined area (127.3 μm by 127.3 μm) among all X–Y sections is shown in the upper row (X–Y). The overview of biofilms in the same area of each X–Y section is shown as 3D image in the lower row (3D). The volume of each 3D image (μm 3 ) in the area of the X–Y planes was quantified and the mean ± SD obtained from 3 different areas chosen at random are denoted in the upper-right corners. The data shown are representative microphotographs of two independent experiments. (F) Quantification of eDNA from BW25113 and RN102 strains. The bars represent the ratio of extracellular DNA to intracellular DNA (eDNA/iDNA). Results are shown as the mean ± SD from 3 independent experiments. * P
Figure Legend Snippet: Contribution of eDNA to biofilm structure formed by RN102. (A–E) CLSM images of biofilms formed by strains: (A) BW25113 (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (D) RN102/pNT3( hldE ). Images of biofilms stained with acrydine orange are shown as digital CLSM images. In each strain, a section which has the largest sum of signals in the defined area (127.3 μm by 127.3 μm) among all X–Y sections is shown in the upper row (X–Y). The overview of biofilms in the same area of each X–Y section is shown as 3D image in the lower row (3D). The volume of each 3D image (μm 3 ) in the area of the X–Y planes was quantified and the mean ± SD obtained from 3 different areas chosen at random are denoted in the upper-right corners. The data shown are representative microphotographs of two independent experiments. (F) Quantification of eDNA from BW25113 and RN102 strains. The bars represent the ratio of extracellular DNA to intracellular DNA (eDNA/iDNA). Results are shown as the mean ± SD from 3 independent experiments. * P

Techniques Used: Confocal Laser Scanning Microscopy, Staining

Biofilm formation and growth of a series of LPS mutants. (A) Biofilm formation by a series of core OS LPS mutants when compared to the parental strain, BW25113. The mean ± SD of results from 3 independent experiments are shown. Statistical analysis was performed using ANOVA. * P
Figure Legend Snippet: Biofilm formation and growth of a series of LPS mutants. (A) Biofilm formation by a series of core OS LPS mutants when compared to the parental strain, BW25113. The mean ± SD of results from 3 independent experiments are shown. Statistical analysis was performed using ANOVA. * P

Techniques Used:

Loss of flagella in RN102. Fourty eight-hour-cultured biofilms were collected and analyzed by TEM, and by Western blot for FliC. (A–E) TEM images of the bacterial cells and the cell appendages are shown for strains (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). Flagella found in figures (A), (C), and (E) are shown by arrowheads. Representative electron-microphotographs of each strain are shown. A 1-μm-long bar is shown in the lower left corner. (F) Supernatants were collected from 48-hour bacterial cultures. Result of Western blot using anti-FliC antiserum is shown. Lanes; 1, BW25113; 2, RN102; 3, BW25113/pNTR-SD; 4, RN102/pNTR-SD; 5, RN102/pNT3( hldE ); 6, RN110.
Figure Legend Snippet: Loss of flagella in RN102. Fourty eight-hour-cultured biofilms were collected and analyzed by TEM, and by Western blot for FliC. (A–E) TEM images of the bacterial cells and the cell appendages are shown for strains (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). Flagella found in figures (A), (C), and (E) are shown by arrowheads. Representative electron-microphotographs of each strain are shown. A 1-μm-long bar is shown in the lower left corner. (F) Supernatants were collected from 48-hour bacterial cultures. Result of Western blot using anti-FliC antiserum is shown. Lanes; 1, BW25113; 2, RN102; 3, BW25113/pNTR-SD; 4, RN102/pNTR-SD; 5, RN102/pNT3( hldE ); 6, RN110.

Techniques Used: Cell Culture, Transmission Electron Microscopy, Western Blot

6) Product Images from "Phevalin (aureusimine B)Production by Staphylococcus aureus Biofilm and Impacts on Human Keratinocyte Gene Expression"

Article Title: Phevalin (aureusimine B)Production by Staphylococcus aureus Biofilm and Impacts on Human Keratinocyte Gene Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040973

S. aureus biofilms produce more phevalin than their planktonic counterparts. (A) HPLC-MS analysis of organic extracts from S. aureus biofilm, planktonic, and growth medium control revealed that biofilms produce more phevalin (aureusimine B) than planktonic cultures (arrow). A compound that is likely tyrvalin (aureusimine A) was also present at higher levels in the biofilm (*). (B) Phevalin production was detected directly in samples without prior organic extraction. Samples were normalized to cell density (optical density, 600 nm, OD 600 ) in biofilm (OD 600 0.9), resuspended biofilm (OD 600 1.4), and planktonic cultures (OD 600 0.66). Data represent means ± SEM, n = 3, ***p
Figure Legend Snippet: S. aureus biofilms produce more phevalin than their planktonic counterparts. (A) HPLC-MS analysis of organic extracts from S. aureus biofilm, planktonic, and growth medium control revealed that biofilms produce more phevalin (aureusimine B) than planktonic cultures (arrow). A compound that is likely tyrvalin (aureusimine A) was also present at higher levels in the biofilm (*). (B) Phevalin production was detected directly in samples without prior organic extraction. Samples were normalized to cell density (optical density, 600 nm, OD 600 ) in biofilm (OD 600 0.9), resuspended biofilm (OD 600 1.4), and planktonic cultures (OD 600 0.66). Data represent means ± SEM, n = 3, ***p

Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry

7) Product Images from "A Distinguishable Role of eDNA in the Viscoelastic Relaxation of Biofilms"

Article Title: A Distinguishable Role of eDNA in the Viscoelastic Relaxation of Biofilms

Journal: mBio

doi: 10.1128/mBio.00497-13

(a) Coefficients (a ij ) of the initial time constant ranges (C i ) for each principal component (PC j ) according to the equation P C j = Σ i = 1 7 α i j × E ¯ i (see also the last equation in Materials and Methods). (b) Assignment of matrix chemistries to the three principal components ( PC j ) as distinguished for the different biofilms involved in this study. Principal components are expressed as a function of relaxation time constants based on positive correlations with matrix chemistries defined in Table 1. The matrix chemistries positively associated with PC 1 include water and soluble polysaccharides, while the matrix chemistry for PC 2 includes other EPS polymers, like insoluble polysaccharides (i.e. glucans). PC 3 includes only intact eDNA.
Figure Legend Snippet: (a) Coefficients (a ij ) of the initial time constant ranges (C i ) for each principal component (PC j ) according to the equation P C j = Σ i = 1 7 α i j × E ¯ i (see also the last equation in Materials and Methods). (b) Assignment of matrix chemistries to the three principal components ( PC j ) as distinguished for the different biofilms involved in this study. Principal components are expressed as a function of relaxation time constants based on positive correlations with matrix chemistries defined in Table 1. The matrix chemistries positively associated with PC 1 include water and soluble polysaccharides, while the matrix chemistry for PC 2 includes other EPS polymers, like insoluble polysaccharides (i.e. glucans). PC 3 includes only intact eDNA.

Techniques Used:

Relative importance of the individual Maxwell elements of different biofilms as a function of their characteristic relaxation time constants in relation to the different matrix chemistries according to Table 1 . Each data point represents one Maxwell element with its time constant plotted against its relative importance. Each individual biofilm possessed an average of four or five Maxwell elements. Similar biofilms were grown and investigated minimally three times with separate initial bacterial cultures. Maxwell elements with 0% relative importance have no accompanying time constant and are not plotted, while characteristic time constants exceeding 7,000 s have been assigned a value of 7,000 s. Vertical lines indicate divisions of relaxation time constant ranges (C i ). Panels: a, P. aeruginosa biofilms; b, S. mutans biofilms; c, S. aureus and S. epidermidis biofilms.
Figure Legend Snippet: Relative importance of the individual Maxwell elements of different biofilms as a function of their characteristic relaxation time constants in relation to the different matrix chemistries according to Table 1 . Each data point represents one Maxwell element with its time constant plotted against its relative importance. Each individual biofilm possessed an average of four or five Maxwell elements. Similar biofilms were grown and investigated minimally three times with separate initial bacterial cultures. Maxwell elements with 0% relative importance have no accompanying time constant and are not plotted, while characteristic time constants exceeding 7,000 s have been assigned a value of 7,000 s. Vertical lines indicate divisions of relaxation time constant ranges (C i ). Panels: a, P. aeruginosa biofilms; b, S. mutans biofilms; c, S. aureus and S. epidermidis biofilms.

Techniques Used:

Panels a to c represent the measured stress relaxation of a P. aeruginosa SG81 biofilm as a function of time, together with model fits to the data, obtained by using two (panels a and b) or five (panel c) Maxwell elements. Note that panel a extends over 100 s, while panels b and c refer only to the first 5 s of the relaxation process. Panel d represents the quality of the fit, indicated by chi-square values, as a function of the number of Maxwell elements used for the fit.
Figure Legend Snippet: Panels a to c represent the measured stress relaxation of a P. aeruginosa SG81 biofilm as a function of time, together with model fits to the data, obtained by using two (panels a and b) or five (panel c) Maxwell elements. Note that panel a extends over 100 s, while panels b and c refer only to the first 5 s of the relaxation process. Panel d represents the quality of the fit, indicated by chi-square values, as a function of the number of Maxwell elements used for the fit.

Techniques Used:

8) Product Images from "Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms"

Article Title: Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms141019434

Anti-biofilm effects of the flavonoids collection when added prior-to ( a ) or post-biofilm formation ( b ). Strains 1 and 2 are S. aureus ATCC 25923 and Newman clinical strains, respectively. Highly active flavonoids are those present in both shadowed areas. Primary screening results are presented in Table S1.
Figure Legend Snippet: Anti-biofilm effects of the flavonoids collection when added prior-to ( a ) or post-biofilm formation ( b ). Strains 1 and 2 are S. aureus ATCC 25923 and Newman clinical strains, respectively. Highly active flavonoids are those present in both shadowed areas. Primary screening results are presented in Table S1.

Techniques Used:

Summary of the anti-biofilm screening and selection criteria applied to the collection in this study. Highly and moderately active flavonoids are listed in Tables S2 and S3, respectively.
Figure Legend Snippet: Summary of the anti-biofilm screening and selection criteria applied to the collection in this study. Highly and moderately active flavonoids are listed in Tables S2 and S3, respectively.

Techniques Used: Selection

The four most active anti-biofilm flavonoids identified in this contribution.
Figure Legend Snippet: The four most active anti-biofilm flavonoids identified in this contribution.

Techniques Used:

Key structural features present in the anti-biofilm chalcones and flavones (moderately and highly active ones).
Figure Legend Snippet: Key structural features present in the anti-biofilm chalcones and flavones (moderately and highly active ones).

Techniques Used:

9) Product Images from "Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism"

Article Title: Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099204

PittGG biofilm formation is stimulated by ampicillin in a narrow concentration range. Crystal violet assay of 1-day PittGG biofilms, formed in the presence of increasing amounts of ampicillin up 300 ng/mL, showed a stimulation of biofilm formation at 170 ng/mL concentration.
Figure Legend Snippet: PittGG biofilm formation is stimulated by ampicillin in a narrow concentration range. Crystal violet assay of 1-day PittGG biofilms, formed in the presence of increasing amounts of ampicillin up 300 ng/mL, showed a stimulation of biofilm formation at 170 ng/mL concentration.

Techniques Used: Concentration Assay, Crystal Violet Assay

Sub-inhibitory concentrations of ampicillin result in an increase in dead NTHi bacteria in newly formed biofilms. Confocal laser scanning microscopy (cLSM) of biofilms formed by NTHi strains 2019 and PittGG in the absence (− amp) and presence (+ amp) of sub-inhibitory concentrations of ampicillin were stained using the LIVE/DEAD viability assay. 2019 were exposed to 90 ng/mL ampicillin and PittGG to 170 ng/mL ampicillin. Live bacteria were colored green and dead bacteria red. From above, biofilms formed in the absence of ampicillin were mostly green (A C), indicating living (or intact) NTHi bacteria. Clumps of bacteria in both biofilms stained red (A C) indicating the presence of dead, or structurally compromised bacteria. Biofilms formed by 2019 (A) contained fewer aggregates of red bacteria than did PittGG (C) contained more. In the presence of ampicillin (B D) the bacteria in the biofilms were mostly red, indicating a large number of dead bacteria. Green-stained bacteria were still present in both biofilms but appeared more aggregated in the presence of antibiotic (B D). Large amounts of aggregated red bacteria were present in both biofilms, with the aggregates being larger in the PittGG biofilm (Fig. D). Z-stack projections of the biofilms (E–H) showed that all the biofilms were denser at the base of the biofilm, whether exposed to ampicillin or not. In the absence of ampicillin, 2019 (E) and PittGG (G) biofilms comprised green, or intact bacteria. In the presence of ampicillin the biofilm contained mostly structurally compromised bacterial cells, which were colored red or yellow. The 2019 (F) and PittGG (H) biofilms formed in the presence of ampicillin were higher than the comparable biofilms formed without exposure to ampicillin (E G). All images; scale bar (G) = 20 µm.
Figure Legend Snippet: Sub-inhibitory concentrations of ampicillin result in an increase in dead NTHi bacteria in newly formed biofilms. Confocal laser scanning microscopy (cLSM) of biofilms formed by NTHi strains 2019 and PittGG in the absence (− amp) and presence (+ amp) of sub-inhibitory concentrations of ampicillin were stained using the LIVE/DEAD viability assay. 2019 were exposed to 90 ng/mL ampicillin and PittGG to 170 ng/mL ampicillin. Live bacteria were colored green and dead bacteria red. From above, biofilms formed in the absence of ampicillin were mostly green (A C), indicating living (or intact) NTHi bacteria. Clumps of bacteria in both biofilms stained red (A C) indicating the presence of dead, or structurally compromised bacteria. Biofilms formed by 2019 (A) contained fewer aggregates of red bacteria than did PittGG (C) contained more. In the presence of ampicillin (B D) the bacteria in the biofilms were mostly red, indicating a large number of dead bacteria. Green-stained bacteria were still present in both biofilms but appeared more aggregated in the presence of antibiotic (B D). Large amounts of aggregated red bacteria were present in both biofilms, with the aggregates being larger in the PittGG biofilm (Fig. D). Z-stack projections of the biofilms (E–H) showed that all the biofilms were denser at the base of the biofilm, whether exposed to ampicillin or not. In the absence of ampicillin, 2019 (E) and PittGG (G) biofilms comprised green, or intact bacteria. In the presence of ampicillin the biofilm contained mostly structurally compromised bacterial cells, which were colored red or yellow. The 2019 (F) and PittGG (H) biofilms formed in the presence of ampicillin were higher than the comparable biofilms formed without exposure to ampicillin (E G). All images; scale bar (G) = 20 µm.

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Viability Assay

A sub-inhibitory concentration of ampicillin changes the composition of forming NTHi biofilms. Two strains of NTHi, 2019 and PittGG, exposed to sub-inhibitory concentrations (90 ng/mL: 2019; 170 ng/mL: PittGG) of ampicillin for 24 hr, showed increases in biofilm biomass as measured by dry weight (A. Biomass), and percent protein content (B. Protein Content). In contrast, the presence of the antibiotic during biofilm formation resulted in a decrease in viable (or culturable) bacteria (C. Viable Bacteria). Strain PittGG showed the most noticeable changes in dry weight, protein content and numbers of viable bacteria resulting from exposure to 170 ng/mL ampicillin (t-test p =
Figure Legend Snippet: A sub-inhibitory concentration of ampicillin changes the composition of forming NTHi biofilms. Two strains of NTHi, 2019 and PittGG, exposed to sub-inhibitory concentrations (90 ng/mL: 2019; 170 ng/mL: PittGG) of ampicillin for 24 hr, showed increases in biofilm biomass as measured by dry weight (A. Biomass), and percent protein content (B. Protein Content). In contrast, the presence of the antibiotic during biofilm formation resulted in a decrease in viable (or culturable) bacteria (C. Viable Bacteria). Strain PittGG showed the most noticeable changes in dry weight, protein content and numbers of viable bacteria resulting from exposure to 170 ng/mL ampicillin (t-test p =

Techniques Used: Concentration Assay, T-Test

Biofilm formation is stimulated by beta-lactam antibiotics. Crystal violet assays of 1-day NTHi biofilms formed in the presence of amoxicillin or amoxicillin. Strains 2019, 9274 and PittEE reacted to inhibitory concentrations of antibiotic (grey vertical bars, Biomass OD 600 ) by producing more crystal violet stainable biofilm (line graph). This stimulatory effect was different for each bacterial strain. PittGG did not react with amoxicillin and produced an ambiguous reaction to ampicillin. PittAA and PittII were not affected by amoxicillin or ampicillin in the concentration ranges studied. Cefuroxime (0–950 ng/mL range) showed a biofilm-stimulatory effect on all the NTHi strains under study. Each strain exhibited biofilm stimulation in different concentrations of antibiotic. Strain 2019 was maximally stimulated at 100 ng/mL cefuroxime, PittGG and PittII at 220 ng/mL, 9274, PittAA at 300 ng/mL and PittEE at 525 ng/mL.
Figure Legend Snippet: Biofilm formation is stimulated by beta-lactam antibiotics. Crystal violet assays of 1-day NTHi biofilms formed in the presence of amoxicillin or amoxicillin. Strains 2019, 9274 and PittEE reacted to inhibitory concentrations of antibiotic (grey vertical bars, Biomass OD 600 ) by producing more crystal violet stainable biofilm (line graph). This stimulatory effect was different for each bacterial strain. PittGG did not react with amoxicillin and produced an ambiguous reaction to ampicillin. PittAA and PittII were not affected by amoxicillin or ampicillin in the concentration ranges studied. Cefuroxime (0–950 ng/mL range) showed a biofilm-stimulatory effect on all the NTHi strains under study. Each strain exhibited biofilm stimulation in different concentrations of antibiotic. Strain 2019 was maximally stimulated at 100 ng/mL cefuroxime, PittGG and PittII at 220 ng/mL, 9274, PittAA at 300 ng/mL and PittEE at 525 ng/mL.

Techniques Used: Produced, Concentration Assay

Sub-inhibitory concentrations of ampicillin change the ultrastructure of newly formed NTHi biofilms. Scanning electron microscopy (SEM) images comparing NTHi biofilms formed in the absence and presence of ampicillin on Thermanox coverslips. A–C: SEM of 2019 biofilms. A) At low power the 2019 biofilm is seen as a mat covering the Thermanox substrate. Bar = 500 µm. B) higher magnification shows the biofilm to be composed of partitions forming empty spaces, or cells, covered with a film of amorphous material (arrow). Bar = 10 µm. C) the partitions within the biofilm are composed primarily of bacterial cells aggregated into flat sheets. Bar = 5 µm. D–F: SEM of 2019 biofilms formed in 90 ng/mL ampicillin. D) The 2019 biofilm formed with ampicillin covers the substrate. Bar = 500 µm. E) The biofilm is composed of partitions around empty spaces with a flat sheet of amorphous material (arrow) over the top. Bar = 50 µm F) The biofilm partitions are composed of aggregated bacteria embedded in sheets of amorphous material. Bar = 5 µm. G–I: SEM of PittGG biofilms. G) The PittGG bacteria form a biofilm over the Thermanox surface. Bar = 500 µm. H) The biofilm is composed of bacterial cells aggregated into poorly defined partitions and covered with a layer of amorphous material (arrow). Bar = 10 µm. I) The biofilm is composed of bacterial cells aggregated into widely spaced strands, and empty space. Bar = 5 µm. J–L: SEM of PittGG biofilms formed in 170 ng/mL ampicillin. J) In the presence of ampicillin the PittGG bacteria form a biofilm comprised of a thick mat, which appears to be strongly attached to itself but less well attached to the substrate. Bar = 500 µm. K) The biofilm mats appear to be composed mostly of amorphous material, a layer of which covers the biofilm (arrow), and arranged into tightly-packed thin partitions. Bar = 50 µm. L) the biofilm is composed of amorphous material formed into thin partitions. The few bacteria detected were embedded in the thin partitions. Bar = 5 µm.
Figure Legend Snippet: Sub-inhibitory concentrations of ampicillin change the ultrastructure of newly formed NTHi biofilms. Scanning electron microscopy (SEM) images comparing NTHi biofilms formed in the absence and presence of ampicillin on Thermanox coverslips. A–C: SEM of 2019 biofilms. A) At low power the 2019 biofilm is seen as a mat covering the Thermanox substrate. Bar = 500 µm. B) higher magnification shows the biofilm to be composed of partitions forming empty spaces, or cells, covered with a film of amorphous material (arrow). Bar = 10 µm. C) the partitions within the biofilm are composed primarily of bacterial cells aggregated into flat sheets. Bar = 5 µm. D–F: SEM of 2019 biofilms formed in 90 ng/mL ampicillin. D) The 2019 biofilm formed with ampicillin covers the substrate. Bar = 500 µm. E) The biofilm is composed of partitions around empty spaces with a flat sheet of amorphous material (arrow) over the top. Bar = 50 µm F) The biofilm partitions are composed of aggregated bacteria embedded in sheets of amorphous material. Bar = 5 µm. G–I: SEM of PittGG biofilms. G) The PittGG bacteria form a biofilm over the Thermanox surface. Bar = 500 µm. H) The biofilm is composed of bacterial cells aggregated into poorly defined partitions and covered with a layer of amorphous material (arrow). Bar = 10 µm. I) The biofilm is composed of bacterial cells aggregated into widely spaced strands, and empty space. Bar = 5 µm. J–L: SEM of PittGG biofilms formed in 170 ng/mL ampicillin. J) In the presence of ampicillin the PittGG bacteria form a biofilm comprised of a thick mat, which appears to be strongly attached to itself but less well attached to the substrate. Bar = 500 µm. K) The biofilm mats appear to be composed mostly of amorphous material, a layer of which covers the biofilm (arrow), and arranged into tightly-packed thin partitions. Bar = 50 µm. L) the biofilm is composed of amorphous material formed into thin partitions. The few bacteria detected were embedded in the thin partitions. Bar = 5 µm.

Techniques Used: Electron Microscopy

NTHi biofilms protect against a lethal dose of cefuroxime. Biofilms of NTHi strains 2019 and PittGG were formed overnight in the absence of antibiotic (Untreated), or in the presence of 170 ng/mL ampicillin (AMP), 230 ng/mL amoxicillin (AMOX) or 170 ng/mL cefuroxime (CEF). The amounts of antibiotic were chosen for their ability to stimulate biofilm formation in these two NTHi strains. After washing, the biofilms were exposed to 10 µg/mL of cefuroxime for a further 24 hr. The percentages of viable bacteria present in each biofilm are tabulated here. Not shown: planktonic NTHi bacteria are killed in the presence of 10 µg/mL cefuroxime. Although all of the formed biofilms (in the presence or absence of antibiotic) protected against cefuroxime, the amoxicillin-stimulated biofilm was able to protect bacteria from the lethal effects of the cefuroxime. p-values are documented in Table S3 .
Figure Legend Snippet: NTHi biofilms protect against a lethal dose of cefuroxime. Biofilms of NTHi strains 2019 and PittGG were formed overnight in the absence of antibiotic (Untreated), or in the presence of 170 ng/mL ampicillin (AMP), 230 ng/mL amoxicillin (AMOX) or 170 ng/mL cefuroxime (CEF). The amounts of antibiotic were chosen for their ability to stimulate biofilm formation in these two NTHi strains. After washing, the biofilms were exposed to 10 µg/mL of cefuroxime for a further 24 hr. The percentages of viable bacteria present in each biofilm are tabulated here. Not shown: planktonic NTHi bacteria are killed in the presence of 10 µg/mL cefuroxime. Although all of the formed biofilms (in the presence or absence of antibiotic) protected against cefuroxime, the amoxicillin-stimulated biofilm was able to protect bacteria from the lethal effects of the cefuroxime. p-values are documented in Table S3 .

Techniques Used:

mRNA analysis of PittGG biofilms. A. Principal component analysis of microarray gene expression of the PittGG biofilm bacteria (NTHi) exposed to 170 ng/mL ampicillin. The major variations (PC1, PC2, and PC3) are visualized in 3-dimensions. The three PCs together accounted for about 85% of the variations present in the entire data set. A distinguishable grouping difference can be seen between ampicillin treated (in blue) and non-treated (in red) samples. B. Hierarchical cluster of significantly differentially expressed genes in the PittGG biofilm bacteria (NTHi) treated with 170 ng/mL ampicillin. Heatmap visualization of 59 significantly regulated genes by ampicillin treatment. Results from the 3 replicate experiments (R1, R2 and R3) of ampicillin-treated and untreated biofilms are presented. A hierarchical clustering was analysed on the probe sets representing the 59 genes including 8 up and 51 down regulated gene transcripts with filter criteria of at least 1.5 folds change plus P≤0.05 and FDR less than 0.05. Rows: samples; columns: genes. The up-regulated genes relating to carbohydrate metabolism are indicated.
Figure Legend Snippet: mRNA analysis of PittGG biofilms. A. Principal component analysis of microarray gene expression of the PittGG biofilm bacteria (NTHi) exposed to 170 ng/mL ampicillin. The major variations (PC1, PC2, and PC3) are visualized in 3-dimensions. The three PCs together accounted for about 85% of the variations present in the entire data set. A distinguishable grouping difference can be seen between ampicillin treated (in blue) and non-treated (in red) samples. B. Hierarchical cluster of significantly differentially expressed genes in the PittGG biofilm bacteria (NTHi) treated with 170 ng/mL ampicillin. Heatmap visualization of 59 significantly regulated genes by ampicillin treatment. Results from the 3 replicate experiments (R1, R2 and R3) of ampicillin-treated and untreated biofilms are presented. A hierarchical clustering was analysed on the probe sets representing the 59 genes including 8 up and 51 down regulated gene transcripts with filter criteria of at least 1.5 folds change plus P≤0.05 and FDR less than 0.05. Rows: samples; columns: genes. The up-regulated genes relating to carbohydrate metabolism are indicated.

Techniques Used: Microarray, Expressing

Ultrastructural visualization of glycogen in PittGG biofilms. Resin-embedded thin sections of PittGG biofilms formed in the presence or absence of ampicillin (+/− Am) and treated with a glycogen stain or a no stain control (+/− gly). A–D Biofilms with ampicillin, sections stained for glycogen (periodic acid and sodium chlorite). The glycogen stain reacted with an extracellular granular substance (arrows) that was associated with biofilm bacteria. Exposure to antibiotic resulted in a bacterial size increase with some bacteria (A–C). Extracellular areas that were not in close proximity to bacteria cells (asterisk) did not contain the extracellular granular substance (B–D). E. Biofilm with antibiotic but only treated with sodium chlorite did not reveal extracellular granular substance. Large bacterial cells were present. F. Biofilm with no antibiotic, sections stained for glycogen. No extracellular granular substance was detected and bacterial cells had a normal diameter (approx. 500 nm). Some lysed cells were detected. G. Biofilm with no antibiotic, treated with sodium chlorite (no periodic acid). Bacterial cells appear normal with a few dying cells present (arrowhead). Scale bars = 500 nm.
Figure Legend Snippet: Ultrastructural visualization of glycogen in PittGG biofilms. Resin-embedded thin sections of PittGG biofilms formed in the presence or absence of ampicillin (+/− Am) and treated with a glycogen stain or a no stain control (+/− gly). A–D Biofilms with ampicillin, sections stained for glycogen (periodic acid and sodium chlorite). The glycogen stain reacted with an extracellular granular substance (arrows) that was associated with biofilm bacteria. Exposure to antibiotic resulted in a bacterial size increase with some bacteria (A–C). Extracellular areas that were not in close proximity to bacteria cells (asterisk) did not contain the extracellular granular substance (B–D). E. Biofilm with antibiotic but only treated with sodium chlorite did not reveal extracellular granular substance. Large bacterial cells were present. F. Biofilm with no antibiotic, sections stained for glycogen. No extracellular granular substance was detected and bacterial cells had a normal diameter (approx. 500 nm). Some lysed cells were detected. G. Biofilm with no antibiotic, treated with sodium chlorite (no periodic acid). Bacterial cells appear normal with a few dying cells present (arrowhead). Scale bars = 500 nm.

Techniques Used: Staining

Biofilm formation is variable between NTHi strains. Biofilm quantification using crystal violet assay showed the variability in biofilm formation between the NTHi strains in this study. Relative Biofilm Quantity was measured as OD 600 of crystal violet in DMSO; higher absorbance indicated more biofilm production. The PittAA and PittEE strains showed the least amount of biofilm attachment to the growth surface while PittII showed the most. The other three strains (2019, 9274 and PittGG) formed intermediate amounts of biofilm material (t-test analysis in Table S2 ).
Figure Legend Snippet: Biofilm formation is variable between NTHi strains. Biofilm quantification using crystal violet assay showed the variability in biofilm formation between the NTHi strains in this study. Relative Biofilm Quantity was measured as OD 600 of crystal violet in DMSO; higher absorbance indicated more biofilm production. The PittAA and PittEE strains showed the least amount of biofilm attachment to the growth surface while PittII showed the most. The other three strains (2019, 9274 and PittGG) formed intermediate amounts of biofilm material (t-test analysis in Table S2 ).

Techniques Used: Crystal Violet Assay, T-Test

10) Product Images from "Use of lectins to in situ visualize glycoconjugates of extracellular polymeric substances in acidophilic archaeal biofilms"

Article Title: Use of lectins to in situ visualize glycoconjugates of extracellular polymeric substances in acidophilic archaeal biofilms

Journal: Microbial Biotechnology

doi: 10.1111/1751-7915.12188

Maximum intensity projections of biofilms from A cidianus sp. DSM 29099 on elemental sulfur. Samples were stained by lectins AAL-Alexa488 (A), Peanut agglutinin (PNA)-fluorescein isothiocyanate (FITC) (B), Erythrina cristagalli agglutinin (ECA)-FITC (C) and GS-I (D). Two distinguished lectin binding patterns became visible, tightly bound “capsular” EPS staining (A and B) and loosely bound “colloidal” (C and D). Color allocation: green = lectins, grey = reflection.
Figure Legend Snippet: Maximum intensity projections of biofilms from A cidianus sp. DSM 29099 on elemental sulfur. Samples were stained by lectins AAL-Alexa488 (A), Peanut agglutinin (PNA)-fluorescein isothiocyanate (FITC) (B), Erythrina cristagalli agglutinin (ECA)-FITC (C) and GS-I (D). Two distinguished lectin binding patterns became visible, tightly bound “capsular” EPS staining (A and B) and loosely bound “colloidal” (C and D). Color allocation: green = lectins, grey = reflection.

Techniques Used: Staining, Binding Assay

XYZ projection (A) and isosurface projection (B) of A cidianus sp. DSM 29099 biofilms on pyrite stained by GS-I-fluorescein isothiocyanate (FITC) and counter stained by Syto64. Color allocation: green = GS-I-FITC, red = Syto 64, grey = reflection. Grid size in B = 10 μm.
Figure Legend Snippet: XYZ projection (A) and isosurface projection (B) of A cidianus sp. DSM 29099 biofilms on pyrite stained by GS-I-fluorescein isothiocyanate (FITC) and counter stained by Syto64. Color allocation: green = GS-I-FITC, red = Syto 64, grey = reflection. Grid size in B = 10 μm.

Techniques Used: Staining

Maximum intensity projections (A, B), XYZ projection (C, D) and isosurface projection (E) of A cidianus sp. DSM 29099 biofilms on pyrite stained by Con A (B) and AAL (D, E), and counter stained by and SybrGreen (A) and Syto 64 (D, E). Color allocation: green = SybrGreen/AAL-fluorescein isothiocyanate (FITC), red = Syto 64/Con A-tetramethyl rhodamine isothiocyanate (TRITC), grey = reflection.
Figure Legend Snippet: Maximum intensity projections (A, B), XYZ projection (C, D) and isosurface projection (E) of A cidianus sp. DSM 29099 biofilms on pyrite stained by Con A (B) and AAL (D, E), and counter stained by and SybrGreen (A) and Syto 64 (D, E). Color allocation: green = SybrGreen/AAL-fluorescein isothiocyanate (FITC), red = Syto 64/Con A-tetramethyl rhodamine isothiocyanate (TRITC), grey = reflection.

Techniques Used: Staining

Maximum intensity projections of A cidianus sp. DSM 29099 biofilms on pyrite stained by Syto 64 (A), SybrGreen (B) and SyproRed (C). S ulfolobus metallicus T biofilms on pyrite stained by Syto 64 (D) and SyproRed (E). Color allocation: green = SybrGreen, red = Syto 64/SyproRed, grey = reflection.
Figure Legend Snippet: Maximum intensity projections of A cidianus sp. DSM 29099 biofilms on pyrite stained by Syto 64 (A), SybrGreen (B) and SyproRed (C). S ulfolobus metallicus T biofilms on pyrite stained by Syto 64 (D) and SyproRed (E). Color allocation: green = SybrGreen, red = Syto 64/SyproRed, grey = reflection.

Techniques Used: Staining

Maximum intensity projections of biofilms from A cidianus sp. DSM 29099 on elemental sulfur. Samples were stained by SybrGreen (A), SyproOrange (B) and Syto 64 (C). Color allocation: green = SybrGreen, red = Syto 64/SyproOrange, grey = reflection. Cells formed a thin biofilm in shallow regions, cracks or holes when sulfur prills were used (A). A clear tendency of cells to attach to physical defects on sulfur coupons is also evident (B and C). Arrows show preferential attachment sites with distortions.
Figure Legend Snippet: Maximum intensity projections of biofilms from A cidianus sp. DSM 29099 on elemental sulfur. Samples were stained by SybrGreen (A), SyproOrange (B) and Syto 64 (C). Color allocation: green = SybrGreen, red = Syto 64/SyproOrange, grey = reflection. Cells formed a thin biofilm in shallow regions, cracks or holes when sulfur prills were used (A). A clear tendency of cells to attach to physical defects on sulfur coupons is also evident (B and C). Arrows show preferential attachment sites with distortions.

Techniques Used: Staining

XYZ projection (A) and isosurface projection (B) of F . acidiphilum DSM 28986 biofilms on pyrite stained by LPA-fluorescein isothiocyanate (FITC) and counter stained by FM4-64. Color allocation: green = LPA-FITC, red = FM4-64, grey = reflection. Grid size in B = 10 μm.
Figure Legend Snippet: XYZ projection (A) and isosurface projection (B) of F . acidiphilum DSM 28986 biofilms on pyrite stained by LPA-fluorescein isothiocyanate (FITC) and counter stained by FM4-64. Color allocation: green = LPA-FITC, red = FM4-64, grey = reflection. Grid size in B = 10 μm.

Techniques Used: Staining

Maximum intensity projections of F . acidiphilum DSM 28986 biofilms on pyrite stained by SybrGreen (A), Syto 9 (B), Syto 64 (C), SyproRed (D) and FM4-64 (E). Color allocation: green = SybrGreen/Syto 9, red = SyproRed/FM4-64. The pyrite surface is shown in reflection mode (= grey).
Figure Legend Snippet: Maximum intensity projections of F . acidiphilum DSM 28986 biofilms on pyrite stained by SybrGreen (A), Syto 9 (B), Syto 64 (C), SyproRed (D) and FM4-64 (E). Color allocation: green = SybrGreen/Syto 9, red = SyproRed/FM4-64. The pyrite surface is shown in reflection mode (= grey).

Techniques Used: Staining

11) Product Images from "The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm"

Article Title: The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002623

Time-lapse imaging of bacterial viability after exposure of the biofilm to 0.12% (v/v) chlorhexidine (CHX). Panel ( A ) shows the relative po pulations of live (SYTO 9-labeled; depicted in green) and dead (propidium iodide-labeled; depicted in red) cells over time in the mixed-species biofilm formed with the parental strain (A-1; cells within EPS-microcolony complex and A-2; cells outside EPS-microcolony complex) and the Δ gtfBC ::kan mutant (A-3) after exposure to CHX. Panel ( B ) shows the survival rate of live cells. The data shown are mean values ± s.d. (n = 6). The asterisks (*) indicate that the differences between the conditions tested (cells within EPS-microcolony complex vs. cells outside EPS-microcolony complex or cells not forming such structures, i.e. biofilm formed with Δ gtfBC ::kan) are statistically significant ( P
Figure Legend Snippet: Time-lapse imaging of bacterial viability after exposure of the biofilm to 0.12% (v/v) chlorhexidine (CHX). Panel ( A ) shows the relative po pulations of live (SYTO 9-labeled; depicted in green) and dead (propidium iodide-labeled; depicted in red) cells over time in the mixed-species biofilm formed with the parental strain (A-1; cells within EPS-microcolony complex and A-2; cells outside EPS-microcolony complex) and the Δ gtfBC ::kan mutant (A-3) after exposure to CHX. Panel ( B ) shows the survival rate of live cells. The data shown are mean values ± s.d. (n = 6). The asterisks (*) indicate that the differences between the conditions tested (cells within EPS-microcolony complex vs. cells outside EPS-microcolony complex or cells not forming such structures, i.e. biofilm formed with Δ gtfBC ::kan) are statistically significant ( P

Techniques Used: Imaging, Labeling, Mutagenesis

Relationship of in situ pH at sHA surface with size of surface-attached EPS-microcolony complexes. This figure depicts mixed-species biofilms (67 h) formed after the introduction of 1% (w/v) sucrose, which were processed for measurement of in situ pH at the interface between sHA surface and microcolonies. All the pH values were sorted by ( A ) the diameter and ( B ) height (distance from surface to fluid-phase) of the surface-attached microcolonies. The data shown are values from three separate experiments (n = 90). Correlation analysis showed that there is a linear relationship between pH and microcolony diameter (Pearson's test, P
Figure Legend Snippet: Relationship of in situ pH at sHA surface with size of surface-attached EPS-microcolony complexes. This figure depicts mixed-species biofilms (67 h) formed after the introduction of 1% (w/v) sucrose, which were processed for measurement of in situ pH at the interface between sHA surface and microcolonies. All the pH values were sorted by ( A ) the diameter and ( B ) height (distance from surface to fluid-phase) of the surface-attached microcolonies. The data shown are values from three separate experiments (n = 90). Correlation analysis showed that there is a linear relationship between pH and microcolony diameter (Pearson's test, P

Techniques Used: In Situ

Expression of Streptococcus mutans EPS-associated genes and proteins during mixed-species biofilm development. ( A ) This panel shows RT-qPCR analysis of gtfB , gtfC , gtfD , fruA , ftf and dexA gene expression by S. mutans in mixed-species biofilms at specific time points after introduction of 1% (w/v) sucrose. The data shown are mean values ± s.d. (n = 12). The asterisks (*) indicate that the expression level of gtfB , gtfC (at 67 h) is significantly different from other time points ( P
Figure Legend Snippet: Expression of Streptococcus mutans EPS-associated genes and proteins during mixed-species biofilm development. ( A ) This panel shows RT-qPCR analysis of gtfB , gtfC , gtfD , fruA , ftf and dexA gene expression by S. mutans in mixed-species biofilms at specific time points after introduction of 1% (w/v) sucrose. The data shown are mean values ± s.d. (n = 12). The asterisks (*) indicate that the expression level of gtfB , gtfC (at 67 h) is significantly different from other time points ( P

Techniques Used: Expressing, Quantitative RT-PCR

Structural arrangement between EPS and bacterial cells during the assembly of surface-attached EPS-microcolony complex. ( A ) This figure gives representative images of 3D renderings of mixed-species biofilms after introduction of 1% (w/v) sucrose. Panel (a) shows the dynamic evolution of surface-attached microcolonies over time. Panels (b–m) show cross sectional images of selected area for close-up views of the structural organization of EPS (red) and bacterial cells (green) during the development of an EPS-microcolony complex. The arrows indicate EPS bridging (i, j) and providing support for aggregation of multiple microcolonies (l, m). ( B ) The amount of co-localization between bacteria and EPS was calculated using DUOSTAT. The graph shows the percentage of bacteria and EPS colocalized within the biofilm over time (mean values ± s.d; n = 15). The asterisks (*) indicate that the values from each time point are significantly different from each other ( P
Figure Legend Snippet: Structural arrangement between EPS and bacterial cells during the assembly of surface-attached EPS-microcolony complex. ( A ) This figure gives representative images of 3D renderings of mixed-species biofilms after introduction of 1% (w/v) sucrose. Panel (a) shows the dynamic evolution of surface-attached microcolonies over time. Panels (b–m) show cross sectional images of selected area for close-up views of the structural organization of EPS (red) and bacterial cells (green) during the development of an EPS-microcolony complex. The arrows indicate EPS bridging (i, j) and providing support for aggregation of multiple microcolonies (l, m). ( B ) The amount of co-localization between bacteria and EPS was calculated using DUOSTAT. The graph shows the percentage of bacteria and EPS colocalized within the biofilm over time (mean values ± s.d; n = 15). The asterisks (*) indicate that the values from each time point are significantly different from each other ( P

Techniques Used:

Mixed-species biofilms formed with S. mutans Δ gtfBC ::kan or with parental strain UA159 treated with mutanase. The biofilms were formed in the presence of the gtfBC null mutant (A) or parental strain UA159 (B) with 1% (w/v) sucrose. Selected areas in ( A ) and ( B ) show detailed views of separated and merged confocal images of bacterial cells (green) and EPS (red). The biomass values of EPS and bacterial cells in the biofilms were calculated using COMSTAT. The data shown are mean values ± s.d. (n = 15).
Figure Legend Snippet: Mixed-species biofilms formed with S. mutans Δ gtfBC ::kan or with parental strain UA159 treated with mutanase. The biofilms were formed in the presence of the gtfBC null mutant (A) or parental strain UA159 (B) with 1% (w/v) sucrose. Selected areas in ( A ) and ( B ) show detailed views of separated and merged confocal images of bacterial cells (green) and EPS (red). The biomass values of EPS and bacterial cells in the biofilms were calculated using COMSTAT. The data shown are mean values ± s.d. (n = 15).

Techniques Used: Mutagenesis

Dynamics of the morphogenesis, 3D architecture development and microbial population shifts of mixed-species biofilms. ( A ) Representative 3D rendering images of mixed-species biofilms at distinct time points. Images (a–c) represent biofilms formed after the introduction of 1% (w/v) glucose. Images (d–f) represent biofilms formed in the presence of 0.1% (w/v) sucrose. Images (g–i) represent biofilms formed after the introduction of 1% (w/v) sucrose. The EPS channel is depicted in red, while the cells are depicted in green. At the upper left of each panel, the two channels are displayed separately, while the merged image is displayed at the bottom right. Lateral (side) views of each biofilm are displayed at the bottom left, while a magnified (close-up) view of each small box depicted in the merged image is positioned in the upper right corner of each panel. ( B ) The data are mean values ± s.d. (n = 30). The asterisks (*) indicate that the each of the values (EPS and bacterial biomass) in the 1% sucrose group are significantly different from others ( P
Figure Legend Snippet: Dynamics of the morphogenesis, 3D architecture development and microbial population shifts of mixed-species biofilms. ( A ) Representative 3D rendering images of mixed-species biofilms at distinct time points. Images (a–c) represent biofilms formed after the introduction of 1% (w/v) glucose. Images (d–f) represent biofilms formed in the presence of 0.1% (w/v) sucrose. Images (g–i) represent biofilms formed after the introduction of 1% (w/v) sucrose. The EPS channel is depicted in red, while the cells are depicted in green. At the upper left of each panel, the two channels are displayed separately, while the merged image is displayed at the bottom right. Lateral (side) views of each biofilm are displayed at the bottom left, while a magnified (close-up) view of each small box depicted in the merged image is positioned in the upper right corner of each panel. ( B ) The data are mean values ± s.d. (n = 30). The asterisks (*) indicate that the each of the values (EPS and bacterial biomass) in the 1% sucrose group are significantly different from others ( P

Techniques Used:

Three-dimensional pH mapping of intact mixed-species biofilm. ( A ) This figure displays representative images of the pH profile throughout the biofilm 3D architecture and the spatial distribution of pH within the selected EPS-microcolony complex in mixed-species biofilms formed after introduction of 1% sucrose. Dark areas are indicative of regions of low pH, while white or light areas are indicative of regions of pH that are close to neutral, as indicated by the scale bar. The EPS channel is depicted in red, while the cells are depicted in green. Arrows indicate various acidic pH regions within the microcolony and at the sHA interface. Red arrows indicate acidic pH regions (i) across the structure and (ii) at the microcolony/sHA interface. Yellow arrows indicate pH close to neutral at the microcolony/fluid phase interface. White arrows indicate the corresponding pH landmarks in the merged panel. ( B ) This panel shows temporal changes of in situ pH across the EPS-microcolony complex after the biofilm was exposed to sodium phosphate-based buffer (pH 7.0) for a total of 120 min.
Figure Legend Snippet: Three-dimensional pH mapping of intact mixed-species biofilm. ( A ) This figure displays representative images of the pH profile throughout the biofilm 3D architecture and the spatial distribution of pH within the selected EPS-microcolony complex in mixed-species biofilms formed after introduction of 1% sucrose. Dark areas are indicative of regions of low pH, while white or light areas are indicative of regions of pH that are close to neutral, as indicated by the scale bar. The EPS channel is depicted in red, while the cells are depicted in green. Arrows indicate various acidic pH regions within the microcolony and at the sHA interface. Red arrows indicate acidic pH regions (i) across the structure and (ii) at the microcolony/sHA interface. Yellow arrows indicate pH close to neutral at the microcolony/fluid phase interface. White arrows indicate the corresponding pH landmarks in the merged panel. ( B ) This panel shows temporal changes of in situ pH across the EPS-microcolony complex after the biofilm was exposed to sodium phosphate-based buffer (pH 7.0) for a total of 120 min.

Techniques Used: In Situ

12) Product Images from "Inhibition of Biofilm Formation, Quorum Sensing and Infection in Pseudomonas aeruginosa by Natural Products-Inspired Organosulfur Compounds"

Article Title: Inhibition of Biofilm Formation, Quorum Sensing and Infection in Pseudomonas aeruginosa by Natural Products-Inspired Organosulfur Compounds

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038492

Panel A: Optical micrographs of bacterial biofilms. The films were grown in the presence of small molecule inhibitors 7 and 12 at 1 mM final concentration. Scale bar = 100 µm. Panel B: Laser scanning confocal micrographs of biofilms. The films were grown in the presence of 1 mM compound 7 (right) or without inhibitor (left). Top down images are shown in the upper views, with vertical and horizontal cross-sections shown to the right and below, respectively. Three dimensional reconstructions are shown in the bottom views. Scale bars for top down and 3D views are 50 µm, while scale bars for cross sections are 10 µm.
Figure Legend Snippet: Panel A: Optical micrographs of bacterial biofilms. The films were grown in the presence of small molecule inhibitors 7 and 12 at 1 mM final concentration. Scale bar = 100 µm. Panel B: Laser scanning confocal micrographs of biofilms. The films were grown in the presence of 1 mM compound 7 (right) or without inhibitor (left). Top down images are shown in the upper views, with vertical and horizontal cross-sections shown to the right and below, respectively. Three dimensional reconstructions are shown in the bottom views. Scale bars for top down and 3D views are 50 µm, while scale bars for cross sections are 10 µm.

Techniques Used: Concentration Assay

Cell viability within microplate-established biofilms as determined by the MTT assay. Biofilms were grown in the presence of 1 mM of compounds 7 , 12 and NPO. ANOVA (p
Figure Legend Snippet: Cell viability within microplate-established biofilms as determined by the MTT assay. Biofilms were grown in the presence of 1 mM of compounds 7 , 12 and NPO. ANOVA (p

Techniques Used: MTT Assay

Panel A: Inhibition of P. aeruginosa PAO1 biofilm formation by small molecule inhibitors. Panel B: Concentration dependence for inhibition of P. aeruginosa PAO1 biofilm formation by small molecule inhibitors. For 2A and 2B, average OD 600nm measurements of crystal violet stained biofilms (top) and planktonic cells (bottom) are shown with error bars representing one standard deviation (n = 3). ANOVA (p
Figure Legend Snippet: Panel A: Inhibition of P. aeruginosa PAO1 biofilm formation by small molecule inhibitors. Panel B: Concentration dependence for inhibition of P. aeruginosa PAO1 biofilm formation by small molecule inhibitors. For 2A and 2B, average OD 600nm measurements of crystal violet stained biofilms (top) and planktonic cells (bottom) are shown with error bars representing one standard deviation (n = 3). ANOVA (p

Techniques Used: Inhibition, Concentration Assay, Staining, Standard Deviation

13) Product Images from "Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA"

Article Title: Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA

Journal: Pathogens

doi: 10.3390/pathogens3020404

Confocal microscopy of MRSA (NRS 385) biofilm without treatment ( A ) and after treatment with octenidine hydrochloride ( B ).
Figure Legend Snippet: Confocal microscopy of MRSA (NRS 385) biofilm without treatment ( A ) and after treatment with octenidine hydrochloride ( B ).

Techniques Used: Confocal Microscopy

Inactivation of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on stainless steel by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM (Standard Error of Mean).
Figure Legend Snippet: Inactivation of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on stainless steel by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM (Standard Error of Mean).

Techniques Used:

Inactivation of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on urinary catheters by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM (Standard Error of Mean).
Figure Legend Snippet: Inactivation of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on urinary catheters by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM (Standard Error of Mean).

Techniques Used:

Inactivation of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on polystyrene by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM(Standard Error of Mean).
Figure Legend Snippet: Inactivation of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on polystyrene by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM(Standard Error of Mean).

Techniques Used:

Inhibition of S . aureus (ATCC 35556), vancomycin-resistant S . aureus (VRSA) (VRS 8) and MRSA (NRS 123) biofilm formation on polystyrene by octenidine hydrochloride (OH). Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM.
Figure Legend Snippet: Inhibition of S . aureus (ATCC 35556), vancomycin-resistant S . aureus (VRSA) (VRS 8) and MRSA (NRS 123) biofilm formation on polystyrene by octenidine hydrochloride (OH). Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM.

Techniques Used: Inhibition

Inhibition of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on stainless steel by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM (Standard Error of Mean).
Figure Legend Snippet: Inhibition of S . aureus (ATCC 35556), VRSA (VRS 8) and MRSA (NRS 123) biofilm on stainless steel by octenidine hydrochloride. Duplicate samples were used for each treatment, and the experiment was replicated three times. Data are represented as the mean ± SEM (Standard Error of Mean).

Techniques Used: Inhibition

14) Product Images from "Parallel Evolution in Streptococcus pneumoniae Biofilms"

Article Title: Parallel Evolution in Streptococcus pneumoniae Biofilms

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evw072

COMSTAT analysis of biofilm-derived colony variants. Biofilm formation of the biofilm-derived variants quantified using COMSTAT 1 and the program Matlab (R2012a) ( Heydorn et al. 2000 ) for triplicate 3-day-old biofilms of each variant type, grown in MatTek culture plates under static conditions. Graph depicts ( A ) the mean average thickness and maximum thickness, ( B ) mean biomass, and ( C ) mean surface area. Error bars represent 95% confidence intervals.
Figure Legend Snippet: COMSTAT analysis of biofilm-derived colony variants. Biofilm formation of the biofilm-derived variants quantified using COMSTAT 1 and the program Matlab (R2012a) ( Heydorn et al. 2000 ) for triplicate 3-day-old biofilms of each variant type, grown in MatTek culture plates under static conditions. Graph depicts ( A ) the mean average thickness and maximum thickness, ( B ) mean biomass, and ( C ) mean surface area. Error bars represent 95% confidence intervals.

Techniques Used: Derivative Assay, Variant Assay

CFU enumeration of biofilm-derived pneumococcal variants. Cells were harvested from triplicate pneumococcal biofilms from two independent experiments at time points 1, 3, 6, and 9 days and plated onto CBA to assess for changes in colony morphology. Variants were defined as follows: SCVs (
Figure Legend Snippet: CFU enumeration of biofilm-derived pneumococcal variants. Cells were harvested from triplicate pneumococcal biofilms from two independent experiments at time points 1, 3, 6, and 9 days and plated onto CBA to assess for changes in colony morphology. Variants were defined as follows: SCVs (

Techniques Used: Derivative Assay, Crocin Bleaching Assay

Mean growth rate of biofilm-derived SCV phenotype. The growth rate was assessed for SCVs from the exponential growth phase (between 4 and 8 h of growth) from triplicate growth experiments. SCV rate was calculated using pooled data from all 12 sequenced SCVs. A two-sample t -test was used to compare SCV rate to the WT rate. Numbers signify mean values. Error bars represent 95% confidence intervals.
Figure Legend Snippet: Mean growth rate of biofilm-derived SCV phenotype. The growth rate was assessed for SCVs from the exponential growth phase (between 4 and 8 h of growth) from triplicate growth experiments. SCV rate was calculated using pooled data from all 12 sequenced SCVs. A two-sample t -test was used to compare SCV rate to the WT rate. Numbers signify mean values. Error bars represent 95% confidence intervals.

Techniques Used: Derivative Assay

Biofilm formation of the biofilm-derived colony variants. Three-day-old biofilms of ( A ) the WT ancestral strain, ( B ) SCV, ( C ) MCV, and ( D ) TCV were visualized on a Leica TCS SP5 confocal laser scanning microscope using Bac Light live/dead stain. Green cells indicate live cells and red cells indicate dead cells. Z-Scans were performed every 0.3 µm on each field of view. White bars represent 25 µm.
Figure Legend Snippet: Biofilm formation of the biofilm-derived colony variants. Three-day-old biofilms of ( A ) the WT ancestral strain, ( B ) SCV, ( C ) MCV, and ( D ) TCV were visualized on a Leica TCS SP5 confocal laser scanning microscope using Bac Light live/dead stain. Green cells indicate live cells and red cells indicate dead cells. Z-Scans were performed every 0.3 µm on each field of view. White bars represent 25 µm.

Techniques Used: Derivative Assay, Laser-Scanning Microscopy, BAC Assay, Staining

Colony quantification of serotype 22F biofilm-derived colony variants. Diameter values of the three distinct biofilm-derived colony variant populations harvested from pneumococcal biofilms quantified using ImageJ analysis software. A total of nine colony variants from triplicate biofilms were measured. Numbers signify mean values.
Figure Legend Snippet: Colony quantification of serotype 22F biofilm-derived colony variants. Diameter values of the three distinct biofilm-derived colony variant populations harvested from pneumococcal biofilms quantified using ImageJ analysis software. A total of nine colony variants from triplicate biofilms were measured. Numbers signify mean values.

Techniques Used: Derivative Assay, Variant Assay, Software

15) Product Images from "Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation"

Article Title: Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17071033

The confocal laser scanning microscope (CLSM) images and relative gene expression of C. albicans in biofilms: ( a ) The CLSM images of C. albicans in multi-species biofilms, C. albicans dyed red, representative hyphae form of C. albicans was marked with white arrows; ( b ) Relative gene expression in C. albicans . Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: The confocal laser scanning microscope (CLSM) images and relative gene expression of C. albicans in biofilms: ( a ) The CLSM images of C. albicans in multi-species biofilms, C. albicans dyed red, representative hyphae form of C. albicans was marked with white arrows; ( b ) Relative gene expression in C. albicans . Values are significantly different when labelled with different letters ( p

Techniques Used: Laser-Scanning Microscopy, Confocal Laser Scanning Microscopy, Expressing

Biofilm structural and metabolic analyses: ( a ) The 3D reconstruction of 72 h multi-species biofilm in different DMADDM containing groups, live microbes dyed green, dead microbes dyed red, adjacent live and dead microbes were presented as yellow when they were merged; ( b ) The biomass of 72 h multi-species biofilm; ( c ) The thickness of 72 h multi-species biofilms in different DMADDM containing groups; ( d ) The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results of multi-species biofilms formed on different DMADDM containing denture base resin. Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: Biofilm structural and metabolic analyses: ( a ) The 3D reconstruction of 72 h multi-species biofilm in different DMADDM containing groups, live microbes dyed green, dead microbes dyed red, adjacent live and dead microbes were presented as yellow when they were merged; ( b ) The biomass of 72 h multi-species biofilm; ( c ) The thickness of 72 h multi-species biofilms in different DMADDM containing groups; ( d ) The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results of multi-species biofilms formed on different DMADDM containing denture base resin. Values are significantly different when labelled with different letters ( p

Techniques Used:

Colony forming unit (CFU) counts of multi-species biofilms: ( a ) The total CFU counts of 72 h multi-species biofilms in different dimethylaminododecyl methacrylate (DMADDM) containing groups; ( b ) The C. albicans CFU counts of 72 h multi-species biofilms in different group. Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: Colony forming unit (CFU) counts of multi-species biofilms: ( a ) The total CFU counts of 72 h multi-species biofilms in different dimethylaminododecyl methacrylate (DMADDM) containing groups; ( b ) The C. albicans CFU counts of 72 h multi-species biofilms in different group. Values are significantly different when labelled with different letters ( p

Techniques Used:

Cell damage based on lactate dehydrogenase (LDH) activity and relative gene expression of TR-146 cell: ( a ) Cell damage based on LDH activity after co-culture with biofilm formed on different DMADDM containing groups; ( b ) Relative gene expression of TR-146 cell after it was co-cultured with biofilms of different groups. Values are significantly different when labelled with different letters ( p
Figure Legend Snippet: Cell damage based on lactate dehydrogenase (LDH) activity and relative gene expression of TR-146 cell: ( a ) Cell damage based on LDH activity after co-culture with biofilm formed on different DMADDM containing groups; ( b ) Relative gene expression of TR-146 cell after it was co-cultured with biofilms of different groups. Values are significantly different when labelled with different letters ( p

Techniques Used: Activity Assay, Expressing, Co-Culture Assay, Cell Culture

16) Product Images from "Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology"

Article Title: Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163966

Variation in the cell index (CI) during biofilm formation, at 37°C under 5% CO 2 , of S . mutans NCTC10449 (type strain) and CI2366 (clinical isolate) depending on the carbon source added to the culture medium. At 8 h of incubation, the presence of the asterisk indicates that CI values were statistically different according to one-way ANOVA tests (p
Figure Legend Snippet: Variation in the cell index (CI) during biofilm formation, at 37°C under 5% CO 2 , of S . mutans NCTC10449 (type strain) and CI2366 (clinical isolate) depending on the carbon source added to the culture medium. At 8 h of incubation, the presence of the asterisk indicates that CI values were statistically different according to one-way ANOVA tests (p

Techniques Used: Incubation

Removal of 8 h preformed S . aureus 15981 biofilms by endolysin LysH5 (added to TSBG from 0.05 to 2.88 μM) reported as variation of the normalized cell index (CI). At the final time, values having distinct letter are statistically different (p
Figure Legend Snippet: Removal of 8 h preformed S . aureus 15981 biofilms by endolysin LysH5 (added to TSBG from 0.05 to 2.88 μM) reported as variation of the normalized cell index (CI). At the final time, values having distinct letter are statistically different (p

Techniques Used:

Schematic drawing of the basis to monitor bacterial biofilm formation by the xCellingence equipment. Standard E-plates (16-wells) and magnification of one of the wells coated with gold microelectrodes ( A ). Evolution of the “cell index” (CI), which derived from the electric impedance, throughout the different steps of bacterial biofilm formation by the strain S . aureus 15981 ( B ).
Figure Legend Snippet: Schematic drawing of the basis to monitor bacterial biofilm formation by the xCellingence equipment. Standard E-plates (16-wells) and magnification of one of the wells coated with gold microelectrodes ( A ). Evolution of the “cell index” (CI), which derived from the electric impedance, throughout the different steps of bacterial biofilm formation by the strain S . aureus 15981 ( B ).

Techniques Used: Derivative Assay

Variation in the cell index (CI) during S . mutans NCTC10449 biofilm formation, at 37°C under 5% CO 2 , depending on the carbon source added to the culture medium. At 8 h of incubation time, those values that have not a common letter are statistically different according to the Duncan mean comparison test (p
Figure Legend Snippet: Variation in the cell index (CI) during S . mutans NCTC10449 biofilm formation, at 37°C under 5% CO 2 , depending on the carbon source added to the culture medium. At 8 h of incubation time, those values that have not a common letter are statistically different according to the Duncan mean comparison test (p

Techniques Used: Incubation

Inhibition of the biofilm formation by S . aureus 15981 due to the addition of endolysin LysH5 (0.15 μ M) to the culture medium TSBG, and by S . epidermidis F12 due to the addition of the bacteriophage phi-IPLA7 (MOI 100) to TSBG expressed as variation of the CI during biofilm treatment ; asterisks show the first incubation time after which two or more consecutive values of CI were statistically different (p
Figure Legend Snippet: Inhibition of the biofilm formation by S . aureus 15981 due to the addition of endolysin LysH5 (0.15 μ M) to the culture medium TSBG, and by S . epidermidis F12 due to the addition of the bacteriophage phi-IPLA7 (MOI 100) to TSBG expressed as variation of the CI during biofilm treatment ; asterisks show the first incubation time after which two or more consecutive values of CI were statistically different (p

Techniques Used: Inhibition, Incubation

Variation in the cell index (CI) during biofilm formation at 37°C of different S . aureus and S . epidermidis biofilm producers (ISP479r, 15981, 132 or V320 and F12, respectively) and no-biofilm producers (CH1368 and CH48, respectively). TSB+0.25% glucose was the culture medium used in the experiment ( A ). Absorbance (595 nm) measured after crystal violet staining of samples collected at different times during the biofilm formation in E-plates of the strains under study ( B ). Counts (Log CFU/ml) of cells collected from the biofilms formed in the E-plates by the strains under study ( C ). Statistical differences among strains at three sampling points (8, 16 and 24 h) are collected in S1 Table , which also shows representative mean and SD values.
Figure Legend Snippet: Variation in the cell index (CI) during biofilm formation at 37°C of different S . aureus and S . epidermidis biofilm producers (ISP479r, 15981, 132 or V320 and F12, respectively) and no-biofilm producers (CH1368 and CH48, respectively). TSB+0.25% glucose was the culture medium used in the experiment ( A ). Absorbance (595 nm) measured after crystal violet staining of samples collected at different times during the biofilm formation in E-plates of the strains under study ( B ). Counts (Log CFU/ml) of cells collected from the biofilms formed in the E-plates by the strains under study ( C ). Statistical differences among strains at three sampling points (8, 16 and 24 h) are collected in S1 Table , which also shows representative mean and SD values.

Techniques Used: Staining, Sampling

17) Product Images from "Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization"

Article Title: Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization

Journal: BMC Microbiology

doi: 10.1186/s12866-016-0678-7

Visualisation of the antibacterial effect of CORM-2 and cefotaxime on established biofilm. Effect of CORM-2 and cefotaxime on bacterial viability within an established biofilm (biofilm grown for 24 h) evaluated by a live/dead viability staining assay using confocal microscopy. Live bacteria with intact cell membrane are stained green (SYTO9) and dead bacteria with damaged cell membrane are stained red (propidium iodine). Photographs show representative areas from the chamber slides; from left to right isolate UPEC isolate 2, ESBL isolate 1 and K-12 strain TG1 and from top-down a controls, b 24 h CORM-2 (500 μM), c 24 h cefotaxime (0.512 μg/ml). Scale bar = 10 μm. Representative photographs from three independent experiments are shown
Figure Legend Snippet: Visualisation of the antibacterial effect of CORM-2 and cefotaxime on established biofilm. Effect of CORM-2 and cefotaxime on bacterial viability within an established biofilm (biofilm grown for 24 h) evaluated by a live/dead viability staining assay using confocal microscopy. Live bacteria with intact cell membrane are stained green (SYTO9) and dead bacteria with damaged cell membrane are stained red (propidium iodine). Photographs show representative areas from the chamber slides; from left to right isolate UPEC isolate 2, ESBL isolate 1 and K-12 strain TG1 and from top-down a controls, b 24 h CORM-2 (500 μM), c 24 h cefotaxime (0.512 μg/ml). Scale bar = 10 μm. Representative photographs from three independent experiments are shown

Techniques Used: Staining, Confocal Microscopy

The antibacterial effect of CORM-2 on biofilm formation. Effect of CORM-2 (250 μM) on biofilm formation in non-ESBL-producing UPEC isolates (UPEC 2 and 3), in an ESBL-producing isolate (ESBL1) and in the non-pathogenic E. coli K-12 strains MG1655 and TG1. Biofilm formation was measured by the crystal violet method 18 h after exposure to CORM-2 and expressed as relative changes compared to untreated controls. The data are presented as mean ± SEM from at least three independent experiments. * P
Figure Legend Snippet: The antibacterial effect of CORM-2 on biofilm formation. Effect of CORM-2 (250 μM) on biofilm formation in non-ESBL-producing UPEC isolates (UPEC 2 and 3), in an ESBL-producing isolate (ESBL1) and in the non-pathogenic E. coli K-12 strains MG1655 and TG1. Biofilm formation was measured by the crystal violet method 18 h after exposure to CORM-2 and expressed as relative changes compared to untreated controls. The data are presented as mean ± SEM from at least three independent experiments. * P

Techniques Used:

18) Product Images from "Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo"

Article Title: Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2016.06.022

IHF NTHI tip-directed MAbs disrupted biofilms formed by clinically-relevant bacterial species. (A) Confocal scanning microscopy images of biofilms (green) formed by multiple clinical isolates of NTHI revealed disruption of pre-formed biofilms by incubation with the MAbs IhfA5 NTHI and mIhfB4 NTHI , a result not observed with the MAbs IhfA3 NTHI and IhfB2 NTHI . (B) This observation was further quantitated as a reduction in biofilm biomass by COMSTAT2 analyses. (C) Representative images showed disruption of biofilms formed by the bacterial pathogens M. catarrhalis , B. ceneocepacia , P. aeruginosa , and S. aureus by treatment with the IHF NTHI tip-directed MAbs and (D) quantitated as significant reduction in mean biofilm biomass. Images are representative of three independent experiments. Scale bars, 20 μm. *, p ≤ 0.05.
Figure Legend Snippet: IHF NTHI tip-directed MAbs disrupted biofilms formed by clinically-relevant bacterial species. (A) Confocal scanning microscopy images of biofilms (green) formed by multiple clinical isolates of NTHI revealed disruption of pre-formed biofilms by incubation with the MAbs IhfA5 NTHI and mIhfB4 NTHI , a result not observed with the MAbs IhfA3 NTHI and IhfB2 NTHI . (B) This observation was further quantitated as a reduction in biofilm biomass by COMSTAT2 analyses. (C) Representative images showed disruption of biofilms formed by the bacterial pathogens M. catarrhalis , B. ceneocepacia , P. aeruginosa , and S. aureus by treatment with the IHF NTHI tip-directed MAbs and (D) quantitated as significant reduction in mean biofilm biomass. Images are representative of three independent experiments. Scale bars, 20 μm. *, p ≤ 0.05.

Techniques Used: Immunohistofluorescence, Microscopy, Incubation

MAbs directed against the DNA-binding tip regions of IHF NTHI disrupted biofilms formed by NTHI 86-028NP in vitro . (A) Images obtained by confocal scanning microscopy demonstrating top-down and side views of representative 24-h biofilms formed by NTHI strain #86-028NP maintained in medium, or incubated for an additional 16 h with 5 μg murine MAbs as indicated. Bacteria within biofilms were stained with FM1-43FX membrane stain (green). Images are representative of three independent experiments. Scale bars, 20 μm. (B) Mean biofilm biomass ± SEM remaining after treatment as determined by COMSTAT2 analyses of three independent experiments. ***, p ≤ 0.001; ****, p ≤ 0.0001.
Figure Legend Snippet: MAbs directed against the DNA-binding tip regions of IHF NTHI disrupted biofilms formed by NTHI 86-028NP in vitro . (A) Images obtained by confocal scanning microscopy demonstrating top-down and side views of representative 24-h biofilms formed by NTHI strain #86-028NP maintained in medium, or incubated for an additional 16 h with 5 μg murine MAbs as indicated. Bacteria within biofilms were stained with FM1-43FX membrane stain (green). Images are representative of three independent experiments. Scale bars, 20 μm. (B) Mean biofilm biomass ± SEM remaining after treatment as determined by COMSTAT2 analyses of three independent experiments. ***, p ≤ 0.001; ****, p ≤ 0.0001.

Techniques Used: Binding Assay, Immunohistofluorescence, In Vitro, Microscopy, Incubation, Staining

Clearance of NTHI from the middle ear in a chinchilla model of experimental otitis media. NTHI biofilms were established within the middle ears of chinchillas prior to infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine MAbs. (A) CFU NTHI per mg mucosal biofilm and NTHI adherent to the middle ear mucosal epithelium. The limit of detection is indicated by a dashed horizontal line. (B) Clearance of NTHI from middle ear fluids. CFU NTHI in middle ear effusions after infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine MAbs. (C) Resolution of established NTHI biofilms from the chinchilla middle ear. Images of each middle ear were blindly ranked to determine the relative quantity of mucosal biofilm that remained after infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine MAbs. An established mucosal biofilm scale was employed ( Novotny et al., 2011 ), whereby 0 = no mucosal biofilm visible, 1 ≤ 25% of middle ear space occluded by mucosal biofilm, 2 = ≥ 25–50% occluded, 3 = ≥ 50–75% occluded, 4 = ≥ 75–100% occluded. (D) Representative images of chinchilla middle ears after infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine monoclonal antibodies. The mucosal biofilm score is indicated in the bottom right corner of each image. TM - tympanic membrane, S - bony septae, MEM - middle ear mucosa, B - mucosal biofilm. For bar graphs, values for each individual middle ear is shown (circles; 6 middle ears per cohort) and the mean ± SEM for the cohort presented (bars).*, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.
Figure Legend Snippet: Clearance of NTHI from the middle ear in a chinchilla model of experimental otitis media. NTHI biofilms were established within the middle ears of chinchillas prior to infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine MAbs. (A) CFU NTHI per mg mucosal biofilm and NTHI adherent to the middle ear mucosal epithelium. The limit of detection is indicated by a dashed horizontal line. (B) Clearance of NTHI from middle ear fluids. CFU NTHI in middle ear effusions after infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine MAbs. (C) Resolution of established NTHI biofilms from the chinchilla middle ear. Images of each middle ear were blindly ranked to determine the relative quantity of mucosal biofilm that remained after infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine MAbs. An established mucosal biofilm scale was employed ( Novotny et al., 2011 ), whereby 0 = no mucosal biofilm visible, 1 ≤ 25% of middle ear space occluded by mucosal biofilm, 2 = ≥ 25–50% occluded, 3 = ≥ 50–75% occluded, 4 = ≥ 75–100% occluded. (D) Representative images of chinchilla middle ears after infusion of the middle ear space with IgG-enriched polyclonal rabbit sera or murine monoclonal antibodies. The mucosal biofilm score is indicated in the bottom right corner of each image. TM - tympanic membrane, S - bony septae, MEM - middle ear mucosa, B - mucosal biofilm. For bar graphs, values for each individual middle ear is shown (circles; 6 middle ears per cohort) and the mean ± SEM for the cohort presented (bars).*, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

Techniques Used:

19) Product Images from "In vivo biofilm formation on stainless steel bonded retainers during different oral health-care regimens"

Article Title: In vivo biofilm formation on stainless steel bonded retainers during different oral health-care regimens

Journal: International Journal of Oral Science

doi: 10.1038/ijos.2014.69

Scanning electron micrographs of 1-week-old biofilms formed in vivo during use of a toothpaste without antibacterial claims. ( a – c ) Single-strand wire. ( d – f ) Multi-strand wire.
Figure Legend Snippet: Scanning electron micrographs of 1-week-old biofilms formed in vivo during use of a toothpaste without antibacterial claims. ( a – c ) Single-strand wire. ( d – f ) Multi-strand wire.

Techniques Used: In Vivo

20) Product Images from "Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms"

Article Title: Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms

Journal: mBio

doi: 10.1128/mBio.01593-17

BONCAT analysis of protein synthesis during ciprofloxacin treatment. (A) Experimental timeline of biofilm treatment and proteome labeling. Biofilms were grown in silicone rubber tubing for 4 days and then treated with 60 μg/ml ciprofloxacin (gray). Control biofilms were untreated. For each condition, biofilms were treated with Anl at the designated time point for 1.5 h (cross-hatched portion), harvested, and lysed. (B) Survival of biofilm cells following exposure to ciprofloxacin for the indicated treatment time and to 1 mM Anl as indicated. (C) Visualization of Anl incorporation in lysates treated with alkyne-TAMRA. Coomassie staining was used to verify equal protein loading. (D) Anl incorporation was quantified by dividing the TAMRA fluorescence by the Coomassie intensity for four gel regions (means + standard deviations; n = 4). Welch’s t-test results are indicated: *, P
Figure Legend Snippet: BONCAT analysis of protein synthesis during ciprofloxacin treatment. (A) Experimental timeline of biofilm treatment and proteome labeling. Biofilms were grown in silicone rubber tubing for 4 days and then treated with 60 μg/ml ciprofloxacin (gray). Control biofilms were untreated. For each condition, biofilms were treated with Anl at the designated time point for 1.5 h (cross-hatched portion), harvested, and lysed. (B) Survival of biofilm cells following exposure to ciprofloxacin for the indicated treatment time and to 1 mM Anl as indicated. (C) Visualization of Anl incorporation in lysates treated with alkyne-TAMRA. Coomassie staining was used to verify equal protein loading. (D) Anl incorporation was quantified by dividing the TAMRA fluorescence by the Coomassie intensity for four gel regions (means + standard deviations; n = 4). Welch’s t-test results are indicated: *, P

Techniques Used: Labeling, Staining, Fluorescence

Targeted proteomic analysis of a biofilm subpopulation. (A) Detection of mCherry fluorescence (green) in live biofilms was used to locate cells expressing the NLL-MetRS–mCherry fusion. Biofilms were counterstained with SYTO9 (magenta) immediately before imaging. (B) Following Anl treatment, BONCAT labeling in biofilms was visualized by treating fixed biofilms with DBCO-TAMRA (green). Biofilms were counterstained with SYTO9 (magenta). Colocalization of fluorescent signals is displayed in white. For panels A and B, cross-sections were reconstructed from confocal image stacks. (C) Proteins identified following BONCAT enrichment from P rpoS : nll-mc and P trc : nll-mc strains. (D) Quantification of relative protein abundances following enrichment from both strains. Ribosomal proteins are shown in orange. Proteins discussed in the text are indicated by gene name. The complete set of LFQ values, ratios, and adjusted P values is provided in Data Set S1 . (E) Spatial distribution of GFP expression (green) under control of the rpoS or algP promoters in live biofilms. Biofilms were counterstained with SYTO62 (magenta).
Figure Legend Snippet: Targeted proteomic analysis of a biofilm subpopulation. (A) Detection of mCherry fluorescence (green) in live biofilms was used to locate cells expressing the NLL-MetRS–mCherry fusion. Biofilms were counterstained with SYTO9 (magenta) immediately before imaging. (B) Following Anl treatment, BONCAT labeling in biofilms was visualized by treating fixed biofilms with DBCO-TAMRA (green). Biofilms were counterstained with SYTO9 (magenta). Colocalization of fluorescent signals is displayed in white. For panels A and B, cross-sections were reconstructed from confocal image stacks. (C) Proteins identified following BONCAT enrichment from P rpoS : nll-mc and P trc : nll-mc strains. (D) Quantification of relative protein abundances following enrichment from both strains. Ribosomal proteins are shown in orange. Proteins discussed in the text are indicated by gene name. The complete set of LFQ values, ratios, and adjusted P values is provided in Data Set S1 . (E) Spatial distribution of GFP expression (green) under control of the rpoS or algP promoters in live biofilms. Biofilms were counterstained with SYTO62 (magenta).

Techniques Used: Fluorescence, Expressing, Imaging, Labeling

21) Product Images from "Interstrain Cooperation in Meningococcal Biofilms: Role of Autotransporters NalP and AutA"

Article Title: Interstrain Cooperation in Meningococcal Biofilms: Role of Autotransporters NalP and AutA

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00434

Interbacterial interactions in dual-strain biofilms. (A) Biofilms constituted of two differently labeled derivatives of strain HB-1, α14 or BB-1. In the enlargements of the BB-1 biofilms, the positions of three clusters are highlighted, one consisting exclusively of GFP-labeled cells, one consisting exclusively of RFP-labeled cells, and one intermixed cluster consisting of both GFP- and RFP-labeled cells. (B) Combinations of α14 and BB-1 and several mutant derivatives. (C) Combinations of α14 or its autA mutant derivative with HB-1. (D) Combinations of HB-1 and BB-1 and several mutant derivatives. Strain names at the left are shown in green or red, reflecting the expression of GFP or RFP, respectively. Designations AutA + and NalP + indicate expression of AutA and NalP from plasmids pFPAutA and pEN300, respectively. For clarity, strain HB-3Δ nhbA Δ iga (Table S1 ) is indicated here as HB-1Δ nalP Δ nhbA Δ iga . However, in contrast to strain HB-1Δ nalP , the isogenic strain HB-3 contains markerless deletions of the nalP gene and the capsule locus, which was necessary to combine multiple mutations into a single strain (Arenas et al., 2013a ). Individual and combined fluorescence is displayed.
Figure Legend Snippet: Interbacterial interactions in dual-strain biofilms. (A) Biofilms constituted of two differently labeled derivatives of strain HB-1, α14 or BB-1. In the enlargements of the BB-1 biofilms, the positions of three clusters are highlighted, one consisting exclusively of GFP-labeled cells, one consisting exclusively of RFP-labeled cells, and one intermixed cluster consisting of both GFP- and RFP-labeled cells. (B) Combinations of α14 and BB-1 and several mutant derivatives. (C) Combinations of α14 or its autA mutant derivative with HB-1. (D) Combinations of HB-1 and BB-1 and several mutant derivatives. Strain names at the left are shown in green or red, reflecting the expression of GFP or RFP, respectively. Designations AutA + and NalP + indicate expression of AutA and NalP from plasmids pFPAutA and pEN300, respectively. For clarity, strain HB-3Δ nhbA Δ iga (Table S1 ) is indicated here as HB-1Δ nalP Δ nhbA Δ iga . However, in contrast to strain HB-1Δ nalP , the isogenic strain HB-3 contains markerless deletions of the nalP gene and the capsule locus, which was necessary to combine multiple mutations into a single strain (Arenas et al., 2013a ). Individual and combined fluorescence is displayed.

Techniques Used: Labeling, Mutagenesis, Expressing, Fluorescence

Biofilm organization in various fluorescent Neisseria strains. (A) Biofilms of various strains of Nm and Nl . The clonal complex (cc) of the Nm strains is indicated. (B) Influence of nalP inactivation on biofilm architecture. Wild-type phenotypes are shown in (A) . HB-1Δ nalP/ NalP + and BB-1Δ nalP/ NalP + represent nalP mutants overexpressing NalP from plasmid pEN300. All strains harbor an opaB P H -gfp or opaB P H -rfp inserted into hrtA locus. Representative pictures from at least four experiments with two technical replicates are shown.
Figure Legend Snippet: Biofilm organization in various fluorescent Neisseria strains. (A) Biofilms of various strains of Nm and Nl . The clonal complex (cc) of the Nm strains is indicated. (B) Influence of nalP inactivation on biofilm architecture. Wild-type phenotypes are shown in (A) . HB-1Δ nalP/ NalP + and BB-1Δ nalP/ NalP + represent nalP mutants overexpressing NalP from plasmid pEN300. All strains harbor an opaB P H -gfp or opaB P H -rfp inserted into hrtA locus. Representative pictures from at least four experiments with two technical replicates are shown.

Techniques Used: Plasmid Preparation

Characteristics of single- and dual-strain biofilms . Biofilms were formed of various combinations of Nm strains HB-1 and BB-1 and their nalP mutant derivatives, and α14 and its autA mutant derivative and biomass and CFU were determined. The strains present in the consortium are shown at the bottom of each panel. The data shown are for the strain indicated in bold. (A) The biomass of biofilms was calculated for each strain using COMSTAT. The biomass for each strain in the consortium is shown separately. (B) Numbers of CFU. The CFU for each strain in the consortium is shown. To allow for strain discrimination in dual-strain biofilms, strains with different antibiotic-resistance markers were used as indicated in Table S1 . The CFU for each strain were determined by plating on GC medium containing kanamycin, rifampicin or gentamicin and overnight incubation. Results are means and standard deviations of three independent experiments. Statistically significant differences are marked with one ( P
Figure Legend Snippet: Characteristics of single- and dual-strain biofilms . Biofilms were formed of various combinations of Nm strains HB-1 and BB-1 and their nalP mutant derivatives, and α14 and its autA mutant derivative and biomass and CFU were determined. The strains present in the consortium are shown at the bottom of each panel. The data shown are for the strain indicated in bold. (A) The biomass of biofilms was calculated for each strain using COMSTAT. The biomass for each strain in the consortium is shown separately. (B) Numbers of CFU. The CFU for each strain in the consortium is shown. To allow for strain discrimination in dual-strain biofilms, strains with different antibiotic-resistance markers were used as indicated in Table S1 . The CFU for each strain were determined by plating on GC medium containing kanamycin, rifampicin or gentamicin and overnight incubation. Results are means and standard deviations of three independent experiments. Statistically significant differences are marked with one ( P

Techniques Used: Mutagenesis, Incubation

Biofilm formation under nutrient limitation. (A) Biofilms of various strains of Nm grown on RPMI medium. (B) Biomass (left panel) and CFU (right panel) in biofilms were calculated for each strain in single- and dual-strain biofilms as in Figure 3 . The strains present in the consortium are shown at the bottom of each panel. The data shown are for the strain indicated in bold. Statistically significant differences are marked with one asterisk ( P
Figure Legend Snippet: Biofilm formation under nutrient limitation. (A) Biofilms of various strains of Nm grown on RPMI medium. (B) Biomass (left panel) and CFU (right panel) in biofilms were calculated for each strain in single- and dual-strain biofilms as in Figure 3 . The strains present in the consortium are shown at the bottom of each panel. The data shown are for the strain indicated in bold. Statistically significant differences are marked with one asterisk ( P

Techniques Used:

22) Product Images from "Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans"

Article Title: Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020434

The degree of cell damage post-PDI in C. albicans biofilms. ( A ) The growth ability of biofilm cells was measured at different time points post PDI (2.5 mM TBO plus 50 J/cm 2 ). Cells obtained from PDI-treated biofilms were suspended in YPD medium and subjected to a plate count. Each point is the mean of results obtained from three independent experiments and shown as mean ± SD. ( B ) Effect of PDI on hyphal formation in C. albicans biofilms. Cells obtained from PDI-treated biofilms were incubated in YPD medium supplemented with 10% FBS for 3 h. The ability of hyphal formation was observed and counted under microscope to determine the ratio of hyphal formation. The scale bar in the left panel is 20 µm. ☐: non-PDI. ■: PDI. Each value is obtained from three independent experiments and shown as the mean ± SD. *** p
Figure Legend Snippet: The degree of cell damage post-PDI in C. albicans biofilms. ( A ) The growth ability of biofilm cells was measured at different time points post PDI (2.5 mM TBO plus 50 J/cm 2 ). Cells obtained from PDI-treated biofilms were suspended in YPD medium and subjected to a plate count. Each point is the mean of results obtained from three independent experiments and shown as mean ± SD. ( B ) Effect of PDI on hyphal formation in C. albicans biofilms. Cells obtained from PDI-treated biofilms were incubated in YPD medium supplemented with 10% FBS for 3 h. The ability of hyphal formation was observed and counted under microscope to determine the ratio of hyphal formation. The scale bar in the left panel is 20 µm. ☐: non-PDI. ■: PDI. Each value is obtained from three independent experiments and shown as the mean ± SD. *** p

Techniques Used: Incubation, Microscopy

The effect of treatment combining PDI and an azole class of antifungal agents against C. albicans biofilms. Biofilm cells were incubated with 2.5 mM TBO and then exposed to 50 J/cm 2 of light. After PDI, different concentrations of fluconazole ( A ) and posaconazole ( B ) were added and incubated for 24 h, then subjected to a plate count. ■: antifungal agents only. ☐: PDI plus antifungal agents. Each value is the mean obtained from three independent experiments ± SD. *** p
Figure Legend Snippet: The effect of treatment combining PDI and an azole class of antifungal agents against C. albicans biofilms. Biofilm cells were incubated with 2.5 mM TBO and then exposed to 50 J/cm 2 of light. After PDI, different concentrations of fluconazole ( A ) and posaconazole ( B ) were added and incubated for 24 h, then subjected to a plate count. ■: antifungal agents only. ☐: PDI plus antifungal agents. Each value is the mean obtained from three independent experiments ± SD. *** p

Techniques Used: Incubation

The antifungal activity against C. albicans biofilms by combining PDI and echinocandin. Biofilm cells were incubated with 2.5 mM TBO and exposed to 50 J/cm 2 of light. After PDI, different concentrations of caspofungin were added to the cells, which were further incubated for 24 h and then subjected to a plate count. ■: caspofungin only. ☐: PDI plus caspofungin. Each value is the mean obtained from three independent experiments and shown as mean ± SD, X represents the complete eradication of cells. *** p
Figure Legend Snippet: The antifungal activity against C. albicans biofilms by combining PDI and echinocandin. Biofilm cells were incubated with 2.5 mM TBO and exposed to 50 J/cm 2 of light. After PDI, different concentrations of caspofungin were added to the cells, which were further incubated for 24 h and then subjected to a plate count. ■: caspofungin only. ☐: PDI plus caspofungin. Each value is the mean obtained from three independent experiments and shown as mean ± SD, X represents the complete eradication of cells. *** p

Techniques Used: Activity Assay, Incubation

The thickness of biofilm extracellular polymeric substances (EPSs) in C. albicans . ( A ) Biofilms treated with PDI were subjected to a measurement of the thickness of EPS by using SYPRO ® Ruby Biofilm Matrix Stain. The fluorescence images were observed under a Leica SP2 confocal scanning fluorescence microscope. The Sagittal XZ section represents the biofilm’s thickness. ( B ) The quantified value of EPS thickness was measured by using the LAS AF lite software. The value of the EPS thickness is the mean from three independent experiments ± SD. *** p
Figure Legend Snippet: The thickness of biofilm extracellular polymeric substances (EPSs) in C. albicans . ( A ) Biofilms treated with PDI were subjected to a measurement of the thickness of EPS by using SYPRO ® Ruby Biofilm Matrix Stain. The fluorescence images were observed under a Leica SP2 confocal scanning fluorescence microscope. The Sagittal XZ section represents the biofilm’s thickness. ( B ) The quantified value of EPS thickness was measured by using the LAS AF lite software. The value of the EPS thickness is the mean from three independent experiments ± SD. *** p

Techniques Used: Staining, Fluorescence, Microscopy, Software

23) Product Images from "Phenalen-1-one-Mediated Antimicrobial Photodynamic Therapy: Antimicrobial Efficacy in a Periodontal Biofilm Model and Flow Cytometric Evaluation of Cytoplasmic Membrane Damage"

Article Title: Phenalen-1-one-Mediated Antimicrobial Photodynamic Therapy: Antimicrobial Efficacy in a Periodontal Biofilm Model and Flow Cytometric Evaluation of Cytoplasmic Membrane Damage

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00688

Exemplary visualization of polymicrobial biofilms by means of scanning electron microscopy. Exemplary SEM visualization of randomly selected fields of untreated biofilms (PS-L–), biofilms treated with aPDT using SAPYR (SAPYR+L+) or SAGUA (SAGUA+L+) or treated with CHX (CHX 0.2%) in 3,000-fold, 12,000-fold, and 24,000-fold magnification. In the CHX-treated biofilms white arrows show debris most likely originating from killed cells on the top layer of the biofilms.
Figure Legend Snippet: Exemplary visualization of polymicrobial biofilms by means of scanning electron microscopy. Exemplary SEM visualization of randomly selected fields of untreated biofilms (PS-L–), biofilms treated with aPDT using SAPYR (SAPYR+L+) or SAGUA (SAGUA+L+) or treated with CHX (CHX 0.2%) in 3,000-fold, 12,000-fold, and 24,000-fold magnification. In the CHX-treated biofilms white arrows show debris most likely originating from killed cells on the top layer of the biofilms.

Techniques Used: Electron Microscopy

Spectroscopic measurements for release of nucleic acids. OD medians, 1st and 3rd quartiles of the supernatants of biofilms treated with phenalen-1-one mediated aPDT (groups: PS-L–, PS-L+, SAPYR+L-, SAPYR+L+, SAGUA+L-, and SAGUA+L+) or positive control (lysozyme treatment followed by Proteinase K digestion), as measured at 260 nm for release of nucleic acids.
Figure Legend Snippet: Spectroscopic measurements for release of nucleic acids. OD medians, 1st and 3rd quartiles of the supernatants of biofilms treated with phenalen-1-one mediated aPDT (groups: PS-L–, PS-L+, SAPYR+L-, SAPYR+L+, SAGUA+L-, and SAGUA+L+) or positive control (lysozyme treatment followed by Proteinase K digestion), as measured at 260 nm for release of nucleic acids.

Techniques Used: Positive Control

24) Product Images from "Gene Acquisition by a Distinct Phyletic Group within Streptococcus pneumoniae Promotes Adhesion to the Ocular Epithelium"

Article Title: Gene Acquisition by a Distinct Phyletic Group within Streptococcus pneumoniae Promotes Adhesion to the Ocular Epithelium

Journal: mSphere

doi: 10.1128/mSphere.00213-17

Functional analyses of the sspB gene encoding the predicted SspB adhesin. (A) Schematic of the predicted SspB protein (GenBank accession no. KGI30072 and OYL08640.1 ) illustrating domains implicated in adhesion and biofilm formation. (B) Gene expression of sspB in S. pneumoniae B1599 (black bars); for comparison, we show the expression of a second related protein with SspC-C2 domains (WP_050568636) (gray bars). The y axis displays N 0, the number of fluorescence units representing the RNA amount in the respective samples. (C) Role of sspB in attachment to HCLE cells. HCLE cells were exposed to S. pneumoniae strain B1599, B1599 Δ sspB , or B1599 Δ sspB :: sspB for 30 min, and HCLE cells with bacteria attached were enumerated. Experiments were performed in triplicate, and a total of 460 HCLE cells were analyzed for presence of bacteria and the number of bacteria attached. The results are plotted using a violin plot, generated in the R statistical package. The violin plot displays the distribution of the data: the pink areas display the density plot, the thick black bars represent the midspread of the data (interquartile range), the thin black lines display the 95% confidence interval, and the white circles correspond to the median.
Figure Legend Snippet: Functional analyses of the sspB gene encoding the predicted SspB adhesin. (A) Schematic of the predicted SspB protein (GenBank accession no. KGI30072 and OYL08640.1 ) illustrating domains implicated in adhesion and biofilm formation. (B) Gene expression of sspB in S. pneumoniae B1599 (black bars); for comparison, we show the expression of a second related protein with SspC-C2 domains (WP_050568636) (gray bars). The y axis displays N 0, the number of fluorescence units representing the RNA amount in the respective samples. (C) Role of sspB in attachment to HCLE cells. HCLE cells were exposed to S. pneumoniae strain B1599, B1599 Δ sspB , or B1599 Δ sspB :: sspB for 30 min, and HCLE cells with bacteria attached were enumerated. Experiments were performed in triplicate, and a total of 460 HCLE cells were analyzed for presence of bacteria and the number of bacteria attached. The results are plotted using a violin plot, generated in the R statistical package. The violin plot displays the distribution of the data: the pink areas display the density plot, the thick black bars represent the midspread of the data (interquartile range), the thin black lines display the 95% confidence interval, and the white circles correspond to the median.

Techniques Used: Functional Assay, Expressing, Fluorescence, Generated

Conjunctivitis-associated strains B1599 and B1567 exhibit aggregates in planktonic culture and form abundant chain-like structures in biofilms. (A) Planktonic cultures of strains B1599, B1567, and D39. Conjunctivitis isolates precipitate at the bottom of the test tube, but no precipitate is observed for model strain D39. (B) Confocal images of 72-h biofilms fixed and stained with Syto59. Boxes on the bottom right display a magnified view. Conjunctivitis isolates B1599 and B1567 form chain-like structures (white arrows point to examples of chain-like structures); however, equivalent chains are not observed in the other eye-associated strains. The scale bar is the same for all micrographs.
Figure Legend Snippet: Conjunctivitis-associated strains B1599 and B1567 exhibit aggregates in planktonic culture and form abundant chain-like structures in biofilms. (A) Planktonic cultures of strains B1599, B1567, and D39. Conjunctivitis isolates precipitate at the bottom of the test tube, but no precipitate is observed for model strain D39. (B) Confocal images of 72-h biofilms fixed and stained with Syto59. Boxes on the bottom right display a magnified view. Conjunctivitis isolates B1599 and B1567 form chain-like structures (white arrows point to examples of chain-like structures); however, equivalent chains are not observed in the other eye-associated strains. The scale bar is the same for all micrographs.

Techniques Used: Staining

25) Product Images from "The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315"

Article Title: The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315

Journal: mSphere

doi: 10.1128/mSphere.00579-17

Expression of ncS35. (A) Northern blot assays. At the upper left is a Northern blot assay of ncS35 in wild-type (WT) B. cenocepacia J2315. Expression was evaluated in biofilms, stationary phase, and exponential phase. Responses to stress were evaluated in various media: LBB with 0.2% H 2 O 2 added 15 min prior to harvesting, LBB supplemented with 0.005% (wt/vol) SDS (membrane stress), and M9 supplemented with either 10 mM glucose (M9gl) or 0.2% (wt/vol) Casamino Acids (M9ca; lower nutrient availability). 5S RNA was used as a loading control. At the upper right is a Northern blot assay of the wild type and ΔncS35 (Δ) that confirms the deletion of ncS35 and the specificity of probe hybridization. The lower parts of panel A are 5S rRNA loading controls. Full-size images of Northern blot assays are depicted in Fig. S6 . (B) qPCR. Expression of ncS35 in B. cenocepacia J2315 was evaluated in exponential phase (Exp), stationary phase (Stat), and biofilms (BF) for full-length ncS35 (blue bars) and for both species combined (red bars). The locations of the primer pairs used are depicted in Fig. S1A . Fold changes were calculated relative to a cDNA standard (mixture of cDNA from all of the samples used in the experiment). Error bars represent standard deviations. ncS35 expression was significantly higher in biofilms than under all other conditions (*, P
Figure Legend Snippet: Expression of ncS35. (A) Northern blot assays. At the upper left is a Northern blot assay of ncS35 in wild-type (WT) B. cenocepacia J2315. Expression was evaluated in biofilms, stationary phase, and exponential phase. Responses to stress were evaluated in various media: LBB with 0.2% H 2 O 2 added 15 min prior to harvesting, LBB supplemented with 0.005% (wt/vol) SDS (membrane stress), and M9 supplemented with either 10 mM glucose (M9gl) or 0.2% (wt/vol) Casamino Acids (M9ca; lower nutrient availability). 5S RNA was used as a loading control. At the upper right is a Northern blot assay of the wild type and ΔncS35 (Δ) that confirms the deletion of ncS35 and the specificity of probe hybridization. The lower parts of panel A are 5S rRNA loading controls. Full-size images of Northern blot assays are depicted in Fig. S6 . (B) qPCR. Expression of ncS35 in B. cenocepacia J2315 was evaluated in exponential phase (Exp), stationary phase (Stat), and biofilms (BF) for full-length ncS35 (blue bars) and for both species combined (red bars). The locations of the primer pairs used are depicted in Fig. S1A . Fold changes were calculated relative to a cDNA standard (mixture of cDNA from all of the samples used in the experiment). Error bars represent standard deviations. ncS35 expression was significantly higher in biofilms than under all other conditions (*, P

Techniques Used: Expressing, Northern Blot, Hybridization, Real-time Polymerase Chain Reaction

Cell aggregation in biofilms and planktonic culture. (A) Confocal laser scanning images. Shown are z-stack images of 24-h-old biofilms of the wild type (WT) and ΔncS35 after LIVE/DEAD staining. Scale bars, 50 µm. (B) Flow cytometry size/granularity plots of wild-type vector control (WT + pM2), ΔncS35 vector control (ΔncS35 + pM2), and complemented ΔncS35 (ΔncS35 + pM2 + ncS35) biofilm cells grown in LBB with 0.2% rhamnose and Tp at 600 µg/ml. The x axis represents forward scatter (FSC) and indicates cell size. The y axis represents side scatter (SSC) and shows cell granularity. Gate R10 represents all cells, and dots outside this gate are background fluorescence.
Figure Legend Snippet: Cell aggregation in biofilms and planktonic culture. (A) Confocal laser scanning images. Shown are z-stack images of 24-h-old biofilms of the wild type (WT) and ΔncS35 after LIVE/DEAD staining. Scale bars, 50 µm. (B) Flow cytometry size/granularity plots of wild-type vector control (WT + pM2), ΔncS35 vector control (ΔncS35 + pM2), and complemented ΔncS35 (ΔncS35 + pM2 + ncS35) biofilm cells grown in LBB with 0.2% rhamnose and Tp at 600 µg/ml. The x axis represents forward scatter (FSC) and indicates cell size. The y axis represents side scatter (SSC) and shows cell granularity. Gate R10 represents all cells, and dots outside this gate are background fluorescence.

Techniques Used: Staining, Flow Cytometry, Cytometry, Plasmid Preparation, Fluorescence

26) Product Images from "Effects of Long-Term Water-Aging on Novel Anti-Biofilm and Protein-Repellent Dental Composite"

Article Title: Effects of Long-Term Water-Aging on Novel Anti-Biofilm and Protein-Repellent Dental Composite

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010186

Representative live/dead images of two-day biofilms on composites: ( A , B ) Commercial control at 1 and 180 days (d), ( C , D ) 3MPC at 1 and 180 d, ( E , F ) 1.5DMAHDM at 1 and 180 d, and ( G , H ) 3MPC + 1.5DMAHDM at 1 and 180 d. Live bacteria were stained green, and dead bacteria were stained red. When live/dead bacteria were in close proximity of each other, the staining exhibited yellow/orange colors. In ( A ), the commercial control had mostly live bacteria. In contrast, in ( C ), the 3MPC composite had much less bacterial attachment. In ( E ), the composite with 1.5% DMAHDM had substantial amounts of dead bacteria with red staining. In ( G ), the 3MPC + 1.5DMAHDM composite had less bacterial coverage, and the biofilms consisted of mostly compromised bacteria. As shown in ( B , D , F , H ), there was no significant difference between 1 and 180 d.
Figure Legend Snippet: Representative live/dead images of two-day biofilms on composites: ( A , B ) Commercial control at 1 and 180 days (d), ( C , D ) 3MPC at 1 and 180 d, ( E , F ) 1.5DMAHDM at 1 and 180 d, and ( G , H ) 3MPC + 1.5DMAHDM at 1 and 180 d. Live bacteria were stained green, and dead bacteria were stained red. When live/dead bacteria were in close proximity of each other, the staining exhibited yellow/orange colors. In ( A ), the commercial control had mostly live bacteria. In contrast, in ( C ), the 3MPC composite had much less bacterial attachment. In ( E ), the composite with 1.5% DMAHDM had substantial amounts of dead bacteria with red staining. In ( G ), the 3MPC + 1.5DMAHDM composite had less bacterial coverage, and the biofilms consisted of mostly compromised bacteria. As shown in ( B , D , F , H ), there was no significant difference between 1 and 180 d.

Techniques Used: Staining

27) Product Images from "Skin-bacteria communication: Involvement of the neurohormone Calcitonin Gene Related Peptide (CGRP) in the regulation of Staphylococcus epidermidis virulence"

Article Title: Skin-bacteria communication: Involvement of the neurohormone Calcitonin Gene Related Peptide (CGRP) in the regulation of Staphylococcus epidermidis virulence

Journal: Scientific Reports

doi: 10.1038/srep35379

Effect of CGRP (10 −6 M) on S. epidermidis biofilm formation under dynamic conditions. Biofilm formation by S. epidermidis in the absence or presence of CGRP was studied by confocal laser scanning microscopy using a flow-cell system. Two dimensional (2D) images collected at 1 μm intervals were used to reconstruct ortho cuts (3D/z) and three-dimensional (3D) images. Pictures are representative of three independent experiments. Scale bars = 20 μm.
Figure Legend Snippet: Effect of CGRP (10 −6 M) on S. epidermidis biofilm formation under dynamic conditions. Biofilm formation by S. epidermidis in the absence or presence of CGRP was studied by confocal laser scanning microscopy using a flow-cell system. Two dimensional (2D) images collected at 1 μm intervals were used to reconstruct ortho cuts (3D/z) and three-dimensional (3D) images. Pictures are representative of three independent experiments. Scale bars = 20 μm.

Techniques Used: Confocal Laser Scanning Microscopy, Flow Cytometry

28) Product Images from "Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis, et al. Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis"

Article Title: Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis, et al. Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis

Journal: Clinical and Experimental Dental Research

doi: 10.1002/cre2.65

Effect of AM404 on Porphyromonas gingivalis biofilms formed on titanium disks. (a) Reduction of P . gingivalis biofilm formation in the presence of AM404, as determined by colony‐forming unit (CFU) counts. (b) Reduction of P . gingivalis biofilm formation in the presence of AM404, as determined by LIVE/DEAD staining. (c) Eradication of preformed biofilms of P. gingivalis by AM404, as determined by CFU counts. (d) Eradication of preformed biofilms of P. gingivalis by AM404, as determined by LIVE/DEAD staining. In (a) and (c), values are means ± SEM of three independent experiments. *** p
Figure Legend Snippet: Effect of AM404 on Porphyromonas gingivalis biofilms formed on titanium disks. (a) Reduction of P . gingivalis biofilm formation in the presence of AM404, as determined by colony‐forming unit (CFU) counts. (b) Reduction of P . gingivalis biofilm formation in the presence of AM404, as determined by LIVE/DEAD staining. (c) Eradication of preformed biofilms of P. gingivalis by AM404, as determined by CFU counts. (d) Eradication of preformed biofilms of P. gingivalis by AM404, as determined by LIVE/DEAD staining. In (a) and (c), values are means ± SEM of three independent experiments. *** p

Techniques Used: Staining

29) Product Images from "Effects of Terminalia catappa Linn. Extract on Candida albicans biofilms developed on denture acrylic resin discs"

Article Title: Effects of Terminalia catappa Linn. Extract on Candida albicans biofilms developed on denture acrylic resin discs

Journal: Journal of Clinical and Experimental Dentistry

doi: 10.4317/jced.54776

Representative microscope images of C. albicans biofilms developed on denture acrylic discs and immersed for 8 hours at PBS (A), TCE at MIC (B), 5XMIC (C) or 10XMIC (D). SYTO-9 and propidium iodide labeled live and dead cells in green or yellow/red, respectively.
Figure Legend Snippet: Representative microscope images of C. albicans biofilms developed on denture acrylic discs and immersed for 8 hours at PBS (A), TCE at MIC (B), 5XMIC (C) or 10XMIC (D). SYTO-9 and propidium iodide labeled live and dead cells in green or yellow/red, respectively.

Techniques Used: Microscopy, Labeling

Cell count of C. albicans biofilms developed on denture acrylic discs and were immersed for 8 hours at PBS, TCE at MIC, 5XMIC or 10XMIC. Different symbols (*,**) represent statically significant differences between groups (ANOVA one-way followed by a Tukey test, p
Figure Legend Snippet: Cell count of C. albicans biofilms developed on denture acrylic discs and were immersed for 8 hours at PBS, TCE at MIC, 5XMIC or 10XMIC. Different symbols (*,**) represent statically significant differences between groups (ANOVA one-way followed by a Tukey test, p

Techniques Used: Cell Counting

30) Product Images from "Novel dental composite with capability to suppress cariogenic species and promote non-cariogenic species in oral biofilms"

Article Title: Novel dental composite with capability to suppress cariogenic species and promote non-cariogenic species in oral biofilms

Journal: Materials science & engineering. C, Materials for biological applications

doi: 10.1016/j.msec.2018.10.004

Colony-forming unit (CFU) counts of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). Note the log scale for the y-axis. Biofilm CFU counts of 3% DMAHDM group were approximately 2 to
Figure Legend Snippet: Colony-forming unit (CFU) counts of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). Note the log scale for the y-axis. Biofilm CFU counts of 3% DMAHDM group were approximately 2 to

Techniques Used:

MTT metabolic activity of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). The metabolic activity of biofilms decreased with increasing DMAHDM content. Values with dissimilar letters
Figure Legend Snippet: MTT metabolic activity of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). The metabolic activity of biofilms decreased with increasing DMAHDM content. Values with dissimilar letters

Techniques Used: MTT Assay, Activity Assay

Polysaccharides production of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). The polysaccharides production of biofilms decreased with increasing DMAHDM content. Values with dissimilar
Figure Legend Snippet: Polysaccharides production of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). The polysaccharides production of biofilms decreased with increasing DMAHDM content. Values with dissimilar

Techniques Used:

Representative live/dead staining images of biofilms: (A, B) commercial control, (C, D) 1.5% DMHADM, (E, F) 2.25% DMHADM, and (G, H) 3% DMHADM. All images had the same magnification as (A). Live bacteria were stained green, and compromised bacteria were
Figure Legend Snippet: Representative live/dead staining images of biofilms: (A, B) commercial control, (C, D) 1.5% DMHADM, (E, F) 2.25% DMHADM, and (G, H) 3% DMHADM. All images had the same magnification as (A). Live bacteria were stained green, and compromised bacteria were

Techniques Used: Staining

Lactic acid production of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). Values with dissimilar letters are significantly different from each other ( p
Figure Legend Snippet: Lactic acid production of (A) 48 h biofilms and (B) 72 h biofilms on composites with different DMAHDM mass fractions (mean ± sd; n = 6). Values with dissimilar letters are significantly different from each other ( p

Techniques Used:

31) Product Images from "Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells. Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells"

Article Title: Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells. Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells

Journal: Stem Cells Translational Medicine

doi: 10.1002/sctm.19-0092

Killing of planktonic and biofilm bacteria by mesenchymal stem cells (MSC) conditioned medium (CM) as assessed by cell membrane permeability assay and immunocytochemical staining. A, Bacterial killing of S. aureus (USA‐300 strain) in the planktonic phase of growth was quantitated using the LIVE/DEAD BacLight kit for quantitation of bacterial cell membrane permeability, as noted in the Materials and Methods section. Representative flow cytometry plots from time points pretreatment, 15, 90, and 180 minutes after incubation with MSC CM. Dead and live quadrants are labeled in bottom left and top right. Figures are representations of results obtained from three different donor MSC CM. B, Percentage of dead bacteria as determined by flow cytometry at different time points, MSC CM incubated bacteria shown as the red line, whereas control medium depicted as blue line. Error bars represent mean and SD from three replicates. C, Bacteria (USA‐300) grown as biofilms were incubated with control medium and MSC CM for the indicated time points, then stained with LIVE/DEAD BacLight kit as described in the Materials and Methods section to identify live and dead bacterial colonies, as revealed by immunohistochemical staining and evaluation by confocal microscopy. Green (SYTO9) represents live bacterial clusters, whereas red clusters represent dead bacteria stained with propidium iodide. Merged channels show yellow color as red and green overlap. Right column “MSC‐CM” shows MRSA biofilm incubated for 2 (top) or 24 (bottom) hour with MSC conditioned medium. Left column “MRSA biofilm” grown in DMEM medium only with additives matched to MSC‐CM. Images taken with ×10 objective. 4D, Prevention of biofilm formation by MSC CM (compared with control medium) as assessed using S. aureus biofilm assays, as noted in the Materials and Methods section. The Y axis depicts bacterial colonies, quantitated using crystal violet staining after 72 hours in culture. Blue shows the biofilm grown in DMEM media with all additives, red shows biofilm with the addition of MSC CM. E, Effects of MSC CM on pre‐formed biofilms following 2 or 24 hours of exposure. Bars depict the ratio of live vs dead bacteria in biofilms, as quantitated using ImageJ software, as described in the Materials and Methods section. Statistical significance was determined for * P ≤ .05, ** P ≤ .01, *** P ≤ .001, **** P ≤ .0001 as assessed by one‐way ANOVA and Tukey multiple means post‐test. Each experiment was conducted using CM from three different donor MSC. The figures depicted are representative of the results obtained in three independent experiments
Figure Legend Snippet: Killing of planktonic and biofilm bacteria by mesenchymal stem cells (MSC) conditioned medium (CM) as assessed by cell membrane permeability assay and immunocytochemical staining. A, Bacterial killing of S. aureus (USA‐300 strain) in the planktonic phase of growth was quantitated using the LIVE/DEAD BacLight kit for quantitation of bacterial cell membrane permeability, as noted in the Materials and Methods section. Representative flow cytometry plots from time points pretreatment, 15, 90, and 180 minutes after incubation with MSC CM. Dead and live quadrants are labeled in bottom left and top right. Figures are representations of results obtained from three different donor MSC CM. B, Percentage of dead bacteria as determined by flow cytometry at different time points, MSC CM incubated bacteria shown as the red line, whereas control medium depicted as blue line. Error bars represent mean and SD from three replicates. C, Bacteria (USA‐300) grown as biofilms were incubated with control medium and MSC CM for the indicated time points, then stained with LIVE/DEAD BacLight kit as described in the Materials and Methods section to identify live and dead bacterial colonies, as revealed by immunohistochemical staining and evaluation by confocal microscopy. Green (SYTO9) represents live bacterial clusters, whereas red clusters represent dead bacteria stained with propidium iodide. Merged channels show yellow color as red and green overlap. Right column “MSC‐CM” shows MRSA biofilm incubated for 2 (top) or 24 (bottom) hour with MSC conditioned medium. Left column “MRSA biofilm” grown in DMEM medium only with additives matched to MSC‐CM. Images taken with ×10 objective. 4D, Prevention of biofilm formation by MSC CM (compared with control medium) as assessed using S. aureus biofilm assays, as noted in the Materials and Methods section. The Y axis depicts bacterial colonies, quantitated using crystal violet staining after 72 hours in culture. Blue shows the biofilm grown in DMEM media with all additives, red shows biofilm with the addition of MSC CM. E, Effects of MSC CM on pre‐formed biofilms following 2 or 24 hours of exposure. Bars depict the ratio of live vs dead bacteria in biofilms, as quantitated using ImageJ software, as described in the Materials and Methods section. Statistical significance was determined for * P ≤ .05, ** P ≤ .01, *** P ≤ .001, **** P ≤ .0001 as assessed by one‐way ANOVA and Tukey multiple means post‐test. Each experiment was conducted using CM from three different donor MSC. The figures depicted are representative of the results obtained in three independent experiments

Techniques Used: Permeability, Staining, Quantitation Assay, Flow Cytometry, Incubation, Labeling, Immunohistochemistry, Confocal Microscopy, Software

Treatment of chronic biofilm infection by activated mesenchymal stem cells (MSC). Mice (n = 6 per group) were implanted with S. aureus infected mesh, then treated with activated MSC and amoxi‐clav, or amoxi‐clav only, as described in the Materials and Methods section. A, Bacterial bioburden in wound tissues (CFU/wound tissue) at the end of the 12 day study. Results pooled from four independent experiments. B, Luminescent imaging of wound bioburden, determined using an IVIS unit, and converted to area under the curve (AUC) Results pooled from three independent experiments. C, Mean measured wound area (mm 2 ) for each treatment group of mice. Results pooled from four independent experiments. D, Representative digital camera images of wound tissues immediately following euthanasia, obtained from treated and control mice. Lower two images showing representative IVIS imaging from treated and control mice. With red circle showing the field used to calculate radiance in ROI, radiance color scale shown in right bar. * denotes P
Figure Legend Snippet: Treatment of chronic biofilm infection by activated mesenchymal stem cells (MSC). Mice (n = 6 per group) were implanted with S. aureus infected mesh, then treated with activated MSC and amoxi‐clav, or amoxi‐clav only, as described in the Materials and Methods section. A, Bacterial bioburden in wound tissues (CFU/wound tissue) at the end of the 12 day study. Results pooled from four independent experiments. B, Luminescent imaging of wound bioburden, determined using an IVIS unit, and converted to area under the curve (AUC) Results pooled from three independent experiments. C, Mean measured wound area (mm 2 ) for each treatment group of mice. Results pooled from four independent experiments. D, Representative digital camera images of wound tissues immediately following euthanasia, obtained from treated and control mice. Lower two images showing representative IVIS imaging from treated and control mice. With red circle showing the field used to calculate radiance in ROI, radiance color scale shown in right bar. * denotes P

Techniques Used: Infection, Mouse Assay, Bioburden Testing, Imaging

32) Product Images from "Human Bile-Mediated Regulation of Salmonella Curli Fimbriae"

Article Title: Human Bile-Mediated Regulation of Salmonella Curli Fimbriae

Journal: Journal of Bacteriology

doi: 10.1128/JB.00055-19

Curli fimbria activation in human bile (HB). Representative images of 7-day biofilms of S . Typhimurium grown in M9 plus glucose (Glu) (control), M9 plus human bile (HB), and M9 plus ox bile (OB). Biofilms were incubated with Congo red for 5 min to stain curli fimbriae, rinsed twice with KPi buffer, and subsequently fixed in 2% paraformaldehyde (PFA). Green represents GFP-expressing Salmonella . All images are at ×20 magnification. The upper panels are top-down views of the biofilm, while the bottom panels are three-dimensional (3-D) Z-stacks.
Figure Legend Snippet: Curli fimbria activation in human bile (HB). Representative images of 7-day biofilms of S . Typhimurium grown in M9 plus glucose (Glu) (control), M9 plus human bile (HB), and M9 plus ox bile (OB). Biofilms were incubated with Congo red for 5 min to stain curli fimbriae, rinsed twice with KPi buffer, and subsequently fixed in 2% paraformaldehyde (PFA). Green represents GFP-expressing Salmonella . All images are at ×20 magnification. The upper panels are top-down views of the biofilm, while the bottom panels are three-dimensional (3-D) Z-stacks.

Techniques Used: Activation Assay, Incubation, Staining, Expressing

(a) Graphical representation of curli operons. RNA-Seq fold changes are presented under each gene. (b) qRT-PCR log 2 fold change in csgA expression from biofilms grown in M9 supplemented with human bile (HB; 10%), mouse bile (MB; 2.5%), or ox bile (OB; 2.5%) with respect to that in M9 supplemented with glucose (10 mM); rpoB was used as a reference. (c) Western blot analysis of the CsgA protein expressed in biofilms grown in M9 minimal medium with glucose (Glu) or HB (lanes 1 and 2 correspond to biological replicates under the same growth conditions).
Figure Legend Snippet: (a) Graphical representation of curli operons. RNA-Seq fold changes are presented under each gene. (b) qRT-PCR log 2 fold change in csgA expression from biofilms grown in M9 supplemented with human bile (HB; 10%), mouse bile (MB; 2.5%), or ox bile (OB; 2.5%) with respect to that in M9 supplemented with glucose (10 mM); rpoB was used as a reference. (c) Western blot analysis of the CsgA protein expressed in biofilms grown in M9 minimal medium with glucose (Glu) or HB (lanes 1 and 2 correspond to biological replicates under the same growth conditions).

Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Western Blot

33) Product Images from "Role of (p)ppGpp in Biofilm Formation by Enterococcus faecalis"

Article Title: Role of (p)ppGpp in Biofilm Formation by Enterococcus faecalis

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.07036-11

Plot graph showing biofilm biovolume accumulation over time (6 to 72 h) by E. faecalis OG1RF and mutant strains JAL1 (Δ relA ), JAL2 (Δ relQ ), and JAL3 (Δ relA Δ relQ ).
Figure Legend Snippet: Plot graph showing biofilm biovolume accumulation over time (6 to 72 h) by E. faecalis OG1RF and mutant strains JAL1 (Δ relA ), JAL2 (Δ relQ ), and JAL3 (Δ relA Δ relQ ).

Techniques Used: Mutagenesis

Representative 3D reconstructions of biofilm sections stained with Live/Dead BacLight stain after different incubation periods.
Figure Legend Snippet: Representative 3D reconstructions of biofilm sections stained with Live/Dead BacLight stain after different incubation periods.

Techniques Used: Staining, Incubation

Representative CSLM micrographs of E. faecalis strains after a 48-h biofilm formation period showing proteolytic activity (%). Proteolytic active cells are fluorescent yellow due to the dual staining with the casein substrate (green) and the SYTO62 counterstain
Figure Legend Snippet: Representative CSLM micrographs of E. faecalis strains after a 48-h biofilm formation period showing proteolytic activity (%). Proteolytic active cells are fluorescent yellow due to the dual staining with the casein substrate (green) and the SYTO62 counterstain

Techniques Used: Activity Assay, Staining

34) Product Images from "Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms"

Article Title: Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

Journal: Infection and Immunity

doi: 10.1128/IAI.02601-14

Evidence that the nuclease is the factor responsible for dispersal of NTHI biofilms. (A) “Comet tails” caused by the release of organisms from NTHI 2019 microcolonies/nascent biofilms as organisms transition from the biofilm to planktonic phase over a 24-h period. (B) Study performed on 2019Δ nuc , which had no evidence of microcolony formation or dispersal of organisms. (C) Partial complementation in cis of 2019Δ nuc because the expression of nuc is unregulated in the complemented strain. Small microcolonies were seen with the comet tail configurations similar to those seen in panel A.
Figure Legend Snippet: Evidence that the nuclease is the factor responsible for dispersal of NTHI biofilms. (A) “Comet tails” caused by the release of organisms from NTHI 2019 microcolonies/nascent biofilms as organisms transition from the biofilm to planktonic phase over a 24-h period. (B) Study performed on 2019Δ nuc , which had no evidence of microcolony formation or dispersal of organisms. (C) Partial complementation in cis of 2019Δ nuc because the expression of nuc is unregulated in the complemented strain. Small microcolonies were seen with the comet tail configurations similar to those seen in panel A.

Techniques Used: Expressing

A 50-image stacked z-series at ×20 magnification of 24-h biofilms grown in continuous flow chambers. The samples were stained with propidium iodide (red) and MAb 6E4 (green) prior to visualization. At 24 h, the NTHI 2019Δ nuc biofilm contains increased amounts of eDNA and large aggregates of organisms (B). This compares to lesser amounts of eDNA and diffuse arrangement of organisms within the biofilm of NTHI 2019 (A) and NTHI 2019Δ nuc :: nuc (C).
Figure Legend Snippet: A 50-image stacked z-series at ×20 magnification of 24-h biofilms grown in continuous flow chambers. The samples were stained with propidium iodide (red) and MAb 6E4 (green) prior to visualization. At 24 h, the NTHI 2019Δ nuc biofilm contains increased amounts of eDNA and large aggregates of organisms (B). This compares to lesser amounts of eDNA and diffuse arrangement of organisms within the biofilm of NTHI 2019 (A) and NTHI 2019Δ nuc :: nuc (C).

Techniques Used: Flow Cytometry, Staining

Cryo sections through 24-h biofilms from NTHI 2019 wild type (A), NTHI 2019Δ nuc (B), and NTHI 2019Δ nuc :: nuc (C). The DNA matrix is stained with DAPI, and the NTHI strains are stained with MAb 6E4. Scale bar, 20 μm. The organisms in the parent strain and in the complemented mutant are clearly dispersed throughout the biofilm, whereas they are clustered in the Δ nuc mutant.
Figure Legend Snippet: Cryo sections through 24-h biofilms from NTHI 2019 wild type (A), NTHI 2019Δ nuc (B), and NTHI 2019Δ nuc :: nuc (C). The DNA matrix is stained with DAPI, and the NTHI strains are stained with MAb 6E4. Scale bar, 20 μm. The organisms in the parent strain and in the complemented mutant are clearly dispersed throughout the biofilm, whereas they are clustered in the Δ nuc mutant.

Techniques Used: Staining, Mutagenesis

Confocal microscopy analysis of 10-μm-thick sections of biofilm formation at day 5 in the chinchilla middle ear after infection with NTHI 2019, NTHI 2019Δ nuc , and NTHI 2019Δ nuc :: nuc . The NTHI are stained with MAb 6E4 (green), and DNA is stained with DRAQ5 (blue). There is an aggregation of organisms staining with MAb 6E4 in NTHI 2019Δ nuc (B) compared to the more diffuse display of organisms in the wild type and the complemented mutant (A and C).
Figure Legend Snippet: Confocal microscopy analysis of 10-μm-thick sections of biofilm formation at day 5 in the chinchilla middle ear after infection with NTHI 2019, NTHI 2019Δ nuc , and NTHI 2019Δ nuc :: nuc . The NTHI are stained with MAb 6E4 (green), and DNA is stained with DRAQ5 (blue). There is an aggregation of organisms staining with MAb 6E4 in NTHI 2019Δ nuc (B) compared to the more diffuse display of organisms in the wild type and the complemented mutant (A and C).

Techniques Used: Confocal Microscopy, Infection, Staining, Mutagenesis

Lateral views and stacked z-series of 48-h biofilms stained with Live/Dead stain (green-red) and DRAQ-5 (blue). (A and C) Biofilm images of NTHI 2019 demonstrating a predominance of live organisms (green), while the images of NTHI 2019Δ nuc biofilm (B and D) demonstrate that the majority of organisms were dead (red) by 48 h.
Figure Legend Snippet: Lateral views and stacked z-series of 48-h biofilms stained with Live/Dead stain (green-red) and DRAQ-5 (blue). (A and C) Biofilm images of NTHI 2019 demonstrating a predominance of live organisms (green), while the images of NTHI 2019Δ nuc biofilm (B and D) demonstrate that the majority of organisms were dead (red) by 48 h.

Techniques Used: Staining

35) Product Images from "Synergistic Effects of Sodium Chloride, Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains ▿ Serotype 1/2a and 4b Strains ▿ † Serotype 1/2a and 4b Strains ▿ † ‡"

Article Title: Synergistic Effects of Sodium Chloride, Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains ▿ Serotype 1/2a and 4b Strains ▿ † Serotype 1/2a and 4b Strains ▿ † ‡

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02185-09

Time course monitoring of cell vitality in biofilms and absorbance values of CV from destained biofilms (A and C) or cell vitality and pH in cell suspension (B and D) during biofilm formation in TSBYE (A and B) or TSBYE gluc1%+NaCl2%
Figure Legend Snippet: Time course monitoring of cell vitality in biofilms and absorbance values of CV from destained biofilms (A and C) or cell vitality and pH in cell suspension (B and D) during biofilm formation in TSBYE (A and B) or TSBYE gluc1%+NaCl2%

Techniques Used:

Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of glucose at each temperature. (Data for
Figure Legend Snippet: Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of glucose at each temperature. (Data for

Techniques Used:

Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of sodium chloride at each temperature.
Figure Legend Snippet: Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of sodium chloride at each temperature.

Techniques Used:

Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of ethanol at each temperature. (Data for
Figure Legend Snippet: Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of ethanol at each temperature. (Data for

Techniques Used:

Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of glucose and sodium chloride at each temperature.
Figure Legend Snippet: Box plot of absorbance ( A 580 ) of crystal violet from destained L. monocytogenes biofilms formed by serotype 1/2a ( n = 18) and 4b ( n = 18) strains in TSBYE containing the indicated concentrations of glucose and sodium chloride at each temperature.

Techniques Used:

36) Product Images from "Impact of Silver-Containing Wound Dressings on Bacterial Biofilm Viability and Susceptibility to Antibiotics during Prolonged Treatment ▿"

Article Title: Impact of Silver-Containing Wound Dressings on Bacterial Biofilm Viability and Susceptibility to Antibiotics during Prolonged Treatment ▿

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00825-10

SEM images of MRSA biofilms developed on the peg surface before exposure (left) to NCPE, MSN, SCMC, MSAL, and MSPU and after 1 day (middle) and 7 days (right) of the treatment in a daily transfer assay. Bar, 5 μm.
Figure Legend Snippet: SEM images of MRSA biofilms developed on the peg surface before exposure (left) to NCPE, MSN, SCMC, MSAL, and MSPU and after 1 day (middle) and 7 days (right) of the treatment in a daily transfer assay. Bar, 5 μm.

Techniques Used:

Time-dependent effects of silver-containing dressings on biofilm bacterial viability and colony morphology.
Figure Legend Snippet: Time-dependent effects of silver-containing dressings on biofilm bacterial viability and colony morphology.

Techniques Used:

SEM images of P. aeruginosa biofilms developed on the peg surface before exposure (left) to Acticoat nanocrystalline silver on polyethylene mesh (NCPE), Silverlon metallic silver on nylon core (MSN), Aquacel Ag silver carboxymethylcellulose (SCMC), SilverCel
Figure Legend Snippet: SEM images of P. aeruginosa biofilms developed on the peg surface before exposure (left) to Acticoat nanocrystalline silver on polyethylene mesh (NCPE), Silverlon metallic silver on nylon core (MSN), Aquacel Ag silver carboxymethylcellulose (SCMC), SilverCel

Techniques Used:

SEM images of E. coli biofilms developed on the peg surface before exposure (left) to NCPE, MSN, SCMC, MSAL, and MSPU and after 1 day (middle) and 7 days (right) of the treatment in a daily transfer assay. Bar, 5 μm.
Figure Legend Snippet: SEM images of E. coli biofilms developed on the peg surface before exposure (left) to NCPE, MSN, SCMC, MSAL, and MSPU and after 1 day (middle) and 7 days (right) of the treatment in a daily transfer assay. Bar, 5 μm.

Techniques Used:

Antibiotic susceptibilities of biofilms pretreated with silver dressings.
Figure Legend Snippet: Antibiotic susceptibilities of biofilms pretreated with silver dressings.

Techniques Used:

Accumulation of silver in the biofilms.
Figure Legend Snippet: Accumulation of silver in the biofilms.

Techniques Used:

37) Product Images from "Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans"

Article Title: Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans

Journal: Scientific Reports

doi: 10.1038/s41598-018-24295-1

Scanning electron microscopy images of biofilms and planktonic cell morphology. Biofilm images were obtained at 5000×, 10000× and 20000×. Planktonic cell images were obtained at 10000×.
Figure Legend Snippet: Scanning electron microscopy images of biofilms and planktonic cell morphology. Biofilm images were obtained at 5000×, 10000× and 20000×. Planktonic cell images were obtained at 10000×.

Techniques Used: Electron Microscopy

Bacterial cell multiplication and EPS synthesis by confocal laser scanning microscopy (CLSM). ( A ) The three-dimensional reconstruction of biofilms. Reconstruction of the biofilms was performed with IMARIS 7.0. Bacterial cells were labelled with the SYTO 9 green fluorescent dye (left column), and EPS was labelled with the Alexa Fluor 647 red fluorescent dye (middle column). ( B ) The EPS and bacteria distributions on the reconstructed biofilm. ( C ) The EPS/bacteria ratio. The asterisks indicate significant differences compared to the parental S . mutans strain group. The error bars represent the standard deviation (SD). * P
Figure Legend Snippet: Bacterial cell multiplication and EPS synthesis by confocal laser scanning microscopy (CLSM). ( A ) The three-dimensional reconstruction of biofilms. Reconstruction of the biofilms was performed with IMARIS 7.0. Bacterial cells were labelled with the SYTO 9 green fluorescent dye (left column), and EPS was labelled with the Alexa Fluor 647 red fluorescent dye (middle column). ( B ) The EPS and bacteria distributions on the reconstructed biofilm. ( C ) The EPS/bacteria ratio. The asterisks indicate significant differences compared to the parental S . mutans strain group. The error bars represent the standard deviation (SD). * P

Techniques Used: Confocal Laser Scanning Microscopy, Standard Deviation

Biofilm biomass assay by crystal violet staining. The absorbance of the crystal violet-stained S . mutans biofilm at 600 nm is shown with the mean plus standard deviation (SD). The asterisks indicate the significant differences compared to the parental S . mutans strain group. The error bars represent the SD. * P
Figure Legend Snippet: Biofilm biomass assay by crystal violet staining. The absorbance of the crystal violet-stained S . mutans biofilm at 600 nm is shown with the mean plus standard deviation (SD). The asterisks indicate the significant differences compared to the parental S . mutans strain group. The error bars represent the SD. * P

Techniques Used: Staining, Standard Deviation

38) Product Images from "An In Vitro Model for Candida albicans–Streptococcus gordonii Biofilms on Titanium Surfaces"

Article Title: An In Vitro Model for Candida albicans–Streptococcus gordonii Biofilms on Titanium Surfaces

Journal: Journal of Fungi

doi: 10.3390/jof4020066

Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using CSLM and 3D reconstruction software. Biofilms were grown for 24 h in BMM synthetic saliva in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.
Figure Legend Snippet: Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using CSLM and 3D reconstruction software. Biofilms were grown for 24 h in BMM synthetic saliva in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.

Techniques Used: Software, Staining

Metabolic activity after 24 h incubation of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium. Biofilms were grown in RPMI 1640, THB + 0.02% YE, 1:1 v / v RPMI/THB + YE and BMM synthetic saliva in a 48-well plate containing titanium discs for 24 h. After incubation, discs were placed inside a new, sterile 48-well plate and viability was measured by Presto Blue TM fluorescence; n = 3. Error bars represent standard deviations. * indicates statistically significant differences.
Figure Legend Snippet: Metabolic activity after 24 h incubation of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium. Biofilms were grown in RPMI 1640, THB + 0.02% YE, 1:1 v / v RPMI/THB + YE and BMM synthetic saliva in a 48-well plate containing titanium discs for 24 h. After incubation, discs were placed inside a new, sterile 48-well plate and viability was measured by Presto Blue TM fluorescence; n = 3. Error bars represent standard deviations. * indicates statistically significant differences.

Techniques Used: Activity Assay, Incubation, Fluorescence

SEM micrographs of C. albicans and S. gordonii single- and dual-species biofilms grown in 1:1 v / v RPMI/THB + 0.02% YE media (upper panel) and BMM synthetic saliva (lower panel) formed on titanium discs. Magnification ×500.
Figure Legend Snippet: SEM micrographs of C. albicans and S. gordonii single- and dual-species biofilms grown in 1:1 v / v RPMI/THB + 0.02% YE media (upper panel) and BMM synthetic saliva (lower panel) formed on titanium discs. Magnification ×500.

Techniques Used:

Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using confocal laser scanning microscopy (CLSM) and 3D reconstruction software. Biofilms were grown for 24 h in 1:1 v / v RPMI/THB + YE in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.
Figure Legend Snippet: Characterization of C. albicans and S. gordonii single- and dual-species biofilms formed on titanium using confocal laser scanning microscopy (CLSM) and 3D reconstruction software. Biofilms were grown for 24 h in 1:1 v / v RPMI/THB + YE in a 48-well plate containing titanium discs, and stained with Concanavalin A-Alexa Fluor ® 488 conjugate, 1 x FilmTracer™ SYPRO ® Ruby Biofilm Matrix Stain, and DAPI. Scale bar corresponds to 50 µm. Included in the figure are the xy , xz , and yz views of the resulting biofilm.

Techniques Used: Confocal Laser Scanning Microscopy, Software, Staining

Antimicrobial susceptibility patterns of preformed dual-species C. albicans/S. gordonii biofilms formed in 1:1 v / v RPMI/THB + 0.02% YE media (1:1, upper panels) or BMM synthetic saliva (BMM, lower panels) using an antibacterial/antifungal combination therapy.
Figure Legend Snippet: Antimicrobial susceptibility patterns of preformed dual-species C. albicans/S. gordonii biofilms formed in 1:1 v / v RPMI/THB + 0.02% YE media (1:1, upper panels) or BMM synthetic saliva (BMM, lower panels) using an antibacterial/antifungal combination therapy.

Techniques Used:

39) Product Images from "Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni"

Article Title: Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087312

Effect of increased ahpC expression on biofilm formation. Biofilm formation levels were determined with crystal violet staining after 24 h incubation of samples. The results show the means and standard deviations of a representative experiment with triplicate samples. The experiment was repeated six times and all produced similar results. The statistical differences between the wild type and each mutant were determined by t -test. NS: non-significant, * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001.
Figure Legend Snippet: Effect of increased ahpC expression on biofilm formation. Biofilm formation levels were determined with crystal violet staining after 24 h incubation of samples. The results show the means and standard deviations of a representative experiment with triplicate samples. The experiment was repeated six times and all produced similar results. The statistical differences between the wild type and each mutant were determined by t -test. NS: non-significant, * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001.

Techniques Used: Expressing, Staining, Incubation, Produced, Mutagenesis

Confocal microscopy analysis of biofilms of the wild type (A), the ahpC mutant (B), and the complementation strain (C). Biofilms were grown 24 h and stained with the LIVE/DEAD Biofilm Viability kit (Life Technologies, US).
Figure Legend Snippet: Confocal microscopy analysis of biofilms of the wild type (A), the ahpC mutant (B), and the complementation strain (C). Biofilms were grown 24 h and stained with the LIVE/DEAD Biofilm Viability kit (Life Technologies, US).

Techniques Used: Confocal Microscopy, Mutagenesis, Staining

Determination of total ROS level (A) and lipid hydroperoxide (LPO; B) in the ahpC mutant, and reduced biofilm formation in the ahpC mutant by treatment with antioxidants (C). The assays were carried out with 24 h old samples. Antioxidants were treated to a final concentration of 1nM. The results show the means and standard deviations of a representative experiment with triplicate samples. The assays were repeated at least three times and similar results were reproducible in all the experiments. Statistical significance was analyzed with t -test (Fig. 3A and 3B) and two-way ANOVA (Fig. 3C). * P ≤0.05, **** P ≤0.0001.
Figure Legend Snippet: Determination of total ROS level (A) and lipid hydroperoxide (LPO; B) in the ahpC mutant, and reduced biofilm formation in the ahpC mutant by treatment with antioxidants (C). The assays were carried out with 24 h old samples. Antioxidants were treated to a final concentration of 1nM. The results show the means and standard deviations of a representative experiment with triplicate samples. The assays were repeated at least three times and similar results were reproducible in all the experiments. Statistical significance was analyzed with t -test (Fig. 3A and 3B) and two-way ANOVA (Fig. 3C). * P ≤0.05, **** P ≤0.0001.

Techniques Used: Mutagenesis, Concentration Assay

Biofilm formation of oxidative stress resistance mutants. Biofilm formation was measured using crystal violet staining. The results show the means and standard deviations of a representative assay with triplicate samples. The experiment was repeated six times and all produced similar results. Statistical significance was analyzed using one-way analysis of variance (ANOVA). * P ≤0.05, ** P ≤0.01, **** P ≤0.0001.
Figure Legend Snippet: Biofilm formation of oxidative stress resistance mutants. Biofilm formation was measured using crystal violet staining. The results show the means and standard deviations of a representative assay with triplicate samples. The experiment was repeated six times and all produced similar results. Statistical significance was analyzed using one-way analysis of variance (ANOVA). * P ≤0.05, ** P ≤0.01, **** P ≤0.0001.

Techniques Used: Staining, Produced

40) Product Images from "Unsaturated Fatty Acid, cis-2-Decenoic Acid, in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria"

Article Title: Unsaturated Fatty Acid, cis-2-Decenoic Acid, in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria

Journal: PLoS ONE

doi: 10.1371/journal.pone.0101677

Effect of CDA combined antimicrobial treatments on killing of pre-established biofilms. CDA treatment reverses biofilm formation in pre-established biofilms and cells remaining on the surface are easily removed and killed various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment, then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+CDA) for 1 h and stained with LIVE/DEAD staining to allow analysis using fluorescence microscopy. The images show microscopic pictures of the biofilms on the surface of cover slip after combinatorial treatments. Images are top-down views (x-y plane); scale bars: 50 µm. Results are representative of 3 separate experiments.
Figure Legend Snippet: Effect of CDA combined antimicrobial treatments on killing of pre-established biofilms. CDA treatment reverses biofilm formation in pre-established biofilms and cells remaining on the surface are easily removed and killed various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment, then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+CDA) for 1 h and stained with LIVE/DEAD staining to allow analysis using fluorescence microscopy. The images show microscopic pictures of the biofilms on the surface of cover slip after combinatorial treatments. Images are top-down views (x-y plane); scale bars: 50 µm. Results are representative of 3 separate experiments.

Techniques Used: Flow Cytometry, Staining, Fluorescence, Microscopy

Induction of planktonic mode of growth in pre-established biofilms formed by food pathogens using CDA. (A) Biofilms were grown for 5 days in petri dishes in which the medium was replaced every 24 h. Dispersion induction was tested by replacing the growth medium with fresh medium containing three different concentrations of CDA (100, 310 or 620 nM) or just the carrier as a control and the cells were incubated for a further 1 h. Medium containing dispersed cells was then homogenized and cell density was determined by measuring the optical density. (B) After 5 days of biofilm growth in flow cell continuous cultures, the influent medium was switched from fresh medium in the test lines to three indicated concentrations of CDA and control lines were switched to new lines containing just the carrier. Effluent runoffs were then collected and cell density was determined by measuring the OD. Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value
Figure Legend Snippet: Induction of planktonic mode of growth in pre-established biofilms formed by food pathogens using CDA. (A) Biofilms were grown for 5 days in petri dishes in which the medium was replaced every 24 h. Dispersion induction was tested by replacing the growth medium with fresh medium containing three different concentrations of CDA (100, 310 or 620 nM) or just the carrier as a control and the cells were incubated for a further 1 h. Medium containing dispersed cells was then homogenized and cell density was determined by measuring the optical density. (B) After 5 days of biofilm growth in flow cell continuous cultures, the influent medium was switched from fresh medium in the test lines to three indicated concentrations of CDA and control lines were switched to new lines containing just the carrier. Effluent runoffs were then collected and cell density was determined by measuring the OD. Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value

Techniques Used: Incubation, Flow Cytometry

Effect of CDA combined antimicrobial treatments on eradication and killing of pre-established biofilms. (A) After 120 h of growth on the surface of SS discs, biofilms were treated for 1 h with biocides alone or combined with 310 nM CDA; CFU plate counts were then performed to assess the viability of the bacteria. (B) The amount of biofilm remaining was determined by the absorbance at 590 nm of crystal violet after staining the 120 h different biofilms in a microtiter plate assay after treatment with tested concentrations of antibiotics alone (- CDA) or in combination with 310 nM CDA (+CDA) for 1 h. All readings are corrected to reflect 0% and 100% controls (blank well, 0%; biofilms without any treatments, 100%). Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value
Figure Legend Snippet: Effect of CDA combined antimicrobial treatments on eradication and killing of pre-established biofilms. (A) After 120 h of growth on the surface of SS discs, biofilms were treated for 1 h with biocides alone or combined with 310 nM CDA; CFU plate counts were then performed to assess the viability of the bacteria. (B) The amount of biofilm remaining was determined by the absorbance at 590 nm of crystal violet after staining the 120 h different biofilms in a microtiter plate assay after treatment with tested concentrations of antibiotics alone (- CDA) or in combination with 310 nM CDA (+CDA) for 1 h. All readings are corrected to reflect 0% and 100% controls (blank well, 0%; biofilms without any treatments, 100%). Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value

Techniques Used: Staining

Effect of CDA combined disinfectant or antibiotic treatments on biofilms surface area. Following dispersion of biofilms by CDA, cells remaining on the surface are easily killed and removed by various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment and then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+ CDA) for 1 h, stained with LIVE/DEAD staining and quantified (percent surface coverage) using digital image analysis. The bars show the levels of biofilm biomass after treatment with antimicrobials alone or combined with 310 nM CDA. Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value
Figure Legend Snippet: Effect of CDA combined disinfectant or antibiotic treatments on biofilms surface area. Following dispersion of biofilms by CDA, cells remaining on the surface are easily killed and removed by various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment and then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+ CDA) for 1 h, stained with LIVE/DEAD staining and quantified (percent surface coverage) using digital image analysis. The bars show the levels of biofilm biomass after treatment with antimicrobials alone or combined with 310 nM CDA. Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value

Techniques Used: Flow Cytometry, Staining

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Labeling:

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Article Snippet: .. Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter). .. The plate was then placed on the motorized stage of an inverted confocal microscope (TCS SP8 AOBS, Leica Microsystems) at INRA-MIMA2 platform .

Incubation:

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Confocal Laser Scanning Microscopy:

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Modification:

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Staining:

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    Fatty acids in <t>biofilms/planktonic</t> cultures. Strain 630Δ erm was grown either as biofilms in microfermentors (like in Figure 1 ) or as planktonic cultures in Falcon tubes. Supernatants of biofilms (in gray) and planktonic cultures (in white) were recovered and their composition in volatile (A) and non-volatile fatty acids (B) was determined. Relevant fatty acids, as inferred from our transcriptomic study, are shown. ∗ indicates a statistically significant difference ( p -value
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    Fatty acids in biofilms/planktonic cultures. Strain 630Δ erm was grown either as biofilms in microfermentors (like in Figure 1 ) or as planktonic cultures in Falcon tubes. Supernatants of biofilms (in gray) and planktonic cultures (in white) were recovered and their composition in volatile (A) and non-volatile fatty acids (B) was determined. Relevant fatty acids, as inferred from our transcriptomic study, are shown. ∗ indicates a statistically significant difference ( p -value

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Fatty acids in biofilms/planktonic cultures. Strain 630Δ erm was grown either as biofilms in microfermentors (like in Figure 1 ) or as planktonic cultures in Falcon tubes. Supernatants of biofilms (in gray) and planktonic cultures (in white) were recovered and their composition in volatile (A) and non-volatile fatty acids (B) was determined. Relevant fatty acids, as inferred from our transcriptomic study, are shown. ∗ indicates a statistically significant difference ( p -value

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques:

    Intact biofilm architecture of the parental strain and CD2214–CD2215 mutant. The biofilms of the parental strain 630Δ erm (A–D) and its CD2214–CD2215 mutant (E–H) are shown. They were grown for 48h in 96-well polystyrene micro-titer plates, in TYt medium freshly added onto adhesive starter cells ( Supplementary Figure S5 ). After live dead staining of intact biofilms directly in the micro-titer plates, their microscopic architecture was observed in situ by CLSM. Images representative of three independent experiments (each using three clones) are shown. Raw confocal z-stacks were treated using IMARIS software. This allowed obtaining both a 3D projection upside view, with its shadow on the right (A,E) , and a section view close to the surface (B,F) , in which the white bar indicates the scale (50 μm). For the section view of each strain, magnifications of micro-aggregated forms (C,G) and rods (D,H) are also provided, with the corresponding scale bar in white (5, 10, or 15 μm as indicated). After data recovery, biovolume, maximum coverage, mean thickness and biovolume/surface ratio were quantified and a statistical analysis was performed (see Supplementary Figure S6 ).

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Intact biofilm architecture of the parental strain and CD2214–CD2215 mutant. The biofilms of the parental strain 630Δ erm (A–D) and its CD2214–CD2215 mutant (E–H) are shown. They were grown for 48h in 96-well polystyrene micro-titer plates, in TYt medium freshly added onto adhesive starter cells ( Supplementary Figure S5 ). After live dead staining of intact biofilms directly in the micro-titer plates, their microscopic architecture was observed in situ by CLSM. Images representative of three independent experiments (each using three clones) are shown. Raw confocal z-stacks were treated using IMARIS software. This allowed obtaining both a 3D projection upside view, with its shadow on the right (A,E) , and a section view close to the surface (B,F) , in which the white bar indicates the scale (50 μm). For the section view of each strain, magnifications of micro-aggregated forms (C,G) and rods (D,H) are also provided, with the corresponding scale bar in white (5, 10, or 15 μm as indicated). After data recovery, biovolume, maximum coverage, mean thickness and biovolume/surface ratio were quantified and a statistical analysis was performed (see Supplementary Figure S6 ).

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Mutagenesis, Staining, In Situ, Confocal Laser Scanning Microscopy, Software

    Intact biofilm architecture of the parental strain over-expressing or not dccA . Biofilms were grown in TYt medium freshly added onto adhesive starter cells in 96-well polystyrene micro-titer plates. The procedure was essentially as described in Figure 8 , except that growth was for only 24 h and that anhydro-tetracycline was added to induce P tet promoter and dccA expression. At the end of growth, intact biofilms were stained and observed by CLMS as described in Figure 8 . Representative images are shown. For each strain, a 3D projection upside view, with its shadow on the right (A,C) , and a section view close to the surface (B,D) are shown, with the white bar indicating the scale (50 μm). The biofilm of the parental strain over-expressing dccA (630Δ erm p dccA in C,D ) and that of the control strain (630Δ erm p in A,B ) are shown. After data recovery, the same four parameters as in Figure 8 were quantified and a statistical analysis was performed (see Supplementary Figure S9 ).

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Intact biofilm architecture of the parental strain over-expressing or not dccA . Biofilms were grown in TYt medium freshly added onto adhesive starter cells in 96-well polystyrene micro-titer plates. The procedure was essentially as described in Figure 8 , except that growth was for only 24 h and that anhydro-tetracycline was added to induce P tet promoter and dccA expression. At the end of growth, intact biofilms were stained and observed by CLMS as described in Figure 8 . Representative images are shown. For each strain, a 3D projection upside view, with its shadow on the right (A,C) , and a section view close to the surface (B,D) are shown, with the white bar indicating the scale (50 μm). The biofilm of the parental strain over-expressing dccA (630Δ erm p dccA in C,D ) and that of the control strain (630Δ erm p in A,B ) are shown. After data recovery, the same four parameters as in Figure 8 were quantified and a statistical analysis was performed (see Supplementary Figure S9 ).

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Expressing, Staining

    Sugar transport and metabolism in biofilms compared to planktonic cultures. A model for sugar transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in sugar transport and metabolism, and their variation between micro-fermentor biofilms ( Figure 1 ) and planktonic cultures ( Supplementary Table S2 ). The name/short identification number of proteins is indicated in green or red when their gene is down- or up-regulated during biofilm/planktonic growth, respectively (see Supplementary Table S2 for protein activity/function). For simplicity, the number of molecules is not indicated in metabolic reactions (e.g., for glycolysis, each molecule of glucose leads to two molecules of glyceraldehyde 3-phosphate and all subsequent products). PEP, Phospho-Enol-Pyruvate; THF, TetraHydroFolate; CoA, Co-enzyme A; P, phosphate.

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Sugar transport and metabolism in biofilms compared to planktonic cultures. A model for sugar transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in sugar transport and metabolism, and their variation between micro-fermentor biofilms ( Figure 1 ) and planktonic cultures ( Supplementary Table S2 ). The name/short identification number of proteins is indicated in green or red when their gene is down- or up-regulated during biofilm/planktonic growth, respectively (see Supplementary Table S2 for protein activity/function). For simplicity, the number of molecules is not indicated in metabolic reactions (e.g., for glycolysis, each molecule of glucose leads to two molecules of glyceraldehyde 3-phosphate and all subsequent products). PEP, Phospho-Enol-Pyruvate; THF, TetraHydroFolate; CoA, Co-enzyme A; P, phosphate.

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Expressing, Activity Assay

    Protein export in biofilms compared to planktonic cultures. A model for protein export pathways and exported protein production in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in protein export or encoding exported proteins, and drawn as in Figure 2 . PSII, surface polysaccharide II and atypical teichoic acid.

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Protein export in biofilms compared to planktonic cultures. A model for protein export pathways and exported protein production in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in protein export or encoding exported proteins, and drawn as in Figure 2 . PSII, surface polysaccharide II and atypical teichoic acid.

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Expressing

    Amino acid transport and metabolism in biofilms compared to planktonic cultures. A model for amino acid transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in amino acid transport and metabolism, and drawn as in Figure 2 . BCAA, Branched Chain Amino Acids; Fe-S, Iron-Sulfur cluster.

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Amino acid transport and metabolism in biofilms compared to planktonic cultures. A model for amino acid transport and utilization pathways in biofilm compared to planktonic cells is proposed. It is based on the expression of genes involved in amino acid transport and metabolism, and drawn as in Figure 2 . BCAA, Branched Chain Amino Acids; Fe-S, Iron-Sulfur cluster.

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Expressing

    Comparison between CD2214–CD2215 regulon and the set of genes differentially expressed in biofilm/planktonic growth. (A) Overlap. Transcriptomes are drawn as elipses: the set of genes differentially expressed during biofilm/planktonic growth ( Supplementary Table S2 ) is on the left and the set of genes differentially expressed in strain 630Δ erm / CD2214–CD2215 mutant ( Supplementary Table S4 ) is on the right. The number of shared genes (whose expression varies in both transcriptomes) is indicated above the overlap region. As shared genes can vary in the same direction in the two transcriptomes or in opposite directions, the overlap region is divided into two parts. The number of shared genes whose expression varies in opposite directions is indicated in the dark gray part, while the number of genes whose expression varies in the same direction is in the light gray part. (B) Functions. Genes common to the two transcriptomes are shown as a pie chart. The main functional categories of genes regulated in the same direction in the two transcriptomes appear in capital letters in light gray slices. A unique dark gray slice is shown for all genes regulated in opposite directions in the two transcriptomes, whatever the functional category they belong to. The function and the product of main shared genes (designated by their name or short identification number) are indicated beside the pie chart. Protein names/short identification numbers are in green or red depending on whether their genes are, respectively, down- or up-regulated in both transcriptomes. Proteins whose genes are controlled by a c-di-GMP riboswitch ( Soutourina et al., 2013 ) are underlined.

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Comparison between CD2214–CD2215 regulon and the set of genes differentially expressed in biofilm/planktonic growth. (A) Overlap. Transcriptomes are drawn as elipses: the set of genes differentially expressed during biofilm/planktonic growth ( Supplementary Table S2 ) is on the left and the set of genes differentially expressed in strain 630Δ erm / CD2214–CD2215 mutant ( Supplementary Table S4 ) is on the right. The number of shared genes (whose expression varies in both transcriptomes) is indicated above the overlap region. As shared genes can vary in the same direction in the two transcriptomes or in opposite directions, the overlap region is divided into two parts. The number of shared genes whose expression varies in opposite directions is indicated in the dark gray part, while the number of genes whose expression varies in the same direction is in the light gray part. (B) Functions. Genes common to the two transcriptomes are shown as a pie chart. The main functional categories of genes regulated in the same direction in the two transcriptomes appear in capital letters in light gray slices. A unique dark gray slice is shown for all genes regulated in opposite directions in the two transcriptomes, whatever the functional category they belong to. The function and the product of main shared genes (designated by their name or short identification number) are indicated beside the pie chart. Protein names/short identification numbers are in green or red depending on whether their genes are, respectively, down- or up-regulated in both transcriptomes. Proteins whose genes are controlled by a c-di-GMP riboswitch ( Soutourina et al., 2013 ) are underlined.

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Mutagenesis, Expressing, Functional Assay

    Biofilm of strain 630Δ erm after growth in a continuous-flow micro-fermentor. After anaerobic growth for 72 h in TYt medium, macro-colonies and biofilms can be observed on micro-fermentor walls. The medium is clear, as expected in the absence of planktonic growth. A representative picture of independent experiments is shown (A) . Magnifications (B,C) allow observing macro-colonies (arrows).

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Biofilm of strain 630Δ erm after growth in a continuous-flow micro-fermentor. After anaerobic growth for 72 h in TYt medium, macro-colonies and biofilms can be observed on micro-fermentor walls. The medium is clear, as expected in the absence of planktonic growth. A representative picture of independent experiments is shown (A) . Magnifications (B,C) allow observing macro-colonies (arrows).

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Flow Cytometry

    Biofilm and planktonic cells of strain 630Δ erm . Biofilms (A–D) and planktonic cultures (E,F) of strain 630Δ erm were grown in parallel in TYt medium, respectively, for 48 h in 24-well polystyrene micro-titer plates and for 24 h in Falcon tubes. After having been recovered, fixed and washed, biofilm and planktonic cells were observed by Transmitted Light Microscopy. Representative images are shown, with a white bar indicating the scale (10 μm). In biofilm (A–D) , (i) elongated rods (∼20–30 μm) are shown by white horizontal arrows, and (ii) cells aligned side by side along the width and tightly packed into micro-aggregates are indicated by black vertical arrows. In a planktonic culture (F) , a refracting spore is indicated by a gray oblique arrow.

    Journal: Frontiers in Microbiology

    Article Title: Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture

    doi: 10.3389/fmicb.2018.02084

    Figure Lengend Snippet: Biofilm and planktonic cells of strain 630Δ erm . Biofilms (A–D) and planktonic cultures (E,F) of strain 630Δ erm were grown in parallel in TYt medium, respectively, for 48 h in 24-well polystyrene micro-titer plates and for 24 h in Falcon tubes. After having been recovered, fixed and washed, biofilm and planktonic cells were observed by Transmitted Light Microscopy. Representative images are shown, with a white bar indicating the scale (10 μm). In biofilm (A–D) , (i) elongated rods (∼20–30 μm) are shown by white horizontal arrows, and (ii) cells aligned side by side along the width and tightly packed into micro-aggregates are indicated by black vertical arrows. In a planktonic culture (F) , a refracting spore is indicated by a gray oblique arrow.

    Article Snippet: Confocal Laser Scanning Microscopy (CLSM) After growth in micro-titer plates, biofilms were anaerobically stained for 2 h by the FilmtracerTM LIVE/DEAD® Biofilm Viability Kit (Thermo Fisher Scientific) at a 1/350 dilution (DNA-dyes SYTO 9 at 9.5 μM and propidium iodide at 57.1 μM, both labeling DNA, but only in damaged-membrane cells for the latter).

    Techniques: Light Microscopy

    Loss of outer membrane integrity in strain RN102. Bacterial samples were collected from 48-hour-cultured biofilms for TEM analysis. TEM images of the bacterial cells and the cell appendages are shown for strains: (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). The outer membranes are indicated by arrows. Representative electron-microphotographs of each strain are shown. A 500-nm-long bar is shown in the lower left corner of each eclectron-micrograph. (F) Western blot analysis of supernatants from BW25113 and RN102. Supernatants were harvested by centrifugation from bacterial liquid culture grown for 48 hours under static conditions. Results of Western blot using anti-Crp, anti-DsbA, anti-OmpC, and anti-OmpA antisera are shown. (G) Supernatants from bacterial liquid cultures of BW25113 or RN102 grown for 48 hours under static conditions were serially diluted with TE. The diluted samples were used as template DNA for PCR using E. coli atoS gene-specific primer pairs. Lanes: 1, without dilution; 2, 10 −1 dilution; 3, 10 −2 dilution; 4, 10 −3 dilution; 5, 10 −4 dilution; 6, 10 −5 dilution; 7, 10 −6 dilution.

    Journal: PLoS ONE

    Article Title: Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

    doi: 10.1371/journal.pone.0051241

    Figure Lengend Snippet: Loss of outer membrane integrity in strain RN102. Bacterial samples were collected from 48-hour-cultured biofilms for TEM analysis. TEM images of the bacterial cells and the cell appendages are shown for strains: (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). The outer membranes are indicated by arrows. Representative electron-microphotographs of each strain are shown. A 500-nm-long bar is shown in the lower left corner of each eclectron-micrograph. (F) Western blot analysis of supernatants from BW25113 and RN102. Supernatants were harvested by centrifugation from bacterial liquid culture grown for 48 hours under static conditions. Results of Western blot using anti-Crp, anti-DsbA, anti-OmpC, and anti-OmpA antisera are shown. (G) Supernatants from bacterial liquid cultures of BW25113 or RN102 grown for 48 hours under static conditions were serially diluted with TE. The diluted samples were used as template DNA for PCR using E. coli atoS gene-specific primer pairs. Lanes: 1, without dilution; 2, 10 −1 dilution; 3, 10 −2 dilution; 4, 10 −3 dilution; 5, 10 −4 dilution; 6, 10 −5 dilution; 7, 10 −6 dilution.

    Article Snippet: In order to quantify the amount of biofilm on a 96-well plate, all stain associated with the attached biofilms was dissolved with 95% ethanol, then OD595 absorbance was measured using a microplate reader (Multiskan RC, ThermoFisher, Waltham, MA).

    Techniques: Cell Culture, Transmission Electron Microscopy, Western Blot, Centrifugation, Polymerase Chain Reaction

    Contribution of eDNA to biofilm structure formed by RN102. (A–E) CLSM images of biofilms formed by strains: (A) BW25113 (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (D) RN102/pNT3( hldE ). Images of biofilms stained with acrydine orange are shown as digital CLSM images. In each strain, a section which has the largest sum of signals in the defined area (127.3 μm by 127.3 μm) among all X–Y sections is shown in the upper row (X–Y). The overview of biofilms in the same area of each X–Y section is shown as 3D image in the lower row (3D). The volume of each 3D image (μm 3 ) in the area of the X–Y planes was quantified and the mean ± SD obtained from 3 different areas chosen at random are denoted in the upper-right corners. The data shown are representative microphotographs of two independent experiments. (F) Quantification of eDNA from BW25113 and RN102 strains. The bars represent the ratio of extracellular DNA to intracellular DNA (eDNA/iDNA). Results are shown as the mean ± SD from 3 independent experiments. * P

    Journal: PLoS ONE

    Article Title: Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

    doi: 10.1371/journal.pone.0051241

    Figure Lengend Snippet: Contribution of eDNA to biofilm structure formed by RN102. (A–E) CLSM images of biofilms formed by strains: (A) BW25113 (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (D) RN102/pNT3( hldE ). Images of biofilms stained with acrydine orange are shown as digital CLSM images. In each strain, a section which has the largest sum of signals in the defined area (127.3 μm by 127.3 μm) among all X–Y sections is shown in the upper row (X–Y). The overview of biofilms in the same area of each X–Y section is shown as 3D image in the lower row (3D). The volume of each 3D image (μm 3 ) in the area of the X–Y planes was quantified and the mean ± SD obtained from 3 different areas chosen at random are denoted in the upper-right corners. The data shown are representative microphotographs of two independent experiments. (F) Quantification of eDNA from BW25113 and RN102 strains. The bars represent the ratio of extracellular DNA to intracellular DNA (eDNA/iDNA). Results are shown as the mean ± SD from 3 independent experiments. * P

    Article Snippet: In order to quantify the amount of biofilm on a 96-well plate, all stain associated with the attached biofilms was dissolved with 95% ethanol, then OD595 absorbance was measured using a microplate reader (Multiskan RC, ThermoFisher, Waltham, MA).

    Techniques: Confocal Laser Scanning Microscopy, Staining

    Biofilm formation and growth of a series of LPS mutants. (A) Biofilm formation by a series of core OS LPS mutants when compared to the parental strain, BW25113. The mean ± SD of results from 3 independent experiments are shown. Statistical analysis was performed using ANOVA. * P

    Journal: PLoS ONE

    Article Title: Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

    doi: 10.1371/journal.pone.0051241

    Figure Lengend Snippet: Biofilm formation and growth of a series of LPS mutants. (A) Biofilm formation by a series of core OS LPS mutants when compared to the parental strain, BW25113. The mean ± SD of results from 3 independent experiments are shown. Statistical analysis was performed using ANOVA. * P

    Article Snippet: In order to quantify the amount of biofilm on a 96-well plate, all stain associated with the attached biofilms was dissolved with 95% ethanol, then OD595 absorbance was measured using a microplate reader (Multiskan RC, ThermoFisher, Waltham, MA).

    Techniques:

    Loss of flagella in RN102. Fourty eight-hour-cultured biofilms were collected and analyzed by TEM, and by Western blot for FliC. (A–E) TEM images of the bacterial cells and the cell appendages are shown for strains (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). Flagella found in figures (A), (C), and (E) are shown by arrowheads. Representative electron-microphotographs of each strain are shown. A 1-μm-long bar is shown in the lower left corner. (F) Supernatants were collected from 48-hour bacterial cultures. Result of Western blot using anti-FliC antiserum is shown. Lanes; 1, BW25113; 2, RN102; 3, BW25113/pNTR-SD; 4, RN102/pNTR-SD; 5, RN102/pNT3( hldE ); 6, RN110.

    Journal: PLoS ONE

    Article Title: Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

    doi: 10.1371/journal.pone.0051241

    Figure Lengend Snippet: Loss of flagella in RN102. Fourty eight-hour-cultured biofilms were collected and analyzed by TEM, and by Western blot for FliC. (A–E) TEM images of the bacterial cells and the cell appendages are shown for strains (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3( hldE ). Flagella found in figures (A), (C), and (E) are shown by arrowheads. Representative electron-microphotographs of each strain are shown. A 1-μm-long bar is shown in the lower left corner. (F) Supernatants were collected from 48-hour bacterial cultures. Result of Western blot using anti-FliC antiserum is shown. Lanes; 1, BW25113; 2, RN102; 3, BW25113/pNTR-SD; 4, RN102/pNTR-SD; 5, RN102/pNT3( hldE ); 6, RN110.

    Article Snippet: In order to quantify the amount of biofilm on a 96-well plate, all stain associated with the attached biofilms was dissolved with 95% ethanol, then OD595 absorbance was measured using a microplate reader (Multiskan RC, ThermoFisher, Waltham, MA).

    Techniques: Cell Culture, Transmission Electron Microscopy, Western Blot

    Anti-biofilm effects of the flavonoids collection when added prior-to ( a ) or post-biofilm formation ( b ). Strains 1 and 2 are S. aureus ATCC 25923 and Newman clinical strains, respectively. Highly active flavonoids are those present in both shadowed areas. Primary screening results are presented in Table S1.

    Journal: International Journal of Molecular Sciences

    Article Title: Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

    doi: 10.3390/ijms141019434

    Figure Lengend Snippet: Anti-biofilm effects of the flavonoids collection when added prior-to ( a ) or post-biofilm formation ( b ). Strains 1 and 2 are S. aureus ATCC 25923 and Newman clinical strains, respectively. Highly active flavonoids are those present in both shadowed areas. Primary screening results are presented in Table S1.

    Article Snippet: After exposing biofilms to the flavonoids (at 37 °C for 18 h), the planktonic phase was transferred to sterile 96-microtiter well plates, and quantitative readouts were obtained (at λ = 620 nm) using the Varioskan Flash Multimode Plate Reader (2.4.3.37 software, Thermo Fisher Scientific Oy, Vantaa, Finland, 2004), as in [ ].

    Techniques:

    Summary of the anti-biofilm screening and selection criteria applied to the collection in this study. Highly and moderately active flavonoids are listed in Tables S2 and S3, respectively.

    Journal: International Journal of Molecular Sciences

    Article Title: Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

    doi: 10.3390/ijms141019434

    Figure Lengend Snippet: Summary of the anti-biofilm screening and selection criteria applied to the collection in this study. Highly and moderately active flavonoids are listed in Tables S2 and S3, respectively.

    Article Snippet: After exposing biofilms to the flavonoids (at 37 °C for 18 h), the planktonic phase was transferred to sterile 96-microtiter well plates, and quantitative readouts were obtained (at λ = 620 nm) using the Varioskan Flash Multimode Plate Reader (2.4.3.37 software, Thermo Fisher Scientific Oy, Vantaa, Finland, 2004), as in [ ].

    Techniques: Selection

    The four most active anti-biofilm flavonoids identified in this contribution.

    Journal: International Journal of Molecular Sciences

    Article Title: Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

    doi: 10.3390/ijms141019434

    Figure Lengend Snippet: The four most active anti-biofilm flavonoids identified in this contribution.

    Article Snippet: After exposing biofilms to the flavonoids (at 37 °C for 18 h), the planktonic phase was transferred to sterile 96-microtiter well plates, and quantitative readouts were obtained (at λ = 620 nm) using the Varioskan Flash Multimode Plate Reader (2.4.3.37 software, Thermo Fisher Scientific Oy, Vantaa, Finland, 2004), as in [ ].

    Techniques:

    Key structural features present in the anti-biofilm chalcones and flavones (moderately and highly active ones).

    Journal: International Journal of Molecular Sciences

    Article Title: Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

    doi: 10.3390/ijms141019434

    Figure Lengend Snippet: Key structural features present in the anti-biofilm chalcones and flavones (moderately and highly active ones).

    Article Snippet: After exposing biofilms to the flavonoids (at 37 °C for 18 h), the planktonic phase was transferred to sterile 96-microtiter well plates, and quantitative readouts were obtained (at λ = 620 nm) using the Varioskan Flash Multimode Plate Reader (2.4.3.37 software, Thermo Fisher Scientific Oy, Vantaa, Finland, 2004), as in [ ].

    Techniques:

    The confocal laser scanning microscope (CLSM) images and relative gene expression of C. albicans in biofilms: ( a ) The CLSM images of C. albicans in multi-species biofilms, C. albicans dyed red, representative hyphae form of C. albicans was marked with white arrows; ( b ) Relative gene expression in C. albicans . Values are significantly different when labelled with different letters ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation

    doi: 10.3390/ijms17071033

    Figure Lengend Snippet: The confocal laser scanning microscope (CLSM) images and relative gene expression of C. albicans in biofilms: ( a ) The CLSM images of C. albicans in multi-species biofilms, C. albicans dyed red, representative hyphae form of C. albicans was marked with white arrows; ( b ) Relative gene expression in C. albicans . Values are significantly different when labelled with different letters ( p

    Article Snippet: 4.3. pH Measurement For pH measurement, the 2 mL supernatant of biofilms was collected and measured by pH meter (Thermo Scientific, Waltham, MA, USA) at 24, 48, and 72 h ( ).

    Techniques: Laser-Scanning Microscopy, Confocal Laser Scanning Microscopy, Expressing

    Biofilm structural and metabolic analyses: ( a ) The 3D reconstruction of 72 h multi-species biofilm in different DMADDM containing groups, live microbes dyed green, dead microbes dyed red, adjacent live and dead microbes were presented as yellow when they were merged; ( b ) The biomass of 72 h multi-species biofilm; ( c ) The thickness of 72 h multi-species biofilms in different DMADDM containing groups; ( d ) The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results of multi-species biofilms formed on different DMADDM containing denture base resin. Values are significantly different when labelled with different letters ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation

    doi: 10.3390/ijms17071033

    Figure Lengend Snippet: Biofilm structural and metabolic analyses: ( a ) The 3D reconstruction of 72 h multi-species biofilm in different DMADDM containing groups, live microbes dyed green, dead microbes dyed red, adjacent live and dead microbes were presented as yellow when they were merged; ( b ) The biomass of 72 h multi-species biofilm; ( c ) The thickness of 72 h multi-species biofilms in different DMADDM containing groups; ( d ) The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) results of multi-species biofilms formed on different DMADDM containing denture base resin. Values are significantly different when labelled with different letters ( p

    Article Snippet: 4.3. pH Measurement For pH measurement, the 2 mL supernatant of biofilms was collected and measured by pH meter (Thermo Scientific, Waltham, MA, USA) at 24, 48, and 72 h ( ).

    Techniques:

    Colony forming unit (CFU) counts of multi-species biofilms: ( a ) The total CFU counts of 72 h multi-species biofilms in different dimethylaminododecyl methacrylate (DMADDM) containing groups; ( b ) The C. albicans CFU counts of 72 h multi-species biofilms in different group. Values are significantly different when labelled with different letters ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation

    doi: 10.3390/ijms17071033

    Figure Lengend Snippet: Colony forming unit (CFU) counts of multi-species biofilms: ( a ) The total CFU counts of 72 h multi-species biofilms in different dimethylaminododecyl methacrylate (DMADDM) containing groups; ( b ) The C. albicans CFU counts of 72 h multi-species biofilms in different group. Values are significantly different when labelled with different letters ( p

    Article Snippet: 4.3. pH Measurement For pH measurement, the 2 mL supernatant of biofilms was collected and measured by pH meter (Thermo Scientific, Waltham, MA, USA) at 24, 48, and 72 h ( ).

    Techniques:

    Cell damage based on lactate dehydrogenase (LDH) activity and relative gene expression of TR-146 cell: ( a ) Cell damage based on LDH activity after co-culture with biofilm formed on different DMADDM containing groups; ( b ) Relative gene expression of TR-146 cell after it was co-cultured with biofilms of different groups. Values are significantly different when labelled with different letters ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Effect of Antimicrobial Denture Base Resin on Multi-Species Biofilm Formation

    doi: 10.3390/ijms17071033

    Figure Lengend Snippet: Cell damage based on lactate dehydrogenase (LDH) activity and relative gene expression of TR-146 cell: ( a ) Cell damage based on LDH activity after co-culture with biofilm formed on different DMADDM containing groups; ( b ) Relative gene expression of TR-146 cell after it was co-cultured with biofilms of different groups. Values are significantly different when labelled with different letters ( p

    Article Snippet: 4.3. pH Measurement For pH measurement, the 2 mL supernatant of biofilms was collected and measured by pH meter (Thermo Scientific, Waltham, MA, USA) at 24, 48, and 72 h ( ).

    Techniques: Activity Assay, Expressing, Co-Culture Assay, Cell Culture