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ibidi biofilms
Quantitative analysis of macrophage migration within 48 h <t>biofilms</t> prepared from C. albicans strains CAI4, pmr1 Δ, and pmr1 Δ+ PMR1 . The chart shows mean relative macrophage velocity + SD relative to macrophage velocity of J774.1 macrophages added in suspension to CAI4 (wild type biofilms). Migration data over a 30 min period are shown. No statistical significance of mean differences was determined using one-way analysis of variance (ANOVA) and Tukey Multiple Analysis Comparison Tests.
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Images

1) Product Images from "Macrophage Migration Is Impaired within Candida albicans Biofilms"

Article Title: Macrophage Migration Is Impaired within Candida albicans Biofilms

Journal: Journal of Fungi

doi: 10.3390/jof3030031

Quantitative analysis of macrophage migration within 48 h biofilms prepared from C. albicans strains CAI4, pmr1 Δ, and pmr1 Δ+ PMR1 . The chart shows mean relative macrophage velocity + SD relative to macrophage velocity of J774.1 macrophages added in suspension to CAI4 (wild type biofilms). Migration data over a 30 min period are shown. No statistical significance of mean differences was determined using one-way analysis of variance (ANOVA) and Tukey Multiple Analysis Comparison Tests.
Figure Legend Snippet: Quantitative analysis of macrophage migration within 48 h biofilms prepared from C. albicans strains CAI4, pmr1 Δ, and pmr1 Δ+ PMR1 . The chart shows mean relative macrophage velocity + SD relative to macrophage velocity of J774.1 macrophages added in suspension to CAI4 (wild type biofilms). Migration data over a 30 min period are shown. No statistical significance of mean differences was determined using one-way analysis of variance (ANOVA) and Tukey Multiple Analysis Comparison Tests.

Techniques Used: Migration

Representative single time point images acquired using Volocity software, showing J774.1 macrophages stained with LysoTracker Red (LTR; red), and C. albicans stained with Calcofluor White (CFW; blue). Macrophages were added in suspension to ( a ) planktonic yeast (40× objective), or ( b ) and ( c ) 48 h biofilms (20× objective); presented with ( a ) and ( b ) opacity 3D rendering, and ( c ) x , y and z at 20 μm depth. White grid unit or bar represents ( a ) 18.05 μm, ( b ) 36.17 μm, and ( c ) 40 μm.
Figure Legend Snippet: Representative single time point images acquired using Volocity software, showing J774.1 macrophages stained with LysoTracker Red (LTR; red), and C. albicans stained with Calcofluor White (CFW; blue). Macrophages were added in suspension to ( a ) planktonic yeast (40× objective), or ( b ) and ( c ) 48 h biofilms (20× objective); presented with ( a ) and ( b ) opacity 3D rendering, and ( c ) x , y and z at 20 μm depth. White grid unit or bar represents ( a ) 18.05 μm, ( b ) 36.17 μm, and ( c ) 40 μm.

Techniques Used: Software, Staining

Quantitative analysis of macrophage migration in the presence of different wildtype C. albicans . This chart shows mean relative macrophage velocity + SD, relative to macrophage velocity of pre-adhered J774.1 (Adh-macs) responding to planktonic yeast. Relative velocities of J774.1 macrophages added in suspension (Susp-macs) to either yeast or a 48 h biofilm over a 30 min period are shown. Statistical significance was evaluated using one-way analysis of variance (ANOVA) and Tukey Multiple Analysis Comparison Tests. ** p
Figure Legend Snippet: Quantitative analysis of macrophage migration in the presence of different wildtype C. albicans . This chart shows mean relative macrophage velocity + SD, relative to macrophage velocity of pre-adhered J774.1 (Adh-macs) responding to planktonic yeast. Relative velocities of J774.1 macrophages added in suspension (Susp-macs) to either yeast or a 48 h biofilm over a 30 min period are shown. Statistical significance was evaluated using one-way analysis of variance (ANOVA) and Tukey Multiple Analysis Comparison Tests. ** p

Techniques Used: Migration, Magnetic Cell Separation

2) Product Images from "Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase"

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Journal: Viruses

doi: 10.3390/v10080438

Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Flow Cytometry, Staining

Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.
Figure Legend Snippet: Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.

Techniques Used: Staining, Confocal Laser Scanning Microscopy

Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Staining, Concentration Assay, Flow Cytometry

Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.
Figure Legend Snippet: Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.

Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Staining

Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.
Figure Legend Snippet: Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.

Techniques Used: Activity Assay, Staining, Concentration Assay

3) Product Images from "Phenotypical Characteristics of the Black Yeast Exophiala dermatitidis Are Affected by Pseudomonas aeruginosa in an Artificial Sputum Medium Mimicking Cystic Fibrosis–Like Conditions"

Article Title: Phenotypical Characteristics of the Black Yeast Exophiala dermatitidis Are Affected by Pseudomonas aeruginosa in an Artificial Sputum Medium Mimicking Cystic Fibrosis–Like Conditions

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.00471

Exophiala dermatitidis (Ed) biofilm formed after 24 or 48 h at 36°C in artificial sputum medium (ASM). (A) Biofilms formed in mono- (Ed pure) and co-cultures with Pseudomonas aeruginosa (Pa). Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms in Transwell permeable support. Ed suspension for biofilm formation was placed into the well, whereas Pa was placed in the insert. As a control, pure ASM was used. Unpaired t -test, * P
Figure Legend Snippet: Exophiala dermatitidis (Ed) biofilm formed after 24 or 48 h at 36°C in artificial sputum medium (ASM). (A) Biofilms formed in mono- (Ed pure) and co-cultures with Pseudomonas aeruginosa (Pa). Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms in Transwell permeable support. Ed suspension for biofilm formation was placed into the well, whereas Pa was placed in the insert. As a control, pure ASM was used. Unpaired t -test, * P

Techniques Used:

Survival of Galleria mellonella after infection with Exophiala dermatitidis (Ed; Strain P2) with or without Pseudomonas aeruginosa (Pa) culture filtrate of strain PA07 (A/B), PA14 wild-type (wt; C/D), PA14 ΔlasR (E/F), and PA14 ΔrhlR (G/H). As a control, sterile phosphate-buffered saline (PBS) was used. (A, C, E, G) Planktonic culture filtrate was added to injection suspension (S). (B, D, F, H) Biofilm culture filtrate (BFS) 24 or 48 h old was added to the injection suspension. N = 30. Log-rank (Mantel-Cox) test for statistical significance: * P
Figure Legend Snippet: Survival of Galleria mellonella after infection with Exophiala dermatitidis (Ed; Strain P2) with or without Pseudomonas aeruginosa (Pa) culture filtrate of strain PA07 (A/B), PA14 wild-type (wt; C/D), PA14 ΔlasR (E/F), and PA14 ΔrhlR (G/H). As a control, sterile phosphate-buffered saline (PBS) was used. (A, C, E, G) Planktonic culture filtrate was added to injection suspension (S). (B, D, F, H) Biofilm culture filtrate (BFS) 24 or 48 h old was added to the injection suspension. N = 30. Log-rank (Mantel-Cox) test for statistical significance: * P

Techniques Used: Infection, Injection

Extracellular matrix (ECM) thickness (μm) of Exophiala dermatitidis mono- (Ed pure) and co-culture biofilms with Pseudomonas aeruginosa (Pa). N = 10.
Figure Legend Snippet: Extracellular matrix (ECM) thickness (μm) of Exophiala dermatitidis mono- (Ed pure) and co-culture biofilms with Pseudomonas aeruginosa (Pa). N = 10.

Techniques Used: Co-Culture Assay

Exophiala dermatitidis (Ed) biofilm formation after 48 h at 36°C with culture filtrates of planktonic (plankt) or biofilm (BF) culture from Pseudomonas aeruginosa (Pa). Also, Ed culture filtrate was added. As a control, biofilm formation in pure ASM was assessed. N = 3.
Figure Legend Snippet: Exophiala dermatitidis (Ed) biofilm formation after 48 h at 36°C with culture filtrates of planktonic (plankt) or biofilm (BF) culture from Pseudomonas aeruginosa (Pa). Also, Ed culture filtrate was added. As a control, biofilm formation in pure ASM was assessed. N = 3.

Techniques Used:

Exophiala dermatitidis (Ed) biofilms formed after 24 h (gray) or 48 h (black) at 36°C in artificial sputum medium (ASM). (A) Biofilms in mono- (Ed pure) and co-culture with Pseudomonas aeruginosa (Pa) wild-type (WT) strains PA07 and PA14, as well as two quorum-sensing (QS) mutants lacking LasR and RhlR. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms of Exophiala dermatitidis (Ed, P2) and P. aeruginosa (Pa, PA14 WT) after 24 h of incubation at 35°C in ASM treated with N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) in a concentration gradient from 0 to 100 μM in DMSO. Biomass in biofilm was measured by staining with 0.1% crystal violet. For correction of biomass in biofilm, difference between the DMSO and the DMSO plus 3-oxo C12 HSL treated biofilms has been estimated and subtracted from the positive control. OD, optical density. Unpaired t -test, * P > 0.05; ** P
Figure Legend Snippet: Exophiala dermatitidis (Ed) biofilms formed after 24 h (gray) or 48 h (black) at 36°C in artificial sputum medium (ASM). (A) Biofilms in mono- (Ed pure) and co-culture with Pseudomonas aeruginosa (Pa) wild-type (WT) strains PA07 and PA14, as well as two quorum-sensing (QS) mutants lacking LasR and RhlR. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms of Exophiala dermatitidis (Ed, P2) and P. aeruginosa (Pa, PA14 WT) after 24 h of incubation at 35°C in ASM treated with N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) in a concentration gradient from 0 to 100 μM in DMSO. Biomass in biofilm was measured by staining with 0.1% crystal violet. For correction of biomass in biofilm, difference between the DMSO and the DMSO plus 3-oxo C12 HSL treated biofilms has been estimated and subtracted from the positive control. OD, optical density. Unpaired t -test, * P > 0.05; ** P

Techniques Used: Co-Culture Assay, Incubation, Concentration Assay, Staining, Positive Control

(A) Pseudomonas aeruginosa (Pa) and Exophiala dermatitidis (Ed) biofilm formation in monocultures (pure) after 48 h of biofilm formation at 35°C under aerobic (black) or anaerobic (gray) conditions. Biofilms were quantified after biofilm detachment with 0.1% dithiothreitol. (B) Ed biofilm formed after 48 h at 36°C in ASM under hypoxic conditions in monoculture (ED pure) or co-culture with Pa. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). Paired t -test, * P
Figure Legend Snippet: (A) Pseudomonas aeruginosa (Pa) and Exophiala dermatitidis (Ed) biofilm formation in monocultures (pure) after 48 h of biofilm formation at 35°C under aerobic (black) or anaerobic (gray) conditions. Biofilms were quantified after biofilm detachment with 0.1% dithiothreitol. (B) Ed biofilm formed after 48 h at 36°C in ASM under hypoxic conditions in monoculture (ED pure) or co-culture with Pa. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). Paired t -test, * P

Techniques Used: Co-Culture Assay

4) Product Images from "Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella"

Article Title: Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella

Journal: mBio

doi: 10.1128/mBio.01973-18

Effect of enzymatic treatments on preformed biofilms. H. pylori SS1 was allowed to form biofilms for 3 days in BB2. The medium was then removed and replaced with either fresh medium or medium containing DNase I or proteinase K. Cells were reincubated for 24 h and then analyzed for the remaining biofilm using the crystal violet assay. The data shown here represent the percentage of remaining biofilm compared to the untreated control. Experiments were performed three times independently with at least 8 technical replicates for each. Statistical analysis was performed using ANOVA (*, P
Figure Legend Snippet: Effect of enzymatic treatments on preformed biofilms. H. pylori SS1 was allowed to form biofilms for 3 days in BB2. The medium was then removed and replaced with either fresh medium or medium containing DNase I or proteinase K. Cells were reincubated for 24 h and then analyzed for the remaining biofilm using the crystal violet assay. The data shown here represent the percentage of remaining biofilm compared to the untreated control. Experiments were performed three times independently with at least 8 technical replicates for each. Statistical analysis was performed using ANOVA (*, P

Techniques Used: Crystal Violet Assay

Confocal scanning laser microscopy (CSLM) images of H. pylori SS1 biofilm. Shown are representative CSLM images of 3-day-old SS1 biofilms grown in BB2 and stained with (A) FM 1–43 to stain total bacterial cells, (B) SYPRO RUBY to stain extracellular proteins, (C) BOBO-3 to stain extracellular DNA, and (D to F) live-dead staining with live cells represented by the green fluorescent SYTO 9 and dead/damaged cells represented by the red fluorescent propidium. Scale bar = 30 µm.
Figure Legend Snippet: Confocal scanning laser microscopy (CSLM) images of H. pylori SS1 biofilm. Shown are representative CSLM images of 3-day-old SS1 biofilms grown in BB2 and stained with (A) FM 1–43 to stain total bacterial cells, (B) SYPRO RUBY to stain extracellular proteins, (C) BOBO-3 to stain extracellular DNA, and (D to F) live-dead staining with live cells represented by the green fluorescent SYTO 9 and dead/damaged cells represented by the red fluorescent propidium. Scale bar = 30 µm.

Techniques Used: Microscopy, Staining

Flagella play integral roles in H. pylori biofilms. (A) Scanning electron microscope (SEM) images of biofilms formed by H. pylori wild-type SS1 (SS1 WT), the isogenic nonmotile but flagellated Δ motB mutant (SS1 Δ motB ), and the isogenic aflagellated Δ fliM mutant (SS1 Δ fliM ). Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori SS1 wild type and Δ motB and Δ fliM mutants. Strains were grown in BB2 medium for 3 days, followed by biofilm evaluation using the crystal violet assay. Experiments were performed three times independently with 6 to 9 technical replicates for each. Statistical analysis was performed using ANOVA (**, P
Figure Legend Snippet: Flagella play integral roles in H. pylori biofilms. (A) Scanning electron microscope (SEM) images of biofilms formed by H. pylori wild-type SS1 (SS1 WT), the isogenic nonmotile but flagellated Δ motB mutant (SS1 Δ motB ), and the isogenic aflagellated Δ fliM mutant (SS1 Δ fliM ). Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori SS1 wild type and Δ motB and Δ fliM mutants. Strains were grown in BB2 medium for 3 days, followed by biofilm evaluation using the crystal violet assay. Experiments were performed three times independently with 6 to 9 technical replicates for each. Statistical analysis was performed using ANOVA (**, P

Techniques Used: Microscopy, Mutagenesis, Crystal Violet Assay

Functional classification of genes differentially expressed in H. pylori SS1 biofilm. Black and gray bars represent upregulated and downregulated genes, respectively, that were significantly differentially expressed ( P
Figure Legend Snippet: Functional classification of genes differentially expressed in H. pylori SS1 biofilm. Black and gray bars represent upregulated and downregulated genes, respectively, that were significantly differentially expressed ( P

Techniques Used: Functional Assay

H. pylori G27 biofilm contains structurally important flagella. (A) Scanning electron microscope (SEM) images of wild-type G27 H. pylori biofilms. Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori G27 wild type (WT), the nonmotile flagellated motB mutant, the nonmotile fliA mutant that is reported to have either truncated flagella or no flagella, and the aflagellated and nonmotile flgS mutant. Biofilms were evaluated using the crystal violet assay. Experiments were performed 2 times independently with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (**, P
Figure Legend Snippet: H. pylori G27 biofilm contains structurally important flagella. (A) Scanning electron microscope (SEM) images of wild-type G27 H. pylori biofilms. Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori G27 wild type (WT), the nonmotile flagellated motB mutant, the nonmotile fliA mutant that is reported to have either truncated flagella or no flagella, and the aflagellated and nonmotile flgS mutant. Biofilms were evaluated using the crystal violet assay. Experiments were performed 2 times independently with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (**, P

Techniques Used: Microscopy, Mutagenesis, Crystal Violet Assay

qPCR validation of the transcription of selected differentially expressed genes. The data indicate the fold change in expression of genes in H. pylori biofilm cells compared to planktonic cells. Fold changes in gene expressions were calculated after normalization of each gene with the constitutively expressed gene control gapB . Bars represent the mean and error bars the standard error of the mean. Black and gray bars represent qPCR and RNA-seq results, respectively. Statistical analyses were performed using threshold cycle (2 −ΔΔ CT ) values, and all results with an asterisk were statistically significant ( P
Figure Legend Snippet: qPCR validation of the transcription of selected differentially expressed genes. The data indicate the fold change in expression of genes in H. pylori biofilm cells compared to planktonic cells. Fold changes in gene expressions were calculated after normalization of each gene with the constitutively expressed gene control gapB . Bars represent the mean and error bars the standard error of the mean. Black and gray bars represent qPCR and RNA-seq results, respectively. Statistical analyses were performed using threshold cycle (2 −ΔΔ CT ) values, and all results with an asterisk were statistically significant ( P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, RNA Sequencing Assay

H. pylori SS1 forms robust biofilms after 3 days of growth in BB2. H. pylori strain SS1 was grown in the indicated media, and biofilm formation was assessed by crystal violet absorbance at 595 nm. (A) H. pylori SS1 was grown for 3 days in BB media supplemented with different concentrations of FBS (BB10, 10%; BB6, 6%; and BB2, 2%). (B) H. pylori SS1 was grown for 3 days in BB media or Ham’s F-12 supplemented with 10% (HAMS10) or 2% (HAMS2) FBS. (C) H. pylori SS1 was grown in BB medium supplemented with 2% FBS, and biofilm formation was evaluated at different time points. Experiments were performed three independent times with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (*, P
Figure Legend Snippet: H. pylori SS1 forms robust biofilms after 3 days of growth in BB2. H. pylori strain SS1 was grown in the indicated media, and biofilm formation was assessed by crystal violet absorbance at 595 nm. (A) H. pylori SS1 was grown for 3 days in BB media supplemented with different concentrations of FBS (BB10, 10%; BB6, 6%; and BB2, 2%). (B) H. pylori SS1 was grown for 3 days in BB media or Ham’s F-12 supplemented with 10% (HAMS10) or 2% (HAMS2) FBS. (C) H. pylori SS1 was grown in BB medium supplemented with 2% FBS, and biofilm formation was evaluated at different time points. Experiments were performed three independent times with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (*, P

Techniques Used:

Biofilm-grown cells and planktonic cells show distinct transcriptional profiles. (A) Principal-component analysis (PCA) of gene expression obtained by RNA-seq between biofilm ( n = 3) and planktonic ( n = 3) populations. (B) Volcano plot of gene expression data. The y axis is the negative log 10 of P values (a higher value indicates greater significance), and the x axis is the log 2 fold change in difference in abundance between two population (positive values represent the upregulated genes in biofilm, and negative values represent downregulated genes). The dashed red line shows where P = 0.01, with points above the line having a P value of
Figure Legend Snippet: Biofilm-grown cells and planktonic cells show distinct transcriptional profiles. (A) Principal-component analysis (PCA) of gene expression obtained by RNA-seq between biofilm ( n = 3) and planktonic ( n = 3) populations. (B) Volcano plot of gene expression data. The y axis is the negative log 10 of P values (a higher value indicates greater significance), and the x axis is the log 2 fold change in difference in abundance between two population (positive values represent the upregulated genes in biofilm, and negative values represent downregulated genes). The dashed red line shows where P = 0.01, with points above the line having a P value of

Techniques Used: Expressing, RNA Sequencing Assay

5) Product Images from "Phenotypical Characteristics of the Black Yeast Exophiala dermatitidis Are Affected by Pseudomonas aeruginosa in an Artificial Sputum Medium Mimicking Cystic Fibrosis–Like Conditions"

Article Title: Phenotypical Characteristics of the Black Yeast Exophiala dermatitidis Are Affected by Pseudomonas aeruginosa in an Artificial Sputum Medium Mimicking Cystic Fibrosis–Like Conditions

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.00471

Exophiala dermatitidis (Ed) biofilm formed after 24 or 48 h at 36°C in artificial sputum medium (ASM). (A) Biofilms formed in mono- (Ed pure) and co-cultures with Pseudomonas aeruginosa (Pa). Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms in Transwell permeable support. Ed suspension for biofilm formation was placed into the well, whereas Pa was placed in the insert. As a control, pure ASM was used. Unpaired t -test, * P
Figure Legend Snippet: Exophiala dermatitidis (Ed) biofilm formed after 24 or 48 h at 36°C in artificial sputum medium (ASM). (A) Biofilms formed in mono- (Ed pure) and co-cultures with Pseudomonas aeruginosa (Pa). Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms in Transwell permeable support. Ed suspension for biofilm formation was placed into the well, whereas Pa was placed in the insert. As a control, pure ASM was used. Unpaired t -test, * P

Techniques Used:

Survival of Galleria mellonella after infection with Exophiala dermatitidis (Ed; Strain P2) with or without Pseudomonas aeruginosa (Pa) culture filtrate of strain PA07 (A/B), PA14 wild-type (wt; C/D), PA14 ΔlasR (E/F), and PA14 ΔrhlR (G/H). As a control, sterile phosphate-buffered saline (PBS) was used. (A, C, E, G) Planktonic culture filtrate was added to injection suspension (S). (B, D, F, H) Biofilm culture filtrate (BFS) 24 or 48 h old was added to the injection suspension. N = 30. Log-rank (Mantel-Cox) test for statistical significance: * P
Figure Legend Snippet: Survival of Galleria mellonella after infection with Exophiala dermatitidis (Ed; Strain P2) with or without Pseudomonas aeruginosa (Pa) culture filtrate of strain PA07 (A/B), PA14 wild-type (wt; C/D), PA14 ΔlasR (E/F), and PA14 ΔrhlR (G/H). As a control, sterile phosphate-buffered saline (PBS) was used. (A, C, E, G) Planktonic culture filtrate was added to injection suspension (S). (B, D, F, H) Biofilm culture filtrate (BFS) 24 or 48 h old was added to the injection suspension. N = 30. Log-rank (Mantel-Cox) test for statistical significance: * P

Techniques Used: Infection, Injection

Extracellular matrix (ECM) thickness (μm) of Exophiala dermatitidis mono- (Ed pure) and co-culture biofilms with Pseudomonas aeruginosa (Pa). N = 10.
Figure Legend Snippet: Extracellular matrix (ECM) thickness (μm) of Exophiala dermatitidis mono- (Ed pure) and co-culture biofilms with Pseudomonas aeruginosa (Pa). N = 10.

Techniques Used: Co-Culture Assay

Exophiala dermatitidis (Ed) biofilm formation after 48 h at 36°C with culture filtrates of planktonic (plankt) or biofilm (BF) culture from Pseudomonas aeruginosa (Pa). Also, Ed culture filtrate was added. As a control, biofilm formation in pure ASM was assessed. N = 3.
Figure Legend Snippet: Exophiala dermatitidis (Ed) biofilm formation after 48 h at 36°C with culture filtrates of planktonic (plankt) or biofilm (BF) culture from Pseudomonas aeruginosa (Pa). Also, Ed culture filtrate was added. As a control, biofilm formation in pure ASM was assessed. N = 3.

Techniques Used:

Exophiala dermatitidis (Ed) biofilms formed after 24 h (gray) or 48 h (black) at 36°C in artificial sputum medium (ASM). (A) Biofilms in mono- (Ed pure) and co-culture with Pseudomonas aeruginosa (Pa) wild-type (WT) strains PA07 and PA14, as well as two quorum-sensing (QS) mutants lacking LasR and RhlR. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms of Exophiala dermatitidis (Ed, P2) and P. aeruginosa (Pa, PA14 WT) after 24 h of incubation at 35°C in ASM treated with N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) in a concentration gradient from 0 to 100 μM in DMSO. Biomass in biofilm was measured by staining with 0.1% crystal violet. For correction of biomass in biofilm, difference between the DMSO and the DMSO plus 3-oxo C12 HSL treated biofilms has been estimated and subtracted from the positive control. OD, optical density. Unpaired t -test, * P > 0.05; ** P
Figure Legend Snippet: Exophiala dermatitidis (Ed) biofilms formed after 24 h (gray) or 48 h (black) at 36°C in artificial sputum medium (ASM). (A) Biofilms in mono- (Ed pure) and co-culture with Pseudomonas aeruginosa (Pa) wild-type (WT) strains PA07 and PA14, as well as two quorum-sensing (QS) mutants lacking LasR and RhlR. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). (B) Biofilms of Exophiala dermatitidis (Ed, P2) and P. aeruginosa (Pa, PA14 WT) after 24 h of incubation at 35°C in ASM treated with N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) in a concentration gradient from 0 to 100 μM in DMSO. Biomass in biofilm was measured by staining with 0.1% crystal violet. For correction of biomass in biofilm, difference between the DMSO and the DMSO plus 3-oxo C12 HSL treated biofilms has been estimated and subtracted from the positive control. OD, optical density. Unpaired t -test, * P > 0.05; ** P

Techniques Used: Co-Culture Assay, Incubation, Concentration Assay, Staining, Positive Control

(A) Pseudomonas aeruginosa (Pa) and Exophiala dermatitidis (Ed) biofilm formation in monocultures (pure) after 48 h of biofilm formation at 35°C under aerobic (black) or anaerobic (gray) conditions. Biofilms were quantified after biofilm detachment with 0.1% dithiothreitol. (B) Ed biofilm formed after 48 h at 36°C in ASM under hypoxic conditions in monoculture (ED pure) or co-culture with Pa. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). Paired t -test, * P
Figure Legend Snippet: (A) Pseudomonas aeruginosa (Pa) and Exophiala dermatitidis (Ed) biofilm formation in monocultures (pure) after 48 h of biofilm formation at 35°C under aerobic (black) or anaerobic (gray) conditions. Biofilms were quantified after biofilm detachment with 0.1% dithiothreitol. (B) Ed biofilm formed after 48 h at 36°C in ASM under hypoxic conditions in monoculture (ED pure) or co-culture with Pa. Biofilms were estimated by detachment of biofilm with 0.1% dithiothreitol and subsequent count of colony-forming units (CFU per mL). Paired t -test, * P

Techniques Used: Co-Culture Assay

6) Product Images from "Surface-associated MUC5B mucins promote protease activity in Lactobacillus fermentum biofilms"

Article Title: Surface-associated MUC5B mucins promote protease activity in Lactobacillus fermentum biofilms

Journal: BMC Oral Health

doi: 10.1186/1472-6831-13-43

Surface proteins identified in biofilm cells of L. fermentum. (a) Western blotting of two-dimensional electrophoresis (2DE) of surface proteins. Samples were subjected to IEF in a pH 4–7 gradient followed by SDS-PAGE in 14% gels. After electroblotting onto PVDF membranes, gpr, O-sialoglycoprotein endopeptidase was detected using the polyclonal antiserum MOB-LFGE-2. (b) Gel stained with Coomassie Brilliant Blue after 2DE of surface proteins. Spots were excised from the stained gel, subjected to in-gel tryptic digestion and proteins identified by LC-MS/MS analysis. The proteins identified were: arg , arginine deiminase; ATPase , ATP synthase pyruvate dehydrogenase; DnaK ; Ef-G , elongation factor G; Ef-Tu , elongation factor Tu; fum , fumarate hydratase; GroEl ; ldh , lactate dehydrogenase; ket , phosphoketolase; orn , ornithine carbamoyltransferase; pdh , pyruvate dehydrogenase and pph , phophopyruvate hydratase.
Figure Legend Snippet: Surface proteins identified in biofilm cells of L. fermentum. (a) Western blotting of two-dimensional electrophoresis (2DE) of surface proteins. Samples were subjected to IEF in a pH 4–7 gradient followed by SDS-PAGE in 14% gels. After electroblotting onto PVDF membranes, gpr, O-sialoglycoprotein endopeptidase was detected using the polyclonal antiserum MOB-LFGE-2. (b) Gel stained with Coomassie Brilliant Blue after 2DE of surface proteins. Spots were excised from the stained gel, subjected to in-gel tryptic digestion and proteins identified by LC-MS/MS analysis. The proteins identified were: arg , arginine deiminase; ATPase , ATP synthase pyruvate dehydrogenase; DnaK ; Ef-G , elongation factor G; Ef-Tu , elongation factor Tu; fum , fumarate hydratase; GroEl ; ldh , lactate dehydrogenase; ket , phosphoketolase; orn , ornithine carbamoyltransferase; pdh , pyruvate dehydrogenase and pph , phophopyruvate hydratase.

Techniques Used: Western Blot, Electrophoresis, Two-Dimensional Gel Electrophoresis, Electrofocusing, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Proteolytic activities of Lactobacillus fermentum biofilm populations in the presence or absence of MUC5B mucins. CSLM images showing biofilm cells growing in (a) the presence of a MUC5B environment or (b) a nutrient broth environment. Proteolytic activity was visualized by incubation with a FITC-conjugated casein substrate (green) for 1 hour. Cells were then counterstained using Syto 24 (red). The bar represents 20 μm and the inserts show four-fold enlargements of the main micrographs.
Figure Legend Snippet: Proteolytic activities of Lactobacillus fermentum biofilm populations in the presence or absence of MUC5B mucins. CSLM images showing biofilm cells growing in (a) the presence of a MUC5B environment or (b) a nutrient broth environment. Proteolytic activity was visualized by incubation with a FITC-conjugated casein substrate (green) for 1 hour. Cells were then counterstained using Syto 24 (red). The bar represents 20 μm and the inserts show four-fold enlargements of the main micrographs.

Techniques Used: Activity Assay, Incubation

Proteolytic activities of Lactobacillus fermentum biofilm populations in the presence of (a) surface-associated or (b) fluid-phase MUC5B mucins. CSLM images showing biofilm cells growing in (a) the presence of a surface-coat of MUC5B mucins or (b) in the presence of MUC5B mucins in the fluid phase. Proteolytic activity was visualized by incubation with a FITC-conjugated casein substrate (green) for 1 hour. Cells were then counterstained using Syto 24 (red). The bar represents 20 μm and the inserts show four-fold enlargements of the main micrographs.
Figure Legend Snippet: Proteolytic activities of Lactobacillus fermentum biofilm populations in the presence of (a) surface-associated or (b) fluid-phase MUC5B mucins. CSLM images showing biofilm cells growing in (a) the presence of a surface-coat of MUC5B mucins or (b) in the presence of MUC5B mucins in the fluid phase. Proteolytic activity was visualized by incubation with a FITC-conjugated casein substrate (green) for 1 hour. Cells were then counterstained using Syto 24 (red). The bar represents 20 μm and the inserts show four-fold enlargements of the main micrographs.

Techniques Used: Activity Assay, Incubation

Degradation of the MUC5B polypeptide backbone by proteases from biofilms of L. fermentum. A control sample of MUC5B (a) and MUC5B which had been in contact with biofilm cells for 24 hours (b) were subjected to SDS-PAGE on 4–12% gels. After electro-blotting onto PVDF membranes, MUC5B was detected using the LUM5B-14 antiserum. The bottom of the well is indicated.
Figure Legend Snippet: Degradation of the MUC5B polypeptide backbone by proteases from biofilms of L. fermentum. A control sample of MUC5B (a) and MUC5B which had been in contact with biofilm cells for 24 hours (b) were subjected to SDS-PAGE on 4–12% gels. After electro-blotting onto PVDF membranes, MUC5B was detected using the LUM5B-14 antiserum. The bottom of the well is indicated.

Techniques Used: SDS Page, Western Blot

Proportion of proteolytically-active Lactobacillus fermentum cells in planktonic and biofilm culture. Graphs showing the percentage of proteolytically active cells in populations of (a) planktonic or (b) biofilm cells of L. fermentum in a MUC5B- or a nutrient broth-environment as revealed using a fluorescent substrate combined with CSLM. The bars represent the mean value of three independent experiments ± SE (** p
Figure Legend Snippet: Proportion of proteolytically-active Lactobacillus fermentum cells in planktonic and biofilm culture. Graphs showing the percentage of proteolytically active cells in populations of (a) planktonic or (b) biofilm cells of L. fermentum in a MUC5B- or a nutrient broth-environment as revealed using a fluorescent substrate combined with CSLM. The bars represent the mean value of three independent experiments ± SE (** p

Techniques Used:

7) Product Images from "Essential Roles of the sppRA Fructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression by Streptococcus mutans"

Article Title: Essential Roles of the sppRA Fructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression by Streptococcus mutans

Journal: Journal of Bacteriology

doi: 10.1128/JB.00586-18

Fructose metabolism alters biofilm development by S. mutans . BM containing 2 mM sucrose and 18 mM glucose (BMGS) or 18 mM fructose (BMFS) was used for biofilm development. (A) Wild-type UA159 and its isogenic levD and fruI mutants were each incubated for 24 h, and the amounts of biofilms formed were visualized and quantified by crystal violet (C.V.) staining. (B) Biofilms formed by UA159 were treated with a LIVE/DEAD vitality stain and visualized under a confocal laser scanning microscope, with green indicating live cells and red indicating dead cells. (C) eDNA released from 48-h biofilms formed by strains UA159/pIB184 and UA159/pIB508 were quantified using a DNA dye (Sytox green) and a standard curve. (D) The results were normalized using total CFU from each sample. Error bars represent standard deviations. Statistical analysis was performed using Student’s t test, with asterisks denoting P values: *,
Figure Legend Snippet: Fructose metabolism alters biofilm development by S. mutans . BM containing 2 mM sucrose and 18 mM glucose (BMGS) or 18 mM fructose (BMFS) was used for biofilm development. (A) Wild-type UA159 and its isogenic levD and fruI mutants were each incubated for 24 h, and the amounts of biofilms formed were visualized and quantified by crystal violet (C.V.) staining. (B) Biofilms formed by UA159 were treated with a LIVE/DEAD vitality stain and visualized under a confocal laser scanning microscope, with green indicating live cells and red indicating dead cells. (C) eDNA released from 48-h biofilms formed by strains UA159/pIB184 and UA159/pIB508 were quantified using a DNA dye (Sytox green) and a standard curve. (D) The results were normalized using total CFU from each sample. Error bars represent standard deviations. Statistical analysis was performed using Student’s t test, with asterisks denoting P values: *,

Techniques Used: Incubation, Staining, Laser-Scanning Microscopy

8) Product Images from "The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection"

Article Title: The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00138

KEGG pathway analysis of genes downregulated in Streptococcus pneumoniae D39Δ luxS biofilms compared with the D39 wild-type biofilms.
Figure Legend Snippet: KEGG pathway analysis of genes downregulated in Streptococcus pneumoniae D39Δ luxS biofilms compared with the D39 wild-type biofilms.

Techniques Used:

In vitro biofilm growth of Streptococcus pneumoniae D39 wild-type and D39Δ luxS strains at different time-points after inoculation (6, 12, 18, and 24 h). The error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p
Figure Legend Snippet: In vitro biofilm growth of Streptococcus pneumoniae D39 wild-type and D39Δ luxS strains at different time-points after inoculation (6, 12, 18, and 24 h). The error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p

Techniques Used: In Vitro, Standard Deviation

Scanning electron microscopy (SEM) images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A–C) are representative SEM images of the D39 wild-type strain. The wild-type strain biofilms were thick and organized with a significant depth. (D–F) are SEM images of the D39Δ luxS strain. The D39Δ luxS biofilms were thin and disorganized, and extracellular polymeric substance (EPS) was absent.
Figure Legend Snippet: Scanning electron microscopy (SEM) images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A–C) are representative SEM images of the D39 wild-type strain. The wild-type strain biofilms were thick and organized with a significant depth. (D–F) are SEM images of the D39Δ luxS strain. The D39Δ luxS biofilms were thin and disorganized, and extracellular polymeric substance (EPS) was absent.

Techniques Used: Electron Microscopy, In Vitro

Confocal microscopy images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A) Confocal microscopy image of the D39 wild-type strain biofilm. (B) Confocal microscopy image of the D39Δ luxS strain biofilm.
Figure Legend Snippet: Confocal microscopy images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A) Confocal microscopy image of the D39 wild-type strain biofilm. (B) Confocal microscopy image of the D39Δ luxS strain biofilm.

Techniques Used: Confocal Microscopy, In Vitro

Scanning electron microscopy (SEM) images of rat bullae inoculated with Streptococcus pneumoniae D39 wild-type and D39Δ luxS . (A–C) are representative SEM images of rat bullae inoculated with the D39 wild-type strain. In rats colonized with the wild-type strain, dense biofilm/cell debris was deposited on the cilia, and the cilia were coagulated and completely covered with biofilm debris. (D–F) are representative SEM images of rat bullae inoculated with the D39Δ luxS strain. In rat bulla colonized with the D39Δ luxS strain, less biofilm debris was visible, although the cilia were coagulated. (G–I) are representative SEM images of rat bullae inoculated with medium (vehicle control). The vehicle control rat bulla were clean.
Figure Legend Snippet: Scanning electron microscopy (SEM) images of rat bullae inoculated with Streptococcus pneumoniae D39 wild-type and D39Δ luxS . (A–C) are representative SEM images of rat bullae inoculated with the D39 wild-type strain. In rats colonized with the wild-type strain, dense biofilm/cell debris was deposited on the cilia, and the cilia were coagulated and completely covered with biofilm debris. (D–F) are representative SEM images of rat bullae inoculated with the D39Δ luxS strain. In rat bulla colonized with the D39Δ luxS strain, less biofilm debris was visible, although the cilia were coagulated. (G–I) are representative SEM images of rat bullae inoculated with medium (vehicle control). The vehicle control rat bulla were clean.

Techniques Used: Electron Microscopy

Streptococcus pneumoniae D39 wild-type and D39Δ luxS planktonic and biofilm growth. (A) Planktonic growth optical density at 600 nm. (B) Quantification of biomass of in vitro biofilms grown for 18 h, using a CV-microplate assay. (C) Colony-forming unit (CFU) counts of in vitro biofilms grown for 18 h. Error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p
Figure Legend Snippet: Streptococcus pneumoniae D39 wild-type and D39Δ luxS planktonic and biofilm growth. (A) Planktonic growth optical density at 600 nm. (B) Quantification of biomass of in vitro biofilms grown for 18 h, using a CV-microplate assay. (C) Colony-forming unit (CFU) counts of in vitro biofilms grown for 18 h. Error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p

Techniques Used: In Vitro, Standard Deviation

9) Product Images from "Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation"

Article Title: Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation

Journal: Journal of Bacteriology

doi: 10.1128/JB.00171-15

Gene transfer efficiency throughout biofilm development. Fraction of transformants (wt) as a function of time in a developing biofilm. recA , recA -inducible strain in the absence of induction. White, planktonic growth; gray, biofilm. n ≥ 3 for each condition; error bars indicate standard deviations.
Figure Legend Snippet: Gene transfer efficiency throughout biofilm development. Fraction of transformants (wt) as a function of time in a developing biofilm. recA , recA -inducible strain in the absence of induction. White, planktonic growth; gray, biofilm. n ≥ 3 for each condition; error bars indicate standard deviations.

Techniques Used:

Distribution of free DNA in gonococcal biofilms and DNase treatment. (a) Typical confocal plane after 3 h, 6 h, 24 h, and 46 h of growth on flat surface after staining with propidium iodide. (b) Average projection of typical confocal stack after 46 h of growth. Green, bacteria; red, extracellular DNA. Scale bars, 10 μm. (c) Fraction of transformants under continuous treatment with DNase between 0 and 5 h and between 5 and 22 h compared to untreated biofilm. DNase was applied at a concentration of 10 U/ml. n ≥ 3 for each condition; error bars indicate standard deviations.
Figure Legend Snippet: Distribution of free DNA in gonococcal biofilms and DNase treatment. (a) Typical confocal plane after 3 h, 6 h, 24 h, and 46 h of growth on flat surface after staining with propidium iodide. (b) Average projection of typical confocal stack after 46 h of growth. Green, bacteria; red, extracellular DNA. Scale bars, 10 μm. (c) Fraction of transformants under continuous treatment with DNase between 0 and 5 h and between 5 and 22 h compared to untreated biofilm. DNase was applied at a concentration of 10 U/ml. n ≥ 3 for each condition; error bars indicate standard deviations.

Techniques Used: Staining, Concentration Assay

Gene transfer efficiency does not depend on biofilm roughness and density. Fraction of transformants as a function of time. Gray, flat surface; white, 1-μm grooves. n ≥ 3 for each condition; error bars indicate standard deviations.
Figure Legend Snippet: Gene transfer efficiency does not depend on biofilm roughness and density. Fraction of transformants as a function of time. Gray, flat surface; white, 1-μm grooves. n ≥ 3 for each condition; error bars indicate standard deviations.

Techniques Used:

Effect of pilin antigenic variation on the gene transfer probability. (a) Fraction of bacteria with a nonpiliated phenotype. (b) Fraction of transformants (wt) as a function of time in a developing biofilm. Gray, wt; white, pilin antigenic-variation-deficient avd strain. n ≥ 3 for each condition; error bars indicate standard deviations.
Figure Legend Snippet: Effect of pilin antigenic variation on the gene transfer probability. (a) Fraction of bacteria with a nonpiliated phenotype. (b) Fraction of transformants (wt) as a function of time in a developing biofilm. Gray, wt; white, pilin antigenic-variation-deficient avd strain. n ≥ 3 for each condition; error bars indicate standard deviations.

Techniques Used:

Spreading of multiresistant clones under selective pressure. Biofilms (strain 1 and strain 2 mixed at a 1:1 ratio) were grown without antibiotics for 24 h and subsequently treated with 2.5 μg/ml erythromycin and 100 μg/ml spectinomycin. Confocal volume plot on flat surface (a) and 1-μm grooves (b) and orthogonal views on flat surface (c) or 1-μm grooves (d). (e) Biomass; (f) roughness coefficient (*, P
Figure Legend Snippet: Spreading of multiresistant clones under selective pressure. Biofilms (strain 1 and strain 2 mixed at a 1:1 ratio) were grown without antibiotics for 24 h and subsequently treated with 2.5 μg/ml erythromycin and 100 μg/ml spectinomycin. Confocal volume plot on flat surface (a) and 1-μm grooves (b) and orthogonal views on flat surface (c) or 1-μm grooves (d). (e) Biomass; (f) roughness coefficient (*, P

Techniques Used: Clone Assay

Biofilm architecture is influenced by surface structure. (a) Confocal volume plot near the glass coverslip after 22 h and 46 h of growth of strain 1 and strain 2 mixed at a 1:1 ratio on a flat surface (left column) and on 1-μm grooves (right column). (b) Biofilm mass; (c) roughness coefficient. Gray, flat PDMS; white, 1-μm-deep grooves (**, P
Figure Legend Snippet: Biofilm architecture is influenced by surface structure. (a) Confocal volume plot near the glass coverslip after 22 h and 46 h of growth of strain 1 and strain 2 mixed at a 1:1 ratio on a flat surface (left column) and on 1-μm grooves (right column). (b) Biofilm mass; (c) roughness coefficient. Gray, flat PDMS; white, 1-μm-deep grooves (**, P

Techniques Used:

10) Product Images from "The spherical nanoparticle-encapsulated chlorhexidine enhances anti-biofilm efficiency through an effective releasing mode and close microbial interactions"

Article Title: The spherical nanoparticle-encapsulated chlorhexidine enhances anti-biofilm efficiency through an effective releasing mode and close microbial interactions

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S105681

Absorbance (490 nm) of 1-day-old microorganism biofilms after being treated with free and two different shaped nanoparticle-encapsulated CHX (XTT assay). Notes: ( A ) S . sobrinus , ( B ) S . mutans , and ( C ) C . albicans . S- and W-CHX encapsulated with the same drug amount as the free CHX. S- and W-MSNs were equivalent to S- and W-CHX at each concentration. The values on the x-axes refer to the concentrations of free or nanoparticle-encapsulated CHX. The lowercase letters above each bar (a, b, c, and d) indicate significant differences ( P
Figure Legend Snippet: Absorbance (490 nm) of 1-day-old microorganism biofilms after being treated with free and two different shaped nanoparticle-encapsulated CHX (XTT assay). Notes: ( A ) S . sobrinus , ( B ) S . mutans , and ( C ) C . albicans . S- and W-CHX encapsulated with the same drug amount as the free CHX. S- and W-MSNs were equivalent to S- and W-CHX at each concentration. The values on the x-axes refer to the concentrations of free or nanoparticle-encapsulated CHX. The lowercase letters above each bar (a, b, c, and d) indicate significant differences ( P

Techniques Used: XTT Assay, Concentration Assay

Confocal images of RITC-S-MSNs and RITC-W-MSNs (50 μg/mL for bacteria and 100 μg/mL for yeast) in 1-day-old microorganism biofilms after 24 hours of incubation. Note: S . sobrinus ( A-i and ii ), S . mutans ( B-i and ii ), and C . albicans ( C-i and ii ). Abbreviations: RITC-S-MSNs, rhodamine B isothiocyanate-labeled spherical mesoporous silica nanoparticles; RITC-W-MSNs, rhodamine B isothiocyanate-labeled wire mesoporous silica nanoparticles; S . sobrinus , Streptococcus sobrinus ; S . mutans , Streptococcus mutans ; C . albicans , Candida albicans .
Figure Legend Snippet: Confocal images of RITC-S-MSNs and RITC-W-MSNs (50 μg/mL for bacteria and 100 μg/mL for yeast) in 1-day-old microorganism biofilms after 24 hours of incubation. Note: S . sobrinus ( A-i and ii ), S . mutans ( B-i and ii ), and C . albicans ( C-i and ii ). Abbreviations: RITC-S-MSNs, rhodamine B isothiocyanate-labeled spherical mesoporous silica nanoparticles; RITC-W-MSNs, rhodamine B isothiocyanate-labeled wire mesoporous silica nanoparticles; S . sobrinus , Streptococcus sobrinus ; S . mutans , Streptococcus mutans ; C . albicans , Candida albicans .

Techniques Used: Incubation, Labeling

11) Product Images from "Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation *"

Article Title: Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.674929

PslG is not required for Psl biosynthesis and biofilm formation. A , crystal violet biofilm assay and Western blot analysis using specific anti-PslG and RNA polymerase ( RNAP ) of wild-type and Δ pslG strains. RNA polymerase was utilized as a loading control in A and C. B , hippeastrum hybrid amaryllis lectin staining to probe for the presence of the Psl polysaccharide in PAO1 Δ pelF P BAD psl , PAO1 Δ pelF P BAD psl pslG E165Q/E276Q , PAO1 Δ pelF P BAD psl Δ pslG , and PAO1 Δ pslG. C , crystal violet biofilm assay and Western blot analysis biofilm assay for the in trans complementation of pslG in PAO1 Δ pelF P BAD psl Δ pslG strain. D , Psl dot blot to examine Psl levels upon overexpression of pslG. Error bars , S.E. from triplicate experiments.
Figure Legend Snippet: PslG is not required for Psl biosynthesis and biofilm formation. A , crystal violet biofilm assay and Western blot analysis using specific anti-PslG and RNA polymerase ( RNAP ) of wild-type and Δ pslG strains. RNA polymerase was utilized as a loading control in A and C. B , hippeastrum hybrid amaryllis lectin staining to probe for the presence of the Psl polysaccharide in PAO1 Δ pelF P BAD psl , PAO1 Δ pelF P BAD psl pslG E165Q/E276Q , PAO1 Δ pelF P BAD psl Δ pslG , and PAO1 Δ pslG. C , crystal violet biofilm assay and Western blot analysis biofilm assay for the in trans complementation of pslG in PAO1 Δ pelF P BAD psl Δ pslG strain. D , Psl dot blot to examine Psl levels upon overexpression of pslG. Error bars , S.E. from triplicate experiments.

Techniques Used: Biofilm Production Assay, Western Blot, Staining, Dot Blot, Over Expression

PslG displays glycoside hydrolase activity that is not required for Psl biosynthesis and biofilm formation. A , reducing sugar assay of cell-associated Psl with PslG(31–442) and catalytic variants. Error bars , S.E. from triplicate experiments. B , crystal violet biofilm assay with P. aeruginosa PAO1 Δ pelF P BAD psl and pslG E165Q , pslG E276Q , and pslG E165Q/E276Q chromosomal variants. PAO1Δ pelF P BAD psl Δ pslD was utilized as a negative control. C , Psl dot blot for strains listed in B .
Figure Legend Snippet: PslG displays glycoside hydrolase activity that is not required for Psl biosynthesis and biofilm formation. A , reducing sugar assay of cell-associated Psl with PslG(31–442) and catalytic variants. Error bars , S.E. from triplicate experiments. B , crystal violet biofilm assay with P. aeruginosa PAO1 Δ pelF P BAD psl and pslG E165Q , pslG E276Q , and pslG E165Q/E276Q chromosomal variants. PAO1Δ pelF P BAD psl Δ pslD was utilized as a negative control. C , Psl dot blot for strains listed in B .

Techniques Used: Activity Assay, Biofilm Production Assay, Negative Control, Dot Blot

12) Product Images from "Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase"

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Journal: Viruses

doi: 10.3390/v10080438

Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Flow Cytometry, Staining

Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.
Figure Legend Snippet: Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.

Techniques Used: Activity Assay, Staining, Concentration Assay

Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Staining, Concentration Assay, Flow Cytometry

Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.
Figure Legend Snippet: Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.

Techniques Used: Staining, Confocal Laser Scanning Microscopy

Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.
Figure Legend Snippet: Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.

Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Staining

13) Product Images from "Eradication of Candida albicans persister cell biofilm by the membranotropic peptide gH625"

Article Title: Eradication of Candida albicans persister cell biofilm by the membranotropic peptide gH625

Journal: Scientific Reports

doi: 10.1038/s41598-020-62746-w

CLSM images (x400 magnification) of the primary biofilm of C1 strain before ( a ) and after AmphB treatment at time 0 ( b ) and the progressive formation of persister-derived biofilms after 2d ( c ), 4d ( d ), 6d ( e ), 8d ( f ) of incubation. In the graphs, substrate coverage ( g ), volume ( h ), bio-volume ( i ), mean thickness (panel j), maximal thickness ( k ), and roughness coefficient ( l ) of the C1 strain persister-derived biofilm are reported, as compared to the corresponding parameters of the primary biofilm (control). In panel h, white bar represents the volume of the dead cells. Significance noted as: *p
Figure Legend Snippet: CLSM images (x400 magnification) of the primary biofilm of C1 strain before ( a ) and after AmphB treatment at time 0 ( b ) and the progressive formation of persister-derived biofilms after 2d ( c ), 4d ( d ), 6d ( e ), 8d ( f ) of incubation. In the graphs, substrate coverage ( g ), volume ( h ), bio-volume ( i ), mean thickness (panel j), maximal thickness ( k ), and roughness coefficient ( l ) of the C1 strain persister-derived biofilm are reported, as compared to the corresponding parameters of the primary biofilm (control). In panel h, white bar represents the volume of the dead cells. Significance noted as: *p

Techniques Used: Confocal Laser Scanning Microscopy, Derivative Assay, Incubation

Biofilm profiles of the primary biofilm ( a ) and of the persister-derived biofilm at 8 d ( b ). In the latter, it is possible to visualize biofilm fragmentation. Bars correspond to 10 μm.
Figure Legend Snippet: Biofilm profiles of the primary biofilm ( a ) and of the persister-derived biofilm at 8 d ( b ). In the latter, it is possible to visualize biofilm fragmentation. Bars correspond to 10 μm.

Techniques Used: Derivative Assay

Detection of persisters in Candida albicans biofilms. ( a ) Survival of Candida albicans cells in biofilms of ATTC 90028 and C1 strain following treatment with AmphB at different concentrations. CLSM images of C1 biofilm before ( b ) and after ( c ) treatment with 100 µg mL −1 AmphB. In the untreated biofilm cells appear green (live), in the treated biofilm the majority of cells are red (dead). Arrows indicate the few persisters.
Figure Legend Snippet: Detection of persisters in Candida albicans biofilms. ( a ) Survival of Candida albicans cells in biofilms of ATTC 90028 and C1 strain following treatment with AmphB at different concentrations. CLSM images of C1 biofilm before ( b ) and after ( c ) treatment with 100 µg mL −1 AmphB. In the untreated biofilm cells appear green (live), in the treated biofilm the majority of cells are red (dead). Arrows indicate the few persisters.

Techniques Used: Confocal Laser Scanning Microscopy

Cell details from C1 persister-derived biofilm after 30 min ( a – f ) and 180 min ( g – l ) from peptide treatment. Calcofluor-white dye (in blue: a , g ), Concanavalin-A (in green: b , h ), peptide (in red: c , i ). 3D reconstruction of a Candida hemicell, to allow a better visualization of the peptide penetration inside the cells. After 30 minutes, the peptide penetrated mainly into the cell wall ( d ), after 180 minutes the peptide was found in a massive way inside the cells ( j ). Highlights of the EPS matrix in treated biofilms ( e , k ) and in the control ( f , l ). Bar scales correspond to 1 μm.
Figure Legend Snippet: Cell details from C1 persister-derived biofilm after 30 min ( a – f ) and 180 min ( g – l ) from peptide treatment. Calcofluor-white dye (in blue: a , g ), Concanavalin-A (in green: b , h ), peptide (in red: c , i ). 3D reconstruction of a Candida hemicell, to allow a better visualization of the peptide penetration inside the cells. After 30 minutes, the peptide penetrated mainly into the cell wall ( d ), after 180 minutes the peptide was found in a massive way inside the cells ( j ). Highlights of the EPS matrix in treated biofilms ( e , k ) and in the control ( f , l ). Bar scales correspond to 1 μm.

Techniques Used: Derivative Assay

Development of a new biofilm from persisters. Viable cells increase during formation of the biofilm generating from cells of the primary biofilm survived at AmphB treatment at time = 0. Data with different letters ( a , b ) are significantly different (p
Figure Legend Snippet: Development of a new biofilm from persisters. Viable cells increase during formation of the biofilm generating from cells of the primary biofilm survived at AmphB treatment at time = 0. Data with different letters ( a , b ) are significantly different (p

Techniques Used:

14) Product Images from "Targeting of Streptococcus mutans Biofilms by a Novel Small Molecule Prevents Dental Caries and Preserves the Oral Microbiome"

Article Title: Targeting of Streptococcus mutans Biofilms by a Novel Small Molecule Prevents Dental Caries and Preserves the Oral Microbiome

Journal: Journal of Dental Research

doi: 10.1177/0022034517698096

Small molecule 3F1 selectively disperses Streptococcus mutans biofilms in vitro. ( A ) Chemical structure of small molecule 3F1 and its structural analogue, 3F2. 3F2 is a structural isoform of 3F1 and only varies in the side projection of the distal propyl oxy group from the center aromatic ring. ( B ) S. mutans UA159 biofilms were formed for 6 h. Biofilms were then washed and treated with dimethyl sulfoxide (DMSO), 5 µM 3F1, or 5 µM 3F2 for 14 h. After 14 h, cells released into the media were measured at OD 470 . Loosely adhered cells were then washed off and remaining biomass quantitated by crystal violet staining at OD 562 . ( C ) S. mutans UA159 biofilms were formed for 6 h, washed, and treated with DMSO, 5 µM 3F1, or 5 µM 3F2 for 14 h. Loosely adhered cells were then washed off and remaining biomass quantitated by crystal violet staining at OD 562 . ( D ) Planktonic S. mutans UA159 cells were treated for 14 h with DMSO or 5 µM 3F1. Viable cells were enumerated by colony-forming unit (CFU) plating. ( E ) Oral commensal species, Streptococcus sanguinis and Streptococcus gordonnii , were allowed to form biofilms for 6 h before treatment with DMSO or 5 µM 3F1 for 14 h. Bars represent the mean of 3 independent experiments. Differences were statistically compared by analysis of variance. Error bars represent standard error. * P
Figure Legend Snippet: Small molecule 3F1 selectively disperses Streptococcus mutans biofilms in vitro. ( A ) Chemical structure of small molecule 3F1 and its structural analogue, 3F2. 3F2 is a structural isoform of 3F1 and only varies in the side projection of the distal propyl oxy group from the center aromatic ring. ( B ) S. mutans UA159 biofilms were formed for 6 h. Biofilms were then washed and treated with dimethyl sulfoxide (DMSO), 5 µM 3F1, or 5 µM 3F2 for 14 h. After 14 h, cells released into the media were measured at OD 470 . Loosely adhered cells were then washed off and remaining biomass quantitated by crystal violet staining at OD 562 . ( C ) S. mutans UA159 biofilms were formed for 6 h, washed, and treated with DMSO, 5 µM 3F1, or 5 µM 3F2 for 14 h. Loosely adhered cells were then washed off and remaining biomass quantitated by crystal violet staining at OD 562 . ( D ) Planktonic S. mutans UA159 cells were treated for 14 h with DMSO or 5 µM 3F1. Viable cells were enumerated by colony-forming unit (CFU) plating. ( E ) Oral commensal species, Streptococcus sanguinis and Streptococcus gordonnii , were allowed to form biofilms for 6 h before treatment with DMSO or 5 µM 3F1 for 14 h. Bars represent the mean of 3 independent experiments. Differences were statistically compared by analysis of variance. Error bars represent standard error. * P

Techniques Used: In Vitro, Staining

15) Product Images from "Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?"

Article Title: Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

Journal: Clinical Orthopaedics and Related Research

doi: 10.1007/s11999-017-5266-0

( A ) The S aureus biofilm after 6 hours of incubation shows grape-like groups and suspected eDNA filaments (arrow) before spreading. ( B ) A magnification is shown of Illustration A, using a filter to better observe the filamentous bonds (arrow). ( C ) After 24 hours of incubation, a biofilm layer is forming. ( D ) The S epidermidis biofilm after 6 hours of incubation shows scattered cells over the surface. ( E ) After 24 hours of incubation, suspected eDNA filaments (arrow) are produced. (Original magnification, ×1000; scale bar: 5 µm).
Figure Legend Snippet: ( A ) The S aureus biofilm after 6 hours of incubation shows grape-like groups and suspected eDNA filaments (arrow) before spreading. ( B ) A magnification is shown of Illustration A, using a filter to better observe the filamentous bonds (arrow). ( C ) After 24 hours of incubation, a biofilm layer is forming. ( D ) The S epidermidis biofilm after 6 hours of incubation shows scattered cells over the surface. ( E ) After 24 hours of incubation, suspected eDNA filaments (arrow) are produced. (Original magnification, ×1000; scale bar: 5 µm).

Techniques Used: Incubation, Produced

Clinical S epidermidis biofilms after ( A ) 6 hours and ( B ) 24 hours, and control isolates of S epidermidis biofilms after ( C ) 6 hours ( D ) and 24 hours are shown. The amount of dead cells DNA (red) measured after 6 hours was higher (p
Figure Legend Snippet: Clinical S epidermidis biofilms after ( A ) 6 hours and ( B ) 24 hours, and control isolates of S epidermidis biofilms after ( C ) 6 hours ( D ) and 24 hours are shown. The amount of dead cells DNA (red) measured after 6 hours was higher (p

Techniques Used:

16) Product Images from "Breaking the Vicious Cycle of Antibiotic Killing and Regrowth of Biofilm-Residing Pseudomonas aeruginosa"

Article Title: Breaking the Vicious Cycle of Antibiotic Killing and Regrowth of Biofilm-Residing Pseudomonas aeruginosa

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01635-18

Concentration- and time-dependent time point of killing and regrowth. (A) Relationship between antibiotic concentration and the time to kill 5% of the biofilm population ( t eff5 ). The dashed line represents the t eff5 of the killing control, ethanol. Asterisks indicate data of one single experiment (see Table S1). (B) Biofilm-active interval (interval [in h] between the time point when the antibiotic starts to kill and the time point when regrowth becomes visible) of 48-h-old PA14 biofilms treated for 1 h and 4 h with tobramycin and/or colistin at the indicated concentrations (in micrograms per milliliter). Asterisks indicate that for CST at 2.5 µg ml −1 and TOB at 5 µg ml −1 (4-h treatment), a BAI could not be determined for all experiments. Data are means ± SDs of two positions in two independent experiments.
Figure Legend Snippet: Concentration- and time-dependent time point of killing and regrowth. (A) Relationship between antibiotic concentration and the time to kill 5% of the biofilm population ( t eff5 ). The dashed line represents the t eff5 of the killing control, ethanol. Asterisks indicate data of one single experiment (see Table S1). (B) Biofilm-active interval (interval [in h] between the time point when the antibiotic starts to kill and the time point when regrowth becomes visible) of 48-h-old PA14 biofilms treated for 1 h and 4 h with tobramycin and/or colistin at the indicated concentrations (in micrograms per milliliter). Asterisks indicate that for CST at 2.5 µg ml −1 and TOB at 5 µg ml −1 (4-h treatment), a BAI could not be determined for all experiments. Data are means ± SDs of two positions in two independent experiments.

Techniques Used: Concentration Assay

Killing dynamics of P. aeruginosa biofilm cells. Biofilms were cultivated for 48 h and treated with 10 µg ml −1 of colistin, 20 µg ml −1 of tobramycin, or 70% EtOH (killing control) for a period of 4 h. The first image series was acquired at the time of the flow start. The maximum antibiotic concentration was reached after approximately 90 min. Following a 4-h administration of the drug, the antibiotics were washed off the system within approximately 2 h. (A) Maximum-intensity projections of untreated control (CTRL, first row), colistin-treated (second row), tobramycin-treated (third row), and ethanol-treated (fourth row) biofilms at given intervals are shown. Green, GFP fluorescence; red, PI fluorescence. Scale bar: 50 µm. (B and C) Increase of the relative red biovolume (PI signal) over time (B) and relevant parameters describing the killing curve (C). t eff5 and t eff50 , time points when 5% and 50%, respectively, of the biofilm cells are killed (slope of the sigmoidal curve and maximal killing rate). Data are means ± SDs for two positions in two independent experiments.
Figure Legend Snippet: Killing dynamics of P. aeruginosa biofilm cells. Biofilms were cultivated for 48 h and treated with 10 µg ml −1 of colistin, 20 µg ml −1 of tobramycin, or 70% EtOH (killing control) for a period of 4 h. The first image series was acquired at the time of the flow start. The maximum antibiotic concentration was reached after approximately 90 min. Following a 4-h administration of the drug, the antibiotics were washed off the system within approximately 2 h. (A) Maximum-intensity projections of untreated control (CTRL, first row), colistin-treated (second row), tobramycin-treated (third row), and ethanol-treated (fourth row) biofilms at given intervals are shown. Green, GFP fluorescence; red, PI fluorescence. Scale bar: 50 µm. (B and C) Increase of the relative red biovolume (PI signal) over time (B) and relevant parameters describing the killing curve (C). t eff5 and t eff50 , time points when 5% and 50%, respectively, of the biofilm cells are killed (slope of the sigmoidal curve and maximal killing rate). Data are means ± SDs for two positions in two independent experiments.

Techniques Used: Flow Cytometry, Concentration Assay, Fluorescence

Killing dynamics in biofilms treated over a period of 48 h. The biofilms were exposed four times for 1 h to 10 µg ml −1 of colistin, 20 µg ml −1 of tobramycin, or a combination of the two. Two treatment intervals were tested: the first set was treated every 8 h and the second set every 12 h. Representative maximum-intensity projections of biofilms before treatment and 1 h after the time points of antibiotic addition are shown. Green, GFP fluorescence; red, PI fluorescence. Scale bar: 50 µm.
Figure Legend Snippet: Killing dynamics in biofilms treated over a period of 48 h. The biofilms were exposed four times for 1 h to 10 µg ml −1 of colistin, 20 µg ml −1 of tobramycin, or a combination of the two. Two treatment intervals were tested: the first set was treated every 8 h and the second set every 12 h. Representative maximum-intensity projections of biofilms before treatment and 1 h after the time points of antibiotic addition are shown. Green, GFP fluorescence; red, PI fluorescence. Scale bar: 50 µm.

Techniques Used: Fluorescence

17) Product Images from "Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase"

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Journal: Viruses

doi: 10.3390/v10080438

Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Flow Cytometry, Staining

Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.
Figure Legend Snippet: Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.

Techniques Used: Activity Assay, Staining, Concentration Assay

Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Staining, Concentration Assay, Flow Cytometry

Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.
Figure Legend Snippet: Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.

Techniques Used: Staining, Confocal Laser Scanning Microscopy

Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.
Figure Legend Snippet: Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.

Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Staining

18) Product Images from "Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species"

Article Title: Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors of Streptococcus Species

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00620-19

Images of dual-species biofilms. (A to C) Selected 3D reconstructions of maximum-intensity z-section confocal microcopy images of dual-species biofilms of either S. mutans /pDL278_P23-sfgfp and S. mutans /pDL278_P23-DsRed-Express2 (control biofilm) (A), S. mutans /pDL278_P23-sfgfp and S. gordonii /pDL278_P23-DsRed-Express2 (B), and S. mutans /pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 (C) at the 24, 48-, and 72-h time points. Images are of 128-μm sections of the fluorescence range within the biofilm collected at 1-μm intervals using a 63× oil objective lens (numerical aperture, 1.40). Biofilms were grown in chemically defined medium (CDM) supplemented with 20 mM glucose and 5 mM sucrose. At 24 h and every 12 h thereafter the spent medium was replaced with fresh medium for the length of the experiment. The biofilm images from time course experiment were reconstructed with Imaris software (6.4.0) using shadow projections. (D and E) The biofilm images from the time course experiment were analyzed by the Comstat2 (v2.1) program for biomass (D) and maximum thickness (E). Green line, S. mutans /pDL278_P23-sfgfp and S. mutans /pDL278_P23-DsRed-Express2 dual-species biofilms (control biofilm); orange line, S. mutans /pDL278_P23-sfgfp and S. gordonii /pDL278_P23-DsRed-Express2 dual-species biofilms; blue line, S. mutans /pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 dual-species biofilms.
Figure Legend Snippet: Images of dual-species biofilms. (A to C) Selected 3D reconstructions of maximum-intensity z-section confocal microcopy images of dual-species biofilms of either S. mutans /pDL278_P23-sfgfp and S. mutans /pDL278_P23-DsRed-Express2 (control biofilm) (A), S. mutans /pDL278_P23-sfgfp and S. gordonii /pDL278_P23-DsRed-Express2 (B), and S. mutans /pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 (C) at the 24, 48-, and 72-h time points. Images are of 128-μm sections of the fluorescence range within the biofilm collected at 1-μm intervals using a 63× oil objective lens (numerical aperture, 1.40). Biofilms were grown in chemically defined medium (CDM) supplemented with 20 mM glucose and 5 mM sucrose. At 24 h and every 12 h thereafter the spent medium was replaced with fresh medium for the length of the experiment. The biofilm images from time course experiment were reconstructed with Imaris software (6.4.0) using shadow projections. (D and E) The biofilm images from the time course experiment were analyzed by the Comstat2 (v2.1) program for biomass (D) and maximum thickness (E). Green line, S. mutans /pDL278_P23-sfgfp and S. mutans /pDL278_P23-DsRed-Express2 dual-species biofilms (control biofilm); orange line, S. mutans /pDL278_P23-sfgfp and S. gordonii /pDL278_P23-DsRed-Express2 dual-species biofilms; blue line, S. mutans /pDL278_P23-sfgfp and Streptococcus A12/pDL278_P23-DsRed-Express2 dual-species biofilms.

Techniques Used: Fluorescence, Software

19) Product Images from "Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans"

Article Title: Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00370-19

Dual-species biofilms formed using clinical commensals and S. mutans . Each commensal was mixed with UA159-Km in equal proportions and inoculated into BM supplemented with 18 mM Glc and 2 mM sucrose (BMGS) to form biofilms overnight on glass coverslips. BMGS was then replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc and incubated for another 24 h before visualization by confocal laser scanning microscopy (CLSM) after LIVE/DEAD staining (A) and CFU quantification of both species (B). Biofilms formed by UA159-Km alone were included for comparison. Values show the percentages of the populations constituted by commensal bacteria (top) and S. mutans (bottom), respectively. Asterisks indicate statistically significant differences in the viable counts of S. mutans compared to that on BMGlc. *, P
Figure Legend Snippet: Dual-species biofilms formed using clinical commensals and S. mutans . Each commensal was mixed with UA159-Km in equal proportions and inoculated into BM supplemented with 18 mM Glc and 2 mM sucrose (BMGS) to form biofilms overnight on glass coverslips. BMGS was then replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc and incubated for another 24 h before visualization by confocal laser scanning microscopy (CLSM) after LIVE/DEAD staining (A) and CFU quantification of both species (B). Biofilms formed by UA159-Km alone were included for comparison. Values show the percentages of the populations constituted by commensal bacteria (top) and S. mutans (bottom), respectively. Asterisks indicate statistically significant differences in the viable counts of S. mutans compared to that on BMGlc. *, P

Techniques Used: Gas Chromatography, Incubation, Confocal Laser Scanning Microscopy, Staining

Amino sugars impact the survival of S. mutans in CCS-derived biofilms. (A and B) To initiate a biofilm, UA159-Km and cell-containing saliva (CCS) were used simultaneously (groups a and d) or 24 h apart (groups b and c) to inoculate BM supplemented with 2 mM sucrose and 18 mM various other carbohydrates. After 24 h of incubation, the biofilms were washed and resupplied with fresh medium and additional bacteria as specified (see Materials and Methods for details). (B) At the end of 2-day incubations, biofilms were processed to quantify the number of CFU of UA159-Km. Asterisks indicate statistically significant differences compared to cultures prepared with Glc. *, P
Figure Legend Snippet: Amino sugars impact the survival of S. mutans in CCS-derived biofilms. (A and B) To initiate a biofilm, UA159-Km and cell-containing saliva (CCS) were used simultaneously (groups a and d) or 24 h apart (groups b and c) to inoculate BM supplemented with 2 mM sucrose and 18 mM various other carbohydrates. After 24 h of incubation, the biofilms were washed and resupplied with fresh medium and additional bacteria as specified (see Materials and Methods for details). (B) At the end of 2-day incubations, biofilms were processed to quantify the number of CFU of UA159-Km. Asterisks indicate statistically significant differences compared to cultures prepared with Glc. *, P

Techniques Used: Derivative Assay, Incubation, Gas Chromatography

20) Product Images from "Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides"

Article Title: Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides

Journal: Microorganisms

doi: 10.3390/microorganisms4030026

Biofilm (BF) production of KpTs101 in the presence of sub-inhibitory concentrations of Bac7(1–35) ( A ); and BMAP-27 ( B ). The BF index in the presence of AMPs after 24 h incubation was reported as percentage with respect to the control. Results are the mean of three independent experiments ± SD.
Figure Legend Snippet: Biofilm (BF) production of KpTs101 in the presence of sub-inhibitory concentrations of Bac7(1–35) ( A ); and BMAP-27 ( B ). The BF index in the presence of AMPs after 24 h incubation was reported as percentage with respect to the control. Results are the mean of three independent experiments ± SD.

Techniques Used: Incubation

3D images of KpTs101 BF treated with AMPs. The biofilm formed after 24 h growth by KpTs101 on glass was incubated for further a 24 h in saline solution alone ( A ); or with addition of 64 µM Bac7(1–35) ( B ); or BMAP-27 ( C ), gently washed and stained with the live/dead kit: live bacteria were stained green and dead bacteria were stained red. Z-stacks were acquired with a 60×/1.4 oil objective at 0.33 µm intervals.
Figure Legend Snippet: 3D images of KpTs101 BF treated with AMPs. The biofilm formed after 24 h growth by KpTs101 on glass was incubated for further a 24 h in saline solution alone ( A ); or with addition of 64 µM Bac7(1–35) ( B ); or BMAP-27 ( C ), gently washed and stained with the live/dead kit: live bacteria were stained green and dead bacteria were stained red. Z-stacks were acquired with a 60×/1.4 oil objective at 0.33 µm intervals.

Techniques Used: Incubation, Staining

Total biofilm produced by KpTs101 ( A ); and KpTs113 ( B ) on glass slides. After 24 h growth the biofilms were fixed and stained with acridine orange 1% w / v . Z-stacks were acquired with a 20×/0.5 air objective at 1 µm intervals. Orthogonal views of Z-stacks are shown in the right and lower sides of each frame.
Figure Legend Snippet: Total biofilm produced by KpTs101 ( A ); and KpTs113 ( B ) on glass slides. After 24 h growth the biofilms were fixed and stained with acridine orange 1% w / v . Z-stacks were acquired with a 20×/0.5 air objective at 1 µm intervals. Orthogonal views of Z-stacks are shown in the right and lower sides of each frame.

Techniques Used: Produced, Staining

21) Product Images from "Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms"

Article Title: Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02416-12

Cross-section of oral biofilm on bovine enamel slabs. Blue reference beads were applied on the biofilm surface for instrument setup and calibration.
Figure Legend Snippet: Cross-section of oral biofilm on bovine enamel slabs. Blue reference beads were applied on the biofilm surface for instrument setup and calibration.

Techniques Used:

Overviews of tiled images constructed from the individual Scan∧R maximum-projection images of the supporting enamel surface of representative 5-day-old in situ oral biofilms on bovine enamel plates. Oral biofilm was hybridized with three different specific probes (EUB 338, FUS 664, and STR 405). Shown are results obtained with green eubacterium-specific probe (EUB 338) (a), red Streptococcus -specific probe (STR 405) (b), and blue Fusobacterium nucleatum -specific probe (FUS 664) (c) after 5 days and all three probes after 5 days (d). Scale bars, 20 μm.
Figure Legend Snippet: Overviews of tiled images constructed from the individual Scan∧R maximum-projection images of the supporting enamel surface of representative 5-day-old in situ oral biofilms on bovine enamel plates. Oral biofilm was hybridized with three different specific probes (EUB 338, FUS 664, and STR 405). Shown are results obtained with green eubacterium-specific probe (EUB 338) (a), red Streptococcus -specific probe (STR 405) (b), and blue Fusobacterium nucleatum -specific probe (FUS 664) (c) after 5 days and all three probes after 5 days (d). Scale bars, 20 μm.

Techniques Used: Construct, In Situ

FISH-Scan∧R image and graphical distribution of different biofilm bacteria (eubacteria, Streptococcus spp., and F. nucleatum ) of a 3-day-old oral biofilm grown in situ on enamel slabs. The image seen in panel A is a tiled overview of the microbial colonization pattern showing an overlay of all three staining patterns. Oral biofilm was stained simultaneously with the all-bacterium-specific EUB 338 probe (green), the Streptococcus -specific EUB 405 probe (red), and the F. nucleatum -specific FUS 664 probe (blue). Panels B, C, and D graphically depict the spatial distribution of positive subobjects after thresholding and analysis in the Scan∧R analysis program, containing eubacteria, Streptococcus spp., and F. nucleatum and labeled with FITC, Cy3, and Cy5, respectively. x and y axis values represent the different positions on the microscope stage.
Figure Legend Snippet: FISH-Scan∧R image and graphical distribution of different biofilm bacteria (eubacteria, Streptococcus spp., and F. nucleatum ) of a 3-day-old oral biofilm grown in situ on enamel slabs. The image seen in panel A is a tiled overview of the microbial colonization pattern showing an overlay of all three staining patterns. Oral biofilm was stained simultaneously with the all-bacterium-specific EUB 338 probe (green), the Streptococcus -specific EUB 405 probe (red), and the F. nucleatum -specific FUS 664 probe (blue). Panels B, C, and D graphically depict the spatial distribution of positive subobjects after thresholding and analysis in the Scan∧R analysis program, containing eubacteria, Streptococcus spp., and F. nucleatum and labeled with FITC, Cy3, and Cy5, respectively. x and y axis values represent the different positions on the microscope stage.

Techniques Used: Fluorescence In Situ Hybridization, In Situ, Staining, Labeling, Microscopy

Box plots depicting percentages of the different bacterial members in 3- and 5-day-old oral biofilm as detected by FISH. The central line is the median; whiskers indicate minimum and maximum. Each box indicates the front (f), middle (m), or back (b) position of the enamel slabs on the right (r) and left (l) sides of the oral cavity, respectively.
Figure Legend Snippet: Box plots depicting percentages of the different bacterial members in 3- and 5-day-old oral biofilm as detected by FISH. The central line is the median; whiskers indicate minimum and maximum. Each box indicates the front (f), middle (m), or back (b) position of the enamel slabs on the right (r) and left (l) sides of the oral cavity, respectively.

Techniques Used: Fluorescence In Situ Hybridization

22) Product Images from "Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella"

Article Title: Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella

Journal: mBio

doi: 10.1128/mBio.01973-18

Effect of enzymatic treatments on preformed biofilms. H. pylori SS1 was allowed to form biofilms for 3 days in BB2. The medium was then removed and replaced with either fresh medium or medium containing DNase I or proteinase K. Cells were reincubated for 24 h and then analyzed for the remaining biofilm using the crystal violet assay. The data shown here represent the percentage of remaining biofilm compared to the untreated control. Experiments were performed three times independently with at least 8 technical replicates for each. Statistical analysis was performed using ANOVA (*, P
Figure Legend Snippet: Effect of enzymatic treatments on preformed biofilms. H. pylori SS1 was allowed to form biofilms for 3 days in BB2. The medium was then removed and replaced with either fresh medium or medium containing DNase I or proteinase K. Cells were reincubated for 24 h and then analyzed for the remaining biofilm using the crystal violet assay. The data shown here represent the percentage of remaining biofilm compared to the untreated control. Experiments were performed three times independently with at least 8 technical replicates for each. Statistical analysis was performed using ANOVA (*, P

Techniques Used: Crystal Violet Assay

Confocal scanning laser microscopy (CSLM) images of H. pylori SS1 biofilm. Shown are representative CSLM images of 3-day-old SS1 biofilms grown in BB2 and stained with (A) FM 1–43 to stain total bacterial cells, (B) SYPRO RUBY to stain extracellular proteins, (C) BOBO-3 to stain extracellular DNA, and (D to F) live-dead staining with live cells represented by the green fluorescent SYTO 9 and dead/damaged cells represented by the red fluorescent propidium. Scale bar = 30 µm.
Figure Legend Snippet: Confocal scanning laser microscopy (CSLM) images of H. pylori SS1 biofilm. Shown are representative CSLM images of 3-day-old SS1 biofilms grown in BB2 and stained with (A) FM 1–43 to stain total bacterial cells, (B) SYPRO RUBY to stain extracellular proteins, (C) BOBO-3 to stain extracellular DNA, and (D to F) live-dead staining with live cells represented by the green fluorescent SYTO 9 and dead/damaged cells represented by the red fluorescent propidium. Scale bar = 30 µm.

Techniques Used: Microscopy, Staining

Flagella play integral roles in H. pylori biofilms. (A) Scanning electron microscope (SEM) images of biofilms formed by H. pylori wild-type SS1 (SS1 WT), the isogenic nonmotile but flagellated Δ motB mutant (SS1 Δ motB ), and the isogenic aflagellated Δ fliM mutant (SS1 Δ fliM ). Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori SS1 wild type and Δ motB and Δ fliM mutants. Strains were grown in BB2 medium for 3 days, followed by biofilm evaluation using the crystal violet assay. Experiments were performed three times independently with 6 to 9 technical replicates for each. Statistical analysis was performed using ANOVA (**, P
Figure Legend Snippet: Flagella play integral roles in H. pylori biofilms. (A) Scanning electron microscope (SEM) images of biofilms formed by H. pylori wild-type SS1 (SS1 WT), the isogenic nonmotile but flagellated Δ motB mutant (SS1 Δ motB ), and the isogenic aflagellated Δ fliM mutant (SS1 Δ fliM ). Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori SS1 wild type and Δ motB and Δ fliM mutants. Strains were grown in BB2 medium for 3 days, followed by biofilm evaluation using the crystal violet assay. Experiments were performed three times independently with 6 to 9 technical replicates for each. Statistical analysis was performed using ANOVA (**, P

Techniques Used: Microscopy, Mutagenesis, Crystal Violet Assay

H. pylori G27 biofilm contains structurally important flagella. (A) Scanning electron microscope (SEM) images of wild-type G27 H. pylori biofilms. Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori G27 wild type (WT), the nonmotile flagellated motB mutant, the nonmotile fliA mutant that is reported to have either truncated flagella or no flagella, and the aflagellated and nonmotile flgS mutant. Biofilms were evaluated using the crystal violet assay. Experiments were performed 2 times independently with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (**, P
Figure Legend Snippet: H. pylori G27 biofilm contains structurally important flagella. (A) Scanning electron microscope (SEM) images of wild-type G27 H. pylori biofilms. Arrows indicate flagella. (B) Quantification of biofilm formation by the H. pylori G27 wild type (WT), the nonmotile flagellated motB mutant, the nonmotile fliA mutant that is reported to have either truncated flagella or no flagella, and the aflagellated and nonmotile flgS mutant. Biofilms were evaluated using the crystal violet assay. Experiments were performed 2 times independently with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (**, P

Techniques Used: Microscopy, Mutagenesis, Crystal Violet Assay

qPCR validation of the transcription of selected differentially expressed genes. The data indicate the fold change in expression of genes in H. pylori biofilm cells compared to planktonic cells. Fold changes in gene expressions were calculated after normalization of each gene with the constitutively expressed gene control gapB . Bars represent the mean and error bars the standard error of the mean. Black and gray bars represent qPCR and RNA-seq results, respectively. Statistical analyses were performed using threshold cycle (2 −ΔΔ CT ) values, and all results with an asterisk were statistically significant ( P
Figure Legend Snippet: qPCR validation of the transcription of selected differentially expressed genes. The data indicate the fold change in expression of genes in H. pylori biofilm cells compared to planktonic cells. Fold changes in gene expressions were calculated after normalization of each gene with the constitutively expressed gene control gapB . Bars represent the mean and error bars the standard error of the mean. Black and gray bars represent qPCR and RNA-seq results, respectively. Statistical analyses were performed using threshold cycle (2 −ΔΔ CT ) values, and all results with an asterisk were statistically significant ( P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, RNA Sequencing Assay

H. pylori SS1 forms robust biofilms after 3 days of growth in BB2. H. pylori strain SS1 was grown in the indicated media, and biofilm formation was assessed by crystal violet absorbance at 595 nm. (A) H. pylori SS1 was grown for 3 days in BB media supplemented with different concentrations of FBS (BB10, 10%; BB6, 6%; and BB2, 2%). (B) H. pylori SS1 was grown for 3 days in BB media or Ham’s F-12 supplemented with 10% (HAMS10) or 2% (HAMS2) FBS. (C) H. pylori SS1 was grown in BB medium supplemented with 2% FBS, and biofilm formation was evaluated at different time points. Experiments were performed three independent times with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (*, P
Figure Legend Snippet: H. pylori SS1 forms robust biofilms after 3 days of growth in BB2. H. pylori strain SS1 was grown in the indicated media, and biofilm formation was assessed by crystal violet absorbance at 595 nm. (A) H. pylori SS1 was grown for 3 days in BB media supplemented with different concentrations of FBS (BB10, 10%; BB6, 6%; and BB2, 2%). (B) H. pylori SS1 was grown for 3 days in BB media or Ham’s F-12 supplemented with 10% (HAMS10) or 2% (HAMS2) FBS. (C) H. pylori SS1 was grown in BB medium supplemented with 2% FBS, and biofilm formation was evaluated at different time points. Experiments were performed three independent times with at least 6 technical replicates for each. Statistical analysis was performed using ANOVA (*, P

Techniques Used:

Biofilm-grown cells and planktonic cells show distinct transcriptional profiles. (A) Principal-component analysis (PCA) of gene expression obtained by RNA-seq between biofilm ( n = 3) and planktonic ( n = 3) populations. (B) Volcano plot of gene expression data. The y axis is the negative log 10 of P values (a higher value indicates greater significance), and the x axis is the log 2 fold change in difference in abundance between two population (positive values represent the upregulated genes in biofilm, and negative values represent downregulated genes). The dashed red line shows where P = 0.01, with points above the line having a P value of
Figure Legend Snippet: Biofilm-grown cells and planktonic cells show distinct transcriptional profiles. (A) Principal-component analysis (PCA) of gene expression obtained by RNA-seq between biofilm ( n = 3) and planktonic ( n = 3) populations. (B) Volcano plot of gene expression data. The y axis is the negative log 10 of P values (a higher value indicates greater significance), and the x axis is the log 2 fold change in difference in abundance between two population (positive values represent the upregulated genes in biofilm, and negative values represent downregulated genes). The dashed red line shows where P = 0.01, with points above the line having a P value of

Techniques Used: Expressing, RNA Sequencing Assay

23) Product Images from "Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase"

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Journal: Viruses

doi: 10.3390/v10080438

Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Efficacy of LysK against dynamic S. aureus SA113 biofilms grown on glass surfaces in the Biostream flow cell. Biofilms were grown for 20 h at 19 °C under continuous flow of medium. Glass slides were then submerged in solutions of LysK at different concentrations or buffer as a control for 5 h ( A , C ) or 2 h ( B , D ), and stained with crystal violet. The stain was dissolved in 96% ethanol and the A 595nm measured spectrophotometrically ( A , B ). All values were normalized to the control. Error bars indicate standard deviations from at least three independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Flow Cytometry, Staining

Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.
Figure Legend Snippet: Confocal laser scanning micrographs of S. aureus biofilms treated with DA7, LysK, or a combination of both agents. SA113 biofilms were grown in IBIDI µ-slides for 20 h at 19 °C and then treated for 2 h with: buffer ( A ); 0.625 nM DA7 ( B ); 1.25 µM LysK ( C ); or a combination of 0.156 nM DA7 and 0.938 µM LysK ( D ). Residual biofilms after treatment were stained with LIVE/DEAD stain and then visualized by CLSM. Side views ( top ); and top views ( bottom ) of biofilm 3D reconstructions are shown. Live cells are depicted in green and dead cells as well as extracellular DNA in red.

Techniques Used: Staining, Confocal Laser Scanning Microscopy

Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p
Figure Legend Snippet: Synergistic effect of DA7 and LysK against S. aureus biofilms in static and dynamic models. ( A ) SA113 biofilms grown statically in 96-well plates were treated with two-fold serial dilutions of DA7, LysK, and mixtures of both enzymes at the ratios 50:50, 75:25, and 25:75, with the highest concentrations on the right and the lowest concentrations on the left of each row. After treatment, residual biofilms were stained with crystal violet. For each individual enzyme, the highest concentration used was the respective minimum biofilm eradication concentration (MBEC) and was defined as 100%. Highest concentrations of enzymes within mixtures are expressed as percentages of the respective MBECs. ( B ) Residual viable S. aureus on the glass surface of the Biostream flow cell after a 2 h treatment of dynamic biofilms with buffer (control), DA7 (0.625 nM), LysK (1.25 µM), or a combination of both (0.156 nM DA7 + 0.938 µM LysK, corresponding to a ratio of 25:75). Error bars represent standard deviations from at least seven independent experiments. Bars with different letters are significantly different from each other ( p

Techniques Used: Staining, Concentration Assay, Flow Cytometry

Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.
Figure Legend Snippet: Purification and anti-biofilm activity of the polysaccharide depolymerase DA7. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified DA7 protein. The band of interest (expected molecular weight: 42.1 kDa) is marked by an arrow. ( B ) Static S. aureus SA113 biofilms were grown at 30 °C for 24 h, treated with increasing concentrations of DA7 ( top ) or buffer as a control ( bottom ) for 30 min at 30 °C, and stained with crystal violet (CV). ( C ) Relative A 595nm values measured after dissolving the CV stain on residual biofilms in 96% ethanol after DA7 treatment as shown in panel B. Values are normalized to the control, and error bars represent standard errors of the mean from three independent experiments.

Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Molecular Weight, Staining

Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.
Figure Legend Snippet: Activity of LysK against biofilms of various S. aureus strains in a static model. ( A ) Biofilm formation and susceptibility to LysK treatment of various S. aureus strains. Biofilms were grown statically in 96-well plates for 24 h at 30 °C and stained with CV. The strains were rated for their biofilm forming ability based on A 595nm measurements of solubilized CV as follows: +++, very strong (A 595nm > 3); ++, strong (A 595nm > 1); +, moderate (A 595nm > 0.2); (+), weak (A 595nm ≤ 0.2). At least three experiments were conducted, and one representative well for each strain is shown ( top ). Efficacy of LysK against biofilms of each strain was determined as exemplified for SA113 in ( B ) and rated as follows ( bottom ): +++, > 40% reduction in A 595nm compared to the control at a concentration (c) ≤ 0.08 µM; ++, > 40% reduction at c ≤ 0.31 µM; +, > 40% reduction at c ≤ 1.25 µM; (+), no reduction > 40% even at c > 1.25 µM. ( B ) S. aureus SA113 biofilms grown in a 96-well plate for 24 h at 30 °C were treated with LysK at different concentrations or buffer as a control (0) for 2.5 h, and residual biofilms after treatment were stained with crystal violet (CV). After dissolving the CV in 96% ethanol, the A 595nm of each well was measured spectrophotometrically. All values were normalized to the control. Error bars indicate standard deviations from three independent experiments.

Techniques Used: Activity Assay, Staining, Concentration Assay

24) Product Images from "Role of Hydrophobins in Aspergillus fumigatus"

Article Title: Role of Hydrophobins in Aspergillus fumigatus

Journal: Journal of Fungi

doi: 10.3390/jof4010002

Localization of RodA and RodC. Immunofluorescence localization of RodA on conidia ( a ) and biofilm ( b ) cell walls; and on phialides ( c ) of ku80 (∆ rodA was used as a negative control) using the anti-recombinant RodA polyclonal antiserum; Immunoblotting localization of formic acid-soluble material of RodC-Flag and ku80 (negative control) conidia using an anti-Flag monoclonal antibody ( d ).
Figure Legend Snippet: Localization of RodA and RodC. Immunofluorescence localization of RodA on conidia ( a ) and biofilm ( b ) cell walls; and on phialides ( c ) of ku80 (∆ rodA was used as a negative control) using the anti-recombinant RodA polyclonal antiserum; Immunoblotting localization of formic acid-soluble material of RodC-Flag and ku80 (negative control) conidia using an anti-Flag monoclonal antibody ( d ).

Techniques Used: Immunofluorescence, Negative Control, Recombinant

25) Product Images from "The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection"

Article Title: The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00138

In vitro biofilm growth of Streptococcus pneumoniae D39 wild-type and D39Δ luxS strains at different time-points after inoculation (6, 12, 18, and 24 h). The error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p
Figure Legend Snippet: In vitro biofilm growth of Streptococcus pneumoniae D39 wild-type and D39Δ luxS strains at different time-points after inoculation (6, 12, 18, and 24 h). The error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p

Techniques Used: In Vitro, Standard Deviation

Scanning electron microscopy (SEM) images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A–C) are representative SEM images of the D39 wild-type strain. The wild-type strain biofilms were thick and organized with a significant depth. (D–F) are SEM images of the D39Δ luxS strain. The D39Δ luxS biofilms were thin and disorganized, and extracellular polymeric substance (EPS) was absent.
Figure Legend Snippet: Scanning electron microscopy (SEM) images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A–C) are representative SEM images of the D39 wild-type strain. The wild-type strain biofilms were thick and organized with a significant depth. (D–F) are SEM images of the D39Δ luxS strain. The D39Δ luxS biofilms were thin and disorganized, and extracellular polymeric substance (EPS) was absent.

Techniques Used: Electron Microscopy, In Vitro

Confocal microscopy images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A) Confocal microscopy image of the D39 wild-type strain biofilm. (B) Confocal microscopy image of the D39Δ luxS strain biofilm.
Figure Legend Snippet: Confocal microscopy images of Streptococcus pneumoniae in vitro biofilms grown for 18 h. (A) Confocal microscopy image of the D39 wild-type strain biofilm. (B) Confocal microscopy image of the D39Δ luxS strain biofilm.

Techniques Used: Confocal Microscopy, In Vitro

Streptococcus pneumoniae D39 wild-type and D39Δ luxS planktonic and biofilm growth. (A) Planktonic growth optical density at 600 nm. (B) Quantification of biomass of in vitro biofilms grown for 18 h, using a CV-microplate assay. (C) Colony-forming unit (CFU) counts of in vitro biofilms grown for 18 h. Error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p
Figure Legend Snippet: Streptococcus pneumoniae D39 wild-type and D39Δ luxS planktonic and biofilm growth. (A) Planktonic growth optical density at 600 nm. (B) Quantification of biomass of in vitro biofilms grown for 18 h, using a CV-microplate assay. (C) Colony-forming unit (CFU) counts of in vitro biofilms grown for 18 h. Error bars are the standard deviation from the mean. Statistical significance was calculated using the Student's t -test, * p

Techniques Used: In Vitro, Standard Deviation

26) Product Images from "A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation"

Article Title: A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006495

Meningococcal biofilm formation of non piliated strains onto epithelial cells. (A) Quantification of the biomass covering cells infected with pilE mutants (Z5463 gfp Δ pilE and Z5463 gfp Δ pilE ΔMDA). Mutants were grown onto FaDu epithelial cells for 18 hours under constant flow. At least three independent experiments were performed. The results are normalized as a percentage of the mean biomass of Z5463 gfp Δ pilE , which was set to 100%. Error bars indicate the standard errors of the mean (SEM). * p
Figure Legend Snippet: Meningococcal biofilm formation of non piliated strains onto epithelial cells. (A) Quantification of the biomass covering cells infected with pilE mutants (Z5463 gfp Δ pilE and Z5463 gfp Δ pilE ΔMDA). Mutants were grown onto FaDu epithelial cells for 18 hours under constant flow. At least three independent experiments were performed. The results are normalized as a percentage of the mean biomass of Z5463 gfp Δ pilE , which was set to 100%. Error bars indicate the standard errors of the mean (SEM). * p

Techniques Used: Infection, Flow Cytometry

27) Product Images from "Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance"

Article Title: Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201505088

Mutation of genes encoding RpoE (HI0628) and MclA (HI0629) has similar effects on biofilm formation and antibiotic tolerance in H. influenzae Biofilms of different H. influenzae strains were developed after 24 h in μ-well chambers in sBHI medium and were the n treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type; (ii) rpoE ( HI0628 ); (iii) mclA ( HI0629 ); (iv) H. influenzae wild-type treated with 150 μg/ml azithromycin; (v) rpoE ( HI0628 ) treated with 150 μg/ml azithromycin; (vi) mclA ( HI0629 ) treated with 150 μg/ml azithromycin. For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bars representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P
Figure Legend Snippet: Mutation of genes encoding RpoE (HI0628) and MclA (HI0629) has similar effects on biofilm formation and antibiotic tolerance in H. influenzae Biofilms of different H. influenzae strains were developed after 24 h in μ-well chambers in sBHI medium and were the n treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type; (ii) rpoE ( HI0628 ); (iii) mclA ( HI0629 ); (iv) H. influenzae wild-type treated with 150 μg/ml azithromycin; (v) rpoE ( HI0628 ) treated with 150 μg/ml azithromycin; (vi) mclA ( HI0629 ) treated with 150 μg/ml azithromycin. For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bars representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P

Techniques Used: Mutagenesis, Standard Deviation, Two Tailed Test

Addition of corticosteroids affects biofilm formation and antibiotic tolerance of H. influenzae H. influenzae biofilms were developed after 24 h in μ-well chambers in sBHI medium with and without corticosteroid and were then treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type with DMSO (solvent control); (ii) H. influenzae wild-type treated with 1 μM beclomethasone (BEC); (iii) H. influenzae wild-type treated with 150 μg/ml azithromycin (AZO); and (iv) H. influenzae wild-type treated with 1 μM beclomethasone (BEC) and 150 μg/ml azithromycin (AZO). For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bar representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P
Figure Legend Snippet: Addition of corticosteroids affects biofilm formation and antibiotic tolerance of H. influenzae H. influenzae biofilms were developed after 24 h in μ-well chambers in sBHI medium with and without corticosteroid and were then treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type with DMSO (solvent control); (ii) H. influenzae wild-type treated with 1 μM beclomethasone (BEC); (iii) H. influenzae wild-type treated with 150 μg/ml azithromycin (AZO); and (iv) H. influenzae wild-type treated with 1 μM beclomethasone (BEC) and 150 μg/ml azithromycin (AZO). For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bar representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P

Techniques Used: Standard Deviation, Two Tailed Test

28) Product Images from "The Effect of Novel Heterocyclic Compounds on Cryptococcal Biofilm"

Article Title: The Effect of Novel Heterocyclic Compounds on Cryptococcal Biofilm

Journal: Journal of Fungi

doi: 10.3390/jof3030042

Effect of S-8 and NA-8 on the viability of C. neoformans and C. gattii biofilms. ( a ) Treatment of C. neoformans H-99 with S-8 and NA-8 during biofilm formation reduces nucleic acid staining by SYTO9. Treatment with S-8 also causes yeast death in the biofilm; ( b ) Treatment of C. gattii R-272 with S-8 and NA-8 during biofilm formation reduces nucleic acid staining by Syto9. Medium with 0.5% DMSO was used as a control. ×400. MIC, minimum inhibitory concentration. DMSO, dimethyl sulfoxide.
Figure Legend Snippet: Effect of S-8 and NA-8 on the viability of C. neoformans and C. gattii biofilms. ( a ) Treatment of C. neoformans H-99 with S-8 and NA-8 during biofilm formation reduces nucleic acid staining by SYTO9. Treatment with S-8 also causes yeast death in the biofilm; ( b ) Treatment of C. gattii R-272 with S-8 and NA-8 during biofilm formation reduces nucleic acid staining by Syto9. Medium with 0.5% DMSO was used as a control. ×400. MIC, minimum inhibitory concentration. DMSO, dimethyl sulfoxide.

Techniques Used: Staining, Concentration Assay

Effect of sub-inhibitory concentrations of S-8 and NA-8 on the biofilm thickness of C. neoformans ( a ) Treatment of C. neoformans H-99 during biofilm formation with NA-8 but not S-8 reduced biofilm thickness; ( b ) Treatment of C. gattii R-272 during biofilm formation with S-8 and NA-8 reduced biofilm thickness. Medium with 0.5% DMSO was used as a control. Mean ± SE. ×40.
Figure Legend Snippet: Effect of sub-inhibitory concentrations of S-8 and NA-8 on the biofilm thickness of C. neoformans ( a ) Treatment of C. neoformans H-99 during biofilm formation with NA-8 but not S-8 reduced biofilm thickness; ( b ) Treatment of C. gattii R-272 during biofilm formation with S-8 and NA-8 reduced biofilm thickness. Medium with 0.5% DMSO was used as a control. Mean ± SE. ×40.

Techniques Used:

The effect of S-8 and NA-8 on biofilm metabolic activity. C. neoformans H-99 ( a ) and C. gattii R-272 ( b ) biofilms were grown in 96-well microtiter plates as described in the Methods section. The inhibition of biofilm formation by S-8 and NA-8 were examined by measuring metabolic activity (XTT assay). Mean ± standard error (SE).
Figure Legend Snippet: The effect of S-8 and NA-8 on biofilm metabolic activity. C. neoformans H-99 ( a ) and C. gattii R-272 ( b ) biofilms were grown in 96-well microtiter plates as described in the Methods section. The inhibition of biofilm formation by S-8 and NA-8 were examined by measuring metabolic activity (XTT assay). Mean ± standard error (SE).

Techniques Used: Activity Assay, Inhibition, XTT Assay

29) Product Images from "Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans"

Article Title: Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00370-19

Dual-species biofilms formed using clinical commensals and S. mutans . Each commensal was mixed with UA159-Km in equal proportions and inoculated into BM supplemented with 18 mM Glc and 2 mM sucrose (BMGS) to form biofilms overnight on glass coverslips. BMGS was then replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc and incubated for another 24 h before visualization by confocal laser scanning microscopy (CLSM) after LIVE/DEAD staining (A) and CFU quantification of both species (B). Biofilms formed by UA159-Km alone were included for comparison. Values show the percentages of the populations constituted by commensal bacteria (top) and S. mutans (bottom), respectively. Asterisks indicate statistically significant differences in the viable counts of S. mutans compared to that on BMGlc. *, P
Figure Legend Snippet: Dual-species biofilms formed using clinical commensals and S. mutans . Each commensal was mixed with UA159-Km in equal proportions and inoculated into BM supplemented with 18 mM Glc and 2 mM sucrose (BMGS) to form biofilms overnight on glass coverslips. BMGS was then replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc and incubated for another 24 h before visualization by confocal laser scanning microscopy (CLSM) after LIVE/DEAD staining (A) and CFU quantification of both species (B). Biofilms formed by UA159-Km alone were included for comparison. Values show the percentages of the populations constituted by commensal bacteria (top) and S. mutans (bottom), respectively. Asterisks indicate statistically significant differences in the viable counts of S. mutans compared to that on BMGlc. *, P

Techniques Used: Gas Chromatography, Incubation, Confocal Laser Scanning Microscopy, Staining

Amino sugars impact the survival of S. mutans in CCS-derived biofilms. (A and B) To initiate a biofilm, UA159-Km and cell-containing saliva (CCS) were used simultaneously (groups a and d) or 24 h apart (groups b and c) to inoculate BM supplemented with 2 mM sucrose and 18 mM various other carbohydrates. After 24 h of incubation, the biofilms were washed and resupplied with fresh medium and additional bacteria as specified (see Materials and Methods for details). (B) At the end of 2-day incubations, biofilms were processed to quantify the number of CFU of UA159-Km. Asterisks indicate statistically significant differences compared to cultures prepared with Glc. *, P
Figure Legend Snippet: Amino sugars impact the survival of S. mutans in CCS-derived biofilms. (A and B) To initiate a biofilm, UA159-Km and cell-containing saliva (CCS) were used simultaneously (groups a and d) or 24 h apart (groups b and c) to inoculate BM supplemented with 2 mM sucrose and 18 mM various other carbohydrates. After 24 h of incubation, the biofilms were washed and resupplied with fresh medium and additional bacteria as specified (see Materials and Methods for details). (B) At the end of 2-day incubations, biofilms were processed to quantify the number of CFU of UA159-Km. Asterisks indicate statistically significant differences compared to cultures prepared with Glc. *, P

Techniques Used: Derivative Assay, Incubation, Gas Chromatography

30) Product Images from "Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents"

Article Title: Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents

Journal: Scientific Reports

doi: 10.1038/srep42886

Confocal laser scan microscopy images of C. albicans (ATCC 90028) biofilm grown for 48 hours at 35 °C. The DNA of the cells was stained by 0.01% acridine orange for 2 minutes. A laser with a wavelength of 488 nm was used. ( A ) Matured biofilm in a 2D image. Scale bar equals 50 μm. ( B ) Matured biofilm a 2.5 D image.
Figure Legend Snippet: Confocal laser scan microscopy images of C. albicans (ATCC 90028) biofilm grown for 48 hours at 35 °C. The DNA of the cells was stained by 0.01% acridine orange for 2 minutes. A laser with a wavelength of 488 nm was used. ( A ) Matured biofilm in a 2D image. Scale bar equals 50 μm. ( B ) Matured biofilm a 2.5 D image.

Techniques Used: Microscopy, Staining

Growth (mean with standard deviation in %) of E. dermatitidis (P2) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P
Figure Legend Snippet: Growth (mean with standard deviation in %) of E. dermatitidis (P2) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P

Techniques Used: Standard Deviation, XTT Assay

Growth (mean with standard deviation in %) of E. dermatitidis (CF2) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P
Figure Legend Snippet: Growth (mean with standard deviation in %) of E. dermatitidis (CF2) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P

Techniques Used: Standard Deviation, XTT Assay

Confocal laser scan microscopy images of Exophiala dermatitidis biofilm grown for 48 hours at 35 °C. The DNA of the cells was stained by 0.01% acridine orange for 2 minutes. ( A ) Matured biofilm of isolate P2 (CBS 116372) in a 2D image. ( B ) Matured biofilm of isolate CF2 (CBS 552.90) in a 2D image. ( C ) Matured biofilm of isolate P2 in a 2.5 D image. ( D ) Matured biofilm of isolate CF2 in a 2.5D image. Scale bar equals 50 μm. A laser with a wavelength of 488 nm was used.
Figure Legend Snippet: Confocal laser scan microscopy images of Exophiala dermatitidis biofilm grown for 48 hours at 35 °C. The DNA of the cells was stained by 0.01% acridine orange for 2 minutes. ( A ) Matured biofilm of isolate P2 (CBS 116372) in a 2D image. ( B ) Matured biofilm of isolate CF2 (CBS 552.90) in a 2D image. ( C ) Matured biofilm of isolate P2 in a 2.5 D image. ( D ) Matured biofilm of isolate CF2 in a 2.5D image. Scale bar equals 50 μm. A laser with a wavelength of 488 nm was used.

Techniques Used: Microscopy, Staining

Exophiala dermatitidis biofilm formation. Optical density at 620 nm (OD 620 ) as measured after staining with crystal violet (CV), and optical density at 492 nm (OD 492 ) after XTT reduction. Each data point represents the mean of at least three independent experiments. P1, P2, P10 = invasive E. dermatitidis isolates from non-CF patients. CF1, CF2, CF4, CF39, CF38 = E. dermatitidis isolates from CF patients. C.a. = Candida albicans. CFU = colony-forming units.
Figure Legend Snippet: Exophiala dermatitidis biofilm formation. Optical density at 620 nm (OD 620 ) as measured after staining with crystal violet (CV), and optical density at 492 nm (OD 492 ) after XTT reduction. Each data point represents the mean of at least three independent experiments. P1, P2, P10 = invasive E. dermatitidis isolates from non-CF patients. CF1, CF2, CF4, CF39, CF38 = E. dermatitidis isolates from CF patients. C.a. = Candida albicans. CFU = colony-forming units.

Techniques Used: Staining

Relative biofilm formation of Candida albicans (ATCC 90028) and various isolates of Exophiala dermatitidis. Box whiskers with 10–90 percentile. E = environmental origin. CF = isolate from cystic fibrosis (CF) patient. P = invasive isolate from non-CF patient. Relative biofilm formation detected by staining with crystal violet (0.1%) for 20 minutes after biofilm formation at 35 °C over 48 hours. Each data point represents the mean of at least three independent experiments. * P
Figure Legend Snippet: Relative biofilm formation of Candida albicans (ATCC 90028) and various isolates of Exophiala dermatitidis. Box whiskers with 10–90 percentile. E = environmental origin. CF = isolate from cystic fibrosis (CF) patient. P = invasive isolate from non-CF patient. Relative biofilm formation detected by staining with crystal violet (0.1%) for 20 minutes after biofilm formation at 35 °C over 48 hours. Each data point represents the mean of at least three independent experiments. * P

Techniques Used: Staining

Confocal laser scan microscopy images of E. dermatitidis isolate P2 (CBS 116372) biofilm, formed in the presence of antiinfective agents. Biofilm was grown for 48 hours at 35 °C in the presence of ( A , C ) 64 mg/L colistin, ( B , D ) 8 mg/L micafungin. 2D ( A , B ) and 2.5 D ( C , D ) images were taken. The DNA of the cells was stained by 0.01% acridine orange for 2 minutes. Scale bar equals 50 μm. A laser with a wavelength of 488 nm was used.
Figure Legend Snippet: Confocal laser scan microscopy images of E. dermatitidis isolate P2 (CBS 116372) biofilm, formed in the presence of antiinfective agents. Biofilm was grown for 48 hours at 35 °C in the presence of ( A , C ) 64 mg/L colistin, ( B , D ) 8 mg/L micafungin. 2D ( A , B ) and 2.5 D ( C , D ) images were taken. The DNA of the cells was stained by 0.01% acridine orange for 2 minutes. Scale bar equals 50 μm. A laser with a wavelength of 488 nm was used.

Techniques Used: Microscopy, Staining

Relative biofilm formation of Candida al bicans (ATCC 90028) and various isolates of Exophiala dermatitidis . Box whiskers with 10–90 percentile. The biofilm formation wasdetected by staining with crystal violet (0.1%) for 20 minutes. Biofilm formation at 35 °C over 24 and 48 hours. Each data point represents the mean of at least three independent experiments. * P
Figure Legend Snippet: Relative biofilm formation of Candida al bicans (ATCC 90028) and various isolates of Exophiala dermatitidis . Box whiskers with 10–90 percentile. The biofilm formation wasdetected by staining with crystal violet (0.1%) for 20 minutes. Biofilm formation at 35 °C over 24 and 48 hours. Each data point represents the mean of at least three independent experiments. * P

Techniques Used: Staining

Growth (mean with standard deviation in %) of C. albicans (ATCC 90028) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P
Figure Legend Snippet: Growth (mean with standard deviation in %) of C. albicans (ATCC 90028) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P

Techniques Used: Standard Deviation, XTT Assay

Growth (mean with standard deviation in %) of E. dermatitidis (P1) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P
Figure Legend Snippet: Growth (mean with standard deviation in %) of E. dermatitidis (P1) biofilm after treatment with voriconazole ( A ), micafungin ( B ), colistin ( C ), farnesol ( D ), epigallocatechin gallate (EGCG, E ) and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG, F ) in the concentrations indicated on the x -axis. Control = growth control without treatment. Adhesion = addition of drug at time point 0. Biofilm formation = drug addition after 2 hours. Mature biofilm = drug addition after 48 hours of biofilm formation. Growth was evaluated by XTT assay; optical density readings at 492 nm (OD 492 ) were measured. * P

Techniques Used: Standard Deviation, XTT Assay

31) Product Images from "Streptococcus mutans Copper Chaperone, CopZ, is critical for biofilm formation and competitiveness"

Article Title: Streptococcus mutans Copper Chaperone, CopZ, is critical for biofilm formation and competitiveness

Journal: Molecular oral microbiology

doi: 10.1111/omi.12150

S. mutans UA159 copZ mutant is defective in biofilm formation Single-gene mutants of copYAZ and the parent strain were allowed to form biofilms for 18 hours in biofilm media supplemented with 1% sucrose. Biomass was quantitated by crystal violet staining and reading at O.D. 562 . Complement strain of ΔcopZ restored biofilm formation (ΔcopZ/copZpVPT). **=p
Figure Legend Snippet: S. mutans UA159 copZ mutant is defective in biofilm formation Single-gene mutants of copYAZ and the parent strain were allowed to form biofilms for 18 hours in biofilm media supplemented with 1% sucrose. Biomass was quantitated by crystal violet staining and reading at O.D. 562 . Complement strain of ΔcopZ restored biofilm formation (ΔcopZ/copZpVPT). **=p

Techniques Used: Mutagenesis, Staining

32) Product Images from "Downregulation of Autolysin-Encoding Genes by Phage-Derived Lytic Proteins Inhibits Biofilm Formation in Staphylococcus aureus"

Article Title: Downregulation of Autolysin-Encoding Genes by Phage-Derived Lytic Proteins Inhibits Biofilm Formation in Staphylococcus aureus

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.02724-16

Confocal laser scanning micrographs of S. aureus biofilms treated or not with CHAPSH3b. Biofilms of strain ISP479r were grown for 24 h at 37°C and then treated with NaPi buffer alone (A) or containing 4× MIC of CHAPSH3b (250.24 μg/ml).
Figure Legend Snippet: Confocal laser scanning micrographs of S. aureus biofilms treated or not with CHAPSH3b. Biofilms of strain ISP479r were grown for 24 h at 37°C and then treated with NaPi buffer alone (A) or containing 4× MIC of CHAPSH3b (250.24 μg/ml).

Techniques Used:

Biofilm formation by S. aureus strains in the presence of subinhibitory concentrations of CHAPSH3b. Biofilms of strains IPLA 1 (A and C) and ISP479r (B and D) were grown for 24 h at 25°C (A and B) or 37°C (C and D). The planktonic phase
Figure Legend Snippet: Biofilm formation by S. aureus strains in the presence of subinhibitory concentrations of CHAPSH3b. Biofilms of strains IPLA 1 (A and C) and ISP479r (B and D) were grown for 24 h at 25°C (A and B) or 37°C (C and D). The planktonic phase

Techniques Used:

Effect of subinhibitory concentrations of CHAPSH3b on S. aureus IPLA 1 biofilm formation. (A to D) Micrographs obtained by CLSM of biofilms grown in the absence (A and B) or presence (C and D) of 0.98 μg/ml CHAPSH3b at 25°C. The samples
Figure Legend Snippet: Effect of subinhibitory concentrations of CHAPSH3b on S. aureus IPLA 1 biofilm formation. (A to D) Micrographs obtained by CLSM of biofilms grown in the absence (A and B) or presence (C and D) of 0.98 μg/ml CHAPSH3b at 25°C. The samples

Techniques Used: Confocal Laser Scanning Microscopy

Effect of subinhibitory concentrations of CHAPSH3b on S. aureus ISP479r biofilm formation. (A to D) Micrographs obtained by CLSM of biofilms grown in the absence (A and B) or presence (C and D) of 0.98 μg/ml CHAPSH3b at 25°C. The samples
Figure Legend Snippet: Effect of subinhibitory concentrations of CHAPSH3b on S. aureus ISP479r biofilm formation. (A to D) Micrographs obtained by CLSM of biofilms grown in the absence (A and B) or presence (C and D) of 0.98 μg/ml CHAPSH3b at 25°C. The samples

Techniques Used: Confocal Laser Scanning Microscopy

Treatment of preformed biofilms of S. aureus IPLA 1 and ISP479r with the fusion protein CHAPSH3b. Biofilms were formed for 24 h and then treated with 4× MIC of CHAPSH3b for 6 h at 25°C (A and B) or 37°C (C and D). Control wells
Figure Legend Snippet: Treatment of preformed biofilms of S. aureus IPLA 1 and ISP479r with the fusion protein CHAPSH3b. Biofilms were formed for 24 h and then treated with 4× MIC of CHAPSH3b for 6 h at 25°C (A and B) or 37°C (C and D). Control wells

Techniques Used:

Effect of atl mutation on the antibiofilm effect of CHAPSH3b. Biofilms of S. aureus SA113 (black), its isogenic atl mutant (white), and the complemented strain (gray) were formed at 25°C in the presence of increasing concentrations of the lysin
Figure Legend Snippet: Effect of atl mutation on the antibiofilm effect of CHAPSH3b. Biofilms of S. aureus SA113 (black), its isogenic atl mutant (white), and the complemented strain (gray) were formed at 25°C in the presence of increasing concentrations of the lysin

Techniques Used: Mutagenesis

33) Product Images from "Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans"

Article Title: Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00370-19

Dual-species biofilms formed using clinical commensals and S. mutans . Each commensal was mixed with UA159-Km in equal proportions and inoculated into BM supplemented with 18 mM Glc and 2 mM sucrose (BMGS) to form biofilms overnight on glass coverslips. BMGS was then replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc and incubated for another 24 h before visualization by confocal laser scanning microscopy (CLSM) after LIVE/DEAD staining (A) and CFU quantification of both species (B). Biofilms formed by UA159-Km alone were included for comparison. Values show the percentages of the populations constituted by commensal bacteria (top) and S. mutans (bottom), respectively. Asterisks indicate statistically significant differences in the viable counts of S. mutans compared to that on BMGlc. *, P
Figure Legend Snippet: Dual-species biofilms formed using clinical commensals and S. mutans . Each commensal was mixed with UA159-Km in equal proportions and inoculated into BM supplemented with 18 mM Glc and 2 mM sucrose (BMGS) to form biofilms overnight on glass coverslips. BMGS was then replaced by BM supplemented with 20 mM Glc, GlcN, or GlcNAc and incubated for another 24 h before visualization by confocal laser scanning microscopy (CLSM) after LIVE/DEAD staining (A) and CFU quantification of both species (B). Biofilms formed by UA159-Km alone were included for comparison. Values show the percentages of the populations constituted by commensal bacteria (top) and S. mutans (bottom), respectively. Asterisks indicate statistically significant differences in the viable counts of S. mutans compared to that on BMGlc. *, P

Techniques Used: Gas Chromatography, Incubation, Confocal Laser Scanning Microscopy, Staining

Amino sugars impact the survival of S. mutans in CCS-derived biofilms. (A and B) To initiate a biofilm, UA159-Km and cell-containing saliva (CCS) were used simultaneously (groups a and d) or 24 h apart (groups b and c) to inoculate BM supplemented with 2 mM sucrose and 18 mM various other carbohydrates. After 24 h of incubation, the biofilms were washed and resupplied with fresh medium and additional bacteria as specified (see Materials and Methods for details). (B) At the end of 2-day incubations, biofilms were processed to quantify the number of CFU of UA159-Km. Asterisks indicate statistically significant differences compared to cultures prepared with Glc. *, P
Figure Legend Snippet: Amino sugars impact the survival of S. mutans in CCS-derived biofilms. (A and B) To initiate a biofilm, UA159-Km and cell-containing saliva (CCS) were used simultaneously (groups a and d) or 24 h apart (groups b and c) to inoculate BM supplemented with 2 mM sucrose and 18 mM various other carbohydrates. After 24 h of incubation, the biofilms were washed and resupplied with fresh medium and additional bacteria as specified (see Materials and Methods for details). (B) At the end of 2-day incubations, biofilms were processed to quantify the number of CFU of UA159-Km. Asterisks indicate statistically significant differences compared to cultures prepared with Glc. *, P

Techniques Used: Derivative Assay, Incubation, Gas Chromatography

Related Articles

Flow Cytometry:

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase
Article Snippet: .. Despite our efforts to apply similar shear forces to growing biofilms in the Biostream flow cell and the IBIDI µ-slides used for CLSM, it cannot be ruled out that biofilms produced in the two systems are different, particularly regarding their 3D structure. .. However, the finding that the results obtained in the CLSM experiments for the different treatments are largely in agreement with those obtained with the Biostream flow cell argues against a significant impact of the flow cell system used on the susceptibility of the produced biofilms to the enzymes.

Produced:

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase
Article Snippet: .. Despite our efforts to apply similar shear forces to growing biofilms in the Biostream flow cell and the IBIDI µ-slides used for CLSM, it cannot be ruled out that biofilms produced in the two systems are different, particularly regarding their 3D structure. .. However, the finding that the results obtained in the CLSM experiments for the different treatments are largely in agreement with those obtained with the Biostream flow cell argues against a significant impact of the flow cell system used on the susceptibility of the produced biofilms to the enzymes.

Incubation:

Article Title: Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance
Article Snippet: .. Cultivation and image analysis of biofilms H. influenzae strains were allowed to form biofilm in static μ-well chambers (Ibidi-iTreat) containing sBHI media incubated at 37°C as previously described (Jurcisek et al , ). ..

Confocal Laser Scanning Microscopy:

Article Title: Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella
Article Snippet: .. Biofilms of H. pylori SS1 were prepared as described above using BB2; however, for confocal laser scanning microscopy (CLSM), μ-Slide 8-well glass bottom chamber slides (ibidi, Germany) were used instead of 96-well microtiter plates. .. Three-day-old biofilms were stained with FilmTracer FM 1–43 (Invitrogen), BOBO-3 (Invitrogen), Filmtracer SYPRO Ruby biofilm matrix stain (Invitrogen), or the FilmTracer LIVE/DEAD biofilm viability kit (Invitrogen) according to the manufacturer’s instructions.

Article Title: Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase
Article Snippet: .. Despite our efforts to apply similar shear forces to growing biofilms in the Biostream flow cell and the IBIDI µ-slides used for CLSM, it cannot be ruled out that biofilms produced in the two systems are different, particularly regarding their 3D structure. .. However, the finding that the results obtained in the CLSM experiments for the different treatments are largely in agreement with those obtained with the Biostream flow cell argues against a significant impact of the flow cell system used on the susceptibility of the produced biofilms to the enzymes.

Imaging:

Article Title: Macrophage Migration Is Impaired within Candida albicans Biofilms
Article Snippet: .. Biofilms were prepared by seeding µ-Slide 8-well chambers (ibidi GmbH, Munich, Germany) with 3 × 105 Leicestershire live C. albicans yeast in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Slough, UK), supplemented with 10% (v /v ) heat-inactivated FCS (Biosera, Ringmer, UK), 200 U/mL penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK), and 2 mM L-glutamine (Invitrogen, Paisley, UK), at 37 °C for 48 h. There were two assay designs, with planktonic yeast added to either imaging dishes containing macrophages, or to fungal biofilms with macrophages added in. ..

Modification:

Article Title: Macrophage Migration Is Impaired within Candida albicans Biofilms
Article Snippet: .. Biofilms were prepared by seeding µ-Slide 8-well chambers (ibidi GmbH, Munich, Germany) with 3 × 105 Leicestershire live C. albicans yeast in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Slough, UK), supplemented with 10% (v /v ) heat-inactivated FCS (Biosera, Ringmer, UK), 200 U/mL penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK), and 2 mM L-glutamine (Invitrogen, Paisley, UK), at 37 °C for 48 h. There were two assay designs, with planktonic yeast added to either imaging dishes containing macrophages, or to fungal biofilms with macrophages added in. ..

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  • 85
    ibidi biofilms biofilms
    Extrinsic DNA localizes to cellular polysaccharides. <t>Biofilms</t> were grown for 24 h in RPMI with 10% [v/v] FCS and 1 mg ml −1 sheared DNA and stained for total polysaccharides using Concavalin A coupled to fluorescent Alexa-fluor 488 (CAAF) or for nucleic acid using propidium iodide (PI). The two right panels represent an overlay of CLSM images from the CAAF and PI channel and the according brightfield images (BF), respectively. White bars indicate 20 μm.
    Biofilms Biofilms, supplied by ibidi, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ibidi biofilm
    Mutation of genes encoding RpoE (HI0628) and MclA (HI0629) has similar effects on <t>biofilm</t> formation and antibiotic tolerance in H. influenzae Biofilms of different H. influenzae strains were developed after 24 h in μ-well chambers in sBHI medium and were the n treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type; (ii) rpoE ( HI0628 ); (iii) mclA ( HI0629 ); (iv) H. influenzae wild-type treated with 150 μg/ml azithromycin; (v) rpoE ( HI0628 ) treated with 150 μg/ml azithromycin; (vi) mclA ( HI0629 ) treated with 150 μg/ml azithromycin. For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bars representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P
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    90
    ibidi confocal microscopy psl biofilms
    The glycoside hydrolases PslG h and PelA h hydrolyze the exopolysaccharides Pel and <t>Psl</t> in a biofilm. Representative confocal images of Psl <t>biofilms</t> grown statically for 24 hours ( top ) and Pel biofilms cultivated for 48 hours ( bottom ) under flow conditions and treated with wild-type hydrolases or hydrolases that have point mutations to catalytic residues. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pel-specific lectin (red). Scale bars, 30 μm.
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    Extrinsic DNA localizes to cellular polysaccharides. Biofilms were grown for 24 h in RPMI with 10% [v/v] FCS and 1 mg ml −1 sheared DNA and stained for total polysaccharides using Concavalin A coupled to fluorescent Alexa-fluor 488 (CAAF) or for nucleic acid using propidium iodide (PI). The two right panels represent an overlay of CLSM images from the CAAF and PI channel and the according brightfield images (BF), respectively. White bars indicate 20 μm.

    Journal: Frontiers in Microbiology

    Article Title: Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus

    doi: 10.3389/fmicb.2013.00141

    Figure Lengend Snippet: Extrinsic DNA localizes to cellular polysaccharides. Biofilms were grown for 24 h in RPMI with 10% [v/v] FCS and 1 mg ml −1 sheared DNA and stained for total polysaccharides using Concavalin A coupled to fluorescent Alexa-fluor 488 (CAAF) or for nucleic acid using propidium iodide (PI). The two right panels represent an overlay of CLSM images from the CAAF and PI channel and the according brightfield images (BF), respectively. White bars indicate 20 μm.

    Article Snippet: Microscopy imaging of biofilms Biofilms for microscopic observation were generated for 24 h on ibiTreat plastic 35 mm high μ-dishes (Ibidi GmbH, Munich, Germany) with an initial inoculum density of 104 spores in a total volume of 1.0 ml in RPMI supplemented with inducers as mentioned above.

    Techniques: Staining, Confocal Laser Scanning Microscopy

    Polysaccharides provide the main stability in early DNA induced biofilms. Biofilms were grown with DNA over a time of 24 h and DNase I was added during early biofilm development (bottom panel). (A) Biofilms were imaged by CLSM after staining with CAAF for polysaccharides, with fluorescence brightener (FB28) for chitin and PI for DNA. White bars indicate 20 μm. Note that DNase I treatment completely diminishes the PI signal without changing the CAAF signal. Three dimensional reconstruction of DNA induced biofilms displaying the bedding layer network structure of polysaccharide and DNA before (B) and after DNase I treatment (C) . The white arrow indicates DNA entirely covered by polysaccharide and hence, presumably escaped enzymatic digestion.

    Journal: Frontiers in Microbiology

    Article Title: Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus

    doi: 10.3389/fmicb.2013.00141

    Figure Lengend Snippet: Polysaccharides provide the main stability in early DNA induced biofilms. Biofilms were grown with DNA over a time of 24 h and DNase I was added during early biofilm development (bottom panel). (A) Biofilms were imaged by CLSM after staining with CAAF for polysaccharides, with fluorescence brightener (FB28) for chitin and PI for DNA. White bars indicate 20 μm. Note that DNase I treatment completely diminishes the PI signal without changing the CAAF signal. Three dimensional reconstruction of DNA induced biofilms displaying the bedding layer network structure of polysaccharide and DNA before (B) and after DNase I treatment (C) . The white arrow indicates DNA entirely covered by polysaccharide and hence, presumably escaped enzymatic digestion.

    Article Snippet: Microscopy imaging of biofilms Biofilms for microscopic observation were generated for 24 h on ibiTreat plastic 35 mm high μ-dishes (Ibidi GmbH, Munich, Germany) with an initial inoculum density of 104 spores in a total volume of 1.0 ml in RPMI supplemented with inducers as mentioned above.

    Techniques: Confocal Laser Scanning Microscopy, Staining, Fluorescence

    Kinetics of biofilm formation. Biofilms at conidial densities of 10 5 per well were grown in RPMI alone (blue), in the presence of 10% [v/v] FCS (red) or 1 mg/ml of DNA (green) and fungal viability was monitored over time. Metabolic reduction of resazurin was measured as the decrease in absorbance at a wavelength of 600 nm. Data are expressed as the change in absorbance relative to an untreated control and represent the means and SDs of a biological triplicate. Time periods of linear growth kinetics for all three conditions are displayed in the inset.

    Journal: Frontiers in Microbiology

    Article Title: Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus

    doi: 10.3389/fmicb.2013.00141

    Figure Lengend Snippet: Kinetics of biofilm formation. Biofilms at conidial densities of 10 5 per well were grown in RPMI alone (blue), in the presence of 10% [v/v] FCS (red) or 1 mg/ml of DNA (green) and fungal viability was monitored over time. Metabolic reduction of resazurin was measured as the decrease in absorbance at a wavelength of 600 nm. Data are expressed as the change in absorbance relative to an untreated control and represent the means and SDs of a biological triplicate. Time periods of linear growth kinetics for all three conditions are displayed in the inset.

    Article Snippet: Microscopy imaging of biofilms Biofilms for microscopic observation were generated for 24 h on ibiTreat plastic 35 mm high μ-dishes (Ibidi GmbH, Munich, Germany) with an initial inoculum density of 104 spores in a total volume of 1.0 ml in RPMI supplemented with inducers as mentioned above.

    Techniques:

    Extrinsic DNA increases ECM formation in biofilms. (A) CAAF fluorescence of total fungal polysaccharides was determined in biofilms seeded at an initial density of 10 4 per well and used as a measure for ECM production in biofilms in the presence of 1 mg ml −1 DNA from indicated sources (sDNA, sheared herring sperm DNA; gDNA, genomic DNA from A. fumigatus ; PC, phenol chloroform extracted DNA; pDNA; purified plasmid DNA). Displayed values represent the means and SDs of three independent experiments and an asterisks indicate p

    Journal: Frontiers in Microbiology

    Article Title: Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus

    doi: 10.3389/fmicb.2013.00141

    Figure Lengend Snippet: Extrinsic DNA increases ECM formation in biofilms. (A) CAAF fluorescence of total fungal polysaccharides was determined in biofilms seeded at an initial density of 10 4 per well and used as a measure for ECM production in biofilms in the presence of 1 mg ml −1 DNA from indicated sources (sDNA, sheared herring sperm DNA; gDNA, genomic DNA from A. fumigatus ; PC, phenol chloroform extracted DNA; pDNA; purified plasmid DNA). Displayed values represent the means and SDs of three independent experiments and an asterisks indicate p

    Article Snippet: Microscopy imaging of biofilms Biofilms for microscopic observation were generated for 24 h on ibiTreat plastic 35 mm high μ-dishes (Ibidi GmbH, Munich, Germany) with an initial inoculum density of 104 spores in a total volume of 1.0 ml in RPMI supplemented with inducers as mentioned above.

    Techniques: Fluorescence, Purification, Plasmid Preparation

    Extrinsic DNA influences ECM distribution. Peripheries of biofilms grown with RPMI with either FCS or sDNA were imaged by CLSM displaying the tips of terminal hyphae. Note the cohesive properties of ECM polysaccharides in the presence of FCS and the network-like distribution of the ECM in response to DNA. White bars indicate 20 μm.

    Journal: Frontiers in Microbiology

    Article Title: Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus

    doi: 10.3389/fmicb.2013.00141

    Figure Lengend Snippet: Extrinsic DNA influences ECM distribution. Peripheries of biofilms grown with RPMI with either FCS or sDNA were imaged by CLSM displaying the tips of terminal hyphae. Note the cohesive properties of ECM polysaccharides in the presence of FCS and the network-like distribution of the ECM in response to DNA. White bars indicate 20 μm.

    Article Snippet: Microscopy imaging of biofilms Biofilms for microscopic observation were generated for 24 h on ibiTreat plastic 35 mm high μ-dishes (Ibidi GmbH, Munich, Germany) with an initial inoculum density of 104 spores in a total volume of 1.0 ml in RPMI supplemented with inducers as mentioned above.

    Techniques: Confocal Laser Scanning Microscopy

    Surface adhesion and biofilm formation with extrinsic DNA. A. fumigatus conidia were grown either in RPMI without additives (RPMI), with addition of 10% [v/v] fetal calf serum (FCS) or with 1 mg ml −1 sheared herring sperm DNA (sDNA) in submerged, shaken culture (A) or in static biofilm inducing conditions (B) . Growth was monitored after 48 h and expressed as total biomass formation during shaking (blue) and static (red) culture conditions (C) . Displayed values represent the means and SDs of three independent experiments and asterisks indicate p

    Journal: Frontiers in Microbiology

    Article Title: Extrinsic extracellular DNA leads to biofilm formation and colocalizes with matrix polysaccharides in the human pathogenic fungus Aspergillus fumigatus

    doi: 10.3389/fmicb.2013.00141

    Figure Lengend Snippet: Surface adhesion and biofilm formation with extrinsic DNA. A. fumigatus conidia were grown either in RPMI without additives (RPMI), with addition of 10% [v/v] fetal calf serum (FCS) or with 1 mg ml −1 sheared herring sperm DNA (sDNA) in submerged, shaken culture (A) or in static biofilm inducing conditions (B) . Growth was monitored after 48 h and expressed as total biomass formation during shaking (blue) and static (red) culture conditions (C) . Displayed values represent the means and SDs of three independent experiments and asterisks indicate p

    Article Snippet: Microscopy imaging of biofilms Biofilms for microscopic observation were generated for 24 h on ibiTreat plastic 35 mm high μ-dishes (Ibidi GmbH, Munich, Germany) with an initial inoculum density of 104 spores in a total volume of 1.0 ml in RPMI supplemented with inducers as mentioned above.

    Techniques:

    Mutation of genes encoding RpoE (HI0628) and MclA (HI0629) has similar effects on biofilm formation and antibiotic tolerance in H. influenzae Biofilms of different H. influenzae strains were developed after 24 h in μ-well chambers in sBHI medium and were the n treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type; (ii) rpoE ( HI0628 ); (iii) mclA ( HI0629 ); (iv) H. influenzae wild-type treated with 150 μg/ml azithromycin; (v) rpoE ( HI0628 ) treated with 150 μg/ml azithromycin; (vi) mclA ( HI0629 ) treated with 150 μg/ml azithromycin. For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bars representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P

    Journal: EMBO Molecular Medicine

    Article Title: Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance

    doi: 10.15252/emmm.201505088

    Figure Lengend Snippet: Mutation of genes encoding RpoE (HI0628) and MclA (HI0629) has similar effects on biofilm formation and antibiotic tolerance in H. influenzae Biofilms of different H. influenzae strains were developed after 24 h in μ-well chambers in sBHI medium and were the n treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type; (ii) rpoE ( HI0628 ); (iii) mclA ( HI0629 ); (iv) H. influenzae wild-type treated with 150 μg/ml azithromycin; (v) rpoE ( HI0628 ) treated with 150 μg/ml azithromycin; (vi) mclA ( HI0629 ) treated with 150 μg/ml azithromycin. For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bars representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P

    Article Snippet: Cultivation and image analysis of biofilms H. influenzae strains were allowed to form biofilm in static μ-well chambers (Ibidi-iTreat) containing sBHI media incubated at 37°C as previously described (Jurcisek et al , ).

    Techniques: Mutagenesis, Standard Deviation, Two Tailed Test

    Addition of corticosteroids affects biofilm formation and antibiotic tolerance of H. influenzae H. influenzae biofilms were developed after 24 h in μ-well chambers in sBHI medium with and without corticosteroid and were then treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type with DMSO (solvent control); (ii) H. influenzae wild-type treated with 1 μM beclomethasone (BEC); (iii) H. influenzae wild-type treated with 150 μg/ml azithromycin (AZO); and (iv) H. influenzae wild-type treated with 1 μM beclomethasone (BEC) and 150 μg/ml azithromycin (AZO). For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bar representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P

    Journal: EMBO Molecular Medicine

    Article Title: Haemophilus influenzae responds to glucocorticoids used in asthma therapy by modulation of biofilm formation and antibiotic resistance

    doi: 10.15252/emmm.201505088

    Figure Lengend Snippet: Addition of corticosteroids affects biofilm formation and antibiotic tolerance of H. influenzae H. influenzae biofilms were developed after 24 h in μ-well chambers in sBHI medium with and without corticosteroid and were then treated with the antibiotic azithromycin as specified. (i) H. influenzae wild-type with DMSO (solvent control); (ii) H. influenzae wild-type treated with 1 μM beclomethasone (BEC); (iii) H. influenzae wild-type treated with 150 μg/ml azithromycin (AZO); and (iv) H. influenzae wild-type treated with 1 μM beclomethasone (BEC) and 150 μg/ml azithromycin (AZO). For these experiments, H. influenzae was visualized with SYTO9 (green strain), as described in Materials and Methods. Scale bars = 20 μm. Images shown are representative of 12 images from five independent experiments. The biofilm biomass after treatments was quantified using COMSTAT. Data are presented as the average of five replicates, with error bar representing the standard deviation of the data. Statistical significance by two-tailed Student’s t -test is indicated: ** P

    Article Snippet: Cultivation and image analysis of biofilms H. influenzae strains were allowed to form biofilm in static μ-well chambers (Ibidi-iTreat) containing sBHI media incubated at 37°C as previously described (Jurcisek et al , ).

    Techniques: Standard Deviation, Two Tailed Test

    Biofilm formation by E. coli in monocultures or in cocultures with E. faecalis . (A and B) Confocal laser scanning microscopy of static biofilms formed by E. coli (expressing mCherry) grown individually (A) or in a mixed culture with E. faecalis (expressing enhanced GFP [EGFP]) (B). Scale bars, 40 μm. The mixed culture was initially inoculated at 1:1 ratio. (C) Side views of the mixed E. coli - E. faecalis biofilm. Scale bars, 20 μm. (D) Distribution of microcolony volumes in static single- and double-species biofilms of E. coli . The P value for the difference between single- and double-species biofilms was calculated using an unpaired t test (the data distribution was confirmed to be normal).

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Biofilm formation by E. coli in monocultures or in cocultures with E. faecalis . (A and B) Confocal laser scanning microscopy of static biofilms formed by E. coli (expressing mCherry) grown individually (A) or in a mixed culture with E. faecalis (expressing enhanced GFP [EGFP]) (B). Scale bars, 40 μm. The mixed culture was initially inoculated at 1:1 ratio. (C) Side views of the mixed E. coli - E. faecalis biofilm. Scale bars, 20 μm. (D) Distribution of microcolony volumes in static single- and double-species biofilms of E. coli . The P value for the difference between single- and double-species biofilms was calculated using an unpaired t test (the data distribution was confirmed to be normal).

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Confocal Laser Scanning Microscopy, Expressing

    Aggregation of E. coli during early stages of biofilm formation in single- or double-species cultures. (A to C) Aggregates formed at the well surface by E. coli cells (expressing EGFP) grown in monoculture (A) or cocultured with unlabeled E. faecalis (B and C). Cells of E. faecalis can be seen in the phase-contrast channel as distinct chains of round cells or as parts of E. coli - E. faecalis aggregates. Scale bars, 30 μm (A and B) or 20 μm (C). White arrows in panel C indicate chains and aggregates of E. faecalis . (D) Sizes of E. coli aggregates in monoculture or in coculture with E. faecalis . Means of at least four independent replicates are shown; error bars indicate standard deviations. P values for the differences between single- and double-species biofilms were calculated using Mann-Whitney tests. **, P

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Aggregation of E. coli during early stages of biofilm formation in single- or double-species cultures. (A to C) Aggregates formed at the well surface by E. coli cells (expressing EGFP) grown in monoculture (A) or cocultured with unlabeled E. faecalis (B and C). Cells of E. faecalis can be seen in the phase-contrast channel as distinct chains of round cells or as parts of E. coli - E. faecalis aggregates. Scale bars, 30 μm (A and B) or 20 μm (C). White arrows in panel C indicate chains and aggregates of E. faecalis . (D) Sizes of E. coli aggregates in monoculture or in coculture with E. faecalis . Means of at least four independent replicates are shown; error bars indicate standard deviations. P values for the differences between single- and double-species biofilms were calculated using Mann-Whitney tests. **, P

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Expressing, MANN-WHITNEY

    Proposed model of a static dual-species biofilm, in comparison to a single-species E. coli biofilm. E. faecalis is an active AI-2 producer, and its aggregates attract E. coli cells expressing LsrB. Cocultivation of E. coli with E. faecalis in static systems results in higher levels of extracellular AI-2, which helps E. coli cells to maintain lsr operon expression at low cell densities and to coaggregate effectively with E. faecalis , which creates nucleation zones for subsequent enhanced aggregate growth and biofilm formation. These coaggregates of E. coli and E. faecalis are more resistant to stress.

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Proposed model of a static dual-species biofilm, in comparison to a single-species E. coli biofilm. E. faecalis is an active AI-2 producer, and its aggregates attract E. coli cells expressing LsrB. Cocultivation of E. coli with E. faecalis in static systems results in higher levels of extracellular AI-2, which helps E. coli cells to maintain lsr operon expression at low cell densities and to coaggregate effectively with E. faecalis , which creates nucleation zones for subsequent enhanced aggregate growth and biofilm formation. These coaggregates of E. coli and E. faecalis are more resistant to stress.

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Expressing

    E. coli biofilm formation in conditioned media and in the presence of exogenous DPD/AI-2. (A) Confocal laser scanning microscopy of static E. coli (expressing EGFP) biofilms grown in TB, in conditioned medium (CM) from E. coli or E. faecalis , or in TB supplemented with 50 μM synthetic DPD/AI-2, as indicated. Scale bars, 40 μm. (B) Distribution of microcolony volumes in the indicated biofilms. P values for the differences from E. coli biofilms grown in TB were calculated using unpaired t tests (the data distribution was confirmed to be normal). (C) Aggregate sizes (assayed as in Fig. 2 ) of E. coli cells grown in TB (in monoculture or in coculture with E. faecalis ), in conditioned medium from E. coli or E. faecalis , or in TB supplemented with 50 μM synthetic DPD/AI-2, as indicated. Means of at least three independent replicates are shown; error bars indicate standard deviations. P values for the differences from E. coli biofilms grown in TB or between indicated cultures were calculated using Mann-Whitney tests. ****, P

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: E. coli biofilm formation in conditioned media and in the presence of exogenous DPD/AI-2. (A) Confocal laser scanning microscopy of static E. coli (expressing EGFP) biofilms grown in TB, in conditioned medium (CM) from E. coli or E. faecalis , or in TB supplemented with 50 μM synthetic DPD/AI-2, as indicated. Scale bars, 40 μm. (B) Distribution of microcolony volumes in the indicated biofilms. P values for the differences from E. coli biofilms grown in TB were calculated using unpaired t tests (the data distribution was confirmed to be normal). (C) Aggregate sizes (assayed as in Fig. 2 ) of E. coli cells grown in TB (in monoculture or in coculture with E. faecalis ), in conditioned medium from E. coli or E. faecalis , or in TB supplemented with 50 μM synthetic DPD/AI-2, as indicated. Means of at least three independent replicates are shown; error bars indicate standard deviations. P values for the differences from E. coli biofilms grown in TB or between indicated cultures were calculated using Mann-Whitney tests. ****, P

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

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

    Dependence of coaggregation and mixed biofilm formation on AI-2 chemotaxis. (A) Confocal laser scanning microscopy of static biofilms of E. coli Δ cheY and Δ lsrB (expressing mCherry) grown in monoculture or mixed with E. faecalis (expressing EGFP), initially inoculated at a 1:1 ratio. Scale bars, 40 μm. (B) Distribution of microcolony volumes in the biofilms. The P values for the differences between single- and double-species biofilms were calculated using unpaired t tests (the data distribution was confirmed to be normal). ns, not significant. (C) Time-lapse fluorescence microscopy of E. coli Δ lsrB (expressing EGFP) grown with E. faecalis (unlabeled). The white arrows indicate an aggregate of E. faecalis .

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Dependence of coaggregation and mixed biofilm formation on AI-2 chemotaxis. (A) Confocal laser scanning microscopy of static biofilms of E. coli Δ cheY and Δ lsrB (expressing mCherry) grown in monoculture or mixed with E. faecalis (expressing EGFP), initially inoculated at a 1:1 ratio. Scale bars, 40 μm. (B) Distribution of microcolony volumes in the biofilms. The P values for the differences between single- and double-species biofilms were calculated using unpaired t tests (the data distribution was confirmed to be normal). ns, not significant. (C) Time-lapse fluorescence microscopy of E. coli Δ lsrB (expressing EGFP) grown with E. faecalis (unlabeled). The white arrows indicate an aggregate of E. faecalis .

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Chemotaxis Assay, Confocal Laser Scanning Microscopy, Expressing, Fluorescence, Microscopy

    Growth of E. coli and E. faecalis in single- or double-species cultures. (A) Growth rates of static E. coli and E. faecalis single-species cultures (red and magenta dots, respectively) and of mixed E. coli - E. faecalis cultures (blue dots). (B) Composition of static E. coli - E. faecalis biofilm cultures during the first 24 h of incubation. Means of three independent experiments are shown; error bars indicate standard deviations.

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Growth of E. coli and E. faecalis in single- or double-species cultures. (A) Growth rates of static E. coli and E. faecalis single-species cultures (red and magenta dots, respectively) and of mixed E. coli - E. faecalis cultures (blue dots). (B) Composition of static E. coli - E. faecalis biofilm cultures during the first 24 h of incubation. Means of three independent experiments are shown; error bars indicate standard deviations.

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Incubation

    Survival of E. coli and E. faecalis in single- or double-species biofilms under oxidative stress. Single-species or mixed ( Ec : Ef ) biofilm cultures of E. coli and E. faecalis were exposed to 0.5% H 2 O 2 as described in Materials and Methods. E. coli cultures incubated under nonaggregating conditions (shaking at 270 rpm) were used as controls. Means of at least five independent replicates are shown; error bars indicate standard deviations. P values for the differences between single- and double-species biofilms were calculated using Mann-Whitney tests. ***, P

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Survival of E. coli and E. faecalis in single- or double-species biofilms under oxidative stress. Single-species or mixed ( Ec : Ef ) biofilm cultures of E. coli and E. faecalis were exposed to 0.5% H 2 O 2 as described in Materials and Methods. E. coli cultures incubated under nonaggregating conditions (shaking at 270 rpm) were used as controls. Means of at least five independent replicates are shown; error bars indicate standard deviations. P values for the differences between single- and double-species biofilms were calculated using Mann-Whitney tests. ***, P

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Incubation, MANN-WHITNEY

    Dependence of P lsr-egfp activity on growth stage and AI-2 signaling. The activity of the lsr operon was measured using flow cytometry. (A to E) E. coli cells carrying the P lsr-egfp reporter plasmid pVS1723 grown in TB alone (A) or with E. faecalis at a 1:1 ratio (B), grown in conditioned medium (CM) from E. coli (C) or E. faecalis (D), or grown in TB supplemented with 50 μM synthetic DPD/AI-2 (E). Dashed lines distinguish GFP-positive (induced E. coli ) and GFP-negative (uninduced E. coli , as well as unlabeled E. faecalis in panel B) subpopulations. Note that, since E. coli constitutes only 50% of the population at 0 h in panel B, the overall fraction of GFP-positive bacteria appears lower than for E. coli monocultures. (F) Percentage of GFP-positive cells in each population. Means of four independent replicates are shown; error bars indicate standard deviations. P values for the difference from E. coli biofilms grown in TB were calculated using Mann-Whitney tests. *, P

    Journal: Applied and Environmental Microbiology

    Article Title: Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

    doi: 10.1128/AEM.02638-17

    Figure Lengend Snippet: Dependence of P lsr-egfp activity on growth stage and AI-2 signaling. The activity of the lsr operon was measured using flow cytometry. (A to E) E. coli cells carrying the P lsr-egfp reporter plasmid pVS1723 grown in TB alone (A) or with E. faecalis at a 1:1 ratio (B), grown in conditioned medium (CM) from E. coli (C) or E. faecalis (D), or grown in TB supplemented with 50 μM synthetic DPD/AI-2 (E). Dashed lines distinguish GFP-positive (induced E. coli ) and GFP-negative (uninduced E. coli , as well as unlabeled E. faecalis in panel B) subpopulations. Note that, since E. coli constitutes only 50% of the population at 0 h in panel B, the overall fraction of GFP-positive bacteria appears lower than for E. coli monocultures. (F) Percentage of GFP-positive cells in each population. Means of four independent replicates are shown; error bars indicate standard deviations. P values for the difference from E. coli biofilms grown in TB were calculated using Mann-Whitney tests. *, P

    Article Snippet: For dual-species biofilm cultivation, the same amounts of E. coli and E. faecalis cells were coinoculated, resulting in a final OD600 of 0.06; 400 μl of each sample was cultivated for 24 h at 37°C in 8-well glass-bottom slides (μ-Slide, 8-well glass bottom; ibidi).

    Techniques: Activity Assay, Flow Cytometry, Cytometry, Plasmid Preparation, MANN-WHITNEY

    The glycoside hydrolases PslG h and PelA h hydrolyze the exopolysaccharides Pel and Psl in a biofilm. Representative confocal images of Psl biofilms grown statically for 24 hours ( top ) and Pel biofilms cultivated for 48 hours ( bottom ) under flow conditions and treated with wild-type hydrolases or hydrolases that have point mutations to catalytic residues. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pel-specific lectin (red). Scale bars, 30 μm.

    Journal: Science Advances

    Article Title: Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

    doi: 10.1126/sciadv.1501632

    Figure Lengend Snippet: The glycoside hydrolases PslG h and PelA h hydrolyze the exopolysaccharides Pel and Psl in a biofilm. Representative confocal images of Psl biofilms grown statically for 24 hours ( top ) and Pel biofilms cultivated for 48 hours ( bottom ) under flow conditions and treated with wild-type hydrolases or hydrolases that have point mutations to catalytic residues. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pel-specific lectin (red). Scale bars, 30 μm.

    Article Snippet: Confocal microscopy Psl biofilms were grown overnight at room temperature in uncoated 15 μ-Slide VI0.4 flow cell chambers (ibidi GmbH).

    Techniques: Flow Cytometry, Staining

    Glycoside hydrolases potentiate antibiotics and increase human neutrophil killing. ( A ) CFUs for PAO1 Δ wspF Δ psl P BAD pel (left) and PAO1 Δ pelF P BAD psl (right) following growth in the presence or absence of glycoside hydrolases before treatment with colistin. The mean was calculated from LB agar plate counts from three independent experiments. ( B ) CFUs for biofilm cultures in (A) after treatment with glycoside hydrolases and colistin for 5 hours. The mean was calculated from LB agar plate counts from three independent experiments. ( C ) HL-60 neutrophil killing of strain PAO1 Δ wspF Δ psl P BAD pel and PAO1 Δ pelF P BAD psl following biofilm formation and treatment with PelA h and PslG h and their catalytic variants, respectively. Percent killing was normalized to a no-treatment control. Error bars indicate SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. NS, no significant difference.

    Journal: Science Advances

    Article Title: Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

    doi: 10.1126/sciadv.1501632

    Figure Lengend Snippet: Glycoside hydrolases potentiate antibiotics and increase human neutrophil killing. ( A ) CFUs for PAO1 Δ wspF Δ psl P BAD pel (left) and PAO1 Δ pelF P BAD psl (right) following growth in the presence or absence of glycoside hydrolases before treatment with colistin. The mean was calculated from LB agar plate counts from three independent experiments. ( B ) CFUs for biofilm cultures in (A) after treatment with glycoside hydrolases and colistin for 5 hours. The mean was calculated from LB agar plate counts from three independent experiments. ( C ) HL-60 neutrophil killing of strain PAO1 Δ wspF Δ psl P BAD pel and PAO1 Δ pelF P BAD psl following biofilm formation and treatment with PelA h and PslG h and their catalytic variants, respectively. Percent killing was normalized to a no-treatment control. Error bars indicate SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. NS, no significant difference.

    Article Snippet: Confocal microscopy Psl biofilms were grown overnight at room temperature in uncoated 15 μ-Slide VI0.4 flow cell chambers (ibidi GmbH).

    Techniques: