Structured Review

Carl Zeiss biofilms
Catalytic and magnetic iron oxide nanoparticles (NPs) as building blocks for small-scale robots designed for biofilm killing and removal. ). (b) Diagram depicting the magnetic-catalytic NPs and their bacterial killing and EPS degradation mechanisms via reactive free radicals generated from hydrogen peroxide (H 2 O 2 ) via peroxidase-like activity. The EPS degrading activity is enhanced by addition of mutanase/dextranase to digest extracellular glucans. (c) To kill and remove <t>biofilms,</t> catalytic-magnetic NPs in suspension serve as multifunctional building blocks to form Catalytic Antimicrobial Robots (CARs). In the first CAR platform, biohybrid CARs are assembled from NPs suspended in H 2 O 2 and mutanase/dextranase solution by a permanent magnet attached to a micromanipulator, and used to remove biofilms from accessible surfaces. The NPs form bristle-like structures that can be moved in a controlled manner for biofilm removal. In a second platform, catalytic-magnetic NPs are embedded into gels to form 3D molded CARs having specialized vane and helicoid structures. 3D molded CARs are designed to remove clogs and biofilms in confined and inaccessible locations.
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1) Product Images from "Catalytic antimicrobial robots for biofilm eradication"

Article Title: Catalytic antimicrobial robots for biofilm eradication

Journal: Science robotics

doi: 10.1126/scirobotics.aaw2388

Catalytic and magnetic iron oxide nanoparticles (NPs) as building blocks for small-scale robots designed for biofilm killing and removal. ). (b) Diagram depicting the magnetic-catalytic NPs and their bacterial killing and EPS degradation mechanisms via reactive free radicals generated from hydrogen peroxide (H 2 O 2 ) via peroxidase-like activity. The EPS degrading activity is enhanced by addition of mutanase/dextranase to digest extracellular glucans. (c) To kill and remove biofilms, catalytic-magnetic NPs in suspension serve as multifunctional building blocks to form Catalytic Antimicrobial Robots (CARs). In the first CAR platform, biohybrid CARs are assembled from NPs suspended in H 2 O 2 and mutanase/dextranase solution by a permanent magnet attached to a micromanipulator, and used to remove biofilms from accessible surfaces. The NPs form bristle-like structures that can be moved in a controlled manner for biofilm removal. In a second platform, catalytic-magnetic NPs are embedded into gels to form 3D molded CARs having specialized vane and helicoid structures. 3D molded CARs are designed to remove clogs and biofilms in confined and inaccessible locations.
Figure Legend Snippet: Catalytic and magnetic iron oxide nanoparticles (NPs) as building blocks for small-scale robots designed for biofilm killing and removal. ). (b) Diagram depicting the magnetic-catalytic NPs and their bacterial killing and EPS degradation mechanisms via reactive free radicals generated from hydrogen peroxide (H 2 O 2 ) via peroxidase-like activity. The EPS degrading activity is enhanced by addition of mutanase/dextranase to digest extracellular glucans. (c) To kill and remove biofilms, catalytic-magnetic NPs in suspension serve as multifunctional building blocks to form Catalytic Antimicrobial Robots (CARs). In the first CAR platform, biohybrid CARs are assembled from NPs suspended in H 2 O 2 and mutanase/dextranase solution by a permanent magnet attached to a micromanipulator, and used to remove biofilms from accessible surfaces. The NPs form bristle-like structures that can be moved in a controlled manner for biofilm removal. In a second platform, catalytic-magnetic NPs are embedded into gels to form 3D molded CARs having specialized vane and helicoid structures. 3D molded CARs are designed to remove clogs and biofilms in confined and inaccessible locations.

Techniques Used: Generated, Activity Assay

Platform 1: Biofilm removal using biohybrid CARs. (a) Orthogonal view of biofilms treated with biohybrid CARs reveals the rod-like superstructure forming pillars (highlighted by white dashed lines) through the compromised biofilms. After incubation, biohybrid CARs are manipulated precisely using a magnetic field gradient to completely remove biofilms and biofilm debris including dead bacteria and degraded EPS from surfaces. (b) Diagram depicting cleaning of large areas of a biofilm coated surface by magnetically controlled sweeping of biohybrid CARs (after bacterial killing/EPS degradation); (b1) The biohybrid CAR morphs into C-shaped aggregate (see arrows) as it moves over the surface to plow through and remove biofilm (labelled with SYTO9-green fluorescence). (b2) Fluorescent images indicating complete cleaning of an S. mutans biofilm (labeled in green) grown on a glass surface by using biohybrid CARs sweeping effect. (c) Biofilm-removed surfaces were incubated for additional 24 h with the biofilm growth medium (supplemented with 1% sucrose) to assess bacterial regrowth using confocal microscopy and culturing methods. There is no biofilm re-growth on biofilm-removed surfaces by CARs even after 24 h incubation. Control and NP treated biofilms were also incubated using the same conditions, both showing abundant bacterial cells (in green) and EPS (in red); quantitative analyses show high amounts of biomass and high number of viable cells. Quantitative data also confirm that there is no detectable biomass or viable cells with treatment by CARs. (d) The controlled movement of focused biohybrid CARs results in a precise removal of biofilms from surfaces with defined geometrical shapes. (d1) Fluorescent microscopy confirms the complete removal of bacteria from the surface.
Figure Legend Snippet: Platform 1: Biofilm removal using biohybrid CARs. (a) Orthogonal view of biofilms treated with biohybrid CARs reveals the rod-like superstructure forming pillars (highlighted by white dashed lines) through the compromised biofilms. After incubation, biohybrid CARs are manipulated precisely using a magnetic field gradient to completely remove biofilms and biofilm debris including dead bacteria and degraded EPS from surfaces. (b) Diagram depicting cleaning of large areas of a biofilm coated surface by magnetically controlled sweeping of biohybrid CARs (after bacterial killing/EPS degradation); (b1) The biohybrid CAR morphs into C-shaped aggregate (see arrows) as it moves over the surface to plow through and remove biofilm (labelled with SYTO9-green fluorescence). (b2) Fluorescent images indicating complete cleaning of an S. mutans biofilm (labeled in green) grown on a glass surface by using biohybrid CARs sweeping effect. (c) Biofilm-removed surfaces were incubated for additional 24 h with the biofilm growth medium (supplemented with 1% sucrose) to assess bacterial regrowth using confocal microscopy and culturing methods. There is no biofilm re-growth on biofilm-removed surfaces by CARs even after 24 h incubation. Control and NP treated biofilms were also incubated using the same conditions, both showing abundant bacterial cells (in green) and EPS (in red); quantitative analyses show high amounts of biomass and high number of viable cells. Quantitative data also confirm that there is no detectable biomass or viable cells with treatment by CARs. (d) The controlled movement of focused biohybrid CARs results in a precise removal of biofilms from surfaces with defined geometrical shapes. (d1) Fluorescent microscopy confirms the complete removal of bacteria from the surface.

Techniques Used: Incubation, Fluorescence, Labeling, Confocal Microscopy, Microscopy

Platform 2: Small scale CARs with functional shapes for specific biofilm disruption tasks. (a) Model representations of vane-like and helicoid shaped CARs fabricated from 3D micromolding agar gel embedded with NPs. Both vanes and helicoids measure 5mm in diameter and 10mm long. The final robot composition is 3% (w/v) agar and 10 % (w/v) NPs. (b) Helmholtz coil system used to drive 3D molded CARs through cylindrical tubes. Sinusoidal time-varying currents (I x , I z ) are applied to each coil pair to generate uniform, rotating magnetic fields. Robots are driven using fields measuring 3.4mT and rotating at 4Hz. (b1) Robots are driven through the cylindrical tube with linear velocity v , which is generated by applying a torque T to the robot body. The torque is generated when the magnetic dipole moment M , seeks to align with the rotating field B . (c) Schematic diagrams of biofilms on the wall of cylindrical tube, and wall cleaning by vane CAR. Vane CARs are used to clean the curved surfaces of glass tubes. Vane CARs are rotated with an applied magnetic torque T , and are driven forward in the tube at a velocity v by applying a force using a magnetic field gradient. (c1) Vane CARs advance forward in the tube, sweeping the walls and generating a pile of debris behind them. The integrated fluorescence intensity from the biofilm debris increases with time behind the advancing robot. (Left) Accumulated biomass behind the vane CAR as it moves forward through the tube. (d) Schematic diagrams of biofilm clogs in cylindrical tubes, and drilling by helicoid CARs. Helicoid CARs are used to clean biofilms clogs at various locations within the glass tube. The robot is propelled along linear paths within glass tubes through an applied magnetic torque T . Their forward propulsion is enabled by their chirality. (d1) Helicoid CARs advance forward in the tube to drill through the biomass clog. (Left) Accumulated biomass behind the helicoid CAR as it moves forward through the tube increases with time. The robot drills by overcoming the biofilm mechanical resistance associated with the conical shape of the biofilm (for t
Figure Legend Snippet: Platform 2: Small scale CARs with functional shapes for specific biofilm disruption tasks. (a) Model representations of vane-like and helicoid shaped CARs fabricated from 3D micromolding agar gel embedded with NPs. Both vanes and helicoids measure 5mm in diameter and 10mm long. The final robot composition is 3% (w/v) agar and 10 % (w/v) NPs. (b) Helmholtz coil system used to drive 3D molded CARs through cylindrical tubes. Sinusoidal time-varying currents (I x , I z ) are applied to each coil pair to generate uniform, rotating magnetic fields. Robots are driven using fields measuring 3.4mT and rotating at 4Hz. (b1) Robots are driven through the cylindrical tube with linear velocity v , which is generated by applying a torque T to the robot body. The torque is generated when the magnetic dipole moment M , seeks to align with the rotating field B . (c) Schematic diagrams of biofilms on the wall of cylindrical tube, and wall cleaning by vane CAR. Vane CARs are used to clean the curved surfaces of glass tubes. Vane CARs are rotated with an applied magnetic torque T , and are driven forward in the tube at a velocity v by applying a force using a magnetic field gradient. (c1) Vane CARs advance forward in the tube, sweeping the walls and generating a pile of debris behind them. The integrated fluorescence intensity from the biofilm debris increases with time behind the advancing robot. (Left) Accumulated biomass behind the vane CAR as it moves forward through the tube. (d) Schematic diagrams of biofilm clogs in cylindrical tubes, and drilling by helicoid CARs. Helicoid CARs are used to clean biofilms clogs at various locations within the glass tube. The robot is propelled along linear paths within glass tubes through an applied magnetic torque T . Their forward propulsion is enabled by their chirality. (d1) Helicoid CARs advance forward in the tube to drill through the biomass clog. (Left) Accumulated biomass behind the helicoid CAR as it moves forward through the tube increases with time. The robot drills by overcoming the biofilm mechanical resistance associated with the conical shape of the biofilm (for t

Techniques Used: Functional Assay, Generated, Fluorescence

Characterization and formation of biohybrid CARs. (a) NP actuation in biofilm in presence of magnetic field. Streptococcus mutans , a biofilm-forming model organism and a bacterial oral pathogen, was used to form biofilms. NPs, absent catalysis, do not aggregate owing to adhesive interactions with the biofilm. Conversely, NPs with catalysis degrade the biofilm and move toward the magnet to assemble into a cluster. (a1) Quantitative imaging analysis shows the size of cluster in NP with and without catalysis (as shown in a). (b) Data from bacterial viability assays using fluorescence imaging and culturing methods. Green fluorescence indicates live bacteria and red indicates dead bacteria, and the number of viable cells as determined by colony-forming units (CFU) counting. The data show that NPs alone are devoid of antibacterial activity, whereas NPs with catalysis potently kill bacteria. (b1) Biohybrid CAR superstructure is revealed by high resolution confocal microscopy. Absent catalytic activity, only small NPs assemblages form (top panel). With catalytic activity, biohybrid CARs form; they consist of a complex bio-inorganic hybrid assemblage comprising spatially oriented, rod-like, micron-size superstructures of NP enmeshed with bacterial mass and debris (bottom and right panels).
Figure Legend Snippet: Characterization and formation of biohybrid CARs. (a) NP actuation in biofilm in presence of magnetic field. Streptococcus mutans , a biofilm-forming model organism and a bacterial oral pathogen, was used to form biofilms. NPs, absent catalysis, do not aggregate owing to adhesive interactions with the biofilm. Conversely, NPs with catalysis degrade the biofilm and move toward the magnet to assemble into a cluster. (a1) Quantitative imaging analysis shows the size of cluster in NP with and without catalysis (as shown in a). (b) Data from bacterial viability assays using fluorescence imaging and culturing methods. Green fluorescence indicates live bacteria and red indicates dead bacteria, and the number of viable cells as determined by colony-forming units (CFU) counting. The data show that NPs alone are devoid of antibacterial activity, whereas NPs with catalysis potently kill bacteria. (b1) Biohybrid CAR superstructure is revealed by high resolution confocal microscopy. Absent catalytic activity, only small NPs assemblages form (top panel). With catalytic activity, biohybrid CARs form; they consist of a complex bio-inorganic hybrid assemblage comprising spatially oriented, rod-like, micron-size superstructures of NP enmeshed with bacterial mass and debris (bottom and right panels).

Techniques Used: Imaging, Fluorescence, Activity Assay, Confocal Microscopy

Potential applications for CARs platforms. (a) CARs can be used to treat biofilms on biotic (e.g. teeth) and abiotic (catheter or implant) surfaces. (b) Demonstration of using CARs to access the interior of human teeth. Cross section of the tooth canal shows the isthmus, which is a narrow gap (300–600 micrometers in width) between the root canals. Longitudinal section (across the tooth length) shows the tooth canal. Biohybrid CARs can access isthmus, one of the most challenging anatomical areas of teeth, where bacterial biofilms are commonly found. Aggregated nanoparticles can readily transvers the isthmus as directed by the external magnetic field. Lower middle panels show disruption of biofilms in the isthmus by CARS using fluorescently labeled biofilms. (c) For 3D molded CARs, miniaturized 3D molded helicoidal robots can be magnetically actuated through the canal of the tooth, another common location of dental biofilm formation.
Figure Legend Snippet: Potential applications for CARs platforms. (a) CARs can be used to treat biofilms on biotic (e.g. teeth) and abiotic (catheter or implant) surfaces. (b) Demonstration of using CARs to access the interior of human teeth. Cross section of the tooth canal shows the isthmus, which is a narrow gap (300–600 micrometers in width) between the root canals. Longitudinal section (across the tooth length) shows the tooth canal. Biohybrid CARs can access isthmus, one of the most challenging anatomical areas of teeth, where bacterial biofilms are commonly found. Aggregated nanoparticles can readily transvers the isthmus as directed by the external magnetic field. Lower middle panels show disruption of biofilms in the isthmus by CARS using fluorescently labeled biofilms. (c) For 3D molded CARs, miniaturized 3D molded helicoidal robots can be magnetically actuated through the canal of the tooth, another common location of dental biofilm formation.

Techniques Used: Labeling

2) Product Images from "Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni"

Article Title: Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00533

Biofilm produced by C. jejuni 81-176 on cover glass after 48 h at 37°C under microaerobic conditions and visualized by Field Emission Scanning Electron Microscopy. (A) C. jejuni 81-176 wild-type, (B) C. jejuni 81-176Δ cj0588 , (C) the complemented C. jejuni strain 81-176Δ cj0588 :: 0588 . Experiments were performed in triplicate and representative micrographs are shown.
Figure Legend Snippet: Biofilm produced by C. jejuni 81-176 on cover glass after 48 h at 37°C under microaerobic conditions and visualized by Field Emission Scanning Electron Microscopy. (A) C. jejuni 81-176 wild-type, (B) C. jejuni 81-176Δ cj0588 , (C) the complemented C. jejuni strain 81-176Δ cj0588 :: 0588 . Experiments were performed in triplicate and representative micrographs are shown.

Techniques Used: Produced, Electron Microscopy

3) Product Images from "Competition in Biofilms between Cystic Fibrosis Isolates of Pseudomonas aeruginosa Is Shaped by R-Pyocins"

Article Title: Competition in Biofilms between Cystic Fibrosis Isolates of Pseudomonas aeruginosa Is Shaped by R-Pyocins

Journal: mBio

doi: 10.1128/mBio.01828-18

Strain competition in biofilms. Shown are 15-h biofilms developed in a microfluidic BioFlux device as a result of competition between A026 and A018. A026 dominated in the biofilm competition of A018 versus A026 and A018 ΔR versus A026 regardless of the fluorophore (mCherry or GFP) used to tag either strain (A to D). However, A018 dominated when in competition with the null-R mutant of A026 (A026 ΔR) (E and F). (A) A026_mCherry versus A018_GFP. (B) A026_GFP versus A018_mCherry. (C) A026_GFP versus A018 ΔR_mCherry. (D) A026_mCherry versus A018 ΔR_GFP. (E) A026 ΔR_GFP versus A018_mCherry. (F) A026 ΔR_mCherry versus A018_GFP.
Figure Legend Snippet: Strain competition in biofilms. Shown are 15-h biofilms developed in a microfluidic BioFlux device as a result of competition between A026 and A018. A026 dominated in the biofilm competition of A018 versus A026 and A018 ΔR versus A026 regardless of the fluorophore (mCherry or GFP) used to tag either strain (A to D). However, A018 dominated when in competition with the null-R mutant of A026 (A026 ΔR) (E and F). (A) A026_mCherry versus A018_GFP. (B) A026_GFP versus A018_mCherry. (C) A026_GFP versus A018 ΔR_mCherry. (D) A026_mCherry versus A018 ΔR_GFP. (E) A026 ΔR_GFP versus A018_mCherry. (F) A026 ΔR_mCherry versus A018_GFP.

Techniques Used: Mutagenesis

Single static treatment of biofilms of A018 or A026 grown on polystyrene beads using R-pyocins of a competitor (A026 or A018, respectively). Hour 0 readings are the CFU/ml values of the 24-h biofilm grown on the beads. In one set of beads, growth was allowed to proceed unhindered (unbroken lines), while in the other set, three beads were harvested every hour, their biofilms were treated for 1 h using purified R-pyocins, and the CFU count was recorded after treatment (broken lines). Beads in panel A were treated with R-pyocins from the wild type of competitor, while the ones in panel B were treated with cell extracts from R-pyocin mutants. *, P
Figure Legend Snippet: Single static treatment of biofilms of A018 or A026 grown on polystyrene beads using R-pyocins of a competitor (A026 or A018, respectively). Hour 0 readings are the CFU/ml values of the 24-h biofilm grown on the beads. In one set of beads, growth was allowed to proceed unhindered (unbroken lines), while in the other set, three beads were harvested every hour, their biofilms were treated for 1 h using purified R-pyocins, and the CFU count was recorded after treatment (broken lines). Beads in panel A were treated with R-pyocins from the wild type of competitor, while the ones in panel B were treated with cell extracts from R-pyocin mutants. *, P

Techniques Used: Purification

Antibiofilm efficacy of R-pyocins. A 15-h biofilm of A018 was treated with R-pyocins extracted from A026 (A) and A026 ΔR (B). A significant portion of the biomass was killed after 2 h and full-depth lethal effects on the biomass was achieved after 4 h. This effect was absent in the control experiment, which utilized the R-pyocins of A026 ΔR.
Figure Legend Snippet: Antibiofilm efficacy of R-pyocins. A 15-h biofilm of A018 was treated with R-pyocins extracted from A026 (A) and A026 ΔR (B). A significant portion of the biomass was killed after 2 h and full-depth lethal effects on the biomass was achieved after 4 h. This effect was absent in the control experiment, which utilized the R-pyocins of A026 ΔR.

Techniques Used:

Fifteen-hour biofilms showing the distribution of two strains. (A and B) Biofilms of mixed cultures of A018 ΔR_GFP and A026 ΔR_mCherry. Panel A is a split image of the fluorescent green and red channels at ×10 magnification showing a wider distribution of the strains as they form microniches, while panel B shows a closer view of the same biofilms at ×63 magnification. Panel C shows a biofilm of wild-type strains A018_GFP and P013_mCherry, which both have the same subtypes of S- and R-pyocins.
Figure Legend Snippet: Fifteen-hour biofilms showing the distribution of two strains. (A and B) Biofilms of mixed cultures of A018 ΔR_GFP and A026 ΔR_mCherry. Panel A is a split image of the fluorescent green and red channels at ×10 magnification showing a wider distribution of the strains as they form microniches, while panel B shows a closer view of the same biofilms at ×63 magnification. Panel C shows a biofilm of wild-type strains A018_GFP and P013_mCherry, which both have the same subtypes of S- and R-pyocins.

Techniques Used:

4) Product Images from "PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation"

Article Title: PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation

Journal: Journal of Bacteriology

doi: 10.1128/JB.00498-13

Biofilms of Δ pdeB and pdeB eal mutants. Biofilms were grown in hydrodynamic flow chambers with LM for 15 to 16 h at 30°C before imaging. White bars represent 100 µm. Quantification of biofilms is shown in .
Figure Legend Snippet: Biofilms of Δ pdeB and pdeB eal mutants. Biofilms were grown in hydrodynamic flow chambers with LM for 15 to 16 h at 30°C before imaging. White bars represent 100 µm. Quantification of biofilms is shown in .

Techniques Used: Flow Cytometry, Imaging

5) Product Images from "Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein"

Article Title: Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01402-13

Microtiter plate biofilm formation by MS2989 in comparison to MS3640. Strains were grown under shaking conditions at 28°C for 24 h in polyvinyl chloride (PVC) microtiter plates containing M9 supplemented with 0.3% Casamino Acids. Plates were washed
Figure Legend Snippet: Microtiter plate biofilm formation by MS2989 in comparison to MS3640. Strains were grown under shaking conditions at 28°C for 24 h in polyvinyl chloride (PVC) microtiter plates containing M9 supplemented with 0.3% Casamino Acids. Plates were washed

Techniques Used:

Microtiter plate assay demonstrating biofilm formation by MS3007, MS3009, MS3011, and MS3014. Biofilm inhibition was performed by supplementing respective wells with a 1:10 dilution of affinity-purified Bap antibodies in TSB to assess inhibitory effects
Figure Legend Snippet: Microtiter plate assay demonstrating biofilm formation by MS3007, MS3009, MS3011, and MS3014. Biofilm inhibition was performed by supplementing respective wells with a 1:10 dilution of affinity-purified Bap antibodies in TSB to assess inhibitory effects

Techniques Used: Inhibition, Affinity Purification

Flow chamber biofilm formation by MS3007 (A), MS3009 (B), MS3011 (C), and MS3014 (D). Biofilm development was monitored by CLSM 48 h postinoculation. Substratum coverage of each strain is as follows: MS3007, 29.96%; MS3009, 53.26%; MS3011, 60.7% and MS3014,
Figure Legend Snippet: Flow chamber biofilm formation by MS3007 (A), MS3009 (B), MS3011 (C), and MS3014 (D). Biofilm development was monitored by CLSM 48 h postinoculation. Substratum coverage of each strain is as follows: MS3007, 29.96%; MS3009, 53.26%; MS3011, 60.7% and MS3014,

Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy

6) Product Images from "Chitin-Induced Carbotype Conversion in Vibrio vulnificus ▿ ▿ †"

Article Title: Chitin-Induced Carbotype Conversion in Vibrio vulnificus ▿ ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.00158-11

Changes in V. vulnificus biofilm composition in response to a lytic phage. The MO-6/24-Tn and CMCP6 strains were grown in mixed culture on crab shells in 12-well plates for 24 h. The biofilms were then exposed to the MO-6/24-specific lytic phage 152-A10
Figure Legend Snippet: Changes in V. vulnificus biofilm composition in response to a lytic phage. The MO-6/24-Tn and CMCP6 strains were grown in mixed culture on crab shells in 12-well plates for 24 h. The biofilms were then exposed to the MO-6/24-specific lytic phage 152-A10

Techniques Used:

Composition of V. vulnificus biofilms grown on crab shells. The 27562 and CMCP6 strains were grown in mixed culture on crab shells in 12-well plates for 24 h. The structure and composition of the biofilms were examined by CSLM. (A) GFP-tagged cells; (B)
Figure Legend Snippet: Composition of V. vulnificus biofilms grown on crab shells. The 27562 and CMCP6 strains were grown in mixed culture on crab shells in 12-well plates for 24 h. The structure and composition of the biofilms were examined by CSLM. (A) GFP-tagged cells; (B)

Techniques Used:

7) Product Images from "Biofilm Formation by the Fungal Pathogen Candida albicans: Development, Architecture, and Drug Resistance"

Article Title: Biofilm Formation by the Fungal Pathogen Candida albicans: Development, Architecture, and Drug Resistance

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.18.5385-5394.2001

Development of C. albicans biofilm on polymethylmethacrylate strips. Fluorescence microscopy images show the three distinct developmental phases of C. albicans biofilms over a 72-h period: early (a), intermediate (b), and maturation (c) phases. Magnification, ×10.
Figure Legend Snippet: Development of C. albicans biofilm on polymethylmethacrylate strips. Fluorescence microscopy images show the three distinct developmental phases of C. albicans biofilms over a 72-h period: early (a), intermediate (b), and maturation (c) phases. Magnification, ×10.

Techniques Used: Fluorescence, Microscopy

CSLM images of a C. albicans biofilm grown on denture acrylic surface. (a to d) Horizontal ( xy ) view of reconstructed 3-D images at 0 (a), 8 (b), 11 (c), and 48 (d) h. Bar, 20 μm. (e and f) Orthogonal images of C. albicans biofilms grown to early and maturation phases show that early-phase (0 h) biofilm consisted of mostly yeast cells separated by blank spaces (arrows) (e), while maturation-phase (48 h) biofilm showed metabolically active (red, FUN1-stained) cells embedded in the polysaccharide extracellular material (green, ConA-stained, arrows) (f). (g and h) Thickness of the biofilm (≈25 μm) can be observed in the side view of the reconstruction (g), while a horizontally tilted image (with false 3-D cubes) shows the heterogeneity of the biofilm (h). Magnification, ×40.
Figure Legend Snippet: CSLM images of a C. albicans biofilm grown on denture acrylic surface. (a to d) Horizontal ( xy ) view of reconstructed 3-D images at 0 (a), 8 (b), 11 (c), and 48 (d) h. Bar, 20 μm. (e and f) Orthogonal images of C. albicans biofilms grown to early and maturation phases show that early-phase (0 h) biofilm consisted of mostly yeast cells separated by blank spaces (arrows) (e), while maturation-phase (48 h) biofilm showed metabolically active (red, FUN1-stained) cells embedded in the polysaccharide extracellular material (green, ConA-stained, arrows) (f). (g and h) Thickness of the biofilm (≈25 μm) can be observed in the side view of the reconstruction (g), while a horizontally tilted image (with false 3-D cubes) shows the heterogeneity of the biofilm (h). Magnification, ×40.

Techniques Used: Metabolic Labelling, Staining

CSLM images of Calcofluor-stained mature C. albicans biofilms formed on silicone elastomer surface. (a and b) Projection ( xz or side view) of 3-D reconstructed images showing an approximately 450-μm-thick biofilm with a basal layer (10 to 12 μm thick) consisting of yeast cells and a top layer consisting of hyphal elements (arrow). The extracellular material (ECM) is stained with ConA, resulting in the green color. (c and d) Orthogonal images of the basal (c) and upper layers (d). The ECM-derived haziness seen in mature biofilm (e) is absent when the extracellular material is removed (f). Magnification, ×20.
Figure Legend Snippet: CSLM images of Calcofluor-stained mature C. albicans biofilms formed on silicone elastomer surface. (a and b) Projection ( xz or side view) of 3-D reconstructed images showing an approximately 450-μm-thick biofilm with a basal layer (10 to 12 μm thick) consisting of yeast cells and a top layer consisting of hyphal elements (arrow). The extracellular material (ECM) is stained with ConA, resulting in the green color. (c and d) Orthogonal images of the basal (c) and upper layers (d). The ECM-derived haziness seen in mature biofilm (e) is absent when the extracellular material is removed (f). Magnification, ×20.

Techniques Used: Staining, Derivative Assay

Schematic representation of biofilm development in C. albicans . (a and b) Biofilm grown on polymethylmethacrylate (PMA) strips. (c and d) Biofilm grown on silicone elastomer (SE) disks. Panels a and c represent the substrate seen from the top, while panels c and d show the view from the sides of the PMA strip and SE disk, respectively. ECM, extracellular material.
Figure Legend Snippet: Schematic representation of biofilm development in C. albicans . (a and b) Biofilm grown on polymethylmethacrylate (PMA) strips. (c and d) Biofilm grown on silicone elastomer (SE) disks. Panels a and c represent the substrate seen from the top, while panels c and d show the view from the sides of the PMA strip and SE disk, respectively. ECM, extracellular material.

Techniques Used: Stripping Membranes

Northern blot analysis of total RNA extracted from planktonic and biofilm-grown C. albicans cells. Total RNA from planktonic and biofilm-grown cells was loaded in various quantities (20, 30, 40, and 50 μg) on a formaldehyde-agarose gel. The resulting Northern blot was probed with a fragment of the C. albicans ALS1 tandem repeats, which hybridized several genes in the family. Probing with a fragment of the TEF1 gene served as loading control. Molecular size markers (kb) are shown on the left.
Figure Legend Snippet: Northern blot analysis of total RNA extracted from planktonic and biofilm-grown C. albicans cells. Total RNA from planktonic and biofilm-grown cells was loaded in various quantities (20, 30, 40, and 50 μg) on a formaldehyde-agarose gel. The resulting Northern blot was probed with a fragment of the C. albicans ALS1 tandem repeats, which hybridized several genes in the family. Probing with a fragment of the TEF1 gene served as loading control. Molecular size markers (kb) are shown on the left.

Techniques Used: Northern Blot, Agarose Gel Electrophoresis

Comparison of the abilities of C. albicans and S. cerevisiae to form biofilms. (a) Quantitative measurement of dry weight of biofilms formed by C. albicans and S. cerevisiae. (b) Growth profiles of planktonic C. albicans and S. cerevisiae . Fluorescence microscopy images of C. albicans (c) and S. cerevisiae (d) grown on polymethylmethacrylate strips. (e) Fluorescence micrograph showing S. cerevisiae growing on silicone elastomer disk (≈3 to 4 μm in thickness). Magnification, ×10.
Figure Legend Snippet: Comparison of the abilities of C. albicans and S. cerevisiae to form biofilms. (a) Quantitative measurement of dry weight of biofilms formed by C. albicans and S. cerevisiae. (b) Growth profiles of planktonic C. albicans and S. cerevisiae . Fluorescence microscopy images of C. albicans (c) and S. cerevisiae (d) grown on polymethylmethacrylate strips. (e) Fluorescence micrograph showing S. cerevisiae growing on silicone elastomer disk (≈3 to 4 μm in thickness). Magnification, ×10.

Techniques Used: Fluorescence, Microscopy

8) Product Images from "Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics"

Article Title: Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics

Journal: Molecular oral microbiology

doi: 10.1111/omi.12157

Functional complementation of DNABII proteins in S. gordonii and P. gingivalis biofilms. S. gordonii biofilms were treated with either naïve serum alone (A) or αHU Sg antiserum and the HUβ Pg protein (B) and then probed with αHUβ Pg antisera. P. gingivalis biofilms were treated with naïve serum alone (C) or both αHUβ Pg antiserum and the HU Sg protein (D) and probed with αHU Sg antisera. The biofilms were stained with SYTO-9 (shown in green), and DNABII-bound antibodies were labeled with a secondary antibody conjugated to Cy-5 (shown in red). The changes in the average thickness and biomass are plotted for both S. gordonii (E) and P. gingivalis (F).
Figure Legend Snippet: Functional complementation of DNABII proteins in S. gordonii and P. gingivalis biofilms. S. gordonii biofilms were treated with either naïve serum alone (A) or αHU Sg antiserum and the HUβ Pg protein (B) and then probed with αHUβ Pg antisera. P. gingivalis biofilms were treated with naïve serum alone (C) or both αHUβ Pg antiserum and the HU Sg protein (D) and probed with αHU Sg antisera. The biofilms were stained with SYTO-9 (shown in green), and DNABII-bound antibodies were labeled with a secondary antibody conjugated to Cy-5 (shown in red). The changes in the average thickness and biomass are plotted for both S. gordonii (E) and P. gingivalis (F).

Techniques Used: Functional Assay, Staining, Labeling

9) Product Images from "Interactions of Aspergillus fumigatus and Stenotrophomonas maltophilia in an in vitro Mixed Biofilm Model: Does the Strain Matter?"

Article Title: Interactions of Aspergillus fumigatus and Stenotrophomonas maltophilia in an in vitro Mixed Biofilm Model: Does the Strain Matter?

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02850

Reduction of mixed biofilms thicknesses and modification of Af13073 phenotype according to S. maltophilia strains. (A) Thicknesses of mixed biofilms compared with thickness of Af13073 vs. S. maltophilia strains: human (black), animal (gray) and environment (white). (B) Wild type phenotype of Af13073 in the presence of S. maltophilia (C) Modified phenotype of Af13073 in the presence of S. maltophilia . The letters indicate the phenotype observed: b, photo B; c, photo C. ∗ p
Figure Legend Snippet: Reduction of mixed biofilms thicknesses and modification of Af13073 phenotype according to S. maltophilia strains. (A) Thicknesses of mixed biofilms compared with thickness of Af13073 vs. S. maltophilia strains: human (black), animal (gray) and environment (white). (B) Wild type phenotype of Af13073 in the presence of S. maltophilia (C) Modified phenotype of Af13073 in the presence of S. maltophilia . The letters indicate the phenotype observed: b, photo B; c, photo C. ∗ p

Techniques Used: Modification

Modification and measurement of cell wall thickness of A. fumigatus in single and mixed biofilms, by fungal strains. (A,A’) Single fungal biofilm. (B,B’) Mixed biofilm with Sm13637 bacteria (white arrows). (A’,B’) Zoom of fungal cell wall. (C) TEM, measurement of fungal cell wall thickness by A. fumigatus origin: human (black), animal (gray) and environment (white). ECM, extracellular matrix; CW, cell wall. ∗ p
Figure Legend Snippet: Modification and measurement of cell wall thickness of A. fumigatus in single and mixed biofilms, by fungal strains. (A,A’) Single fungal biofilm. (B,B’) Mixed biofilm with Sm13637 bacteria (white arrows). (A’,B’) Zoom of fungal cell wall. (C) TEM, measurement of fungal cell wall thickness by A. fumigatus origin: human (black), animal (gray) and environment (white). ECM, extracellular matrix; CW, cell wall. ∗ p

Techniques Used: Modification, Transmission Electron Microscopy

Growth, adhesion, and biofilm formation of S. maltophilia strains by temperature degree. Culture of S. maltophilia strains originated from human (black), animal (gray), and environment (white), in RPMI + FBS media at 25 and 37°C. (A) Planktonic bacterial growth measured by absorbance (OD 600 nm) after 24 h of culture. (B) Bacterial adhesion on polystyrene support after 4 h measured by crystal violet (OD 550 nm). (C) Biofilm formation after 24 h of culture measured by crystal violet (OD 550 nm); ∗ p
Figure Legend Snippet: Growth, adhesion, and biofilm formation of S. maltophilia strains by temperature degree. Culture of S. maltophilia strains originated from human (black), animal (gray), and environment (white), in RPMI + FBS media at 25 and 37°C. (A) Planktonic bacterial growth measured by absorbance (OD 600 nm) after 24 h of culture. (B) Bacterial adhesion on polystyrene support after 4 h measured by crystal violet (OD 550 nm). (C) Biofilm formation after 24 h of culture measured by crystal violet (OD 550 nm); ∗ p

Techniques Used:

Quantification of fungal and bacterial DNA in mixed biofilms by S. maltophilia strains. (A) Quantification by qPCR of Af13073 (Log CE/mL) in single and mixed biofilms with Sm strains from human (black), animal (gray), and environment (white) (at 10 6 cells/mL). (B) Quantification by qPCR of Sm (Log BE/mL) in the mixed biofilms (Af13073 + Sm at 10 6 cells/mL); ∗ p
Figure Legend Snippet: Quantification of fungal and bacterial DNA in mixed biofilms by S. maltophilia strains. (A) Quantification by qPCR of Af13073 (Log CE/mL) in single and mixed biofilms with Sm strains from human (black), animal (gray), and environment (white) (at 10 6 cells/mL). (B) Quantification by qPCR of Sm (Log BE/mL) in the mixed biofilms (Af13073 + Sm at 10 6 cells/mL); ∗ p

Techniques Used: Real-time Polymerase Chain Reaction

Dose-dependent fungal biomass inhibition in mixed biofilms. (A) Af13073 phenotype in mixed biofilm with S. maltophilia strains from animal (gray) or environment (white). (B) Quantification by qPCR of Af13073 in fungal and mixed biofilms with 10 8 bacteria/mL; ∗ p
Figure Legend Snippet: Dose-dependent fungal biomass inhibition in mixed biofilms. (A) Af13073 phenotype in mixed biofilm with S. maltophilia strains from animal (gray) or environment (white). (B) Quantification by qPCR of Af13073 in fungal and mixed biofilms with 10 8 bacteria/mL; ∗ p

Techniques Used: Inhibition, Real-time Polymerase Chain Reaction

10) Product Images from "NtrC Adds a New Node to the Complex Regulatory Network of Biofilm Formation and vps Expression in Vibrio cholerae"

Article Title: NtrC Adds a New Node to the Complex Regulatory Network of Biofilm Formation and vps Expression in Vibrio cholerae

Journal: Journal of Bacteriology

doi: 10.1128/JB.00025-18

NtrC represses vpsL expression independently of regulators of biofilm formation, except for VpsR and RpoN. Shown are expression levels of P vpsL-luxCADBE (pFY_0950) in the WT, Δ ntrC , Δ vpsR , Δ ntrC Δ vpsR , Δ vpsT , Δ ntrC Δ vpsT , Δ rpoN , Δ ntrC Δ rpoN , Δ hapR , Δ ntrC Δ hapR , Δ crp , and Δ ntrC Δ crp strains. The graph represents the averages and standard deviations of RLU obtained from four technical replicates from at least three independent biological samples. RLU are reported in luminescence counts per minute per milliliter per OD 600 . Pairwise analysis was done by using a two-tailed unpaired t test with Welch's correction. *, P
Figure Legend Snippet: NtrC represses vpsL expression independently of regulators of biofilm formation, except for VpsR and RpoN. Shown are expression levels of P vpsL-luxCADBE (pFY_0950) in the WT, Δ ntrC , Δ vpsR , Δ ntrC Δ vpsR , Δ vpsT , Δ ntrC Δ vpsT , Δ rpoN , Δ ntrC Δ rpoN , Δ hapR , Δ ntrC Δ hapR , Δ crp , and Δ ntrC Δ crp strains. The graph represents the averages and standard deviations of RLU obtained from four technical replicates from at least three independent biological samples. RLU are reported in luminescence counts per minute per milliliter per OD 600 . Pairwise analysis was done by using a two-tailed unpaired t test with Welch's correction. *, P

Techniques Used: Expressing, Two Tailed Test

11) Product Images from "Nonthermal Plasma Jet Treatment Negatively Affects the Viability and Structure of Candida albicans SC5314 Biofilms"

Article Title: Nonthermal Plasma Jet Treatment Negatively Affects the Viability and Structure of Candida albicans SC5314 Biofilms

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01163-18

Thermal image measurements of biofilms taken directly after heating. Images were taken at a distance of 20 cm with an FLIR thermal imaging camera. Biofilms were heated 1 min at 130°C on a plate heater.
Figure Legend Snippet: Thermal image measurements of biofilms taken directly after heating. Images were taken at a distance of 20 cm with an FLIR thermal imaging camera. Biofilms were heated 1 min at 130°C on a plate heater.

Techniques Used: Imaging

Box and whisker plots of CFU, fluorescence, and XTT measurements of biofilms heated on a plate heater. Candida albicans biofilms were heated 1 min at 130°C on a plate heater and used for CFU (A), fluorescence (B), and XTT (C) assays. Biofilms were compared to untreated control biofilms. n = 15.
Figure Legend Snippet: Box and whisker plots of CFU, fluorescence, and XTT measurements of biofilms heated on a plate heater. Candida albicans biofilms were heated 1 min at 130°C on a plate heater and used for CFU (A), fluorescence (B), and XTT (C) assays. Biofilms were compared to untreated control biofilms. n = 15.

Techniques Used: Whisker Assay, Fluorescence

Fluorescence microscopy of plasma-treated biofilms. (A) Control; (B to F) plasma treatment for 30 s (B), 60 s (C), 120 s (D), 180 s (E), and 300 s (F). The biofilms were stained with SYTO9, showing green fluorescence (living cells), and propidium iodide, showing red fluorescence (dead cells).
Figure Legend Snippet: Fluorescence microscopy of plasma-treated biofilms. (A) Control; (B to F) plasma treatment for 30 s (B), 60 s (C), 120 s (D), 180 s (E), and 300 s (F). The biofilms were stained with SYTO9, showing green fluorescence (living cells), and propidium iodide, showing red fluorescence (dead cells).

Techniques Used: Fluorescence, Microscopy, Staining

Fluorescence assay of Candida albicans biofilms. The G/R ratio is the quotient of green fluorescence intensity and red fluorescence intensity. The data points are the mean values for the total population of the quadruple repetition. n = 6 for each repetition. P
Figure Legend Snippet: Fluorescence assay of Candida albicans biofilms. The G/R ratio is the quotient of green fluorescence intensity and red fluorescence intensity. The data points are the mean values for the total population of the quadruple repetition. n = 6 for each repetition. P

Techniques Used: Fluorescence

Atomic-force microscopy images of the plasma-treated biofilms. The left panels show topographical images from the top of the biofilm layer, and the right panels show error signal images of the same section. (A) Control; (B and C) plasma treatment times of 120 s (B) and 300 s (C). The images were taken in contact mode with a cantilever spring constant ( k ) of 0.1 to 0.6 N/m 2 and a frequency of 0.4 Hz, the set point at 8 N/m 2 , and an area of 20 μm 2 .
Figure Legend Snippet: Atomic-force microscopy images of the plasma-treated biofilms. The left panels show topographical images from the top of the biofilm layer, and the right panels show error signal images of the same section. (A) Control; (B and C) plasma treatment times of 120 s (B) and 300 s (C). The images were taken in contact mode with a cantilever spring constant ( k ) of 0.1 to 0.6 N/m 2 and a frequency of 0.4 Hz, the set point at 8 N/m 2 , and an area of 20 μm 2 .

Techniques Used: Microscopy

Fluorescence microscopy of Candida albicans biofilms after heating on a plate heater. Biofilms were heated 1 min at 130°C on a plate heater. Shown are four different biofilms from different locations of the 96-well plate after heating on a plate heater. Green, living cells; red (orange), dead cells.
Figure Legend Snippet: Fluorescence microscopy of Candida albicans biofilms after heating on a plate heater. Biofilms were heated 1 min at 130°C on a plate heater. Shown are four different biofilms from different locations of the 96-well plate after heating on a plate heater. Green, living cells; red (orange), dead cells.

Techniques Used: Fluorescence, Microscopy

Thermal image measurements of biofilms taken directly after plasma treatment. Images were taken at a distance of 20 cm with an FLIR thermal imaging camera at plasma treatment times of 30 s (A), 60 s (B), 120 s (C), 180 s (D), and 300 s (E).
Figure Legend Snippet: Thermal image measurements of biofilms taken directly after plasma treatment. Images were taken at a distance of 20 cm with an FLIR thermal imaging camera at plasma treatment times of 30 s (A), 60 s (B), 120 s (C), 180 s (D), and 300 s (E).

Techniques Used: Imaging

XTT assay of Candida albicans biofilms. The data points are the mean values for the total population of the quadruple repetition. n = 6 for each repetition. P
Figure Legend Snippet: XTT assay of Candida albicans biofilms. The data points are the mean values for the total population of the quadruple repetition. n = 6 for each repetition. P

Techniques Used: XTT Assay

CFU assay of the Candida albicans biofilms after plasma treatment. The orange line represents the detection limit up to which representative values could be counted. The blue line shows the reduction factor (RF) of the different plasma treatment times. The error bars were calculated using the propagation of error and weighted error for the controls and the weighted mean value for the samples. The data points are the weighted mean values for the total population of the quadruple repetition. n = 6 for each repetition. P
Figure Legend Snippet: CFU assay of the Candida albicans biofilms after plasma treatment. The orange line represents the detection limit up to which representative values could be counted. The blue line shows the reduction factor (RF) of the different plasma treatment times. The error bars were calculated using the propagation of error and weighted error for the controls and the weighted mean value for the samples. The data points are the weighted mean values for the total population of the quadruple repetition. n = 6 for each repetition. P

Techniques Used: Colony-forming Unit Assay

Confocal laser scanning microscopy of Live/Dead-stained Candida albicans biofilms after plasma treatment. Left panels show an orthogonal view of the top biofilm layer (horizontal optical sections in the center and vertical optical sections in the flanking pictures). Central and right panels show 3D images with top and bottom views of the biofilms. For each biofilm, an area of 1,272.2 μm by 1,272.2 μm was visualized.
Figure Legend Snippet: Confocal laser scanning microscopy of Live/Dead-stained Candida albicans biofilms after plasma treatment. Left panels show an orthogonal view of the top biofilm layer (horizontal optical sections in the center and vertical optical sections in the flanking pictures). Central and right panels show 3D images with top and bottom views of the biofilms. For each biofilm, an area of 1,272.2 μm by 1,272.2 μm was visualized.

Techniques Used: Confocal Laser Scanning Microscopy, Staining

12) 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

13) Product Images from "Clostridium difficile Biofilm: Remodeling Metabolism and Cell Surface to Build a Sparse and Heterogeneously Aggregated Architecture"

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

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02084

Intact biofilm architecture of the parental strain and CD2214–CD2215 mutant. The biofilms of the parental strain 630Δ erm (A–D) and its CD2214–CD2215 mutant (E–H) ). After live dead staining of intact biofilms directly in the micro-titer plates, their microscopic architecture was observed in situ by CLSM. Images representative of three independent experiments (each using three clones) are shown. Raw confocal z-stacks were treated using IMARIS software. This allowed obtaining both a 3D projection upside view, with its shadow on the right (A,E) , and a section view close to the surface (B,F) , in which the white bar indicates the scale (50 μm). For the section view of each strain, magnifications of micro-aggregated forms (C,G) and rods (D,H) ).
Figure Legend Snippet: Intact biofilm architecture of the parental strain and CD2214–CD2215 mutant. The biofilms of the parental strain 630Δ erm (A–D) and its CD2214–CD2215 mutant (E–H) ). After live dead staining of intact biofilms directly in the micro-titer plates, their microscopic architecture was observed in situ by CLSM. Images representative of three independent experiments (each using three clones) are shown. Raw confocal z-stacks were treated using IMARIS software. This allowed obtaining both a 3D projection upside view, with its shadow on the right (A,E) , and a section view close to the surface (B,F) , in which the white bar indicates the scale (50 μm). For the section view of each strain, magnifications of micro-aggregated forms (C,G) and rods (D,H) ).

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

Comparison between CD2214–CD2215 regulon and the set of genes differentially expressed in biofilm/planktonic growth. (A) ) is on the left and the set of genes differentially expressed in strain 630Δ erm / CD2214–CD2215 ) is on the right. The number of shared genes (whose expression varies in both transcriptomes) is indicated above the overlap region. As shared genes can vary in the same direction in the two transcriptomes or in opposite directions, the overlap region is divided into two parts. The number of shared genes whose expression varies in opposite directions is indicated in the dark gray part, while the number of genes whose expression varies in the same direction is in the light gray part. (B) ) are underlined.
Figure Legend Snippet: Comparison between CD2214–CD2215 regulon and the set of genes differentially expressed in biofilm/planktonic growth. (A) ) is on the left and the set of genes differentially expressed in strain 630Δ erm / CD2214–CD2215 ) is on the right. The number of shared genes (whose expression varies in both transcriptomes) is indicated above the overlap region. As shared genes can vary in the same direction in the two transcriptomes or in opposite directions, the overlap region is divided into two parts. The number of shared genes whose expression varies in opposite directions is indicated in the dark gray part, while the number of genes whose expression varies in the same direction is in the light gray part. (B) ) are underlined.

Techniques Used: Expressing

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

Techniques Used: Flow Cytometry

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

Techniques Used: Light Microscopy

14) Product Images from "Ecosystem Screening Approach for Pathogen-Associated Microorganisms Affecting Host Disease ▿Ecosystem Screening Approach for Pathogen-Associated Microorganisms Affecting Host Disease ▿ †"

Article Title: Ecosystem Screening Approach for Pathogen-Associated Microorganisms Affecting Host Disease ▿Ecosystem Screening Approach for Pathogen-Associated Microorganisms Affecting Host Disease ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.05371-11

Biofilm community and in planta screening. (A) Illustration of a mixed-species biofilm after colonization of a P. parasitica microcolony. (B) For in planta screening, P. parasitica zoospores were used alone ( Pp ) or with spores from isolates Ieuk1, Ieuk2,
Figure Legend Snippet: Biofilm community and in planta screening. (A) Illustration of a mixed-species biofilm after colonization of a P. parasitica microcolony. (B) For in planta screening, P. parasitica zoospores were used alone ( Pp ) or with spores from isolates Ieuk1, Ieuk2,

Techniques Used:

Scheme of microbial community screening for pathogen-associated microorganisms affecting host disease. This represents an example approach to analysis of the rhizosphere community associated, in biofilms, with the plant pathogen P. parasitica .
Figure Legend Snippet: Scheme of microbial community screening for pathogen-associated microorganisms affecting host disease. This represents an example approach to analysis of the rhizosphere community associated, in biofilms, with the plant pathogen P. parasitica .

Techniques Used:

Vorticella-Phytophthora interaction. (A) Vorticella species anchored in a biofilm. The inset illustrates a larger view of the attachment of a ciliate cell to a microcolony. (B) Confocal laser scanning microscopy images of a ciliate cell and a P. parasitica
Figure Legend Snippet: Vorticella-Phytophthora interaction. (A) Vorticella species anchored in a biofilm. The inset illustrates a larger view of the attachment of a ciliate cell to a microcolony. (B) Confocal laser scanning microscopy images of a ciliate cell and a P. parasitica

Techniques Used: Confocal Laser Scanning Microscopy

15) Product Images from "Bacteriological Effects of Dentifrices with and without Active Ingredients of Natural Origin"

Article Title: Bacteriological Effects of Dentifrices with and without Active Ingredients of Natural Origin

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02315-14

Two-factor principal components analysis of PCR-DGGE banding profiles representative of oral biofilms exposed to toothpaste formulations following 1-day (triangles), 3-day (circles), and 5-day (squares) periods. Formulations included the following: Colgate
Figure Legend Snippet: Two-factor principal components analysis of PCR-DGGE banding profiles representative of oral biofilms exposed to toothpaste formulations following 1-day (triangles), 3-day (circles), and 5-day (squares) periods. Formulations included the following: Colgate

Techniques Used: Polymerase Chain Reaction, Denaturing Gradient Gel Electrophoresis

16) Product Images from "Variation in Biofilm Formation among Strains of Listeria monocytogenes"

Article Title: Variation in Biofilm Formation among Strains of Listeria monocytogenes

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.12.7336-7342.2003

SEM images of strain M39503A (high biofilm former) on stainless steel (A) and PVC (C) and strain M35584A (low biofilm former) on stainless steel (B) and PVC (D). The cracks visible under cells in panel B are artifacts in the stainless steel surface. Scale bars, 8.6 μm.
Figure Legend Snippet: SEM images of strain M39503A (high biofilm former) on stainless steel (A) and PVC (C) and strain M35584A (low biofilm former) on stainless steel (B) and PVC (D). The cracks visible under cells in panel B are artifacts in the stainless steel surface. Scale bars, 8.6 μm.

Techniques Used:

17) Product Images from "Targeting the HUβ Protein Prevents Porphyromonas gingivalis from Entering into Preexisting Biofilms"

Article Title: Targeting the HUβ Protein Prevents Porphyromonas gingivalis from Entering into Preexisting Biofilms

Journal: Journal of Bacteriology

doi: 10.1128/JB.00790-17

Preincubation of P. gingivalis with anti-PgHUβ prevents P. gingivalis from entering into streptococcal biofilm. Biofilms were grown for 24 h before addition of P. gingivalis. P. gingivalis was incubated with a 1:50 or 1:10 dilution of anti-PgHUβ antisera for 1 h before addition. Representative biofilm images show results for S. gordonii alone (A) as well as naive serum (B) and a 1:50 dilution of anti-PgHUβ-treated P. gingivalis (C). All cells (green) and P. gingivalis (red) are shown in the top image, while P. gingivalis -only labeling is shown in in the middle image. The bottom image is a side view of the P. gingivalis -only-labeled biofilm. A no- P. gingivalis control biofilm (A) shows little to no red signal, while that treated with naive serum shows significant amounts of P. gingivalis (B). Treatment with a 1:50 dilution of anti-PgHUβ results in a significant decrease in P. gingivalis detected within the biofilm (C). Graphical representations of decrease in P. gingivalis entering into S. mitis (D), S. oralis (E), S. cristatus (F), or S. gordonii (G) indicate dose-dependent decreases in detection of P. gingivalis . Experiments with S. gordonii were repeated with 50% pooled human saliva (H). Total naive serum-treated P. gingivalis entering streptococcal biofilms is shown in panel I, which shows that significantly more P. gingivalis cells enter an S. gordonii biofilm than the other oral streptococci tested. Dual-species biofilms were grown for 16 h in THBHK. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE). P. gingivalis was immunofluorescently labeled with a polyclonal antifimbrial primary antisera and a secondary goat anti-rabbit Alexa Fluor 647-conjugated antibody and visualized using confocal scanning laser microscopy. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Figure Legend Snippet: Preincubation of P. gingivalis with anti-PgHUβ prevents P. gingivalis from entering into streptococcal biofilm. Biofilms were grown for 24 h before addition of P. gingivalis. P. gingivalis was incubated with a 1:50 or 1:10 dilution of anti-PgHUβ antisera for 1 h before addition. Representative biofilm images show results for S. gordonii alone (A) as well as naive serum (B) and a 1:50 dilution of anti-PgHUβ-treated P. gingivalis (C). All cells (green) and P. gingivalis (red) are shown in the top image, while P. gingivalis -only labeling is shown in in the middle image. The bottom image is a side view of the P. gingivalis -only-labeled biofilm. A no- P. gingivalis control biofilm (A) shows little to no red signal, while that treated with naive serum shows significant amounts of P. gingivalis (B). Treatment with a 1:50 dilution of anti-PgHUβ results in a significant decrease in P. gingivalis detected within the biofilm (C). Graphical representations of decrease in P. gingivalis entering into S. mitis (D), S. oralis (E), S. cristatus (F), or S. gordonii (G) indicate dose-dependent decreases in detection of P. gingivalis . Experiments with S. gordonii were repeated with 50% pooled human saliva (H). Total naive serum-treated P. gingivalis entering streptococcal biofilms is shown in panel I, which shows that significantly more P. gingivalis cells enter an S. gordonii biofilm than the other oral streptococci tested. Dual-species biofilms were grown for 16 h in THBHK. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE). P. gingivalis was immunofluorescently labeled with a polyclonal antifimbrial primary antisera and a secondary goat anti-rabbit Alexa Fluor 647-conjugated antibody and visualized using confocal scanning laser microscopy. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Techniques Used: Incubation, Labeling, Staining, Microscopy

18) Product Images from "Targeting the HUβ Protein Prevents Porphyromonas gingivalis from Entering into Preexisting Biofilms"

Article Title: Targeting the HUβ Protein Prevents Porphyromonas gingivalis from Entering into Preexisting Biofilms

Journal: Journal of Bacteriology

doi: 10.1128/JB.00790-17

Preincubation of P. gingivalis with anti-PgHUβ prevents P. gingivalis from entering into streptococcal biofilm. Biofilms were grown for 24 h before addition of P. gingivalis. P. gingivalis was incubated with a 1:50 or 1:10 dilution of anti-PgHUβ antisera for 1 h before addition. Representative biofilm images show results for S. gordonii alone (A) as well as naive serum (B) and a 1:50 dilution of anti-PgHUβ-treated P. gingivalis (C). All cells (green) and P. gingivalis (red) are shown in the top image, while P. gingivalis -only labeling is shown in in the middle image. The bottom image is a side view of the P. gingivalis -only-labeled biofilm. A no- P. gingivalis control biofilm (A) shows little to no red signal, while that treated with naive serum shows significant amounts of P. gingivalis (B). Treatment with a 1:50 dilution of anti-PgHUβ results in a significant decrease in P. gingivalis detected within the biofilm (C). Graphical representations of decrease in P. gingivalis entering into S. mitis (D), S. oralis (E), S. cristatus (F), or S. gordonii (G) indicate dose-dependent decreases in detection of P. gingivalis . Experiments with S. gordonii were repeated with 50% pooled human saliva (H). Total naive serum-treated P. gingivalis entering streptococcal biofilms is shown in panel I, which shows that significantly more P. gingivalis cells enter an S. gordonii biofilm than the other oral streptococci tested. Dual-species biofilms were grown for 16 h in THBHK. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE). P. gingivalis was immunofluorescently labeled with a polyclonal antifimbrial primary antisera and a secondary goat anti-rabbit Alexa Fluor 647-conjugated antibody and visualized using confocal scanning laser microscopy. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
Figure Legend Snippet: Preincubation of P. gingivalis with anti-PgHUβ prevents P. gingivalis from entering into streptococcal biofilm. Biofilms were grown for 24 h before addition of P. gingivalis. P. gingivalis was incubated with a 1:50 or 1:10 dilution of anti-PgHUβ antisera for 1 h before addition. Representative biofilm images show results for S. gordonii alone (A) as well as naive serum (B) and a 1:50 dilution of anti-PgHUβ-treated P. gingivalis (C). All cells (green) and P. gingivalis (red) are shown in the top image, while P. gingivalis -only labeling is shown in in the middle image. The bottom image is a side view of the P. gingivalis -only-labeled biofilm. A no- P. gingivalis control biofilm (A) shows little to no red signal, while that treated with naive serum shows significant amounts of P. gingivalis (B). Treatment with a 1:50 dilution of anti-PgHUβ results in a significant decrease in P. gingivalis detected within the biofilm (C). Graphical representations of decrease in P. gingivalis entering into S. mitis (D), S. oralis (E), S. cristatus (F), or S. gordonii (G) indicate dose-dependent decreases in detection of P. gingivalis . Experiments with S. gordonii were repeated with 50% pooled human saliva (H). Total naive serum-treated P. gingivalis entering streptococcal biofilms is shown in panel I, which shows that significantly more P. gingivalis cells enter an S. gordonii biofilm than the other oral streptococci tested. Dual-species biofilms were grown for 16 h in THBHK. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE). P. gingivalis was immunofluorescently labeled with a polyclonal antifimbrial primary antisera and a secondary goat anti-rabbit Alexa Fluor 647-conjugated antibody and visualized using confocal scanning laser microscopy. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Techniques Used: Incubation, Labeling, Staining, Microscopy

19) Product Images from "Global Identification of Biofilm-Specific Proteolysis in Candida albicans"

Article Title: Global Identification of Biofilm-Specific Proteolysis in Candida albicans

Journal: mBio

doi: 10.1128/mBio.01514-16

Label-free quantitation of proteins identified by shotgun proteomic analysis in conditioned medium from C. albicans cultures grown under biofilm and planktonic conditions. (A, left) Volcano plot depicting the log 2 -fold change in protein abundance (reported as biofilm/planktonic), demonstrating a significant increase in Sap5 and Sap6 levels under biofilm conditions with a log 2 -fold change of 3.1 for Sap5 ( P =3.7 × 10 −4 ) and a log 2 -fold change of 5.3 for Sap6 ( P = 9.1 × 10 −5 ). (A, right) Corresponding histogram showing the relative distribution of proteins identified. (B) Comparison of relative Sap5 and Sap6 peptide levels under biofilm and planktonic conditions with mean log 2 -fold changes indicated. Each data point represents quantification of a unique tryptic peptide for Sap5 or Sap6. Mean values of triplicate samples are reported. Supporting proteomic data are provided in Data Set S1 in the supplemental material.
Figure Legend Snippet: Label-free quantitation of proteins identified by shotgun proteomic analysis in conditioned medium from C. albicans cultures grown under biofilm and planktonic conditions. (A, left) Volcano plot depicting the log 2 -fold change in protein abundance (reported as biofilm/planktonic), demonstrating a significant increase in Sap5 and Sap6 levels under biofilm conditions with a log 2 -fold change of 3.1 for Sap5 ( P =3.7 × 10 −4 ) and a log 2 -fold change of 5.3 for Sap6 ( P = 9.1 × 10 −5 ). (A, right) Corresponding histogram showing the relative distribution of proteins identified. (B) Comparison of relative Sap5 and Sap6 peptide levels under biofilm and planktonic conditions with mean log 2 -fold changes indicated. Each data point represents quantification of a unique tryptic peptide for Sap5 or Sap6. Mean values of triplicate samples are reported. Supporting proteomic data are provided in Data Set S1 in the supplemental material.

Techniques Used: Quantitation Assay

Global protease substrate specificity profiling reveals that Sap5 and Sap6 activities are highly increased in C. albicans under biofilm growth conditions. (A) iceLogo representations of recombinantly produced Sap5 and Sap6 following 240 min of incubation with the MSP-MS peptide library ( P ≤ 0.05 for residues colored by physicochemical property). Further comparison of Sap5 and Sap6 specificity is shown in Fig. S4 in the supplemental material. (B) iceLogo representation of cleavages from the C. albicans biofilm conditioned medium MSP-MS assay that are sensitive to pretreatment with the aspartyl protease inhibitor pepstatin A (10 µM). (C) Assignment of pepstatin-sensitive cleavages in the biofilm and planktonic profiles through comparison to recombinantly produced Sap5 and Sap6. The biofilm (60 min) and planktonic (240 min) time points were chosen to normalize for the higher total proteolytic activity under the biofilm condition. iceLogo representations for unassigned cleavages are distinct from the Sap5 and Sap6 specificity profiles and are provided in Fig. S5 in the supplemental material. (D) Example peptide cleavages from the biofilm (green arrows) and planktonic (yellow arrows) MSP-MS assays are shown with pepstatin-sensitive cleavages indicated by an X and the time point of first appearance noted. Selected cleavages were omitted for clarity. All MSP-MS cleavages identified are provided in Data Set S1 in the supplemental material.
Figure Legend Snippet: Global protease substrate specificity profiling reveals that Sap5 and Sap6 activities are highly increased in C. albicans under biofilm growth conditions. (A) iceLogo representations of recombinantly produced Sap5 and Sap6 following 240 min of incubation with the MSP-MS peptide library ( P ≤ 0.05 for residues colored by physicochemical property). Further comparison of Sap5 and Sap6 specificity is shown in Fig. S4 in the supplemental material. (B) iceLogo representation of cleavages from the C. albicans biofilm conditioned medium MSP-MS assay that are sensitive to pretreatment with the aspartyl protease inhibitor pepstatin A (10 µM). (C) Assignment of pepstatin-sensitive cleavages in the biofilm and planktonic profiles through comparison to recombinantly produced Sap5 and Sap6. The biofilm (60 min) and planktonic (240 min) time points were chosen to normalize for the higher total proteolytic activity under the biofilm condition. iceLogo representations for unassigned cleavages are distinct from the Sap5 and Sap6 specificity profiles and are provided in Fig. S5 in the supplemental material. (D) Example peptide cleavages from the biofilm (green arrows) and planktonic (yellow arrows) MSP-MS assays are shown with pepstatin-sensitive cleavages indicated by an X and the time point of first appearance noted. Selected cleavages were omitted for clarity. All MSP-MS cleavages identified are provided in Data Set S1 in the supplemental material.

Techniques Used: Produced, Incubation, Mass Spectrometry, Protease Inhibitor, Activity Assay

Deletion of SAP5 and SAP6 impairs C. albicans biofilm formation under in vitro growth conditions and in vivo in a rat central venous catheter biofilm model. (A) Biofilm formation in Spider medium after 24 h of growth of the wild-type (WT) reference (SN250) and sap5 Δ/Δ, sap6 Δ/Δ, and sap5/6 ΔΔ/ΔΔ deletion strains. OD 600 readings were measured for adhered biofilms after removal of the medium and normalized to the wild-type strain (OD 600 set to 1.0), and the mean ± SD is shown ( n = 4 for each strain). OD 600 measurements of the sap5 , sap6 , and sap5/6 deletion strains deviated significantly from those of the reference strain (**, P
Figure Legend Snippet: Deletion of SAP5 and SAP6 impairs C. albicans biofilm formation under in vitro growth conditions and in vivo in a rat central venous catheter biofilm model. (A) Biofilm formation in Spider medium after 24 h of growth of the wild-type (WT) reference (SN250) and sap5 Δ/Δ, sap6 Δ/Δ, and sap5/6 ΔΔ/ΔΔ deletion strains. OD 600 readings were measured for adhered biofilms after removal of the medium and normalized to the wild-type strain (OD 600 set to 1.0), and the mean ± SD is shown ( n = 4 for each strain). OD 600 measurements of the sap5 , sap6 , and sap5/6 deletion strains deviated significantly from those of the reference strain (**, P

Techniques Used: In Vitro, In Vivo

Sap5- and Sap6-cleavable fluorogenic peptide substrates distinguish between C. albicans under biofilm and planktonic growth conditions. (A) Fluorogenic substrates were developed on the basis of Sap5 and Sap6 cleavages from the MSP-MS peptide library. An example MS-based time course is provided showing Sap6-favored cleavage of KWLIHPTFSYnRWP, one substrate within a 25-member peptide sublibrary. Complete hydrolysis of the parent substrate and cleavage at a single site allowed for the calculation of k cat / K m values of 4.4 × 10 4 M −1 s −1 (Sap5) and 2.0 × 10 5 M −1 s −1 (Sap6). Cleavage time courses for the remaining peptides used in sequence selection are provided in Fig. S6 in the supplemental material. (B) Activity (relative fluorescence units [RFU] per second per microgram) of the internally quenched fluorogenic substrates VFILWRTE (blue bars) and TFSYnRWP (red bars) against recombinantly produced Sap5 and Sap6. (C) Both VFILWRTE and TFSYnRWP displayed significantly higher activity (***, P
Figure Legend Snippet: Sap5- and Sap6-cleavable fluorogenic peptide substrates distinguish between C. albicans under biofilm and planktonic growth conditions. (A) Fluorogenic substrates were developed on the basis of Sap5 and Sap6 cleavages from the MSP-MS peptide library. An example MS-based time course is provided showing Sap6-favored cleavage of KWLIHPTFSYnRWP, one substrate within a 25-member peptide sublibrary. Complete hydrolysis of the parent substrate and cleavage at a single site allowed for the calculation of k cat / K m values of 4.4 × 10 4 M −1 s −1 (Sap5) and 2.0 × 10 5 M −1 s −1 (Sap6). Cleavage time courses for the remaining peptides used in sequence selection are provided in Fig. S6 in the supplemental material. (B) Activity (relative fluorescence units [RFU] per second per microgram) of the internally quenched fluorogenic substrates VFILWRTE (blue bars) and TFSYnRWP (red bars) against recombinantly produced Sap5 and Sap6. (C) Both VFILWRTE and TFSYnRWP displayed significantly higher activity (***, P

Techniques Used: Mass Spectrometry, Sequencing, Selection, Activity Assay, Fluorescence, Produced

Protease activity profiling enables the detection of biofilm formation from diverse pathogenic Candida species. Both fluorogenic substrates VFILWRTE (blue bars) (A) and TFSYnRWP (red bars) (B) displayed significantly higher activity (relative fluorescence units [RFU] per second) (**, P
Figure Legend Snippet: Protease activity profiling enables the detection of biofilm formation from diverse pathogenic Candida species. Both fluorogenic substrates VFILWRTE (blue bars) (A) and TFSYnRWP (red bars) (B) displayed significantly higher activity (relative fluorescence units [RFU] per second) (**, P

Techniques Used: Activity Assay, Fluorescence

Wild-type C. albicans has distinct global protease substrate specificity profiles under biofilm and planktonic growth conditions. (A) iceLogo substrate specificity representations for 24-h conditioned medium from wild-type C. albicans (SN425) following 240 min of incubation with the MSP-MS peptide library ( P ≤ 0.05 for residues colored by physicochemical property; “n” is norleucine). Specificity was determined with equivalent protein amounts from each condition. Specificity profiles for the 15- and 60-min assay time points are provided in Fig. S1 in the supplemental material. (B) Quantification of the total shared and unique cleavages for the biofilm and planktonic conditions at the 240-min assay time point. (C) Heat map representation of biofilm and planktonic specificity differences at the 240-min time point calculated by using Z score differences at the P4-P4′ positions. Biofilm-favored residues are blue ( Z score, > 0), and planktonic-favored residues are red ( Z score,
Figure Legend Snippet: Wild-type C. albicans has distinct global protease substrate specificity profiles under biofilm and planktonic growth conditions. (A) iceLogo substrate specificity representations for 24-h conditioned medium from wild-type C. albicans (SN425) following 240 min of incubation with the MSP-MS peptide library ( P ≤ 0.05 for residues colored by physicochemical property; “n” is norleucine). Specificity was determined with equivalent protein amounts from each condition. Specificity profiles for the 15- and 60-min assay time points are provided in Fig. S1 in the supplemental material. (B) Quantification of the total shared and unique cleavages for the biofilm and planktonic conditions at the 240-min assay time point. (C) Heat map representation of biofilm and planktonic specificity differences at the 240-min time point calculated by using Z score differences at the P4-P4′ positions. Biofilm-favored residues are blue ( Z score, > 0), and planktonic-favored residues are red ( Z score,

Techniques Used: Incubation, Mass Spectrometry

20) Product Images from "Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa"

Article Title: Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.00102

Purification and identification of active component of EPS273. (A) The profiles of the fractions in the gel filtration, which were collected and monitored for the biofilm formation determined at OD595 nm after crystal violet staining and polysaccharide content determined at OD490 nm after the phenol-sulfuric acid assay. (B) Effects of Proteinase K, DNase I, RNase A and NaIO 4 on the activities of EPS273 inhibiting biofilm formation of P. aeruginosa PAO1. EPS273 (0.1 μg/mL) was, respectively, treated with proteinase K (PrK) (1 mg/mL), DNaseI (100 μg/mL), RNaseA (100 μg/mL) for 1 h or NaIO 4 (10 mM) for 12 h at 37°C, then taken to measure the antibiofilm activity. Error bars represent standard deviations of three independent experiments. Error bars indicate the standard deviations of three measurements. ∗ P
Figure Legend Snippet: Purification and identification of active component of EPS273. (A) The profiles of the fractions in the gel filtration, which were collected and monitored for the biofilm formation determined at OD595 nm after crystal violet staining and polysaccharide content determined at OD490 nm after the phenol-sulfuric acid assay. (B) Effects of Proteinase K, DNase I, RNase A and NaIO 4 on the activities of EPS273 inhibiting biofilm formation of P. aeruginosa PAO1. EPS273 (0.1 μg/mL) was, respectively, treated with proteinase K (PrK) (1 mg/mL), DNaseI (100 μg/mL), RNaseA (100 μg/mL) for 1 h or NaIO 4 (10 mM) for 12 h at 37°C, then taken to measure the antibiofilm activity. Error bars represent standard deviations of three independent experiments. Error bars indicate the standard deviations of three measurements. ∗ P

Techniques Used: Purification, Filtration, Staining, Acid Assay, Activity Assay

Anbibiofilm and antibacterial activity analyses of purified EPS273. (A) Florescence microscopic observation of inhibition on biofilm formation and dispersion on preformed biofilm by EPS273. The images are representative of three separate experiments. (B) Antibacterial activity analyses of EPS273. Error bars indicate the standard deviations of three measurements. ∗ P
Figure Legend Snippet: Anbibiofilm and antibacterial activity analyses of purified EPS273. (A) Florescence microscopic observation of inhibition on biofilm formation and dispersion on preformed biofilm by EPS273. The images are representative of three separate experiments. (B) Antibacterial activity analyses of EPS273. Error bars indicate the standard deviations of three measurements. ∗ P

Techniques Used: Activity Assay, Purification, Inhibition

Effects of Pseudomonas stutzeri 273 supernatant on biofilm formation and dispersion of P. aeruginosa PAO1. (A) Biofilm formation of P. aeruginosa PAO1 in 96-well polystyrene plate wells in the presence or absence of 10% supernatant of P. stutzeri 273 (273S) and duplicate wells are shown. (B) Biofilm formation of P. aeruginosa PAO1 on glass slides in the presence or absence of 10% 273S. As for the controls, the same amount of LB broth was used. (C) Preformed biofilm dispersion of P. aeruginosa PAO1 in 96-well polystyrene plate wells in the presence or absence of 10% 273S and duplicate wells are shown. (D) Preformed biofilm dispersion of P. aeruginosa PAO1 on glass slides in the presence or absence of 10% 273S. As for the controls, the same amount of LB broth was used.
Figure Legend Snippet: Effects of Pseudomonas stutzeri 273 supernatant on biofilm formation and dispersion of P. aeruginosa PAO1. (A) Biofilm formation of P. aeruginosa PAO1 in 96-well polystyrene plate wells in the presence or absence of 10% supernatant of P. stutzeri 273 (273S) and duplicate wells are shown. (B) Biofilm formation of P. aeruginosa PAO1 on glass slides in the presence or absence of 10% 273S. As for the controls, the same amount of LB broth was used. (C) Preformed biofilm dispersion of P. aeruginosa PAO1 in 96-well polystyrene plate wells in the presence or absence of 10% 273S and duplicate wells are shown. (D) Preformed biofilm dispersion of P. aeruginosa PAO1 on glass slides in the presence or absence of 10% 273S. As for the controls, the same amount of LB broth was used.

Techniques Used:

21) Product Images from "Anode potential influences the structure and function of anodic electrode and electrolyte-associated microbiomes"

Article Title: Anode potential influences the structure and function of anodic electrode and electrolyte-associated microbiomes

Journal: Scientific Reports

doi: 10.1038/srep39114

Representative turnover cyclic voltammograms at a scan rate of 1 mV s −1 of biofilms metabolizing volatile fatty acids. Red, blue, and green lines indicate CVs performed on the BES operated at 300 mV, 550 mV, and 800 mV, respectively. Solid lines indicate forward scans (from negative to positive potentials) and dashed lines indicate backward scans (from positive to negative potentials). Dotted line indicates a CV measured on a sterile electrode immersed in the same electrolyte as the other BESs. Putative electron transfer sites (E f ) were determined by first derivative analysis (FDA) and are indicated with dots in the CVs. E f were centered at −158 mV and at −140 mV for the BES operated at 300 mV and 550 mV, respectively. The BES operated at 800 mV displayed a less clear current vs potential response with two putative electron transfer sites centered at +150 mV and +390 mV.
Figure Legend Snippet: Representative turnover cyclic voltammograms at a scan rate of 1 mV s −1 of biofilms metabolizing volatile fatty acids. Red, blue, and green lines indicate CVs performed on the BES operated at 300 mV, 550 mV, and 800 mV, respectively. Solid lines indicate forward scans (from negative to positive potentials) and dashed lines indicate backward scans (from positive to negative potentials). Dotted line indicates a CV measured on a sterile electrode immersed in the same electrolyte as the other BESs. Putative electron transfer sites (E f ) were determined by first derivative analysis (FDA) and are indicated with dots in the CVs. E f were centered at −158 mV and at −140 mV for the BES operated at 300 mV and 550 mV, respectively. The BES operated at 800 mV displayed a less clear current vs potential response with two putative electron transfer sites centered at +150 mV and +390 mV.

Techniques Used:

Degradation of Volatile Fatty Acids at different anode potentials over time. The grey vertical lines indicate the times that the reactors were paused for biofilm sampling.
Figure Legend Snippet: Degradation of Volatile Fatty Acids at different anode potentials over time. The grey vertical lines indicate the times that the reactors were paused for biofilm sampling.

Techniques Used: Sampling

( A ) Anodic biofilm thickness at different potentials (SHE) over time. ( B ) Anodic biofilm thickness as a function of charge after 38 days at different potentials. ( C ) Biofilm thickness per unit charge after 38 days at different potentials. Error bars represent standard deviations and the asterisk in panel C signifies that significantly (P = 0.03) more biofilm thickness was produced per unit charge at 800 mV than at 300 mV or 550 mV.
Figure Legend Snippet: ( A ) Anodic biofilm thickness at different potentials (SHE) over time. ( B ) Anodic biofilm thickness as a function of charge after 38 days at different potentials. ( C ) Biofilm thickness per unit charge after 38 days at different potentials. Error bars represent standard deviations and the asterisk in panel C signifies that significantly (P = 0.03) more biofilm thickness was produced per unit charge at 800 mV than at 300 mV or 550 mV.

Techniques Used: Produced

Chronoamperometric profiles at different anode potentials. The chronoamperometric output was from all 15 anodes within each reactor. The grey vertical lines indicate the times that the reactors were paused for biofilm sampling.
Figure Legend Snippet: Chronoamperometric profiles at different anode potentials. The chronoamperometric output was from all 15 anodes within each reactor. The grey vertical lines indicate the times that the reactors were paused for biofilm sampling.

Techniques Used: Sampling

22) Product Images from "RpoN Regulates Virulence Factors of Pseudomonas aeruginosa via Modulating the PqsR Quorum Sensing Regulator"

Article Title: RpoN Regulates Virulence Factors of Pseudomonas aeruginosa via Modulating the PqsR Quorum Sensing Regulator

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms161226103

( A ) Images of biofilm co-cultures of S. aureus 15981/pSB2019 with (i) PAO1; (ii) ∆ rpoN ; (iii) Δ rpoN COM and (iv) Δ rpoN /pME6032 -pqsR , respectively. S. aureus 15981/pSB2019 appeared green due to GFP expression whereas P. aeruginosa strains were stained with red fluorescent dye CYTO62 used to generate the simulated 3D images (Bitplane, AG). Scale bar, 20 μm; ( B ) Biomass ratios of S. aureus to P. aeruginosa strains from different biofilm co-cultures were calculated using Imaris and shown in the histogram. Means and S.D. from triplicate experiments are shown. Student’s t -test was performed for testing differences between groups. * p ≤ 0.05.
Figure Legend Snippet: ( A ) Images of biofilm co-cultures of S. aureus 15981/pSB2019 with (i) PAO1; (ii) ∆ rpoN ; (iii) Δ rpoN COM and (iv) Δ rpoN /pME6032 -pqsR , respectively. S. aureus 15981/pSB2019 appeared green due to GFP expression whereas P. aeruginosa strains were stained with red fluorescent dye CYTO62 used to generate the simulated 3D images (Bitplane, AG). Scale bar, 20 μm; ( B ) Biomass ratios of S. aureus to P. aeruginosa strains from different biofilm co-cultures were calculated using Imaris and shown in the histogram. Means and S.D. from triplicate experiments are shown. Student’s t -test was performed for testing differences between groups. * p ≤ 0.05.

Techniques Used: Expressing, Staining

23) Product Images from "A new mercury‐accumulating Mucor hiemalis strain EH8 from cold sulfidic spring water biofilms"

Article Title: A new mercury‐accumulating Mucor hiemalis strain EH8 from cold sulfidic spring water biofilms

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.368

Biodiversity of Marching spring biofilm shown by electron microscopy and fluorescence in situ hybridization ( FISH ). (a, b) Electron microscope images of Marching biofilm showing (a) occurrence of, for example, coccoid archaea/bacteria (C), exopolymeric structures ( EPS ) structures (E), fungal hypha EH 8 (F), and (b) diatoms (D), microalgae (A); (c–e) FISH labeling of (c) coccoidae archaea (Arch915), (d) euryarchaeota (Eury498), (e) high G‐C content bacteria ( HGC ), (f) ß‐proteobacteria (beta42a), (g) γ ‐proteobacteria ( GAM 42a), and (h) Mucor hiemalis EH 8 ( MH 1) with spores are shown. The FISH ‐probes applied are denoted in c–h and in legend (see above and Experimental Procedures).
Figure Legend Snippet: Biodiversity of Marching spring biofilm shown by electron microscopy and fluorescence in situ hybridization ( FISH ). (a, b) Electron microscope images of Marching biofilm showing (a) occurrence of, for example, coccoid archaea/bacteria (C), exopolymeric structures ( EPS ) structures (E), fungal hypha EH 8 (F), and (b) diatoms (D), microalgae (A); (c–e) FISH labeling of (c) coccoidae archaea (Arch915), (d) euryarchaeota (Eury498), (e) high G‐C content bacteria ( HGC ), (f) ß‐proteobacteria (beta42a), (g) γ ‐proteobacteria ( GAM 42a), and (h) Mucor hiemalis EH 8 ( MH 1) with spores are shown. The FISH ‐probes applied are denoted in c–h and in legend (see above and Experimental Procedures).

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

Association of biofilm with moss in the spring water of Marching. (A) Spring Marching with moss–microbial biofilm, (B) Moss Brachythecium rivulare (green part) is interfaced with the microbial biofilm (white part), and (C) microbial biofilm with fungus‐like filaments (F) interfacing moss leaf (Lf) after magnification using stereo microscopy.
Figure Legend Snippet: Association of biofilm with moss in the spring water of Marching. (A) Spring Marching with moss–microbial biofilm, (B) Moss Brachythecium rivulare (green part) is interfaced with the microbial biofilm (white part), and (C) microbial biofilm with fungus‐like filaments (F) interfacing moss leaf (Lf) after magnification using stereo microscopy.

Techniques Used: Microscopy

Phylogenetic tree based on ITS 1‐5.8S‐ ITS 2 sequence data showing the position of aquatic M. hiemalis EH 8 (Spring clone EH 8) from the sulfidic‐sulfurous Marching spring biofilms in comparison to related fungal strains and some other known fungal species of the class zygomycetes (see Experimental Procedures ). Bootstrap values greater than 50% are shown at the nodes. Bar = 10% estimated difference in nucleotide sequences.
Figure Legend Snippet: Phylogenetic tree based on ITS 1‐5.8S‐ ITS 2 sequence data showing the position of aquatic M. hiemalis EH 8 (Spring clone EH 8) from the sulfidic‐sulfurous Marching spring biofilms in comparison to related fungal strains and some other known fungal species of the class zygomycetes (see Experimental Procedures ). Bootstrap values greater than 50% are shown at the nodes. Bar = 10% estimated difference in nucleotide sequences.

Techniques Used: Sequencing

Growth of EH 8 on expanded clay. (1) Sterilized expanded clay (4–8 mm diameter) before cultivation, (2) EH 8's biofilm cultivated on expanded clay for in situ or ex situ treatment of mercury‐contaminated water even at low‐temperature sulfur‐reducing conditions.
Figure Legend Snippet: Growth of EH 8 on expanded clay. (1) Sterilized expanded clay (4–8 mm diameter) before cultivation, (2) EH 8's biofilm cultivated on expanded clay for in situ or ex situ treatment of mercury‐contaminated water even at low‐temperature sulfur‐reducing conditions.

Techniques Used: In Situ, Ex Situ

24) Product Images from "The Emerging Pathogen Candida auris: Growth Phenotype, Virulence Factors, Activity of Antifungals, and Effect of SCY-078, a Novel Glucan Synthesis Inhibitor, on Growth Morphology and Biofilm Formation"

Article Title: The Emerging Pathogen Candida auris: Growth Phenotype, Virulence Factors, Activity of Antifungals, and Effect of SCY-078, a Novel Glucan Synthesis Inhibitor, on Growth Morphology and Biofilm Formation

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.02396-16

Formation of biofilms by C. albicans and C. auris strains. Confocal scanning laser micrographs show top-down three-dimensional views (A to C) and side views (D to F) of biofilms formed by C. albicans (A, D), C. auris MRL 31102 (B, E), and C. auris MRL 31103 (C, F). Magnifications, ×100. (G) Thickness of biofilms formed by the tested isolates. *, P value compared to the thickness of C. albicans biofilms. A P value of
Figure Legend Snippet: Formation of biofilms by C. albicans and C. auris strains. Confocal scanning laser micrographs show top-down three-dimensional views (A to C) and side views (D to F) of biofilms formed by C. albicans (A, D), C. auris MRL 31102 (B, E), and C. auris MRL 31103 (C, F). Magnifications, ×100. (G) Thickness of biofilms formed by the tested isolates. *, P value compared to the thickness of C. albicans biofilms. A P value of

Techniques Used:

Quantification of biofilms formed by C. albicans and C. auris strains. The metabolic activity (A) and dry weight (B) of the biofilms formed by C. albicans , C. auris MRL 31102 (control), and 14 CBS C. auris strains are shown. *, P value compared to the results for C. albicans . A P value of
Figure Legend Snippet: Quantification of biofilms formed by C. albicans and C. auris strains. The metabolic activity (A) and dry weight (B) of the biofilms formed by C. albicans , C. auris MRL 31102 (control), and 14 CBS C. auris strains are shown. *, P value compared to the results for C. albicans . A P value of

Techniques Used: Activity Assay

Confocal scanning laser microscopy analyses of the effect of SCY-078 on biofilms formed by C. auris . Biofilms formed by C. auris MRL 31102 were exposed to no drug (control) (A, E) or SCY-078 at different concentrations: 0.5 mg/liter (B, F), 2 mg/liter (C, G), or 4 mg/liter (D, H). Top-down views (A to D) and side views (E to H) of untreated and treated biofilms are shown. Magnifications, ×25. (I and J) The thickness (I) and metabolic activity (J) of untreated (control) and SCY-078-treated biofilms. *, P value compared to the results for the untreated control (no drug). A P value of
Figure Legend Snippet: Confocal scanning laser microscopy analyses of the effect of SCY-078 on biofilms formed by C. auris . Biofilms formed by C. auris MRL 31102 were exposed to no drug (control) (A, E) or SCY-078 at different concentrations: 0.5 mg/liter (B, F), 2 mg/liter (C, G), or 4 mg/liter (D, H). Top-down views (A to D) and side views (E to H) of untreated and treated biofilms are shown. Magnifications, ×25. (I and J) The thickness (I) and metabolic activity (J) of untreated (control) and SCY-078-treated biofilms. *, P value compared to the results for the untreated control (no drug). A P value of

Techniques Used: Microscopy, Activity Assay

25) Product Images from "Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection"

Article Title: Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008421

Comparison of biofilm formation of wild type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains on polysterene and silicone surfaces. Metabolic activity of the cells was measured by XTT assay on polysterene plates (A), and plates containing silicone disk (B). Microscopic analysis of biofilm structures of the WT (C) and KO (D) strains formed after 48 hours on polysterene plates. XTT assay was measured at 492 nm. Experiments were performed twice with triplicates that reproduced similar results. Error bars indicate standard deviation. * P
Figure Legend Snippet: Comparison of biofilm formation of wild type (WT), heterozygous (HET), homozygous (KO), and reconstituted (RE) mutant strains on polysterene and silicone surfaces. Metabolic activity of the cells was measured by XTT assay on polysterene plates (A), and plates containing silicone disk (B). Microscopic analysis of biofilm structures of the WT (C) and KO (D) strains formed after 48 hours on polysterene plates. XTT assay was measured at 492 nm. Experiments were performed twice with triplicates that reproduced similar results. Error bars indicate standard deviation. * P

Techniques Used: Mutagenesis, Activity Assay, XTT Assay, Standard Deviation

26) Product Images from "The Role of msa in Staphylococcus aureus Biofilm Formation"

Article Title: The Role of msa in Staphylococcus aureus Biofilm Formation

Journal: BMC Microbiology

doi: 10.1186/1471-2180-8-221

Biofilm formation in the msa mutant in flow cells . The wild type strain COL, the msa mutant and the complemented msa mutant were used to inoculate flow cells. TSB supplemented with NaCl and glucose was provided at a flow rate of 0.5 ml/minute. Biofilm formation was monitored for 36 hours. Arrow indicates the direction of flow of medium.
Figure Legend Snippet: Biofilm formation in the msa mutant in flow cells . The wild type strain COL, the msa mutant and the complemented msa mutant were used to inoculate flow cells. TSB supplemented with NaCl and glucose was provided at a flow rate of 0.5 ml/minute. Biofilm formation was monitored for 36 hours. Arrow indicates the direction of flow of medium.

Techniques Used: Mutagenesis, Flow Cytometry

Biofilm formation in the msa mutant in microtiter plates . The wild type strain COL, the msa mutant and the complemented msa mutant were grown in TSB supplemented with NaCl and glucose. Cultures were incubated for 6, 12 and 24 hours in the wells of microtiter plates with pre-coating with plasma proteins. Biofilm was quantitated by staining with crystal violet and elution with ethanol as described in text. All values have been normalized to wild type levels which were arbitrarily set as 100%.
Figure Legend Snippet: Biofilm formation in the msa mutant in microtiter plates . The wild type strain COL, the msa mutant and the complemented msa mutant were grown in TSB supplemented with NaCl and glucose. Cultures were incubated for 6, 12 and 24 hours in the wells of microtiter plates with pre-coating with plasma proteins. Biofilm was quantitated by staining with crystal violet and elution with ethanol as described in text. All values have been normalized to wild type levels which were arbitrarily set as 100%.

Techniques Used: Mutagenesis, Incubation, Staining

Confocal microscopy images of biofilm . The msa mutant and the wild type strain COL were imaged 6 hours post inoculation of flow cells. The panels on the left are an overlay of multiple slices, and the side frames of the panels on the right show the z-stack showing the thickness and the architecture of the biofilm. The line in the z-stack indicates the level at which the photograph of the x-y plane was taken. Photographs were taken at a magnification of ×600.
Figure Legend Snippet: Confocal microscopy images of biofilm . The msa mutant and the wild type strain COL were imaged 6 hours post inoculation of flow cells. The panels on the left are an overlay of multiple slices, and the side frames of the panels on the right show the z-stack showing the thickness and the architecture of the biofilm. The line in the z-stack indicates the level at which the photograph of the x-y plane was taken. Photographs were taken at a magnification of ×600.

Techniques Used: Confocal Microscopy, Mutagenesis, Flow Cytometry

27) Product Images from "Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †"

Article Title: Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.00466-08

Analysis of the cAMP-CRP contribution to VpsR regulation of rbmC and bap1 expression. (A to C) β-Galactosidase assays of wild-type, Δ crp , Δ vpsT , Δ crp Δ vpsT , Δ vpsR , and Δ crp Δ vpsR strains harboring (A) vpsL-lacZ , (B) rbmC-lacZ , and (C) bap1-lacZ fusion constructs. The data are representative of at least two independent experiments. The error bars indicate standard deviations. (D) Quantitative comparison of biofilm formation by wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. The data are representative of two independent experiments. The error bars indicate standard deviations. (E) Biofilms formed after 8 h of incubation at 30°C in a non-flow-cell system by the wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. Biofilms were stained with SYTO9, and images were acquired by CLSM. The large images are images of the upper surfaces of biofilms, and the images below and to right of the large images are orthogonal views. Bars = 40 μm.
Figure Legend Snippet: Analysis of the cAMP-CRP contribution to VpsR regulation of rbmC and bap1 expression. (A to C) β-Galactosidase assays of wild-type, Δ crp , Δ vpsT , Δ crp Δ vpsT , Δ vpsR , and Δ crp Δ vpsR strains harboring (A) vpsL-lacZ , (B) rbmC-lacZ , and (C) bap1-lacZ fusion constructs. The data are representative of at least two independent experiments. The error bars indicate standard deviations. (D) Quantitative comparison of biofilm formation by wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. The data are representative of two independent experiments. The error bars indicate standard deviations. (E) Biofilms formed after 8 h of incubation at 30°C in a non-flow-cell system by the wild-type, Δ crp , Δ vps -I, Δ crp Δ vps -I, Δ vps -I Δ vps -II, Δ crp Δ vps -I Δ vps -II, Δ rbmA , Δ crp Δ rbmA , Δ bap1 , and Δ crp Δ bap1 strains. Biofilms were stained with SYTO9, and images were acquired by CLSM. The large images are images of the upper surfaces of biofilms, and the images below and to right of the large images are orthogonal views. Bars = 40 μm.

Techniques Used: Expressing, Construct, Incubation, Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

Phenotypic characterization of GGDEF deletion mutants and GGDEF crp double-deletion mutants. (A) Pellicle formation, (B) quantitative comparison of biofilm formation, and (C) motility assays for the wild type, for Δ crp and GGDEF single-deletion mutants, and for mutants with GGDEF deletions generated in the Δ crp genetic background. The data are representative of two independent experiments. The error bars indicate standard deviations.
Figure Legend Snippet: Phenotypic characterization of GGDEF deletion mutants and GGDEF crp double-deletion mutants. (A) Pellicle formation, (B) quantitative comparison of biofilm formation, and (C) motility assays for the wild type, for Δ crp and GGDEF single-deletion mutants, and for mutants with GGDEF deletions generated in the Δ crp genetic background. The data are representative of two independent experiments. The error bars indicate standard deviations.

Techniques Used: Generated

Model of cAMP-CRP regulation of biofilm formation in V. cholerae . cAMP-CRP regulates biofilm formation at multiple levels. Expression of vps and rbm genes is negatively regulated by cAMP-CRP through positive regulation of hapR expression and through negative regulation of vpsR and vpsT expression. In turn, VpsR and VpsT positively regulate vps and rbm gene expression, while HapR negatively regulates vps and rbm gene expression. VpsR, VpsT, and HapR regulation of vps and rbm gene expression also involves c-di-GMP signaling, where cdgA expression is negatively regulated by HapR and positively regulated by VpsR and VpsT. An increase in cdgA transcription leads to an increase in the c-di-GMP level, which in turn could interact with an effector protein(s) to positively regulate biofilm formation. We have a very limited understanding of the link between c-di-GMP pools and the signaling that leads to biofilm formation in V. cholerae . In addition, cAMP-CRP may also directly regulate vpsR , cdgA , and rbmC expression and indirectly regulate vpsT expression.
Figure Legend Snippet: Model of cAMP-CRP regulation of biofilm formation in V. cholerae . cAMP-CRP regulates biofilm formation at multiple levels. Expression of vps and rbm genes is negatively regulated by cAMP-CRP through positive regulation of hapR expression and through negative regulation of vpsR and vpsT expression. In turn, VpsR and VpsT positively regulate vps and rbm gene expression, while HapR negatively regulates vps and rbm gene expression. VpsR, VpsT, and HapR regulation of vps and rbm gene expression also involves c-di-GMP signaling, where cdgA expression is negatively regulated by HapR and positively regulated by VpsR and VpsT. An increase in cdgA transcription leads to an increase in the c-di-GMP level, which in turn could interact with an effector protein(s) to positively regulate biofilm formation. We have a very limited understanding of the link between c-di-GMP pools and the signaling that leads to biofilm formation in V. cholerae . In addition, cAMP-CRP may also directly regulate vpsR , cdgA , and rbmC expression and indirectly regulate vpsT expression.

Techniques Used: Expressing

28) Product Images from "OmpR and RcsB abolish temporal and spatial changes in expression of flhD in Escherichia coli Biofilm"

Article Title: OmpR and RcsB abolish temporal and spatial changes in expression of flhD in Escherichia coli Biofilm

Journal: BMC Microbiology

doi: 10.1186/1471-2180-13-182

Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD :: gfp ), BP1432 (AJW678 ompR :: gfp ), and BP1462 (AJW678 rcsB :: gfp ), respectively. The red line is the temporal expression profile of BP1531 ( flhD :: gfp ompR :: Tn 10), the orange line that of BP1532 ( flhD :: gfp rcsB :: Tn5 ). The purple line is our housekeeping strain BP1437 which contains the aceK :: gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating the highest and the lowest level of the total fluorescence intensity. The color code is identical to A.
Figure Legend Snippet: Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD :: gfp ), BP1432 (AJW678 ompR :: gfp ), and BP1462 (AJW678 rcsB :: gfp ), respectively. The red line is the temporal expression profile of BP1531 ( flhD :: gfp ompR :: Tn 10), the orange line that of BP1532 ( flhD :: gfp rcsB :: Tn5 ). The purple line is our housekeeping strain BP1437 which contains the aceK :: gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating the highest and the lowest level of the total fluorescence intensity. The color code is identical to A.

Techniques Used: Expressing, Mutagenesis, Fluorescence, Plasmid Preparation

CV assay to quantify the biofilm amounts of the ompR and rcsB mutants in comparison to the parent strain. The biofilm biomass was determined for BP1470 (AJW678 pPS71), BP1531 ( ompR :: Tn 10 pPS71) and BP1532 ( rcsB :: Tn 5 pKK12). This was done at four different time points, which are indicated on the x-axis. The yellow bars are the biofilm biomass of the parent strain, the red bars are for the ompR mutant, and the orange bars are for the rcsB mutant. Averages and standard deviations were calculated across three replicate experiments.
Figure Legend Snippet: CV assay to quantify the biofilm amounts of the ompR and rcsB mutants in comparison to the parent strain. The biofilm biomass was determined for BP1470 (AJW678 pPS71), BP1531 ( ompR :: Tn 10 pPS71) and BP1532 ( rcsB :: Tn 5 pKK12). This was done at four different time points, which are indicated on the x-axis. The yellow bars are the biofilm biomass of the parent strain, the red bars are for the ompR mutant, and the orange bars are for the rcsB mutant. Averages and standard deviations were calculated across three replicate experiments.

Techniques Used: Mutagenesis

Fluorescence images of flhD::gfp, ompR::gfp, rcsB::gfp in AJW678 and flhD in the ompR and rcsB mutant strains. Biofilms of BP1470, BP1432, BP1462, BP1531, and BP1532 were grown in flow cells and subjected to fluorescence microscopy. Four time points were selected for each strain; these are printed on top of the respective images. At the very top of each column, promoter names are printed. Images were taken at 1,000 fold magnification.
Figure Legend Snippet: Fluorescence images of flhD::gfp, ompR::gfp, rcsB::gfp in AJW678 and flhD in the ompR and rcsB mutant strains. Biofilms of BP1470, BP1432, BP1462, BP1531, and BP1532 were grown in flow cells and subjected to fluorescence microscopy. Four time points were selected for each strain; these are printed on top of the respective images. At the very top of each column, promoter names are printed. Images were taken at 1,000 fold magnification.

Techniques Used: Fluorescence, Mutagenesis, Flow Cytometry, Microscopy

Spatial gene expression of flhD in the parent strain. (A) and (B) are the 3D images constructed from the z- stacked images (bright field and fluorescence) at 12 hours (A) and 51 hours (B) , using BP1470 (AJW678 pPS71). (C) is the quantitative representation of the spatial gene expression of flhD at 12 hours (dashed yellow line) and 51 hours (solid yellow line) of biofilm formation. The purple line is the spatial expression profile from the aceK :: gfp fusion at 34 h.
Figure Legend Snippet: Spatial gene expression of flhD in the parent strain. (A) and (B) are the 3D images constructed from the z- stacked images (bright field and fluorescence) at 12 hours (A) and 51 hours (B) , using BP1470 (AJW678 pPS71). (C) is the quantitative representation of the spatial gene expression of flhD at 12 hours (dashed yellow line) and 51 hours (solid yellow line) of biofilm formation. The purple line is the spatial expression profile from the aceK :: gfp fusion at 34 h.

Techniques Used: Expressing, Construct, Fluorescence

Spatial gene expression of flhD in the ompR and rcsB mutant strains. (A) is the 3D image of the 33 h biofilm from BP1531 ( ompR :: Tn 10 pPS71), (B) is the respective image from the 51 h biofilm from BP1532 ( rcsB :: Tn 5 pKK12). (C) is the quantitative representation of the spatial gene expression of flhD in the ompR mutant (red line) and the rcsB mutant (orange line) at the times points represented in A and B .
Figure Legend Snippet: Spatial gene expression of flhD in the ompR and rcsB mutant strains. (A) is the 3D image of the 33 h biofilm from BP1531 ( ompR :: Tn 10 pPS71), (B) is the respective image from the 51 h biofilm from BP1532 ( rcsB :: Tn 5 pKK12). (C) is the quantitative representation of the spatial gene expression of flhD in the ompR mutant (red line) and the rcsB mutant (orange line) at the times points represented in A and B .

Techniques Used: Expressing, Mutagenesis

29) Product Images from "Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces"

Article Title: Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-11-125

Root attachment assays used to study the ability of rhizobia to form biofilms on Trifolium repens . ( A-C , E-G ) Confocal laser scanning microscopy of propidium iodide-stained roots inoculated with gfp-tagged ANU843 and its derivatives showing biofilm formation along the root surface at different magnifications. ( D ) Number of colony-forming units (cfu) per gram of root tissue after washing and sonicating the roots. Each datum point is the average of at least 9 determinations. Error bars indicate the standard error from the mean. Root biofilms with either wild-type or celC mutant bacteria were harvested 72 h post-inoculation. Fluorescence ( H-J ) and phase-contrast ( K ) microcopy show root hair colonization in detail. The wild-type strain ( A , E , H ) forms three-dimensional biofilms that cover both root surface and root hairs forming distinct “caps” on the tip (H). In contrast, ANU843ΔC2 ( B , F , I ) establishes aggregates that cover the root irregularly and forms a thicker cap on the root hairs ( I ) whereas ANU843C2 + ( C , G , J ) appears to coat the root surface without cap formation ( J ). Nevertheless, sufficient adhesion of individual bacteria occurs on the tip to produce the hot (hole on the tip) phenotype ( K ).
Figure Legend Snippet: Root attachment assays used to study the ability of rhizobia to form biofilms on Trifolium repens . ( A-C , E-G ) Confocal laser scanning microscopy of propidium iodide-stained roots inoculated with gfp-tagged ANU843 and its derivatives showing biofilm formation along the root surface at different magnifications. ( D ) Number of colony-forming units (cfu) per gram of root tissue after washing and sonicating the roots. Each datum point is the average of at least 9 determinations. Error bars indicate the standard error from the mean. Root biofilms with either wild-type or celC mutant bacteria were harvested 72 h post-inoculation. Fluorescence ( H-J ) and phase-contrast ( K ) microcopy show root hair colonization in detail. The wild-type strain ( A , E , H ) forms three-dimensional biofilms that cover both root surface and root hairs forming distinct “caps” on the tip (H). In contrast, ANU843ΔC2 ( B , F , I ) establishes aggregates that cover the root irregularly and forms a thicker cap on the root hairs ( I ) whereas ANU843C2 + ( C , G , J ) appears to coat the root surface without cap formation ( J ). Nevertheless, sufficient adhesion of individual bacteria occurs on the tip to produce the hot (hole on the tip) phenotype ( K ).

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Mutagenesis, Fluorescence

Attachment on glass (A) and polystyrene plates (B-C). ( A ) Ring formation at the glass-air-liquid interface after bacterial static growth . From left to right ANU843 (wild-type strain) and its derivatives ANU843ΔC2 (ΔC2), ANU843C2 + (C2+), ANU843ΔC2 complemented (ΔC2comp), and ANU843emptyvector (EV). Note the formation of a thick visible ring in ΔC2. ( B - C ) The data show absorbance values of CV-stained biofilms following growth in minimal media and TY at different times after inoculation. The culture media of the static culture was removed and differences in biofilm matrix development were measured by the intensity of CV staining. Before CV staining, the OD 600 of the broth cultures were measured in a Microtiter Plate reader to verify that no differences in growth rate among the wells had occurred. Each datum point is the average of at least 20 wells. Error bars indicate the standard deviation. Each experiment was repeated at least three times. Values followed by the same letter do not differ significantly according to Fisher protected LSD test at P = 0.01. The degree of biofilm formation was significantly different among the strains tested, although these differences were less pronounced in the complex medium.
Figure Legend Snippet: Attachment on glass (A) and polystyrene plates (B-C). ( A ) Ring formation at the glass-air-liquid interface after bacterial static growth . From left to right ANU843 (wild-type strain) and its derivatives ANU843ΔC2 (ΔC2), ANU843C2 + (C2+), ANU843ΔC2 complemented (ΔC2comp), and ANU843emptyvector (EV). Note the formation of a thick visible ring in ΔC2. ( B - C ) The data show absorbance values of CV-stained biofilms following growth in minimal media and TY at different times after inoculation. The culture media of the static culture was removed and differences in biofilm matrix development were measured by the intensity of CV staining. Before CV staining, the OD 600 of the broth cultures were measured in a Microtiter Plate reader to verify that no differences in growth rate among the wells had occurred. Each datum point is the average of at least 20 wells. Error bars indicate the standard deviation. Each experiment was repeated at least three times. Values followed by the same letter do not differ significantly according to Fisher protected LSD test at P = 0.01. The degree of biofilm formation was significantly different among the strains tested, although these differences were less pronounced in the complex medium.

Techniques Used: Staining, Standard Deviation

Test for adhesion to sand (A-C) and biofilm formation on PVC (D-F) tabs of the studied strains marked with GFP. The wild-type strain formed three-dimensional structures ( A , D ) whereas ANU843ΔC2 established microcolonies forming a layer that coated the surface ( B , E ), and ANU843C2 + barely adhered ( C , F ). Bar ( A - F ) 500 μm. ( G ) We also evaluated attachment quantitatively, by counting cfus of bacteria attached to sand grains. Error bars indicate the standard deviation. Each experiment was repeated three times. Values followed by the same letter do not differ significantly according to the Fisher protected LSD test at P = 0.01.
Figure Legend Snippet: Test for adhesion to sand (A-C) and biofilm formation on PVC (D-F) tabs of the studied strains marked with GFP. The wild-type strain formed three-dimensional structures ( A , D ) whereas ANU843ΔC2 established microcolonies forming a layer that coated the surface ( B , E ), and ANU843C2 + barely adhered ( C , F ). Bar ( A - F ) 500 μm. ( G ) We also evaluated attachment quantitatively, by counting cfus of bacteria attached to sand grains. Error bars indicate the standard deviation. Each experiment was repeated three times. Values followed by the same letter do not differ significantly according to the Fisher protected LSD test at P = 0.01.

Techniques Used: Standard Deviation

30) Product Images from "Oral Mycobiome Analysis of HIV-Infected Patients: Identification of Pichia as an Antagonist of Opportunistic Fungi"

Article Title: Oral Mycobiome Analysis of HIV-Infected Patients: Identification of Pichia as an Antagonist of Opportunistic Fungi

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003996

Biochemical characterization of Pichia spent medium. (A) Metabolic activity of Candida biofilms exposed to metabolites extracted from spent media of Pichia, Penicillium, or media control. (B) Effect of PSM exposed to proteinase, heat, or alkali on Candida biofilms. Confocal images show architecture of Candida biofilms exposed to (C) no PSM (control), (D) untreated PSM, (E) proteinase-K treated PSM, (F) heat, or (G) alkali.
Figure Legend Snippet: Biochemical characterization of Pichia spent medium. (A) Metabolic activity of Candida biofilms exposed to metabolites extracted from spent media of Pichia, Penicillium, or media control. (B) Effect of PSM exposed to proteinase, heat, or alkali on Candida biofilms. Confocal images show architecture of Candida biofilms exposed to (C) no PSM (control), (D) untreated PSM, (E) proteinase-K treated PSM, (F) heat, or (G) alkali.

Techniques Used: Activity Assay

Activity of Pichia spent medium (PSM) against fungal biofilms. (A) Effect of Pichia cells on the ability of Candida to form biofilms. Candida and Pichia were co-incubated [ Candida ∶ Pichia (C∶P) = 3∶1, 1∶1, or 1∶3] and biofilm formation was monitored (* P ≤.002, compared to Candida or Pichia controls). (B) Effect of media supernatant obtained from Pichia, Penicillium, or Cladosporium on Candida biofilms. Mean ± SD of ≥3 separate experiments. (C–E) Confocal microscopy images of Candida biofilms formed in presence of (C) no media supernatant, (D) Penicillium supernatant or (E) Pichia supernatant. (F) Thickness of biofilms formed in presence of media supernatant of Pichia or Penicillium .
Figure Legend Snippet: Activity of Pichia spent medium (PSM) against fungal biofilms. (A) Effect of Pichia cells on the ability of Candida to form biofilms. Candida and Pichia were co-incubated [ Candida ∶ Pichia (C∶P) = 3∶1, 1∶1, or 1∶3] and biofilm formation was monitored (* P ≤.002, compared to Candida or Pichia controls). (B) Effect of media supernatant obtained from Pichia, Penicillium, or Cladosporium on Candida biofilms. Mean ± SD of ≥3 separate experiments. (C–E) Confocal microscopy images of Candida biofilms formed in presence of (C) no media supernatant, (D) Penicillium supernatant or (E) Pichia supernatant. (F) Thickness of biofilms formed in presence of media supernatant of Pichia or Penicillium .

Techniques Used: Activity Assay, Incubation, Confocal Microscopy

Dose dependent activity of PSM, and its effect on Candida germination and adhesion. Effect of undiluted and diluted (50%) PSM on (A) metabolic activity and (B) thickness of Candida biofilms was assessed. (C) Germination in Candida grown in SDB, exposed to fetal bovine serum, (D) Stunted germ tubes formed by Candida exposed to Pichia supernatant (Magnification 20X), (E) Effect of PSM on the ability of Candida (grown in SDB) to adhere to solid substrates. (SDB - Sabouraud dextrose broth.)
Figure Legend Snippet: Dose dependent activity of PSM, and its effect on Candida germination and adhesion. Effect of undiluted and diluted (50%) PSM on (A) metabolic activity and (B) thickness of Candida biofilms was assessed. (C) Germination in Candida grown in SDB, exposed to fetal bovine serum, (D) Stunted germ tubes formed by Candida exposed to Pichia supernatant (Magnification 20X), (E) Effect of PSM on the ability of Candida (grown in SDB) to adhere to solid substrates. (SDB - Sabouraud dextrose broth.)

Techniques Used: Activity Assay

31) Product Images from "Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa"

Article Title: Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091813

Susceptibility of Δ katA mutant biofilms to A-NO 2 − . Biofilms of wild-type P. aeruginosa PAO1, its Δ katA mutant, and the complemented mutant Δ katA :: katA were grown anaerobically in LBN broth for 24 hrs. After gently washing off the planktonic cells, the biofilm cells were then cultured anaerobically in fresh LB, pH 6.5 with 15 mM KNO 3 , or 15 mM KNO 3 + 15 mM NaNO 2 , or 15 mM KNO 3 + 15 mM NaNO 2 + 10 mM c-PTIO for additional 48 hrs. The biofilms were then stained with a viability stain containing the DNA binding agent Syto 9 (live, green cells) and dead (propidium iodide, red) according to the Materials and Methods, and such bacteria were observed by confocal laser scanning microscopy. A. Biofilm formation of P. aeruginosa PAO1, Δ katA mutant and Δ katA::katA in the absence of NaNO 2 (top panel, top and sagittal views, serving as control biofilms) and in the presence of NaNO 2 (lower panel, top and sagittal views, treated biofilms). Biofilms of the Δ katA mutant treated with 10 mM c-PTIO under both conditions were shown in far right panels. B. The ratio of dead/live cells in treated biofilms in Fig. 6A was calculated and normalized using ImageJ. The experiment was performed three times independently. The average values were plotted with standard error.
Figure Legend Snippet: Susceptibility of Δ katA mutant biofilms to A-NO 2 − . Biofilms of wild-type P. aeruginosa PAO1, its Δ katA mutant, and the complemented mutant Δ katA :: katA were grown anaerobically in LBN broth for 24 hrs. After gently washing off the planktonic cells, the biofilm cells were then cultured anaerobically in fresh LB, pH 6.5 with 15 mM KNO 3 , or 15 mM KNO 3 + 15 mM NaNO 2 , or 15 mM KNO 3 + 15 mM NaNO 2 + 10 mM c-PTIO for additional 48 hrs. The biofilms were then stained with a viability stain containing the DNA binding agent Syto 9 (live, green cells) and dead (propidium iodide, red) according to the Materials and Methods, and such bacteria were observed by confocal laser scanning microscopy. A. Biofilm formation of P. aeruginosa PAO1, Δ katA mutant and Δ katA::katA in the absence of NaNO 2 (top panel, top and sagittal views, serving as control biofilms) and in the presence of NaNO 2 (lower panel, top and sagittal views, treated biofilms). Biofilms of the Δ katA mutant treated with 10 mM c-PTIO under both conditions were shown in far right panels. B. The ratio of dead/live cells in treated biofilms in Fig. 6A was calculated and normalized using ImageJ. The experiment was performed three times independently. The average values were plotted with standard error.

Techniques Used: Mutagenesis, Cell Culture, Staining, Binding Assay, Confocal Laser Scanning Microscopy

32) Product Images from "Streptococcus gordonii glucosyltransferase promotes biofilm interactions with Candida albicans"

Article Title: Streptococcus gordonii glucosyltransferase promotes biofilm interactions with Candida albicans

Journal: Journal of Oral Microbiology

doi: 10.3402/jom.v6.23419

A gtfG deletion mutant has an attenuated C. albicans biofilm binding phenotype. S. gordonii CH1 wild type, mutant and complemented gtfG strains were tested in their ability to adhere to a preformed 4 h C. albicans biofilm in saliva-supplemented media. Panel (a) depicts S. gordonii wild type (CH1), mutant (ΔGtf) and complemented (Gtf+) (red) strains binding to C. albicans (green), after immuno-FISH staining. Bar=50 µm. Panel (b) depicts the mean ratio of red/green fluorescence signal in eight microscopic fields per condition, set up in duplicate, after image J quantification. *p=0.0003 and **p=0.04, for a comparison with wild type and complemented strains, respectively.
Figure Legend Snippet: A gtfG deletion mutant has an attenuated C. albicans biofilm binding phenotype. S. gordonii CH1 wild type, mutant and complemented gtfG strains were tested in their ability to adhere to a preformed 4 h C. albicans biofilm in saliva-supplemented media. Panel (a) depicts S. gordonii wild type (CH1), mutant (ΔGtf) and complemented (Gtf+) (red) strains binding to C. albicans (green), after immuno-FISH staining. Bar=50 µm. Panel (b) depicts the mean ratio of red/green fluorescence signal in eight microscopic fields per condition, set up in duplicate, after image J quantification. *p=0.0003 and **p=0.04, for a comparison with wild type and complemented strains, respectively.

Techniques Used: Mutagenesis, Binding Assay, Fluorescence In Situ Hybridization, Staining, Fluorescence

Sucrose promotes dual biofilm development under static conditions. C. albicans SC5314 (Ca) and S. gordonii CH1 (Sg CH1) were allowed to form single and dual static biofilms for 24 h, in 96 well plates, in salivary flow media supplemented with 1% sucrose (wt/vol), 1% glucose (wt/vol) or 1% PBS control (vol/vol). Biofilm biomass was measured using the crystal violet assay. *p=0.0002 and **p=0.005 for a comparison between sucrose and glucose.
Figure Legend Snippet: Sucrose promotes dual biofilm development under static conditions. C. albicans SC5314 (Ca) and S. gordonii CH1 (Sg CH1) were allowed to form single and dual static biofilms for 24 h, in 96 well plates, in salivary flow media supplemented with 1% sucrose (wt/vol), 1% glucose (wt/vol) or 1% PBS control (vol/vol). Biofilm biomass was measured using the crystal violet assay. *p=0.0002 and **p=0.005 for a comparison between sucrose and glucose.

Techniques Used: Flow Cytometry, Crystal Violet Assay

S. gordonii CH1 forms robust dual species biofilms with C. albicans when inoculated simultaneously under conditions of salivary flow. Biofilms were allowed to develop in flow cells for 12–14 h in saliva-supplemented medium. Panel (a) depicts 3-D reconstructions of representative confocal images of biofilms. C. albicans SC5314 (green) was visualized after staining with an FITC-conjugated anti- Candida antibody. S. gordonii CH1 was visualized after fluorescence in situ hybridization (FISH) with a Streptococcus sp.-specific probe conjugated to Alexa 546. Panel (b) depicts the average biovolumes (in µm 3 ) for each species as measured in eight different CLSM image stacks from two independent experiments. Bottom panel: S. gordonii= CH1, C. albicans= Ca. * indicates a p-value of less than 0.05 when S. gordonii mono-species biovolumes were compared to mixed-species biovolumes by t -test.
Figure Legend Snippet: S. gordonii CH1 forms robust dual species biofilms with C. albicans when inoculated simultaneously under conditions of salivary flow. Biofilms were allowed to develop in flow cells for 12–14 h in saliva-supplemented medium. Panel (a) depicts 3-D reconstructions of representative confocal images of biofilms. C. albicans SC5314 (green) was visualized after staining with an FITC-conjugated anti- Candida antibody. S. gordonii CH1 was visualized after fluorescence in situ hybridization (FISH) with a Streptococcus sp.-specific probe conjugated to Alexa 546. Panel (b) depicts the average biovolumes (in µm 3 ) for each species as measured in eight different CLSM image stacks from two independent experiments. Bottom panel: S. gordonii= CH1, C. albicans= Ca. * indicates a p-value of less than 0.05 when S. gordonii mono-species biovolumes were compared to mixed-species biovolumes by t -test.

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

Dual S. gordonii – C. albicans biofilms are disrupted by dextranase digestion. Organisms were allowed to form single and dual species biofilms in 96 well plates for 24 h, in 1% sucrose-supplemented salivary flow media, and were subsequently digested by dextranase for 1 h or 4 h. The remaining biofilm mass was assessed by the crystal violet staining assay. Results represent mean percent reduction of biofilm signal, compared to buffer control. Means±SD of two independent experiments are shown, with each condition set up in triplicate. *p=0.04 and **p=0.02, for a comparison between the buffer and dextranase signals in dual S. gordonii–C. albicans (CaSg) and single S. gordonii (Sg) biofilms, respectively.
Figure Legend Snippet: Dual S. gordonii – C. albicans biofilms are disrupted by dextranase digestion. Organisms were allowed to form single and dual species biofilms in 96 well plates for 24 h, in 1% sucrose-supplemented salivary flow media, and were subsequently digested by dextranase for 1 h or 4 h. The remaining biofilm mass was assessed by the crystal violet staining assay. Results represent mean percent reduction of biofilm signal, compared to buffer control. Means±SD of two independent experiments are shown, with each condition set up in triplicate. *p=0.04 and **p=0.02, for a comparison between the buffer and dextranase signals in dual S. gordonii–C. albicans (CaSg) and single S. gordonii (Sg) biofilms, respectively.

Techniques Used: Flow Cytometry, Staining

A gtfG deletion mutant has an attenuated dual biofilm phenotype. Dual biofilms of C. albicans SC5314 with an S. gordonii gtfG deletion mutant (strain AMS12, ΔGtf) or complemented (AMS12/pAMS40, Gtf + ) strains were allowed to develop in flow cells for 12–14 h in saliva-supplemented medium. Panel (a) depicts 3-D reconstructions of representative confocal images of single and dual biofilms. C. albicans SC5314 (green) and S. gordonii (red) strains were visualized by immuno-FISH staining, as above. Panels (b,c) depict the average biovolumes (in µm 3 ) for each strain separately in single and dual biofilms, as measured in eight different CLSM image stacks from two independent experiments. Bottom panel: C. albicans= Ca, mutant strain AMS12=ΔGtf, complemented strain AMS12/pAMS40=Gtf+. * indicates a p-value of less than 0.05 for a comparison between the mutant and complemented strains.
Figure Legend Snippet: A gtfG deletion mutant has an attenuated dual biofilm phenotype. Dual biofilms of C. albicans SC5314 with an S. gordonii gtfG deletion mutant (strain AMS12, ΔGtf) or complemented (AMS12/pAMS40, Gtf + ) strains were allowed to develop in flow cells for 12–14 h in saliva-supplemented medium. Panel (a) depicts 3-D reconstructions of representative confocal images of single and dual biofilms. C. albicans SC5314 (green) and S. gordonii (red) strains were visualized by immuno-FISH staining, as above. Panels (b,c) depict the average biovolumes (in µm 3 ) for each strain separately in single and dual biofilms, as measured in eight different CLSM image stacks from two independent experiments. Bottom panel: C. albicans= Ca, mutant strain AMS12=ΔGtf, complemented strain AMS12/pAMS40=Gtf+. * indicates a p-value of less than 0.05 for a comparison between the mutant and complemented strains.

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

Sucrose promotes dual biofilm development under flow conditions. C. albicans SC5314 (Ca, green) and S. gordonii CH1 (Sg CH1, red) were allowed to form single and dual biofilms for 12–14 h in flow cells. Salivary flow media were supplemented with 1% sucrose (wt/vol), 1% glucose (wt/vol) or 1% PBS control (vol/vol). Biofilm biomass was measured using confocal imaging after immuno-FISH staining of C. albicans SC5314 and S. gordonii CH1, as described above. Panels (a,c) depict 3-D reconstructions of representative confocal images of single (a) and dual (c) biofilms. Panels (b,d) depict the average biovolumes (in µm 3 ) of each microorganism in single (b) and dual biofilms (d). In panel b, C. albicans =Ca, S. gordonii CH1= Sg Ch1. In panel d, C. albicans + S. gordonii CH1=CaSgCH1. Panel (b): *p=0.002 and **p=0.003, for a comparison to glucose and PBS, respectively. Panel (d): *p=0.004 and **p=0.002, for a comparison to glucose and PBS, respectively.
Figure Legend Snippet: Sucrose promotes dual biofilm development under flow conditions. C. albicans SC5314 (Ca, green) and S. gordonii CH1 (Sg CH1, red) were allowed to form single and dual biofilms for 12–14 h in flow cells. Salivary flow media were supplemented with 1% sucrose (wt/vol), 1% glucose (wt/vol) or 1% PBS control (vol/vol). Biofilm biomass was measured using confocal imaging after immuno-FISH staining of C. albicans SC5314 and S. gordonii CH1, as described above. Panels (a,c) depict 3-D reconstructions of representative confocal images of single (a) and dual (c) biofilms. Panels (b,d) depict the average biovolumes (in µm 3 ) of each microorganism in single (b) and dual biofilms (d). In panel b, C. albicans =Ca, S. gordonii CH1= Sg Ch1. In panel d, C. albicans + S. gordonii CH1=CaSgCH1. Panel (b): *p=0.002 and **p=0.003, for a comparison to glucose and PBS, respectively. Panel (d): *p=0.004 and **p=0.002, for a comparison to glucose and PBS, respectively.

Techniques Used: Flow Cytometry, Imaging, Fluorescence In Situ Hybridization, Staining

C. albicans enhances the ability of S. gordonii strain CH107 to form biofilms. Biofilms were allowed to develop in flow cells for 12–14 h in saliva-supplemented medium. Panel (a) depicts 3-D reconstructions of representative confocal images of biofilms with C. albicans SC5314 (green) and S. gordonii CH107 (red), stained with immuno-FISH as described above. Panel (b) depicts the average biovolumes (in µm 3 ) for strain CH107 in single and double biofilms as measured in eight different CLSM image stacks from two independent experiments. Bottom panel: S. gordonii CH107= CH107, C. albicans= Ca. *p=0.009 compared to single-species biovolumes by t -test.
Figure Legend Snippet: C. albicans enhances the ability of S. gordonii strain CH107 to form biofilms. Biofilms were allowed to develop in flow cells for 12–14 h in saliva-supplemented medium. Panel (a) depicts 3-D reconstructions of representative confocal images of biofilms with C. albicans SC5314 (green) and S. gordonii CH107 (red), stained with immuno-FISH as described above. Panel (b) depicts the average biovolumes (in µm 3 ) for strain CH107 in single and double biofilms as measured in eight different CLSM image stacks from two independent experiments. Bottom panel: S. gordonii CH107= CH107, C. albicans= Ca. *p=0.009 compared to single-species biovolumes by t -test.

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

33) Product Images from "The Staphylococcus aureus SrrAB Two-Component System Promotes Resistance to Nitrosative Stress and Hypoxia"

Article Title: The Staphylococcus aureus SrrAB Two-Component System Promotes Resistance to Nitrosative Stress and Hypoxia

Journal: mBio

doi: 10.1128/mBio.00696-13

Biofilm formation by wild-type and mutant S. aureus . Wild-type S. aureus UAMS-1 or isogenic srrAB , hmp , and sarA mutants were grown in TSBGN medium for 3 to 5 days in either 24-well or 6-well tissue culture plates with sterile coverslips pretreated with 20% human plasma. (A) Biofilms were quantified with crystal violet staining, and the absorbance at 595 nm was read for 3-fold dilutions of the crystal violet elution. Shown is the result from 3 independent experiments with at least 6 wells per strain; error bars = SD. (B) A minimum of 3 different coverslips were analyzed for each strain on each day. Biofilms were stained with SYTO-9 and propidium iodide and imaged with a Zeiss 510-Meta confocal microscope. The orthogonal images are representative of biofilms from days 2 and 4. (C) Split-color biofilm profile images were analyzed using ImageJ for integrated density to determine the relative percentage of live and dead cells in each image. At least 7 profile images were analyzed for each strain on each day; error bars = SD. Significance for panels A and C was calculated using Student’s t test comparing means to the WT for each day (*, P
Figure Legend Snippet: Biofilm formation by wild-type and mutant S. aureus . Wild-type S. aureus UAMS-1 or isogenic srrAB , hmp , and sarA mutants were grown in TSBGN medium for 3 to 5 days in either 24-well or 6-well tissue culture plates with sterile coverslips pretreated with 20% human plasma. (A) Biofilms were quantified with crystal violet staining, and the absorbance at 595 nm was read for 3-fold dilutions of the crystal violet elution. Shown is the result from 3 independent experiments with at least 6 wells per strain; error bars = SD. (B) A minimum of 3 different coverslips were analyzed for each strain on each day. Biofilms were stained with SYTO-9 and propidium iodide and imaged with a Zeiss 510-Meta confocal microscope. The orthogonal images are representative of biofilms from days 2 and 4. (C) Split-color biofilm profile images were analyzed using ImageJ for integrated density to determine the relative percentage of live and dead cells in each image. At least 7 profile images were analyzed for each strain on each day; error bars = SD. Significance for panels A and C was calculated using Student’s t test comparing means to the WT for each day (*, P

Techniques Used: Mutagenesis, Staining, Microscopy

34) Product Images from "Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm"

Article Title: Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm

Journal: Journal of Applied Oral Science

doi: 10.1590/1678-7757-2019-0516

Effect of different medicaments on E. faecalis biofilms. (a) Bacterial thickness, (b) EPS thickness, (c-f) representative CLSM images of EPS; (c) Control, (d) Calasept Plus™, (e) Calcipex II®, and (F) CleaniCal®. One-way ANOVA and Tukey test were performed. *Statistical significance was determined at p
Figure Legend Snippet: Effect of different medicaments on E. faecalis biofilms. (a) Bacterial thickness, (b) EPS thickness, (c-f) representative CLSM images of EPS; (c) Control, (d) Calasept Plus™, (e) Calcipex II®, and (F) CleaniCal®. One-way ANOVA and Tukey test were performed. *Statistical significance was determined at p

Techniques Used: Confocal Laser Scanning Microscopy

(a-d) Representative FE-SEM images after removal of pastes from E. faecalis biofilms formed on the specimens (5,000X). (a’-d’) Stereomicroscopic images of bovine tooth blocks showing remaining nail polish; Control (a and a’), Calasept Plus™ (b and b’), Calcipex II® (c and c’), and CleaniCal® (d and d’)
Figure Legend Snippet: (a-d) Representative FE-SEM images after removal of pastes from E. faecalis biofilms formed on the specimens (5,000X). (a’-d’) Stereomicroscopic images of bovine tooth blocks showing remaining nail polish; Control (a and a’), Calasept Plus™ (b and b’), Calcipex II® (c and c’), and CleaniCal® (d and d’)

Techniques Used:

35) Product Images from "Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture"

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

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02638-17

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).
Figure Legend 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).

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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 .
Figure Legend 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 .

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

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

36) Product Images from "Resistance to leukocytes ties benefits of quorum-sensing dysfunctionality to biofilm infection"

Article Title: Resistance to leukocytes ties benefits of quorum-sensing dysfunctionality to biofilm infection

Journal: Nature microbiology

doi: 10.1038/s41564-019-0413-x

QS-dysfunctional S. aureus forms compact biofilms that prevent neutrophil penetration. a, Confocal laser scanning microscopy (CLSM) images of biofilms [stained with PI (red), which stains extracellular DNA, and SYTO 9 (green) staining live cells] formed by the indicated S. aureus , with and without application of neutrophils (yellow). Biofilms were grown for 48 h and neutrophils were added at equal numbers. The top rows show biofilm structures without neutrophils, and the other three rows with neutrophils from two different angles (“diamond” view from above and side view), as well as with only SYTO 9 staining in side view. CLSM experiments were repeated at least once giving similar results. b , Neutrophil penetration into the biofilm, based on SYTO 9-only stain. The total number of neutrophils for every strain in the shown field of view was analyzed [n=24 (LAC), n=13 (LACΔ agr ), n=8 (LACΔ psm ), n=12 (252), n=8 (252Δ agr ), n=16 (N315)]. Statistical analysis is by 1-way ANOVA with Dunnett’s post-test versus WT (for LAC series), and by unpaired two-tailed t-tests for 252 versus 252Δ agr . *, P
Figure Legend Snippet: QS-dysfunctional S. aureus forms compact biofilms that prevent neutrophil penetration. a, Confocal laser scanning microscopy (CLSM) images of biofilms [stained with PI (red), which stains extracellular DNA, and SYTO 9 (green) staining live cells] formed by the indicated S. aureus , with and without application of neutrophils (yellow). Biofilms were grown for 48 h and neutrophils were added at equal numbers. The top rows show biofilm structures without neutrophils, and the other three rows with neutrophils from two different angles (“diamond” view from above and side view), as well as with only SYTO 9 staining in side view. CLSM experiments were repeated at least once giving similar results. b , Neutrophil penetration into the biofilm, based on SYTO 9-only stain. The total number of neutrophils for every strain in the shown field of view was analyzed [n=24 (LAC), n=13 (LACΔ agr ), n=8 (LACΔ psm ), n=12 (252), n=8 (252Δ agr ), n=16 (N315)]. Statistical analysis is by 1-way ANOVA with Dunnett’s post-test versus WT (for LAC series), and by unpaired two-tailed t-tests for 252 versus 252Δ agr . *, P

Techniques Used: Confocal Laser Scanning Microscopy, Staining, Two Tailed Test

QS in biofilm- and non-biofilm-associated infection. a , Non-biofilm (skin abscess) model. Mice were subcutaneously injected with 1 × 10 7 CFU and CFU in the abscesses were determined at day 6 post infection. WT, wild-type USA300 LAC (n=10); Δ agr , Δ psm , isogenic agr and psm deletion mutants (n=8 each). See Supplementary Fig. 2 for abscess and open lesion sizes over the course of the experiment. b , Biofilm (catheter) model. 1-cm catheter pieces coated with approximately equal numbers of bacteria (between 10 3 and 10 4 CFU per catheter piece) were inserted under the dorsum of mice (2 per mouse; n=10 mice per strain). CFU on the catheter and directly surrounding tissue were determined at day 6 post infection. Statistical analysis in the experiments shown in panels a and b is by 1-way ANOVA with Dunnett’s post-test versus data obtained with WT. c - e , The device-related biofilm and the non-biofilm skin abscess model were performed using ~ 1:1 mixed inocula of wild-type and Δ agr (QS-deficient) bacteria. In both models, CFU were determined in the abscesses or on the catheters, respectively, 6 days after injection. c , Skin infection model. The model was performed with n = 10 mice. The inoculum contained 5 × 10 6 CFU each of WT and Δ agr bacteria. d , Catheter infection model. The model was performed with n = 5 mice and 2 catheters implanted per mouse on the left and right dorsum. One catheter fell out during the model and was not counted (total n=9). Statistical analysis in the experiments shown in panels c and d is by two-tailed paired t-tests. e , Data achieved per abscess or catheter in panels c and d expressed as percentage of Δ agr among total bacteria. Bacteria attached in vitro to control catheters (“catheter inoculum”) were measured to estimate the inoculated bacteria (n=5). Statistical analysis is by two-tailed unpaired t-tests (skin infection versus catheter infection; catheter infection versus control catheters.) a - e , Error bars show the mean ± SD.
Figure Legend Snippet: QS in biofilm- and non-biofilm-associated infection. a , Non-biofilm (skin abscess) model. Mice were subcutaneously injected with 1 × 10 7 CFU and CFU in the abscesses were determined at day 6 post infection. WT, wild-type USA300 LAC (n=10); Δ agr , Δ psm , isogenic agr and psm deletion mutants (n=8 each). See Supplementary Fig. 2 for abscess and open lesion sizes over the course of the experiment. b , Biofilm (catheter) model. 1-cm catheter pieces coated with approximately equal numbers of bacteria (between 10 3 and 10 4 CFU per catheter piece) were inserted under the dorsum of mice (2 per mouse; n=10 mice per strain). CFU on the catheter and directly surrounding tissue were determined at day 6 post infection. Statistical analysis in the experiments shown in panels a and b is by 1-way ANOVA with Dunnett’s post-test versus data obtained with WT. c - e , The device-related biofilm and the non-biofilm skin abscess model were performed using ~ 1:1 mixed inocula of wild-type and Δ agr (QS-deficient) bacteria. In both models, CFU were determined in the abscesses or on the catheters, respectively, 6 days after injection. c , Skin infection model. The model was performed with n = 10 mice. The inoculum contained 5 × 10 6 CFU each of WT and Δ agr bacteria. d , Catheter infection model. The model was performed with n = 5 mice and 2 catheters implanted per mouse on the left and right dorsum. One catheter fell out during the model and was not counted (total n=9). Statistical analysis in the experiments shown in panels c and d is by two-tailed paired t-tests. e , Data achieved per abscess or catheter in panels c and d expressed as percentage of Δ agr among total bacteria. Bacteria attached in vitro to control catheters (“catheter inoculum”) were measured to estimate the inoculated bacteria (n=5). Statistical analysis is by two-tailed unpaired t-tests (skin infection versus catheter infection; catheter infection versus control catheters.) a - e , Error bars show the mean ± SD.

Techniques Used: Infection, Mouse Assay, Injection, Two Tailed Test, In Vitro

Benefits o f QS mutants in biofilm-associated infection are due to interaction with leukocytes. The biofilm (catheter) model was performed with cyclophosphamide (CY), which destroys leukocytes, to determine to which extent the achieved in-vivo results are due to biofilm-mediated resistance to leukocytes. Mice received an initial CY dose of 150 mg/kg body weight and three further doses of 100 mg/kg body weight in 48-h intervals before inoculation of the bacteria the day after the last dose. Results from Fig. 1 are included for direct comparison in panels a - c . All experimental details are the same as in Fig. 1 . a , Catheter model (separate infections). Statistical analysis is by 1-way ANOVA with Dunnett’s comparison post-test versus data obtained with WT. n=10 mice with 2 catheters each. b , Catheter model (1:1 WT/Δ agr infection; n=9). Statistical analysis is by two-tailed paired t-tests. c , Data achieved in the experiment shown in panel b (and the corresponding experiment without CY treatment from Fig. 1 ) expressed as percentage of Δ agr among total bacteria. Statistical analysis is by two-tailed unpaired t-test. a - c , Error bars show the mean ± SD.
Figure Legend Snippet: Benefits o f QS mutants in biofilm-associated infection are due to interaction with leukocytes. The biofilm (catheter) model was performed with cyclophosphamide (CY), which destroys leukocytes, to determine to which extent the achieved in-vivo results are due to biofilm-mediated resistance to leukocytes. Mice received an initial CY dose of 150 mg/kg body weight and three further doses of 100 mg/kg body weight in 48-h intervals before inoculation of the bacteria the day after the last dose. Results from Fig. 1 are included for direct comparison in panels a - c . All experimental details are the same as in Fig. 1 . a , Catheter model (separate infections). Statistical analysis is by 1-way ANOVA with Dunnett’s comparison post-test versus data obtained with WT. n=10 mice with 2 catheters each. b , Catheter model (1:1 WT/Δ agr infection; n=9). Statistical analysis is by two-tailed paired t-tests. c , Data achieved in the experiment shown in panel b (and the corresponding experiment without CY treatment from Fig. 1 ) expressed as percentage of Δ agr among total bacteria. Statistical analysis is by two-tailed unpaired t-test. a - c , Error bars show the mean ± SD.

Techniques Used: Infection, In Vivo, Mouse Assay, Two Tailed Test

QS-dysfunctional biofilms prevent killing by leukocytes. Quantitative analysis of survival of biofilms formed by the same strains as in Fig. 2 upon incubation with neutrophils, and biofilms formed by LAC wild-type and its isogenic Δ agr and Δ psm mutants upon incubation with M1 or M2 macrophages. Biofilms were grown in TSBg for 48 h and the leukocytes were added at equal numbers. Experiments were performed in triplicate (n=3). Statistical analysis in the graphs showing experiments with LAC is by 2-way ANOVA with Tukey’s multiple comparison post-test; shown comparisons are versus WT. Values obtained with 252 and 252 Δ agr are compared by two-tailed unpaired t-tests at every time point. Values are shown as percentages as compared to the value at t=0 min for every strain, which was set to 100%. Error bars show the mean ± SD.
Figure Legend Snippet: QS-dysfunctional biofilms prevent killing by leukocytes. Quantitative analysis of survival of biofilms formed by the same strains as in Fig. 2 upon incubation with neutrophils, and biofilms formed by LAC wild-type and its isogenic Δ agr and Δ psm mutants upon incubation with M1 or M2 macrophages. Biofilms were grown in TSBg for 48 h and the leukocytes were added at equal numbers. Experiments were performed in triplicate (n=3). Statistical analysis in the graphs showing experiments with LAC is by 2-way ANOVA with Tukey’s multiple comparison post-test; shown comparisons are versus WT. Values obtained with 252 and 252 Δ agr are compared by two-tailed unpaired t-tests at every time point. Values are shown as percentages as compared to the value at t=0 min for every strain, which was set to 100%. Error bars show the mean ± SD.

Techniques Used: Incubation, Two Tailed Test

37) Product Images from "The Porphyromonas gingivalis Ferric Uptake Regulator Orthologue Binds Hemin and Regulates Hemin-Responsive Biofilm Development"

Article Title: The Porphyromonas gingivalis Ferric Uptake Regulator Orthologue Binds Hemin and Regulates Hemin-Responsive Biofilm Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111168

P. gingivalis biofilm development. Orthogonal projections of CLSM images showing a representative region of the x-y plane over the depth of the biofilm in both xz and yz dimensions of the ATCC 33277 wild-type, har mutant ECR455 and har complement ECR 475 strains grown in excess hemin ( A ) or hemin-limitation ( B ). Comparison of the Biovolume ( C ), Average Thickness ( D ) and SA:Biovolume ( E ) calculated for each strain's biofilm growth in either excess hemin (dark bars) or limited hemin (light bars) over three independent experiments. All biometric parameters analysed for the biofilms formed by ATCC 33277 and ECR475 were significantly (p
Figure Legend Snippet: P. gingivalis biofilm development. Orthogonal projections of CLSM images showing a representative region of the x-y plane over the depth of the biofilm in both xz and yz dimensions of the ATCC 33277 wild-type, har mutant ECR455 and har complement ECR 475 strains grown in excess hemin ( A ) or hemin-limitation ( B ). Comparison of the Biovolume ( C ), Average Thickness ( D ) and SA:Biovolume ( E ) calculated for each strain's biofilm growth in either excess hemin (dark bars) or limited hemin (light bars) over three independent experiments. All biometric parameters analysed for the biofilms formed by ATCC 33277 and ECR475 were significantly (p

Techniques Used: Confocal Laser Scanning Microscopy, Mutagenesis

38) Product Images from "Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073"

Article Title: Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073

Journal: Infection and Immunity

doi: 10.1128/IAI.05322-11

Biofilm formation by E. coli OS56 cells harboring plasmids expressing UpaB and UpaC. The effect of AT expression on biofilm formation was assessed in E. coli OS56 (MG1655Δ flu , Gfp+) cells containing the following plasmids pBAD, pUpaB, or pUpaC.
Figure Legend Snippet: Biofilm formation by E. coli OS56 cells harboring plasmids expressing UpaB and UpaC. The effect of AT expression on biofilm formation was assessed in E. coli OS56 (MG1655Δ flu , Gfp+) cells containing the following plasmids pBAD, pUpaB, or pUpaC.

Techniques Used: Expressing

39) Product Images from "Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea"

Article Title: Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147114

Quantitative analysis of the biofilm structure of C . ochracea . The height (A) and % volume occupied by C . ochracea in total volume (B) are shown. The height was evaluated by using Zen 2009 software and the volume by use of IMARIS software. Data are presented as means ± SD of OD 405 . The valuation was performed at six points. *, P
Figure Legend Snippet: Quantitative analysis of the biofilm structure of C . ochracea . The height (A) and % volume occupied by C . ochracea in total volume (B) are shown. The height was evaluated by using Zen 2009 software and the volume by use of IMARIS software. Data are presented as means ± SD of OD 405 . The valuation was performed at six points. *, P

Techniques Used: Software

Effect of luxS complementation on biofilm formation by C . ochracea . C . ochracea ATCC27872, LKT7 ( luxS ∷ tetQ ), and luxS-C3 ( luxS complemented strain) were incubated in TS broth for 48 h under anaerobic conditions. Biofilm formation was then assayed by means of crystal violet staining. Data are presented as means ± SD (n = 6) *, P
Figure Legend Snippet: Effect of luxS complementation on biofilm formation by C . ochracea . C . ochracea ATCC27872, LKT7 ( luxS ∷ tetQ ), and luxS-C3 ( luxS complemented strain) were incubated in TS broth for 48 h under anaerobic conditions. Biofilm formation was then assayed by means of crystal violet staining. Data are presented as means ± SD (n = 6) *, P

Techniques Used: Incubation, Staining

Representative SEM images of biofilms of C . ochracea wild type and TmAI2. C . ochracea ATCC27872 (A) and TmAI2 (B) were incubated anaerobically on coverglasses in 12-well polystyrene plates for 48 h. Representative images of the biofilms are shown at the indicated magnifications. Arrowheads indicate partially vacant spaces.
Figure Legend Snippet: Representative SEM images of biofilms of C . ochracea wild type and TmAI2. C . ochracea ATCC27872 (A) and TmAI2 (B) were incubated anaerobically on coverglasses in 12-well polystyrene plates for 48 h. Representative images of the biofilms are shown at the indicated magnifications. Arrowheads indicate partially vacant spaces.

Techniques Used: Incubation

Biofilm formation by C . ochracea wild type and TmAI2. C . ochracea ATCC27872 and TmAI2 were incubated in (A) 1.0 x TS broth for 24 h, (B) 1.0 x TS broth for 48 h, (C) 0.5 × TS broth for 24 h, or (D) 0.5 × TS broth for 48 h, in a 12-well plate under anaerobic conditions. Biofilm formation was then assayed by crystal violet staining. Data are presented as means ± SD (n = 10). *, P
Figure Legend Snippet: Biofilm formation by C . ochracea wild type and TmAI2. C . ochracea ATCC27872 and TmAI2 were incubated in (A) 1.0 x TS broth for 24 h, (B) 1.0 x TS broth for 48 h, (C) 0.5 × TS broth for 24 h, or (D) 0.5 × TS broth for 48 h, in a 12-well plate under anaerobic conditions. Biofilm formation was then assayed by crystal violet staining. Data are presented as means ± SD (n = 10). *, P

Techniques Used: Incubation, Staining

Representative CSLM images of biofilms of C . ochracea wild type and TmAI2. C . ochracea ATCC27872 (A) and TmAI2 (B) were incubated in 12-well polystyrene plates containing coverglass plates for 48 h, and CSLM images of the cells that attached to the coverglass were obtained. The upper panels (i) show each x - y images, and the lower panels (ii) show the center of each x - z reconstructions. Scale bars, 50 μm.
Figure Legend Snippet: Representative CSLM images of biofilms of C . ochracea wild type and TmAI2. C . ochracea ATCC27872 (A) and TmAI2 (B) were incubated in 12-well polystyrene plates containing coverglass plates for 48 h, and CSLM images of the cells that attached to the coverglass were obtained. The upper panels (i) show each x - y images, and the lower panels (ii) show the center of each x - z reconstructions. Scale bars, 50 μm.

Techniques Used: Incubation

Effect of extrinsic AI-2 on biofilm formation by the luxS -deficient mutant and wild-type strain. (A) Assessment of biofilm formation by mutant and wild-type strains by using a two-compartment system. C . ochracea ATCC 27872 and TmAI2 were inoculated into TS broth in the indicated compartments (upper or lower well) in the wells of a 12-well polystyrene plate, and were then incubated for 48 h. Biofilm formation by the strain in the lower compartment of each well was stained with crystal violet staining. After rinsing and ethanol extraction, the mass of biofilm was quantified as OD 595 of extracted stain. Data are presented as means ± SD (n = 10). *, P
Figure Legend Snippet: Effect of extrinsic AI-2 on biofilm formation by the luxS -deficient mutant and wild-type strain. (A) Assessment of biofilm formation by mutant and wild-type strains by using a two-compartment system. C . ochracea ATCC 27872 and TmAI2 were inoculated into TS broth in the indicated compartments (upper or lower well) in the wells of a 12-well polystyrene plate, and were then incubated for 48 h. Biofilm formation by the strain in the lower compartment of each well was stained with crystal violet staining. After rinsing and ethanol extraction, the mass of biofilm was quantified as OD 595 of extracted stain. Data are presented as means ± SD (n = 10). *, P

Techniques Used: Mutagenesis, Incubation, Staining

40) Product Images from "Expression of UME6, a Key Regulator of Candida albicans Hyphal Development, Enhances Biofilm Formation via Hgc1- and Sun41-Dependent Mechanisms"

Article Title: Expression of UME6, a Key Regulator of Candida albicans Hyphal Development, Enhances Biofilm Formation via Hgc1- and Sun41-Dependent Mechanisms

Journal: Eukaryotic Cell

doi: 10.1128/EC.00163-12

UME6 expression promotes hyphal growth in C. albicans biofilms. CSLM was used to visualize cells in the bottommost layer of biofilms formed on 6-well polystyrene plates by the indicated strains in the presence or absence of 20 μg/ml Dox. C. albicans cells were stained with concanavalin A for 1 h in the dark at 37°C. Bar = 25 μm.
Figure Legend Snippet: UME6 expression promotes hyphal growth in C. albicans biofilms. CSLM was used to visualize cells in the bottommost layer of biofilms formed on 6-well polystyrene plates by the indicated strains in the presence or absence of 20 μg/ml Dox. C. albicans cells were stained with concanavalin A for 1 h in the dark at 37°C. Bar = 25 μm.

Techniques Used: Expressing, Staining

Hgc1 and Sun41 are important for UME6 -driven hyphal growth in C. albicans biofilms. CSLM was used to visualize cells in the top layer of biofilms formed on 6-well polystyrene plates by the indicated strains in the presence or absence of 20 μg/ml Dox. C. albicans cells were stained with concanavalin A for 1 h in the dark at 37°C. Bar = 25 μm.
Figure Legend Snippet: Hgc1 and Sun41 are important for UME6 -driven hyphal growth in C. albicans biofilms. CSLM was used to visualize cells in the top layer of biofilms formed on 6-well polystyrene plates by the indicated strains in the presence or absence of 20 μg/ml Dox. C. albicans cells were stained with concanavalin A for 1 h in the dark at 37°C. Bar = 25 μm.

Techniques Used: Staining

UME6 expression level is correlated with increased biofilm formation. Suspensions of 1 × 10 6 ). Error bars represent standard deviations ( n = 4).
Figure Legend Snippet: UME6 expression level is correlated with increased biofilm formation. Suspensions of 1 × 10 6 ). Error bars represent standard deviations ( n = 4).

Techniques Used: Expressing

Model for roles of Efg1, Hgc1, and Sun41 in UME6 -driven enhanced C. albicans biofilm formation. Ume6 functions downstream of Efg1 and upstream of both Hgc1 and Sun41 to promote biofilm development. UME6 ). UME6 expression also appears to cause a slight increase in SUN41 transcript levels. SUN41 , in turn, may function indirectly in a positive-feedback loop to increase UME6 ). Both the physical process of hyphal development and Sun41-mediated cell wall integrity therefore appear to play important roles in UME6 -driven enhanced biofilm formation. In addition, we cannot exclude the possibility that Sun41 at least partly contributes to UME6 -driven biofilm growth by playing a role in hyphal development (dashed line). Finally, it is important to note that an additional mechanism(s), which at this point has not yet been determined, may also contribute to UME6 -driven enhanced biofilm formation.
Figure Legend Snippet: Model for roles of Efg1, Hgc1, and Sun41 in UME6 -driven enhanced C. albicans biofilm formation. Ume6 functions downstream of Efg1 and upstream of both Hgc1 and Sun41 to promote biofilm development. UME6 ). UME6 expression also appears to cause a slight increase in SUN41 transcript levels. SUN41 , in turn, may function indirectly in a positive-feedback loop to increase UME6 ). Both the physical process of hyphal development and Sun41-mediated cell wall integrity therefore appear to play important roles in UME6 -driven enhanced biofilm formation. In addition, we cannot exclude the possibility that Sun41 at least partly contributes to UME6 -driven biofilm growth by playing a role in hyphal development (dashed line). Finally, it is important to note that an additional mechanism(s), which at this point has not yet been determined, may also contribute to UME6 -driven enhanced biofilm formation.

Techniques Used: Expressing

Hgc1 and Sun41 are important for the ability of UME6 expression to cause enhanced biofilm formation. Suspensions of 1 × 10 6 ). Error bars represent standard deviations ( n = 8).
Figure Legend Snippet: Hgc1 and Sun41 are important for the ability of UME6 expression to cause enhanced biofilm formation. Suspensions of 1 × 10 6 ). Error bars represent standard deviations ( n = 8).

Techniques Used: Expressing

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Article Snippet: .. Next, the internal structure of a ∼30-μm-thick, 48-h biofilm was visualized by staining cells with acridine orange and examining the biofilm with confocal laser scanning microscopy (CLSM) using a Zeiss LSM 410 microscope containing a ×40 1.2-numerical aperture C-Apochromat objective lens. ..

Confocal Laser Scanning Microscopy:

Article Title: Periodicity of Cell Attachment Patterns during Escherichia coli Biofilm Development
Article Snippet: .. Next, the internal structure of a ∼30-μm-thick, 48-h biofilm was visualized by staining cells with acridine orange and examining the biofilm with confocal laser scanning microscopy (CLSM) using a Zeiss LSM 410 microscope containing a ×40 1.2-numerical aperture C-Apochromat objective lens. ..

Laser-Scanning Microscopy:

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Microscopy:

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    Carl Zeiss m smegmatis biofilm
    CNPs treatments disintegrate the M. <t>smegmatis</t> cell wall. (A) Scanning electron microscopic images showing disruption in cell wall morphology of single M. smegmatis (control) and after exposure to as-synthesized CNP-L (100 μg/ml) for 36 h. (B) (i) Control M. smegmatis <t>biofilm</t> (untreated), (ii) CNP treatment inhibits the biofilm formation, and (iii) CNP-L treatment at the same dose showed more biofilm inhibition after 36 h. (C) Biofilms were grown and treated with CNP and CNP-L followed by incubation with XTT for 1 h, absorbance at 495 nm was measured for biofilm inhibition. Increased killing of M. smegmatis was observed at 200 μg/ml dose of CNP-L. Note : scanning electron microscopy analysis was performed on a SU1510 scanning electron microscope (Hitachi, Tokyo, Japan).
    M Smegmatis Biofilm, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FFT analysis of cell distribution at the glass surface. (a) A digitized micrograph (×32 objective) of a <t>biofilm</t> at 4 h of growth. (b) Two-dimensional FFT spectrum of the data from panel a. (c) One-dimensional FFT spectrum, obtained from an intensity profile measured by a horizontal line that bisects the image of panel b. (d) The cell density pattern obtained by low-pass and reverse-transform operations. Note four distinct regions of cell density, highlighted in color, and the corresponding regions that are indicated in the micrograph of panel a. (e) An example of a quasi-random cell distribution: micrograph of the biofilm (×40 objective) obtained after 24 h of growth with obscured multiple layers of cells. (f) Two-dimensional FFT spectrum of data from panel e. (g) One-dimensional FFT spectrum obtained from an intensity profile of image of panel f, generated as in panel c.
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    CNPs treatments disintegrate the M. smegmatis cell wall. (A) Scanning electron microscopic images showing disruption in cell wall morphology of single M. smegmatis (control) and after exposure to as-synthesized CNP-L (100 μg/ml) for 36 h. (B) (i) Control M. smegmatis biofilm (untreated), (ii) CNP treatment inhibits the biofilm formation, and (iii) CNP-L treatment at the same dose showed more biofilm inhibition after 36 h. (C) Biofilms were grown and treated with CNP and CNP-L followed by incubation with XTT for 1 h, absorbance at 495 nm was measured for biofilm inhibition. Increased killing of M. smegmatis was observed at 200 μg/ml dose of CNP-L. Note : scanning electron microscopy analysis was performed on a SU1510 scanning electron microscope (Hitachi, Tokyo, Japan).

    Journal: Frontiers in Microbiology

    Article Title: Selective Targeting of 4SO4-N-Acetyl-Galactosamine Functionalized Mycobacterium tuberculosis Protein Loaded Chitosan Nanoparticle to Macrophages: Correlation With Activation of Immune System

    doi: 10.3389/fmicb.2018.02469

    Figure Lengend Snippet: CNPs treatments disintegrate the M. smegmatis cell wall. (A) Scanning electron microscopic images showing disruption in cell wall morphology of single M. smegmatis (control) and after exposure to as-synthesized CNP-L (100 μg/ml) for 36 h. (B) (i) Control M. smegmatis biofilm (untreated), (ii) CNP treatment inhibits the biofilm formation, and (iii) CNP-L treatment at the same dose showed more biofilm inhibition after 36 h. (C) Biofilms were grown and treated with CNP and CNP-L followed by incubation with XTT for 1 h, absorbance at 495 nm was measured for biofilm inhibition. Increased killing of M. smegmatis was observed at 200 μg/ml dose of CNP-L. Note : scanning electron microscopy analysis was performed on a SU1510 scanning electron microscope (Hitachi, Tokyo, Japan).

    Article Snippet: The treated M. smegmatis biofilm was washed with PBS, fixed with 4% formaldehyde, and stained for SYTO-9/PI staining for 1 h followed by washing step to remove unbound biofilm and dye, observed with a confocal microscope (LSM710, Zeiss, Germany).

    Techniques: Synthesized, Inhibition, Incubation, Electron Microscopy, Microscopy

    Confocal microscope image showing M. smegmatis biofilm inhibition by CNPs as visualized in 63X oil immersion magnification: anti-biofilm activity of CNPs was assessed by incubating M. smegmatis with increasing concentrations of CNPs for 36 h in a six-well plate. The treated biofilm was stained with SYTO-9/PI. The addition of increasing concentration (100–200 μg/ml) of CNPs inhibited M. smegmatis biofilm formation. Red-dye showing PI-stain corresponds to killing activity and green dye showing viable bacteria in preformed biofilm. Yellow corresponds to co-localization of merged green and red dye at the same place.

    Journal: Frontiers in Microbiology

    Article Title: Selective Targeting of 4SO4-N-Acetyl-Galactosamine Functionalized Mycobacterium tuberculosis Protein Loaded Chitosan Nanoparticle to Macrophages: Correlation With Activation of Immune System

    doi: 10.3389/fmicb.2018.02469

    Figure Lengend Snippet: Confocal microscope image showing M. smegmatis biofilm inhibition by CNPs as visualized in 63X oil immersion magnification: anti-biofilm activity of CNPs was assessed by incubating M. smegmatis with increasing concentrations of CNPs for 36 h in a six-well plate. The treated biofilm was stained with SYTO-9/PI. The addition of increasing concentration (100–200 μg/ml) of CNPs inhibited M. smegmatis biofilm formation. Red-dye showing PI-stain corresponds to killing activity and green dye showing viable bacteria in preformed biofilm. Yellow corresponds to co-localization of merged green and red dye at the same place.

    Article Snippet: The treated M. smegmatis biofilm was washed with PBS, fixed with 4% formaldehyde, and stained for SYTO-9/PI staining for 1 h followed by washing step to remove unbound biofilm and dye, observed with a confocal microscope (LSM710, Zeiss, Germany).

    Techniques: Microscopy, Inhibition, Activity Assay, Staining, Concentration Assay

    FFT analysis of cell distribution at the glass surface. (a) A digitized micrograph (×32 objective) of a biofilm at 4 h of growth. (b) Two-dimensional FFT spectrum of the data from panel a. (c) One-dimensional FFT spectrum, obtained from an intensity profile measured by a horizontal line that bisects the image of panel b. (d) The cell density pattern obtained by low-pass and reverse-transform operations. Note four distinct regions of cell density, highlighted in color, and the corresponding regions that are indicated in the micrograph of panel a. (e) An example of a quasi-random cell distribution: micrograph of the biofilm (×40 objective) obtained after 24 h of growth with obscured multiple layers of cells. (f) Two-dimensional FFT spectrum of data from panel e. (g) One-dimensional FFT spectrum obtained from an intensity profile of image of panel f, generated as in panel c.

    Journal: Journal of Bacteriology

    Article Title: Periodicity of Cell Attachment Patterns during Escherichia coli Biofilm Development

    doi: 10.1128/JB.185.18.5632-5638.2003

    Figure Lengend Snippet: FFT analysis of cell distribution at the glass surface. (a) A digitized micrograph (×32 objective) of a biofilm at 4 h of growth. (b) Two-dimensional FFT spectrum of the data from panel a. (c) One-dimensional FFT spectrum, obtained from an intensity profile measured by a horizontal line that bisects the image of panel b. (d) The cell density pattern obtained by low-pass and reverse-transform operations. Note four distinct regions of cell density, highlighted in color, and the corresponding regions that are indicated in the micrograph of panel a. (e) An example of a quasi-random cell distribution: micrograph of the biofilm (×40 objective) obtained after 24 h of growth with obscured multiple layers of cells. (f) Two-dimensional FFT spectrum of data from panel e. (g) One-dimensional FFT spectrum obtained from an intensity profile of image of panel f, generated as in panel c.

    Article Snippet: Next, the internal structure of a ∼30-μm-thick, 48-h biofilm was visualized by staining cells with acridine orange and examining the biofilm with confocal laser scanning microscopy (CLSM) using a Zeiss LSM 410 microscope containing a ×40 1.2-numerical aperture C-Apochromat objective lens.

    Techniques: Generated

    CLSM images of the cell distribution within a thick biofilm. Color panels (1 to 16) show optical sections parallel to the substrate starting at a distance of 2 μm and proceeding at 2-μm increments. Density distribution after FFT and low-pass operation for panels 4, 8, 12, and 16 are shown immediately to the right, in black and white.

    Journal: Journal of Bacteriology

    Article Title: Periodicity of Cell Attachment Patterns during Escherichia coli Biofilm Development

    doi: 10.1128/JB.185.18.5632-5638.2003

    Figure Lengend Snippet: CLSM images of the cell distribution within a thick biofilm. Color panels (1 to 16) show optical sections parallel to the substrate starting at a distance of 2 μm and proceeding at 2-μm increments. Density distribution after FFT and low-pass operation for panels 4, 8, 12, and 16 are shown immediately to the right, in black and white.

    Article Snippet: Next, the internal structure of a ∼30-μm-thick, 48-h biofilm was visualized by staining cells with acridine orange and examining the biofilm with confocal laser scanning microscopy (CLSM) using a Zeiss LSM 410 microscope containing a ×40 1.2-numerical aperture C-Apochromat objective lens.

    Techniques: Confocal Laser Scanning Microscopy

    Biofilm patterns in a flow cell. The csrA mutant of MG1655 was grown under laminar flow. The direction of the flow is from left to right in all panels. (a) Micrograph (×10 objective) at 96 h of growth at a flow rate of 15 ml/h of a culture that was inoculated under static conditions. The frame width of the image is 1,200 μm. (b) Same conditions (×100 objective) as in panel a, at a frame width of 120 μm. (c) Biofilm (×10 objective) after 50 h of cultivation at a flow rate of 15 ml/hour, with in-flow inoculation. The frame width of the image is 1,200 μm. (d) Biofilm (×10 objective) after 72 h of cultivation at a flow rate of 22 ml/h, with in-flow inoculation. The frame width of the image is 1,200 μm.

    Journal: Journal of Bacteriology

    Article Title: Periodicity of Cell Attachment Patterns during Escherichia coli Biofilm Development

    doi: 10.1128/JB.185.18.5632-5638.2003

    Figure Lengend Snippet: Biofilm patterns in a flow cell. The csrA mutant of MG1655 was grown under laminar flow. The direction of the flow is from left to right in all panels. (a) Micrograph (×10 objective) at 96 h of growth at a flow rate of 15 ml/h of a culture that was inoculated under static conditions. The frame width of the image is 1,200 μm. (b) Same conditions (×100 objective) as in panel a, at a frame width of 120 μm. (c) Biofilm (×10 objective) after 50 h of cultivation at a flow rate of 15 ml/hour, with in-flow inoculation. The frame width of the image is 1,200 μm. (d) Biofilm (×10 objective) after 72 h of cultivation at a flow rate of 22 ml/h, with in-flow inoculation. The frame width of the image is 1,200 μm.

    Article Snippet: Next, the internal structure of a ∼30-μm-thick, 48-h biofilm was visualized by staining cells with acridine orange and examining the biofilm with confocal laser scanning microscopy (CLSM) using a Zeiss LSM 410 microscope containing a ×40 1.2-numerical aperture C-Apochromat objective lens.

    Techniques: Flow Cytometry, Mutagenesis

    Design of experiment 2. Box 1 shows different treatments A to D, in 4: A) Simulation of kelp forest canopy with seawater and kelp blade; B) Kelp blade and filtered seawater to remove phytoplankton and control for TEP produced in seawater; C) Synthetic kelp blade and filtered seawater to control for EPS on blade and TEP in seawater; D) Filtered seawater to control for surrogate loss due to settling or attachment to surfaces of jar. Box 2 indicates where and when measurements were taken and box 2.1. shows steps to measure parameters on kelp blade biofilm. Box 3 shows parameters measured and box 4 indicates methods used for each parameter.

    Journal: PLoS ONE

    Article Title: A New Pathogen Transmission Mechanism in the Ocean: The Case of Sea Otter Exposure to the Land-Parasite Toxoplasma gondii

    doi: 10.1371/journal.pone.0082477

    Figure Lengend Snippet: Design of experiment 2. Box 1 shows different treatments A to D, in 4: A) Simulation of kelp forest canopy with seawater and kelp blade; B) Kelp blade and filtered seawater to remove phytoplankton and control for TEP produced in seawater; C) Synthetic kelp blade and filtered seawater to control for EPS on blade and TEP in seawater; D) Filtered seawater to control for surrogate loss due to settling or attachment to surfaces of jar. Box 2 indicates where and when measurements were taken and box 2.1. shows steps to measure parameters on kelp blade biofilm. Box 3 shows parameters measured and box 4 indicates methods used for each parameter.

    Article Snippet: Snail pellet content and biofilm organisms from kelp blades on which they had grazed were examined using a Zeiss Axio Imager with phase contrast and a 50 W light source fitted with 2 DAPI bandpass filter sets (wavelength excitation (λex) 350 nm, (wavelength emission) λem > 420 nm and λex 350 nm, λem > 460 nm).

    Techniques: Produced