Journal: Journal of Bacteriology

Article Title: FleQ finetunes the expression of a subset of BrlR-activated genes to enable antibiotic tolerance by Pseudomonas aeruginosa biofilms

doi: 10.1128/jb.00503-24

Figure Lengend Snippet: FleQ contributes to biofilm formation in a manner independent of SagS and SagS downstream signaling. ( A ) Representative confocal images of 3-day-old biofilms formed by P. aeruginosa PAO1, Δ fleQ, and double mutant Δ fleQΔsagS . White bar = 100 µm. ( B ) Quantitative of the biofilm biomass and biofilm height by COMSTAT of biofilms formed by P. aeruginosa PAO1, Δ fleQ, and double mutant Δ fleQΔsagS. * *, ***, significantly different ( P = 0.0001, <0.0001, respectively) relative to PAO1 using ANOVA followed by the Bartlett’s test. Ns, not significant. ( C ) Total cellular c-di-GMP levels in 6-day-old biofilms formed by P. aeruginosa PAO1, Δ fleQ, and ΔsagS, as determined by HPLC quantitative analysis, followed by normalization relative to total cell protein content. *, P < 0.05, relative to PAO1 using ANOVA followed by Dunnett’s T3 multiple comparisons test. ( D ) Representative confocal images of 6-day-old P. aeruginosa Δ fleQ and ΔsagS biofilms and mutant biofilms expressing bfiR and gcbA. gcbA encodes diguanylate cyclase GcbA, bfiR the two-component response regulator BfiR. White bar = 100 µm. All experiments were performed in triplicate. Error bars indicate standard deviations.

Article Snippet: Quantitative analysis of the images was performed using COMSTAT MATLAB package and COMSTAT2 ( , ).

Techniques: Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium

doi: 10.1371/journal.pone.0016791

Figure Lengend Snippet: ( A ) The synthetic symbiotic consortium. The blue population cannot synthesize diaminopimelate or lysine; when cultured without lysine or diaminopimelate, this population forms only a scant biofilm. The yellow population cannot form biofilms alone but is otherwise healthy. It synthesizes C4HSL, which diffuses freely and activates production of diaminopimelate in the blue population. Yellow cells become bound within the biofilm formed by the blue population and rescue growth. Together, the two populations form viable biofilms that persist. ( B ) The symbiotic consortium functions as designed. The blue population control forms a biofilm which eventually dies (blue bars), and the yellow population accumulates very little biomass (insignificant, not shown). When the yellow and blue populations are inoculated into a flow chamber in a 50/50 mixture, more biomass accumulates than in either control (solid yellow areas, total yellow biomass in the biofilm; solid blue areas, total blue biomass in the biofilm; the sum of blue and yellow areas is the overall total biomass in the biofilm at each time point; all errors are s.d.). All biofilm measurements were derived from images quantified with COMSTAT .

Article Snippet: All image-based measurements were calculated using the COMSTAT biofilm image processing package in Matlab .

Techniques: Cell Culture, Control, Derivative Assay

Journal: PLoS ONE

Article Title: Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium

doi: 10.1371/journal.pone.0016791

Figure Lengend Snippet: ( A ) After 80 hours, the blue population remains primarily near the substrate while the yellow population forms clumps attached to the blue population, as shown in this cross-sectional projection taken at 1/3 the total height of the biofilm. ( B ) After 80 hours of growth, the yellow population begins to exhibit a consistently larger biomass median (yellow lines, throughout figure) than the blue population (blue lines, throughout figure), revealing that the yellow population grows further from the substrate while the blue population remains close to the substrate. (The biomass median indicates the average distance from the substrate at which cells of a given population are found.) Gray bars, plotted against the right-hand axis, indicate total biomass accumulation for the entire consortium at each time-point throughout the figure (errors throughout figure are s.d.). ( C ) Maximum total biomass accumulated by the downstream biofilm is double that in the initial biofilm (compare gray bars in [B] and [C]). Additionally, the downstream biofilm assumes the layered structure more quickly: the biomass medians reveal structure after 24 hours of growth. ( D ) When aggregates are disrupted prior to transfer, leaving all else constant, this treated effluent can form biofilms, but they never exhibit the layered structure or growth advantage. ( E ) The layered structure is recovered when the sorted blue-and-yellow aggregate fraction forms downstream biofilms. In fact, the consortium starts with this structure, exhibiting it by 24 hours after inoculation. Further, maximum total biomass accumulation is more than double the highest amount observed in the predecessor (illustrated in [D]), suggesting recovery of the growth advantage. ( F ) The single-cell fraction consists of more than 99% yellow cells, and thus neither the blue population nor the layered structure nor any growth advantage is evident in the downstream biofilm it forms. This biofilm accumulates less biomass than the biofilm formed by the aggregate fraction (compare to grey bars in [E]). ( G ) Here, effluent is taken from the treated biofilms, which are less productive and do not exhibit structure (illustrated in [D]). Although this effluent is left untreated, it forms initially dense, monomorphic, and primarily yellow downstream biofilms that do not exhibit layered structure. These biofilms consistently lose biomass.

Article Snippet: All image-based measurements were calculated using the COMSTAT biofilm image processing package in Matlab .

Techniques:

Journal: Antibiotics

Article Title: Baicalein Inhibits Streptococcus mutans Biofilms and Dental Caries-Related Virulence Phenotypes

doi: 10.3390/antibiotics10020215

Figure Lengend Snippet: ( A ) CLSM (Confocal Laser Scanning Microscopy) micrographs illustrating the effect on the sub-inhibitory concentration of baicalein (200 µM) on biofilm architecture and viability of the biofilm cells through Live/Dead staining. Baicalein treated cell cultures show sparing green color cells with no confluent mass, indicating inhibition of biofilm formation. ( B ) COMSTAT analysis of the CLSM images revealed a reduction in the biofilm biomass, average thickness, and diffusion distances in the baicalein treated biofilms compared to the untreated control. ( C ) ImageJ analysis shows the proportion of viable cells in the biofilm formed with baicalein treatment at the concentration of 200 µM (MBIC 50 ).

Article Snippet: Z-stacks were obtained from at least 5 regions and the obtained images were further analyzed using COMSTAT package in MATLAB R2017b [ ].

Techniques: Confocal Laser Scanning Microscopy, Concentration Assay, Staining, Inhibition, Diffusion-based Assay, Control