Article Figures & Data
Figures
- FIG 1
Planktonic growth curves and MBCs for P. aeruginosa ATCC 39324 in ASM+, ASM−, and LB medium. (a) Numbers of CFU per milliliter in planktonic cultures as a function of time in different growth media. Growth curves were performed in duplicate, with error bars denoting the difference between the two experiments. (b) MBCs for planktonic P. aeruginosa upon 24-h exposure to the different antimicrobials in PBS. MBC values were determined in triplicate with separately grown bacterial cultures, which yielded no differences in MBC values.
- FIG 2
Microscopic images of P. aeruginosa ATCC 39324 biofilms grown in ASM+, ASM−, and LB medium. (a) Two-dimensional, cross-sectional, OCT images. Scale bars, 200 μm. (b) CLSM two-dimensional overlay (left) and three-dimensional (right) images of SYTO9-stained biofilms, yielding green-fluorescent bacteria. Scale bars, 100 μm.
- FIG 3
Characteristics and matrix composition of P. aeruginosa ATCC 39324 biofilms grown in ASM+, ASM−, and LB medium. (a) Thickness of the biofilms measured by OCT. (b) Biovolume of the biofilms obtained from COMSTAT analysis of CLSM images. (c) Metabolic activity of the biofilms measured with MTT. (d) eDNA presence in the biofilms, isolated with phenol-chloroform and measured with the nanodrop method. (e) Polysaccharide presence in the biofilms, measured using the anthrone-sulfuric acid colorimetric assay. (f) Total protein concentration in the biofilms. (g) Water content, obtained from a comparison of the weight of hydrated and dried biofilms and expressed as a percentage of the hydrated biofilm weight. Error bars denote standard deviations over n (numbers given in the columns) different biofilms, taken from different pans in three separate CDFF runs, except for data in panels b and c, which were taken from different pans in two separate CDFF runs. Significant differences (P < 0.05, ANOVA with Tukey’s post hoc analysis) between groups are indicated.
- FIG 4
Stress relaxation analysis of P. aeruginosa ATCC 39324 biofilms grown in ASM+, ASM−, or LB medium. (a) Examples of the normalized stress in compressed biofilms (strain of 0.2) as a function of relaxation time. Stress at time zero amounted 2.2 kPa for all biofilms, regardless of the growth medium. (b) Quality of fitting of the stress relaxation data to a generalized Maxwell model as a function of the number of elements included in the model. The quality of the fit is indicated by chi-squared values. (c) Distribution of the relative importance of individual Maxwell elements (three-element model) for differently grown P. aeruginosa biofilms over different relaxation time constant ranges. Each data point represents a single measurement of 30 biofilms, taking 10 biofilms from different pans in three separate CDFF runs. Median values are indicated by horizontal lines. Significant differences (P < 0.05, ANOVA with Dunn’s post hoc analysis) between groups are indicated.
- FIG 5
Numbers of CFU per square centimeter in P. aeruginosa ATCC 39324 biofilms grown in ASM+, ASM−, or LB medium after 24-h exposure to different concentrations of tobramycin, colistin, or the antimicrobial peptide AA-230 in PBS, with PBS as a control. Error bars denote standard deviations for at least nine different biofilms, taken from different pans in three separate CDFF runs. Significant differences (P < 0.05, ANOVA with Tukey’s post hoc analysis) between groups are indicated.
Tables
- TABLE 1
Summary of statistically significant differences in the characteristics of P. aeruginosa ATCC 39324 biofilms grown in ASM+, ASM−, or LB medium and their killing by antimicrobials (taking tobramycin, colistin, and the antimicrobial peptide AA-230 together)
Biofilm characteristic Differencea Matrix eDNA ASM+ = ASM− > LB Matrix polysaccharides ASM+ > ASM− = LB Stress relaxation time <0.75 s LB > ASM− > ASM+ 0.75 s to <3 s ASM+ > ASM− > LB 3 s to <10 s ASM+ > LB; ASM+ = ASM−; LB = ASM− 10 s to <25 s ASM+ > LB; ASM+ = ASM−; LB = ASM− Antimicrobial killing LB > ASM− ≥ ASM+ ↵a =, no significant difference; >, significant difference (P < 0.05).
