Article Figures & Data
Figures
- FIG 1
The development of 19-h wild-type P. aeruginosa-S. aureus, P. aeruginosa ΔpelA-S. aureus, and P. aeruginosa ΔpslBCD-S. aureus dual-species biofilms imaged using confocal imaging every 6 h. The monospecies wild-type P. aeruginosa and S. aureus biofilms are shown for comparison. P. aeruginosa is mCherry tagged (red), and S. aureus is Gfp tagged (green). Images are representative of four biological replicates. The scale bar is 30 μm.
- FIG 2
The development of 19-h biofilms formed by wild-type and matrix mutants of P. aeruginosa cocultured with S. aureus. Error bars represent standard errors of the means (SEM) (n ≥ 4). *, P < 0.05, α = 0.05 (unpaired two-sided t test with Welch’s correction). (A) Biovolumes per area of P. aeruginosa (solid lines) in the dual-species biofilms every 6 h. (B) Biovolumes per area of S. aureus (dashed lines) in the dual-species biofilms every 6 h. Note that the y axis scales for panels A and B are different. (C) Fitness of P. aeruginosa relative to S. aureus, where a selection constant of rij = 0 means that P. aeruginosa and S. aureus are equally competitive and a rij value of >0 means P. aeruginosa is more competitive than S. aureus.
- FIG 3
Comparison of microcolony sizes in dual-species biofilms using the two-sample, two-sided Kolmogorov-Smirnov test. The red curve shows the difference between the two distributions (*, P < 0.05, **, P < 0.01, ***, P < 0.001; α = 0.05). (Top panel) Changes in size distribution as biofilms progresses from 13 to 19 h. (A to D) P. aeruginosa microcolonies in (A) wild-type P. aeruginosa-S. aureus, (B) mutant ΔpelA-S. aureus, and (C) mutant ΔpslBCD-S. aureus biofilms and S. aureus microcolonies in (D) mutant ΔpslBCD-S. aureus biofilms. (Bottom panel) Differences between P. aeruginosa microcolony size distributions formed by wild-type and matrix mutants of P. aeruginosa cocultured with S. aureus. (E and F) Wild-type P. aeruginosa-S. aureus compared to mutant ΔpelA-S. aureus at (E) 13 h and (F) 19 h. (G and H) Wild-type P. aeruginosa-S. aureus compared to mutant ΔpslBCD-S. aureus at (G) 13 h and (H) 19 h.
- FIG 4
Microrheological measurements of wild-type and matrix mutants of P. aeruginosa cocultured with S. aureus. (A) MSD curves for wild-type P. aeruginosa-S. aureus, mutant ΔpelA-S. aureus, and mutant ΔpslBCD-S. aureus microcolonies. The MSD curve for TSB medium is shown for comparison. SA, S. aureus. The orange dotted lines indicate the line of best fit to the experimentally determined MSD using a power law function for the estimation of α. (B) Representative particle trajectories in wild-type P. aeruginosa-S. aureus, mutant ΔpelA-S. aureus, and mutant ΔpslBCD-S. aureus microcolonies. The red trajectory in the middle panel indicates a particle undergoing directed motion and superdiffusion, whereas the blue and yellow trajectories indicate subdiffusion. Error bars represent SEM.
- FIG 5
Confocal images of 19-h biofilms of P. aeruginosa CdrA and diguanylate cyclase mutants cocultured with S. aureus. (A) P. aeruginosa ΔcdrA-S. aureus. (B) P. aeruginosa ΔsadC-S. aureus. (C) P. aeruginosa ΔsiaD-S. aureus. (D) P. aeruginosa ΔsiaD(siaD)-S. aureus. P. aeruginosa was mCherry tagged (red), and S. aureus was Gfp tagged (green). Images are representative of results from at least three biological replicates. The scale bar is 30 μm.
- FIG 6
MSD curves for P. aeruginosa ΔsiaD-S. aureus, P. aeruginosa ΔcdrA-S. aureus, and P. aeruginosa ΔsadC-S. aureus microcolonies. SA, S. aureus. The orange dotted lines indicate the line of best fit to the experimentally determined MSD using a power law function for the estimation of α. Error bars represent SEM.
- FIG 7
Relative levels of pyoverdine and PQS in 19-h P. aeruginosa-S. aureus biofilms. (A) Pyoverdine, as indicated by its fluorescence at an emission peak of 450 nm. (B). PQS, as indicated by the fluorescence emission of the green fluorescent protein from the PQS biosensor strain, mutant ΔpqsC(pqsA-gfp). Values are means (± standard deviations [SD]) of relative fluorescence units (RFU) determined from three biological replicates.
- FIG 8
Schematic showing how matrix polysaccharides and SiaD contribute to P. aeruginosa predominance in dual-species P. aeruginosa-S. aureus communities. (A) Psl enhances P. aeruginosa competitiveness in early stages, possibly via SiaD activation, whereas Pel enables biofim expansion to increase P. aeruginosa predominance in the later stages. (B) Dominance of wild-type P. aeruginosa and SiaD and SiaD complement mutant over S. aureus, with their corresponding PQS/pyoverdine (PVD) levels.
Tables
- TABLE 1
List of bacterial strainsa
Strain Relevant characteristic(s) Reference
or sourceP. aeruginosa wtPAO1 Wild-type strain 64 P. aeruginosa ΔpelA PAO1 that does not produce the Pel matrix polysaccharide 53 P. aeruginosa ΔpslBCD PAO1 that does not express the Psl matrix polysaccharide 53 P. aeruginosa ΔcdrA PAO1 lacking the extracellular adhesin CdrA This study P. aeruginosa ΔsadC PAO1 lacking the DGC SadC 51 P. aeruginosa ΔsiaD PAO1 lacking the DGC SiaD 51 P. aeruginosa ΔsiaD/pUC18-siaD Carbr; ΔSiaD mutant containing the pUC18-siaD complementation plasmid This study S. aureus 15981 Wild-type strain 53 ↵a wtPAO1, wild-type strain PAO1; Carbr, carboxylate resistance.
- TABLE 2
Biovolumes for single- and dual-species biofilmsa
Biofilm Strain(s) Biovolume per area (μm3 mm−2) 1 h 7 h 13 h 19 h Monospecies: P. aeruginosa wtPAO1 26,286 ± 4,128 542,412 ± 260,428 1,548,912 ± 682,644 277,455 ± 59,146 Monospecies: S. aureus S. aureus 15981 39,168 ± 2,660 495,781 ± 190,117 851,130 ± 292,722 4,068,969 ± 1,335,431 Dual species: P. aeruginosa-
S. aureuswtPAO1 14,388 ± 2,168 73,488 ± 21,746 837,343 ± 104,916 792,562 ± 10,272 S. aureus 15981 14,031 ± 2,418 35,015 ± 18,203 12,785 ± 8,189 10,521 ± 3,878 Dual species: mutant
ΔpelA-S. aureusMutant ΔpelA 14,285 ± 2,693 82,682 ± 32,691 220,077 ± 52,939* 267,374 ± 114,530* S. aureus 15981 13,045 ± 1,993 128,861 ± 62,036 6,927 ± 2,785 14,982 ± 4,887 Dual species: mutant
ΔpslBCD-S. aureusMutant ΔpslBCD 14,722 ± 799 14,992 ± 4,185 178,387 ± 48,490* 1,419,718 ± 432,200 S. aureus 15981 17,662 ± 1,729 62,035 ± 15,636 67,605 ± 31,815 20,726 ± 5,301 Dual species: mutant ΔpelA
ΔpslBCD-S. aureusMutant ΔpelA ΔpslBCD 7,611 ± 513* 23,988 ± 2,379 21,310 ± 4,559* 50,401 ± 21,795* S. aureus 15981 7,691 ± 2,315 17,752 ± 5,103 4,559 ± 1,818 1,868 ± 701 ↵a Data represent results from four biological replicates with each replicate composed of three confocal images of the biofilm in different areas on average. Asterisks (*) indicate a significant difference from the wild-type P. aeruginosa-S. aureus biofilms (P < 0.05) (unpaired t test with Welch’s correction). Error data represent SEM.
- TABLE 3
Competitiveness of P. aeruginosa relative to S. aureus
Biofilm Selection rate constant, rija Correlation of fitness curvesb 7 h 13 h 19 h Biofilm rXY wtPAO1-S. aureus 0.16 ± 0.09 0.43 ± 0.07 0.25 ± 0.04 wtPAO1-S. aureus with ΔpelA-S. aureus 0.97 Mutant ΔpelA-S. aureus −0.04 ± 0.20 0.30 ± 0.06 0.15 ± 0.04 wtPAO1-S. aureus with ΔpslBCD-S. aureus 0.58 Mutant ΔpslBCD-S. aureus −0.21 ± 0.09* 0.12 ± 0.01* 0.23 ± 0.02 ΔpelA-S. aureus with ΔpslBCD-S. aureus 0.76 Mutant ΔpelA ΔpslBCD-S. aureus 0.06 ± 0.02 0.07 ± 0.04* 0.18 ± 0.07 wtPAO1-S. aureus with ΔpelA ΔpslBCD-S. aureus -0.11 Mutant ΔsiaD-S. aureus 0.11 ± 0.06 0.07 ± 0.04* 0.01 ± 0.01* ΔpelA-S. aureus with ΔpelA ΔpslBCD-S. aureus 0.13 Mutant ΔsiaD(siaD)-S. aureus 0.23 ± 0.05 0.19 ± 0.04 0.18 ± 0.05 ΔpslBCD-S. aureus with ΔpelA ΔpslBCD-S. aureus 0.75 ↵a Data represent selection rate constants during 19 h of biofilm development determined from four biological replicates, with each replicate derived from an average of three confocal images. Asterisks (*) indicate a significant difference from the wild-type P. aeruginosa-S. aureus biofilms (P < 0.05) (unpaired t test with Welch’s correction). Error data represent SEM.
↵b Correlation of fitness curves is given by the Pearson correlation coefficient (rXY).
- TABLE 4
Surface coverage, microcolony size, and number of microcolonies in single- and mixed-species biofilmsa
Biofilm Strain(s) Avg surface
coverage (%)Avg no. of microcolony
per area (mm−2)Avg microcolony
biovolume (μm3)13 h 19 h 13 h 19 h 13 h 19 h Monospecies: P. aeruginosa wtPAO1 14 ± 4 10 ± 2 1,757 ± 386 1,974 ± 360 109 ± 46 58 ± 23 Monospecies: S. aureus 15981 S. aureus 15981 20 ± 5 40 ± 10 2,520 ± 552 3,325 ± 507 1,346 ± 3,035 7,356 ± 2,279 Dual species: P. aeruginosa-
S. aureuswtPAO1 19 ± 3 22 ± 0 5,265 ± 489 3,716 ± 462 108 ± 7 499 ± 91 S. aureus 15981 0 ± 2 0 ± 0 Dual species: mutant
ΔpelA-S. aureusMutant ΔpelA 8 ± 1 9 ± 3 1,025 ± 302 2,581 ± 1,117 67 ± 5 111 ± 19 S. aureus 15981 0 ± 0 1 ± 0 Dual species: mutant
ΔpslBCD-S. aureusMutant ΔpslBCD 7 ± 3 33 ± 14 1,043 ± 756 4,661 ± 2,407 50 ± 4 1,400 ± 358 S. aureus 15981 2 ± 1 1 ± 0 207 ± 138 85 ± 85 65 ± 16 65 ± 15 Dual species: mutant ΔpelA
ΔpslBCD-S. aureusMutant ΔpelA ΔpslBCD 3 ± 1 5 ± 2 S. aureus 15981 1 ± 0 0 ± 0 ↵a Values are derived from three biological replicates, with each replicate derived from an average of three confocal images. Error data represent SEM.
- TABLE 5
Viscoelasticity and creep compliance of 19-h biofilms formed by P. aeruginosa and matrix polysaccharide mutants with S. aureus, respectively
Biofilm Region α J(t) (t = 101 s, Pa−1) Medium (negative control) Liquid phase 1.05 11,500 ± 789 wtPAO1-S. aureus Microcolony 0.87 3 ± 2 Mutant ΔpelA-S. aureus Microcolony 0.74 2 ± 0 Mutant ΔpslBCD-S. aureus Loose microcolony 0.41 1,294 ± 109
FIG S1
MSD curve for particles undergoing superdiffusion in wild-type P. aeruginosa-S. aureus microcolonies. The orange dotted line indicates the line of best fit to the experimentally determined MSD curve determined by the use of a power law function for the estimation of α. Error bars represent standard errors of the means (SEM). Download FIG S1, PDF file, 0.2 MB.
Copyright © 2018 Chew et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Fitness of P. aeruginosa mutants ΔcdrA, ΔsadC, and mutant ΔsiaD and complemented strain ΔsiaD(siaD) relative to S. aureus. A selection constant of rij = 0 means that P. aeruginosa and S. aureus are equally competitive and that of rij > 0 means P. aeruginosa is more competitive than S. aureus. Asterisks (*) indicate a significant difference. Download FIG S2, PDF file, 0.01 MB.
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This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
MSD curves for S. aureus microcolonies in mutant ΔsiaD-S. aureus dual-species biofilms compared to monospecies S. aureus microcolonies. SA, S. aureus. The orange dotted lines indicate the line of best fit to the experimentally determined MSD curve determined by the use of a power law function for the estimation of α. Error bars represent SEM. Download FIG S3, PDF file, 0.1 MB.
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TABLE S1
Microcolony formation and microrheological properties of 19-h biofilms formed by mutant ΔcdrA and diguanylate cyclase mutants of P. aeruginosa with S. aureus. Download Table S1, DOCX file, 0.01 MB.
Copyright © 2018 Chew et al.
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TEXT S1
Supplemental Methods. Download Text S1, DOCX file, 0.01 MB.
Copyright © 2018 Chew et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
