Quantification of P85-formulated farnesol effects on pre-formed <i>S</i>. <i>mutans</i> biofilm architecture.

<p>Biofilms described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133886#pone.0133886.g004" target="_blank">Fig 4</a> (8 random fields of view acquired from 2 biological replicates) were analyzed with COMSTAT software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133886#pone.0133886.ref034" target="_blank">34</a>] to quantify CLSM z-stack images for biomass (A) and roughness co-efficient (B). The average CFU/ml (n = 3 biological replicates) of biofilms grown in the presence of each condition was also determined in a parallel experiment (C). Error bars = SEM. *Indicates statistical significance compared to untreated control •Denotes statistically-significant difference compared to farnesol-treated condition (p < 0.05; SNK test).</p

Quantification of P85-formulated farnesol effects on <i>S</i>. <i>mutans</i> biofilm architecture and viability.

<p>Biofilms described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133886#pone.0133886.g001" target="_blank">Fig 1</a> were analyzed with COMSTAT software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133886#pone.0133886.ref034" target="_blank">34</a>] to quantify CLSM z-stack images for biomass (A) and roughness co-efficient (B). Viability (CFU/ml) of disrupted biofilms was determined by serial dilution plating (C). For A and B, data = 12 random fields of view from 3 biological replicates acquired over 2 independent experiments. The average CFU/ml of biofilms grown in the presence of each condition (Panel C) was averaged from 3 biological replicates acquired over 2 independent experiments. Error bars = standard error of the mean (SEM). *Denotes statistically-significant difference relative to untreated condition (p < 0.05); •Denotes statistically-significant difference compared to farnesol-treated condition (p < 0.05). A: Holm-Sidak test (UA159) or Dunnett’s test (BM71 and P1); B: Dunnett's test; C: Student-Newman-Keuls (SNK) test.</p

Effect of <i>gtfBC</i> mutation on <i>S</i>. <i>mutans</i> biofilm architecture and viability in response to P85-formulated farnesol.

<p><i>S</i>. <i>mutans gtf</i>BC mutant was grown for 48 hours in the presence of P85 (A), P85F (B), F alone (C) or untreated (D) in BM media containing 0.25% sucrose and 0.25% glucose. Wells containing adherent biofilm were stained with LIVE/DEAD stain. Biofilm z-stacks of 1 μm cross-sectional images were acquired at 400× magnification by CLSM. Representative orthogonal images of each biofilm are shown. Scale bars = 20 μm. In a parallel experiment (n = 3 biological replicates), the average CFU/ml of each biofilm was also determined (E). All data are representative of 2 independent experiments. Error bars = SEM. *Denotes statistically-significant difference relative to untreated condition (p < 0.05), SNK Test.</p

Effect of P85-formulated farnesol on <i>S</i>. <i>mutans</i> sucrose-independent biofilm architecture.

<p>UA159 static biofilms were grown in BM lacking sucrose (supplemented with 0.5% glucose) in the presence of P85 (A), P85F (B), F alone (C), or untreated (D). After 48 hours of growth, adherent biofilm was stained with LIVE/DEAD stain. Biofilm z-stacks were acquired at 400× magnification by CLSM, and orthogonal images are representative of 12 random fields of view acquired from 2 independent experiments. Scale bars = 20 μm.</p

Effect of pluronics-formulated farnesol treatment on pre-formed biofilm architecture.

<p>UA159 pre-formed biofilms were treated with P85 (A), P85F (B), F alone (C), or left untreated (D) at 24, 48, and 96 hours growth in 5% CO₂ at 37°C. After 96 hours, the supernatant was removed and adherent biofilms were stained with LIVE/DEAD stain. Biofilm z-stacks of 1 μm cross-sectional images were acquired at 400× magnification by confocal laser scanning microscopy (CLSM). Orthogonal images of each biofilm are shown, representing 8 fields of view acquired from 2 biological replicates. Scale bars = 20 μm.</p

Quantification of P85-formulated farnesol effects on sucrose-independent <i>S</i>. <i>mutans</i> biofilms.

<p>Biofilms described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133886#pone.0133886.g006" target="_blank">Fig 6</a> (12 random fields of view acquired from 2 independent experiments) were analyzed with COMSTAT software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133886#pone.0133886.ref034" target="_blank">34</a>] to quantify CLSM z-stack images for biomass (A) and roughness co-efficient (B). The average CFU/ml (n = 3 biological replicates, representative of two independent experiments) of biofilms grown in each condition was also determined in a parallel experiment (C). Error bars = SEM. *Indicates statistical significance compared to untreated control •Denotes statistically-significant difference compared to farnesol-treated condition (p < 0.05; SNK test).</p

Morphology of <i>S</i>. <i>mutans</i> biofilms grown with Pluronics-formulated farnesol.

<p>Strains UA159 (A-D), BM71 (E-H), and P1 (I-L) static biofilms were grown in BM containing 0.25% glucose and 0.25% sucrose, either untreated or in the presence of 2% P85 (P85), 2% P85 formulated with 50 μg/ml farnesol (P85F), or 50 μg/ml farnesol alone (F). After 48 hours of growth at 37°C in 5% CO₂, wells containing adherent biofilm were stained with LIVE/DEAD stain (Life Technologies), whereby live cells fluoresce green and dead/damaged cells fluoresce red. Biofilm z-stacks were acquired at 400× magnification by CLSM. Orthogonal images of each biofilm are shown, representing 12 random fields of view from 3 biological replicates acquired over 2 independent experiments. Scale bars = 20 μm.</p

Detection of intracellular NO/RNS with DAF-FM diacetate.

<p><b>A:</b> Cells harvested from replicate UAMS-1 7 hour static biofilms were resuspended in HBSS containing 5 µM DAF-FM diacetate. After incubation for 1 hour at 37°C, cells were collected by centrifugation, washed, and resuspended in HBSS alone (“untreated”) or HBSS supplemented with 100 µM DEA or 100 µM DEA/NO. Where indicated, 150 µM cPTIO (NO scavenger) was added to cell suspensions during the 1 hour DAF-FM diacetate staining step. Aliquots (200 µl) of each cell suspension were immediately transferred to a 96-well plate, and incubated at 37°C in a Synergy HT fluorescent plate reader. Fluorescence and OD₆₀₀ measurements were recorded after 30 minutes, and data were reported as relative fluorescent units (RFU) per OD₆₀₀ of each well. Data represents the average of n = 3 independent experiments, error bars  =  SEM. *statistical significance compared to untreated UAMS-1 (p<0.05, Tukey Test). <b>B:</b> UAMS-1 (pMK4; wild-type), KR1010 (pMK4; <i>nos</i> mutant), and KR1010 (pMKnos; <i>nos</i> complement) were grown on agar plates for 26 hours, followed by harvesting and resuspension in HBSS +5 µM DAF-FM dicaetate. Fluorescence (RFU) and OD₆₀₀ readings were recorded after 90 minutes incubation in a microplate reader as described above. Data represents the average of n = 3 independent experiments, error bars  =  SEM. *statistical significance compared to UAMS-1 (pMK4) (p<0.05, Tukey Test).</p

Effects of <i>nos</i> and <i>pdt</i> on growth in chemically-defined media.

<p>UAMS-1 (pMK4; wild-type), KR1010 (pMK4; <i>nos</i> mutant), KR1010 (pMKnos; <i>nos</i> complement), KR1013 (pMK4; <i>pdt</i> mutant), and KR1013 (pMKpdt; <i>pdt</i> complement) were grown in chemically-defined media in the presence (A) or absence (B and C) of phenylalanine. Cultures were inoculated to an OD₆₀₀ = 0.02, and grown statically in a 96-well plate for 24 hours at 37°C. OD₆₀₀ readings were measured every 2 hours using a microplate reader. Data represents the average of n = 4 biological replicates acquired in 2 independent experiments. Error bars  =  SEM. *statistical significance of <i>pdt</i> mutant (pMK4) OD₆₀₀ compared to UAMS-1 (pMK4) at t = 24 hours growth (p<0.05, Tukey Test).</p

Genetic arrangement of the <i>S. aureus nos</i> and <i>pdt</i> genes.

<p>The representative bacterial genera and/or species that contain a <i>nos</i> gene are listed below the <i>S. aureus nos-pdt</i> operon diagram. The “+” designation under <i>nos</i> indicates its presence in each respective genome, whereas the “+/−” designation under <i>pdt</i> indicates if this gene is located immediately downstream of <i>nos</i> in each respective bacterial genera/species. The predicted molecular weight (kDa) of the <i>nos</i> and <i>pdt</i> ORFs are indicated above each gene, and the size (bp) of each gene is shown below. Gene designations refer to the MRSA252 genome. The site where the <i>erm</i> resistance cassette was inserted to create the <i>nos</i>::<i>erm</i> mutant (KR1010) is indicated by the black block arrow. *denotes that all sequenced strains in NCBI contain the indicated genes.</p