Colony morphology of <i>S. aureus</i> SH1000 (parental strain) (A), LPVs (B) and WVs (C).

<p>Colony morphology of <i>S. aureus</i> SH1000 (parental strain) (A), LPVs (B) and WVs (C).</p

Schematic representation of the emergence and role of morphological variants in <i>S. aureus</i> biofilms during infection.

<p>Schematic representation of the emergence and role of morphological variants in <i>S. aureus</i> biofilms during infection.</p

Development and validation of the cellulose disk static biofilm model.

<p><b>A</b>. Effect of human plasma on the adherence of <i>S. aureus</i> SH1000 to cellulose disks. On the x-axis, 0 represents no human plasma or buffer and 0(B) is buffer only. Data indicate the proportion of adherent cells, in 48 hr disk cultures, as a percentage of the maximum achieved at 10% (v/v) human plasma (dashed line) (1.8×10¹⁰ cfu/disk). Data from three experimental replicates. Error bars indicate standard error. <b>B.</b> Determination of the proportion of adherent and planktonic cells present in <i>S. aureus</i> SH1000 cellulose disk cultures. (A) Cultures grown for 48 hrs in the absence of human plasma. (B) Cultures grown for 48 hrs with human plasma (4% v/v) added prior to inoculation. (C) Cultures grown for 144 hrs with human plasma (4% v/v) added prior to inoculation. (D) Cultures grown for 144 hrs with human plasma (4% v/v) added prior to inoculation and every 48 hrs. Data from three experimental replicates. Error bars indicate standard error. <b>C.</b> Time-kill curves of <i>S. aureus</i> SH1000 exponential phase planktonic (P) and 48 hr cellulose disk (D) cultures exposed to 0.25 mg/L rifampicin. Data from three experimental replicates. Error bars indicate standard deviations. <b>D.</b> The dissociation of adherent cells of <i>S. aureus</i> SH1000 from cellulose disk cultures in the presence of d-tyrosine (100 µM) and l-tyrosine (100 µM). Data from three experimental replicates. Error bars indicate standard error. Methodology and additional information regarding validation of the cellulose disk model can be found in Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047695#pone.0047695.s001" target="_blank">Information S1</a>.</p

Differentially regulated genes in <i>S. aureus</i> SH1000 biofilms, grown for 48 hrs and 144 hrs, compared with planktonic cultures. ORFs encoding hypothetical proteins not shown.

<p>Differentially regulated genes in <i>S. aureus</i> SH1000 biofilms, grown for 48 hrs and 144 hrs, compared with planktonic cultures. ORFs encoding hypothetical proteins not shown.</p

The effect of catalase and <i>cidC</i> inactivation on <i>S. aureus</i> biofilm mutability.

<p>Mutation frequencies of <i>S. aureus</i> SH1000 planktonic (P) and 96 h biofilm (B) cultures, determined by rifampicin selection. WT indicates wild-type (<i>S. aureus</i> SH1000), +CAT indicates wild-type supplemented with catalase (4 U/ml) and <i>ΔcidC</i> indicates a CidC-deficient mutant of <i>S. aureus</i> SH1000. Data from six experimental replicates. Error bars indicate 95% confidence intervals.</p

Quantification of morphological variants of <i>S. aureus</i> SH1000 arising in planktonic cultures (denoted by ‘P’) and CDS biofilms (denoted by ‘B’).

<p>(A) WVs and (B) LPVs (white and grey bars indicate the proportion of variants present in the adherent and nonadherent phase, respectively). Data are the means of three experimental replicates, and error bars indicate standard error.</p

Mutation frequencies of <i>S. aureus</i> biofilms grown under constant flow.

<p>Mutation frequencies of planktonic and biofilm cultures (grown in the Sorbarod model) of <i>S. aureus</i> SH1000 (<b>A</b>) and <i>S. aureus</i> UAMS-1 (<b>B</b>) determined by rifampicin and mupirocin selection. P indicates planktonic cultures and 96 h and 144 h indicate biofilm (B) incubation time. Biofilm cultures were incubated in the presence (+HP) and absence (−HP) of 4% (v/v) human plasma. Data are based on three experimental replicates. Error bars indicate 95% confidence intervals.</p

The effect of antioxidants on <i>S. aureus</i> biofilm mutability.

<p>Mutation frequencies of <i>S. aureus</i> SH1000 (<b>A</b> & <b>B</b>) and UAMS-1 (<b>C</b> & <b>D</b>) planktonic (P) and biofilm (B) cultures in the presence of antioxidants vanillin (Van) and ascorbic acid (Asc). Mutation frequencies were determined using mupirocin (<b>A</b> & <b>C</b>) and rifampicin (<b>B</b> & <b>D</b>) selection. Data from six experimental replicates. Error bars indicate 95% confidence intervals.</p

Bacterial strains used in this study.

<p>RIF – Rifampicin, MUP – Mupirocin, VAN – Vanillin, ASC – Ascorbic acid. nd – Not determined. NARSA – Network on Antimicrobial Resistance in <i>Staphylococcus aureus</i>.</p

Selected phenotypic properties of <i>S. aureus</i> morphological variants identified in this study.

<p>(A) Assessment of biofilm formation in SH1000 and the variants WV1, WV2, LPV1 and LPV2 (white and grey sectors indicate proportions of adherent and nonadherent cells, respectively), (<b>B</b>) casein proteolysis of milk agar, (<b>C</b>) haemolysis of fresh blood agar, and (<b>D</b>) colony spreading. Error bars indicate standard error for three experimental replicates.</p