Additional file 1: Figure S1. of Enterococcus hirae biofilm formation on hospital material surfaces and effect of new biocides

In vitro effect of LH IDROXI FAST and LH ENZYCLEAN SPRAY on mature biofilm of S. aureus ATCC 6538. Top; the untreated and treated biofilms were analyzed for the biomass production, after 48 h of incubation at 37 °C on polystyrene surface, through Cristal Violet staining method. The results were expressed as average of OD595 values of three experiments (mean value ± SD). Symbol represents result statistically significant (p ˂ 0.05). Down; representative images of the in vitro mature biofilms at 37 °C on polystyrene surface untreated (a) and treated with LH IDROXI FAST (b) and LH ENZYCLEAN SPRAY (c). Biofilms were cultured for 48 h, stained with live/dead reagents, and visualized with the optical microscope fluorescence. Sessile population in biofilms stained in red (propidium iodide) expresses a compromised membrane integrity (damaged), whereas green stained bacteria (SYTO 9) remained viable. Both biocides reduced significantly S. aureus ATCC 6538 biomasses even exerting a killing effect. Original magnification ×1000. (ZIP 114 kb

Antimicrobial evaluation of selected naturally occurring oxyprenylated secondary metabolites

<p>This study tested the antimicrobial activity of eight selected naturally occurring oxyprenylated secondary metabolites against <i>Staphylococcus aureus</i> ATCC 29213, <i>S. epidermidis</i> ATCC 35984, <i>Escherichia coli</i> ATCC 8739, <i>Pseudomonas aeruginosa</i> ATCC 9027 and <i>Candida albicans</i> ATCC 10231. Results showed a moderate antimicrobial activity. The most active compounds were 3-(4-geranyloxyphenyl)-1-ethanol (<b>4</b>) and 3-(4-isopentenyloxyphenyl)-1-propanol (<b>5</b>) that were tested on mature and in-formation biofilms of all micro-organisms, moreover the cytotoxic activity was evaluated. Except for <i>S. epidermidis</i>, both compounds reduced significantly (<i>p</i> < 0.05) the microbial biofilm formation at 1/2 MIC and 1/4 MIC, in particular, compounds <b>4</b> and <b>5</b> at each concentration, inhibited <i>E. coli</i> biofilm formation to a greater extent, the biofilm formation was never more than 44% in respect to the control, moreover both compounds showed a low cytotoxic effect. Oxyprenylated derivatives may be of great interest for the development of novel antimicrobial therapeutic strategies and the synthesis of semi-synthetic analogues with anti-biofilm efficacy.</p

Effect of Chitlac and Chitlac-nAg thermosets on IL-6 and PGE₂ release in HGFs/<i>Streptococcus mitis</i> co-culture model.

<p>Graph represents the mean concentration (pg/ml) ± SD of three different consistent experiments. <b>U</b>: HGFs; <b>Th</b>: HGFs with Chitlac thermoset; <b>Ag</b>: HGFs with Chitlac-nAg thermoset; <b>MSTh</b>: HGFs with Chitlac thermoset, <i>S. mitis</i> and saliva; <b>MSAg</b>: HGFs with Chitlac-nAg thermoset, <i>S. mitis</i> and saliva; *Th, Ag, MSTh and MSAg vs U IL-6, p = 0.0391, p = 0.0299, p = 0.0325, p = 0.0408; § Th, Ag, MSTh and MSAg vs U PGE₂, p = 0.0300, p = 0.0262, p = 0.0472, p = 0.0362.</p

Effect of Chitlac and Chitlac-nAg thermosets on cell migration in HGFs/<i>Streptococcus mitis</i> co-culture model.

<p>The graph represents the mean fold increase of cell number ± SD of three experiments. <b>U</b>: HGFs; <b>Th</b>: HGFs with Chitlac thermoset; <b>Ag</b>: HGFs with Chitlac-nAg thermoset; <b>MTh</b>: HGFs with Chitlac thermoset and <i>S. mitis</i>; <b>MAg</b>: HGFs with Chitlac-nAg thermoset and <i>S. mitis</i>; <b>MSTh</b>: HGFs with Chitlac thermoset, <i>S. mitis</i> and saliva; <b>MSAg</b>: HGFs with Chitlac-nAg thermoset, <i>S. mitis</i> and saliva; <b>M bottom</b>: <i>S. mitis</i> in the bottom chamber: <b>S bottom</b>: Saliva in the bottom chamber; <b>MS bottom</b>: <i>S. mitis</i> and saliva in the bottom chamber. <b>*</b>MTh, M bottom, S bottom vs U, p = 0.0421, p = 0.0397, p = 0.0041; § MSTh vs MTh, p = 0.0461; £ MS bottom vs M bottom, p = 0.0037; °MS bottom vs S bottom, p = 0.0234.</p

Effect of BisGMA/TEGDMA thermosets on LDH release.

<p><b>A</b>: in HGFs. The graph represents the mean percentage ± SD of three different consistent experiments. <b>*</b>BT 24 h and BT 48 h vs U, p = 0.0392 and p = 0.0019. <b>B</b>: in HGFs/<i>Streptococcus mitis</i> co-culture model. The graph represents the mean percentage ± SD of three different consistent experiments. <b>U</b>: HGFs; <b>BT</b>: HGFs with uncoated thermoset; <b>Th</b>: HGFs with Chitlac thermoset; <b>Ag</b>: HGFs with Chitlac-nAg thermoset; <b>MTh</b>: HGFs with Chitlac thermoset and <i>S. mitis</i>; <b>MAg</b>: HGFs with Chitlac-nAg thermoset and <i>S. mitis</i>; <b>MSTh</b>: HGFs with Chitlac thermoset, <i>S. mitis</i> and saliva; <b>MSAg</b>: HGFs with Chitlac-nAg thermoset, <i>S. mitis</i> and saliva. <b>*</b>Th and Ag 24 h vs U 24 h, p = 0.0335 and p = 0.0018; § Th and Ag 48 h vs U 48 h, p = 0.0257 and p = 0.0181; £ MTh 48 h vs Th 48 h, p = 0.0304; °MSTh 48 h vs MTh 48 h, p = 0.0272.</p

Physicochemical properties of carvacrol codrug 4.

<p>^aValues are means of three experiments, standard deviation is given in parentheses.</p><p>Physicochemical properties of carvacrol codrug 4.</p

Transmission electron microscopy demonstrating the effects of carvacrol codrug 4 on <i>S</i>. <i>aureus</i> ATCC 29213 (B), <i>E</i>. <i>coli</i> ATCC 8739 (D), <i>C</i>. <i>albicans</i> ATCC 10231 (F), and untreated cultures (control), respectively (A, C, and E).

<p>Microorganisms incubated for 3 h in media containing 12.5 mg/mL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120937#pone.0120937.ref045" target="_blank">45</a>] of carvacrol codrug <b>4</b> (B, D, and F). Irregular features of septa in <i>S</i>. <i>aureus</i> ATCC 29213 (B: arrows); numerous electron-dense bubbles protrude from the cell surface (higher magnification b 140000x and c 110000x) in <i>E</i>. <i>coli</i> ATCC 8739 treated (D) and electron-dense granules of the substance internalized into the cytoplasm (d 110000x); integrity of the membrane (a 110000x) in the controls (C); disintegration of membrane in <i>C</i>. <i>albicans</i> ATCC 10231 (F).</p

Synthesis of carvacrol codrugs 1–4 and 9.

<p>Reagents and conditions: a) Ac₂O, AcOH, 4 h, rt for compounds <b>11–14</b>; KOH, Propargyl bromide, in dry MeOH, 1 h, 60°C (reflux) for compound <b>15</b>; b) DCC in DMF/DCM, 1 h, rt, then carvacrol, DMAP, 15 h, rt; c) TIPS, TFA in DCM, 48 h, rt under nitrogen atmosphere.</p

Kinetic data for hydrolysis of carvacrol codrug 4 at 37°C.^a

<p>^aValues are means of three experiments, standard deviation is given in parentheses.</p><p>^bAbbreviations: SGF, simulated gastric fluid; SIF, simulated intestinal fluid.</p><p>Kinetic data for hydrolysis of carvacrol codrug 4 at 37°C.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120937#t004fn001" target="_blank">^a</a></p

PAMPA-GI permeability of carvacrol codrug 4 after 16 h of incubation.

<p>^a pH of both donor and acceptor compartment.</p><p>PAMPA-GI permeability of carvacrol codrug 4 after 16 h of incubation.</p