Strains and plasmids used in the present study.

<p>Strains and plasmids used in the present study.</p

Phenol soluble modulins are small peptides expressed from three discrete regions of the <i>S. aureus</i> genome.

<p>(A) Phenol soluble modulins (PSMs) are encoded in two operons, the alpha (<i>αPSM1</i>–<i>4</i>) and beta (<i>βPSM1</i>–<i>2</i>) operons, and δ-toxin is encoded within the Agr regulatory RNA, RNAIII (<i>hld</i>). (B) PSMs are small hydrophobic peptides with highly similar amino acid content.</p

Synthetic phenol soluble modulin peptides bind ThT and polymerize into amyloid-like fibers.

<p>(A) Normalized fluorescence intensity of [white circle] 0.1 mg/mL of each PSM peptide or [black circle] 0.05 mg/mL of each PSM peptide in 2 mM ThT. Fluorescence emission was measured at 495 nm after excitation at 438 nm. Assays were repeated in triplicate and all demonstrated a similar trend. (B) 48 hours after mixing 100 µg/mL each of the seven PSM peptides (α1–4, β1–2, and δ-toxin), fibril structures are readily observed by TEM. (C) PSM fibers [black circle] display a ThT fluorescence peak around 482 nm compared to a ThT-only blank [grey circle]. (D) PSM fibers [black circle] produce a characteristic Congo red (CR) absorbance “red-shift” associated with amyloid binding compared to a CR-only blank [grey circle]. (E) Pelleted PSM fibers [grey circle] display a greater β-sheet content than the remaining supernatant [black circle]. Assays were repeated in triplicate and displayed similar trends. Bar indicates 500 nm.</p

Amyloid fiber formation modulates PSM activity.

<p>(A) <i>S. aureus</i> wildtype biofilms were grown in microtiter plates for 24 hours then washed and exposed to increasing concentrations of soluble αPSM1 or αPSM1 fibers at concentrations of 10, 50 or 100 µg/mL for six hours. Biofilms were then washed, stained and remaining biofilm biomass was visualized (images of wells below graph) and quantitated (OD at A₅₉₅). (B & C) TEM micrographs of αPSM1 samples used in the experiment demonstrate the absence (B) and presence (C) of fibers. * P<0.002 compared to control no αPSM1 treatment. We verified that αPSM1 fibers bind CR (D) and ThT (E) similar to amyloid fibers.</p

Mutants unable to produce α and βPSMs fail to form fibers during biofilm growth.

<p>TEM micrographs of <i>S. aureus</i> biofilm cells grown for five days in PNG media. (A) wildtype (strain SH1000), (B) Δ<i>αβpsm</i> (strain BB2388), (C) Δ<i>αβpsm</i> complemented (strain BB2408). (D–F) TEM micrographs of fiber preparations from wildtype (D), Δ<i>αβpsm</i> (E), and Δ<i>αβpsm</i> complemented (F). Bars indicate 500 nm.</p

Growth media influences biofilm disassembly.

<p>Confocal micrographs of <i>S. aureus</i> SH1000 biofilms grown in TSBg media (A) for 30 hours readily disassemble upon exposure to biofilm matrix degrading enzymes proteinase K, dispersin B, and DNaseI at 0.2 µg/mL each. <i>S. aureus</i> biofilms grown in PNG media (B) for 30 hours fail to disassemble upon exposure to matrix-degrading enzymes. Images are representative of three separate experiments and each side of a grid square represents 20 µm. (C) Biofilms at the air-liquid interface of test tube cultures withstand 1% SDS exposure when grown in PNG media but disassemble when grown in TSBg. Top images show stained test tube biofilms; graph below is quantification of biofilm biomass. * P<0.002 compared to no SDS treatment.</p

An <i>αβPSM</i> mutant forms biofilms susceptible to disassembly by matrix degrading enzymes and mechanical stress.

<p>Confocal micrographs of Δ<i>αβpsm</i> mutant (A) (strain BB2388) versus complemented mutant expressing <i>α</i> and <i>βpsm</i> operons <i>in trans</i> (B) (strain BB2408) flow cell biofilms grown for 30 hours prior to proteinase K, dispersin B, and DNaseI exposure (at 0.2 µg/mL each). Images are representative of three separate experiments and each side of a grid square represents 20 µm. (C) Analysis of biofilm development at the air-liquid interface of test tube cultures in PNG media after vortexing. Graph shows quantification of biofilm biomass (OD A₅₉₅) and images below show stained test tube biofilms. * P<0.005 compared to wildtype.</p

<i>S. aureus</i> produces extracellular fibers during biofilm growth in PNG media.

<p>TEM micrographs of cells from <i>S. aureus</i> SH1000 biofilms grown in TSBg medium (A) versus cells from biofilms grown in PNG media (B). High magnification reveals fibers are associated with the cell wall and approximately 12 nm in width (C). An <i>agr</i> mutant does not produce extracellular fibers (D). Bar length indicates 1 µm in A, B, and D, and 250 nm in C.</p

L-Arginine Destabilizes Oral Multi-Species Biofilm Communities Developed in Human Saliva

<div><p>The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37^oC. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm³/μm²) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of <i>Streptococcus</i> and <i>Veillonella</i> species increased and the proportion of Gram-negative bacteria such as <i>Neisseria</i> and <i>Aggregatibacter</i> species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi-species oral biofilm development and community composition and enhances the activity of CPC. The incorporation of LAHCl into oral healthcare products may be useful for enhanced biofilm control.</p></div

Graphical representation of the overall in-host compartmental CDI model within its human host.

<p>Graphical representation of the overall in-host compartmental CDI model within its human host.</p