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

Differences in architecture of oral biofilms grown a static biofilm system containing different concentrations of L-arginine monohydrochloride (LAHCl).

<p>Images show representative 3D renderings of 22 h-old oral biofilms grown from a cell-containing saliva (CCS) inoculum in the static biofilm system containing cell free saliva (CFS) supplemented with different concentrations of LAHCl. Green signal (Syto 9) indicates viable cells and red signal (propidium iodide) indicates damaged/dead cells. Upper renderings (A₁–F₁) are of the x–y plane. Middle renderings (A₂–F₂) are of the x–z plane. Lower renderings (A₃–F₃) represent an angled view (x–y–z). Bars represent 50 μm. The associated table shows changes in percentage of cell viability with means presented in bold and standard deviations shown in parentheses (each derived from at least 27 images from nine biological replicates). *P<0.05; **P<0.01: significant differences from the CFS control.</p

Effects of flowing saliva supplemented with different LAHCl concentrations on the development of oral biofilms for 20 h in the Bioflux system.

<p>The graph shows differences in biofilm biovolume and representative CLSM 3D renderings are included to highlight differences in biofilm architecture. Light blue colored star symbols embedded in the graph indicate a significant increase in average biofilm biovolume over CFS control and orange colored star symbols embedded in the graph indicate a significant decrease in average biofilm biovolume over CFS control. For the rendered biofilms within the embedded images, green colored cells indicate viable cells and red colored cells indicate damaged/dead cells. Bars represent 20 μm. The associated table shows, in addition to the biovolumes highlighted in the graph, changes in average biofilm thickness, average biofilm roughness, and cell viability. For data presented in the associated table, means are shown in bold and standard deviations are shown in parentheses (each derived from at least 18 images from six biological replicates). *P<0.05; **P<0.01; ***P<0.001: significant differences from the CFS control.</p

LAHCl affects preformed biofilms and enhances antimicrobial effectiveness.

<p>Demonstration that a 60 s exposure of oral multi-species biofilms developed in flowing cell free saliva (CFS) to solutions supplemented with LAHCl reduces biofilm biovolume and enhances antimicrobial efficacy of cetylpyridinium chloride (CPC). Representative biofilm renderings show 20 h oral biofilms developed from pooled CCS in flowing CFS in the Bioflux microfluidic system and subsequently exposed for 60 s to 0.05% or 0.01% CPC with or without 500 mM LAHCl. Green signal (Syto 9) shows viable cells, red signal (propidium iodide) shows damaged/dead cells. Upper images (A₁–F₁) are of the x–y plane. Middle images (A₂–F₂) are of the x–z plane. Lower images (A₃–F₃) are an angled view of each plane (x–y–z). Bars represent 20 μm. Associated table shows changes in cell viability, biofilm biovolume, thickness, and roughness. For data presented in the associated table, means are presented in bold and standard deviations are shown in parentheses (each derived from at least 9 images from three biological replicates). *P<0.05, **P<0.01: significant differences from the H₂O control; ▪P<0.05, ▪▪P<0.01: significant differences from 0.05% CPC treatment; †P<0.05, ††P<0.01: significant differences from 0.01% CPC treatment.</p

A model showing the proposed biofilm destabilizing effects of short-term exposure (transient; minutes) and longer-term exposure (sustained; hours) to high millimolar (≥100mM) LAHCl concentrations in flowing saliva.

<p>The model shows the effect of LAHCl on a well-developed multi-species biofilm (with respect to architecture and species composition) although similar destabilizing effects would occur on multi-species biofilms of younger or older developmental age. Cell shapes and sizes are not to scale. Postulated effects are based on data presented in this manuscript and on previous observations of the effects of L-arginine on bacterial cells and cell-cell interactions, as discussed in the body of the text.</p

Sustained exposure to LAHCl alters biofilm community composition.

<p>Oral biofilms developed in the Bioflux system containing flowing CFS supplemented with 500 mM LAHCl display an altered biofilm community composition compared to those developed in non-supplemented CFS. Data derived from bacterial tag-encoded FLX amplicon pyrosequencing of oral biofilms grown for 20 h in three Bioflux microfluidic channels exposed to flowing CFS (control) and three Bioflux microfluidic channels exposed to flowing CFS supplemented with 500 mM LAHCl. (<b>A</b>) Comparison of OTU (97% identity) richness derived from rarefaction curves for biofilms developed in non-supplemented CFS (black square symbols) and biofilms developed in CFS supplemented with 500 mM LAHCl (grey square symbols). (<b>B</b>) Comparison of differences in abundance of phyla. Black bars represent data derived from the analysis of biofilms developed in flowing non-supplemented CFS while the grey bars represent data derived from the analysis of biofilms developed in CFS supplemented with 500 mM LAHCl. (<b>C</b>) Comparisons of differences in abundances of genera whereby abundances are normalized to biofilm biovolume (calculated from data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121835#pone.0121835.g002" target="_blank">Fig 2</a>). Note color coding (from left to right in the key) follows the order from most abundant in the CFS control. Far right color-coded bar is a magnified view of the community composition of the biofilm developed in CFS supplemented with 500 mM LAHCl. Supporting data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121835#pone.0121835.s004" target="_blank">S1 Table</a>.</p