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

Abstract

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 37oC. 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 Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter 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.

l -Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms

“…In addition to providing bulk and mechanical stability, the EPS matrix also forms a diffusion-limiting milieu that embeds cariogenic bacteria, forming protective niches against antimicrobials (1,6,55). Recently, it was shown that the antimicrobial efficacy of cetylpyridinium chloride (CPC) against oral biofilms was enhanced when used in combination with arginine in vitro (27). The authors suggested potential alterations in the EPS matrix, which could facilitate CPC penetration (27); our data certainly support this clinically relevant observation.…”

“…These findings suggest the possibility of arginine supplementation as a strategy to disrupt pathogenic biofilms. In vitro studies show that exogenous L-arginine can inhibit the adhesion of S. mutans to saliva-coated surfaces (25), influence single-species S. gordonii biofilm formation (26), and destabilize a saliva-derived biofilm community (27). Arginine treatments also increased arcA gene (involved in arginine metabolism) abundance compared to that in untreated biofilms, indicating a potential role for the enrichment of alkali-producing microflora (28).…”

Effects of Arginine on Streptococcus mutans Growth, Virulence Gene Expression, and Stress Tolerance

“…Real-time PCR showed significant reductions in spaP expression (ϳ1.5-fold), while quantitative real-time PCR (qRT-PCR) and RNA-seq confirmed a significant decline in gtfB mRNA (Ͼ2-fold). The results from studies using mixed-species biofilm models of dental plaque containing S. mutans established that treatment with arginine alters the architecture of the biofilms by interfering with the proper production of EPS (mostly glucans and fructans) (27,30,41). Glucans are the key matrix components enabling S. mutans organisms to adhere to each other and to the tooth surfaces.…”

Biofilm Models of Polymicrobial Infection

“…Many models have been developed in an attempt to simulate the oral cavity [55,[60][61][62]. Innovative strategies have been incorporated, such as using a salivary medium and/or teeth as the substratum, to more closely simulate the in vivo environment.…”