L-arginine destabilizes oral multi-species biofilm communities developed in human saliva.

Ethan Kolderman,Alexander H Rickard,Nicholas S Jakubovics,Betsy Foxman,Derek Samarian,Deepti Bettampadi,Scot E Dowd,Ali Al-Ahmad

doi:10.1371/journal.pone.0121835

Ethan Kolderman, Alexander H Rickard + Show 6 more

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https://doi.org/10.1371/journal.pone.0121835

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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.

Highlights

Dental plaque biofilms are surface-associated microbial communities that are bathed in flowing saliva and typically contain tens to hundreds of bacterial species [1]
Microcosm biofilms were developed in a static glass bottom microplate biofilm system that was inoculated with cell-containing saliva (CCS) and used cell-free saliva (CFS) with increasing concentrations of L-arginine monohydrochloride (LAHCl) (50 μM–500 mM) as the growth medium
Visual inspection of the biofilms developed in 50 mM and 500 mM LAHCl suggested that the red signal was likely due to non-responsive/dead cells that are typically seen in multi-species biofilms but their proportion was increased, potentially due to the change in biofilm architecture and biomass (Fig 1E and 1F versus 1A–1D)

Summary

Introduction

Dental plaque biofilms are surface-associated microbial communities that are bathed in flowing saliva and typically contain tens to hundreds of bacterial species [1]. Depending upon the location (supragingival versus subgingival), the biomass (number of bacteria), the species composition (types and relative abundance), and spatial arrangement of the constituent species (in three dimensions), dental plaque biofilms can cause caries or periodontal disease [2,5,6]. Dental plaque biofilm communities are extremely recalcitrant to external chemical and physical perturbations. They are up to 1,000 times less susceptible to antimicrobials compared to their planktonic counterparts, and are typically resistant to abrasive treatments [7,8]. Difficulties in treating oral biofilm communities, especially those causing dental caries and periodontal disease, imparts a considerable health burden in the US: approximately 500 million visits to dental offices and an estimated cost of $108 billion per annum to treat or prevent oral disease [9]

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