Russell P. Pesavento

Dental caries is a multifactorial process attributed to a diet high in fermentable carbohydrates, tooth-adherent plaque comprising acidogenic microbes (Streptococcus mutans), and a host of associated risk factors. We previously reported a nanohybrid aggregate formulation comprising nanoceria (CeO₂-NP) and chondroitin sulfate A (CSA) that limited sucrose-dependent biofilm formation of S. mutans via the induction of nonadherent cell clusters. Herein, we report the chemical and bioactivity analysis of CeO₂-NP-CSA and a related derivative (CeO₂-NP-CSA-B) in a series of preclinical studies culminating in both in vivo efficacy and toxicity studies. Time-dependent sedimentation assays and dynamic light scattering studies in whole human saliva model systems demonstrate changes to aggregate size/zeta potential (activation) of both CeO₂-NP-CSA and CeO₂-NP-CSA-B that correlate with the rapid clearance of S. mutans. Further, CeO₂-NP-CSA-B was found to significantly inhibit tooth-retained rodent plaque and dental caries, similar to an over-the-counter anticaries mouth rinse. Human cell toxicity and maximum tolerated dose studies provided no significant evidence of toxicity of CeO₂-NP-CSA-B as quantified in a series of well-known clinical chemistry and hematological assays. This study summarizes the chemical and biological data supporting the potential of nanohybrid aggregate formulation(s) as future caries prevention agents.

Natural products are ubiquitously produced by many species that we encounter during our daily lives. One genus, Streptococcus , can produce a wide array of quorum sensing linked natural products known as RaS-RiPPs (ribosomally synthesized and post-translationally modified peptides). Their production is triggered by the induction of an Rgg-SHP quorum sensing system, which senses the presence of SHPs (short hydrophobic peptides) and induces the gene expression of these operons. Previous work has found that streptococcal RaS-RiPPs modulate the growth of different streptococci and might play a role in antibiotic tolerance. This is of particular importance to the oral microbiome, where streptococci are a predominant genus. This study examines the impact of the RaS-RiPP Tryglysin A on ex-vivo oral systems and provides the groundwork for determining how producer streptococci might impact the composition and function of oral microbiome communities. We additionally explore important factors to consider when culturing ex-vivo oral systems.

Streptococcus mutans is one of the key etiological factors in tooth-borne biofilm development that leads to dental caries in the presence of fermentable sugars. We previously reported on the ability of acid-stabilized nanoceria (CeO₂-NP) produced by the hydrolysis of ceric salts to limit biofilm adherence of S. mutans via non-bactericidal mechanism(s). Herein, we report a chondroitin sulfate A (CSA) formulation (CeO₂-NP-CSA) comprising nanoceria aggregates that promotes resistance to bulk precipitation under a range of conditions with retention of the biofilm-inhibiting activity, allowing for a more thorough mechanistic study of its bioactivity. The principal mechanism of reduced in vitro biofilm adherence of S. mutans by CeO₂-NP-CSA is the production of nonadherent cell clusters. Additionally, dose-dependent in vitro human cell toxicity studies demonstrated no additional toxicity beyond that of equimolar doses of sodium fluoride, currently utilized in many oral health products. This study represents a unique approach and use of a nanoceria aggregate formulation with implications for promoting oral health and dental caries prevention as an adjunctive treatment.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)—an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a K_D value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure–activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.