Nanoceria Aggregate Formulation Promotes Buffer Stability, Cell Clustering, and Reduction of Adherent Biofilm in Streptococcus mutans.

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.

Lactobacilli and human dental caries: more than mechanical retention.

Lactobacilli have been considered as major contributors to human dental caries for over a century. Recent in vitro model studies have shown that when compared to Streptococcus mutans, a keystone pathogen of human dental caries, the ability of lactobacilli to form biofilms is poor, although differences exist between the different major species. Further studies using molecular and bioinformatics approaches provide evidence that multiple mechanisms, including adhesin-receptor mediated physical contact with S. mutans, facilitate the adherence and establishment of lactobacilli on the tooth surface. There is also evidence that under conditions like continuous sugar consumption, weak acids and other antimicrobials such as bacteriocins from lactobacilli can become detrimental to the microbial community, especially those in the proximity. Details on the underlying mechanisms of how different Lactobacillus sp. establish and persist in the highly complex microbiota on the tooth surface await further investigation.

Antimicrobial-free graphene nanocoating decreases fungal yeast-to-hyphal switching and maturation of cross-kingdom biofilms containing clinical and antibiotic-resistant bacteria.

Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA) synergize in cross-kingdom biofilms to increase the risk of mortality and morbidity due to high resistance to immune and antimicrobial defenses. Biomedical devices and implants made with titanium are vulnerable to infections that may demand their surgical removal from the infected sites. Graphene nanocoating (GN) has promising anti-adhesive properties against C. albicans. Thus, we hypothesized that GN could prevent fungal yeast-to-hyphal switching and the development of cross-kingdom biofilms. Herein, titanium (Control) was coated with high-quality GN (coverage > 99%). Thereafter, mixed-species biofilms (C. albicans combined with S. aureus or MRSA) were allowed to develop on GN and Control. There were significant reductions in the number of viable cells, metabolic activity, and biofilm biomass on GN compared with the Control (CFU counting, XTT reduction, and crystal violet assays). Also, biofilms on GN were sparse and fragmented, whereas the Control presented several bacterial cells co-aggregating with intertwined hyphal elements (confocal and scanning electronic microscopy). Finally, GN did not induce hemolysis, an essential characteristic for blood-contacting biomaterials and devices. Thus, GN significantly inhibited the formation and maturation of deadly cross-kingdom biofilms, which can be advantageous to avoid infection and surgical removal of infected devices.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

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 KD 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.

Streptococcus mutans Lacking sufCDSUB Is Viable, but Displays Major Defects in Growth, Stress Tolerance Responses and Biofilm Formation.

Streptococcus mutans appears to possess a sole iron-sulfur (Fe-S) cluster biosynthesis system encoded by the sufCDSUB cluster. This study was designed to examine the role of sufCDSUB in S. mutans physiology. Allelic exchange mutants deficient of the whole sufCDSUB cluster and in individual genes were constructed. Compared to the wild-type, UA159, the sufCDSUB-deficient mutant, Δsuf::kan r , had a significantly reduced growth rate, especially in medium with the absence of isoleucine, leucine or glutamate/glutamine, amino acids that require Fe-S clusters for biosynthesis and when grown with medium adjusted to pH 6.0 and under oxidative and nitrosative stress conditions. Relative to UA159, Δsuf::kan r had major defects in stress tolerance responses with reduced survival rate of > 2-logs following incubation at low pH environment or after hydrogen peroxide challenge. When compared to UA159, Δsuf::kan r tended to form aggregates in broth medium and accumulated significantly less biofilm. As shown by luciferase reporter fusion assays, the expression of sufCDSUB was elevated by > 5.4-fold when the reporter strain was transferred from iron sufficient medium to iron-limiting medium. Oxidative stress induced by methyl viologen increased sufCDSUB expression by > 2-fold, and incubation in a low pH environment led to reduction of sufCDSUB expression by > 7-fold. These results suggest that lacking of SufCDSUB in S. mutans causes major defects in various cellular processes of the deficient mutant, including growth, stress tolerance responses and biofilm formation. In addition, the viability of the deficient mutant also suggests that SUF, the sole Fe-S cluster machinery identified is non-essential in S. mutans, which is not known in any other bacterium lacking the NIF and/or ISC system. However, how the bacterium compensates the Fe-S deficiency and if any novel Fe-S assembly systems exist in this bacterium await further investigation.

Persistent inhibition of Candida albicans biofilm and hyphae growth on titanium by graphene nanocoating.

OBJECTIVES: Candida albicanscolonizes biomaterial surfaces and are highly resistant to therapeutics. Graphene nanocoating on titanium compromises initial biofilm formation. However, its sustained antibiofilm potential is unknown. The objective of this study was to investigate the potential of graphene nanocoating to decrease long-term fungal biofilm development and hyphae growth on titanium. METHODS: Graphene nanocoating was deposited twice (TiGD) or five times (TiGV) on grade 4 titanium with vacuum assisted technique and characterized with Raman spectroscopy and atomic force microscope. The biofilm formation and hyphae growth of C. albicans was monitored for seven days by CFU, XTT, confocal, mean cell density and scanning electronic microscopy (SEM). Uncoated titanium was the Control. All tests had three independent biological samples and were performed in independent triplicates. Data was analyzed with one- or two-way ANOVA and Tukey's HSD (α = 0.05). RESULTS: Both TiGD and TiGV presented less biofilms at all times points compared with Control. The confocal and SEM images revealed few adhered cells on graphene coated samples, absence of hyphae and no features of a mature biofilm architecture. The increase in number of layers of graphene nanocoating did not improve its antibiofilm potential. SIGNIFICANCE: The graphene nanocoating exerted a long-term persistent inhibitory effect on the biofilm formation on titanium. The fewer cells that were able to attach on graphene coated titanium were scattered and unable to form a mature biofilm with hyphae elements. The findings open opportunities to prevent microbial attachment and proliferation on implantable materials without the use of antibiotics.

Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans.

Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4'-phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74-fold (p < .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di-adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p < .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5-fold (p < .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.

Inhibitory effects of xylitol and sorbitol on Streptococcus mutans and Candida albicans biofilms are repressed by the presence of sucrose.

OBJECTIVE: Among the preventive and therapeutic options available for dental caries, sugar alcohols (xylitol and sorbitol) have been widely promoted as oral healthcare products due to its perceived anticariogenic effect. However, the therapeutic efficacy of these sugar alcohols against Streptococcus mutans and Candida albicans in a sucrose supplemented environment, as found in disease-prone conditions in the oral cavity, has not been adequately investigated. METHODS: Single and mixed-species biofilm formation was evaluated in medium with different concentrations of xylitol, sorbitol with or without sucrose supplementation. Biofilm quantification methods such as crystal violet assay, XTT assay, CFU counting complemented with confocal and electron microscopic techniques were used. RESULTS: Under sucrose-free conditions, xylitol and sorbitol demonstrated a significant dose-dependent inhibitory effect on S. mutans biofilms, whereas inhibitory effect on C. albicans biofilm was weak. The presence of 1 % sucrose in the environment diminished the inhibitory effect of both xylitol and sorbitol on S. mutans and C. albicans mono-species biofilms. Sucrose supplementation on pre-formed S. mutans biofilms also reduced the inhibitory effect of xylitol. Xylitol and sorbitol presence reduced mixed-species biofilm formation and altered the biofilm architecture and glucan production. However, sucrose supplementation reduced the inhibitory effect of sugar alcohols and enhanced the mixed-species biofilm formation. CONCLUSIONS: Xylitol and sorbitol exerts an inhibitory effect on S. mutans and C. albicans biofilm formation and this inhibitory effect is repressed by the presence of sucrose.

Lactobacillus Plantarum 108 Inhibits Streptococcus mutans and Candida albicans Mixed-Species Biofilm Formation.

Streptococcus mutans is the principal biofilm forming oral pathogen associated with dental caries. Studies have shown that Candida albicans, a commensal oral fungus is capable of forming pathogenic mixed-species biofilms with S. mutans. The treatment of bacterial and fungal infections using conventional antimicrobial agents has become challenging due to the antimicrobial resistance of the biofilm mode of growth. The present study aimed to evaluate the efficacy of secretory components of Lactobacillus plantarum 108, a potentially promising probiotic strain, against S. mutans and C. albicans single and mixed-species biofilms. L. plantarum 108 supernatant inhibited S. mutans and C. albicans single-species biofilms as shown by XTT reduction assay, crystal violet assay, and colony forming units counting. The probiotic supernatant significantly inhibited the S. mutans and C. albicans mixed-species biofilm formation. The pre-formed mixed-species biofilms were also successfully reduced. Confocal microscopy showed poorly developed biofilm architecture in the probiotic supernatant treated biofilms. Moreover, the expression of S. mutans genes associated with glucosyltransferase activity and C. albicans hyphal specific genes (HWP1, ALS1 and ALS3) were down-regulated in the presence of the probiotic supernatant. Altogether, the data demonstrated the capacity of L. plantarum 108 supernatant to inhibit the S. mutans and C. albicans mixed-species biofilms. Herein, we provide a new insight on the potential of probiotic-based strategies to prevent bacterial-fungal mixed-species biofilms associated with dental caries.

Hydrophobicity of graphene as a driving force for inhibiting biofilm formation of pathogenic bacteria and fungi.

OBJECTIVE: To evaluate the surface and wettability characteristics and the microbial biofilm interaction of graphene coating on titanium. METHODS: Graphene was deposited on titanium (Control) via a liquid-free technique. The transfer was performed once (TiGS), repeated two (TiGD) and five times (TiGV) and characterized by AFM (n=10), Raman spectroscopy (n=10), contact angle and SFE (n=5). Biofilm formation (n=3) to Streptococcus mutans, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans was evaluated after 24h by CV assay, CFU, XTT and confocal microscopy. Statistics were performed by one-way Anova, Tukey's tests and Pearson's correlation analysis at a pre-set significance level of 5 %. RESULTS: Raman mappings revealed coverage yield of 82 % for TiGS and ≥99 % for TiGD and TiGV. Both TiGD and TiGV presented FWHM>44cm-1 and ID/IG ratio<0.12, indicating multiple graphene layers and occlusion of defects. The contact angle was significantly higher for TiGD and TiGV (110° and 117°) comparing to the Control (70°). The SFE was lower for TiGD (13.8mN/m) and TiGV (12.1mN/m) comparing to Control (38.3mN/m). TiGD was selected for biofilm assays and exhibited significant reduction in biofilm formation for all microorganisms compared to Control. There were statistical correlations between the high contact angle and low SFE of TiGD and decreased biofilm formation. SIGNIFICANCE: TiGD presented high quality and coverage and decreased biofilm formation for all species. The increased hydrophobicity of graphene films was correlated with the decreased biofilm formation for various species.

Graphene onto medical grade titanium: an atom-thick multimodal coating that promotes osteoblast maturation and inhibits biofilm formation from distinct species.

The time needed for the osseointegration of titanium implants is deemed too long. Moreover, the bacterial colonization of their surfaces is a major cause of failure. Graphene can overcome these issues but its wet transfer onto substrates employs hazardous chemicals limiting the clinical applications. Alternatively, dry transfer technique has been developed, but the biological properties of this technique remain unexplored. Here, a dry transfer technique based on a hot-pressing method allowed to coat titanium substrates with high-quality graphene and coverage area >90% with a single transfer. The graphene-coated titanium is cytocompatible, did not induce cell membrane damage, induced human osteoblast maturation (gene and protein level), and increased the deposition of mineralized matrix compared to titanium alone. Moreover, graphene decreased the formation of biofilms from Streptococcus mutans, Enterococcus faecalis and even from whole saliva on titanium without killing the bacteria. These findings confirm that coating of titanium with graphene via a dry transfer technique is a promising strategy to improve osseointegration and prevent biofilm formation on implants and devices.

Potential Use of Phenolic Acids as Anti-Candida Agents: A Review.

There has been a sharp rise in the occurrence of Candida infections and associated mortality over the last few years, due to the growing body of immunocompromised population. Limited number of currently available antifungal agents, undesirable side effects and toxicity, as well as emergence of resistant strains pose a considerable clinical challenge for the treatment of candidiasis. Therefore, molecules that derived from natural sources exhibiting considerable antifungal properties are a promising source for the development of novel anti-candidal therapy. Phenolic compounds isolated from natural sources possess antifungal properties of interest. Particularly, phenolic acids have shown promising in vitro and in vivo activity against Candida species. However, studies on their mechanism of action alone or in synergism with known antifungals are still scarce. This review attempts to discuss the potential use, proposed mechanisms of action and limitations of the phenolic acids in anti-candidal therapy.