Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans.

Bacterial pathogens have remained a major public health concern for several decades. This study investigated the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica serovar Typhimurium TISTR 292 and Streptococcus mutans DMST 18777. The potential of Polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans DMST 18777 and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MIC of 0.29 and 0.72 mg/mL for nonfilamentous fungi (NFP) Miang and filamentous-fungi-processed (FFP) Miang respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially, in NFP and FFP Miang. Characterization of the PVPP-treated extracts using High performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, Crystal violet-staining also showed better IC50 values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) against S. mutans DMST 18777 biofilms in vitro. Homology modeling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans DMST 18777 sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP Miang might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products.

Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Evaluating Streptococcus mutans Colonization on 3D-Printed, Milled, and Conventional Acrylic Resin Materials: An In Vitro Study.

Purpose: To compare surface roughness and bacterial colonization of Streptococcus mutans to 3D printed (3DP), milled (M), and conventional (CV) acrylic resin. Methods: Thirty-six discs (n equals 12 per group) were fabricated from 3DP, M, and CV materials. One surface of sample was polished (Po); the opposite surface was left unpolished (UPo). Surface roughness (µm) was assessed using a contact profilometer. The specimens were placed in S. mutans suspension and incubated at 37 degrees Celsius overnight. The attached colonies were separated using a sonicator, and the resulting solution was diluted to 10-3 to assess colony-forming units per milliliter (CFU/ml) after 48 hours. The colonies were categorized into a quantitative S. mutans (QS) index. Data were analyzed using one-way ANOVA, chi-squares, and multivariate analysis of variance analysis with the least significant difference (LSD) post-hoc test (P<0.05). Results: Roughness average (Ra) values of CV were higher than 3DP and M for UPo surfaces (P<0.001; 3DP=0.10; M=0.13; CV=0.26 µm, respectively). For Po and UPo surfaces, the CV harbored more S. mutans colonies than M and 3DP (P<0.001; 3DP=5.2x10 6 ; M=4.7x10 6 ; CV=1.49x10 7 CFU/ml, respectively). M group had the lowest range of QS scores, while CV had the highest range (P<0.001). Conclusions: Digitally manufactured material provides smoother surfaces than the conventional group, resulting in fewer Streptococcus mutans colonies. However, all the material groups must still be adequately polished to prevent the colonization of S. mutans, regardless of the manufacturing methods, as higher S. mutans counts were observed with an increase in surface roughness values.

Catechin/ß-cyclodextrin complex modulates physicochemical properties of pre-gelatinized starch-based orally disintegrating films.

The study aimed to develop pre-gelatinized starch-based orally disintegrating films (ODFs) containing catechin/ß-cyclodextrin (CAT/ß-CD) complex and to evaluate the influence of the complex on the physicochemical properties of the ODFs. SEM images showed that a compacter and more homogeneous ODFs were formed due to interactions between starch matrix and CAT/ß-CD. FTIR spectra demonstrated that the interactions between starches or starch and CAT/ß-CD were enhanced by hydrogen bonds. Thermal stability of ODFs was improved by incorporating CAT/ß-CD, its peak decomposition temperature was enhanced from 310.74 to 321.83 °C. Tensile strength was increased from 11.597 ± 0.153 to 22.172 ± 0.752 MPa, while elongation at break decreased by from 11.233% ± 1.079% to 3.633% ± 0.058%. The prepared ODFs have an acceptable in vitro disintegration time, which were between 9.03 ± 0.79 s and 42.23 ± 1.76 s. Antimicrobial test showed that ODFs incorporating CAT/ß-CD inhibited the growth of S. aureus and S. mutans successfully. The limited release of CAT molecules from the ODFs was also found. In addition, the ODFs have excellent antioxidant capacity. Its antioxidant activity remained at around 70% after 28 days of storage. The study indicated that the combination of ODFs and ß-CD complex have a high potential for the delivery of natural active ingredients.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization.

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 µg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R2 = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes.

Effects of cigarette smoking on the growth of Streptococcus mutans biofilms: An in vitro study.

The increased incidence of dental caries by cigarette smoking (CS) has been widely reported in epidemiological studies, but the relationship between CS and cariogenic biofilm growth has been rarely studied. This study aims to investigate the effects of CS exposure on the growth and virulence of Streptococcus mutans biofilms (S. mutans). Briefly, S. mutans biofilms were formed on saliva-coated hydroxyapatite disks, which were exposed to CS 1, 3, and 6 times per day, respectively. In addition, S. mutans biofilms without CS exposure were considered as the control group. Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides (IPSs) were analyzed and confocal laser scanning microscopy (CLSM) images of 74-h-old S. mutans biofilms were obtained. The lowest accumulation of biofilms and EPSs were detected in the 6 times/day CS exposure group compared with those of the control group and other CS exposure groups in 74-h-old S. mutans biofilms. CLSM also revealed the lowest bacterial count (live and dead cells) and EPSs biovolume in the six times/day CS exposure group in 74-h-old S. mutans biofilms. CS exposure inhibited the growth of S. mutans biofilm in vitro study, the anti-cariogenic biofilm formation was enhanced with a dose (frequency)-dependent at which frequency has more influence in the present findings.

Effects of brief sodium fluoride treatments on the growth of early and mature cariogenic biofilms.

Although fluoride has been widely used as a preventive agent for dental caries, the effects of fluoride on the activities of biofilms in different stages of cariogenic biofilm formation are less studied. This study was designed to investigate the antibiofilm activity of sodium fluoride during the early and mature stages of Streptococcus mutans (S. mutans) biofilm formation. S. mutans biofilms were formed on saliva-coated hydroxyapatite disks. In the early (0-46 h) and mature (46-94 h) biofilm stages, the biofilms were treated with different concentrations of fluoride (250, 500, 1000, 2000 ppm; 5 times in total, 1 min/treatment). Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides were analysed, and confocal laser scanning microscopy images were obtained of the two stages of biofilms to determine antibiofilm activities of fluoride at varying concentrations during the formation of early and mature biofilms. In the early stages of cariogenic biofilm formation, test groups with all fluoride concentrations significantly inhibited the growth of S. mutans biofilms. The antibiofilm and anti-EPS formation activities of the brief fluoride treatments increased with a concentration-dependent pattern. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited biofilm accumulation, activity, and intracellular/extracellular polysaccharide content compared with those of the control and other fluoride treatment groups. The antimicrobial effect of fluoride treatment on the growth of S. mutans biofilms was linked with the stage of cariogenic biofilm formation. The inhibition of S. mutans biofilm growth by fluoride treatment was easier in the early formation stage than in the mature stage. Fluoride treatment in the early stage of cariogenic biofilm formation may be an effective approach to controlling cariogenic biofilm development and preventing dental caries.

The transcription regulator BrsR serves as a network hub of natural competence protein-protein interactions in Streptococcus mutans.

Genome evolution is an essential and stringently regulated aspect of biological fitness. For bacteria, natural competence is one of the principal mechanisms of genome evolution and is frequently subject to multiple layers of regulation derived from a plethora of environmental and physiological stimuli. Here, we present a regulatory mechanism that illustrates how such disparate stimuli can be integrated into the Streptococcus mutans natural competence phenotype. S. mutans possesses an intriguing, but poorly understood ability to coordinately control its independently regulated natural competence and bacteriocin genetic pathways as a means to acquire DNA released from closely related, bacteriocin-susceptible streptococci. Our results reveal how the bacteriocin-specific transcription activator BrsR directly mediates this coordination by serving as an anti-adaptor protein responsible for antagonizing the proteolysis of the inherently unstable, natural competence-specific alternative sigma factor ComX. This BrsR ability functions entirely independent of its transcription regulator function and directly modulates the timing and severity of the natural competence phenotype. Additionally, many of the DNA uptake proteins produced by the competence system were surprisingly found to possess adaptor abilities, which are employed to terminate the BrsR regulatory circuit via negative feedback. BrsR-competence protein heteromeric complexes directly inhibit nascent brsR transcription as well as stimulate the Clp-dependent proteolysis of extant BrsR proteins. This study illustrates how critical genetic regulatory abilities can evolve in a potentially limitless variety of proteins without disrupting their conserved ancestral functions. These unrecognized regulatory abilities are likely fundamental for transducing information through complex genetic networks.

Antibacterial Photodynamic Inactivation of Fagopyrin F from Tartary Buckwheat (Fagopyrum tataricum) Flower against Streptococcus mutans and Its Biofilm.

The objective of this study was to determine reactive oxygen species (ROS) produced by fagopyrin F-rich fraction (FFF) separated from Tartary buckwheat flower extract exposed to lights and to investigate its antibacterial photodynamic inactivation (PDI) against Streptococcus mutans and its biofilm. ROS producing mechanisms involving FFF with light exposure were determined using a spectrophotometer and a fluorometer. S. mutans and its biofilm inactivation after PDI treatment of FFF using blue light (BL; 450 nm) were determined by plate count method and crystal violet assay, respectively. The biofilm destruction by ROS produced from FFF after exposure to BL was visualized using confocal laser scanning microscopy (CLSM) and field emission scanning electron microscope (FE-SEM). BL among 3 light sources produced type 1 ROS the most when applying FFF as a photosensitizer. FFF exposed to BL (5 and 10 J/cm2) significantly more inhibited S. mutans viability and biofilm formation than FFF without the light exposure (p < 0.05). In the PDI of FFF exposed to BL (10 J/cm2), an apparent destruction of S. mutans and its biofilm were observed by the CLSM and FE-SEM. Antibacterial PDI effect of FFF was determined for the first time in this study.

Function-adaptive clustered nanoparticles reverse Streptococcus mutans dental biofilm and maintain microbiota balance.

Dental plaques are biofilms that cause dental caries by demineralization with acidogenic bacteria. These bacteria reside inside a protective sheath which makes any curative treatment challenging. We propose an antibiotic-free strategy to disrupt the biofilm by engineered clustered carbon dot nanoparticles that function in the acidic environment of the biofilms. In vitro and ex vivo studies on the mature biofilms of Streptococcus mutans revealed >90% biofilm inhibition associated with the contact-mediated interaction of nanoparticles with the bacterial membrane, excessive reactive oxygen species generation, and DNA fragmentation. An in vivo examination showed that these nanoparticles could effectively suppress the growth of S. mutans. Importantly, 16S rRNA analysis of the dental microbiota showed that the diversity and richness of bacterial species did not substantially change with nanoparticle treatment. Overall, this study presents a safe and effective approach to decrease the dental biofilm formation without disrupting the ecological balance of the oral cavity.

Oxygen radical antioxidant capacity (ORAC) and antibacterial properties of Melicope glabra bark extracts and isolated compounds.

Melicope glabra (Blume) T. G. Hartley from the Rutaceae family is one of the richest sources of plant secondary metabolites, including coumarins and flavanoids. This study investigates the free radical scavenging and antibacterial activities of M. glabra and its isolated compounds. M. glabra ethyl acetate and methanol extracts were prepared using the cold maceration technique. The isolation of compounds was performed with column chromatography. The free radical scavenging activity of the extracts and isolated compounds were evaluated based on their oxygen radical absorbance capacity (ORAC) activities. The extracts and compounds were also subjected to antibacterial evaluation using bio-autographic and minimal inhibitory concentration (MIC) techniques against two oral pathogens, Enterococcus faecalis and Streptococcus mutans. Isolation of phytoconstituents from ethyl acetate extract successfully yielded quercetin 3, 5, 3'-trimethyl ether (1) and kumatakenin (2), while the isolation of the methanol extract resulted in scoparone (3), 6, 7, 8-trimethoxycoumarin (4), marmesin (5), glabranin (6), umbelliferone (7), scopoletin (8), and sesamin (9). The study is the first to isolate compound (1) from Rutaceae plants, and also the first to report the isolation of compounds (2-5) from M. glabra. The ORAC evaluation showed that the methanol extract is stronger than the ethyl acetate extract, while umbelliferone (7) exhibited the highest ORAC value of 24 965 µmolTE/g followed by glabranin (6), sesamin (9) and scopoletin (8). Ethyl acetate extract showed stronger antibacterial activity towards E. faecalis and S. mutans than the methanol extract with MIC values of 4166.7 ± 1443.4 µg/ml and 8303.3 ± 360.8 µg/ml respectively. Ethyl acetate extract inhibited E. faecalis growth, as shown by the lowest optical density value of 0.046 at a concentration of 5.0 mg/mL with a percentage inhibition of 95%. Among the isolated compounds tested, umbelliferone (7) and sesamin (9) exhibited promising antibacterial activity against S. mutans with both exhibiting MIC values of 208.3 ± 90.6 µg/ml. Findings from this study suggests M. glabra as a natural source of potent antioxidant and antibacterial agents.

Mutanofactin promotes adhesion and biofilm formation of cariogenic Streptococcus mutans.

Cariogenic Streptococcus mutans is known as a predominant etiological agent of dental caries due to its exceptional capacity to form biofilms. From strains of S. mutans isolated from dental plaque, we discovered, in the present study, a polyketide/nonribosomal peptide biosynthetic gene cluster, muf, which directly correlates with a strong biofilm-forming capability. We then identified the muf-associated bioactive product, mutanofactin-697, which contains a new molecular scaffold, along with its biosynthetic logic. Further mode-of-action studies revealed that mutanofactin-697 binds to S. mutans cells and also extracellular DNA, increases bacterial hydrophobicity, and promotes bacterial adhesion and subsequent biofilm formation. Our findings provided an example of a microbial secondary metabolite promoting biofilm formation via a physicochemical approach, highlighting the importance of secondary metabolism in mediating critical processes related to the development of dental caries.

A concerted probiotic activity to inhibit periodontitis-associated bacteria.

Periodontitis can result in tooth loss and the associated chronic inflammation can provoke several severe systemic health risks. Adjunctive to mechanical treatment of periodontitis and as alternatives to antibiotics, the use of probiotic bacteria was suggested. In this study, the inhibitory effect of the probiotic Streptococcus salivarius subsp. salivarius strains M18 and K12, Streptococcus oralis subsp. dentisani 7746, and Lactobacillus reuteri ATCC PTA 5289 on anaerobic periodontal bacteria and Aggregatibacter actinomycetemcomitans was tested. Rarely included in other studies, we also quantified the inverse effect of pathogens on probiotic growth. Probiotics and periodontal pathogens were co-incubated anaerobically in a mixture of autoclaved saliva and brain heart infusion broth. The resulting genome numbers of the pathogens and of the probiotics were measured by quantitative real-time PCR. Mixtures of the streptococcal probiotics were also used to determine their synergistic, additive, or antagonistic effects. The overall best inhibitor of the periodontal pathogens was L. reuteri ATCC PTA 5289, but the effect is coenzyme B12-, anaerobiosis-, as well as glycerol-dependent, and further modulated by L. reuteri strain DSM 17938. Notably, in absence of glycerol, the pathogen-inhibitory effect could even turn into a growth spurt. Among the streptococci tested, S. salivarius M18 had the most constant inhibitory potential against all pathogens, followed by K12 and S. dentisani 7746, with the latter still having significant inhibitory effects on P. intermedia and A. actinomycetemcomitans. Overall, mixtures of the streptococcal probiotics did inhibit the growth of the pathogens equally or-in the case of A. actinomycetemcomitans- better than the individual strains. P. gingivalis and F. nucleatum were best inhibited by pure cultures of S. salivarius K12 or S. salivarius M18, respectively. Testing inverse effects, the growth of S. salivarius M18 was enhanced when incubated with the periodontal pathogens minus/plus other probiotics. In contrast, S. oralis subsp. dentisani 7746 was not much influenced by the pathogens. Instead, it was significantly inhibited by the presence of other streptococcal probiotics. In conclusion, despite some natural limits such as persistence, the full potential for probiotic treatment is by far not utilized yet. Especially, further exploring concerted activity by combining synergistic strains, together with the application of oral prebiotics and essential supplements and conditions, is mandatory.

Understanding the Basis of METH Mouth Using a Rodent Model of Methamphetamine Injection, Sugar Consumption, and Streptococcus mutans Infection.

"METH mouth" is a common consequence of chronic methamphetamine (METH) use, resulting in tooth decay and painful oral tissue inflammation that can progress to complete tooth loss. METH reduces the amount of saliva in the mouth, promoting bacterial growth, tooth decay, and oral tissue damage. This oral condition is worsened by METH users' compulsive behavior, including high rates of consumption of sugary drinks, recurrent tooth grinding, and a lack of frequent oral hygiene. Streptococcus mutans is a Gram-positive bacterium found in the oral cavity and associated with caries in humans. Hence, we developed a murine model of METH administration, sugar intake, and S. mutans infection to mimic METH mouth in humans and to investigate the impact of this drug on tooth colonization. We demonstrated that the combination of METH and sucrose stimulates S. mutans tooth adhesion, growth, and biofilm formation in vivo METH and sucrose increased the expression of S. mutans glycosyltransferases and lactic acid production. Moreover, METH contributes to the low environmental pH and S. mutans sucrose metabolism, providing a plausible mechanism for bacterium-mediated tooth decay. Daily oral rinse treatment with chlorhexidine significantly reduces tooth colonization in METH- and sucrose-treated mice. Furthermore, human saliva inhibits S. mutans colonization and biofilm formation after exposure to either sucrose or the combination of METH and sucrose. These findings suggest that METH might increase the risk of microbial dental disease in users, information that may help in the development of effective public health strategies to deal with this scourge in our society.IMPORTANCE "METH mouth" is characterized by severe tooth decay and gum disease, which often causes teeth to break or fall out. METH users are also prone to colonization by cariogenic bacteria such as Streptococcus mutans In addition, this oral condition is aggravated by METH users' compulsive behavior, including the consumption of beverages with high sugar content, recurrent tooth grinding, and a lack of frequent oral hygiene. We investigated the effects of METH and sugar consumption on S. mutans biofilm formation and tooth colonization. Using a murine model of METH administration, sucrose ingestion, and oral infection, we found that the combination of METH and sucrose increases S. mutans adhesion and biofilm formation on the teeth of C57BL/6 mice. However, daily chlorhexidine-based oral rinse treatment reduces S. mutans tooth colonization. Similarly, METH has been associated with dry mouth or hyposalivation in users. Hence, we assessed the impact of human saliva on biofilm formation and demonstrated that surface preconditioning with saliva substantially reduces S. mutans biofilm formation. Our results are significant because to our knowledge, this is the first basic science study focused on elucidating the fundamentals of METH mouth using a rodent model of prolonged drug injection and S. mutans oral infection. Our findings may have important translational implications for the development of treatments for the management of METH mouth and more effective preventive public health strategies that can be applied to provide effective dental care for METH users in prisons, drug treatment centers, and health clinics.

Bacterial Adhesion of Streptococcus mutans to Dental Material Surfaces.

The aim of this study was to investigate and understand bacterial adhesion to different dental material surfaces like amalgam, Chromasit, an Co-Cr alloy, an IPS InLine ceramic, yttrium stabilized tetragonal polycrystalline zirconia (TPZ), a resin-based composite, an Au-Pt alloy, and a tooth. For all materials, the surface roughness was assessed by profilometry, the surface hydrophobicity was determined by tensiometry, and the zeta potential was measured by electrokinetic phenomena. The arithmetic average roughness was the lowest for the TPZ ceramic (Ra = 0.23 µm ± 0.02 µm), while the highest value was observed for the Au-Pt alloy (Ra = 0.356 µm ± 0.075 µm). The hydrophobicity was the lowest on the TPZ ceramic and the highest on the Co-Cr alloy. All measured streaming potentials were negative. The most important cause of tooth caries is the bacterium Streptococcus mutans, which was chosen for this study. The bacterial adhesion to all material surfaces was determined by scanning electron microscopy. We showed that the lowest bacterial extent was on the amalgam, whereas the greatest extent was on tooth surfaces. In general, measurements showed that surface properties like roughness, hydrophobicity and charge have a significant influence on bacterial adhesion extent. Therefore, dental material development should focus on improving surface characteristics to reduce the risk of secondary caries.

The impact of medical cannabis consumption on the oral flora and saliva.

OBJECTIVE: To evaluate the effect of medical cannabis consumption on oral flora and saliva. DESIGN: A clinical prospective study, at the rheumatology clinic of the Nazareth Hospital in Nazareth, recruiting consecutively patients approved for medical cannabis, evaluating their saliva flow, pH and microbial load of Streptococcus mutans and Lactobacillus, prior to and under medical cannabis treatment. METHODS: Patients recently licensed for medical cannabis treatment, were recruited just prior to starting medical cannabis consumption (week 0), 1 and 4 weeks later, patients provided 5-minute time saliva samples, which were measured for their volume and pH, and cultured on a special microbial kit, evaluating the growth of Streptococcus mutans and Lactobacillus. RESULTS: Out of 16 patients enrolled, 14 were female and had fibromyalgia. The mean age of the patients was 52.8±12.9 years. The mean saliva flow at week 0, week 1 and week 4 were 5.38±3.36 ml/5-minutes, 6 (p = 0.769) and 5.45 (p = 0.391), respectively, and for saliva pH were 6.28, 5.94 (p = 0.51) and 5.5 (p = 0.07) respectively also. The mean Streptococcus mutans growth score at weeks 0, 1 and 4 was1.8±0.75, 1.6±0.83 (p = 0.234), and 2.4±0.84 (p = 0.058), respectively. The mean Lactobacilli growth score at weeks 0, 1 and 4 was 2.59±0.88, 3.1±0.69 (p = 0.033) and 3.3±0.67 (p = 0.025), respectively. CONCLUSIONS: The results of this study show that medical cannabis consumption has no significant effect on saliva volume or pH, but it may be associated with changes in salivary levels of oral microbes such as Streptococcus mutans and Lactobacilli.

PspC domain-containing protein (PCP) determines Streptococcus mutans biofilm formation through bacterial extracellular DNA release and platelet adhesion in experimental endocarditis.

Bacterial extracellular DNA (eDNA) and activated platelets have been found to contribute to biofilm formation by Streptococcus mutans on injured heart valves to induce infective endocarditis (IE), yet the bacterial component directly responsible for biofilm formation or platelet adhesion remains unclear. Using in vivo survival assays coupled with microarray analysis, the present study identified a LiaR-regulated PspC domain-containing protein (PCP) in S. mutans that mediates bacterial biofilm formation in vivo. Reverse transcriptase- and chromatin immunoprecipitation-polymerase chain reaction assays confirmed the regulation of pcp by LiaR, while PCP is well-preserved among streptococcal pathogens. Deficiency of pcp reduced in vitro and in vivo biofilm formation and released the eDNA inside bacteria floe along with reduced bacterial platelet adhesion capacity in a fibrinogen-dependent manner. Therefore, LiaR-regulated PCP alone could determine release of bacterial eDNA and binding to platelets, thus contributing to biofilm formation in S. mutans-induced IE.

Development of an enzyme-enhancer system to improve laccase biological activities.

The present investigation reports an in-vitro study using combination of laccase and an enhancer capable of inhibiting the growth of pathogenic microorganisms, preventing biofilm formation, and whitening teeth. Laccase-cinnamic acid system remarkably inhibited the growth of Aggregatibacter actinomycetemcomitans, Candida albicans, S. aureus, and Streptococcus mutans whilst showed no significant effects on Gram-negative bacteria. Data presented that cinnamic acid (10 mM) with laccase (0.125 U ml-1) led to a maximum decrease of about 90%, in S. mutans biofilm formation. The confocal laser scanning microscopy showed considerable detachment of S. mutans cells from glass substratum. The combined laccase-cinnamic acid system could remove teeth discoloration caused by coffee. SEM of the teeth surface exhibited no damages such as surface cracking or fracture. Liquid chromatography-tandem mass spectrometry (LC-MS) and cyclic voltammetry (CV) studies showed that laccase can catalyze the one-electron oxidation of cinnamic acid to the respective radical. This radical can then undergo several fates, including recombination with another radical to form a dimeric species, dismutation of the radical back to cinnamic acid or decarboxylation to give various reduced oxygen species. Therefore, the redox potential values of phenolic monomers/oligomers are related with their biological activities.

Design, synthesis and evaluation of carbazole derivatives as potential antimicrobial agents.

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5-2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure-activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.

Repression of the TreR transcriptional regulator in Streptococcus mutans by the global regulator, CcpA.

Streptococcus mutans, the etiologic agent of dental caries in humans, is considered a dominating force in the oral microbiome due to its highly-evolved propensity for survival. The oral pathogen encodes an elaborate array of regulatory elements, including the carbon catabolite-responsive regulator, CcpA, a global regulator key in the control of sugar metabolism and in stress tolerance response mechanisms. The recently characterized trehalose utilization operon, integral for the catabolism of the disaccharide trehalose, is controlled by a local regulator, TreR, which has been implicated in a number of cellular functions outside of trehalose catabolism. Electrophoretic mobility shift assays demonstrated that CcpA bound a putative cre site in the treR promoter. Loss of ccpA resulted in elevated expression of treR in cultures of the organism grown in glucose or trehalose, indicating that CcpA not only acts as a repressor of trehalose catabolism genes, but also the local regulator. The loss of both CcpA and TreR in S. mutans resulted in an impaired growth rate and fitness response, supporting the hypothesis that these regulators are involved in carbon catabolism control and in induction of components of the organism's stress response.