Chemical composition, antifungal, antibiofilm, and molecular docking studies of Syzygium dyerianum essential oil.

The current study describes the chemical composition, antifungal, antibiofilm, antibacterial and molecular docking studies of Syzygium dyerianum growing in Malaysia. The essential oil was obtained through hydrodistillation and characterized using gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The antifungal and antibacterial activities were developed using the broth microdilution assay, whereas the effect on the microbial biofilms was determined using a semi-quantitative static biofilm assay. A total of 31 components were identified, which represent 99.5 % of the essential oil. The results revealed that the essential oil consisted mainly of ß-pinene (15.6 %), α-terpineol (13.3 %), α-pinene (11.1 %), caryophyllene oxide (8.8 %), limonene (8.1 %), borneol (6.0 %) and viridiflorol (5.1 %). The results of the microdilution method showed that essential oil exhibited activity against Candida albicans and Streptococcus mutans with minimal inhibitory concentration values of 125 and 250 µg/mL, respectively. Furthermore, essential oil decreased the biofilm of C. albicans and S. mutans by 20.11 ± 0.27 % and 32.10 ± 4.81 % when treated with 250 µg/mL. The best docking energy was observed with viridiflorol (-29.7 kJ/mol). This study highlights that essential oil can potentially be a natural antifungal, antibacterial, and antibiofilm agent that could be applied in the pharmaceutical and food industries.

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.

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.

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.

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.

Multiple biosurfactant production by Aureobasidium pullulans strain YTP6-14 in aqueous and heavy oil layers.

Aureobasidium pullulans YTP6-14 was demonstrated to be an excellent multiple biosurfactant producer utilizing cheap carbon sources available in Thailand, including glycerol and cassava flour hydrolysate. A. pullulans YTP6-14 maximally produced 1.81 g/l biosurfactant in an aqueous layer (BS-AQ) in a medium containing glycerol, and 7.37 or 6.37 g/l biosurfactant in a heavy oil layer (BS-HO) in cassava flour hydrolysate or a glucose containing medium, respectively. Each BS-AQ and BS-HO had critical micelle concentration values of 41.32 mg/l and 13.51 mg/l, and both biosurfactants formed a stable food oil emulsion and reduced the amount of biofilms formed by Streptococcus sobrinus and Streptococcus mutans. BS-AQ and BS-HO were mainly composed of liamocins or exophilins and massoia lactone, respectively.

Recuento de Streptococcus mutans sobre restauraciones realizadas con adhesivo con y sin carga de nanopartículas de óxido de zinc y cobre / Streptococcus mutans count on restorations made with adhesive with and without loading zinc and copper oxide nanoparticles

Objetivo: Determinar las propiedades antimicrobianas de la incorporación de nanopartículas de óxido de zinc y cobre en un adhesivo de grabado y lavado total sobre Streptococcus mutans en pacientes con restauraciones de resina compuesta confeccionadas con adhesivo cargado. Métodos: Estudio experimental, randomizado, la muestra estuvo conformada por 25 pacientes, de ambos sexos, pertenecientes al posgrado de Ortodoncia de la Facultad de Odontología de la Universidad de Chile, en los cuales se confirmó presencia de Streptococcus mutans en saliva. Se confeccionaron restauraciones de resina compuesta oclusales, en premolares superiores con indicación de exodoncia por el tratamiento de ortodoncia, con adhesivo cargado (cuya composición fue 5/0,2 por ciento ZnO y Cu, respectivamente) y control (sin presencia de nanopartículas en su composición), según el listado de aleatorización. Se tomaron muestras microbiológicas en tres tiempos con la técnica de la cubeta (antes, 1 semana y 4 semanas posterior a la confección de las restauraciones). Se obtuvieron, aislaron e identificaron colonias de Streptococcus mutans a partir de las muestras obtenidas. Se usó el test de Mann-Whitney mediante el paquete estadístico SPSS v.21 Resultados: El promedio del recuento de UFC de Streptococcus mutans en el grupo experimental fue mayor posterior a la confección de las restauraciones de resina compuesta. Los resultados de la identificación molecular por PCR demuestran la presencia de Streptococcus mutans en 20 de 25 muestras. Conclusiones: No existen diferencias en el recuento de Streptococcus mutans antes y después de la aplicación del adhesivo sobre las restauraciones de resina compuesta(AU)

Objective: To determine the antimicrobial properties of the incorporation of zinc and copper oxide nanoparticles in an etching and total wash adhesive on Streptococcus mutans in patients with composite resin restorations made with loaded adhesive. Methods: Experimental and randomized trial, the sample were 25 patients, of both sexes, belonging to the FOUCH Orthodontic postgraduate program, in whom the presence of Streptococcus mutans in saliva was confirmed. Occlusal composite resin restorations were made in upper premolars with indication of extraction by orthodontic treatment, with loaded adhesive (whose composition is 5 / 0.2% ZnO and Cu respectively) and control (without the presence of nanoparticles in their composition), according to the scrambling listing. Microbiological samples were taken in three stages with the cuvette technique (before, 1 week and 4 weeks after the restoration was made). Colonies of Streptococcus mutans were obtained, isolated and identified from the samples obtained. The statistical analysis used the SPSS v.21 software, the data was analyzed by Mann Whitney test Results: The average CFU count of Streptococcus mutans in the experimental group (adhesive modified with zinc oxide and copper nanoparticles) was higher after the fabrication of composite resin restorations. The results of molecular identification by PCR demonstrate the presence of Streptococcus mutans in 20 of 25 samples. Conclusions: There are no differences in the count of Streptococcus mutans before and after the application of the adhesive on the composite resin restorations(AU)

Efecto antimicrobiano sobre Streptococcus mutans de sistema adhesivo con nanopartículas de óxido de zinc y cobre / Antimicrobial effect against Streptococcus mutans of an adhesive system with copper and zinc oxide nanoparticles

Objetivo: Determinar las propiedades antimicrobianas de la incorporación de nanopartículas de óxido de zinc y cobre en un adhesivo de grabado y lavado total sobre Streptococcus mutans en pacientes con restauraciones de resina compuesta confeccionadas con adhesivo cargado. Métodos: Este estudio clínico randomizado, participaron 25 pacientes, de ambos sexos, pertenecientes al posgrado de Ortodoncia de la Facultad de Odontología de la Universidad de Chile, en los cuales se confirmó presencia de Streptococcus mutans en saliva. Se confeccionaron restauraciones de resina compuesta oclusales, en premolares superiores con indicación de exodoncia por el tratamiento de ortodoncia, con adhesivo cargado (cuya composición es 5 / 0.2 por ciento ZnO y Cu respectivamente) y control (sin presencia de nanopartículas en su composición), según el listado de aleatorización. Se tomaron muestras microbiológicas en tres tiempos con la Técnica de la cubeta (antes, 1 semana y 4 semanas posterior a la confección de las restauraciones). Se obtuvieron, aislaron e identificaron colonias de Streptococcus mutans a partir de las muestras obtenidas. Los datos fueron analizados por el test de Mann Whittney. Resultados: El promedio del recuento de UFC (unidades formadoras de colonias) de Streptococcus mutans en el grupo experimental fue mayor posterior a la confección de las restauraciones de resina compuesta.Los resultados de la identificación molecular por PCR demuestran la presencia de Streptococcus mutans en 20 de 25 muestras. Conclusiones: No existen diferencias en el recuento de Streptococcus mutans antes y después de la aplicación del adhesivo sobre las restauraciones de resina compuesta(AU)

Objective: Determine the antimicrobial properties of the incorporation of copper and zinc oxide nanoparticles into a total rinse and etch adhesive against Streptococcus mutans in patients with composite resin restorations made with loaded adhesive. Methods: A randomized clinical study was conducted of 25 patients of both sexes from the orthodontics graduate course taught at the Dental School of the University of Chile, in whom the presence of Streptococcus mutans was confirmed in saliva. Occlusal composite resin restorations were performed in upper premolars with exodontia indicated as part of the orthodontic treatment, using loaded adhesive (composition 5 / 0.2 percent ZnO and Cu, respectively) and control (without nanoparticles in its composition), according to the randomization list. Microbiological samples were taken at three moments applying the tray technique (before, 1 week after and 4 weeks after the restorations). Streptococcus mutans were obtained, isolated and identified from the samples taken. Data analysis was based on the Mann-Whitney test. Results: Mean Streptococcus mutans CFU count in the experimental group was higher after the composite resin restorations were made. Results of PCR molecular identification show the presence of Streptococcus mutans in 20 of 25 samples. Conclusions: No differences were found in the Streptococcus mutans count before and after application of the adhesive over the composite resin restorations(AU)

Characterization of the Streptococcus mutans SMU.1703c-SMU.1702c Operon Reveals Its Role in Riboflavin Import and Response to Acid Stress.

Streptococcus mutans utilizes numerous metabolite transporters to obtain essential nutrients in the "feast or famine" environment of the human mouth. S. mutans and most other streptococci are considered auxotrophic for several essential vitamins including riboflavin (vitamin B2), which is used to generate key cofactors and to perform numerous cellular redox reactions. Despite the well-known contributions of this vitamin to central metabolism, little is known about how S. mutans obtains and metabolizes B2 The uncharacterized protein SMU.1703c displays high sequence homology to the riboflavin transporter RibU. Deletion of SMU.1703c hindered S. mutans growth in complex and defined medium in the absence of saturating levels of exogenous riboflavin, whereas deletion of cotranscribed SMU.1702c alone had no apparent effect on growth. Expression of SMU.1703c in a Bacillus subtilis riboflavin auxotroph functionally complemented growth in nonsaturating riboflavin conditions. S. mutans was also able to grow on flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) in an SMU.1703c-dependent manner. Deletion of SMU.1703c and/or SMU.1702c impacted S. mutans acid stress tolerance, as all mutants showed improved growth at pH 5.5 compared to that of the wild type when medium was supplemented with saturating riboflavin. Cooccurrence of SMU.1703c and SMU.1702c, a hypothetical PAP2 family acid phosphatase gene, appears unique to the streptococci and may suggest a connection of SMU.1702c to the acquisition or metabolism of flavins within this genus. Identification of SMU.1703c as a RibU-like riboflavin transporter furthers our understanding of how S. mutans acquires essential micronutrients within the oral cavity and how this pathogen successfully competes within nutrient-starved oral biofilms.IMPORTANCE Dental caries form when acid produced by oral bacteria erodes tooth enamel. This process is driven by the fermentative metabolism of cariogenic bacteria, most notably Streptococcus mutans Nutrient acquisition is key in the competitive oral cavity, and many organisms have evolved various strategies to procure carbon sources or necessary biomolecules. B vitamins, such as riboflavin, which many oral streptococci must scavenge from the oral environment, are necessary for survival within the competitive oral cavity. However, the primary mechanism and proteins involved in this process remain uncharacterized. This study is important because it identifies a key step in S. mutans riboflavin acquisition and cofactor generation, which may enable the development of novel anticaries treatment strategies via selective targeting of metabolite transporters.

Bactericidal activity and recovery effect of hydroxyl radicals generated by ultraviolet irradiation and silver ion application on an infected titanium surface.

This study investigated the bactericidal effect, the underlying mechanisms of treatment, and recovery of biocompatibility of the infected titanium surface using a combination treatment of silver ion application and ultraviolet-A (UV-A) light irradiation. Streptococcus mutans and Aggregatibacter actinomycetemcomitans were used in suspension and as a biofilm on a titanium surface to test for the bactericidal effect. The bactericidal effect of the combination treatment was significantly higher than that of silver ion application or UV-A light irradiation alone. The bactericidal effect of the combination treatment was attributable to hydroxyl radicals, which generated from the bacterial cell wall and whose yield increased with the silver concentration. To assess the biocompatibility, proliferation and calcification of MC3T3E1 cells were evaluated on the treated titanium surface. The treated titanium screws were implanted into rat tibias and the removal torques were measured 28 days post-surgery. The titanium surface that underwent the combination treatment exhibited recovery of biocompatibility by allowing cellular proliferation or calcification at levels observed in the non-infected titanium surfaces. The removal torque 28 days after surgery was also comparable to the control values. This approach is a novel treatment option for peri-implantitis.

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature.

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p < 0.05). Hot steeping significantly reduced bacterial growth (p < 0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.

Genetic Analysis of Mutacin B-Ny266, a Lantibiotic Active against Caries Pathogens.

Bacteriocins are ribosomally synthesized proteinaceous antibacterial peptides. They selectively interfere with the growth of other bacteria. The production and secretion of bacteriocins confer a distinct ecological advantage to the producer in competing against other bacteria that are present in the same ecological niche. Streptococcus mutans, a significant contributor to the development of dental caries, is one of the most prolific producers of bacteriocins, known as mutacins in S. mutans In this study, we characterized the locus encoding mutacin B-Ny266, a lantibiotic with a broad spectrum of activity. The chromosomal locus is composed of six predicted operon structures encoding proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. Mutacin B-Ny266 was purified from semisolid cultures, and two inhibitory peptides, LanA and LanA', were detected. Both peptides were highly modified. Such modifications include dehydration of serine and threonine and the formation of a C-terminal aminovinyl-cysteine (AviCys) ring. While LanA peptide alone is absolutely required for antimicrobial activity, the presence of LanA' enhanced the activity of LanA, suggesting that B-Ny266 may function as a two-peptide lantibiotic. The activation of lanAA' expression is most likely controlled by the conserved two-component system NsrRS, which is activated by LanA peptide but not by LanA'. The chromosomal locus encoding mutacin B-Ny266 was not universally conserved in all sequenced S. mutans genomes. Intriguingly, the genes encoding LanAA' peptides were restricted to the most invasive serotypes of S. mutansIMPORTANCE Although dental caries is largely preventable, it remains the most common and costly infectious disease worldwide. Caries is initiated by the presence of dental plaque biofilm that contains Streptococcus mutans, a species extensively characterized by its role in caries development and formation. S. mutans deploys an arsenal of strategies to establish itself within the oral cavity. One of them is the production of bacteriocins that confer a competitive advantage by targeting and killing closely related competitors. In this work, we found that mutacin B-Ny266 is a potent lantibiotic that is effective at killing a wide array of oral streptococci, including nearly all S. mutans strains tested. Lantibiotics produced by oral bacteria could represent a promising strategy to target caries pathogens embedded in dental plaque biofilm.

Growth and viability of Streptococcus mutans in sucrose with different concentrations of Stevia rebaudiana Bertoni.

OBJECTIVE: To evaluate total absorbance, planktonic growth, biofilm formation, viability, metabolic activity, and pH of Streptococcus mutans UA159 cultures when different dilutions of Stevia rebaudiana Bertoni were applied and to determine the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) of Stevia on S. mutans. MATERIALS AND METHODS: The effects of different dilutions of Stevia (0-400 mg/ml) on S. mutans total growth, planktonic growth, biofilm formation, viability, metabolic activity, and pH during a 72-h growth period were evaluated in this in vitro study. A stock solution was prepared by mixing 10 ml of tryptic soy broth (TSB) supplemented with 1% sucrose (TSBS) and 4 g of Stevia. RESULTS: S. mutans total growth and biofilm formation decreased with reduced concentrations of Stevia. Furthermore, the MIC was 25 mg/ml and the MBIC was 6.25 mg/ml. Complete eradication of S. mutans was not observed with any of the Stevia concentrations. Planktonic growth of S. mutans was not repressed by high concentrations of Stevia and most of the Stevia concentrations generated an increased pH. CONCLUSION: Because Stevia reduces biofilm and acid production, Stevia can be considered a non-cariogenic sweetener. CLINICAL RELEVANCE: This study confirms the anticariogenic effect of Stevia, like it has been previously reported, but more studies on the most effective concentration are needed, and in the present study, the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) was determined in the presence of sucrose. Additionally, this is the first study to evaluate the effect of Stevia on S. mutans metabolic activity.

Regulatory circuits controlling Spx levels in Streptococcus mutans.

Spx is a major regulator of stress responses in Firmicutes. In Streptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation of clpP or simultaneous inactivation of clpC and clpE stabilized the GFP::SpxA2tail fusion protein. Addition of acidic amino acids to the GFP::SpxA2tail chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1tail . Promoter reporter fusions revealed that spxA1 transcription is co-repressed by the metalloregulators PerR and SloR while spxA2 transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement with spxA2 being part of the LiaR regulon, SpxA2 was found to be critical for the growth of S. mutans under envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.

A salivary metabolite signature that reflects gingival host-microbe interactions: instability predicts gingivitis susceptibility.

Several proteins and peptides in saliva were shown to stimulate gingival wound repair, but the role of salivary metabolites in this process remains unexplored. In vitro gingival re-epithelialization kinetics were determined using unstimulated saliva samples from healthy individuals collected during an experimental gingivitis study. Elastic net regression with stability selection identified a specific metabolite signature in a training dataset that was associated with the observed re-epithelialization kinetics and enabled its prediction for all saliva samples obtained in the clinical study. This signature encompassed ten metabolites, including plasmalogens, diacylglycerol and amino acid derivatives, which reflect enhanced host-microbe interactions. This association is in agreement with the positive correlation of the metabolite signature with the individual's gingival bleeding index. Remarkably, intra-individual signature-variation over time was associated with elevated risk for gingivitis development. Unravelling how these metabolites stimulate wound repair could provide novel avenues towards therapeutic approaches in patients with impaired wound healing capacity.

Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance.

The MarR-like transcriptional regulator and two ABC transporters encoded by the rcrRPQ operon in the dental caries pathogen Streptococcus mutans have important regulatory roles related to oxidative stress tolerance, genetic competence and (p)ppGpp metabolism. A unique feature of the rcrRPQ operon, when compared to other bacteria, is the presence of two peptides, designated Pep1 and Pep2, encoded in alternative reading frames at the 3' end of rcrQ. Here, we show that the rcrRPQ operon, including Pep1 and 2, is essential for S. mutans to survive and maintain viability at elevated temperatures. No major changes in the levels of the heat shock proteins DnaK or GroEL that could account for the thermosensitivity of rcrRPQ mutants were observed. By introducing a single amino acid substitution into the comX gene that deletes an internally encoded peptide, XrpA, we found that XrpA is a contributing factor to the thermosensitive phenotype of a ΔrcrR strain. Overexpression of XrpA on a plasmid also caused a significant growth defect at 42 °C. Interestingly, loss of the gene for the RelA/SpoT homologue (RSH) enzyme, relA, restored growth of the ΔrcrR strain at 42 °C. During heat stress and when a stringent response was induced, levels of (p)ppGpp were elevated in the ΔrcrR strain. Deletion of relA in the ΔrcrR strain lowered the basal levels of (p)ppGpp to those observed in wild-type S. mutans. Thus, (p)ppGpp pools are dysregulated in ΔrcrR, which likely leads to aberrant control of transcriptional/translational processes and the thermosensitive phenotype. In summary, the genes and peptides encoded in the rcrRPQ operon are critical for thermotolerance, and in some strains these phenotypes are related to altered (p)ppGpp metabolism and increased production of the XrpA peptide.

Activity of Sodium Lauryl Sulfate, Rhamnolipids, and N-Acetylcysteine Against Biofilms of Five Common Pathogens.

Bacteria in biofilms are more resistant to antibacterial agents than bacteria in planktonic form. Hence, antibacterial agents should be able to eradicate biofilms to ensure the best outcomes. Little is known about how well many antibacterial agents can disrupt biofilms. In this study, we compared sodium lauryl sulfate (SDS), rhamnolipids (RHL), and N-acetylcysteine (NAC) for their ability to eradicate mature biofilms and inhibit new biofilm formation against Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans. SDS and RHL effectively inhibited formation of five bacterial biofilms in a dose-dependent manner, even at concentrations below the minimal inhibitory concentrations (MICs), suggesting that their antibiofilm activities are unrelated to their antibacterial activities. In contrast, NAC at certain concentrations promoted biofilm formation by all bacteria except P. aeruginosa, whereas at supra-MIC concentrations, it inhibited biofilm formation against the four bacteria, suggesting that its antibiofilm activity depends on its antibacterial activity. NAC was ineffective at eradicating mature H. pylori biofilms, and it actually promoted their formation at concentrations >10 mg/mL. Our results suggest that RHL is superior at eradicating biofilms of H. pylori, E. coli, and S. mutans; SDS is more effective against S. aureus biofilms; and NAC is more effective against P. aeruginosa biofilms. Our results may help determine which antibiofilm agents are effective against certain bacterial strains and develop agents effective against specific bacterial threats.

A novel non-cytotoxic synthetic peptide, Pug-1, exhibited an antibiofilm effect on Streptococcus mutans adhesion.

The aim of this study was to evaluate the effects of synthetic peptides on the growth and biofilm formation of Streptococcus mutans and their cytotoxicity on a human keratinocyte cell line. Four pomegranate-derived peptides; Pug-1, Pug-2, Pug-3 and Pug-4 were synthesized. Streptococcus mutans (ATCC 25175 and DMST 41283) were used. The antibacterial and anti-adherence activities of the peptides were evaluated. The effect of the peptides on S. mutans glucosyltransferase (Gtf) activity was measured. The peptides' cytotoxicity was evaluated on a HaCaT cell line using an MTT assay. The bactericidal penetration depth of Pug-1 into the biofilm was investigated using confocal laser scanning microscopy (CLSM). Among the four peptides, Pug-1 (200 µg ml-1 ) exhibited antibacterial activity on S. mutans (DMST 41283). All peptides demonstrated anti-adherence activity. Streptococcus mutans Gtf activity was significantly inhibited by Pug-1. Interestingly, Pug-1 peptide was not cytotoxic to the HaCaT cell line. Pug-1 (100-200 µg ml-1 ) exhibited bactericidal penetration at approximately 50-100% of the biofilm depth. Therefore, Pug-1 might be a candidate of anticariogenic agent. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data highlighted that pomegranate-derived antimicrobial peptides (AMPs) inhibit Streptococcus mutans adhesion, virulence-associated genes and enzymes. This in vitro study supports the hypothesis that pomegranate AMPs would be noncytotoxic to human keratinocytes, with a potent anticariogenic effect via an anti-adherence mode-of-action. The present study provides evidence for further investigation of these AMPs as therapeutic and preventive agents for dental caries.

The influence of nanoporous anodic aluminum oxide on the initial adhesion of Streptococcus mitis and mutans.

The use of nanoscale surface modifications offers a possibility to regulate the bacterial adherence behavior. The aim of this study was to evaluate the influence of nanoporous anodic aluminum oxide of different pore diameters on the bacterial species Streptococcus mitis and Streptococcus mutans. Nanoporous anodic aluminum oxide (AAO) surfaces with an average pore diameter of 15 and 40 nm, polished pure titanium and compact aluminum oxide (alumina) samples as reference material were investigated. S. mitis and mutans were evaluated for initial adhesion and viability after an incubation period of 30 and 120 min. After 30 min a significantly reduced growth of S. mitis and mutans on 15 nm samples compared to specimens with 40 nm pore diameter, alumina and titanium surfaces could be observed (p < .001). Even after 120 min incubation there was a significant difference between the surfaces with 15 nm pore diameter and the remaining samples (p < .001). AAO surfaces with a small pore diameter have an inhibitory effect on the initial adhesion of S. mitis and mutans. The use of such pore dimensions in the area of the implant shoulder represents a possibility to reduce the adhesion behavior of these bacterial species.