Role of Hemin and Growth Media on the Autofluorescence of Streptococcus mutans

poster abstractAbstract Carious lesions fluoresce under blue light. The primary cariogenic bacterium Streptococcus mutans has been shown previously to fluoresce within blue light wavelengths. In this study we wanted to determine the role of hemin and various growth media on the fluorescing properties of S. mutans under planktonic (total biomass) and biofilm (biofilm mass) growth conditions. UA159 was grown for 24 h at 37ºC in Tryptic Soy Broth (TSB), Brain Heart Infusion (BHI) and Todd Hewitt broth (THB) with and without hemin in 5% CO2. Biofilm was grown for 24 h in a 96 well sterile microplate in the above described media with and without hemin. A stock solution of Protoporphyrin –IX was prepared and diluted to concentrations ranging from 1.6-3.1×10-4 g/ml. A SpectraMax (M3) was used to determine the fluorescence from UA159. RFU of total biomass and biofilm mass was assessed by exciting at fixed wavelengths of 385 and 405 nm at a spectral band width of 10 nm. Emission spectra at 770 nm were observed with 385 nm and an emission of 800 and 810 nm with 405 nm. ANOVA on the ranks of the measurements was used, with four different factors including wavelength (770, 800 and 810 nm); total biomass or biofilm mass; various growth media (TSB, BHI, THB) and the presence/absence of hemin and interactions among the factors. The analysis allowed each media-hemin combination to have different variances. Hemin decreased the amount of fluorescence; regardless of the levels of the other factors (p≤0.0001). Without hemin, BHI had more fluorescence than THB (p≤0.0003) and TSB (p≤0.0001). However with hemin, THB had more fluorescence than BHI (p≤0.0001) and TSB (p≤0.0001). The role of hemin and porphyrin-related compounds in the metabolism of S. mutans should be elucidated

In Vitro effects of Plantago major extract, aucubin and baicalein on Candida albicans biofilm formation, metabolic activity and cell surface hydrophobicity

Purpose To determine the in vitro effectiveness of Plantago major extract, along with two of its active components, aucubin and baicalein, on the inhibition of Candida albicans growth, biofilm formation, metabolic activity, and cell surface hydrophobicity. Materials and Methods Twofold dilutions of P. major, aucubin, and baicalein were used to determine the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and the minimum biofilm inhibitory concentration (MBIC) of each solution. Separately, twofold dilutions of P. major, aucubin, and baicalein were used to determine the metabolic activity of established C. albicans biofilm using a 2,3-bis (2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-carboxanilide reduction assay. Twofold dilutions of P. major, aucubin, and baicalein were used to determine the cell surface hydrophobicity of treated C. albicans biofilm by a two-phase assay using hexadecane. The hydrophobicity percentage of the cell surface was then calculated. A mixed-model ANOVA test was used for intergroup comparisons. Results The MICs of P. major extract (diluted 1:2 to 1:8), aucubin (61 to 244 μg/ml), and baicalein (0.0063 to 100 μg/ml) on the total growth of C. albicans were noticeable at their highest concentrations, and the inhibition was dose dependent. The MFC was evaluated after 48 hours of incubation, and aucubin (244 μg/ml) exhibited a strong fungicidal activity at its highest concentration against C. albicans growth. The MBIC indicated no growth or reduced growth of C. albicans biofilm at the highest concentrations of aucubin (61 to 244 μg/ml) and baicalein (25 to 100 μg/ml). Similarly, the effects of these reagents on C. albicans biofilm metabolic activity and hydrophobicity demonstrated high effectiveness at their highest concentrations. Conclusion P. major extract, aucubin, and baicalein caused a dose-dependent reduction on the total growth, biofilm formation, metabolic activity, and cell surface hydrophobicity of C. albicans. This demonstrates their effectiveness as antifungals and suggests their promising potential use as solutions for C. albicans biofilm-related infections

Effect of Commonly Prescribed Liquid Medications on Streptococcus mutans Biofilm. An in vitro study

Objective: This study addressed the effect of pediatric liquid antibiotic medications on Streptococcus mutans UA159. These suspensions commonly contain sugars such as sucrose to make them more palatable for children. The study was designed to evaluate the effects of oral liquid antibiotics on Streptococcus mutans growth and biofilm formation. Study Design: A 24 hour culture of S. mutans was treated with various concentrations of liquid medications commonly prescribed to children for odontogenic or fungal infections– amoxicillin, penicillin VK, clindamycin, and nystatin. The study was conducted in sterile 96-well flat bottom microtiter plates. The minimum inhibitory and biofilm inhibitory concentrations (MIC/MBIC) of S. mutans were determined for each medication. S. mutans was cultured with and without the test drugs, the amount of total growth measured, the biofilms washed, fixed, and stained with crystal violet. The absorbance was determined to evaluate biofilm formation. Results: Higher concentrations of amoxicillin, penicillin VK and clindamycin had decreased biofilm and overall growth than the control. The MICs were 1:2,560 (1.95 ug/ml), 1:2,560 (1.95 ug/ml) and 1:40 (9.375 ug/ml), while the MBIC were 1:640 (7.8 ug/ml), 1:1,280 (3.9 ug/ml) and 1:20 (18.75 ug/ml), respectively. Lower concentrations provided increased biofilm and overall growth. Nystatin induced significantly more biofilm and overall growth than the control at all concentrations. Conclusion: At high concentrations, approximately at the levels expected to be present in the oral cavity of children, amoxicillin, penicillin, and clindamycin inhibited S. mutans biofilm and overall growth due to their antibiotic activity, while at lower concentrations the three antibiotics demonstrated an increase in biofilm and growth. The increase in S. mutans biofilm and overall growth is most likely attributed to the sugar content in the medications. Nystatin provided an increase in biofilm and growth at each concentration tested

In Vitro Effects of Sports and Energy Drinks on Streptococcus mutans Biofilm Formation and Metabolic Activity

Purpose: Sports and energy drinks are being increasingly consumed and contain large amounts of sugars, which are known to increase Streptococcus mutans biofilm formation and metabolic activity. The purpose of this in vitro study was to investigate the effects of sports and energy drinks on S. mutans biofilm formation and metabolic activity. Methods: S. mutans UA159 was cultured with and without a dilution (1:3 ratio) of a variety of sports and energy drinks in bacterial media for 24 hours. The biofilm was washed, fixed, and stained. Biofilm growth was evaluated by reading absorbance of the crystal violet. Biofilm metabolic activity was measured by the biofilm-reducing XTT to a water-soluble orange compound. Results: Gatorade Protein Recovery Shake and Starbucks Doubleshot Espresso Energy were found to significantly increase biofilm (30-fold and 22-fold, respectively) and metabolic activity (2-fold and 3-fold, respectively). However, most of the remaining drinks significantly inhibited biofilm growth and metabolic activity. Conclusions: Several sports and energy drinks, with sugars or sugar substitutes as their main ingredients inhibited S. mutans biofilm formation. Among the drinks evaluated, Gatorade Protein Recovery Chocolate Shake and Starbucks Doubleshot Energy appear to have cariogenic potential since they increased the biofilm formation and metabolic activity of S. mutans

Caries lesion remineralization with fluoride toothpastes and chlorhexidine - effects of application timing and toothpaste surfactant

INTRODUCTION: Habitual toothbrushing with fluoridated toothpaste followed by rinsing with antibacterial mouthwashes is a method to maintain good oral hygiene and to diminish the occurrence and severity of dental caries and periodontal disease. However, our understanding of how antimicrobial agents in mouthwashes affect fluoride-mediated caries lesion remineralization is still poor. OBJECTIVE: The objectives of this in vitro study were a) to determine the effects of the waiting period of chlorhexidine (CHX) rinsing after fluoride toothpaste use and b) to further determine the effect of the type of toothpaste surfactant [sodium dodecyl sulfate (SDS) or cocamidopropyl betaine (CAPB)] on caries lesion remineralization associated with CHX rinsing. MATERIAL AND METHODS: Caries lesions were formed in bovine enamel specimens and assigned to 10 treatment groups (n=18) based on Vickers surface microhardness (VHN). Lesions were then pH-cycled for 10 days with daily regimen comprised of twice daily toothpaste slurry treatments (1150 ppm fluoride, with SDS or CAPB), followed by CHX solution treatments [0, 15, 30 or 60 minutes following slurry treatment or no CHX treatment (negative control)]. VHN was measured again and the extent of lesion remineralization calculated (∆VHN). RESULTS: ∆VHN with SDS-toothpaste was significantly lower than with CAPB-toothpaste, indicating more remineralization for the CAPB-toothpaste. ∆VHN with 0-minute waiting time was significantly lower than with 30-minute waiting time and with negative control. CONCLUSIONS: The absence of CHX as an adjunct to fluoride toothpastes led to greater remineralization of enamel lesions compared with the immediate use of CHX treatment for both SDS- and CAPB-toothpastes. CAPB-toothpastes indicated significantly greater remineralization than SDS-toothpastes, and can be suggested for patients at high risk of caries. A 30-minute waiting time for CHX treatment is recommended after brushing

Caries lesion remineralization with fluoride toothpastes and chlorhexidine - effects of application timing and toothpaste surfactant

Habitual toothbrushing with fluoridated toothpaste followed by rinsing with antibacterial mouthwashes is a method to maintain good oral hygiene and to diminish the occurrence and severity of dental caries and periodontal disease. However, our understanding of how antimicrobial agents in mouthwashes affect fluoride-mediated caries lesion remineralization is still poor. Objective: The objectives of this in vitro study were a) to determine the effects of the waiting period of chlorhexidine (CHX) rinsing after fluoride toothpaste use and b) to further determine the effect of the type of toothpaste surfactant [sodium dodecyl sulfate (SDS) or cocamidopropyl betaine (CAPB)] on caries lesion remineralization associated with CHX rinsing. Material and Methods: Caries lesions were formed in bovine enamel specimens and assigned to 10 treatment groups (n=18) based on Vickers surface microhardness (VHN). Lesions were then pH-cycled for 10 days with daily regimen comprised of twice daily toothpaste slurry treatments (1150 ppm fluoride, with SDS or CAPB), followed by CHX solution treatments [0, 15, 30 or 60 minutes following slurry treatment or no CHX treatment (negative control)]. VHN was measured again and the extent of lesion remineralization calculated (∆VHN). Results: ∆VHN with SDS-toothpaste was significantly lower than with CAPB-toothpaste, indicating more remineralization for the CAPB-toothpaste. ∆VHN with 0-minute waiting time was significantly lower than with 30-minute waiting time and with negative control. Conclusions: The absence of CHX as an adjunct to fluoride toothpastes led to greater remineralization of enamel lesions compared with the immediate use of CHX treatment for both SDS- and CAPB-toothpastes. CAPB-toothpastes indicated significantly greater remineralization than SDS-toothpastes, and can be suggested for patients at high risk of caries. A 30-minute waiting time for CHX treatment is recommended after brushing

The Impact of Nicotine and Cigarette Smoke Condensate on Metabolic Activity and Biofilm Formation of Candida albicans on Acrylic Denture Material

Purpose Smokers have increased denture stomatitis caused primarily by Candida albicans. The primary aim of this study was to demonstrate the impact of a wide range of nicotine and cigarette smoke condensate (CSC) concentrations on biofilm formation and metabolic activity of C. albicans on acrylic denture material. Materials and Methods C. albicans (ATCC strain 10231) was used. Standardized denture acrylic (PMMA) specimens (total of 135 specimens) were incubated with C. albicans and exposed to nicotine and CSC at different concentrations (0, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 mg/ml) and (0, 0.25, 0.5, 1, 2, and 4 mg/ml), respectively. For each experiment, 3 samples per nicotine and CSC concentration and a total of 45 specimens (27 specimens for the nicotine and 18 specimens for the CSC‐treated samples) were used and were selected randomly for each group. The control group consisted of 0 mg/ml of nicotine or CSC. The viability of C. albicans was measured using spiral plating on blood agar plates. The effect of nicotine and CSC concentrations on planktonic cells was were measured using a microplate reader. Metabolic activity of 24‐hour‐old established C. albicans biofilm exposed to nicotine and CSC for 24 hours in microtiter plates was determined using a 2,3‐bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐carboxanilide (XTT) reduction assay. Results The viability of C. albicans increased concomitant with increasing concentrations of CSC and nicotine, particularly at 0.5 and 2 mg/ml, respectively. Concentrations of CSC and nicotine above this resulted in an inhibitory effect on C. albicans viability. CSC and nicotine at 4 and 16 mg/ml, respectively, increased C. albicans biofilm metabolic activity. Conclusion Nicotine and CSC up to certain concentrations caused increases in biofilm formation, metabolic activity, viability, and planktonic cell absorbance of C. albicans. This in vitro study demonstrates the effectiveness of tobacco on promoting the growth of C. albicans and suggests their potential contributing factor in C. albicans biofilm related infections in smokers

Nicotine Upregulates Coaggregation of Candida albicans and Streptococcus mutans

Purpose Denture stomatitis is a condition of painless inflammation of denture-bearing mucosa. Reports indicate that nicotine, the major psychoactive ingredient in tobacco, increases growth of Streptococcus mutans and Candida albicans in denture biofilm. The purpose of this study was to determine the in vitro effects of nicotine on coaggregation of C. albicans with S. mutans. Material and Methods C. albicans strain ATCC 10231, S. mutans strain UA159 (ATTC 700610), and nicotine dilutions (ranging from 0 to 32 mg/ml) were used for this study. Both microorganisms were grown for 24 hours in dilutions of nicotine (0 to 32 mg/ml) made in tryptic soy broth (TSB) or TSB supplemented with 1% sucrose (TSBS; S. mutans) or yeast peptone dextrose broth (YPD; C. albicans). Suspensions of the nicotine-treated cells were prepared, mixed together and incubated for up to 24 hours to determine if there was an increase in coaggregation of nicotine-treated cells compared to the no nicotine control cells. Qualitative analysis of coaggregation was performed using a visual aggregation assay and light microscopic observation. A spectrophotometric assay was used to provide a quantitative analysis of the coaggregation. Results The visual aggregation assay indicated a significant increase in coaggregation between C. albicans and S. mutans with increasing incubation time (0 to 24 hours) and nicotine concentrations (0 to 4 mg/ml). Microbial growth in nicotine at 4 mg/ml demonstrated a significant increase in coaggregation after 24 hours of incubation. The numbers of coaggregated S. mutans/C. albicans cells exhibited a significant increase with incubation time and nicotine concentrations when the samples were examined microscopically. More coaggregation of S. mutans and C. albicans was observed with incubation time and increased nicotine compared to the 0 mg/ml nicotine group. There was a noticeable increase of coaggregation when cells were grown in TSBS compared to TSB. Absorbance of nicotine-treated cells (0.25 to 4 mg/ml) exhibited a decrease in values compared to 0 mg/ml at 0 hours of incubation, confirming increased coaggregation. Conclusion These results demonstrated the effect of nicotine in increasing the coaggregation of S. mutans with C. albicans. Coaggregation increased with incubation time and nicotine concentration. Coaggregation was increased with S. mutans grown in TSBS compared to TSB, suggesting that growth in sucrose media leads to an increase in receptors responsible for coaggregation

The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation

BACKGROUND: Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules. METHODOLOGY/PRINCIPAL FINDINGS: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels