Richard L. Gregory

Inhibitory effect of gels loaded with a low concentration of antibiotics against biofilm formation by Enterococcus faecalis and Porphyromonas gingivalis

We explored longitudinally the inhibitory effect of gels loaded with 1 mg/mL modified triple antibiotic paste (MTAP) or double antibiotic paste (DAP) against biofilm formation by Enterococcus faecalis and Porphyromonas gingivalis. Methylcellulose-based antibiotic gels of MTAP (ciprofloxacin, metronidazole and clindamycin) and DAP (ciprofloxacin and metronidazole) were prepared at a concentration of 1 mg/mL. Individually cultured E. faecalis and P. gingivalis bacterial suspensions were treated with MTAP, DAP, or placebo (vehicle only) gels at different dilutions and allowed to grow in 96-well microtiter plates. Untreated bacterial suspensions served as a negative control. Crystal violet assays were used to evaluate biofilm formation after 48 h. The ability of the gels to inhibit biofilm formation was determined immediately, and at 1 month and 3 months after the gels had been prepared. Data were analyzed using a mixed-model ANOVA. The MTAP and DAP gels significantly reduced biofilm formation by both bacterial species at all time points, regardless of the tested dilution. No-significant differences in biofilm-inhibitory effects between MTAP and DAP gels were observed at the majority of the tested dilutions through various time points. Gels loaded with 1 mg/mL MTAP and DAP demonstrated a significant antibiofilm effect against E.faecalis and P. gingivalis.

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.

Residual antibiofilm effects of various concentrations of double antibiotic paste used during regenerative endodontics after different application times.

OBJECTIVE We investigated the residual antibiofilm effects of different concentrations of double antibiotic paste (DAP) applied on radicular dentin for 1 or 4 weeks. DESIGN Dentin samples were prepared (n=120), sterilized and pretreated for 1 or 4 weeks with the clinically used concentration of DAP (500mg/mL), low concentrations of DAP (1, 5 or 50mg/mL) loaded into a methylcellulose system, calcium hydroxide (Ca(OH)2), or placebo paste. After the assigned treatment time, treatment pastes were rinsed off and the samples were kept independently in phosphate buffered saline for 3 weeks. Pretreated dentin samples were then inoculated with Enterococcus faecalis and bacterial biofilms were allowed to grow for an additional 3 weeks. Biofilms were then retrieved from dentin using biofilm disruption assays, diluted, spiral plated, and quantified. Fishers Exact and Wilcoxon rank sum tests were used for statistical comparisons (α=0.05). RESULTS Dentin pretreatment for 4 weeks with 5, 50 or 500mg/mL of DAP demonstrated significantly higher residual antibiofilm effects and complete eradication of E. faecalis biofilms in comparison to a 1 week pretreatment with similar concentrations. However, dentin pretreated with 1mg/mL of DAP or Ca(OH)2 did not provide a substantial residual antibiofilm effect regardless of the application time. CONCLUSIONS Dentin pretreatment with 5mg/mL of DAP or higher for 4 weeks induced significantly higher residual antibiofilm effects in comparison to a 1 week pretreatment with the same concentrations.

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.