Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial "adhesion strength" to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix' glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans.

BACKGROUND: Dental caries is a biofilm-diet-dependent worldwide public health problem, and approaches against microorganisms in cariogenic biofilms are necessary. METHODS: The antimicrobial and antibiofilm activities of 12 Casearia sylvestris extracts (0.50 mg/mL) from different Brazilian biomes (Atlantic Forest, Cerrado, Caatinga, Pampa, and Pantanal) and varieties (sylvestris, lingua, and intermediate) were tested against two species found in cariogenic biofilms (Streptococcus mutans and Candida albicans). The extracts effective against S. mutans were used to evaluate the "adhesion strength" of this bacterium to the salivary pellicle and initial glucan matrix and the S. mutans-GtfB activity. Also, the antimicrobial activity against S. mutans of three fractions (methanol, ethyl acetate, and hexane; 0.25 mg/mL) from the extracts was evaluated. RESULTS: Three extracts from the Atlantic Forest variety sylvestris (FLO/SC, GUA/CE, PRE/SP) reduced ≥50% (> 3 logs) S. mutans viable population (p < 0.0001 vs. vehicle), while two extracts from the same biome and variety (PAC/CE, PRE/SP) decreased ≥50% of the viable counts of C. albicans (p < 0.0001 vs. vehicle). For S. mutans biofilms, three extracts (GUA/CE, PAC/CE, PRE/SP) reduced the biomass by ≥91% (p > 0.0001 vs. vehicle) and 100% of the microbial population (p < 0.0001 vs. vehicle). However, for the fungal biofilm, two extracts (PAC/CE, PRE/SP) reduced the viable counts by ≥52% (p < 0.0001 vs. vehicle), but none reduced biomass. The extracts with higher antimicrobial and antibiofilm activities presented higher content of clerodane-type diterpenes and lower content of glycosylated flavonoids than the less active extracts. The extracts had no effect on the removal of cells adhered to the pellicle (p > 0.05 vs. vehicle) while promoted the detachment of a larger number of S. mutans cells from GtfB-glucan matrix (p < 0.0031 vs. vehicle), and FLO/SC, GUA/CE and PRE/SP reduced the quantity of glucans (p < 0.0136 vs. vehicle). Only the ethyl acetate fractions reduced the microbial population of S. mutans (p < 0.0001 vs. vehicle), except for one (PAC/CE). Among the ethyl acetate fractions, three from var. lingua (two from Cerrado, and one from Cerrado/Caatinga) reduced ≥83% of the microbial population. CONCLUSIONS: C. sylvestris extracts from Atlantic Forest var. sylvestris and ethyl acetate fractions from Cerrado and Cerrado/Caatinga var. lingua may be used as a strategy against cariogenic microorganisms.

Antimicrobial photodynamic therapy alone or in combination with antibiotic local administration against biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

Antimicrobial photodynamic therapy (aPDT) kills several planktonic pathogens. However, the susceptibility of biofilm-derived anaerobic bacteria to aPDT is poorly characterized. Here, we evaluated the effect of Photodithazine (PDZ)-mediated aPDT on Fusobacterium nucleatum and Porphyromonas gingivalis biofilms. In addition, aPDT was tested with metronidazole (MTZ) to explore the potential antimicrobial effect of the treatment. The minimum inhibitory concentration (MIC) of MTZ was defined for each bacterial species. Single-species biofilms of each species were grown on polystyrene plates under anaerobic conditions for five days. aPDT was performed by applying PDZ at concentrations of 50, 75 and 100 mg/L, followed by exposure to 50 J/cm2 LED light (660 nm) with or without MTZ. aPDT exhibited a significant reduction in bacterial viability at a PDZ concentration of 100 mg/L, with 1.12 log10 and 2.66 log10 reductions for F. nucleatum and P. gingivalis in biofilms, respectively. However, the antimicrobial effect against F. nucleatum was achieved only when aPDT was combined with MTZ at 100× MIC. Regarding P. gingivalis, the combination of PDZ-mediated aPDT at 100 mg/L with MTZ 100× MIC resulted in a 5 log10 reduction in the bacterial population. The potential antimicrobial effects of aPDT in combination with MTZ for both single pathogenic biofilms were confirmed by live/dead staining. These results suggest that localized antibiotic administration may be an adjuvant to aPDT to control F. nucleatum and P. gingivalis biofilms.