Name: Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants Extracts and Fractions to Prevent Dental Caries (Video) | JoVE Journal
Uploaded: 2021-03-31T12:30:00.000+00:00
Duration: 8 min 20 s
Description: Discover a streamlined protocol for screening plant extracts to identify antimicrobial compounds that inhibit Streptococcus mutans biofilms. This method enables researchers to develop natural product-based approaches for preventing dental caries.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We express our gratitude to N&#250;cleo de Bioensaios, Bioss&#237;ntese e Ecofisiologia de Produtos Naturais (NuBBE) of the Chemistry Institute of UNESP, Araraquara/SP for providing the laboratories for preparing plant material. We also thank the Applied Microbiology Laboratory of the Department of Dental Materials and Prosthodontics, UNESP, Araraquara/SP. This research was supported by a research grant from the S&#227;o Paulo Research Foundation (FAPESP #2013/07600&#8211;3 to AJC) and scholarships plus overhead funds (FAPESP #2017/07408&#8211;6 and FAPESP #2019/23175-7 to SMR; #2011/21440&#8211;3 and #2012/21921&#8211;4 to PCPB). The National Council for Scientific and Technological Development in association with FAPESP provided additional support (INCT CNPq #465637/2014&#8211;0 and FAPESP #2014/50926&#8211;0 to AJC).

No conflicts of interest declared.

The main challenges related to the work with natural crude extracts comprise their complex composition and the inadequacies of classic bio-guided isolation studies. Although this process is slow, it is effective and has led to major findings in NP research. To rationalize, prioritization-driven studies are needed to rationalize. Thus, the use of modern chemical profiling approaches for the analysis of CE and dereplication before isolation are important to characterize the studied material and especially useful to avoid r...

The earliest models of medicine used in societies were based on natural products (NPs). Since then, humans have been searching for new chemicals in nature that can be transformed into drugs1. This search caused a continuous improvement of technologies and methods for ethnobotanical screening1,2,3. NPs offer a rich source of structurally diverse substances, with a wide range of biological activities useful for developing alternative or adjuvant therapies. However, the inherent complex plant matrix makes the isolation and identification of the active compounds a challenging and time-consuming task4.
NPs-based drugs or formulations can be used to prevent and/or treat several conditions affecting oral, including dental caries4. Dental caries, one of the most prevalent chronic diseases globally, derives from the interaction of sugar-rich diet and microbial biofilms (dental plaque) formed on the tooth surface that leads to demineralization caused by organic acids derived from microbial metabolism, and if not treated, leads to teeth loss5,6. Although other microorganisms may be associated7, Streptococcus mutans is a critical cariogenic bacterium because it is acidogenic, aciduric, and an extracellular matrix builder. This species encodes multiple exoenzymes (e.g., glycosyltransferases or Gtfs) that use sucrose as a substrate8 to build the extracellular matrix rich in exopolysaccharides, which are a virulence determinant9. Also, the fungus Candida albicans can drive up the production of that extracellular matrix7. Albeit fluoride, administered in various modalities, remains the basis for preventing dental caries10, new approaches are needed as adjuvants to increase its effectiveness. In addition, the available anti-plaque modalities are based on the use of broad-spectrum microbicidal agents (e.g., chlorhexidine)11. As an alternative, NPs are potential therapies for controlling biofilms and preventing dental caries12,13.
The further advance in the discovery of new bioactive compounds from plants includes necessary steps or approaches such as: (i) the use of reliable and reproducible protocols for sampling, considering that plants often show intraspecific variability; (ii) the preparation of comprehensive extracts and their respective fractions in small scale; (iii) the characterization and/or dereplication of their chemical profiles thought the acquisition of multidimensional data such as GC-MS, LC-DAD-MS, or NMR, for example; (iv) the use of viable and high-yield models to assess bioactivity; (v) the selection of potential new hits based on multivariate data analysis or other statistical tools; (vi) to perform the isolation and purification of the targeted compounds or promising candidates; and (vii) the validation of the corresponded biological activities using the isolated compounds2,14.
Dereplication is the process of rapidly identifying known compounds in crude extract and allows differentiating novel compounds from those that have already been studied. Besides, this process prevents isolation when bioactivity has already been described for certain compounds, and it is particularly helpful to detect “frequent hitters”. It has been used in different untargeted workflows ranging from major compound identification or the acceleration of activity-guided fractionation up to the chemical profiling of collections of extracts. It can be fully integrated with metabolomic studies for the untargeted chemical profiling of CE or the targeted identification of metabolites. All of this ultimately leads to prioritizing extracts before the isolation procedures1,15,16,17.
Therefore, in the present manuscript, we describe a systematic approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions. It includes four multidisciplinary steps: (1) collection of plant material; (2) preparation of crude extracts (CE) and fractions (CEF), followed by their chemical profile analysis; (3) bioassays; and (4) biological and chemical data analyses (Figure 1). Thus, we present the protocol developed to analyze of the antimicrobial and antibiofilm activities of Casearia sylvestris extracts and fractions against Streptococcus mutans and Candida albicans13, as well as the procedures for the phytochemical characterization and data analysis. For simplicity, the focus here is to demonstrate the approach for screening natural compounds using the bacterium.
<img alt="Plant extract analysis: Diagram of UHPLC-DAD-HRMS profiling, bioassays, and data analysis steps." class="xfigimg" src="/files/ftp_upload/61773/61773fig01v2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Flow-chart of the Systematic Approach to identify active molecules from plants extracts and fractions. <a href="https://www.jove.com/files/ftp_upload/61773/61773fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>96-well microplates&amp;nbsp;</td><td>Kasvi</td><td>Flat bottom</td><td></td></tr><tr><td>Activated carbon</td><td>LABSYNTH</td><td>Clean up and/or fractionation step</td><td></td></tr><tr><td>Analytical mill</td><td>Ika LabortechniK</td><td>Model A11 Basic</td><td></td></tr><tr><td>Blood agar plates</td><td>Laborclin</td><td></td><td></td></tr><tr><td>Chromatographic column C18</td><td>Phenomenex Kinetex</td><td>150 &amp;times; 2.1 mm, 2.6 &amp;micro;m, 100&amp;Acirc;</td><td></td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Vehicle solution</td><td></td></tr><tr><td>ELISA plate reader</td><td>Biochrom Ez</td><td></td><td></td></tr><tr><td>Ethanol</td><td>J. T. Baker</td><td>For extraction and fractionation steps, and mobile phase composition</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>Vehicle solution</td><td></td></tr><tr><td>Ethyl acetate</td><td>J. T. Baker</td><td>Fractionation step</td><td></td></tr><tr><td>GraphPad Software</td><td>La Jolla</td><td>GraphPad Prism7</td><td></td></tr><tr><td>Hexane</td><td>J. T. Baker</td><td>Fractionation step</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>J. T. Baker</td><td>For extraction step</td><td></td></tr><tr><td>Lyophilizer (a freeze dryer)</td><td>Savant</td><td>Modulyo</td><td></td></tr><tr><td>Nylon Millipore</td><td>LAC</td><td>0.22 &amp;micro;m x 13 mm</td><td></td></tr><tr><td>Orbital shaker</td><td>Quimis</td><td>Model G816&amp;thinsp;M20</td><td></td></tr><tr><td>Polyamide solid phase extraction cartridge</td><td>Macherey-Nagel</td><td>Clean up and/or fractionation step</td><td></td></tr><tr><td>Silica gel</td><td>Merck</td><td>40&amp;ndash;63&amp;thinsp;&amp;mu;m, 60&amp;thinsp;&amp;Acirc;</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Synth</td><td>0,89% in water</td><td></td></tr><tr><td>Solid phase extraction cartridges (SPE)</td><td>Macherey-Nagel</td><td>Clean up and/or fractionation step</td><td></td></tr><tr><td>Tryptone</td><td>Difco</td><td></td><td></td></tr><tr><td>UHPLC-DAD</td><td>Dionex</td><td>Ultimate 3000 RS</td><td></td></tr><tr><td>Ultrasonic bath</td><td>UNIQUE</td><td>Model USC 2800</td><td></td></tr><tr><td>Yeast extract</td><td>Difco</td><td></td><td></td></tr></tbody></table>

systematic approach to identify novel antimicrobial and antibiofilm molecules from plants' extracts and fractions to prevent dental caries

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 &#34;adhesion strength&#34; 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&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

We provide an example of using a systematic approach to screen the biological activity of plant extracts and fractions to identify potentially active molecules for possible new anti-caries therapies: antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans13.
Background 
Complex interactions between specific oral microorganisms-...

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

systematic-approach-to-identify-novel-antimicrobial-antibiofilm

Research

JoVE Journal

Biology

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque. Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue.   We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Discover a streamlined protocol for screening plant extracts to identify antimicrobial compounds that inhibit Streptococcus mutans biofilms. This method enables researchers to develop natural product-based approaches for preventing dental caries.

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants Extracts and Fractions to Prevent Dental Caries (Video) | JoVE Journal

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

Explore More Videos