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 &#8220;frequent hitters&#8221;. 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="Figure 1" class="xfigimg" src="/files/ftp_upload/61773/61773fig01v2.jpg" /> 
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

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 microorganism...

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

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 ...

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6.2K Views.  São Paulo State University (UNESP). Het gepresenteerde protocol wordt gebruikt voor het correct screenen en analyseren van bioactieve kandidaten in natuurlijke producten om orale biofilms te beheersen. Het kan ook worden aangepast voor toepassingen in andere biofilmonderzoeksgebieden. Deze meest zetmeelscreenings- en analysemethode maakt het mogelijk om bioactieve componenten tegelijkertijd te verwijderen.Tandcariës is een biofilmdieet afgeleid van, zeer voorkomende, chronische ziekte. Dit protocol kan helpen om natuurl...

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

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

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

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.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Immunology and Infection

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

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

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.

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Medicine

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

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.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Environment

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

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species 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.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Bioengineering

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

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

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

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.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

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

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

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

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

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

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.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

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

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