The authors are grateful to Joachim Theussl (Center of Medical Research, Medical University of Graz) for the excellent technical assistance in the video production and to Monica Farrell (Research Management, Medical University of Graz) as voice talent.

Protocol and video shooting were approved by the institutional review board of the Medical University of Graz (Votum 27-126ex14/15). Written consent for this video publication was obtained from the child and her parent.
1. Instruments and Materials
<ol>
	<li>Cut calibrated (ISO 015/02) and sterile paper points, usually used in endodontic therapy, at the first ring mark in order to standardize their length (Figure 1).</li>
	<li>Apply UV light irradiation at 260 nm for 30 min in order to avoid DNA and RNA contamination of the paper points (Figure 2).</li>
	<li>Autoclave the tubes for storage at 120 &#176;C and 1.2 bar for 20 min and UV-irradiate them as well or employ ready-to-use gamma irradiated tubes.</li>
</ol>
2. Preparation of Subjects
Note: Written informed consent was obtained from the child and her parent prior to enrollment.
<ol>
	<li>Apply cocoa butter to the lips.</li>
	<li>Mount the cheek and tongue retractor for full access to both dental arches.</li>
	<li>Stain the dental plaque with plaque disclosure.</li>
	<li>Remove the dry field apparatus.</li>
	<li>Rinse the mouth with water until the water is colorless.</li>
	<li>Brush teeth thoroughly with an electric tooth brush at 45 &#176; angulation. Apply water only but no toothpaste, as this would alter the oral biofilm.</li>
	<li>Mount the dry field apparatus again, including the tongue guard to keep the mouth open and dry.</li>
	<li>Clean and dry the index teeth with sterile cotton swabs to avoid absorption of supragingival fluid during paper point sampling.</li>
</ol>
3. Biofilm Sampling
<ol>
	<li>Paper point sampling of the subgingival sulcus
	<ol>
		<li>Grasp the paper points with sterile dental tweezers.</li>
		<li>Insert paper points tangentially up to a defined length of 4 mm. Take special care not to traumatize the junctional epithelium (Figure 3).</li>
		<li>Remove paper points after 20 s.</li>
		<li>Collect paper points directly into prepared tubes: place the paper point tip directly in a sterile and DNA-free vial, and cut it at the third mark to a standardized length of 4 mm (Figure 4).</li>
		<li>If indicated by the study protocols, insert two paper points parallel and simultaneously at the same site (Figure 5A).</li>
		<li>Alternatively, collect paper points from several sites if the study protocol requires &#34;pooled samples&#34; (Figure 5B).</li>
		<li>Remove the dry field apparatus for mucosal and saliva sampling.</li>
	</ol>
	</li>
	<li>Mucosal paper point sampling
	<ol>
		<li>Apply paper points to the vestibular fold of the upper cheek (Figure 6).</li>
		<li>Close the cheek and massage it briefly.</li>
		<li>Open the cheek and remove the paper points after 20 s.</li>
		<li>Cut paper points at the third mark and place them in a sterile tube as indicated for subgingival sampling.</li>
	</ol>
	</li>
	<li>Saliva sampling
	<ol>
		<li>Let the child spit unstimulated saliva into a sterilized collection vessel (Figure 7).</li>
	</ol>
	</li>
</ol>
4. Transfer and Storage
<ol>
	<li>Collect paper points as single, pooled, or parallel samples depending on the study design (Figure 8).</li>
	<li>Apply a color-coded storage system to facilitate further sample management (Figure 9).</li>
	<li>Finally, store the samples at &#8722;80 &#176;C pending microbiome analysis.</li>
</ol>

<ol>
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The authors report no conflicts of interest related to this scientific video production.

Bacteria are found at all sites within the oral cavity24,25,26. Many studies have focused on the use of saliva as a sampling medium to map oral colonization as it can easily be sampled through spitting27,28,29,30,31,32. However, salivary biofilm does not reflect subgingival biofilm composition. Thus, the use of other sampling methods is crucial for creating the whole picture, and will evoke new discussions in dental research.Several articles compare the use of curettes and paper points for subgingival sampling of periodontally diseased subjects8,33,34. Yet no research group is known to have focused on the application of standardized sampling devices for different age groups, especially for children19.
In this video, oral biofilm sampling of three oral habitats in healthy children is presented.
Modifications and Troubleshooting:
The aim of the manuscript focuses on the clinical protocol for subgingival biofilm sampling of healthy children. The method of choice refers to sound subgingival sulci where limited space makes sampling especially challenging. In this video the method was applied to early permanent dentition. It can also be applied to mixed or mature dentition, and to children and/or adults. Our sampling method allows for modifications such as single or pooled samples. The latter might become a crucial factor for molecular analyses due to the small amount of DNA collectable from the healthy subgingival sulcus. The choice of the index teeth may be modified on demand; in particular, another tooth may be sampled as an alternative when the epithelium is traumatized and bleeding, thus making the paper point unusable. Parallel sampling using two paper points simultaneously at one site renders sample replicates in one step, in addition to shortening the chair time for children.
With slight modifications diseased periodontal pockets can be sampled. Here paper points will need to be inserted to the full length of the pocket, which can reach up to 10 mm. The length and conus of paper points as well as the depth of insertion must be adapted to the intraoral habitat of interest, but should always be standardized by material and dimensions. Samples can also be taken from the mucosa using paper points. Identical protocols allow comparisons of different oral habitats and various dental materials. Patient preparation and sample storage can be performed as described in this video. For metatranscriptome research, RNA could be sampled with the same method. Thus, after sampling, the paper points should be inserted directly in RNA stabilization solution.
Limitations of the Technique:
Regardless of modifications, the clinical sampling method we describe is limited to the sound subgingival sulcus. Other sampling sites, as for example periodontally diseased pockets, were not part of our study design. As sampling to the full depth of such pockets is needed, paper points might not be large enough to collect samples sufficiently. If the experimental goal is to compare data sampled from sound subgingival sulci and periodontally diseased pockets, it is important to be aware of the inherent bias that is associated with different clinical sampling methods. It therefore is not advisable to compare such data.
A limitation of the method presented here is sampling without the subsequent use of propidium monoazide. Adding this agent directly after sampling allows for specific analysis of only the living cells in the biofilm analyzed. The sampling method described here reflects numbers of living and dead cells. For research of severe periodontitis, paper points will probably need to be replaced by curettes, as biofilm formation in deep pockets is strong. Paper point sampling might not reflect the entire microbial spectrum in these cases, as their surface might be saturated too quickly.
Significance with Respect to Existing Methods:
The proposed manuscript is the first of its kind to standardize subgingival sampling in the healthy sulcus with paper points. Other reports have referred to the use of metal curettes35,36 or paper points 37,38,39, but did not describe the processes that take place prior to sampling, such as plaque control, tooth cleaning, tooth isolation, and drying, as well as the processes that result from inadequate specifications on the sampling technique and time lines.
In two previous articles, we show that the use of paper points is a reproducible method for subgingival biofilm sampling in children22,40. Due to their design, curettes are too large for the shallow sulcus with limited space and too sharp for the tender junctional epithelium. It is crucial to access the subgingival sulcus without traumatizing the epithelium. In this way, bias arising from bleeding is avoided. Thus, the use of the slim and tender paper points is preferred for this area. Paper points are functional to monitor changes in biofilm composition during orthodontic treatment21,41. They are slim and flexible enough to fit between the elements of fixed orthodontic appliances. This is a major advantage to other sampling methods like curettes. Atraumatic sampling is simplified and sampling of small amounts of DNA is possible.
Future Applications:
In this video we demonstrated the method on non-diseased, early, permanent dentition. It also can be applied to mixed or mature dentition. With some adaptations it is possible to sample periodontally diseased sites like teeth or implants and other niches due to dental materials by following the same protocol. Caries research could benefit from this standardized protocol, in particular studies on root caries. The most important lecture from our sampling video is standardization of the protocols to make data comparable, no matter what and how samples are measured. Future video demonstrations could enhance the field of clinical sampling in general.
Oral biofilm obtained as shown in the video is used in clinics and for scientific investigations. This in vivo approach represents a good addition to in vitro molecular techniques such as fluorescence in situ hybridization and confocal laser scanning microscopy15. NGS methods, as the pyrosequencing shown here, applied to the collected biofilm, allow the analysis of the complete microbiota. Calculating the relative abundances of certain bacterial species can be used to support treatments or to compare different patients or different treatments. Together with a standardized sequencing protocol and sequence analysis workflow that we have previously described40, this sampling method allows sequence analysis down to the genus level. Further &#34;meta&#34;-studies such as metatranscriptomic or metabolomic&#160;studies are possible based on the sampling protocol presented in this video.
Critical Steps:
Avoiding contamination is a critical step: sterile instruments have to be applied in a non-sterile in vivo environment. The transfer from the mouth to the bench has to be performed quickly and securely by an experienced examiner to keep chair time for study participants as short as possible. The most critical step in the protocol is the atraumatic insertion of the paper point into the subgingival sulcus. Disruption of the junctional epithelium and bleeding absolutely have to be avoided. Further care has to be taken in order to not contaminate paper points and storage vials with exogenous DNA or RNA. The storage itself then also is a critical step. Samples need to be frozen directly, at best at &#8722;80 &#176;C. This avoids changes in bacterial composition post sampling, which would falsify sequencing results.

Human oral biofilm comprises a broad community consisting mostly of commensals and beneficial microorganisms1,2,3. Species found here colonize all niches that the oral cavity offers4,5,6. Biofilm composition in these niches varies as widely as the habitats. Saliva for example displays different bacterial profiles than plaque samples. In plaque samples of healthy adults, the relative abundance of Actinobacteria is over 20%, while less than 7% is found in saliva. Bacteroidetes and Firmicutes in contrast appear in much higher numbers in saliva7. Thus, it is important to sample at different locations in the mouth in order to get the whole picture. Additionally, various factors such as geographic and ethnic differences, age, sex, and many other factors make it difficult to identify general rules for biofilm development and disease onset8,9. For many years the investigation of periopathogenic and cariogenic biofilm has been a central concern8,10,11,12,13.
In recent years, research on healthy subjects has gained importance not only for a broader understanding of disease but also for the implementation of preventive measures14. New technologies and molecular analyses further elucidate oral biofilm formation and function15,16, and enable the complete profiling of microbial diversity17,18. This is expected to also lead to a new understanding of microbial changes during orthodontic therapy19. An impact on the development of orthodontic biomaterials is foreseeable. The enhanced perspective will shed new light on complications of orthodontic therapy associated with biofilm, such as enamel demineralization and periodontal diseases12,20,21. To permit worldwide data comparisons, it is crucial to standardize all steps in data generation. Minor changes in laboratory procedures can strongly influence the results. In addition, the use of varying computational platforms in data processing can lead to non-comparable datasets. Lastly, the choice of statistical tests and corrections has an influence on the results. However, sampling bias can already occur long before any lab work or bio-computing commences. Non-standardized sampling methods lead to an inherently biased study. In view of low to moderate levels of standardization in the relevant studies, little evidence exists on the relationship between orthodontics and microbiomes19. Therefore, the development of standardized methods would facilitate qualified comparison of data in the field.
This article presents standardized oral biofilm sampling procedures. The protocol is intended to contribute towards generating globally comparable collections of microbial sequence data. A step-by-step protocol for oral biofilm sampling in healthy children is presented. As the method of choice, paper points are inserted atraumatically into the sound subgingival sulcus. To facilitate habitat comparisons, cheek mucosa is sampled according to the same protocol. This article additionally demonstrates parallel and pooled sampling. Furthermore, saliva sampling is shown. A simple color-coded transport and storage system is also presented, facilitating specimen management for further processing. The choice of downstream operations regarding storage medium, metagenomic analyses, and bioinformatics depends on the clinical questions raised by different fields in oral research. In this manuscript, DNA sampling for NGS has been chosen as an example of a possible application for orthodontic research.

<table><tbody><tr><td>Paper Points Taper .02, sterilised</td><td>VDW Dental</td><td>550 029 015</td><td>http://www.vdw-dental.com/en/products/root-canal-drying/paper-points.html?seminarNo=24&amp;amp;cHash=&lt;br/&gt; 1107d79ab05653b454fd90a954dc92e2</td></tr><tr><td>GC Cocoa Butter, Tube of 10 g</td><td>GC EUROPE N.V.</td><td>000387</td><td>http://www.gceurope.com/products/detail.php?id=22</td></tr><tr><td>Rondells blue</td><td>DIRECTA AB</td><td>Rondell Tablets</td><td>http://www.directadental.com/exego.aspx?p_id=767</td></tr><tr><td>Great Lakes Nola Dry Field System</td><td>Great Lakes Ortho</td><td>300-401</td><td>http://www.greatlakesortho.com/commerce/detail/?nPID=1626</td></tr><tr><td>tooth brush</td><td>Oral-B</td><td></td><td>http://www.oralb-blendamed.de/de-DE/pro</td></tr><tr><td>micro-Ident plus11</td><td>Hain Lifescience GmbH</td><td>23312</td><td>http://www.hain-lifescience.de/en/products/microbiology/dental-diagnostics/micro-ident-und-micro-identplus.html</td></tr><tr><td>ParoCheck</td><td>Greiner Bio One International GmbH</td><td>460020</td><td>https://shop.gbo.com/en/row/articles/catalogue/article/0150_00020/12705/</td></tr><tr><td>Carpegen Perio Diagnostik</td><td>Carpegen GmbH</td><td></td><td>http://www.carpegen.de/en/products-and-services/carpegen-perio-diagnostics.html</td></tr><tr><td>tubes Reaktionsgef&amp;auml;&amp;szlig;e 1,5 ml</td><td>Lactan</td><td>CH77.1&amp;nbsp; to CH81.1</td><td>www.lactan</td></tr><tr><td>cotton swabs</td><td>Henry Schein</td><td>9003182</td><td>www.henryschein.at</td></tr></tbody></table>

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.

In the present publication, DNA sampling for NGS is demonstrated as an example of a possible application for orthodontic research. Bacterial DNA is extracted directly from the paper point samples. This DNA can then be used in studies of the microbial composition for clinical or research questions (as summarized in Figure 10).
Modern molecular analysis methods such as the NGS method 454-pyrosequencing give an overview of the complete microbiota of a sample. The DNA of thousands of bacteria is identified simultaneously at different phylogenetic levels over the 16s rRNA sequence differences. Bioinformatic platforms need to be generated as a means of structuring into clusters the information retrieved from the large data sets. Statistical analysis can then be applied to microbiota datasets.
The overall relative abundance of certain bacterial species can be analyzed as can shifts in the microbiota due to changing habitat conditions. Bar charts (Figure 11) and heatmaps (Figure 12) display the subgingival bacterial composition at phylum level or at order level. Various individuals and treatments can be compared by descriptive and inferential statistics. Figure 13 presents a histogram comparison of two modes of subgingival paper point sampling at different taxonomic levels. Table 1 shows the shift in biofilm composition during an in vitro study comparing two time points (T1 and T3). Statistically significant changes were calculated with Wilcoxon signed-rank test and following Bonferroni correction from the 454-pyrosequencing data.
Finally, Principal Coordinate Analysis (PCoA) enables direct 3D comparison at Operational Taxonomic Unit (OTU) level as identified in different samples. The PCoA plots in Figure 14 display intra- and inter-individual differences of the subgingival microbiome in a case series of five children. Figure 15 shows the results of an orthodontic case-control study: pink to red dots are the cases, light to dark blue dots are the controls, all sampled repeatedly over a period of four months. A clear clustering of the cases after the orthodontic intervention (red dots) represents a shift in the microbiome. Blue dots, representing the control group, are evenly distributed.
<img alt="Thin-layer chromatography band cutting; sample preparation; laboratory process; chromatography analysis." class="xfigimg" src="/files/ftp_upload/56320/56320fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Prepare paper points of standardized length. Sterile calibrated paper points are cut at the first ring mark to standardize their length. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Petri dish with samples under UV light in a sterile hood for microbial growth analysis." class="xfigimg" src="/files/ftp_upload/56320/56320fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Sterilize and free paper points from DNA. Standardized paper points are sterilized and UV irradiated in a clean bench. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dental tool demonstrating interdental cleaning technique on canine teeth close-up." class="xfigimg" src="/files/ftp_upload/56320/56320fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Subgingival paper point insertion. Sterile calibrated paper points are inserted tangentially into the subgingival sulcus until the 4 mm ring mark for 20 s.
<img alt="Gloved hand handling microtube with tweezers, DNA sample preparation, laboratory process." class="xfigimg" src="/files/ftp_upload/56320/56320fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Cutting paper point into tube. Directly after sampling, paper points are cut at the third ring mark (4 mm) into a sterile and DNA-free 1.5 mL vial. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Orthodontic procedure demonstrating dental brace wire placement using pliers, two-step method." class="xfigimg" src="/files/ftp_upload/56320/56320fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Pooled and parallel sampling. Two paper points are inserted simultaneously into the subgingival sulcus of one tooth (A) or several paper points into the subgingival sulci of several teeth (B). <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Submucosal dental implant diagram highlighting placement for orthodontic correction steps." class="xfigimg" src="/files/ftp_upload/56320/56320fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Mucosal sampling. Sterile calibrated paper points are laid into the mucosa of the vestibular fold. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Oral dosage measurement with plastic cup, illustrating medication administration process." class="xfigimg" src="/files/ftp_upload/56320/56320fig7.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Saliva collection. Unstimulated saliva is spit into a sterile beaker. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dental plaque sampling diagram; single, pooled, and parallel collection tubes shown below." class="xfigimg" src="/files/ftp_upload/56320/56320fig8.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Sampling scheme. Samples can be collected as single (left side, one tooth each), pooled (several to all stripes in one vial), or parallel (blue stripes, two paper points at one tooth) samples. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Color-coded sample containers for chemical analysis, organized in a box, used in lab experiments." class="xfigimg" src="/files/ftp_upload/56320/56320fig9.jpg" data-altupdatedat="1747911167000" data-alt="Figure 9"> 
Figure 9: Color-coded storage. A referring color code for sheets and vials facilitates further sample handling. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Clinical sampling, DNA extraction, sequencing diagram; shows steps: sampling, extraction, sequencing." class="xfigimg" src="/files/ftp_upload/56320/56320fig10.jpg" data-altupdatedat="1747911167000" data-alt="Figure 10"> 
Figure 10: Subgingival biofilm analysis. Schematic presentation of clinical biofilm sampling (A), DNA extraction (B), and different NGS technologies (C). <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial community composition; stacked bar chart; taxonomic distribution analysis; microbial diversity." class="xfigimg" src="/files/ftp_upload/56320/56320fig11.jpg" data-altupdatedat="1747911167000" data-alt="Figure 11"> 
Figure 11: Bar chart. Showing relative abundance of bacteria at phylum level analyzed using 454-pyrosequencing. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microbial diversity heatmap; genus-level comparison, sample analysis, color-coded abundance data." class="xfigimg" src="/files/ftp_upload/56320/56320fig12.jpg" data-altupdatedat="1747911167000" data-alt="Figure 12"> 
Figure 12: Heatmap. Showing relative abundances of bacterial orders analyzed using 454-pyrosequencing. Dark red represents a high relative abundance (&gt; 10%). Dark blue represents a very low to no relative abundance of the respective bacterial order. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Comparative taxonomy profile charts of two modes across phylum, class, family, genus levels." class="xfigimg" src="/files/ftp_upload/56320/56320fig13.jpg" data-altupdatedat="1747911167000" data-alt="Figure 13"> 
Figure 13: Histogram. Comparison of two modes of paper point sampling (modes A and B) at different taxonomic levels. Data generated with 454-pyrosequencing. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Principal component analysis (PCA) graphs illustrating P1, P2, P3 variance across patient data." class="xfigimg" src="/files/ftp_upload/56320/56320fig14.jpg" data-altupdatedat="1747911167000" data-alt="Figure 14"> 
Figure 14: 2D PCoA plot. A case series (n=5) showing intra-individual and inter-individual differences as a result of two subgingival sampling methods (1 color is 1 subject).Data generated with 454-pyrosequencing. Image modified from Santigli et al.22 <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="3D PCA plot diagram showing data clustering; principal component analysis, statistical method." class="xfigimg" src="/files/ftp_upload/56320/56320fig15.jpg" data-altupdatedat="1747911167000" data-alt="Figure 15"> 
Figure 15: Snapshot of a 3D PCoA plot animation. Showing subgingival microbiome shifts in an orthodontic case control study (n = 16; each group, 3 points in time; cases: pink to red, controls: light to dark blue). Data generated with 454-pyrosequencing. Unpublished data from working group Santigli. <a href="https://cloudflare.jove.com/files/ftp_upload/56320/56320fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Taxon: Bacteriae</td>
			<td colspan="3">Time point 1 [%]</td>
			<td colspan="3">Time point 3 [%]</td>
			<td colspan="2">Wilcoxon signed-rank test</td>
		</tr>
		<tr>
			<td colspan="2">Phylum/Genus</td>
			<td>25th</td>
			<td>Median</td>
			<td>75th</td>
			<td>25th</td>
			<td>Median</td>
			<td>75th</td>
			<td>p-value</td>
			<td>p-value adjusted#</td>
		</tr>
		<tr>
			<td colspan="2">Other</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Other</td>
			<td>0.70</td>
			<td>1.07</td>
			<td>1.28</td>
			<td>1.32</td>
			<td>1.63</td>
			<td>1.93</td>
			<td>.000</td>
			<td>.005</td>
		</tr>
		<tr>
			<td colspan="2">Actinobacteria</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Actinomyces</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.13</td>
			<td>0.06</td>
			<td>0.30</td>
			<td>0.97</td>
			<td>.001</td>
			<td>.021</td>
		</tr>
		<tr>
			<td colspan="2">Rothia</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.05</td>
			<td>0.00</td>
			<td>0.13</td>
			<td>0.32</td>
			<td>.001</td>
			<td>.009</td>
		</tr>
		<tr>
			<td colspan="2">Bacteroidetes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Prevotella</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.22</td>
			<td>0.15</td>
			<td>1.02</td>
			<td>2.44</td>
			<td>.000</td>
			<td>.006</td>
		</tr>
		<tr>
			<td colspan="2">Firmicutes</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Other (Bacilli)</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.06</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.10</td>
			<td>.706</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Staphylococcus</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.12</td>
			<td>0.00</td>
			<td>0.07</td>
			<td>0.17</td>
			<td>.548</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">(Gemellaceae)</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.07</td>
			<td>0.12</td>
			<td>0.77</td>
			<td>1.24</td>
			<td>.005</td>
			<td>.091</td>
		</tr>
		<tr>
			<td colspan="2">(Lactobacillales)</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.00</td>
			<td>0.12</td>
			<td>1.50</td>
			<td>.004</td>
			<td>.073</td>
		</tr>
		<tr>
			<td colspan="2">Granulicatella</td>
			<td>0.09</td>
			<td>0.23</td>
			<td>0.37</td>
			<td>0.64</td>
			<td>1.59</td>
			<td>2.28</td>
			<td>.000</td>
			<td>.003</td>
		</tr>
		<tr>
			<td colspan="2">Lactobacillus</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.18</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>.700</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Other (Streptococcaceae)</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.13</td>
			<td>0.00</td>
			<td>0.10</td>
			<td>0.19</td>
			<td>.768</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">(Streptococcaceae)</td>
			<td>0.04</td>
			<td>0.10</td>
			<td>0.23</td>
			<td>0.12</td>
			<td>0.37</td>
			<td>0.69</td>
			<td>.005</td>
			<td>.083</td>
		</tr>
		<tr>
			<td colspan="2">Streptococcus</td>
			<td>53.42</td>
			<td>61.96</td>
			<td>88.38</td>
			<td>48.44</td>
			<td>60.83</td>
			<td>73.97</td>
			<td>.055</td>
			<td>.930</td>
		</tr>
		<tr>
			<td colspan="2">Other (Lachnospiraceae)</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.01</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.11</td>
			<td>.003</td>
			<td>.058</td>
		</tr>
		<tr>
			<td colspan="2">Dialister</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.04</td>
			<td>.240</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Veillonella</td>
			<td>3.43</td>
			<td>27.15</td>
			<td>42.78</td>
			<td>6.69</td>
			<td>13.38</td>
			<td>27.05</td>
			<td>.157</td>
			<td>1.000</td>
		</tr>
		<tr>
			<td colspan="2">Proteobacteria</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Haemophilus</td>
			<td>0.00</td>
			<td>0.00</td>
			<td>0.03</td>
			<td>0.31</td>
			<td>0.84</td>
			<td>2.38</td>
			<td>.000</td>
			<td>.008</td>
		</tr>
		<tr>
			<td colspan="10">#p-value Wilcoxon signed-rank test adjusted according Bonferroni correction</td>
		</tr>
		<tr>
			<td colspan="10">A p&lt;0.0026 was considered significant</td>
		</tr>
	</tbody>
</table>
Table 1: Statistical analysis. Differences in the oral microbiome of healthy children at two points in time. Data generated with 454-pyrosequencing. Image modified from Klug et al.23

Watch this Scientific Journal Video about Oral Biofilm Sampling for Microbiome Analysis in Healthy Children at JoVE.com

Oral Biofilm Sampling for Microbiome Analysis in Healthy 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-sampling-for-microbiome-analysis-in-healthy-children

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JoVE Journal

Medicine

17.0K Views.  Medical University of Graz. Veranderingen in de mondelinge microbiome tijdens jeugd zijn van toenemende belangstelling. Vergelijking van verschillende microbiome studies blijkt een gebrek aan gestandaardiseerde bemonstering protocollen. Beperkte ruimte maakt het geluid subgingivale Sulcus (hersenanatomie) van kinderen uitdagende bemonstering. Papieren punt bemonstering wordt hier in detail gepresenteerd als de methode van keuze voor dit gebied.

Video: Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

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 ...

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 ...

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

Breath Collection from Children for Disease Biomarker Discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

Chemistry

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

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.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

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.

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 ...

Biology

Specimen Collection and Analysis of the Duodenal Microbiome

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut microbiome has been typically studied through fecal specimen collections. The ease of obtaining fecal samples has resulted in many studies that have revealed information concerning the distal luminal gastrointestinal tract. However, few studies have addressed the importance of the microbiome in the proximal gut. Given that the duodenum is a major site for digestion and absorption, its microbiome is relevant to nutrition and liver disease and warrants further investigation. Here we detail a novel method for sampling the proximal luminal and mucosal gut microbiome in human subjects undergoing upper endoscopy by obtaining duodenal aspirate and biopsies. Specimen procurement is facile and unaffected by artifacts such as patient preparatory adherence, as might be the case in obtaining colonic samples during colonoscopy. The preliminary results show that the luminal and mucosal microbiomes differ significantly, which is likely related to environmental conditions and barrier functions. Therefore, a combination of duodenal aspirate and biopsies reveal a more comprehensive picture of the microbiome in the duodenum. Biopsies are obtained from the descending and horizontal segments of the duodenum, which are anatomically close to the liver and biliary tree. This is important in studying the role of bile acid biology and the gut-liver axis in liver disease. Biopsies and aspirate can be used for 16S ribosomal RNA sequencing, metabolomics, and other similar applications.

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

Shifts in the microbiome have been correlated with the physiology and pathophysiology of many organ systems both in humans and in mouse models. The gut ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

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