Name: oral biofilm sampling for microbiome analysis in healthy children
Uploaded: 2017-12-31T15:00:00.000+00:00
Duration: 10 min 42 s
Description: Watch this Scientific Journal Video about Oral Biofilm Sampling for Microbiome Analysis in Healthy Children at JoVE.com

作者感谢 Theussl (格拉茨医科大学医学研究中心) 在视频制作方面的优秀技术援助, 并向莫尼卡-法雷尔 (格拉茨医科大学研究管理) 作为配音人才。

格拉茨医科大学机构审查委员会 (Votum 27-126ex14/15) 批准了礼宾和视频拍摄。此视频出版物的书面同意书是从孩子和她的父母获得的。
1. 仪器和材料
<ol>
	<li>切割校准 (ISO 015/02) 和无菌纸点, 通常用于牙髓治疗, 在第一环标记, 以规范其长度 (图 1)。</li>
	<li>应用 UV 光照射在 260 nm 30 分钟, 以避免 DNA 和 RNA 污染的纸张点 (图 2)。</li>
	<li>蒸压管在120° c 和1.2 酒吧为存贮在 20 min 和紫外-照射他们以及或使用可利用的伽玛辐照管。</li>
</ol>
2. 专题的编写
注: 书面知情同意是从孩子和她的父母在入学前获得的。
<ol>
	<li>把可可黄油涂在嘴唇上。</li>
	<li>安装脸颊和舌牵引器, 完全进入两个牙科拱门。</li>
	<li>在牙菌斑上染色。</li>
	<li>卸下干燥的现场设备。</li>
	<li>用水冲洗嘴, 直到水无色。</li>
	<li>用电刷在45°角度彻底刷牙。只用水, 但不使用牙膏, 因为这将改变口腔生物膜。</li>
	<li>再装上干场仪, 包括舌部保持口张开和干燥。</li>
	<li>用无菌棉签清洁和干燥索引牙, 避免在纸点取样时吸收龈液。</li>
</ol>
3. 生物膜取样
<ol>
	<li>龈下沟的纸点取样
	<ol>
		<li>用无菌牙科镊子抓纸点。</li>
		<li>将纸点切至 4 mm 的定义长度。特别注意不要伤害连接上皮 (图 3)。</li>
		<li>二十年代后移除纸张点。</li>
		<li>将纸点直接收集到准备好的管子中: 将纸点尖端直接放在无菌和无 DNA 的瓶子中, 并将其在第三个标记处切割为 4 mm 的标准长度 (图 4)。</li>
		<li>如果由研究协议指示, 在同一站点 (图 5A) 并行插入两个纸点。</li>
		<li>或者, 如果研究协议需要 "汇集样本" (图 5B), 则从多个站点收集纸张点。</li>
		<li>取出干场装置进行黏膜和唾液取样。</li>
	</ol>
	</li>
	<li>黏膜纸点取样
	<ol>
		<li>将纸张点应用于上颊的前庭褶皱 (图 6)。</li>
		<li>闭上脸颊, 简单地按摩一下。</li>
		<li>在二十年代以后打开面颊并且去除纸点。</li>
		<li>在第三个标记处切纸点, 并将其放置在无菌管中, 如龈下取样所示。</li>
	</ol>
	</li>
	<li>唾液取样
	<ol>
		<li>让孩子吐出刺激唾液到一个灭菌的收集容器 (图 7)。</li>
	</ol>
	</li>
</ol>
4. 转让和储存
<ol>
	<li>根据研究设计 (图 8), 收集纸点作为单个、池或并行示例。</li>
	<li>应用 color-coded 存储系统以便于进一步的示例管理 (图 9)。</li>
	<li>最后, 将样品存放在−80° c 等待微生物分析。</li>
</ol>

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+a+healthy+oral+microbiome+through+metagenomics.">Identifying a healthy oral microbiome through metagenomics.</a> Clin Microbiol Infec. 18 (11), 54-57 (2012).</li><li>Dewhirst, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Oral+Microbiome.">The Human Oral Microbiome.</a> J Bacteriol. 192, 5002-5017 (2010).</li><li>Keijser, B. J. 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作者报告没有与这一科学视频制作相关的利益冲突。

在口腔内的所有部位都有细菌24,25,26。许多研究集中在使用唾液作为取样媒介来映射口腔定植, 因为它可以很容易地通过随地吐痰27,28,29,30,31, 32。然而, 唾液生物膜并不能反映龈下生物膜的组成。因此, 使用其他抽样方法是至关重要的创造整个图片, 并将唤起新的讨论, 在牙科研究。几篇文章比较了刮和纸点对周病患者龈下取样的使用情况8,33,34。然而, 目前还没有一个研究小组关注于对不同年龄组的标准化取样设备的应用, 特别是对儿童19。
在这段视频中, 我们介绍了口腔生物膜在健康儿童中的三口腔生境的取样。
修改和故障排除:
本论文的目的是对健康儿童龈下生物膜取样的临床方案进行研究。选择的方法是指在有限的空间使取样特别具有挑战性的声音龈下沟。在这个视频中, 该方法适用于早期恒牙。它也可以应用于混合或成熟的牙列, 以及儿童和/或成人。我们的抽样方法允许修改, 如单一或汇集样本。后者可能成为一个关键因素的分子分析, 由于少量的 DNA 收集从健康的龈下沟。可根据需要修改指标牙的选择;特别是, 另一颗牙齿可以作为替代品, 当上皮细胞受到创伤和出血, 从而使纸张的点不可用。平行抽样使用两个纸点同时在一个站点上呈现样本复制的一个步骤, 除了缩短儿童的椅子时间。
与轻微的修改病牙周袋可以取样。这里的纸点将需要插入到口袋的全长, 这可以达到10毫米。纸张的长度和圆锥, 以及插入深度必须适应的口内栖息地的利益, 但应始终是标准化的材料和尺寸。样品也可以从粘膜上使用纸点。相同的协议允许比较不同的口腔生境和各种牙科材料。病人准备和样品存储可以按照本视频中的描述进行。对于 metatranscriptome 的研究, RNA 可以用同样的方法取样。因此, 在取样后, 应直接在 RNA 稳定液中插入纸点。
技术的局限性:
无论如何修改, 我们描述的临床取样方法仅限于龈下沟。其他抽样地点, 例如周病的口袋, 不是我们的研究设计的一部分。由于需要对这些口袋的全部深度进行取样, 纸张点可能不够大, 无法充分收集样本。如果实验目的是比较从龈下沟和周病囊取样的数据, 重要的是要知道与不同的临床取样方法相关的固有偏见。因此, 不宜比较这些数据。
在这里提出的方法的一个限制是抽样不使用碘 monoazide。在取样后直接添加该剂, 可以对生物膜中的活细胞进行具体分析。这里所描述的取样方法反映了活细胞和死体细胞的数量。对于严重牙周炎的研究, 纸点可能需要被刮取代, 因为在深口袋里的生物膜形成强烈。在这些情况下, 纸点取样可能无法反映整个微生物谱, 因为它们的表面可能饱和得太快。
关于现有方法的意义:
本文提出的原稿是在健康沟中用纸点规范龈下取样的第一种方法。其他报告提到使用金属刮35、36或纸张点37、38、39, 但没有描述在取样之前发生的过程, 如作为斑块控制, 牙齿清洁, 牙齿隔离, 干燥, 以及由于取样技术和时间线的规格不足而导致的过程。
在前两篇文章中, 我们表明, 使用纸点是一种可重现的方法龈下生物膜取样在儿童22,40。由于其设计, 刮太大, 对于狭窄的沟, 空间有限, 对嫩连接上皮来说过于尖锐。这是至关重要的, 进入龈下沟没有创伤的上皮。这样, 避免了因出血引起的偏倚。因此, 使用薄和招标的纸张点是首选的这一领域。纸点功能, 以监测在正畸治疗过程中生物膜组成的变化21,41。他们是苗条和足够灵活的, 以适应之间的固定矫治器具的元素。这是一个主要的优势, 其他抽样方法, 如刮。创伤取样简化, 少量的 DNA 取样是可能的。
未来应用:
在这个视频中, 我们展示了 non-diseased、早、恒牙列的方法。它也可以用于混合或成熟的牙列。随着一些适应, 它是可能的样本周生病的网站, 如牙齿或植入物和其他利基由于牙科材料遵循相同的协议。龋病研究可以受益于这一标准化的协议, 特别是对根龋的研究。从我们的采样视频最重要的讲座是标准化的协议, 使数据可比较, 无论是什么和如何测量样本。未来的视频演示可以提高临床取样领域的总体情况。
如录像所示的口腔生物膜用于诊所和科学调查。这个体内方法代表了一个良好的除了体外分子技术, 如荧光原位杂交和共焦激光扫描显微镜15。NGS 方法, 如序所示, 适用于收集的生物膜, 允许分析完整的微生物。计算某些细菌种类的相对丰度可用于支持治疗或比较不同的患者或不同的治疗方法。与我们先前描述的40的标准化排序协议和序列分析工作流一起, 此抽样方法允许序列分析下至属级别。进一步 "元"-研究例如 metatranscriptomic 或代谢研究是可能的根据在这个录影提供的抽样协议。
关键步骤:
避免污染是一个关键的步骤: 不育仪器必须应用在一个消毒的在体内环境。从口腔到工作台的转移必须由有经验的考官快速而安全地进行, 以便尽可能缩短学习参与者的时间。协议中最关键的步骤是将纸点创伤插入龈下沟。连接上皮和出血的破坏绝对必须避免。为了不沾染带有外源 DNA 或 RNA 的纸点和储存瓶, 必须进一步注意。存储本身也是一个关键步骤。样品需要直接冷冻, 至多在−80° c。这避免了细菌组成后取样的变化, 这将伪造测序结果。

人体口腔生物膜包括主要由 commensals 和有益微生物组成的广泛群落1,2,3。在这里找到的物种殖民地的所有利基, 口腔提供4,5,6。在这些壁龛中, 生物膜的组成变化与生境一样广泛。唾液例如显示不同的细菌外形比匾样品。在健康成人的斑块样本中,放线菌的相对丰度超过 20%, 而唾液中却发现少于7%。Bacteroidetes和厚的对比度在唾液7中出现的数字要高得多。因此, 它是重要的样本在不同的位置, 在口中, 以获得整个图片。此外, 不同的因素, 如地理和种族差异, 年龄, 性别, 和许多其他因素, 使得很难确定一般规则的生物膜发展和疾病发作8,9。多年来, periopathogenic 和龋生物膜的研究一直是一个中心问题8,10,11,12,13。
近年来, 对健康学科的研究不仅对疾病的广泛了解, 而且对预防措施的实施也具有重要意义14。新技术和分子分析进一步阐明口腔生物膜的形成和功能15,16, 并启用微生物多样性的完整分析17,18。这也将导致对正畸治疗期间的微生物变化的新理解19。对正畸生物材料的发展的影响是可预见的。增强的视角将会对与生物膜有关的正畸治疗的并发症产生新的认识, 如釉质脱矿和牙周疾病12,20,21。为了允许世界范围内的数据比较, 关键是要标准化数据生成的所有步骤。实验室程序的细微变化会对结果产生强烈影响。此外, 在数据处理中使用不同的计算平台可以导致 non-comparable 数据集。最后, 统计检验和修正的选择对结果有影响。然而, 在任何实验室工作或生物计算开始之前, 取样偏差已经发生很长时间了。非标准化抽样方法导致了一项固有的偏颇研究。鉴于相关研究中的标准化程度低到中等水平, 正畸与微生物的关系有很小的证据19。因此, 标准化方法的发展将有助于对实地数据进行合格的比较。
本文介绍了标准化的口腔生物膜取样程序。该议定书旨在促进产生全球可比的微生物序列数据的收集。介绍了一种用于健康儿童口腔生物膜取样的 step-by 步协议。作为选择的方法 , 纸点入 atraumatically 入声音龈下沟。为了便于生境比较, 根据同一协议对颊粘膜进行取样。本文还演示了并行和池式取样。此外, 唾液取样显示。还提出了一个简单的 color-coded 运输和存储系统, 便于标本管理, 进一步处理。关于存储介质、基因分析和生物信息学的下游操作选择取决于口腔研究中不同领域提出的临床问题。在这份手稿中, NGS 的 DNA 取样被选为一个可能用于正畸研究的例子。

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oral biofilm sampling for microbiome analysis in healthy children

口腔生物膜及其分子分析为调查各种牙科研究和临床问题提供了依据。对生物膜组成的认识, 可以更好地了解龋和 periopathogenic 机制。在儿童时期口腔内发生的微生物变化有几个原因引起了关注。儿童口腔微生物的演变和其组成的变化需要进一步分析, 以了解和可能预防疾病的发病。同时, 还需要对口腔生物膜的天然成分进行深入的了解。早期的无龋永久牙列与健康的牙龈提供了一个广泛不受影响的龈下栖息地, 可以作为一个原位基线, 研究口腔健康和疾病的特点。儿童口腔生物膜在生命不同阶段的分析是这一领域的一个重要课题。现代分子分析方法可以为生物膜的细菌多样性提供全面的信息。为了实现微生物数据的比较, 对分子数据生成过程中的每个步骤进行标准化是非常重要的。这个过程从临床取样, 下一代测序 (NGS), 生物数据处理, 到分类学解释。其中最关键的因素是生物膜取样。儿童取样更具挑战性, 特别是由于龈下地区的空间有限。因此, 我们的重点是使用纸点龈下取样。本文提供了一个详细的协议, 口腔生物膜取样的龈下沟, 黏膜, 和唾液的儿童。

在本刊物中, NGS 的 DNA 取样被证明是一个可能应用于正畸研究的例子。细菌 DNA 直接从纸点样品中提取。这种 DNA 可以用于临床或研究问题的微生物组成的研究 (如图 10中所总结的)。
现代分子分析方法, 如 NGS 方法 454-序概述了完整的微生物的样本。数千种细菌的 DNA 在不同的系统发育水平上在 16s rRNA 序列差异中同时被识别。需要生成生物平台, 以便将从大型数据集中检索到的信息构建成集群。统计分析可以应用到微生物数据集。
某些细菌种类的总相对丰度可以被分析, 因为由于生境条件的改变, 微生物可能发生变化。条形图 (图 11) 和 heatmaps (图 12) 在门级或按顺序显示龈下细菌成分。不同的个体和治疗可以通过描述和推断统计来比较。图 13给出了两种不同分类级别的龈下纸点取样模式的直方图比较。表 1显示了在比较两个时间点 (T1 和 T3) 的体外研究中生物膜组成的变化。统计显著的变化是计算与魏氏签名秩测试和以下 Bonferroni 校正从 454-序数据。
最后, 主坐标分析 (动脉) 使在操作分类学单元 (OTU) 级别的直接3D 比较能够在不同的样本中识别出来。在图 14中的动脉图形显示了龈下微生物在五儿童系列中的个体差异。图 15显示了正畸病例对照研究的结果: 粉红色到红色点的情况下, 光到深蓝色的点是控制, 所有取样重复超过四月。在正畸干预 (红点) 后, 明确的病例聚类是微生物群的一个转变。代表控制组的蓝点分布均匀。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56320/56320fig1.jpg"> 
图 1: 准备标准长度的纸张点.在第一个环标记处切割无菌校准的纸点, 以使其长度标准化。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56320/56320fig2.jpg"> 
图 2:从 DNA 中消毒和释放纸张点.标准化的纸张点是消毒和紫外线照射在一个干净的长凳。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56320/56320fig3.jpg"> 
图 3: 龈下纸张点插入.无菌校准的纸点入到龈下的沟中 , 直到 4 毫米圆环标记二十年代。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56320/56320fig4.jpg"> 
图 4: 将纸张切割成管子.直接取样后, 在第三环标记 (4 mm) 上将纸张点切割成无菌和无 DNA 的1.5 毫升瓶。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56320/56320fig5.jpg"> 
图 5: 池和并行取样。两个纸点同时插入到龈下沟的一个牙齿 (A) 或几个纸点到龈下沟的几个牙齿 (B)。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56320/56320fig6.jpg"> 
图 6: 粘膜取样.无菌校准的纸点被放置在前庭褶皱的粘膜上。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56320/56320fig7.jpg"> 
图 7: 唾液收集.刺激唾液被吐在一个无菌的烧杯里。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56320/56320fig8.jpg"> 
图 8: 取样方案.样品可以收集为单一 (左侧, 一颗牙齿), 汇集 (几个到所有条纹在一个小瓶), 或平行 (蓝色条纹, 两个纸点在一颗牙齿) 样本。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56320/56320fig9.jpg"> 
图 9:彩色编码存储一个参考色码为板料和小瓶促进进一步样品处理。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig9large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56320/56320fig10.jpg"> 
图 10: 龈下生物膜分析.临床生物膜取样 (A)、DNA 提取 (B) 和不同 NGS 技术 (C) 的示意图演示。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56320/56320fig11.jpg"> 
图 11: 条形图.用 454-序分析了门层细菌的相对丰度。从 Santigli et al.中修改的图像22 <a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56320/56320fig12.jpg"> 
图 12: 图.用 454-序分析了细菌数量的相对丰度。暗红色代表较高的相对丰度 (和 #62; 10%)。深蓝色代表一个非常低到没有各自细菌顺序的相对丰盈。从 Santigli et al.中修改的图像22 <a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56320/56320fig13.jpg"> 
图 13: 直方图.不同分类水平下两种纸点取样方式 (a 和 B) 的比较。用 454-序生成的数据。从 Santigli et al.中修改的图像22 <a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56320/56320fig14.jpg"> 
图 14: 2D 动脉图.一个案例系列 (n=5) 显示个体和个体的差异, 由于两个龈下取样方法 (1 颜色是1主题)。用 454-序生成的数据。从 Santigli et al.中修改的图像22 <a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig14large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/56320/56320fig15.jpg"> 
图 15: 3D 动脉绘图动画的快照.显示龈下菌群转移在正畸病例对照研究 (n = 16; 每组, 3 点的时间; 案例: 粉红色到红色, 控制: 光到深蓝色)。用 454-序生成的数据。工作组 Santigli 未公布的数据。<a href="//ecsource.jove.com/files/ftp_upload/56320/56320fig15large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">分类: Bacteriae</td>
			<td colspan="3">时间点 1 [%]</td>
			<td colspan="3">时间点 3 [%]</td>
			<td colspan="2">魏氏签名测试</td>
		</tr>
		<tr>
			<td colspan="2">门/属</td>
			<td>第二十五</td>
			<td>中</td>
			<td>第七十五</td>
			<td>第二十五</td>
			<td>中</td>
			<td>第七十五</td>
			<td>p-值</td>
			<td>p 值调整 #</td>
		</tr>
		<tr>
			<td colspan="2">其他</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">其他</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">放线菌</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">放线菌</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">菌</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">厚</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">其他 (杆菌)</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">葡萄球菌</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">乳酸</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">其他 (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">链球菌</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">其他 (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">嗜血</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">基于 Bonferroni 校正的 #p 值魏氏符号秩检验</td>
		</tr>
		<tr>
			<td colspan="10">p 和 #60; 0.0026 被认为是重要的</td>
		</tr>
	</tbody>
</table>
表 1:统计分析健康儿童口腔微生物在两个时间点的差异。用 454-序生成的数据。从斯科特·克鲁格et al.中修改的图像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

在整个儿童时期, 口腔微生物的变化越来越引起人们的兴趣。比较不同的微生物研究表明缺乏标准化的抽样协议。有限的空间使取样的儿童的声音龈下沟的挑战。本文详细地介绍了纸点取样作为该领域的选择方法。

口腔生物膜取样在健康儿童中的微生物分析

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

Research

JoVE Journal

Medicine

17.0K 次观看.  Medical University of Graz. 在整个儿童时期, 口腔微生物的变化越来越引起人们的兴趣。比较不同的微生物研究表明缺乏标准化的抽样协议。有限的空间使取样的儿童的声音龈下沟的挑战。本文详细地介绍了纸点取样作为该领域的选择方法。

视频: 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.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

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

科研

免疫与感染

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.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

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

医学

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.

细胞和生物膜细胞外成分的定量并发

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.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

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

生物工程

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.

儿童疾病生物标志物发现的呼吸收集

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

化学

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.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

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.

使用共聚焦显微镜监测跨王国生物膜中的细胞外pH值

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.

牙种植体不同材料的口腔生物膜形成

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.

从植物提取物和分数中识别新型抗菌和抗生物膜分子的系统方法，以防止牙科蛀牙

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

生物学

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 ...