Name: a method to assess bacteriocin effects on the gut microbiota of mice
Uploaded: 2017-07-25T15:00:00
Description: Watch this Scientific Journal Video about A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice at JoVE.com

作者希望感谢EEA授予NILS科学与可持续发展研究人员协调流动性（参考017-ABEL-CM-2013）。 CB和GP-M。得到西班牙经济和竞争力部的AGL2015-70487-P拨款的支持。 OCOU和DBD得到挪威生命科学大学（NMBU）的食品科学研究战略奖学金计划（项目1205051025）的支持。我们还要感谢Inmaculada Noguera对动物护理和抽样的帮助，以及Jesus Dehesa的帮助，确保在动物设施中提供实验室材料。我们也赞赏拉斯古斯塔夫·西尼潘教授的统计建议。

护理和处理必须在专门的动物保健单位进行。此处描述的程序由巴伦西亚大学和地方当局相应的伦理委员会批准，遵循欧盟法律和2010/63 / EU强制实验动物护理的原则和西班牙政府关于动物保护的第53/2013号用于科学目的，以便在动物实验（替代，减量，细化）中遵守3R原则。 1.用于接种小鼠的冷冻细菌培养物<ol><li>在5 mL脑心脏输注（BHI）培养基中接种LAB的单个菌株，并在30℃?...

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这里描述的程序已被用于确定微生物群体的变化是否与健康或年龄有关。方案的不同部分很重要，其中抽样粪便，选择要测序和分析的DNA片段，进行DNA提取和生物信息学分析肯定是最关键的。取样至关重要，因为道德上的原因，小鼠不应该受到压力，因为知道改变肠道细菌的比例。样品必须尽快加工或应冷冻使用，因为一些细菌对脱水和/或氧气非常敏感。从扩增的16S rRNA基因中选择要测序的DNA片?...

细菌素是由大范围的细菌物种1,2的生产抗微生物肽。这些化合物和它们的生产者，尤其是LAB，已探索和几十年来他们在食品保鲜，医药3的潜在应用全球开发。已知有几种细菌素杀死重要的病原体，包括李斯特氏菌，肠球菌属，葡萄球菌属和芽孢杆菌属 。一些细菌素甚至具有调节免疫反应的能力4 。许多细菌素具有相对较窄的光谱，这在一些应用中是非常受赞赏的。例如，可以使用一些窄谱细菌素来指导针对所选择的有问题细菌群体的特异性活性，而不会对分享相同利基的共生或有益菌群产生太大的干扰;这在肠道环境中尤其重要onment，其中有大量的有益微生物中的交互动态方式5茁壮成长。细菌素也可用于预防或益生菌使用非常有吸引力的，因为它们可以抑制病原体，pathobionts，或机会致病菌可能不平衡肠道稳态6,7（下）的生长。 在其性质和物理化学性质方面，细菌素是非常多样化的，因为它们具有不同的结构，靶特异性，作用方式等。大多数细菌素已经在体外设置中进行了非常详细的研究 ，但很少已经在食品中进行了测试矩阵8,9或在体内，如在动物肠道6,10。由于复杂性o， 在体内评估时体外性质可能会有很大的不同肠道环境，以及对有益细菌的推测意外的影响。大多数益生菌是LAB。它们产生一系列其他代谢物，包括已知会影响宿主生理学的短链脂肪酸，以及显示针对某些细菌的抗微生物特性。因此，在产生细菌素的益生菌菌株的情况下，最好建立现实的检测方法，例如具有正常微生物群的健康动物。 在本研究中，我们提供了一种策略，可以评估不同细菌素生产菌株（其细菌素具有不同抑制谱）对健康小鼠的影响。我们的策略包括喂养具有同基因非细菌素突变体的小鼠，这使得能够将细菌素介导的作用从非细菌素介导的作用分化。测序16S rDNA允许跟踪肠道中细菌群体​​的动态变化。替补均衡统计分析破译细菌物种之间的相关性，以及细菌物种与测量的生理参数（ 例如体重，血清生化参数等 ）之间的相关性。我们认为本研究中提出的方案也适用于活体动物中细菌素研究以外的其他益生菌或益生元应用。

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Balb/c mice (female)</td>
			<td style="width:141px;">Harlan</td>
			<td style="width:183px;"></td>
			<td style="width:287px;">Mice should be 6 - 8 weeks of age</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic Petri dish</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:183px;">101VR20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brain-Heart-Infusion broth</td>
			<td style="width:141px;">Conda</td>
			<td style="width:183px;">1400.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">European Bacteriological Agar</td>
			<td style="width:141px;">Pronadisa</td>
			<td style="width:183px;">1800.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose D1 Low EEO</td>
			<td style="width:141px;">Pronadisa</td>
			<td style="width:183px;">8010.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1XTAE buffer</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:183px;">15558042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MRS broth</td>
			<td style="width:141px;">Difco</td>
			<td style="width:183px;">288130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS tablets</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">P4417-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scale</td>
			<td style="width:141px;">Mettler Toledo</td>
			<td style="width:183px;">PB602-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile forceps</td>
			<td style="width:141px;">Levantina de Laboratorios S.L.</td>
			<td style="width:183px;">260-3710014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:141px;">Eppendorf</td>
			<td style="width:183px;">5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:141px;">Hermle</td>
			<td style="width:183px;">Z383K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:141px;">AppliChem Panreac</td>
			<td style="width:183px;">121659.1211</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Realpure SSS Kit</td>
			<td style="width:141px;">Real Life Science Solutions, Durviz, Spain</td>
			<td style="width:183px;">RBME04 (300 ml)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropanol</td>
			<td style="width:141px;">AppliChem Panreac</td>
			<td style="width:183px;">131090.1611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:141px;">AppliChem Panreac</td>
			<td style="width:183px;">131086.1214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit fluorometer</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:183px;">Q32851</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AMPure XP beads</td>
			<td style="width:141px;">Beckman Coulter Genomics, USA</td>
			<td style="width:183px;">A63881 (60 ml)</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PerfeCta NGS library quantification kit</td>
			<td style="width:141px;">Quanta BioSciences, Maryland, USA</td>
			<td style="width:183px;">733-2300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MiSeq v3 reagent kit</td>
			<td style="width:141px;">Illumina, San Diego, California, USA</td>
			<td style="width:183px;">MS-102-3003</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Primers for 16S rRNA gene amplification</td>
			<td style="width:141px;"></td>
			<td style="width:183px;"></td>
			<td style="width:287px;">Primers contain V3-V4 region of bacterial 16S rRNA gene and Illumina overhang adaptors:5&#8217;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3&#8217; and 5&#39;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nextera XT Index kit</td>
			<td style="width:141px;"></td>
			<td style="width:183px;">FC-131-1002</td>
			<td>Indices and Illumina sequencing adaptors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropestle for 1.5 ml tubes, Eppendorf / Sigma , Ref.</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">Z317314-1PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass beads, 0.1 mm diameter</td>
			<td style="width:141px;">Biospec Products</td>
			<td style="width:183px;">11079-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoSpin Gel and PCR Clean-up Kit</td>
			<td style="width:141px;">Macherey-Nagel</td>
			<td style="width:183px;">740609.25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Omni Bead Ruptor 24</td>
			<td style="width:141px;">Omni International Inc.</td>
			<td style="width:183px;">19-040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mutanolysin</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">M9901-10KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lysozyme</td>
			<td style="width:141px;">Roche</td>
			<td style="width:183px;">10837059001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">proteinase K</td>
			<td style="width:141px;">Roche</td>
			<td style="width:183px;">3115887001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase A</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">R4875</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to assess bacteriocin effects on the gut microbiota of mice

随着我们对肠道微生物群落组成的了解和与健康的关系，特别是有助于维持人口平衡的因素，我们提出了非常有趣的问题。然而，有限的可用方法来评估这些因素。细菌素是由许多细菌产生的抗微生物肽，可以赋予食物获取和/或利基建立的竞争优势。许多益生菌乳酸菌（LAB）菌株通过防止病原体的生长具有很大的潜力来促进人类和动物的健康。它们也可用于免疫调节，因为它们产生细菌素。然而，细菌素的拮抗活性通常通过实验室生物测定来确定，与细菌面对来自宿主和数百种微生物物种的多因素影响的人类和动物中的复杂肠道环境相比，在明确定义但过度简化的条件下讨论同样的利基。这项工作描述了一个完整和有效的评估效果的程序 在鼠系统中具有不同靶特异性的各种细菌素。使用组合16S rDNA测序监测细菌治疗期间微生物群组成的变化。我们的方法使用细菌素生产者和其同基因非细菌素产生突变体，后者具有区分细菌素相关的微生物群与非细菌素相关的修饰的能力。粪便DNA提取和16S rDNA测序方法是一致的，并且与生物信息学一起构成了在细菌谱中发现微弱变化并在细菌群体和健康标记之间建立胆固醇和甘油三酯浓度方面的相关性的强大程序。我们的协议是通用的，因此可用于研究其他化合物或营养物质，以改变主机麦克风在研究毒性或有益效果时，可以使用robiota组合。

细菌素的生产被认为是LAB中的益生菌特征，因为它被认为是防止机会性细菌和病原体的生长。这项工作的目的是显示细菌素在小鼠模型中调节肠道微生物群体的能力。为此，开发了一种程序来比较细菌素生产菌株及其同基因非产生菌株的摄入效果。接种的方法和测定的时间延长如图1所示。将小鼠分为十一个笼子：没有任何治疗的对照，用细菌素生产...

Watch this Scientific Journal Video about A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice at JoVE.com

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

评估细菌素对小鼠肠道微生物的影响的方法

细菌素被认为在定义不同生态位的微生物多样性方面发挥关键作用。在这里，我们描述了一种有效的方法来评估细菌素如何影响动物模型中的肠道微生物群组成。

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Research

JoVE Journal

Biology

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

从白蚁肠道微生物（Zootermopsis Angusticollis）中提取的DNA和可视化肠道微生物

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

科研

生物学

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

全自动毛细 Electrophoretic-based 免疫分析方法的程序和关键优化策略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

小鼠间间间禁食的代谢效应评估 2：1 间歇禁食

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice

优化经食管心房起搏以评估小鼠心房颤动易感性

Mouse models of genetic and acquired risk factors for atrial fibrillation (AF) have proven valuable in investigating the molecular determinants of AF. ...