Name: a method to assess bacteriocin effects on the gut microbiota of mice
Uploaded: 2017-07-25T15:00:00.000+00:00
Duration: 7 min 54 s
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℃下将其生长20-24小时（过夜）。 </li><li>通过将2mL过夜培养物转移到200mL BHI中，在BHI中每个细菌培养物稀释100倍。在30°C下生长20-24小时。 注意:本研究中使用的菌株列于表1。 </li><li>收获ce在4℃下以5,000×g离心20分钟。 </li><li>通过将每个细胞沉淀悬浮在100mL冰冷的磷酸盐缓冲盐水（PBS）中并除去上清液并洗涤细胞两次，并如步骤1.3一样收集细胞。 </li><li>第二次洗涤后，将每个细胞沉淀物悬浮于20mL冰冷的15％甘油的PBS溶液中。 </li><li>将细胞悬浮液等分至管中1 mL体积，然后将其储存在-80°C直至使用。 注意:细胞应在1-2个月后使用。 </li><li> 在储存之前和给药前确定细胞数量，以便知道给予小鼠的活细胞数。 <ol><li>对于细胞计数，在冰冷的0.9％氯化钠（NaCl）溶液中进行十倍连续稀释的细胞。将100μL每种稀释液扩散到BHI琼脂平板上。在30℃下孵育过夜（20-24小时）用于细胞生长和集落形成，后者确定集落形成单位（cfu）/ mL。 </li></ol></li><li>为了使含细菌的饮用水，将每个细菌储存培养物的细胞稀释在100mL无菌，过滤的饮用水中，最终平均cfu为10 9 / mL水。对于细菌生存评估，在细菌处理的前两周，在稀释后和24小时后每周一次计数活细胞。 </li></ol>小鼠分析和实验设计注意:本实验需要特异性无病原体（SPF）BALB / c幼小雌性小鼠（6-8周）这里共购买了100只动物。 <ol><li>在整个实验过程中，让老鼠免费进入沉淀的食物和水。 </li><li> 设计实验并计算功率。 <ol><li>如果每个治疗都有自己的控制，应用配对病例对照设计（t检验）。使用初步测定来计算要确定的最重要影响的平均值和标准误差。 </LI&gt; <li>使用不同的方法和免费获得的程序优化测定的力量和每组所需的动物数量11 。 </li><li>为了测试细菌素生产者与非生产菌株的影响，每个实验条件使用9只动物。 注意:该数字是根据实验标准误差确定的，并提供令人满意的功率（0.7 - 1.0）和显着性水平低于0.05。 注意:在给定的例子中，制作了11组小鼠，每笼1只。十个笼子对应于具有5个生物碱生产菌株和相应的5个同基因非产生菌株的处理;第11 个笼子有10只小鼠，构成未处理对照。 </li></ol></li><li>小鼠到达牧场设施后，将其分发到笼子里，并对动物进行耳标，以便进行个人跟踪。将小鼠适应饲养设施和条件一周之前roceeding。 </li><li>如步骤1.8所述，制备含细菌的饮用水，并将饮用水瓶放置在相应的独立笼子中，这些笼子被清楚地识别，使得每个笼子中的小鼠共享同一个水瓶。 注意:对照组必须保持在一个单独的笼子里，与被测细菌无接触。 </li><li>每天用新鲜饮用水（100 mL）准备一个新瓶，并加入新鲜除霜的细菌悬浮液。这样做连续15天。 </li><li>按照细菌处理两周的时间，在此期间，小鼠喝无菌水（ 图1 ）。 </li></ol> 3.收集样品<ol><li> 收集粪便样品。 <ol><li>准备高压灭菌的笼子（空的）和无菌镊子，并为每只小鼠标记无菌的1.5-mL管。 </li><li>在一天的同一时间收集样品，以避免在此期间微生物群组成的变化一天一天为获得最佳效果，请分别收集新鲜样品。 注意:优选地，早晨收集粪便，当小鼠倾向于自发排便时。 </li><li>用70％酒精清洁一个空的笼子，然后放入一只老鼠。使其排便，并使用无菌镊子收集2-4粪便颗粒，将其放入1.5 mL管中。 注意:有时只能将鼠标向上按住;然后可以将粪便从肛门直接收集到管中。 </li><li>在四周的实验中，每周一次收集每只小鼠的粪便样品（ 图1 ）。在小鼠接触细菌之前，先收集第一个样品（T0，时间零）。在第7,14,21和28天收集以下粪便样品。 </li><li>将样品在4℃冷藏，直到其运送到实验室，在-80℃冷冻。提前计划提供足够的储物箱，作为大量的fe在实验过程中将收集cal样品。 </li></ol></li><li>每周在粪便收集日称重所有老鼠。 </li><li>在第 15天，细菌给药的最后一天从所有小鼠收集血液样品，并测定其中甘油三酯，总胆固醇，高密度脂蛋白（HDL）和低密度脂蛋白（LDL）的水平血清。 注意:经验丰富的人员应收集血液以减轻动物的压力。 </li></ol> LAB计数和细菌素活性<ol><li>称量每个样品的一个粪便颗粒在1.5 mL管中，并加入足够体积的冰冷的0.9％NaCl以达到10％（w / v）溶液。使用无菌微塞子1.5 mL管，使粪便悬浮液均质化，并在冰冷的0.9％NaCl中制备十倍连续稀释液。 </li><li> 计算LAB细胞的总数。 <ol><li>将稀释的细胞（100μL）转移到4mL预热的Ma中n Rogosa Sharpe（MRS）软琼脂（50℃，0.8％琼脂）。在将细胞倒入固体MRS琼脂平板（1.5％琼脂）之前通过涡旋混合。 </li><li>通过在无菌罩中取盖5 - 10分钟干燥板，30℃孵育细胞20-24小时，用于细胞生长和集落形成。 </li></ol></li><li> 计数细菌素生产细胞。 <ol><li>将细菌素生产者的稀释细胞（100μL）转移到4mL预热的MRS软琼脂（50℃，0.8％琼脂）中，通过涡旋混合，并将细胞倒入MRS琼脂平板（1.5％琼脂）上;该层被称为第一层。 </li><li>将另外4 mL无细胞的MRS软琼脂（0.8％琼脂）转移到第一层上，将所有细胞嵌入软琼脂中。 注意:此层旨在防止细胞在琼脂平板表面上作为菌落生长。在顶部注入指示剂细胞时，表面生长的细胞容易移位（步骤4.3.4）和因此会使结果的解释复杂化。该层被称为第二层。 </li><li>在无菌罩中盖上盖子5-10分钟，然后在30℃下孵育20-24小时以进行细胞生长和集落形成，干燥板。 </li><li>为了鉴定产生细菌素的菌落，将40μL适当的指示菌株的过夜培养物混合，如表1所示，与4mL预热的软琼脂。将混合物倒入板上;该层被称为第三层。 </li><li>在无菌罩中盖上盖子5-10分钟，然后在30℃下孵育20-24小时以进行细胞生长和集落形成，干燥板。 注意:细菌素生产菌落在菌落周围有明显的（抑制）区域（ 图2 ）。 </li></ol></li></ol> 5. DNA提取，16S rDNA扩增和测序注意:步骤f或DNA提取描述为使用商业试剂盒（ 例如， Realpure"SSS"试剂盒）。 <ol><li>在300μL裂解液中悬浮1〜2只粪便小鼠（〜200mg）。 </li><li>将样品转移到其中预先放置约0.5g玻璃珠（直径:0.1mm）的2mL微量管中。 </li><li>向每个样品加入1μL10 U /μL的溶菌酶和2μL20 mg / mL的溶菌酶，37℃孵育40 - 60 min。继续从粪便样品中提取DNA的标准方案之一12 。 </li><li>应用两个循环的搅拌步骤，每次1分钟。 </li><li>加入2μL蛋白酶K并充分混合。 </li><li>孵育20分钟，每5分钟涡旋。 </li><li>将样品冷却至37℃，加入1μL5μg/ mL RNA酶A至样品。彻底混合</li><li>在37℃下孵育30-60分钟。 </li><li>将样品冷却至室温，加入180μL的蛋白质沉淀溶液。以最大速度剧烈旋涡20-30秒。 </li><li>以16,000 xg离心5分钟。如果上清液中有任何颗粒漂浮，将样品在冰中孵育5分钟，然后再次离心。 </li><li>将含有DNA的上清液转移到含有300μL异丙醇的新型1.5 mL微量管中。 </li><li>通过倒置管20-30次混合，并在-20℃下孵育1小时。或者，将管子孵育一段时间（ 例如，过夜）。 </li><li>以16,000 xg离心2分钟。 </li><li>取出上清液，并将吸收纸上的管干燥。加入300μL70％乙醇洗涤DNA沉淀。 </li><li>以16,000 xg离心1分钟，除去上清液，使颗粒干燥约15分钟。 </li><li>一旦颗粒干燥，加入100μL无菌水或Tris（10 mM）/ EDTA（1 mM）缓冲液，并重新悬浮沉淀。 </li><li>消除任何可能的抑制化合物在随后的步骤中，清洗DNA。将提取的DNA与2体积（200μL）的缓冲液NTI混合以调节步骤5.15的结合条件。 </li><li>将含有PCR清洗柱的二氧化硅膜放入收集管（2mL）中并加载样品。 </li><li>以11,000 x g离心30秒。丢弃流通。 </li><li>用700μL缓冲液NT3洗涤二氧化硅膜，重复步骤5.16。 </li><li>通过以11,000 xg离心1分钟干燥二氧化硅膜，以除去任何残留的含乙醇洗涤缓冲液NT3。 </li><li>将柱转移到新的1.5-mL微管，并在70℃下孵育2-5分钟以完全除去乙醇。 </li><li>加入30μL的PCR级水，并在70℃下孵育5分钟。通过在11,000×g离心1分钟洗脱。 </li><li>使用UV / Vis分光光度计或荧光测定方法对DNA进行定量。 </li><li>正常化并汇集来自ea共享同一笼的小鼠的DNA样品<html> ch时间点。 </li><li>在时间上观察个体差异，在第0,14和28天，来自对照的3只随机选择的小鼠和两只其他随机笼中的序列DNA样品。 </li></ol> 16S rRNA基因扩增和测序<ol><li>准备扩增的16S rRNA基因库。使用具有适当突出适配器的正向和反向引物扩增细菌16S rRNA基因5的V3-V4区域: 5&#39;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3&#39; 5&#39;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3&#39; </li><li>使用1％琼脂糖凝胶电泳检查扩增的条带。 </li><li>使用磁珠清洗聚合酶链反应（PCR）产物用于扩增子纯化。 </li><li>按照制造商的说明，按照使用的大规模测序平台进行以下步骤&gt; 6。 </li></ol>数据分析与统计<ol><li> 质量过滤（通常在排序设施中进行）。 <ol><li>使用设备提供的软件过滤原始读取数据和解复用。 </li><li>使用USEARCH 14 （版本7.0.1090）中实现的UPARSE管道13处理配对端读取。合并配对末端，并使用最大预期误差（maxee）值1.0进行质量过滤。舍弃小于150 nt的序列。重排序列丢弃单身人士。 </li><li>将序列分成具有97％序列同一性的操作分类单位（OTU），并使用UCHIME 15过滤嵌合序列。 </li></ol></li><li> OTU采摘，分类学分配和系统发育重建，以及多样性分析和可视化。 <ol><li>使用Quantitative Insights Int进行OTU处理o微生物生态学（QIIME） 16 。 </li><li>使用最小同一性为75％（默认值）的PyNAST 18 ，使用Greengenes核心集数据库17选择并对齐代表性的OTU。 </li><li>使用信度为0.8的核糖体数据库项目（RDP）分类器程序19将分类法​​分配给对齐的序列。 </li><li>将OTU表规范化为具有最低序列深度的样本的序列计数。 </li><li>在过滤步骤之后使用Fast Tree 20从对齐序列构建系统发生树，以便去除高度可变的区域和全部空白位置。使用该树来计算alpha和β多样性，并计算稀疏曲线和香农指数。 </li><li>要生成和可视化未加权的UniFrac距离度量21 ，请使用主要坐标分析（PCoA）。 注意:对于s可以使用不同的软件包，如R 22或QIIME 16中实现的统计工具。 </li><li>为了比较不同处理之间香农多样性指数，使用ANCOVA，考虑时间作为R中的连续因变量。 </li><li>使用双侧学生t检验比较QIIME中PCoA的治疗距离，并使用Bonferroni校正计算1000个蒙特卡罗置换的非参数p值。 </li><li>使用Pearson相关分析，分析特定OTU的相对丰度与其他测量参数（如血清胆固醇水平，HDL，LDL 等）之间的相关性。为此，使用CoNet 23随后使用Cytoscape 3.1.1 24相关网络可视化。 </li></ol></li></ol>

<ol>
	<li>Papagianni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomally+synthesized+peptides+with+antimicrobial+properties:+biosynthesis,+structure,+function,+and+applications.">Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications.</a> Biotechnol Adv. 21 (6), 465-499 (2003).</li><li>Gillor, O., Kirkup, B. C., Riley, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Applied Microbiology. 54, 129-146 (2004).</li><li>Dzung, B. D., Ingolf, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomally+Synthesized+Antibacterial+Peptides+in+Gram+Positive+Bacteria.">Ribosomally Synthesized Antibacterial Peptides in Gram Positive Bacteria.</a> Curr Drug Targets. 3 (2), 107-122 (2002).</li><li>Dobson, A., Cotter, P. D., Ross, R. P., Hill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocin+Production:+a+Probiotic+Trait?.">Bacteriocin Production: a Probiotic Trait?.</a> Appl Environ Microb. 78 (1), 1-6 (2012).</li><li>Li, D., Wang, P., Wang, P., Hu, X., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiota:+A+treasure+for+human+health.">The gut microbiota: A treasure for human health.</a> Biotechnol Adv. 34 (7), 1210-1224 (2016).</li><li>Millette, M., 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=Capacity+of+Human+Nisin-+and+Pediocin-Producing+Lactic+Acid+Bacteria+To+Reduce+Intestinal+Colonization+by+Vancomycin-Resistant+Enterococci.">Capacity of Human Nisin- and Pediocin-Producing Lactic Acid Bacteria To Reduce Intestinal Colonization by Vancomycin-Resistant Enterococci.</a> Appl Environ Microb. 74 (7), 1997-2003 (2008).</li><li>Cotter, P. D., Ross, R. P., Hill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocins+[mdash]+a+viable+alternative+to+antibiotics?.">Bacteriocins [mdash] a viable alternative to antibiotics?.</a> Nat Rev Microb. 11 (2), 95-105 (2013).</li><li>Leroy, F., Foulqui&#233; Moreno, M. R., De Vuyst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterococcus+faecium+RZS+C5,+an+interesting+bacteriocin+producer+to+be+used+as+a+co-culture+in+food+fermentation.">Enterococcus faecium RZS C5, an interesting bacteriocin producer to be used as a co-culture in food fermentation.</a> Int J Food Microb. 88 (2-3), 235-240 (2003).</li><li>Ananou, S., 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=Combined+effect+of+enterocin+AS-48+and+high+hydrostatic+pressure+to+control+food-borne+pathogens+inoculated+in+low+acid+fermented+sausages.">Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages.</a> Meat Sci. 84 (4), 594-600 (2010).</li><li>Riboulet-Bisson, E., 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=Effect+of+Lactobacillus+salivarius+Bacteriocin+Abp118+on+the+Mouse+and+Pig+Intestinal+Microbiota.">Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota.</a> PLoS One. 7 (2), e31113 (2012).</li><li>Charan, J., Kantharia, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+calculate+sample+size+in+animal+studies?.">How to calculate sample size in animal studies?.</a> J Pharmacol Pharmacother. 4 (4), 303-306 (2013).</li><li>Umu, &. #. 2. 1. 4. ;. C. O., 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+Potential+of+Class+II+Bacteriocins+to+Modify+Gut+Microbiota+to+Improve+Host+Health.">The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health.</a> PLoS One. 11 (10), e0164036 (2016).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UPARSE:+highly+accurate+OTU+sequences+from+microbial+amplicon+reads.">UPARSE: highly accurate OTU sequences from microbial amplicon reads.</a> Nat Meth. 10 (10), 996-998 (2013).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Search+and+clustering+orders+of+magnitude+faster+than+BLAST.">Search and clustering orders of magnitude faster than BLAST.</a> Bioinformatics. 26 (19), 2460-2461 (2010).</li><li>Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCHIME+improves+sensitivity+and+speed+of+chimera+detection.">UCHIME improves sensitivity and speed of chimera detection.</a> Bioinformatics. 27 (16), 2194-2200 (2011).</li><li>Caporaso, J., 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=QIIME+allows+analysis+of+high-throughput+community+sequencing+data.">QIIME allows analysis of high-throughput community sequencing data.</a> Nat Meth. 7 (5), 335-336 (2010).</li><li>DeSantis, T. Z., 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=Greengenes,+a+Chimera-Checked+16S+rRNA+Gene+Database+and+Workbench+Compatible+with+ARB.">Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB.</a> Appl Environ Microb. 72 (7), 5069-5072 (2006).</li><li>Caporaso, J. G., 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=PyNAST:+a+flexible+tool+for+aligning+sequences+to+a+template+alignment.">PyNAST: a flexible tool for aligning sequences to a template alignment.</a> Bioinformatics. 26 (2), 266-267 (2010).</li><li>Wang, Q., Garrity, G. M., Tiedje, J. M., Cole, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+Bayesian+Classifier+for+Rapid+Assignment+of+rRNA+Sequences+into+the+New+Bacterial+Taxonomy.">Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy.</a> Appl Environ Microb. 73 (16), 5261-5267 (2007).</li><li>Price, M. N., Dehal, P. S., Arkin, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FastTree+2+-+Approximately+Maximum-Likelihood+Trees+for+Large+Alignments.">FastTree 2 - Approximately Maximum-Likelihood Trees for Large Alignments.</a> PLoS One. 5 (3), e9490 (2010).</li><li>Lozupone, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+a+New+Phylogenetic+Method+for+Comparing+Microbial+Communities.">UniFrac: a New Phylogenetic Method for Comparing Microbial Communities.</a> Appl Environ Microb. 71 (12), 8228-8235 (2005).</li><li>Faust, K., 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=Microbial+co-occurrence+relationships+in+the+human+microbiome.">Microbial co-occurrence relationships in the human microbiome.</a> PLoS Comput Biol. 8 (7), e1002606 (2012).</li><li>Shannon, P., 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=Cytoscape:+A+Software+Environment+for+Integrated+Models+of+Biomolecular+Interaction+Networks.">Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks.</a> Genome Res. 13 (11), 2498-2504 (2003).</li><li>Klindworth, A., 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=Evaluation+of+general+16S+ribosomal+RNA+gene+PCR+primers+for+classical+and+next-generation+sequencing-based+diversity+studies.">Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.</a> Nucleic Acids Res. 41 (1), e1 (2013).</li><li>Edwards, A. N., Su&#225;rez, J. M., McBride, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+Maintaining+Clostridium+difficile+in+an+Anaerobic+Environment.">Culturing and Maintaining Clostridium difficile in an Anaerobic Environment.</a> J Vis Exp. (79), e50787 (2013).</li><li>Delves-Broughton, J., Blackburn, P., Evans, R. J., Hugenholtz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+bacteriocin,+nisin.">Applications of the bacteriocin, nisin.</a> Antonie Van Leeuwenhoek. 69 (2), 193-202 (1996).</li><li>Allende, A., 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=Growth+and+bacteriocin+production+by+lactic+acid+bacteria+in+vegetable+broth+and+their+effectiveness+at+reducing+Listeria+monocytogenes+in+vitro+and+in+fresh-cut+lettuce.">Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce.</a> Food Microb. 24 (7-8), 759-766 (2007).</li><li>Strompfov&#225;, V., Laukov&#225;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+bacteriocin+production+of+Enterococci+associated+with+chickens.">In vitro study on bacteriocin production of Enterococci associated with chickens.</a> Anaerobe. 13 (5-6), 228-237 (2007).</li><li>Caballero-Guerrero, B., Jim&#233;nez D&#237;az, R., Maldonado-Barrag&#225;n, A., Ruiz-Barba, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coculture+with+specific+bacteria+enhances+survival+of+Lactobacillus+plantarum+NC8,+an+autoinducer-regulated+bacteriocin+producer,+in+olive+fermentations.">Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer, in olive fermentations.</a> Food Microb. 27 (3), 413-417 (2010).</li><li>Drider, D., Fimland, G., H&#233;chard, Y., McMullen, L. M., Pr&#233;vost, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Continuing+Story+of+Class+IIa+Bacteriocins.">The Continuing Story of Class IIa Bacteriocins.</a> Microb. Mol Biol Rev. 70 (2), 564-582 (2006).</li><li>Gao, Y., Jia, S., Gao, Q., Tan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+bacteriocin+with+a+broad+inhibitory+spectrum+produced+by+Lactobacillus+sake+C2,+isolated+from+traditional+Chinese+fermented+cabbage.">A novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus sake C2, isolated from traditional Chinese fermented cabbage.</a> Food Control. 21 (1), 76-81 (2010).</li></ol>

作者没有什么可以披露的。

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片段是至关重要的。在这项工作中，可变区V3 - V4选择25。 DNA提取方法的重要性不能低估，因为样品中存在的细菌细胞不是全部等效的对不同的裂解方案敏感，并且在细菌群体的估计中的偏差可以由不完全溶解引起。因此，需要引入搅拌步骤。同源菌株可以通过遗传敲低或通过质粒凝集来构建，尽管在转化染色体编码的细菌素时难以转录的菌株可能代表一个问题。该方法可以使用不同的特异性培养技术，如厌氧方法26来适应。 该程序可以在大多数步骤中进行修改，具体取决于要研究的因子变化的类型。可以缩短接种程序，但血清参数的变化可能需要一些时间才能发展。该方法的可能限制可能是研究在水中不能存活数小时或可能聚集（或化合物沉淀）的细菌菌株（营养物或化合物）。因此，这是必须的一个因素<html>所以要考虑。 到现在为止，细菌素的体外活性27，28，29或在混合培养物30被普遍确定。该方法允许体内监测来自标准实验室小鼠的全肠微生物群的五种细菌素的活性。可以确定人口的小变化，以及它们对其他细菌群体或生理因素的影响，如血清参数12 。细菌素具有相对较窄的活性谱，抑制分类学相关细菌的生长。然而，用于该测定中的那些是最有效的，因为其中一些对金黄色葡萄球菌 ， 单核细胞增多性李斯特菌 31 ，甚至一些大肠杆菌菌株是活性的，如在萨卡辛Aass ="xref"&gt; 32。当接种在小鼠中时，产生LAB的细菌素在全球微生物群组成中没有显着变化，而是减少了细菌分类群的数量。然而，全球相关网络分析揭示了不同细菌之间有趣的生态相容性和拮抗作用。这种方法可以适应于分析食物成分，药物或其他化合物的影响，尽管预期的结果可能被故意限制以保持健康状况。

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><tbody><tr><td>Balb/c mice (female)</td><td>Harlan</td><td></td><td>Mice should be 6 - 8 weeks of age</td></tr><tr><td>Plastic Petri dish</td><td>Thermo Scientific</td><td>101VR20</td><td></td></tr><tr><td>Brain-Heart-Infusion broth</td><td>Conda</td><td>1400.00</td><td></td></tr><tr><td>European Bacteriological Agar</td><td>Pronadisa</td><td>1800.00</td><td></td></tr><tr><td>Agarose D1 Low EEO</td><td>Pronadisa</td><td>8010.00</td><td></td></tr><tr><td>1x TAE buffer</td><td>Thermo Scientific</td><td>15558042</td><td></td></tr><tr><td>MRS broth</td><td>Difco</td><td>288130</td><td></td></tr><tr><td>PBS tablets</td><td>Sigma</td><td>P4417-100TAB</td><td></td></tr><tr><td>scale</td><td>Mettler Toledo</td><td>PB602-S</td><td></td></tr><tr><td>sterile forceps</td><td>Levantina de Laboratorios S.L.</td><td>260-3710014</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Centrifuge</td><td>Hermle</td><td>Z383K</td><td></td></tr><tr><td>sodium chloride</td><td>AppliChem Panreac</td><td>121659.1211</td><td></td></tr><tr><td>Realpure SSS Kit</td><td>Real Life Science Solutions, Durviz, Spain</td><td>RBME04 (300 ml)</td><td></td></tr><tr><td>Isopropanol</td><td>AppliChem Panreac</td><td>131090.1611</td><td></td></tr><tr><td>Ethanol</td><td>AppliChem Panreac</td><td>131086.1214</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>AMPure XP beads</td><td>Beckman Coulter Genomics, USA</td><td>A63881 (60 ml)</td><td></td></tr><tr><td>PerfeCta NGS library quantification kit</td><td>Quanta BioSciences, Maryland, USA</td><td>733-2300</td><td></td></tr><tr><td>MiSeq v3 reagent kit</td><td>Illumina, San Diego, California, USA</td><td>MS-102-3003</td><td></td></tr><tr><td>Primers for 16S rRNA gene amplification</td><td></td><td></td><td>Primers contain V3-V4 region of bacterial 16S rRNA gene and Illumina overhang adaptors:5&amp;rsquo;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3&amp;rsquo; and 5&#039;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3&amp;rsquo;</td></tr><tr><td>Nextera XT Index kit</td><td></td><td>FC-131-1002</td><td>Indices and Illumina sequencing adaptors</td></tr><tr><td>Micropestle for 1.5 mL tubes, Eppendorf / Sigma , Ref.</td><td>Sigma</td><td>Z317314-1PAK</td><td></td></tr><tr><td>Glass beads, 0.1 mm diameter</td><td>Biospec Products</td><td>11079-101</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-up Kit</td><td>Macherey-Nagel</td><td>740609.25</td><td></td></tr><tr><td>Omni Bead Ruptor 24</td><td>Omni International Inc.</td><td>19-040</td><td></td></tr><tr><td>mutanolysin</td><td>Sigma</td><td>M9901-10KU</td><td></td></tr><tr><td>lysozyme</td><td>Roche</td><td>10837059001</td><td></td></tr><tr><td>proteinase K</td><td>Roche</td><td>3115887001</td><td></td></tr><tr><td>RNase A</td><td>Sigma</td><td>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，用细菌素生产菌株处理的五个笼子，以及用相应的同基因突变体处理的5个笼子，其具有减少或没有细菌素生产。经测试的细菌素生产LAB以及饮用水中的相应计数（cfu / mL）和粪便中的计数总结在<stro表1。从粪便中回收的LAB的细菌素生产的测定如图2所示 。当所有样品和所有细菌组合在一起时，细菌素处理和同基因菌株之间没有显着差异（ 图3 ）。然而， 如图4所示，特殊细菌属独立研究时可能存在差异。此外，Pearson的相关分析揭示了不同细菌属之间的显着连锁关系，以及细菌属和甘油三酯和血清中LDL之间的相关性， 如图5中CoNet 所示 。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56053/56053fig1.jpg" /> 图1:展示实验时间设计的方案riment。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56053/56053fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56053/56053fig2.jpg" /> 图2:通过三层方案检测从粪便中回收的LAB中的细菌素生产（如程序中所述）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56053/56053fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/56053/56053fig3.jpg" /> 图3:细菌处理组成比较。基于未加权的UniFrac矩阵中的计算距离生成PCoA图。来自同一样本的不同时间点et al。 ，2016 12 。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56053/56053fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56053/56053fig4.jpg" /> 图4:处理和后处理期间LAB和细菌素靶向细菌群在种属水平上的相对丰度的变化。更改将相对于时间0获得的处理属的相对丰度与对照组（CON）中的相对丰度进行比较。为了说明的清楚，仅显示四种菌株:加洛蛋白和沙卡辛生产者和非生产菌株（参见图中关于颜色和处理的图例）。误差条代表标准偏差。重要度表示如下:P &lt;0.1，点（。）;一星（*）P &lt;0.05; P &lt;0.01，两星（**）。改编自Umu et al。 ，2016 12 。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56053/56053fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/56053/56053fig5.jpg" /> 图5:第14天OTU相对丰富度与血清水平的相关性网络。相关性为ca在CoNet中使用Pearson相关性计算。只有重要的（P &lt;0.05）显示在网络上。在血清 - 总胆固醇（ Chol ），高密度脂蛋白（ HDL ），低密度脂蛋白（ LDL ）和甘油三酯（ Trig）中确定的参数由一种颜色（灰色）显示，而属于不同家族的OTU由不同的颜色（见图例），绿色边缘显示正相关，红色边缘显示负相关，节点上的OTU用OTU号或它们所属的属性表示，从Umu 等 ，2016 12 。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56053/56053fig5large.jpg" target="_blank">请点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><stroNG&gt;应变</td><td> 接种在饮用水中 </td><td></td><td> 乳酸菌在粪便中计数 </td><td> 指标菌株 </td></tr><tr><td></td><td> CFU / ml（log10） </td><td></td><td> CFU / g（平均log10） </td><td></td></tr><tr><td>控制</td><td> ---- </td><td> 10只小鼠</td><td> ND </td><td></td></tr><tr><td> 乳酸乳杆菌B1500 （sakA +） </td><td> 1.6 </td><td> 9只小鼠</td><td> 8.53±0.18 </td><td> 屎肠球菌P21（LMG 2783） </td></tr><tr><td> 乳酸乳杆菌B1501 （sakA - ） </td><td> 2.0 </td><td> 9只小鼠</td><td> 8.22±0.05 </td><td></td></tr><tr><td> Pediocccus acidilactici B1502（ped +） </td><td> 26 </td><td> 9只小鼠</td><td> 8.73±0.12 </td><td> 屎肠球菌P21（LMG 2783） </td></tr><tr><td> Pediocccu酸奶B1503（ped - ） </td><td> 25 </td><td> 9只小鼠</td><td> 8.75±0.09 </td><td></td></tr><tr><td> 屎肠球菌 B1504（L50重量+） </td><td> 6 </td><td> 9只小鼠</td><td> 8.83±0.14 </td><td> 小球藻（LMG 3397） </td></tr><tr><td> 屎肠球菌 B1505（L50固化） </td><td> 10 </td><td> 9只小鼠</td><td> 8.72±0.14 </td><td></td></tr><tr><td> 植物乳杆菌 B1507（planta +） </td><td> 26 </td><td> 9只小鼠</td><td> 9.14±0.14 </td><td> 乳杆菌属965（LMG 2003） </td></tr><tr><td> 植物乳杆菌 B1508（植物 - ） </td><td> 23 </td><td> 9只小鼠</td><td> 8.60±0.18 </td><td></td></tr><tr><td> 乳酸乳球菌B1515 （GarML +） </td><td> 17 </td><td> 9只小鼠</td><td> 8.41±0.08 </td><td> 乳酸乳球菌IL1403（LMG2705） </EM&gt; </td></tr><tr><td> 乳酸乳球菌B1516 （GarML - ） </td><td> 17 </td><td> 9只小鼠</td><td> 8.05±0.18 </td><td></td></tr><tr><td colspan="5"> 屎肠球菌 P21（LMG 2783），用于坂和PedPA-1， 片球菌属damnosus（LMG 3397），用于enterocins， 乳杆菌属。 965（LMG 2003）和植物乳杆菌乳酸乳球菌 IL1403（LMG2705）。 </td></tr></tbody></table> 表1:接种菌株和粪便样品中的计数。

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Biology

14.2K 次观看.  Consejo Superior de Investigaciones Científicas (CSIC). 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。 

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

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

科研

免疫与感染

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

医学

<meta charset="utf8"></head><body>使用微量移液器引导的给药减轻研究动物的压力

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

<meta charset="utf8"></head><body>在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

癌症研究

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

使用简单、经济高效的DNA分离方法分析狼疮易发小鼠的粪便微生物群动力学

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

厌氧菌小鼠阴道定植模型

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

使用 T4 噬菌体探索小鼠模型中的肠道噬菌体-细菌动力学

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

作者聚焦:研究噬菌体诱导的 Gnotobiotic 小鼠免疫反应

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

评估细菌素对小鼠肠道微生物的影响的方法

摘要

探索更多视频

此视频中的章节

“

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

头孢哌酮治疗小鼠模型的临床相关的

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

使用简单、经济高效的DNA分离方法分析狼疮易发小鼠的粪便微生物群动力学

厌氧菌小鼠阴道定植模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

评估细菌素对小鼠肠道微生物的影响的方法

摘要

探索更多视频

此视频中的章节

“

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

在啮齿动物模型中使用微量移液器引导给药作为口服灌胃的替代方法

头孢哌酮治疗小鼠模型的临床相关的

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

使用简单、经济高效的DNA分离方法分析狼疮易发小鼠的粪便微生物群动力学

厌氧菌小鼠阴道定植模型

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型