我们感谢箴。伯纳德香格里拉斯科拉的有益的技术咨询和amoebal共培养和amoebal充实有趣的讨论。我们也感谢博士文森特托马斯，他在实施该技术在我们实验室的帮助。

1。 Amoebal共培养 1.1样品制备<ol><li> 环境样品 <ol><li> 水样 通过0.22μm孔径的膜过滤水样（500毫升至1升）。然后，摇动膜在页面的阿米巴盐水介质首席助理秘书长（120毫克氯化钠，4毫克硫酸镁4•7H 2 O，4毫克氯化钙2•2H 2 O，142毫克的Na 2 HPO 4，和136毫克的KH 2 PO 4在1升的蒸馏水）。 </li><li> 固体样品 悬浮固体样品像土壤或沙子样品和半固体样品，如活性污泥在蒸馏水或PBS中，通过0.22μm孔径的膜过滤。然后，在摇PAS膜。 </li><li> 样品严重污染的内生阿米巴原虫和 首先通过低速离心（180×g离心）FO沉淀样品-R 10分钟或通过5微米孔径的膜过滤。进一步处理上清液（分别滤液），如在1.1.1。 注:其他净化技术可以用于进一步净化环境样品:将样品加热，在50℃下进行30分钟或治疗用酸性或碱性溶液14。 </li></ol></li><li> 临床样本 处理临床样品根据它们的物理 - 化学性质。过滤或离心液去除大杂质。悬浮固体样品。使用的组织，通过用Dounce homogeneiser或玻璃珠研磨它们，例如，和裂解细胞以释放细胞内的细菌。 </li></ol> 1.2变形虫准备<ol><li> 肉汤准备 准备以下介质:含蛋白胨，酵母提取物和葡萄糖（PYG富媒体;100克胨，10克酵母提取物4.9克MgSO 4干燥•7H 2的输出，5克柠檬酸钠•2H 2 O，0.1g的铁（NH 4）2（SO 4）2•6H 2 O，1.7克KH 2 PO 4，1.97克Na 2 HPO 4的•7H 2 O 45克 ​​葡萄糖，和0.295克的CaCl 2在5升蒸馏水）和非营养性介质，如PAS为棘阿米巴物种。 </li><li> Amoebal文化 <ol><li>在装有30ml PYG培养基的细胞培养瓶中培养变形虫（优先氏棘阿米巴 ATCC 30010或A多食领潮-AP1）在25°C。 </li><li>由烧瓶中剧烈摇晃收获变形虫和离心细胞悬液10分钟，1,500×g的。与PAS中等两次洗涤沉淀。计数细胞在KOVA幻灯片，调整音量，以获得每毫升5×10 5细胞悬浮液。 </li><li>转移amoebal悬挂到微孔板。使用每孔1毫升12 - 和24-W埃尔板，500μl的48孔板中，并加入300微升96孔板中。孵育微孔板至少2小时，在25℃下这允许沉积和附着在变形虫的每个孔的底部。 </li></ol></li></ol> 1.3共培养<ol><li> 样品接种 <ol><li>从2.3.3接种板与试样的从1.1或1.2系列稀释液，通常以10倍稀释系列，从用100μl稀释的样品。 </li><li>离心微孔板在1800×g离心10分钟，沉淀在amoebal草坪上的微生物可能存在于样品中。这增加了接触和微生物的吞噬变形虫。 </li><li>孵育板45分钟，在25℃下并且通过与新鲜的PAS更换介质洗涤三次，PAS。添加每孔1毫升PAS添加或不添加抗生素（链霉素，青霉素，庆大霉素和/或万古霉素）的，取决于bacteri被搜索的人的物种。 </li><li>孵育微孔板在32℃，饱和湿度条件下，以避免阿米巴的包囊。每天用20倍物镜观察每孔检测细菌入侵和裂解变形虫的存在。 </li><li>在溶解的情况下，通过接种100μl的共培养物中，以约10 5变形虫/ cm 2的单分子层上执行新鲜变形虫一个传代培养。要明确地隔离特定的细菌种类，接种设计也为这些细菌（ 即 BCYE琼脂军团菌 ）的具体琼脂培养基。 </li><li>在没有溶解，取100微升共同培养的初次接种后4至7天，接种900微升新鲜amoebal文化在24孔板。如果不带细菌的增殖观察变形虫的快速裂解，病毒可能存在。在这种情况下，过滤上清液在0.22微米和使用该过滤悬浮液感染新鲜的变形虫。 </li></醇&gt; </li></ol> 1.4细菌分离和鉴定<ol><li> 细菌染色 直接在24孔微孔板上执行的玻璃盖玻片上共培养物。取出介质，并进行染色或免疫荧光。 <ol><li>修改Romanowsky染色<ol><li>让盖玻片干。浸入盖玻片在固定液（2毫克/升坚牢绿在甲醇中）5次。 </li><li>沉浸在盖玻片五次染色溶液I（1.22 g / L的曙红的G磷酸盐缓冲液pH值6.6） </li><li>最后，将其浸入盖玻片5倍，在染色溶液II（在磷酸盐缓冲液pH 6.61.1克/ L噻嗪）。 </li><li>冲洗样品用蒸馏水冲洗。让干燥并通过显微镜观察。 </li></ol></li><li>抗酸染色<ol><li>让盖玻片干。盖上施乐品红样品。加热染料具有阻燃，直到蒸气出现。 </li><li>在室温下冷却至少5分钟并用蒸馏水冲洗。覆盖所述样品与在异丙醇中进行2分钟的3％盐酸溶液，并用蒸馏水冲洗。 </li><li>覆盖30秒用亚甲蓝和用蒸馏水冲洗。让干燥并通过显微镜观察22。 </li></ol></li><li>希门尼斯染色<ol><li>通过将100毫升10％碱性品红（10克碱性品红在100毫升95％的乙醇），加入250毫升4％的含水苯酚和650毫升蒸馏水中制备碱性品红贮备液。在37℃，使用前48小时。 </li><li>让盖玻片干燥和修复通过火焰传递给它。盖在2分钟新鲜过滤的碱性品红（4毫升碱性品红储备液在10毫升的M 0.1的磷酸钠缓冲液，pH 7.45）的样品。 </li><li>冲洗样品与水和10秒的孵育中孔雀绿（在蒸馏水中的0.8％）。用清水再次冲洗和重复孔雀石绿染色。用清水再次冲洗。让盖玻片干，M&#39;mount它，并通过显微镜观察23。 </li></ol></li><li>免疫荧光<ol><li>孵化甲醇5分钟，或用多聚甲醛10分钟4％固定盖玻片。 </li><li>用PBS洗涤三次，并用封闭液（5％BSA，0.1％皂苷的PBS中）在室温下孵育2小时。 </li><li>孵育盖玻片1小时阻断含有提出对感兴趣的微生物的抗体溶液。 </li><li>在PBS中再次洗涤三次，并孵育1小时用针对第一抗体和连接到荧光团的第二抗体。用PBS洗三次，安装盖玻片，用荧光显微镜观察。 </li></ol></li></ol></li><li> DNA的PCR检测 从100到200微升amoebal共存的提取DNA。检测微生物与靶向16S rRNA基因或特异性引物对感兴趣的物种，如分枝杆菌24通用引物，军团菌25或Chlamydiales 26的成员。 </li></ol> 2。 Amoebal富集 2.1。样品制备通过涡旋重新悬浮固体和半固体样品中PAS。离心悬浮液以低速（180×g离心）10分钟。这允许在小球自由生活阿米巴富集。上清液可用于amoebal共存，沉淀为amoebal富集21。 2.2。培养基的配制<ol><li>加入1.5克琼脂的至100ml的PAS和高压灭菌该培养基15分钟，在121℃下倒入温暖的介质在培养皿中，让在室温下固化。 </li><li>在LB或巯基乙酸盐肉汤生长的大肠杆菌 （ATCC 25922）中过夜，在37℃下用PBS洗两次的细菌和悬浮在其中的PAS媒体。稀释10倍的PAS和传播2-3毫升这种稀释对的PAS琼脂平板上，并让其干燥。 </li></ol>加一滴样品（或一块滤波器）在板的一侧，并且让它流过培养皿中，形成在盘的中心的直线。 2.4。 Amoebal增长和amoebal亚文化<ol><li>每天观察培养皿中。如果amoebal迁移前被检测到，切出一小块琼脂在迁移前和接种新鲜NNA培养皿盖上E的草坪大肠杆菌 。 </li><li>重复该回接几次取决于样品的纯度，以便具有给定的amoebal菌株的纯培养物。 </li></ol> 2.5。变形虫和细菌鉴定<ol><li>刮细胞，重悬在他们的PAS。 </li><li>提取DNA并确定变形虫和/或细菌共生菌通过PCR和测序（16S rRNA基因的扩增对细菌，呼吸道。18S rRNA基因的变形虫和测序，例如）的身份。 </li> &lt;李&gt;使用在这些PAS刮下细胞接种到新鲜变形虫和执行amoebal共培养，以检测可能存在于样品中的细菌。 </li></ol>

<ol>
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Health. 213, 158-166 (2010).</li><li>La Scola, B., 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=Legionella+drancourtii+sp.+nov.,+a+strictly+intracellular+amoebal+pathogen.">Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen.</a> Int. J. Syst. Evol. Microbiol. 54, 699-703 (2004).</li><li>Thomas, V., Casson, N., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Afipia+and+Bosea+strains+isolated+from+various+water+sources+by+amoebal+co-culture.">New Afipia and Bosea strains isolated from various water sources by amoebal co-culture.</a> Syst. Appl. Microbiol. 30, 572-579 (2007).</li><li>La Scola, B., 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=Amoeba-resisting+bacteria+and+ventilator-associated+pneumonia.">Amoeba-resisting bacteria and ventilator-associated pneumonia.</a> Emerging Infect. Dis. 9, 815-821 (2003).</li><li>Collingro, 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=Recovery+of+an+environmental+Chlamydia+strain+from+activated+sludge+by+co-cultivation+with+Acanthamoeba+sp.">Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp.</a> Microbiology. 151, 301-309 (2005).</li><li>Lienard, J., Croxatto, A., Prod'hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrella+lausannensis,+a+new+star+in+the+Chlamydiales+order.">Estrella lausannensis, a new star in the Chlamydiales order.</a> Microbes Infect. 13, 1232-1241 (2011).</li><li>La Scola, B., 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=A+giant+virus+in+amoebae.">A giant virus in amoebae.</a> Science. 299, 2033 (2003).</li><li>Boyer, 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=Giant+Marseillevirus+highlights+the+role+of+amoebae+as+a+melting+pot+in+emergence+of+chimeric+microorganisms.">Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 21848-21853 (2009).</li><li>Thomas, V., 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=Lausannevirus,+a+giant+amoebal+virus+encoding+histone+doublets.">Lausannevirus, a giant amoebal virus encoding histone doublets.</a> Environ. Microbiol. 13, 1454-1466 (2011).</li><li>Raoult, D., Renesto, P., Brouqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+infection+of+a+technician+by+mimivirus.">Laboratory infection of a technician by mimivirus.</a> Ann. Internal Med. 144, 702-703 (2006).</li><li>Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parachlamydia+acanthamoebae,+an+emerging+agent+of+pneumonia.">Parachlamydia acanthamoebae, an emerging agent of pneumonia.</a> Clin. Microbiol. Infect. 15, 18-28 (2009).</li><li>Lamoth, F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoebal+pathogens+as+emerging+causal+agents+of+pneumonia.">Amoebal pathogens as emerging causal agents of pneumonia.</a> FEMS Microbiol. Rev. 34, 260-280 (2010).</li><li>Lienard, J. G., Ashbolt, K., Sen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+6.">Ch. 6.</a> Environmental microbiology, current technology and water applications. , 143-162 (2011).</li><li>Boughalmi, 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=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ. Microbiol. , (2012).</li><li>Ovrutsky, A. R., 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=Cooccurrence+of+Free-Living+Amoebae+and+Nontuberculous+Mycobacteria+in+Hospital+Water+Networks,+and+Preferential+Growth+of+Mycobacterium+avium+in+Acanthamoeba+lenticulata.">Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata.</a> Appl. Environ. Microbiol. 79, 3185-3192 (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

Amoebal共培养和amoebal富集是使许多新的细菌和amoebal物种的隔离有效的方法。用这些方法得到的结果证实了这两种阿米巴变形虫和抗细菌在环境中无处不在，最有趣的是在被认为通过化学处理，如氯化，臭氧控制人为供水网络。 Amoebal共培养和amoebal富集是必不可少的工具来隔离和培养这些潜在的病原微生物，并获得菌株纯培养，才能再进一步研究其生物学和致病性。最近，这种方法被改编为巨病毒...

分子诊断来临之前，目前在环境壁龛或临床样品中的微生物经培养他们在不同的选择性培养基，主要是在琼脂培养皿中经常发现。细菌菌落和其代谢活性的表现型，让细菌分类在物种水平。肉汤也可以被用于增加检测的灵敏度。然而，这两种技术不允许该生长缓慢或根本没有在这些介质上的细菌的恢复。这就是为什么分子生物学方法被如此广泛地采用了时下的原因。然而，检测DNA提供了对细菌的生存能力没有线索。另外，相反地，以培养，分子生物学方法不会导致菌株，可以进一步表征。 研究病原体生长不良固体培养基上，或需要细胞生长是复杂的。大多数这些"难以生长"的细菌都讲究不及物动词脱细胞的细菌，经常发现其特征如下，因为它是为嗜肺军团菌的情况下大规模爆发。这种细菌的特点如下，一位美国退伍军人大会期间发生的爆发。多达182人被感染，29死于严重肺炎1,2。后来证明，变形虫是这种细菌的自然宿主，而他们在酒店空调系统和供水网络的存在是在所谓的退伍军人症3爆发的起源。 变形虫是目前全球范围内的土壤，空气，水和人类志愿者（4审查）的鼻腔粘膜分离。这些"自由生活"变形虫一般都划分在自主的环境，但有时可能会侵入许可主机5。通过吞噬作用和随后的溶酶体消化HY上的各种微生物饲料阿米巴drolases 6。许多兼性或专性细胞内细菌能够抵抗消化，从而感染和阿米巴划分为例如军团菌 ， 衣原体相关的细菌或分枝杆菌（7审阅和8）。自由生活的阿米巴可能是一个重要的潜在水库已尚未被发现的细胞内细菌。这导致本集团在洛桑实现两个主要的技术，称为amoebal共培养和amoebal富集，这使得不同的群体从不同环境样品9-15隔离了几个新的专性细胞内微生物。 由于变形虫是专业放牧吞噬细胞对细菌，能抵抗吞噬和这些原生生物生长里面的细菌也可能拓殖人的吞噬细胞和致病走向人类。这是部分地显示出对某些衣原体相关的细菌，如Waddlia町ndrophila。 W. chondrophila不仅可以在变形虫而且在一些细胞类型中，如哺乳动物上皮细胞，巨噬细胞，和鱼细胞系16-18成长。该amoebal共培养也出现相关检测细胞内细菌的临床标本19,20，包括大便这是严重污染，不同的细菌种类21。 在这里，我们描述amoebal共培养和amoebal富集的主要步骤，包括:（一）治疗环境或临床标本;变形虫（二）在无菌介质中生长，对大肠杆菌的细菌草坪及（c）选择和表征胞内细菌。

<table><tbody><tr><td>Glucose monohydrate</td><td>Merck, Darmstadt, Germany</td><td>108342</td><td></td></tr><tr><td>0.22 &amp;mu;m pore size membrane</td><td>Merck Millipore, Darmstadt, Germany</td><td>SCVPU11RE</td><td></td></tr><tr><td>proteose peptone</td><td>Becton-Dickinson, Franklin Lakes, NJ</td><td>211693</td><td></td></tr><tr><td>yeast extract</td><td>Becton-Dickinson, Franklin Lakes, NJ</td><td>212750</td><td></td></tr><tr><td>Cell culture flasks</td><td>Becton-Dickinson, Franklin Lakes, NJ</td><td>353135</td><td></td></tr><tr><td>Kova slide</td><td>Hycor, Indianapolis, IN</td><td>87144</td><td></td></tr><tr><td>cell culture microplates</td><td>Corning Inc, Corning, NY</td><td>3524</td><td></td></tr><tr><td>Diff-Quik staining kit</td><td>Siemens Healthcare diagn., Munich, Germany</td><td>130832</td><td></td></tr><tr><td>Ziehl fuchsin</td><td>Fluka, St-Louis, MI</td><td>21820</td><td></td></tr><tr><td>basic fuchsin</td><td>Sigma, St-Louis, MI</td><td>857843</td><td></td></tr><tr><td>Phenol</td><td>Sigma, St-Louis, MI</td><td>P1037</td><td>Corrosive and mutagenic</td></tr><tr><td>malachite green oxalate</td><td>Fluka, St-Louis, MI</td><td>63160</td><td></td></tr><tr><td>Paraformaldehyde 16% solution</td><td>Electron Microscopy Sciences, Hatfield, PA</td><td>15710</td><td></td></tr><tr><td>Saponin</td><td>Sigma, St-Louis, MI</td><td>84510</td><td></td></tr></tbody></table>

discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches

细胞内病原体如军团菌，分枝杆菌和衣原体样生物是难以分离，因为它们往往较差或根本不生长在所有上，通常是用于培养细菌的选择性培养基。由于这个原因，许多这些病原体被发现仅在最近或以下重要的爆发。这些病原体往往与变形虫，它作为宿主细胞，并允许细菌的存活和生长有关。我们打​​算在这里提供了两种技术，使孤立的细胞内病原体和表征目前临床或环境样品中的示范:在amoebal共培养和amoebal富集。 Amoebal共培养使细胞内细菌的恢复由所研究的样品接种到一个amoebal草坪，可感染并裂解由存在于样品中的胞内细菌。 Amoebal富集使得目前临床或环境样品中变形虫的恢复。钍是可能导致发现新的amoebal物种，但也是新的细胞内细菌在这些变形虫专门生长。总之，这两个技术有助于发现能够在变形虫长出新的细胞内细菌。因为他们的感染变形虫和抵制吞噬能力，​​这些细胞内的细菌也可能会被巨噬细胞吞噬逃脱，因此，是致病性高等真核生物。

使用amoebal共培养和amoebal富集，环境和/或致病菌的整个范围被发现（ 表1）。 Amoebal共培养采​​用由本集团及其他分析环境样品，水处理厂和配水系统。范围广泛的微生物可能是孤立使用这种技术。分离amoebal共培养中最常见的细菌是分枝杆菌属的成员，可以从污水处理厂，并从水网13,14,24,27,28回收。 军团菌和α-变形细菌物种也可从污水处?...

Watch this Scientific Journal Video about Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches at JoVE.com

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Amoebal共存是使用贴壁变形虫以选择性地生长细胞内的病原体能够抵抗吞噬细胞如变形虫和巨噬细胞的细胞培养系统。因此，它代表发现新的传染性病原体的关键工具。 Amoebal富集允许发现其特定的细胞内细菌的新amoebal品种和。

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

discovery-new-intracellular-pathogens-amoebal-coculture-amoebal

Research

JoVE Journal

Immunology and Infection

14.4K 次观看.  University Hospital Center and University of Lausanne.  Amoebal共存是使用贴壁变形虫以选择性地生长细胞内的病原体能够抵抗吞噬细胞如变形虫和巨噬细胞的细胞培养系统。因此，它代表发现新的传染性病原体的关键工具。 Amoebal富集允许发现其特定的细胞内细菌的新amoebal品种和。 

视频: Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

听话的哺乳动物细胞原生动物引发的细菌感染

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

科研

免疫与感染

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

生物化学

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

一种基于变形虫的快速图像细菌毒力检测方法

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

生物工程

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

摘要

探索更多视频

此视频中的章节

听话的哺乳动物细胞原生动物引发的细菌感染

上皮损伤制作分析

一种快速战略新Faustoviruses从环境样品使用隔离

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

一种基于变形虫的快速图像细菌毒力检测方法

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

微生物相互作用研究的高通量共培养测定

采用共培养，以检测化学介导的种间相互作用

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

摘要

探索更多视频

此视频中的章节

听话的哺乳动物细胞原生动物引发的细菌感染

上皮损伤制作分析

一种快速战略新Faustoviruses从环境样品使用隔离

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

一种基于变形虫的快速图像细菌毒力检测方法

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

微生物相互作用研究的高通量共培养测定

采用共培养，以检测化学介导的种间相互作用

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作