Name: targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay
Uploaded: 2016-05-05T13:00:00.000+00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1.启动生物膜<ol><li>成长生物膜的产生菌的培养在营养丰富的培养基。接种金黄色葡萄球菌菌株Newman中从一个甘油10毫升的Mueller-Hinton培养基的。执行涉及处理S.各项工作金黄色葡萄球菌手套和生物安全柜中。 </li><li>在37℃下孵育16小时，在旋转摇动器（100-200转）。 </li><li>决定使用分光光度计和标准比色皿（路径长度:1厘米）的文化的OD 600。 </li><li>计算使用下面的公式中chelexed罗斯韦尔园区纪念研究所介质（CRPMI）9来制备20种菌的溶液具有0.1的OD 600（接种物）所需的培养的体积（V）:[V = 20毫升* 0.1 / OD 600（培养）] 注:参考9 CRPMI准备提供详细信息。 <ol><li>通过在10,000×g下旋转1分钟加入计算培养物体积（从上方）到离心管中，并沉淀细胞。为了准备工作重新生物膜接种物，吸在20ml CRPMI所述废生长培养基和悬浮细胞沉淀。 </li></ol></li><li>倾接种到25ml的贮存器。 </li><li>小心地向外径直从无菌96孔挂钩板取下盖子。 注意:必须小心不要污染栓挂从盖向下。盖子可以倒放在干净的表面上的生物安全柜中。 </li><li>用200μl多通道移液器，加入125微升接种物到A行底板的G各孔中。 </li><li>填写排H的每个以及无菌控制125微升无菌CRPMI网上平台。 </li><li>取栓盖并保持其上的板底部朝下钉。小心地将各自的水井个人挂钩。避免在板和钉之间的物理接触。一旦对准，降低盖子仔细了。避免错位的钉后进行调整已经触及底板可续行H.氨化无菌控制</li><li>密封盖子平板用石蜡膜，防止蒸发发生在一个密封的塑料袋，封口严密。保持在袋尽可能低以减少蒸发的空气量。 </li><li>孵育没有在一个5％CO 2培养箱中于37℃振荡48小时。 注:48小时增长已经最佳为S.金黄色葡萄球菌 ，但会发生变化根据所使用的生物体。 注意:虽然静态孵化是一个很好的起点，以优化测定法，以150rpm在微孔板摇床轻轻摇晃是一个可能的变化，可能会提高一些S.生物膜的形成金黄色葡萄球菌菌株。 </li></ol> 2.生物膜挑战板的制作<ol><li>上生物膜挑战的天采取一个新的96孔板中并加入100微升CRPMI的，平衡至室温，以行的每个孔B至H.为了避免不一致的结果，利用96孔的板，其可容纳200μl的媒体当钉在里面，并设有尺寸完全相同生物膜启动板的孔中。 </li><li>稀释最多4试验化合物分别于1.0ml CRPMI的2倍的所需最终浓度，以占步骤2.7的最终稀释度。 注意:一个好的起点是8折浮游细胞的抑菌浓度以上。 </li><li>井A1加入200μl化合物1至A3，化合物2井A4至A6，化合物3井A7至A9和化合物4井A10至A12。 </li><li>通过使用多道移液器连续稀释测试化合物，并从A行转移100微升排B和混合。 </li><li>以这种方式，继续传输100微升很好很好。每次传输后混匀，停止行F. </li><li>混合后，从排出F行100微升到废物容器中。不要任何液体转移到行G和H </li><li>添加100微升CRPMI向板的每个孔用多道移液器向使体积为200微升。 图1所示为最终的板布局。 </li></ol> 3.生物膜挑战<ol><li>加入200μl的磷酸缓冲盐水（PBS）的到设有尺寸完全相同生物膜起始板的孔中新鲜无菌96孔板中。 </li><li>取下培养生物膜启动盘的塑料袋和石蜡膜。 </li><li>掀开锅盖直线上升。避免这些桩和板底部，因为这之间的接触可能会损坏生物膜。保持底部为后续OD 600分析（见3.8）。 </li><li>通过将钉盖到含PBS（步骤3.1）的平板冲洗掉浮游细胞。淹没钉5秒，吊出PBS，并淹没一次，小心不要撞到以及两侧的挂钩。 </li><li>放置冲洗钉盖成挑战板。避免钉和底板，因为这之间的不必要的接触会损伤生物膜导致不一致的结果。 </li><li>密封板用石蜡膜并放置到密封的塑料袋中步骤1.10中描述。 </li><li>孵育所述板没有在5％ 的 CO 2培养箱中于37℃振荡24小时。 </li><li>以生物膜启动盘底3.3。重悬在每个细菌用多道移液器井并测量OD 600与适宜酶标仪确认生长在所有孔中，但在排H的无菌控制发生</li></ol> 4.确定生物膜<ol><li>使用配有孵育室和能够采取动力学荧光读数酶标仪检测刃天青转换。使用530nm的激发和590 nm发射设立了24小时动能荧光测量。 </li><li>设置腔室温度至37℃，并有板阅读每20分钟。设置板器根据制造商的instruc到从板的底部测量系统蒸发散。 </li><li>通过加入200微升的PBS用多道移液器到无菌的96孔板中制备洗涤板。 </li><li>通过加入400微升的0.8毫克制备生物膜回收介质/ ml的刃天青原液至20ml CRPMI培养基至16微克/毫升刃天青的终浓度。 注:刃天青是一种已知的皮肤刺激。在处理使用白大褂，防护眼镜和手套。 CRPMI作为生物膜回收培养基使用，因为它提供了最低的背景信号，但它可以通过的Mueller Hinton培养基如果适当被取代。 </li><li>添加150微升生物膜回收培养基用多道移液器到一个新的96孔板的每个孔中。 </li><li>转移的挑战，从板块到孵化一个生物安全柜，小心地取出塑料袋，封口膜。 </li><li>从挑战板拆下钉盖和3.4中描述的冲洗掉浮游细胞在PBS。保持挑战板的底部后续OD 600 ANA裂解（见4.10）。 </li><li>冲洗后，将钉盖到含有生物膜回收介质板底部。 </li><li>包装用石蜡膜板的一侧，小心不要妨碍周边井的底部。立即包裹后盘，将其放置在酶标仪，并通过启动从步骤4.1以前刃天青动能协议作出重新开始进行24小时的动力读。 </li><li>量化浮游细胞的生长可能会或可能不会挑战过程中发生的是，读取用微型板读数器在整个挑战板底部的OD 600。按照步骤3.8的指示。 注意:仔细悬浮每个的内容以及细胞脱落掉的生物膜倾向于在井的底部中团块生长。阱内的任何气泡会强烈的OD 600读数干扰，必须避免或之前读除去。 </li></ol>

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The authors have nothing to disclose.

这里，我们已经描述了修饰的生物膜测定为S上确定测试抑制剂活性葡萄球菌生物膜注重生物膜有关的细胞的代谢状态。尽管所述生物膜起始和挑战的程序大多模仿制造商的建议，使用刃天青染料来检测和量化存活24小时抑制剂挑战生物膜相关的细胞显着地简化细胞回收（通过水浴超声处理），并随后列举的建议的过程通过的CFU计数的活细胞。该制造商推荐的程序涉及至少5个额外的操作步骤，他们每个人容易出错，劳动强度大，而且不切实际的自动化。简化了栓板测定的读出过程的优点增加了其对于高吞吐量操作和应用的吸引力。 虽然通过的步数冗长协议，该再修改的e是相当简单的概念，很少关键步骤。最关键的是，反复种植同样质量的生物膜的能力是获得可重复的结果至关重要。此外，必须拆卸或更换钉盖时小心。在一个良好的墙上任意点的联系会破坏生物膜，倾斜的结果。更何况，充足的洗濯是至关重要的:失败在任何时候以去除浮游细胞移除任何测量生物膜相关细菌的能力。相反，尽管，洗涤不能太严格;额外的洗涤步骤可以开始删除生物膜，产生不一致的结果。 栓柱板方法具有多种用途，这取决于所期望的结果数据。该法在这里描述为代谢刃天青转化的一个信息丰富的动能测量到的荧光染料11。这种转换是用从蓝色到粉红色的色移（ 图2C），其中，在additi相关到定量荧光读，可以容易初始质读出简单地通过观察转换。除去塞销盖后，这种转换也可以通过读取在600nm的吸光度应该荧光能力不可用定量。虽然荧光最终在每个时最大染料转化高原以及（幸存者），染料转化的发作可以使用相对于未处理的对照（ 图3）的代谢活性幸存者人口来估计。在我们手中，我们已发现，每个〜100分钟的延迟在刃天青转换的开始对应于开始相对于未处理的对照的接种（作为测定浮游细胞培养串联在代谢活性细胞的数量约10倍的降低在使用生物膜恢复介质10倍递增稀释（数据未显示））。 超越动力学读数，虽然，改性栓板方法更适合于高通量药物筛选项目比原始协议。通过测量刃天青的转换作为一个端点代替动力学，用户可以产生样品的二元分析来跟踪化合物简单的活动（ 即，染料转换/不转换），这消除了荧光板读数器的需要24小时，允许传统的孵化。这种方法允许确定的最小生物膜根除浓度无需声处理和电镀。动力学读还跟踪根据处理条件和抑制剂浓度生物膜的代谢状态。数目减少的操纵步骤，增加的再现性，定量读出方法（相对于电镀各孔单独地12），用户可以潜在地筛选数以千计每天化合物在单一浓度屏幕。事实上，栓式板先前已在高通量博士使用微克屏幕生物膜分散剂，虽然刃天青（如这里使用的）是一个更经济的选择比利用13专有的荧光素酶试剂。 而在现有的制造商协议的普遍改善，这里的特色化验确实有一定的局限性必须牢记。本质上，我们只衡量生物膜相关细胞的代谢能力。此法不测量生物膜质量或以其他方式削弱生物膜结构和完整性。这些影响不一定被检测到。代谢不活跃，但仍存活，持留细胞，这已被证明在生物膜14,15存在也将被检测到。进一步，诱导静止而不实际灭菌生物膜可能错误地显示为误报，取决于诱导状态下的长度的化合物。可行性指标是最终替代物更铁一般的测量方法，如流式细胞仪或CFU枚举，并且最终用户必须确定哪些方法是合适的。最后，由于刃天青转换依赖于内源NADH，细胞内贮存的耗竭可能是有害的细菌。虽然我们还没有看到对S.有害影响金黄色葡萄球菌 ，其他细菌物种可能会作出不同的反应。用户必须经验测试和验证此协议的可靠性和重复性给予不同的生物体和实验室环境。

致病微生物可在体内形成生物膜导致非急性慢性感染1。生物膜相关的感染是涉及异物（ 例如，人造骨替换，乳房植入物）或气管导管或导尿管2的安装植入医疗过程的严重危险因素。在这些情况下，抗感染治疗几乎总是必要的，因为基于生物膜的感染很少自行清除，甚至在免疫的个体。 金黄色葡萄球菌是在生物膜相关并发症牵连的最常观察到的病原体的使用期间发生的1微创医疗器械3。 不幸的是，生物膜的性质作为保护屏障，使他们对治疗比浮游细胞1,4-更耐磨，并预测临床疗效的评价是两个初始的关键部分药物开发以及耐药性监测。有越来越多的承认，实验室条件下，专注于浮游文化，可能无法忠实地代表现实世界的疾病5。进一步使问题复杂化，生物膜表型的复制是困难的，现有的生物膜模型繁琐和高之间和内部检测变异6受苦。因此，许多研究人员，通过必要性，默认为药物敏感性的浮游细胞的测定，从而潜在地忽略细菌毒力和疾病的一个重要方面。 在这里，我们描述了协议用 ​​于测定细菌，特别是S.金黄色葡萄球菌 ，在利用96孔基于生物膜系统-7,8-预生长的生物膜。而对生物膜形成和挑战的协议基本上遵循制造商的建议，我们提出一种替代信息丰富的方法用于量化biof的可行性攻击后ILM。简要地说，细菌是在栓板，其中生物膜上附着在板的盖的突出栓形成中培养。经过生物膜形成，钉在一个新的盘子装满PBS去除浮游细胞的井轻轻蘸。栓柱盖，具有附着生物膜，被转移到一个新的挑战板，含有不同浓度的抗生素进行检测。第二次温育后，盖子被再次除去，洗净，并转移至含有刃天青染料，这里它们经受一个最终温育恢复板。刃天青转化动力学上能够记录或作为一个定义的恢复期后端点读数。此染料型定量生物膜的可行性的方法从繁琐CFU（菌落形成单位）有很大不同的原始协议7所述计数基于方法学。药物的挑战板和刃天青转化动力学OD 600测量结果作为可行性读浮游和生物膜细胞，分别出局，提供快速，可靠，且技术上简单的信息丰富的生物膜存活试验。

<table><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></td></tr></tbody></table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

刃天青试验是非常灵敏可靠地检测能力攻击后复制的药物培养基很少活细胞。在这种方法中，我们定义的最小生物膜根除浓度在哪些没有刃天青转化24小时内看到的最低浓度。刃天青检测依赖于它转换染料的颜色由蓝色变为粉红色代谢活跃的细胞中发现的氧化分子。染料转换是这样的细胞生长的一个指标，并且可以使用荧光微板读数器来量化。在本文中，亚铜灵和Cu-亚铜灵（铜亚铜灵络合）为他们的活动进行了测试对生物膜有关S.金黄色葡萄球菌施加是基于图1的布局。该协议的执行过程中获得的数据的OD 600读数生物膜起始板的，它作为一种质量控制（数据未示出）中，板底部的挑战后的OD 600读数已经完成（ 图2A），和刃天青变换的动能记录到回收板（ 图2B）的其荧光代谢物试卤灵。进一步的挑战板的终点图像可以用于刃天青转换（ 图2C）的目视检查，如果有/无刃天青转换评价足够用于实验的目的。另外，如果动能录制功能不可用或多个板被并行运行，荧光可以之后通过单个端点读取24小时来确定。如在图2A中看到的那样，从挑战板的OD读数表明，单独亚铜灵不抑制浮游细胞的生长;然而，铜亚铜灵以1.3μM抑制生长，如预期10。一个类似的模式被从生物膜板的刃天青动力学观察到的，指示只有铜亚铜灵能够消除代谢活性生物膜相关的细胞（ 图2B）。 除了提供生物膜根除浓度，动力学测定法提供了在其上指示的活菌数的生物膜的代谢状态的信息。当比较用庆大霉素的浓度增加对未处理的对照处理的生物膜，刃天青变换的滞后可以看出（ 图3）。为2.5微克/毫升庆大霉素，有一个13小时的延迟，直到井达到刃天青的相同的转换作为未处理的对照。这种滞后可能来自存在于处理生物膜与未处理的活细胞的数目减少。 <img alt="图1" src="/files/ftp_upload/53925/53925fig1.jpg" /> 图1.挑战板布局。为例挑战板电子布局在六个不同浓度的容纳一式三份四种化合物并行测试。未处理的和不育对照也包括在内。布局设计支持方便板准备使用多管移液器连续稀释液。浓度是μM。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53925/53925fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53925/53925fig2.jpg" /> 图2.确定最小生物膜根除浓度。新铜试剂和Cu-亚铜灵被消灭生物膜电位测试。 （A）中的OD 600从挑战板采取除去钉盖的后量化生物膜脱落的细胞的生长。 （B）刃天青转化率在INDIVI动力学监测双口井在24小时的时间内。个人minigraphs显示一段时间（24小时）荧光（RFU）。缺乏刃天青转化表示没有幸存者。刃天青转换表示存活细胞脱落掉的生物膜和在回收培养基中生长的存在。 （3）如果动能读出是不可能或不需要，那么一个简单的是/否的答案活细胞的存在，可以直观地从蓝色刃天青到粉色，简化形式的颜色转换决定。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53925/53925fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53925/53925fig3.jpg" /> 图3.动力学测定刃天青揭示生物膜对细菌有抑制作用。化合物可能无法完全根除生物膜嵌入细胞，但仍可以减少IR可行性。这可以比较刃天青用不同浓度的庆大霉素处理的孔的转化延迟时可以看到。具有在刃天青转换延迟井表示存活细胞的数量减少。黑色（仅媒体），橙（0微克/毫升），绿（0.3微克/毫升），蓝（0.6微克/毫升），紫（2.5微克/毫升），和灰色（5微克/毫升）。 ΔT是指在大致一半最大荧光值未处理和庆大霉素处理的样品呈指数刃天青转换的时间差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53925/53925fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

13.0K 次观看.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

视频: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

科研

免疫与感染

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

生物工程

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

标准化 体外 测定，以可视化和量化人中性粒细胞与 金黄色葡萄球菌 生物膜之间的相互作用

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

改进酶保护测定法研究 金黄色葡萄球菌 内化和抗菌化合物的细胞内效

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

生物学

In Vitro Biofilm Synthesis by Staphylococcus aureus

Source: Rana, P. S. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63773">Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the preparation of in vitro biofilm by S. aureus in a poly-L-Lysine-coated multi-well plate. The biofilm-formed plate can be used for downstream experiments.

金黄色葡萄球菌体外生物膜合成

Source: Rana, P. S. et al., Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus ...

针对相关生物膜

摘要

探索更多视频

此视频中的章节

酶标菜生物膜形成实验

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

使用人工痰介质测试抗生素药效试验

芯片上的压力诱导抗生素易感性测试

适合与天然化合物库的探索抗生物试验的平台

系统抑制剂靶向细胞内的疗效和毒性筛选

标准化体外测定，以可视化和量化人中性粒细胞与金黄色葡萄球菌生物膜之间的相互作用

一种基于四唑的可溶性还原测定，用于评估抗体对热带念珠菌生物膜的影响

改进酶保护测定法研究金黄色葡萄球菌内化和抗菌化合物的细胞内效

金黄色葡萄球菌体外生物膜合成

针对相关生物膜

摘要

探索更多视频

此视频中的章节

酶标菜生物膜形成实验

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

使用人工痰介质测试抗生素药效试验

芯片上的压力诱导抗生素易感性测试

适合与天然化合物库的探索抗生物试验的平台

系统抑制剂靶向细胞内的疗效和毒性筛选

标准化 体外 测定，以可视化和量化人中性粒细胞与 金黄色葡萄球菌 生物膜之间的相互作用

一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响

改进酶保护测定法研究 金黄色葡萄球菌 内化和抗菌化合物的细胞内效

金黄色葡萄球菌体外生物膜合成

标准化体外测定，以可视化和量化人中性粒细胞与金黄色葡萄球菌生物膜之间的相互作用

一种基于四唑的可溶性还原测定，用于评估抗体对热带念珠菌生物膜的影响

改进酶保护测定法研究金黄色葡萄球菌内化和抗菌化合物的细胞内效