Name: high-throughput identification of bacteria repellent polymers for medical devices
Uploaded: 2016-11-05T13:00:00.000+00:00
Duration: 10 min 43 s
Description: Watch this Scientific Journal Video about High-throughput Identification of Bacteria Repellent Polymers for Medical Devices at JoVE.com

The authors thank EASTBIO (the East of Scotland BioScience Doctoral Training Partnership funded by the BBSRC) (S. V.) and the Medical Research Council (P.J.G) for funding.

1.琼脂糖包膜幻灯片的制备注:制造聚合物微阵列之前，氨基烷基硅烷涂覆的玻璃载片涂覆有琼脂糖的IB以减少非特异性背景结合，并允许结合或排斥菌6的聚合物的评价。硅烷涂层有利于琼脂糖以幻灯片的结合。 <ol><li>补2％（重量/体积）琼脂糖ⅠB的在蒸馏水中的250毫升瓶中溶液。 </li><li>松散封盖瓶子后，加热在微波炉的悬浮液在30秒时间段直至溶解。确保该帽不紧紧关闭，以避免任何可能的压力累积。定期取出瓶子，轻轻摇动。 </li><li>确保固体溶解，将溶液是明确的。倒在65℃的水浴此琼脂糖溶液的100毫升烧杯中，并地方保持液体溶液。 </li><li>浸硅烷包被的载玻片放入琼脂糖溶液，以确保均匀的涂层，和WiPE的幻灯片用棉纸背面。 </li><li>干的幻灯片，用涂边了，在无尘的环境中（ 例如 ，一个柜子里或通风柜）最少24小时。 </li><li>确认通过目测均匀的涂层。会形成冷凝非涂布地区 - 涂层可以通过呼吸到幻灯片很容易评估。只有完全和均匀包被的载玻片应该用于微阵列打印。 </li></ol> 2.印刷聚合物溶液的制备<ol><li>选择包括在玻璃小瓶N-甲基吡咯烷酮（NMP）中的聚丙烯酸酯，聚丙烯酰胺和聚氨基甲酸乙酯预形成聚合物的制备溶液（1％重量/体积）。 （制备各1ml聚合物）。涡旋直至聚合物完全溶解。聚合物的合成在别处6中描述。 </li><li>用微型移液管，填充用25微升的聚合物溶液384微孔板的各孔中，以确保不存在交叉CONTA萌发和每个孔含有独特的聚合物。使用NMP作为在至少2个孔的阴性对照。记录上的孔板模板或电子表格文件在每个孔中的聚合物溶液的身份。 </li></ol> 3.印刷聚合物微阵列使用接触打印机注:使用接触打印机进行芯片的打印。关于聚合物微阵列打印具体说明如下。有关使用打印机和安全建议的一般准则，遵循从制造商的用户手册。虽然我们使用一个接触打印机，合适的手动冲压设备也可使用。 <ol><li>作为在打印机的用户手册劝，创建例程（参见步骤3.3），允许的384液体（聚合物溶液或NMP）中一式四份的印刷，使用的是32针microarraying头（设置为8行和4列）。 </li><li>打印排列为48行和32列的1536点。编程例程在12节打印S， 即 ，在时间32的解决方案，从而使打印机能够打印每个部分，以允许销清洗后停止。 </li><li>要创建一个打印程序: <ol><li>打开软件，然后从可用选项中选择"创建一个新的程序"。选择"说明"选项卡，在实验的输入信息。选择"头"选项卡，然后选择&#39;32 -pin microarraying头"。 </li><li>选择"源"选项卡，然后选择板夹（来源板支架（1×5）），板型（板384点¯x6000），总板（1）和源顺序（按列）。 </li><li>在选项卡"幻灯片设计"，选择"3X1&#39;滑&#39;，并选择通过排列田间号"。然后选择"组阵模式"。输入"现货视图"作为"布局"和"布局尺寸"为"行数"（6），"列数"（8），"行间距"（750）和"列间距"（560）。选择一款打印冷杉吨。 </li><li>在"幻灯片版式"选项卡中输入用于打印幻灯片的数量。选择"打印"选项卡输入打印参数（每墨邮票数量:1，每点邮票数量:100毫秒5，冲压时间:200毫秒，墨时间）。 注:现在一式肆份打印384液体（聚合物溶液或NMP），采用32引脚microarraying头（排列为8行4列）创建的程序。总共日常应打印排列为48行和32列具有750微米的行距和560微米的行距1536点。 </li></ol></li><li>放置在平台上的琼脂糖包被的载玻片，并确保良好的真空密封以保持滑动就位。 </li><li>调整打印头，使得所有管脚处于相同的高度。通过手动降头在载玻片上，并确认该引脚在同一时间拉升检查。如果有与高度的任何差异，通过使用黑色PU皮ip调整E在针向上或向下。 </li><li>放置在正确的方向在板夹持器， 即 ，A1孔是在支架的右上角的孔板并确保孔板被牢固地固定。 </li><li>使用高度调节，确保该板保持器的高度是这样的，采摘聚合物溶液时，销不接触孔的底部。 注:这是均一的聚合物斑点重要，也避免了聚合物溶液的蔓延到相邻的井，从而造成交叉污染。 </li><li>选择"开始"选项卡，选择"运行类型"为"正常"。点击"运行"，并确认该幻灯片，孔板等 ，在出现提示时在适当的位置。 </li><li>在一个时间（每次32解决方案; 12节）打印1部分，允许部分之间的引脚清洁。清洁销在丙酮彻底用纸巾浸渍，接着干燥的薄纸，以确保销完全干燥。 </li><li>在微阵列的打印完成时，将它们放置在滑动架和在炉中在45℃以除去NMP中的聚合物斑点真空过夜干燥。用UV光30分钟灭菌后，将微阵列准备好细菌的接种。 </li><li>细菌接种幻灯片之前，请背景荧光的测量（如第5节）。细菌结合的计算中使用这种测量。 </li></ol> 4.聚合物微阵列接种细菌注:确保良好的无菌技术。培养物的所有处理应该在无菌环境中进行的:要么使用本生灯或在流罩。培养物应当用氧的可用性，生长培养基，和温度调节到每个物种的要求来生长。 <ol><li>接种5毫升的Luria-BERTANI肉汤（LB准备过夜培养10克L- -1细菌用蛋白胨，5克L -1 NaCl的，和10克L- &lt;SUP&gt; -1酵母抽提物）与从板的殖民地。在37℃下孵育过夜，以200rpm振荡。 </li><li>通过添加甘油（10％浓度的整体）的准备过夜培养的冷冻库存，并存储在股票-80℃。 </li><li>为了从解冻的细菌种群的样品确定的细胞数，进行各股票的连续稀释并过夜生长的细菌在固体培养基上。在股细胞数量的准确测定可确保各种6的适当接种。 </li><li>微阵列准备接种。单一物种培养物如上述生长并直接施用到微阵列。 BacMix-1是肺炎克雷伯氏菌 （ 肺炎克雷伯 ）， 腐生葡萄球菌 （ 腐生葡萄球菌 ）和金黄色葡萄球菌 （ 金黄色葡萄球菌 ）的细菌混合物。BacMix-2是由链球菌（变形链球菌）的细菌混合物， S.金黄色葡萄球菌 </eM&gt;，K.肺炎和粪肠球菌 （ 粪肠球菌 ）。 </li><li> ，以产生混合培养，在相同体积（3毫升每个）混合过夜并稀释四次新鲜LB（到50 ml终体积）。 </li><li>放置紫外线灭菌的聚合物微阵列在矩形4孔板并用6ml的混合细菌培养物，在37℃，轻轻摇动（30转）5天孵育它们。 </li><li>孵育后，轻轻地用磷酸缓冲盐水冲洗微阵列两次（PBS:137mM的氯化钠，2.7mM的氯化钾，10毫的 Na 2 HPO 4，1.8毫摩尔KH 2 PO 4），并用1微克毫升的溶液孵育4 -1 &#39;，6-二脒基-2-苯基吲哚（DAPI）在PBS中染色30分钟。 </li><li>用PBS覆盖，并轻轻摇动，改变了PBS洗一次在一个新的孔板染色芯片幻灯片。空气流下干燥。应用盖玻片的微阵列及密封的地方用胶水。一旦ðRY，消毒的外表面，用70％（体积/体积）乙醇。 </li><li>根据自己的防护水平的安全文化处分的权利。 </li></ol> 5.芯片成像和分析<ol><li>捕捉每个聚合物点单幅图像在明和DAPI（前/ EM = 358纳米/ 361纳米）的通道。用安装有一20X物镜的荧光显微镜或使用自动荧光显微镜（由图像获取软件控制的XYZ载物台）手动执行此。 注意:请参考手册18操作软件和调整显微镜获得使用明和DAPI通道的图像。确保用于图像捕获的增益和曝光时间对于所有的微阵列是相同的。 </li><li>通过选择的点区域和量化用图像处理软件的荧光获得在DAPI通道中的每个点的平均荧光强度。 </li><li>对于每个点的背景荧光，OBTA在复制芯片聚合物点的图像只能用无细菌培养的媒体。从点的强度扣除背景荧光从细菌获得的荧光。 </li><li>从四个点计算平均归一化值，代表每个聚合物，用于比较细菌各种聚合物6的结合。 </li><li>识别表现出最少的细菌结合（最低荧光）为"重灾区"聚合物6聚合物。 </li></ol> 6.涂层盖玻片为"重灾区"验证<ol><li>与聚合物涂层盖玻片: <ol><li>制备"击中"的聚合物的溶液（〜2％重量/体积）在四氢呋喃（THF），或其它适当的溶剂。 </li><li>用旋涂器以2000rpm进行10秒的聚合物溶液的合适尺寸的旋涂圆形盖玻片。 注意:在不存在旋涂器，盖玻片或其它材料可以是浸涂，虽然旋涂层产生更均匀的表面。 </li><li>干燥的对流烘箱中涂覆的盖玻片在40℃下过夜，并使用UV光进行接种细菌之前30分钟消毒。 </li></ol></li><li>与琼脂糖大衣盖玻片阴性对照: <ol><li>清洁盖玻片或者通过等离子体处理10分钟或在1M氢氧化钠中浸渍4小时。 </li><li>沉浸盖玻片在含有1％（3-氨基丙基）三乙氧基硅烷过夜乙腈。用丙酮清洗盖玻片3次，并放入烘箱（100℃）1小时。 </li><li>浸涂用1％琼脂糖水溶液干燥盖玻片，在60℃的水浴中保持。放置在平坦的表面上的琼脂糖包被的盖玻片在24小时无尘环境条件和使用UV光进行接种细菌之前30分钟消毒。 </li></ol></li></ol> 7.附件和盖玻片利用扫描电镜分析（SEM） <ol><li>后灭菌用UV光30分钟，放置在标准的12孔板或24孔板（取决于盖玻片的大小）的盖玻片（聚合物/琼脂糖涂覆或未涂覆的玻璃）中，用菌孵育作为部分所述4。 </li><li>与细菌培养后，用0.1M二甲胂酸盐缓冲液（pH 7.4）洗涤未涂覆和涂覆的盖玻片两次，然后用2.5％（重量/体积）在0.1M二甲胂酸盐缓冲液戊二醛（pH7.4）中固定2小时。 </li><li>修复交样品用1％（重量/体积）的四氧化锇在室温下1小时，并用连续的乙醇洗涤，在50，70％，90％和100％（V / V），每30分钟脱水。 </li><li>在CO 2的干样品根据标准方法19。干燥时间取决于样品的性质。 </li><li>涂层样品在用溅射镀膜机，在30毫安的电流，并在真空在0.75乇的金/钯合金（60/40％）。电子显微镜按标准协议，20岁以下的检查。 注:少尉URE足够的安全预防措施，以管理来自贮存，处理和处置的四氧化锇导致的风险，因为它是剧毒。 </li><li>直观比较的无涂层盖玻片的图像和那些涂有琼脂糖或"重灾区"的聚合物，以确认聚合物的细菌结合/排斥能力。 注:附件的阻力应尽量减少细胞存在容易看到。 </li><li>选择最有前途的"重灾区"非约束性聚合物用于医疗设备（ 例如 ，中心静脉导管）6涂层的研究。 </li></ol> 8.一种溶剂为涂料导管的选择<ol><li>评估各种溶剂为它们与导管的留置部分的相容性，以及它们以溶解"击中"聚合物的能力。切割导管-1，并进入圆筒件5毫米份的长度，并用数字游标卡尺测量。 </li><li>评估各种溶剂如ACET集成电路酸，氨，丙酮，乙腈，二乙醚，四氢呋喃（THF），二甲基甲酰胺（DMF），N-甲基-2-吡咯烷酮（NMP），乙醇，甲醇，乙二醇，甲苯和二甲苯。沉浸在溶剂导管件12小时，对导管和溶剂清晰度的完整性目视评价。 注意:选择不会导致导管膨胀或崩解或导致混浊溶剂的溶剂。溶剂必须溶解"重灾区"的聚合物。 </li></ol> 9.激光共聚焦显微镜上的导管细菌吸附分析<ol><li>制备2.5％（重量/体积）的丙酮或其它合适的溶剂的聚合物溶液作为第8.将5毫米导管件确定成一个小玻璃小瓶（5毫升）和沉浸其中在1ml的聚合物溶液2分钟。除去多余的聚合物溶液，并在40℃的真空烘箱中过夜干燥所述导管件。 </li><li>通过混合细菌解冻ST接种准备ocks，得到约10 6各自物种的细胞在50ml的LB 6。 </li><li>消毒涂覆和未涂覆的导管件用UV光30分钟，并用1ml在24孔平板接种LB孵育，在37℃下进行3天，用温和搅拌6。 </li><li>温育后，冲洗导管，用PBS（2×2毫升）中，固定用在PBS中的10％的多聚甲醛的菌30分钟，并用PBS（1ml）中洗涤。染色上导管件用DAPI细菌（1微克毫升- 1）20分钟，并用PBS（1ml）中洗涤。 </li><li>获得使用以下设置导管件共聚焦图像:405纳米的蓝色激光二极管，检测范围414到502纳米，销孔 - Airy1，图像大小 - 1,024×1,024像素，像素宽度 - 105.2纳米，Z-栈间距0.5μ米，放大倍率 - 40X 1.25。 </li><li>由Z-堆叠横跨导管的100微米长50幅图像完成共焦成像。 </li><li>分析使用suitab图像乐分析软件:扁平化的Z堆叠图像Z平面，使用功能来扩展景深，创造了导管件的单个图像。 注:然后将该图像进行分析，以获得由细菌覆盖导管的区域中，之后使用"弄平背景&#39;功能背景校正。 </li></ol>通过SEM导管上细菌附着10.分析<ol><li>制备10％的丙酮的聚合物溶液（重量/体积）。 </li><li>按一个枪头（200微升微量）插入切片导管的中间部分，以保持它与浸在聚合物溶液中的一块约30秒。干在环境条件下涂覆的片30分钟。通过再次浸入在环境条件下在聚合物溶液中并干燥过夜施加第二涂层。 </li><li>紫外线处理和培养细菌后洗片（N = 3）用PBS（2×1毫升），并将它们转移到含有1 48孔板在PBS 0％甲醛30分钟。定影后，洗片用PBS（1ml）中，在室温下干燥过夜，安装在用导电性碳磁盘存根和金涂层用溅射涂布机（见步骤7.5）。 </li><li>获得使用扫描电子显微镜6的图像。 </li></ol>

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The authors have no competing financial interests to declare.

细菌附着到一个表面是由依赖于细菌种类广泛的因素决定的一个复杂的过程，从表面上看，周围介质和物理环境的属性。虽然某些化学基团是已知的影响细菌的结合（聚乙二醇，例如，典型地抗蚀附件11），关联的聚合物与它们的化学结构的生物影响是困难的，使聚合物的合理设计为具有挑战性的特定功能。在没有详细的附件机制，其他研究都试图模仿天然驱蚊表面，具有长期和广泛的优化处理21。这里介绍的小型化高通量方法克服了便利数百聚合物的平行筛选，以确定进一步研究的线索这些挑战。 从微阵列方法结果主要用于IDEntify有可能导致的候选者。 图2示出22名候选人与低结合的至少一个品种，而图3说明了在结合能力的明显减少。在图2中所示的全部22个低结合性聚合物被带到正向成比例增加的实验中，在此期间，最好（以排斥和涂层性质方面）被确定为PU83，PA13，和PA515（ 图4和5）。聚丙烯酸酯提供在聚合方法方面具有更大的灵活性，因此将结合最低聚丙烯酸酯，PA13，被选定为导管涂层的研究（ 图6和7）。对其他候选人更详细的进一步开展这项工作，并已在别处6日报道。 通过一些实验性的迭代，我们发现了一些小的步骤是成功的关键和可重复性。以及促进的粘附聚合物的玻璃载玻片，使用琼脂糖下涂层提供了一个干净的背景，如琼脂糖是细菌定植高度抵抗性。同样在聚合物中的一致性掩护本身，都在同一阵列中与阵列之间，是至关重要的，因此，阵列的打印，必须仔细控制。需要在打印头销与384孔板中也均匀填充仔细调整，以确保均匀的斑点。如一些我们所使用的聚合物的表现出一定程度的自体荧光的，取背景荧光数据对每个幻灯片之前孵育细菌是至关重要的。为了说明变化和取得的芯片强大的数据重复建议。 吲哚（DAPI）此处所用的染色剂具有用于细菌种类，结合非特异性DNA的无选择性。因此，一旦细菌培养被引入污染物可能会被忽视，混淆了interpre良好的无菌技术是必不可少的结果的塔季翁。同样是使用扫描电子显微镜，它是仅能够区分杆和球菌但不属或种以后的实验事实。 基因芯片筛选后，有前途的聚合物应选择进一步验证。在这里介绍的实施例中，感兴趣7聚合物进行可以通过明显减少在荧光确定在微阵列和它们附着的抑制通过在较大的表面上涂敷他们证实。 图4和5示出在上玻璃盖玻片上，一个结合所取得的还原实用装置来测试聚合物作为散装涂料，而不是作为微阵列点的行为。随后，这些聚合物被涂覆在医疗器械的完全量化在细菌附着减少。重要的是，该溶剂选择（见协议部分8），用于这些涂料的研究是良性到所需的基板（在此，导管），同时保留ING溶解感兴趣的聚合物，以便允许涂层的能力。这里，我们用丙酮其中，以及属性所提到的，具有低的沸点，并迅速蒸发，留下一个均匀的涂层。 正在研究的选择将取决于具体应用的验证的装置。正如电子和荧光显微镜细胞观察允许单个细胞附着的直接量化，我们选择了这些技术作为补充大量染色芯片分析。结果示于图6和7，其展示的免费验证方法的重要性。 图6中的共焦图象提供各个单元的非常清晰的图像，而扫描电镜具有允许该聚合物，这是这里平滑和均匀的表面的评估的附加益处。这些方法是由视所使用的显微镜​​的领域的限制，因此，它是性重要NT采取一系列快照有结果的可信度。上述方法不能定量细菌粘附在整个表面上，只有从多个小区域的推断覆盖。我们相信，这是足以为所描述的应用程序。减少细菌的结合可以通过使用其他地方22所描述的方法，整个涂层和非涂层的导管件列举表面细菌附着进行评估。然而，这样的方法需要筛选有均匀的表面面积，这是困难的，当分析用的医疗器械，这往往具有复杂的几何形状进行保持生物材料的表面。 显然，用于临床使用的任何设备都必须经过大量的进一步的测试，以确保在人类中的安全性和有效性。这里介绍的方法代表此过程和进一步的工作的开始必须包括在体内活性的确认。在这种情况下，学习静脉Çatheters，初始工作可以调查血液成分和全细胞与聚合物的结合。对细菌结合的血液成分的效果，也应考虑，可能通过在灭活血清或解fibrinated血液23的存在重复结合测定。该技术的明确的测试将在体内模型中，诸如皮下植入感染模型24。 我们表现​​出表面改变聚合物的筛分聚合物微阵列的方法的潜力。这种聚合物（包括抵制和促进细菌的结合）有医药，食品行业和生物技术应用了大量的，这意味着这种方法可能会在研究许多领域是有用的。虽然工作在这里使用的细菌中，该方法可以适用于其他类型的细胞，同样其它化学微阵列。

<html>聚合物微阵列是小型化，其中高达7,000聚合物1被印刷到载玻片于原核或真核细胞2平行分析的高通量平台。这里介绍的方法基础上，与我们于2010年首次3所述。这种筛选系统已被应用于许多类型的细胞，包括人肝细胞4，干细胞5，肾小管上皮细胞2，细菌3,6和原生动物病原体7。在每一种情况下，促进或所研究的细胞的抗蚀剂结合性聚合物被确定8。用合成的聚阳离子聚合物的DNA的复合物也已在基因转染的候选9的高通量筛选的微阵列形式使用。以及筛选细胞-基底相互作用，聚合物微阵列也被用于评估材料特性10。 "&gt;合成聚合物的调节细菌附着于表面的能力是公认的3,6,11。许多因素，包括电荷，疏水性和聚合物表面的表面粗糙度是已知的影响发现生物材料细菌结合。该常规方法该抗蚀剂的细菌通过依次或结合凭经验设计和一次测试一个材料是劳动密集型的，昂贵的和耗时的过程。聚合物微阵列提供规避这种限制一个有吸引力的替代方案。 表面相关的细菌成长为一个复杂的人口称为生物膜 - 这种生物膜对许多环境压力和抗生素高度耐药。这部分是由于它们的稠密细胞外基质（蛋白质，多糖和核酸组成）12，部分由于健壮"persistor"细胞的生物膜13增加的存在。本书虽然是啊表面关联和随后的生物膜形成的确切机制是难以表征，它一般认为有表面生长14的三个不同阶段- 16。最初，可逆附着后跟细胞更强的粘附性，由生产的胞外蛋白和多糖基质和细胞增殖建立生物膜。最后，成熟的生物膜释放自由生活的浮游细胞，它可以在其他地方引发新的感染。菌驱蚊防止细菌的初始附着，从而防止生物膜形成的早期阶段的聚合物，可能代表用于最小化感染的最佳解决方案。给予抗生素抗性的上升（也表面相关的细菌12的内在阻力较大），减少了感染的无抗生素的装置是特别有意义的。在医院环境中，细菌驱蚊聚合物涂层可以在医院内感染，这通常形成围绕植入设备17的减少有直接的医疗应用。 这里，描述了用于381聚合物为针对一系列与院内感染相关的病原性细菌的驱避活性的筛选的高通量方法，随后命中的验证和随后的涂覆和中心静脉导管的材料的测定中，（ 图1）。简要地说，将聚合物点样到由接触印刷琼脂糖包被的载玻片上，干燥和消毒后，小型化的阵列，用临床上重要的细菌培养物温育。孵育后，将微阵列轻轻洗涤和粘附的细菌细胞染色，并通过荧光可视化。接着，其抑制细菌结合聚合物是通过涂覆到玻璃盖玻片上的较大规模研究和利用电子显微镜观察。所选击退然后借给聚合物涂覆到商业导管和示出了近100倍，以减少细菌的附着。

<table><tbody><tr><td>Agarose</td><td>Sigma</td><td>05066&amp;nbsp;</td><td></td></tr><tr><td>Silane-prep slides</td><td>Sigma</td><td>S4651</td><td></td></tr><tr><td>Polymers</td><td>Synthesised in-house</td><td>Not applicable</td><td></td></tr><tr><td>NMP</td><td>Sigma</td><td>494496</td><td></td></tr><tr><td>LB Broth</td><td>Oxoid</td><td>CM1018</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Sigma</td><td>401757</td><td></td></tr><tr><td>(3-aminopropyl) triethoxysilane coated glass slides</td><td>Sigma</td><td>Silane-prep</td><td></td></tr><tr><td>Cacodylate buffer</td><td>Sigma</td><td>97068</td><td></td></tr><tr><td>Catheter 1</td><td>Arrow International</td><td>CS12123E</td><td></td></tr><tr><td>Catheter 2</td><td>Baxter Healthcare</td><td>ECS1320</td><td></td></tr><tr><td>Osmium tetroxide</td><td>Sigma</td><td>201030</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Contact printer</td><td>Genetix</td><td>Qarraymini</td><td></td></tr><tr><td>Microarray microscope</td><td>IMSTAR</td><td>Pathfinder</td><td></td></tr><tr><td>Spin Coater</td><td>Speedline Technologies</td><td>6708D</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>SP5</td><td></td></tr><tr><td>Image analysis software</td><td>Media Cybernetics</td><td>Image-Pro Plus</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Philips</td><td>XL30CP</td><td></td></tr><tr><td>Sputter coater</td><td>Bal-Tec</td><td>SCD050</td><td></td></tr></tbody></table>

high-throughput identification of bacteria repellent polymers for medical devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

图2示出了细菌附着（归一化的荧光强度）与数如通过微阵列分析测定的聚合物。印刷没有聚合物斑点（NMP只）是阴性对照，如琼脂糖强烈抵抗细菌结合6 -记录荧光很低。显示的聚合物是所有低结合虽然在大多数情况下，聚合物的拒水拒油性测试细菌物种之间显著而变化。这反映了在不同种类的附件机制之间的巨大差异。适当聚合物的选择是因此依赖于应用，但低结合性聚合物容易被琼脂糖比较鉴定。高粘合聚合物有可能在一个阵列被识别，并且可以用作用于随后的实验中的阳性对照。为不显示自发荧光的DAPI通道的聚合物，定性共光斑图像的mparison可以视觉进行（ 如图3，突出一个代表性的高结合性聚合物和三个示例低结合性聚合物）。这样的比较，同时提供比统计分析少的信息，可以是计算出的强度值的有用的视觉验证。 使用微阵列鉴定22适当的聚合物，扩大进行了实验用于涂覆大表面时，以确认其细菌排斥性。显示的是表现最好的例子，用于涂层的玻璃盖玻片（用扫描电镜分析（ 图4和图 5））和导管片（共聚焦显微镜（ 图6）和SEM分析（ 图7））。这两种显微方法具有允许在表面上的直接细胞计数，提供明确的数据的好处;减少细胞附着是容易visibl即其中最好保持在按比例放大的驱避性能，以及作为适合于大规模的涂布技术的那些涂层，进行进一步的研究。所示结果表明，从每个阶段表现最好的聚合物。 <img alt="图1" src="/files/ftp_upload/54382/54382fig1.jpg" /> 图 1: 通过聚合物微阵列生物医学应用参与细菌斥聚合物的鉴定步骤 SEM =扫描电子显微镜。 <img alt="图2" src="/files/ftp_upload/54382/54382fig2.jpg" /> 图 2: 细菌在一系列的聚合物，通过微阵列分析测定的结合低细菌结合被认为在许多聚丙烯酸酯/丙烯酰胺（PA）和聚氨酯（PU）的。从聚合物微阵列探测的结果与一些细菌小号<html> pecies（ 空肠弯曲杆菌 ， 艰难梭菌 ， 产气荚膜梭菌 ， 变形链球菌和两个财团; BacMix-1和BacMix-2）被示出。细菌结合表示为背景校正的平均DAPI荧光强度（归一化）。误差棒代表标准偏差。从参考6改编。 <a href="https://www.jove.com/files/ftp_upload/54382/54382fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54382/54382fig3.jpg" /> 图 3:BacMix-2的聚合物点的实施例的图像荧光吲哚（DAPI）和明显微镜图像示出了有代表性的聚合物细菌附着。一个强结合聚合物是包括比较几个非结合性聚合物（PU-20，PA-336和PU-179）。比例尺= 100微米。从参考6改编。://www.jove.com/files/ftp_upload/54382/54382fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54382/54382fig4.jpg" /> 图4: 规模化的实验用玻璃盖片的SEM图像显示在无涂层的玻璃及玻璃涂有"重灾区"聚合物（从表1中挑选的例子）细菌附着。比例尺= 20微米。 <img alt="图5" src="/files/ftp_upload/54382/54382fig5.jpg" /> 图 5: 细菌结合由细胞计数从SEM图像量化每涂覆有最好的"击中"聚合物表面的单位面积附着的细胞的比较。琼脂糖用作对照"非约束&#39;的表面上，玻璃作为对照结合表面。误差棒表示标准偏差。 <img alt="图6" src="/files/ftp_upload/54382/54382fig6.jpg" /> 图6: 光面铜版纸和胶版导管片细菌结合的比较比较孵育后未经处理的导管（导管-1）（A）和导管涂上PA13（B）与BacMix-2共聚焦图像。用40X的目标（比例尺= 20微米）拍摄的图像。从参考6改编。 <img alt="图7" src="/files/ftp_upload/54382/54382fig7.jpg" /> 图 7: 细菌结合对涂覆和未涂覆导管切片比较 SEM图像显示孵育后涂覆有PA13（B）的用细菌鸡尾酒组成BacMix-2的未经处理的导管（导管-1）（A）和导管的比较量表。酒吧= 101，M。从参考6改编。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 聚合物 </td><td> 单体1 </td><td> 单体2 </td><td> 单体3 </td><td colspan="3"> 单体的比率 </td></tr><tr><td> PA465 </td><td> MEMA </td><td> DEAEMA </td><td> HEA </td><td> 8 </td><td> 1 </td><td> 1 </td></tr><tr><td> PA475 </td><td> MEMA </td><td> DEAEA </td><td> HEMA </td><td> 6 </td><td> 1 </td><td> 3 </td></tr><tr><td> PA513 </td><td> MEMA </td><td> DEAEMA </td><td> MMA </td><td> 8 </td><td> 1 </td><td> 1 </td></tr><tr><td> PA515 </td><td> MEMA </td><td> DEAEA </td><td> MMA </td><td> 6 </td><td> 1 </td><td> 3 </td></tr><tr><td> PA13 </td><tD&gt; MMA </td><td> DMAA </td><td> - </td><td> 9 </td><td> 1 </td><td> - </td></tr><tr><td> PU1 </td><td> PEG2000 </td><td> HDI </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr><tr><td> PU16 </td><td> PEG2000 </td><td> MDI </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr><tr><td> PU161 </td><td> PEG2000 </td><td> MDI </td><td> BD </td><td> 2.5 </td><td> 5.2 </td><td> 2.3 </td></tr><tr><td> PU7 </td><td> PEG900 </td><td> BICH </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr><tr><td> PU83 </td><td> PEG900 </td><td> HMDI </td><td> BD </td><td> 2.5 </td><td> 5.2 </td><td> 2.3 </td></tr><tr><td> PU227 </td><td> PPG-PEG-1900 </td><td> HDI </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr><tr><td> PU129</td><td> PPG425 </td><td> BICH </td><td> DMAPD </td><td> 2.5 </td><td> 5.2 </td><td> 2.3 </td></tr><tr><td> PU10 </td><td> PTMG2000 </td><td> BICH </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr><tr><td> PU179 </td><td> PTMG2000 </td><td> HDI </td><td> NMAPD </td><td> 2.5 </td><td> 5.2 </td><td> 2.3 </td></tr><tr><td> PU20 </td><td> PTMG2000 </td><td> MDI </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr><tr><td> PU5 </td><td> PTMG2000 </td><td> HDI </td><td> - </td><td> 4.9 </td><td> 5.2 </td><td> - </td></tr></tbody></table> 表1: 细菌的组成非约束在图2中"命中"的聚合物。

Watch this Scientific Journal Video about High-throughput Identification of Bacteria Repellent Polymers for Medical Devices at JoVE.com

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

high-throughput-identification-bacteria-repellent-polymers-for

Research

JoVE Journal

Bioengineering

9.0K 次观看.  University of Edinburgh. A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

视频: High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

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) ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

环境科学

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

免疫与感染

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

生物工程应用先进材料的抗菌性能研究

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

使用PCR板深孔微孔设备生成更大的细菌生物膜生物质

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

A Novel Method to Determine the Longitudinal Antibacterial Activity of Drug-Eluting Materials

Ultrahigh molecular weight polyethylene (UHMWPE) is widely used in total joint arthroplasties as a load-bearing surface. Periprosthetic joint infections, the majority of which occur shortly after joint replacement, constitute almost 25% of total knee revision surgeries, and the complete eradication of bacterial infection poses a major challenge. A promising way to tackle this problem is to ensure the local sustained delivery of antibiotic concentrations sufficient to inhibit the bacteria to support routine systemic antibiotic prophylaxis. There is increased research into the development of efficient local drug delivery devices. Although established antibacterial testing methods for drugs can be used to test the antibacterial efficacy of drug-eluting materials, they are lacking in terms of providing real-time and longitudinal antibacterial efficacy data that can be correlated to the elution profiles of antibiotics from these devices. Here, we report a direct and versatile methodology to determine the antibacterial efficacy of antibiotic-eluting UHMWPE implants. This methodology can be used as a platform to avoid bacterial culture at each time point of a lengthy experiment and can also be adapted to other local drug delivery devices.

Here, we present a protocol to evaluate the antibacterial efficacy of an antibiotic-eluting polymer to simulate prophylactic clinical application by using a commercially available real-time ATP-based luminescent microbial viability assay. This method enables the monitoring of the longitudinal activity of drug-eluting materials and can be widely adapted to test anti-microbial drug delivery platforms.

一种测定药物洗脱材料纵向抗菌活性的新方法

Ultrahigh molecular weight polyethylene (UHMWPE) is widely used in total joint arthroplasties as a load-bearing surface. Periprosthetic joint infections, ...