This work was supported by grants from the Academic Research Fund of the Ministry of Education, and the National Medical Research Council and the National Research Foundation, Singapore.

生物膜的形成A. 1.结晶紫定量鲍曼不动杆菌 S1 <ol><li>成长5 ml培养A的杆菌中的S1 LB培养基（LB）（胰蛋白胨10克/升，酵母提取物5克/升），在30℃下在振荡培养箱（220转）16小时。 </li><li>调整A.文化杆菌 S1至所希望的OD 600为0.8。使用96孔板，接种细菌培养物（1:100稀释）到新鲜LB含有10微升纯化GKL酶（40毫克/毫升）的;新的文化的最终体积为100微升。 </li><li>准备控制文化为好;这将不包含任何酶。重复类似的状况以产生重复的期望数量。 </li><li>覆盖板用盖，并放入一个密封的10升塑料容器中。前轻轻取出介质孵育板在30℃下3小时。 </li><li>添加另外100微升新鲜LB培养基到井和孵育板21小时，在30℃。 </li&gt; <li>孵化的第二阶段后，轻轻取出所有的媒体。洗净的浮游细菌细胞用200微升无菌水。确保有清洗期间只有最小干扰细胞中。 </li><li>加入100微升的1％结晶紫溶液至每个孔，并孵育15分钟，在RT。通过洗涤井用200μl无菌水去除结晶紫溶液。重复洗两次。 </li><li>加入100微升33％乙酸至每个孔，并孵育15分钟，轻轻摇动;这会溶解染料。 </li><li>定量生物膜的通过测量结晶紫的吸光度在600nm处形成的量。结晶紫的量是成比例的生物膜的形成的量。 </li></ol> A. 2.激光共聚焦显微镜鲍曼不动杆菌 S1生物膜<ol><li>成长5 ml培养A的杆菌中的S1的LB在30℃在振荡培养箱（220rpm下）16小时。 </li><li>广告A的只是培养杆菌 S1至所希望的OD 600为0.8。使用35毫米的玻璃底μ-盘子，接种细菌培养物（1:100稀释）到新鲜LB含有30微升纯化GKL酶（40毫克/毫升）的;新的文化的最终体积为1毫升</li><li>覆盖μ-盘用盖子，并将其放置在一个密封的10升塑料容器中。前轻轻取出介质孵育μ-盘在30℃下3小时。添加30微升纯化GKL酶和新鲜培养基，将它带到1毫升的总体积。孵育另外21小时，在30℃。 </li><li>重复步骤2.3孵育μ-菜另外24小时在30℃。然后，轻轻地取出介质。 </li><li>加入500微升5微克/毫升阿莱克斯的Fluo 488缀合的麦胚凝集素（WGA）溶于Hank平衡盐溶液（HBSS）到μ-盘和孵育在37℃，30分钟;这会污染形成的生物膜。除去染色液和洗涤吨他μ-菜用2毫升的HBSS。重复该洗涤步骤一次。 </li><li>加入500微升3.7％的甲醛溶解在HBSS中，37℃，30分钟;这将解决生物膜到μ-菜。用2毫升的HBSS洗μ-菜一次，然后彻底删除解决方案。 μ-盘固定生物膜可以在4℃之前CLSM成像储存在黑暗中。 </li><li>对于激光共聚焦显微镜成像和分析，用63倍的放大倍率，产生97堆栈每幅图像，每堆0.21微米的间隔。 </li></ol>

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The authors declare that they have no competing financial interests.

在这两组实验中，A杆菌 S1中培养在LB培养基无氯化钠作为高盐浓度可减少生物膜由细菌15形成的量。这种伪影的存在可能会低估生物膜的形成的量，以及仲裁 - 淬火酶在不同的处理条件的影响。使用催化失活酶是重要的，作为阴性对照，以消除酶封存的可能影响。 图1表明，即使不活动酶螯合仲裁分子，生物膜的形成不被中断。 鲍曼不动杆菌 S1形成在96孔板的孔的空气和液体的界面之间的微妙环状的生物膜结构。因此，关键的是要避免在介质取出和清洗步骤过度搅拌，以防止意外除去细菌生物膜的。每次温育后，除去LB培养基消除浮游细胞。此外，96孔板置于10升的塑料容器，以尽量减少在气流扰动，并创造有利于生物膜形成一个微厌氧环境。在一个96孔板生物膜定量也依赖于结晶紫加入每个孔中的量。在过量的结晶紫加入成孔的情况下，洗涤步骤的数目可以增加。该结晶紫染色实验提供了法定淬效率的生物膜分裂的相对比较。虽然结晶紫染色是定量测定生物膜的形成，它是唯一的半定量在仲裁 - 淬火酶的催化效率方面。为了获得准确的统计比较，足够的样本大小是针对不同的治疗组重要。离群值也应删除，以防止结果不实陈述。 共聚焦成像和细菌生物膜的分析是一个USEFUL工具，以评估仲裁淬火酶的定性作用。但是，选择的生物膜进行分析的过程可以是用于一个偏压电位通道，如果用户意识到仲裁淬酶施用（活性或失活）的类型。为了避免偏差的可能性，该实验可被设计为从用户排除酶信息，或使用一个随机的方式进行选择。一个公正的选择策略也使生物膜形态的激光共聚焦显微镜更好的定量比较。尽管如此，这是描述的评价方法，对细菌生物膜仲裁 - 淬火的酶的结果的第一项研究。结晶紫染色已经证明在生物膜分裂仲裁-淬火酶的效用和显示，酶" 改性operandorum不限于减少的细胞数和毒力因子表达的影响。同时，激光共聚焦显微镜分析确定形态的变化也可以提供深入了解可能molecul这些酶的芳目标。虽然目前的实验的目的是要调查对细菌生物膜的酶预处理的效果，该协议可以被修改由细菌生长后加入酶，以评估对预制生物膜的酶的作用，或者通过使用血清的检查其在生理条件下的能力样条件的文化。用于研究GKL酶协议可以扩展到其他仲裁 - 淬火酶和病原体调查其在生物膜分裂关系。

对于感染性疾病的治疗方案已被复杂化在多药耐药性细菌是免疫广泛的抗生素药物1的快速增加。从耐药细菌介导的感染的高发病率和死亡率，有必要升级药物开发过程和/或探索更好抗菌替代改善的治疗选择。近来，抗毒性的方法是获得给出其在经由非杀菌方法防止致病潜力的兴趣，因此减轻耐药机制2的风险。 群体感应是细菌毒力和这种信号中断现象的一个"总开关"是对发病3一个有前途的抗毒性的方法。毒力的发作需要仲裁分子在细胞外环境中的积累达到临界细菌种群密度之后。由于现状朗姆酒分子扩散回进入胞内基质中，与它们的同源受体结合导致的毒力因子的活化，以及与抗生素抗性和生物膜形成4相关的基因。在一般情况下，群体感应中断涉及抑制仲裁分子和受体相互作用，而不会影响主代谢途径。因此，它不具有对细胞生长的任何直接含意。由于健康不受损害，是最小的选择压力细菌进化并获得对这种治疗5阻力。此外，群体感应中断可以与固有的细菌保护机制干扰，如在生物膜形成，它提供了保护从抗菌剂和宿主免疫反应的情况下。 据估计，在复杂的生物膜状矩阵存在99％地球上的微生物，赋予关键存活优势居住在第微生物ESE结构6。更重要的是，形成这些无柄域是最持久和慢性医院获得性感染7的原因。 鲍曼不动杆菌是一个与全球医院获得性感染相关联，其毒力在很大程度上归因于群体感应的主要的人类病原体之一介导的生物膜形成8。仲裁淬酶已被成功地用于通过靶向一组化合物已知为 N -酰基高丝氨酸内酯（信号分子）是由革兰氏阴性细菌产生的9扰乱仲裁介导的信号转导。一些研究也扩大于使用这些酶通过毒力因子的表达和细胞数在生物膜10,11减少到方框细菌发病机理。不幸的是，仍缺乏有效利用仲裁 - 淬火酶对生物膜形成由细菌病原体扪示范的。该重新已经尝试使用定额抑制剂（AHL类似物）代替法定淬火酶，以破坏A.鲍曼不动杆菌生物膜形成12。虽然使用小分子抑制剂的这种方法是一种有效的方法，维持在平利用其生物利用度可能是一个挑战。与此相反，使用催化仲裁 - 淬火酶可以规避生物利用度问题，因为酶是朝固定上用于治疗效果的生物医学装置的表面更适合。 在这里，我们描述了从地芽孢kaustophilus（GKL）13对细菌生物膜的形成工程定额淬火lactonases的影响进行评估，采用结晶紫染色和激光共聚焦扫描显微镜（CLSM）。这项研究是生物膜分裂的在一个临床相关的A中的第一次成功的示范鲍曼不动杆菌菌株S1采用定额淬火酶。的方法中所述在这项研究中的用于评估对致病性革兰氏阴性细菌后续治疗的努力发展其他仲裁淬火酶的效力是有用的。

<table><tbody><tr><td>Tryptone&amp;nbsp;</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>96-well plate</td><td>Costar</td><td>3596</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma-Aldrich</td><td>C6158</td><td></td></tr><tr><td>Acetic Acid</td><td>Lab-Scan</td><td>PLA00654X</td><td>Caution: Flammable</td></tr><tr><td>&amp;mu;-Dish</td><td>Ibidi</td><td>80136</td><td></td></tr><tr><td>Alex Fluo 488-conjugated WGA</td><td>Invitrogen</td><td>W11261</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Invitrogen</td><td>141475095</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Caution: Corrosive&amp;nbsp;</td></tr><tr><td>Synergy HT Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>1X-81 Inverted Fluorescence Microscope</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

在结晶紫定量实验中，两名仲裁淬火酶用于演示的可行性破坏生物膜的形成:野生型GKL和改进的GKL双突变体（E101G / R230C）。两种酶已显示表明内酯酶活性对3-羟基癸酰基-L-高丝氨酸内酯（3-OH-C 10 -HSL），使用由 A.主要仲裁分子鲍曼不动杆菌 S1 14。为生物膜分裂的有效评估，它们各自的无催化活性的酶（先前表明不螯合AHL配体）也包括在内作为立即对照（GKL D266N突变体和GKL E101G / R230C / D266N突变体）。除了 ​​使用野生型A.杆菌 S1中，突变ΔABAI株（AHL合成酶缺陷）的生物膜形成能力也被测试。既仲裁淬酶，野生型GKL和GKL E101G / R230C突变体能够显著减少在预生物膜形成处理A.杆菌 S1培养物（N = 10; p值0.0001≤）（ 图1）。尽管如此，生物膜分裂为两种酶的程度不成正比它们对3-OH-C 10 -HSL功效。在GKL E101G / R230C突变的周转率（K 猫 ）比野生型GKL对快3-OH-C 10 -HSL 14六倍。然而，在酶之间生物膜分裂的程度的差异是仅有的两个倍。 <img alt="图1" src="/files/ftp_upload/53243/53243fig1.jpg" /> 图1. A.鲍曼不动杆菌 生物膜分裂法。生物膜的形成是由结晶紫染色法。红柱代表生物膜的野生型A.生成量杆菌 ，ΔABAI突变体，不加入仲裁-淬火lactonases的。蓝柱代表AMOUNT生物膜由野生型A.形成杆菌中的四个不同GKL酶的存在:闲置GKL D266N突变体，野生型GKL，不活动GKL E101G / R230C / D266N突变体和GKL E101G / R230C突变体。 ****，P-的≤0.0001值。转载自抗菌药物和化疗 58的许可，1802-1805（2014）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53243/53243fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> A.共聚焦成像杆菌 S1生物膜形成是用来提供对这些固着结构域的结构形态仲裁淬火效果的定性和定量测量。改进GKL E101G / R230C突变体和其催化活性的酶被用于比较。差分图像对比（DIC）图像显示，与改进的GKL E101G / R230C突变处理导致生物膜尺寸的减小（FIGURE2）。荧光图像的分析还显示，当与改进GKL E101G / R230C突变体（表1）处理有减少表面积，生物量和生物膜的平均厚度。 <img alt="图2" src="/files/ftp_upload/53243/53243fig2.jpg" /> A的 图2代表共聚焦激光扫描显微镜图像 鲍曼不动杆菌 生物膜。A. 鲍曼不动杆菌生物膜处理和未激活的GKL E101G / R230C / D266N突变体（A）和 GKL E101G / R230C突变体（B）和染色用Alexa 氟488缀合的WGA。生物膜（左）和生物膜（右）的荧光图像的DIC图像示为代表的xy（中心），YZ（右），和XZ（底部）的部分。从抗菌药物和化疗 &lt;授权转载STRONG&gt; 58，1802年至1805年（2014）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53243/53243fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td></td><td colspan="3"> 值±SD 一个 </td></tr><tr><td> 特性 </td><td> 无治疗 </td><td> 治疗无效突变 </td><td> 治疗E101G / R230C突变 </td></tr><tr><td>生物量（微米3 /微米2） </td><td> 2.57±1.65 </td><td> 3.39±1.33 </td><td> ** 1.37±0.20 </td></tr><tr><td>平均厚度（μm） </td><td> 3.68±2.51 </td><td> 3.41±1.31 </td><td> ** 1.21±0.21 </td></tr><tr><td>表面积（微米） </td><td> 235,920.59±79,456.46 </td><td> 209,872.6±115,094.7 </td><td> 115,354.9 *±7,630.3 </td></tr><tr><td colspan="4"> A N = 10图像堆栈。 **，P≤0.001; *，P≤0.05，与非活动E101G / R230C / D266N突变处理相比。 </td></tr></tbody></table> 表1. A.鲍曼不动杆菌 生物膜结构定量。

Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

病原生物膜利用工程定额淬Lactonases防有毒中断

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

anti-virulent-disruption-pathogenic-biofilms-using-engineered-quorum

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.6K 次观看.  Yong Loo Lin School of Medicine. Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

视频: Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

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

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 ...

生物工程

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.

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

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.

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

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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 ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

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 ...

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

使用静态和动态方法相结合的方法评估有机过氧酸根除乳品生物膜的功效

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...

生物学

<meta charset="utf8"></head><body>噻吩磺酰胺在群体感应抑制中的生物学评价

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

<meta charset="utf8"></head><body>作者聚焦：推进治疗人类和水生生物弧菌病的疗法

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

弧菌群体感应抑制剂的合成和测定

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

病原生物膜利用工程定额淬Lactonases防有毒中断

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

通过创建一个的合成Curli的操纵子工程粘附的细菌

针对相关生物膜

方法学研究

适合与天然化合物库的探索抗生物试验的平台

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响

使用PCR板深孔微孔设备生成更大的细菌生物膜生物质

使用静态和动态方法相结合的方法评估有机过氧酸根除乳品生物膜的功效

弧菌群体感应抑制剂的合成和测定

病原生物膜利用工程定额淬Lactonases防有毒中断

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

通过创建一个的合成Curli的操纵子工程粘附的细菌

针对相关生物膜

方法学研究

适合与天然化合物库的探索抗生物试验的平台

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

使用PCR板深孔微孔设备生成更大的细菌生物膜生物质

使用静态和动态方法相结合的方法评估有机过氧酸根除乳品生物膜的功效

弧菌群体感应抑制剂的合成和测定

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响