Name: a platform of anti-biofilm assays suited to the exploration of natural compound libraries
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作者感谢教授保罗COS和LMPH，安特卫普大学，比利时期间，在他的实验室拍摄过程中他的支持。这项工作是由芬兰工程学院（项目272266和282981）和瑞典技术科学院芬兰资助。硕士Janni Kujala的技术贡献得到承认。

1.成长的细菌<ol><li>预培养物在37℃下在胰蛋白酶大豆肉汤（TSB）中的细菌过夜以220rpm振荡（16-18小时）。 </li><li>稀释预培养100-1,000倍（取决于细菌的生长速率;在这里，1000倍，用于金黄色葡萄球菌 ）的新鲜TSB，让它生长在37℃和200rpm下达到指数生长（光密度在0.2和0.6）之间的595纳米（OD 595）。 注:此步骤需要特定应变优化。 </li></ol> 2.生物膜的形成:前和暴露后<ol><li>稀释成倍增长文化的100倍（这相当于每毫升约10 6个菌落形成单位（CFU /毫升））。 </li><li> 预曝光协议 <ol><li>对于未经处理的对照样品，加入200微升每孔稀释细菌培养无菌96微孔板的。 </li><li>添加测试化合物或对照的4微升抗生素（50X原液）和196微升每孔稀释细菌培养。 </li><li>在37℃下以200rpm搅拌18小时孵育它在板振荡器上。 注意:在这里我们使用2毫米的轨道振动筛。 </li></ol></li><li> 暴露后协议 <ol><li>对于所有的样品，加入200微升每孔稀释细菌培养无菌96微孔板的。 </li><li>在37℃下以200rpm搅拌18小时孵育它在板振荡器上。 注意:在这里我们使用2毫米的轨道振动筛。 </li><li>小心地取出整个浮游的解决方案，而不触及生物膜，使用多道移液器。 </li><li>添加4微升测试化合物或控制抗生素（50X原液），并每孔196微升TSB的。 </li><li>在37℃下孵育其上以200rpm板振荡器另外24小时。 注意:在这里我们使用2毫米的轨道振动筛。 </li></ol></li></ol> 3.刃天青染色Protocol对于生物膜的可行性分析<ol><li>制备的在无菌PBS 0.1毫克/毫升刃天青（0.4毫摩尔）的储备溶液。保持这个股票不育，从曝光的保护，并在4℃。 </li><li>稀释刃天青库存1:50在无菌磷酸盐缓冲盐水（PBS），以实现20微米的最终浓度。 </li><li>传送整个浮游液（离开生物膜在孔中）小心，不接触生物膜或产生气泡，要使用多道移液器一个单独的，干净的96孔板中。 </li><li>通过加入每孔200μl的用无菌PBS洗涤生物膜一次，并小心地用多道移液器将其删除。 </li><li>加入200μl每使用多道移液器生物膜板以及稀释刃天青。 </li><li>孵育它在黑暗中，在室温（RT），并以200rpm，约20分钟，振摇直到未处理的生物膜控制是均匀的粉红色。 </li><li>测量在荧光λEXC = 560纳米和λEM =用酶标仪（顶端读数）590纳米。 </li></ol> 4.结晶紫染色协议生物膜生物量定量使用同一板块如步骤3 <ol><li>使用多道移液器井小心取出刃天青染色。 </li><li>固定用200μl甲醇的生物膜15分钟。 </li><li>让板空气干燥10分钟。 </li><li>加入190微升0.02％（体积/体积，在去离子水稀释）龙胆紫液，小心使用多道移液器。避免与同时移液污渍接触孔的侧面和防止空气气泡的形成不按移液器完成吹出。 </li><li>孵育它在RT 5分钟。 </li><li>小心取出色斑，使用多道移液器。 </li><li>用去离子水（200每次微升）洗两次。 </li><li>让各孔在RT干燥5分钟，溶解剩余的污点在96％乙醇或33％乙酸。 </li><li>孵育它在RT 1小时，并在595nm处读取吸光度。 </li></ol> 5.浮游菌评价杀菌效果使用相同的样品板作为生物膜<ol><li>测量在含有从3.3浮游溶液板的595nm处的浊度。 </li><li>加入10微升刃天青股票，每孔用染色的刃天青浮游细菌。吹打拌匀。 </li><li>它孵育在黑暗中在室温约23分钟，直到被处理控件是均匀的粉红色。 </li><li>在测量EXCλ= 560nm的荧光，λ= EM 590纳米。 </li></ol> 6.麦胚凝集素染色矩阵量化和生物膜的荧光显微成像<ol><li> 量化矩阵 <ol><li>制备在无菌PBS WGA的探针的5微克/毫升的溶液。 </li><li>使用并行样品解放军忒这个染色。除去从孔浮游溶液并用无菌PBS（每孔200微升）洗一次，小心用多道移液器而不触及生物膜。 </li><li>加入200μl的WGA解决每个被染色良好。 </li><li>它孵育在黑暗中在4℃下2小时。 </li><li>通过用200μl的PBS清洗3次洗涤各孔除去未结合的污点。 </li><li>让板干燥在室温15分钟。 </li><li>溶解在33％乙酸结合的染色，使用每孔200μl的。 </li><li>密封该孔与带状帽和使用水浴超声波仪在室温30秒和40千赫超声处理它们。 </li><li>将培养板在RT 1小时。 </li><li>重复超声步骤。井可以在超声处理步骤之间保持密封。 </li><li>测量在λEX = 495nm处的荧光，λEM =用酶标仪（顶端读数）520纳米。 </li></ol></li><li> 可视化与基质荧光显微镜<ol><li>使用相同的协议，上面直到步骤6.1.6。 </li><li>在干燥步骤之后，可视化的样品用荧光显微镜，使用FITC标记的过滤器（或其它合适的绿色激发过滤器）。 </li></ol></li></ol>在生物膜内7.染色活细胞和死细胞与荧光显微镜和信号的定量绿色到红色比（G / R）成像<ol><li> 成像与荧光显微镜 <ol><li>制备各探针（一个染色的活细胞，另一种染色死细胞）的溶液中，根据制造商的指导方针。 </li><li>除去从孔浮游溶液，并用无菌PBS洗涤一次（每孔200微升）用多道移液器。 </li><li>加入6微升每孔的染色溶液中。 </li><li>孵育在黑暗中板15分钟。 </li><li>在显微镜之前，除去多余的液态间全线采用多道移液器。 </li><li>捕获使用荧光显微镜图像，使用例如FITC过滤器（绿色荧光，活细胞）或TRITC滤波器（红色荧光，死细胞）。 </li></ol></li><li> 信号作为绿色到红色荧光比的定量（G / R） <ol><li>按照相同的协议，在步骤7.1.1和7.1.2。 </li><li>加入200μl每孔的染色溶液的孵育它们在黑暗中15分钟。 </li><li>测量与激发/发射波长为绿色和红色荧光535分之485nm和635分之485，分别为（顶端读数）读板器的荧光。 </li></ol></li></ol>

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	<li>Costerton, J. W., Stewart, P. S., Greenberg, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Dufour, D., Leung, V., L&#233;vesque, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilm:+structure,+fuction,+and+antimicrobial+resistance.">Bacterial biofilm: structure, fuction, and antimicrobial resistance.</a> Endodontic Topics. 22, 2-16 (2012).</li><li>Donne, J., Dewilde, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenging+World+of+Biofilm+Physiology.">The Challenging World of Biofilm Physiology.</a> Adv. Microb. Physiol. 67, 235-292 (2015).</li><li>Otto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+infections:+mechanisms+of+biofilm+maturation+and+detachment+as+critical+determinants+of+pathogenicity.">Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity.</a> Annu. Rev. Med. 64, 175-188 (2013).</li><li>Cos, P., Tote, K., Horemans, T., Maes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+an+extra+hurdle+for+effective+antimicrobial.">Biofilms: an extra hurdle for effective antimicrobial.</a> Curr. Pharm. Des. 16, 2279-2295 (2010).</li><li>Bjarnsholt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+biofilm.">The in vivo biofilm.</a> Trends Microbiol. 21, 466-474 (2013).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persister+cells.">Persister cells.</a> Annu. Rev. Microbiol. 64, 357-372 (2010).</li><li>Mulcahy, L. R., Burns, J. L., Lory, S., Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+Pseudomonas+aeruginosa+strains+producing+high+levels+of+persister+cells+in+patients+with+cystic+fibrosis.">Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis.</a> J. Bacteriol. 192, 6191-6199 (2010).</li><li>Buckingham-Meyer, K., Goeres, D. M., Hamilton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+biofilm+disinfectant+efficacy+tests.">Comparative evaluation of biofilm disinfectant efficacy tests.</a> J. Microbiol. Methods. 70, 236-244 (2007).</li><li>Goeres, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor.">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nat. Protoc. 4, 783-788 (2009).</li><li>Goeres, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+assessment+of+a+laboratory+method+for+growing+biofilms.">Statistical assessment of a laboratory method for growing biofilms.</a> Microbiology. 151, 757-762 (2005).</li><li>Parker, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruggedness+and+reproducibility+of+the+MBEC+biofilm+disinfectant+efficacy+test.">Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test.</a> J. Microbiol. Methods. 102, 55-64 (2014).</li><li>Ceri, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Calgary+Biofilm+Device:+new+technology+for+rapid+determination+of+antibiotic+susceptibilities+of+bacterial+biofilms.">The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.</a> J. Clin. Microbiol. 37, 1771-1776 (1999).</li><li>Harrison, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+susceptibility+testing+of+microbes+growing+on+peg+lids:+a+miniaturized+biofilm+model+for+high-throughput+screening.">Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening.</a> Nat. Protoc. 5, 1236-1254 (2010).</li><li>Konrat, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bead+Assay+for+Biofilms:+A+Quick,+Easy+and+Robust+Method+for+Testing+Disinfectants.">The Bead Assay for Biofilms: A Quick, Easy and Robust Method for Testing Disinfectants.</a> PLoS One. 11, e0157663 (2016).</li><li>Cragg, G. M., Newman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+products:+A+continuing+source+of+novel+drug+leads.">Natural products: A continuing source of novel drug leads.</a> Biochim Biophys Acta. 1830, 3670-3695 (2013).</li><li>Sandberg, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pros+and+cons+of+using+resazurin+staining+for+quantification+of+viable+Staphylococcus+aureus+biofilms+in+a+screening+assay.">Pros and cons of using resazurin staining for quantification of viable Staphylococcus aureus biofilms in a screening assay.</a> J. Microbiol. Methods. 78, 104-106 (2009).</li><li>Mariscal, A., Lopez-Gigosos, R., Carnero-Varo, M., Fernandez-Crehuet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+assay+based+on+resazurin+for+detection+of+activity+of+disinfectants+against+bacterial+biofilm.">Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm.</a> Appl. Microbiol. Biotechnol. 82, 773-783 (2009).</li><li>Peeters, E., Nelis, H., Coenye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+multiple+methods+for+quantification+of+microbial+biofilms+grown+in+microtiter+plates.">Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.</a> J. Microbiol. Methods. 72, 157-165 (2008).</li><li>Pettit, R., Weber, C., Pettit, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+high+throughput+Alamar+blue+biofilm+susceptibility+assay+to+Staphylococcus+aureus+biofilms.">Application of a high throughput Alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms.</a> Ann. Clin. Microbiol. Antimicrob. 8 (28), (2009).</li><li>Stepanovic, S., Vukovic, D., Dakic, I., Savic, B., Svabic-Vlahovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+microtiter-plate+test+for+quantification+of+staphylococcal+biofilm+formation.">A modified microtiter-plate test for quantification of staphylococcal biofilm formation.</a> J. Microbiol. Methods. 40, 175-179 (2000).</li><li>Stiefel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+biofilm+removal+properly+assessed?+Comparison+of+different+quantification+methods+in+a+96-well+plate+system.">Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.</a> Appl. Microbiol. Biotechnol. , (2016).</li><li>Sandberg, M., M&#228;&#228;tt&#228;nen, A., Peltonen, J., Vuorela, P. M., Fallarero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automating+a+96-well+microtitre+plate+model+for+Staphylococcus+aureus+biofilms:+an+approach+to+screening+of+natural+antimicrobial+compounds.">Automating a 96-well microtitre plate model for Staphylococcus aureus biofilms: an approach to screening of natural antimicrobial compounds.</a> Int. J. Antimicrob. Agents. 32, 233-240 (2008).</li><li>Thomas, V. L., Sanford, B. A., Moreno, R., Ramsay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-linked+lectinsorbent+assay+measures+N-acetyl-D-glucosamine+in+matrix+of+biofilm+produced+by+Staphylococcus+epidermidis.">Enzyme-linked lectinsorbent assay measures N-acetyl-D-glucosamine in matrix of biofilm produced by Staphylococcus epidermidis.</a> Curr. Microbiol. 35, 249-254 (1997).</li><li>Burton, E., Yakandawala, N., LoVetri, K., Madhyastha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microplate+spectrofluorometric+assay+for+bacterial+biofilms.">A microplate spectrofluorometric assay for bacterial biofilms.</a> J. Ind. Microbiol. Biotechnol. 34, 1-4 (2007).</li><li>Skogman, M. E., Vuorela, P. M., Fallarero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+biofilm+matrix+measurements+with+biomass+and+viability+assays+in+susceptibility+assessments+of+antimicrobials+against+Staphylococcus+aureus+biofilms.">Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.</a> J. Antibiot. Tokyo. 65, 453-459 (2012).</li><li>Skogman, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antibacterial+and+anti-biofilm+activities+of+cinchona+alkaloid+derivatives+against+Staphylococcus+aureus).">Evaluation of antibacterial and anti-biofilm activities of cinchona alkaloid derivatives against Staphylococcus aureus).</a> Nat. Prod. Commun. 7, 1173-1176 (2012).</li><li>Fallarero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(+)-+Dehydroabietic+Acid,+an+Abietane-Type+Diterpene,+Inhibits+Staphylococcus+aureus+Biofilms+in+Vitro.">(+)- Dehydroabietic Acid, an Abietane-Type Diterpene, Inhibits Staphylococcus aureus Biofilms in Vitro.</a> Int J Mol Sci. 14, 12054-12072 (2013).</li><li>Manner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+derivatives+of+dehydroabietic+acid+target+planktonic+and+biofilm+bacteria+in+Staphylococcusaureus+and+effectively+disrupt+bacterial+membrane+integrity.">New derivatives of dehydroabietic acid target planktonic and biofilm bacteria in Staphylococcusaureus and effectively disrupt bacterial membrane integrity.</a> Eur. J. Med. Chem. 102, 68-79 (2015).</li><li>Muller, G., Kramer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+index+of+antiseptic+agents+by+parallel+assessment+of+antimicrobial+activity+and+cellular+cytotoxicity.">Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity.</a> J. Antimicrob. Chemother. 61, 1281-1287 (2008).</li><li>Tot&#233;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+efficacy+of+various+antibiotics+on+matrix+and+viable+mass+of+Staphylococcus+aureus+and+Pseudomonas+aeruginosa+biofilms.">Inhibitory efficacy of various antibiotics on matrix and viable mass of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.</a> Int. J. Antimicrob. Agents. 33, 525-531 (2009).</li><li>Djordjevic, D., Wiedmann, M., McLandsborough, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+plate+assay+for+assessment+of+Listeria+monocytogenes+biofilm+formation.">Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation.</a> Appl. Environ. Microbiol. 68, 2950-2958 (2002).</li><li>Li, X., Yan, Z., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+variation+of+biofilms+among+strains+in+natural+populations+of+Candida+albicans.">Quantitative variation of biofilms among strains in natural populations of Candida albicans.</a> Microbiolog. 149, 353-362 (2003).</li><li>Barbosa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+and+simple+micro+assay+for+sulfated+glycosaminoglycans+quantification+in+biological+extracts+and+its+use+in+skin+and+muscle+tissue+studies.">Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies.</a> Glycobiology. 13, 647-653 (2003).</li><li>Tot&#233;, K., Vanden Berghe, D., Maes, L., Cos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+colorimetric+microtitre+model+for+the+detection+of+Staphylococcus+aureus+biofilms.">A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms.</a> Lett. Appl. Microbiol. 46, 249-254 (2008).</li><li>Arciola, C. R., Campoccia, D., Ravaioli, S., Montanaro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+intercellular+adhesin+in+biofilm:+structural+and+regulatory+aspects.">Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects.</a> Front Cell Infect Microbiol. 5 (7), (2015).</li><li>Ausbacher, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+biofilm+susceptibility+to+small+and+potent+beta(2,2)-amino+acid+derivatives.">Staphylococcus aureus biofilm susceptibility to small and potent beta(2,2)-amino acid derivatives.</a> Biofouling. 30, 81-93 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

没有可以同时测量上的可行性，生物质，和生物膜基质的化合物的效果单一方法。因此，有必要在为了检测在三个端点的效果，优选在初次筛选阶段结合测定。 刃天青是仅由加入氧化还原探针的一个非常简单的染色方案。然而，建立与刃天青生物膜的最佳温育时间是该测定的成功是至关重要的。在某些细菌菌株，刃天青探针的粉红色，荧光试卤灵的减少发生得非常快，而在其他情况可以持续几个小时19。此外，同样的细菌菌株中，也可以有浮游细菌和生物膜之间显著差异。在浮游菌通常更容易达到，主要是因为细菌密度是浮游人口超过生物膜婆低pulations，从而促进该反应的速度快的营业额。在生物膜活力染色探针的出版比较，刃天青得出的结论是最准确的，使用简单，和最便宜的测定19之一。而且，它已被证明是适用于一系列细菌和真菌生物体19的。另一方面，结晶紫测定法是最广泛地用于生物膜质量量化19,32,33。这个协议的最重要的步骤是将结晶紫染色溶液的添加和去除。这些步骤必须非常小心地进行，以避免非特异性染色（ 如，由于孔的壁上污渍滴）。一种使用刃天青作为初始染色法的主要优点是，它是无毒的细胞，其能够为结晶紫染色使用相同板的。两种测定的组合显著简化了工作流程，降低了成本，节省了消费冷杉，并最大限度地减少了使用测试化合物，这是在筛选环境特别有价值的。 如前面所指出的，自产生的细胞外多糖基质是生物膜的一个基本组成部分。为了测量矩阵含量（特别是必需的多糖），第三测定包括在这里，是根据荧光标记的WGA。原本WGA的量化被描述为一个酶联凝集素吸附测定（ELLA）24。基于在金黄色葡萄球菌生物膜与二甲基亚甲基蓝（DMMB）的基质染色糖胺聚糖（GAG）的类似试验也被用来31,34。聚糖类似于在葡萄球菌属的基质中发现的多糖胞间黏附（PIA的）。生物膜35。 WGA的测定法类似目标PIA，但WGA更特异性地结合到聚-N-乙酰葡糖胺残基，其在生物膜基质36 &lt;发挥至关重要的作用/ SUP&gt;。靶向基质是具有高度重要性由于这样的事实，如果矩阵被化学或抗生素治疗后留下的生物膜很容易再生。细菌也可以更容易地附加到在基质预先涂布35的表面。已经表明，某些抗生素可有效地降低存活率和生物膜的生物量，但没有对基质的任何影响。在我们的实验中，这是通过环丙沙星31的情况举例说明。一些抗生素甚至可以提高基体制作，在这里与青霉素G 26证明。基于这些变化，抗生素可以分为表2中所列的类别。在我们的筛选研究（ 图2）中的所有命中可以被分类，环丙沙星一起，作为杀死生物膜中的细菌，并减少生物质，但不拆卸或破坏生物膜基质的化合物。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within页="总是"&gt; <tbody><tr><td> 类别 </td><td> 对细菌的影响 </td><td> 矩阵的影响 </td><td> 例如抗生素（目标生物膜） </td><td> 参考 </td></tr><tr><td> 1 </td><td>没有</td><td>没有</td><td>氨苄青霉素（SA）的</td><td> （手提包等，2009） </td></tr><tr><td> 2 </td><td>没有</td><td> - </td><td>头孢噻吩（SA）的</td><td> （手提包等，2009） </td></tr><tr><td> 3 </td><td> - </td><td>没有</td><td>多粘菌素（SA）的</td><td> （手提包等，2009） </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> - </td><td rowspan="2"> - </td><td>卡那霉素（PA） </td><td> （手提包等，2009） </td></tr><tr><td>环丙沙星（SA）的</td><td> （Skogman 等，2012） </td></tr><tr><td> 五 </td><td> - （+） </td><td> - </td><td>多西环素（PA） </td><td> （手提包等，2009） </td></tr><tr><td> 6 </td><td> - </td><td> + </td><td>青霉素G（SA，EC） </td><td> （Skogman 等，2012） </td></tr></tbody></table> 表2: 抗生素分类。的抗生素（使用刃天青染色）和基质（使用WGA或二甲基亚甲基蓝（DMMB）染色）分为基于其上的生物膜细菌的生存能力效果类别。 SA: 金黄色葡萄球菌 ; PA: 铜绿假单胞菌 ; EC: 大肠杆菌 。从手提包等进行修改。 </em&gt; 31 此外，该平台非常适合进行初步筛选活动。最成本和时间有效的策略是采用刃天青和水晶基于紫测定作为一线战略，在二线（后续）的研究移动到WGA矩阵法。这是由于这样的事实，即WGA探针是相当昂贵的，并且该测定包括几个步骤，这使得它更费力和费时。 这个平台的一个相关特征是评估所确定的抗菌药物的长期影响的可能性。如果生物膜基质拆开才能实现长期的化疗效果。已经表明，该生物膜感染的复苏的风险是高得多，如果矩阵被留下。与此平台，它是可能的，如果整体生物膜生物质用结晶紫染色受影响到第一评估。更Ð生物膜矩阵多糖etailed评估，使用WGA检测，可以遵循。值得注意的是，识别单个分子既能拆卸基质和施加抗菌（杀生物）的影响可被认为是低的可能性。的确，迄今为止，我们已经确定了这种类型的唯一化合物是抗微生物肽37。为了解决这个问题，跟进在此平台与任一resazurin-或结晶紫活性命中进行的，因为这种策略允许与单独的杀生物或基质降解活性的化合物的鉴定。这样引线可以是多组分的策略可能有趣。与杀生物或抗生素类化合物基质降解分子的组合可望提供更有效的生物膜根除。 总之，这里提出的平台使用存活率和生物量测量结果与基质quantifi一起提供了抗生物筛选了良好的基础阳离子和可视化。两个刃天青和结晶紫染色测定法，也可用于其他种类的生物膜形成微生物。然而，这些测定需要为生长和染色条件单独的最优化。 WGA的染色法的适用性是有限的那些细菌，其中聚-N-乙酰葡糖胺是生物膜基质的主要成分。其它基质的定量测定（例如基于二甲基亚甲蓝染色的那一个）可在其它情况下被应用。总之，在这个平台上的检测是相当容易执行，所有试剂容易获得以及总体经济实惠。该平台适用于中低吞吐量抗生物筛选，而不需要昂贵的设备投资。

细菌可以两种非常不同的生活方式，浮游和固着，其中生物膜是最常见的例子之间进行切换。在生物膜，细菌形成嵌入在自产矩阵1结构化的社区。此自产矩阵是细菌，它们的外部环境之间的屏障，它保护了的微生物细胞，使它们在接近。生物膜基质的组合物之间，甚至在物种而异，但它主要是由脂多糖，胞外DNA和蛋白质的紧密网。基质用作物理屏障抑制有害剂的入口，但它也可以防止脱水生物膜并防止营养物质逸出的小区2。 生物膜被视为现代生物医学的最具挑战性的课题之一，它们是对抗生素耐受感染3超过80％的据称负责。他们disp已铺设针对外部威胁的固有的高耐受性:湿度，渗透压，机械应力4，热，紫外线辐射5，消毒剂，抗菌剂，和宿主免疫系统1。例如，为了杀死生物膜所需的所需的抗微生物剂的浓度已显示相比较，以杀死浮游细菌所需的要高的1000倍。此更高公差的解释似乎是多因素的。基质提供了降低抗生素渗透到生物膜的物理屏障。另外，生物膜内的细胞表型多样;他们由于氧，营养物，和生物膜6的内和外部分之间的代谢物的现有梯度不同的代谢状态之间转变。因此，在一些生物膜地区，如核心，细菌被剥夺氧气和营养，生活在一个代谢活性较低或完全休眠状态<suP&gt; 7。完全休眠细胞被称为持留细胞，它们是不易于常规抗微生物治疗8。因此，它很可能是生物膜的韧性源自的当前建议等，未知，机制的组合。 葡萄球菌属。仍然是最有问题的革兰氏阳性菌中，引起严重的，通常生物膜相关的，感染4。有人建议，所有的细菌高达99％的生物膜内的关联，使得它的主要细菌生活方式3。但是，所有的现有的抗生素已经开发针对单小区（浮游）细菌。迄今，分子的一个非常有限的剧目存在可在成熟生物膜选择性作用。这种情况已经在推动药物发现一个渐进的转变，其中的抗生物膜搜索已敦促占据了更加突出的位置。 从methodologiCal中的角度来看，额外的挑战存在，因为只有生物膜的方法有限数目已经由标准制定组织，特别是那些适用于化学文库的高通量筛选开发的。所有的标准化测定（只有一个例外）都基于生物膜反应器中，并且这些方法需要大量的工作容积和大量的化合物进行测试，这通常是在早期研究性阶段9-12不可用。现有的唯一标准筛选适用的测定法是所谓的卡尔加里生物膜的装置，从该商购的最小生物膜消除浓度（MBEC）系统的开发13-15。然而，该测定的限制是该生物膜生长在钉，并且不是所有的细菌物种或同一物种内甚至菌株能够形成这个装置上生物膜。而且，可以特别适用于天然化合物的探索方法必要的。天然产品已经在抗菌药物发现在过去的16世纪创新的主要来源。它们可提供具有独特的作用机制也可以是针对持留细胞有效新颖抗生物化合物。因此，自然和自然的启发库的探索有前途的生产和独特的抗生物引线的高机会。 这里，我们提出测定的一个平台，是为使用三个测定法来测量上的可行性，总生物量，和金黄色葡萄球菌生物膜基质的影响抗生物化合物的化学筛选开发的实验细节。第一测定测量生物膜的可行性，并且它是基于刃天青染色。刃天青是氧化还原染色是蓝色和非荧光在其氧化态和由细菌的代谢活性降低时变为粉红色，高荧光试卤灵。这是一个非常简单和快速米ethod适合初级筛查17-20。第二个实验中，根据结晶紫染色，测量总质量的生物膜。结晶紫为在生物膜19,21-23研究细菌和细菌广泛使用的污点。该测定法是基于廉价的试剂和具有简单的吸光度端点读数。最后，第三个测定目标通过小麦胚凝集素生物膜（WGA），其特异性结合聚-N-乙酰葡糖胺残基（PNAG）存在于葡萄球菌生物膜24的基体的胞外聚合物物质（EPS） -矩阵。 WGA的结合有可使用荧光强度读者25被检测的荧光团。我们在座的基本原理和我们开发，包括示例应用程序的平台的详细信息。

<table><tbody><tr><td>Resazurin</td><td>Sigma Aldrich</td><td>R7017</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma Aldrich</td><td>HT90132&amp;nbsp;</td><td></td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate</td><td>Thermo Fisher Scientific</td><td>W11261</td><td></td></tr><tr><td>LIVE/DEAD &lt;em&gt;BacLight&lt;/em&gt;&amp;nbsp;</td><td>Molecular Probes</td><td>L7012</td><td>SYTO 9 for staining viable cells green and propidium iodide for staining dead cells red</td></tr><tr><td>Phosphate Buffered Saline</td><td></td><td></td><td></td></tr><tr><td>Tryptone soy agar</td><td>Lab M, Neogen</td><td>LAB011</td><td></td></tr><tr><td>Tryptine soy broth</td><td>Lab M, Neogen</td><td>LAB004</td><td></td></tr><tr><td>F96 Well Plate Polystyrene Sterile Clear Flat bottom</td><td>Thermo Fisher Scientific</td><td>161093</td><td></td></tr><tr><td>BRAND caps, strips of 8</td><td>Sigma Aldrich</td><td>BR781413-300EA</td><td></td></tr><tr><td>Branson CPX series ultrasonic bath</td><td>Sigma Aldrich</td><td>Z769428-1EA</td><td></td></tr><tr><td>Multipipette</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Multidrop dispenser</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Biomek 3000</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Varioskan Flash Multiplate reader</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC&amp;nbsp;</td><td>25923</td><td></td></tr></tbody></table>

a platform of anti-biofilm assays suited to the exploration of natural compound libraries

生物膜被视为现代生物医学的最具挑战性的课题之一，它们是潜在的负责抗生素耐受性感染的80％以上。生物膜已显示化疗极高的耐受性，这被认为是多因素的。例如，基体提供了降低抗生素渗透到生物膜的物理屏障。另外，生物膜内的细胞的表型多样性。可能的是，生物膜韧性源于这些和其他，未知，机制的组合。所有的现有的抗生素已经开发针对单细胞（浮游）细菌。因此，到目前为止，分子的一个非常有限的剧目存在可在成熟生物膜选择性作用。这种情况已经在推动药物发现一个渐进的转变，其中的抗生物膜搜索已敦促占据了更加突出的位置。一个额外的挑战是，有一非常有限的生物膜研究标准化方法，尤其是那些可用于化学文库的大通量筛选号码。在这里，药剂筛选试验的抗生物平台提出。在聚-N-乙酰氨基葡萄糖，PNAG的它采用三个实验使用小麦胚芽凝集素测量生物膜活力（带刃天青染色），总生物量（用结晶紫染色），和生物膜矩阵（，WGA-荧光染色基础， 分数）。所有的测定用金黄色葡萄球菌作为模型细菌开发的。的平台如何可以用于初步筛选以及用于鉴定抗生物命中功能表征实例。这个实验序列还允许根据测量的终点命中的分类。它还提供了对他们的行动模式的信息，特别是在长期和短期化疗的效果。因此，它是非常ADVAntageous用于快速识别，可以作为起点关于各种生物医学应用的高质量的命中化合物。

在所提出的平台上的可行性，生物质，和生物膜基质的影响量化。在工作序列（ 图1），一个样品板沾有刃天青，随后用结晶紫同时评价对细菌生物膜存活和总生物膜的生物量的影响。这两个实验可以连续在同一个板块，因为它被证明早些时候与刃天青第一染色对结晶紫染色结果（P = 0.4149无统计学显著影响水晶紫激光印版之间最大的信号吸光度单位的比较分别染色或进行后刃天青染色，N = 20），26。浮游细菌的效果可以同时评估。 当命中从任生存能力或基于生物质的测定法鉴定，第二板是圣<html>与WGA探针ained量化生物膜基质的多糖组分（PNAG）的化合物的作用。该工作流可以应用于化学文库的筛选，以及以对命中化合物的效力的测量功能的随访研究，如下举例说明。 <img alt="图1" src="/files/ftp_upload/54829/54829fig1.jpg" /> 图1: 三测定台的工作流程。工作流上生物膜样品三种测定结合的示意图。该平台包括对生存能力，生物量和EPS层的染色效果;使用荧光显微镜成像;和评估对浮游相位的影响。 "A"和"NA"分别代表"积极"和"不活动"。从Skogman 等进行修改。 26JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 用于筛选的三检测平台性能这里，二筛分运行被示为平台的性能的代表性结果。在第一运动（ 图2A），金鸡纳生物碱衍生物的小文库筛选对抗金黄色葡萄球菌的抗生物膜活性的生物膜27，而在第二个例子中（ 图2B），松香烷型的自然和自然的启发库二萜类化合物和衍生物进行了探讨28,29。使用所计算的命中限制（;等式6， 表1阈值），确定了活性命中。在各筛选研究中，相关性很好的前两个实验的结果;命中都能够减少的可行性和生物膜的生物量，W往往微不足道的非活性化合物显示对任一测定没有任何影响。然后所有确定命中分别对第三（WGA）测定中测试，但他们没有基质拆卸（或基质降解）的影响（结果未示出）。 <img alt="图2" src="/files/ftp_upload/54829/54829fig2.jpg" /> 图2: 使用带有 金黄色葡萄球菌 生物膜 的平台筛查活动的代表性成果 。使用该试验平台进行验证的概念验证屏幕的两个示例。该结果表示为与未处理的生物膜控制的百分比，命中化合物以红色表示，而线代表所计算出的匹配的限制。 （A）一金鸡纳生物碱衍生库的筛选确定一种活性化合物。 （B）松香烷型二萜类化合物和衍生物的库中确定了五个行为筛选香港专业教育学院化合物。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54829/54829fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 三分析平台的性能进行后续研究代表性的结果在图3中，其中，两个已知的抗生素在一个非常宽范围的浓度（为0.5nM-5mM的）对金黄色葡萄球菌生物膜试验中可以看出。对浮游细菌参考化合物（从文献或在实验室中进行的）的最低抑制浓度（MIC）值作为选择的浓度来测试针对同一物种的生物膜的准则。此外，在其中在哺乳动物细胞中没有检测到细胞毒性的浓度值的知识也可以帮助在浓度的选择来测试抗生物随访。理想情况下，如果是可用于特定化合物既抗微生物和细胞毒性数据，被称为"生物相容性指数"（BI）的参数可以计算，由米勒和Kramer 30所限定。这里，确定为青霉素G和环丙沙星对浮游金黄色葡萄球菌的MIC值分别为0.04μM和6微米，（未示出结果）。因此，该浓度间隔范围从大约10 5×MIC至10 -2点¯xMIC和分别用于青霉素和环丙沙星，10 3×MIC至10 -4×MIC。两种抗生素可以显著降低存活率，生物质，和生物膜基质PNAG内容时，他们暴露于之前的生物膜形成工艺（ 图3a）的起始单胞菌。然而，尽管对预成形的（18小时）生物膜测试抗生素的浓度非常高，活力和生物量只减少到的t约50％未经处理生物膜（ 图3b）的软管。这里最突出的结果是生物膜基质的增加（超过200％）时，预制生物膜与高浓度青霉素G，当预制生物膜环丙沙星治疗检测生物膜矩阵的内容没有改变对待。 <img alt="图3" src="/files/ftp_upload/54829/54829fig3.jpg" /> 图 3: 使用对 金黄色葡萄球菌 生物膜 两型抗生素抗生物效应的后续研究的例子 。青霉素G和环丙沙星的影响是这里提出:（A）以生物膜形成（预曝光）和（B）后的生物膜形成（暴露后）之前。为使图像简洁，只最低及最高浓度测试被示出。标准偏差不超过20％。 *** EQUALS P &lt;0.01。从Skogman 等进行修改。 26 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54829/54829fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 基于显微荧光成像在400μM结果是青霉素G（以及较高浓度，如在图3b中所示）杀死预制金黄色葡萄球菌生物膜细菌群体的约50％，用另一个生存力染色法确认。绿色到红色（G / R）荧光比率为未处理的生物膜孔中的平均值为2.75，表明活（绿色染色）细胞的优势，用死（红色染色）细胞。然而，在青霉素处理的细胞中的G / R比降低到1.54，对应于对照孔的56％。青霉素治疗后，亲活着剩余细胞duced一个显著较高量的EPS，如从绿（WGA）荧光检测的增加来判断，当与未处理的生物膜进行比较（ 图4b）。我们建议，只要有可能，来执行这些基于成像的实验，以进一步证实平台的结果，尤其是在击中候选表征的阶段。 <img alt="图4" src="/files/ftp_upload/54829/54829fig4.jpg" /> 图4: 荧光显微镜。除了青霉素（400μM）的可行性和生产EPS的影响在这里显示。顶部图像（A）对应于未处理的生物膜和青霉素治疗的生物膜，其中活细胞染绿和死细胞被染成红色。插入的图表说明G / R荧光比率的计算。底部图像（B）中对应于未处理的生物膜和青霉素与WGA探头染色 - 处理的细胞。插入的曲线图给出了处理和未处理的生物膜样品WGA信号的量化，及误差棒表示标准偏差。从Skogman 等进行修改。 26 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54829/54829fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 数据处理和统计分析统计参数计算，以表征测定的质量和筛选运行期间跟随它们的性能。所使用的最重要的参数，以及所获得的值和目标值的方程，都列在表1中 。在所有方程，SD 分钟 ，μ 分钟 ，SD max和μ 最大值代表最小的标准偏差和装置（分钟）和最大（最大）的信号，分别在此处显示的结果，成对的原始值的比较采用非配对t检验与韦尔奇的校正，其中p &lt;0.05被认为是统计学显著完成。 <img alt="图1" src="/files/ftp_upload/54829/54829table1.jpg" /> 表1:用于评价测定的性能的统计参数。在所有方程，SD 分钟 ，μ 分钟 ，SD max和μ 最大分别表示的最小（MIN）的标准偏差和装置和最大（最大）信号。的染色方法，刃天青，结晶紫，和小麦胚芽凝集素，缩写RES，CRV，和WGA，分别。 RFU:相对荧光单位; RAU:相对吸收单位。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54829/Table1.xlsx">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries at JoVE.com

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

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 ...

a-platform-anti-biofilm-assays-suited-to-exploration-natural-compound

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.5K 次观看.  University of Helsinki. 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. 

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

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 ...

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An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

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 ...

<meta charset="utf8"></head><body>用于抗菌检测和微生物动力学分析的适应性 CF 模型

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

从植物提取物和分数中识别新型抗菌和抗生物膜分子的系统方法，以防止牙科蛀牙

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

生物学

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Source: Dalecki, A. G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53925">Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay.</a> J. Vis. Exp. (2016)
This video demonstrates an assay for screening antimicrobial compounds that efficiently eliminate biofilms using a peg-plate. The methodology monitors the biofilm&#39;s metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

用于筛选抗菌剂的 Peg 板生物膜测定

1. Biofilm Initiation

	Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman ...

适合与天然化合物库的探索抗生物试验的平台

摘要

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此视频中的章节

酶标菜生物膜形成实验

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

细胞和生物膜细胞外成分的定量并发

针对相关生物膜

方法学研究

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

从植物提取物和分数中识别新型抗菌和抗生物膜分子的系统方法，以防止牙科蛀牙

用于筛选抗菌剂的 Peg 板生物膜测定