Name: an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm
Uploaded: 2018-04-16T15:00:00.000+00:00
Duration: 5 min 12 s
Description: Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

这项工作由中国国家自然科学基金 (81300810 号)、上海市青年医生培训计划 (20141057 号) 和中国国家自然科学基金 (81671982、81271791和 81571955) 资助。我们感谢 LetPub (www.letpub.com) 在编写本手稿期间提供语言援助。

1. 生物膜形成
<ol><li>
96井微型生物膜的形成
	<ol>
<li>检索金黄色葡萄球菌应变 USA300 和3生物膜形成的临床菌株 (C1-C3), 存储在-80 摄氏度。 注: 临床菌株形成生物膜的能力由前述的微量滴定板检测方法确定(15)。</li>
		<li>在5毫升的胰蛋白胨大豆汤中接种细菌, 并在一个孵化器中培养, 一夜之间以37摄氏度的温度抖动到固定的阶段。</li>
		<li>将隔夜细菌培养在25摄氏度的 4000 x g 上离心, 然后丢弃上清。并用重悬磷酸盐缓冲盐水 (PBS) 中的颗粒, 最终浓度为 2.0 x10 9 CFU/毫升。 注: 通过测量光学密度并进一步确定了细菌浓度, 并通过平板计数16, 揭示了 1 OD600的悬浮液中含有 1.5 x 108 CFU/毫升。</li>
		<li>将细菌悬浮液稀释到含有0.5% 葡萄糖的 1:200 (1.0 x 107 CFU/毫升) 中。接种200µL 细菌悬浮液进入细胞培养处理过的聚苯乙烯 96-井微板块。</li>
		<li>在含氧良好的环境下, 将微板块静态地孵化为24小时37摄氏度。 注: 成熟生物膜形成的潜伏期可能因不同菌株而异;这应在 PDT 实验15之前确定。</li>
		<li>在井中丢弃介质, 用 PBS 轻轻冲洗微板块井三次, 然后丢弃上清。 注意: 该步骤应非常温和地进行, 以避免扰乱形成的生物膜。</li>
	</ol>
</li>
	<li>
碟形中的生物膜形成
	<ol>
<li>将金黄色葡萄球菌菌株接种到5毫升的 USA300 中, 并在一个孵化室中培养, 在37摄氏度一夜之间摇晃到固定相。</li>
		<li>将隔夜细菌培养在25摄氏度的 4000 x g 上离心, 然后丢弃上清。将 PBS 中的颗粒并用重悬到最终浓度为 2.0 x10 9 CFU/毫升。然后, 将细菌悬浮液稀释到含有0.5% 葡萄糖的 1:200 (1.0 x 107 CFU/毫升) 中。将2毫升细菌悬浮液接种到35毫米光学质量的玻璃底部细胞培养皿中, 并以37摄氏度静态孵育24小时。 注: 通过测量光学密度来估计细菌的浓度。</li>
		<li>用吸管吸出培养基, 然后用 PBS 轻轻地冲洗盘子中的生物膜三次, 然后小心地丢弃上清液。 注意: 避免接触到盘子底部的吸管尖端。步骤2.2 应在这一步骤后立即执行, 以防止干燥的形成生物膜。</li>
	</ol>
</li>
</ol>2. 光辐照
<ol><li>在4摄氏度冰箱里存储 5-阿拉巴马。实验前, 用 PBS 稀释 5-阿拉-10 毫米。 注: 5-阿拉氨酸溶液应在实验前进行新的准备。</li>
	<li>在实验组, 添加200µL 10 毫米的微板块或2毫升到养殖皿的每一个井。用铝箔盖住盘子/盘子, 孵化1小时。然后, 用发光二极管 (LED) 照射板/盘, 亮度为100兆瓦/厘米2 , 为 1 h, 在 2 @ 633 nm10的主要波长处达到光剂量 360 J/厘米17 。 注: 为了让光能有效地和均匀地传递到所有水井/盘子中的生物膜, 将光源的峰值与6.0 厘米的井/盘的距离固定, 并将实验区限制在中心辐照区 (10 厘米 x 8cm). 为了确保结果是可重现的, 实验应在相同的室温下进行。 在 led 照射步骤中, 为了避免直接接触到其他光源 (如日光、房间照明或灯光), led 在将盘子/盘子移到辐照区前打开, 光线足够亮, 足以完成操作。</li>
	<li>设置控制组 (在实验中设置了三个控制组)。<ol>
<li>对于第一个控制组 (阿拉巴马 LED), 添加200µL 的 PBS, 每井微板块或2毫升的文化菜。用铝箔盖住盘子/盘子, 孵化2小时。</li>
		<li>对于第二个控制组 (阿拉巴马 + LED), 添加200µL 10 毫米的微板块或2毫升到养殖皿的每一个井。用铝箔盖住盘子/盘子, 孵化2小时。</li>
		<li>对于第三个控制组 (阿拉巴马 LED +), 增加200µL 的 PBS 微板块或2毫升的文化菜。用铝箔盖住盘子/盘子, 孵化1小时。然后将该板暴露在 LED 上, 其 360 J/厘米2光照射波长为 633 @ 10 nm17。 注: 在 LED 照射步骤中, 避免直接接触到其他光源, 如日光、房间照明或灯光。</li>
	</ol>
</li>
</ol>3. PDT 治疗效果的测定
注: 为了确认对金黄色葡萄球菌生物膜的影响, 通过 CFU 计数和活性染色法对具有或不带 pdt 的细胞的生存能力进行了评价。
<ol><li>
剩余存活菌细胞的测定
	<ol>
<li>治疗后, 丢弃在水井中的介质和冲洗与 PBS 三倍的水井, 以消除所有非黏附细胞的实验和对照组。 注意: 这一步应该非常温和地进行。</li>
		<li>用吸管尖端从水井中彻底刮去附着的细菌细胞, 并收集锥形管中的细胞。</li>
		<li>离心机的细菌悬浮在 4000 x g 10 分钟4摄氏度, 然后丢弃上清。</li>
		<li>并用重悬1毫升0.25% 胰酶酶在 PBS 中的细菌和孵育37°c 1.5 小时。</li>
		<li>离心机在 4000 x g 10 分钟;丢弃上清, 然后在 PBS 的200µL 中并用重悬颗粒。</li>
		<li>用 PBS 制作1:10 系列稀释细胞溶液;然后, 将每个系列稀释样品的5µL 添加到胰蛋白胨大豆琼脂 (TSA) 板上。孵育37°c 的 TSA 板16小时;然后, (肉眼) 计数细菌菌落数 (CFU/毫升)。</li>
	</ol>
</li>
	<li>
CLSM 对金黄色葡萄球菌生物膜的观察
	<ol>
<li>光照射后, 用 PBS 将培养基中的生物膜洗净三次。 注意: 这一步应该非常温和地进行。</li>
		<li>添加1毫升的1µM 绿色荧光核和染色体染色, 可渗透到原核细胞膜 (例如, SYTO9) 和1毫升1µM 碘碘 (PI), 以染色生物膜和死细胞。</li>
		<li>观察可行细胞 (绿色荧光, 前/em 485 nm/530 nm) 和死细胞 (红色荧光, 前/em 485 nm/630nm) 下 CLSM 与 63X 1.4-钠油浸泡物镜。</li>
		<li>使用显微软件生成图像。</li>
	</ol>
</li>
</ol>

<ol>
	<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=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrob Agents Chemother. 45 (4), 999-1007 (2001).</li><li>Rabin, N., 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=Biofilm+formation+mechanisms+and+targets+for+developing+antibiofilm+agents.">Biofilm formation mechanisms and targets for developing antibiofilm agents.</a> Future Med Chem. 7 (4), 493-512 (2015).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Sharma, 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=Toluidine+blue-mediated+photodynamic+effects+on+staphylococcal+biofilms.">Toluidine blue-mediated photodynamic effects on staphylococcal biofilms.</a> Antimicrob Agents Chemother. 52 (1), 299-305 (2008).</li><li>Rosa, L. P., da Silva, F. C., Nader, S. A., Meira, G. A., Viana, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effectiveness+of+antimicrobial+photodynamic+therapy+(APDT)+using+a+660+nm+laser+and+malachite+green+dye+in+Staphylococcus+aureus+biofilms+arranged+on+compact+and+cancellous+bone+specimens.">In vitro effectiveness of antimicrobial photodynamic therapy (APDT) using a 660 nm laser and malachite green dye in Staphylococcus aureus biofilms arranged on compact and cancellous bone specimens.</a> Lasers Med Sci. 29 (6), 1959-1965 (2014).</li><li>Rosa, L. P., Silva, F. C., Nader, S. A., Meira, G. A., Viana, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+antimicrobial+photodynamic+therapy+using+a+660+nm+laser+and+methyline+blue+dye+for+inactivating+Staphylococcus+aureus+biofilms+in+compact+and+cancellous+bones:+An+in+vitro+study.">Effectiveness of antimicrobial photodynamic therapy using a 660 nm laser and methyline blue dye for inactivating Staphylococcus aureus biofilms in compact and cancellous bones: An in vitro study.</a> Photodiagnosis Photodyn Ther. 12 (2), 276-281 (2015).</li><li>Mai, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+effect+of+sinoporphyrin+sodium+photodynamic+therapy+on+Staphylococcus+aureus+planktonic+and+biofilm+cultures.">The antibacterial effect of sinoporphyrin sodium photodynamic therapy on Staphylococcus aureus planktonic and biofilm cultures.</a> Lasers Surg Med. 48 (4), 400-408 (2016).</li><li>Gandara, L., Mamone, L., Bohm, G. C., Buzzola, F., Casas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+photodynamic+inactivation+of+Staphylococcus+aureus+biofilms+by+disruptive+strategies.">Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.</a> Lasers Med Sci. 32 (8), 1757-1767 (2017).</li><li>Baltazar, L. 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=Antimicrobial+photodynamic+therapy:+an+effective+alternative+approach+to+control+fungal+infections.">Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections.</a> Front Microbiol. 6, 202 (2015).</li><li>Fernandes, T., Bhavsar, C., Sawarkar, S., D'Souza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+novel+approaches+for+control+of+dental+biofilm.">Current and novel approaches for control of dental biofilm.</a> Int J Pharm. 536 (1), 199-210 (2017).</li><li>De Sordi, L., 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=Development+of+Photodynamic+Antimicrobial+Chemotherapy+(PACT)+for+Clostridium+difficile.">Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.</a> PLoS One. 10 (8), e0135039 (2015).</li><li>Harris, F., Pierpoint, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+based+on+5-aminolevulinic+acid+and+its+use+as+an+antimicrobial+agent.">Photodynamic therapy based on 5-aminolevulinic acid and its use as an antimicrobial agent.</a> Med Res Rev. 32 (6), 1292-1327 (2012).</li><li>Donnelly, R. F., McCarron, P. A., Tunney, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+photodynamic+therapy.">Antifungal photodynamic therapy.</a> Microbiol Res. 163 (1), 1-12 (2008).</li><li>Shi, H., Li, J., Zhang, H., Zhang, J., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+5-aminolevulinic+acid+photodynamic+therapy+on+Candida+albicans+biofilms:+An+in+vitro+study.">Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: An in vitro study.</a> Photodiagnosis Photodyn Ther. 15, 40-45 (2016).</li><li>Zhang, Q. Z., 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=5-aminolevulinic+acid-mediated+photodynamic+therapy+and+its+strain-dependent+combined+effect+with+antibiotics+on+Staphylococcus+aureus+biofilm.">5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm.</a> PLoS One. 12 (3), 0174627 (2017).</li><li>Chang, Y. C., 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=Rapid+single+cell+detection+of+Staphylococcus+aureus+by+aptamer-conjugated+gold+nanoparticles.">Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.</a> Sci Rep. 3, 1863 (2013).</li><li>Barra, F., 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=Photodynamic+and+Antibiotic+Therapy+in+Combination+to+Fight+Biofilms+and+Resistant+Surface+Bacterial+Infections.">Photodynamic and Antibiotic Therapy in Combination to Fight Biofilms and Resistant Surface Bacterial Infections.</a> Int J Mol Sci. 16 (9), 20417-20430 (2015).</li><li>St Denis, T. G., 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=All+you+need+is+light:+antimicrobial+photoinactivation+as+an+evolving+and+emerging+discovery+strategy+against+infectious+disease.">All you need is light: antimicrobial photoinactivation as an evolving and emerging discovery strategy against infectious disease.</a> Virulence. 2 (6), 509-520 (2011).</li><li>O'Neill, J. F., Hope, C. K., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+bacteria+in+multi-species+biofilms+can+be+killed+by+red+light+in+the+presence+of+toluidine+blue.">Oral bacteria in multi-species biofilms can be killed by red light in the presence of toluidine blue.</a> Lasers Surg Med. 31 (2), 86-90 (2002).</li><li>Li, X., 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=Effects+of+5-aminolevulinic+acid-mediated+photodynamic+therapy+on+antibiotic-resistant+staphylococcal+biofilm:+an+in+vitro+study.">Effects of 5-aminolevulinic acid-mediated photodynamic therapy on antibiotic-resistant staphylococcal biofilm: an in vitro study.</a> J Surg Res. 184 (2), 1013-1021 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, 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:+from+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: from the natural environment to infectious diseases.</a> Nat Rev Microbiol. 2 (2), 95-108 (2004).</li><li>Elias, S., Banin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-species+biofilms:+living+with+friendly+neighbors.">Multi-species biofilms: living with friendly neighbors.</a> FEMS Microbiol Rev. 36 (5), 990-1004 (2012).</li><li>Wu, 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=Design+and+Proof+of+Programmed+5-Aminolevulinic+Acid+Prodrug+Nanocarriers+for+Targeted+Photodynamic+Cancer+Therapy.">Design and Proof of Programmed 5-Aminolevulinic Acid Prodrug Nanocarriers for Targeted Photodynamic Cancer Therapy.</a> ACS Appl Mater Interfaces. 9 (17), 14596-14605 (2017).</li></ol>

作者没有什么可透露的

PDT 一直是一个良好的研究治疗癌症治疗, 因为它是发明100年前的18。在过去的十年中, PDT 已被应用作为一种抗菌策略, 并显示了对某些耐药性致病菌的有效性12。与浮游状态相比, 细菌生物膜似乎对抗生素治疗的抗药性更有3, 而对生物膜的影响还没有得到充分的研究。
本文介绍了一种体外PDT 系统, 并对该模型对金?...

金黄色葡萄球菌是一种重要的革兰氏阳性病原体, 殖民了人类寄主的皮肤和粘膜。它形成生物膜的能力被认为是其发病机制的一个重要方面1。细菌生物膜是一种植入自产基质中的细菌群落, 由胞外高分子物质组成, 包括多糖、DNA 和蛋白质。这种基质在细菌感染的持续性中起着重要作用, 对人体免疫系统和目前的抗微生物治疗有很大的抵抗力,2。尽管抗生素对生物膜的影响有限, 但抗生素仍然是生物膜感染的主要治疗方法。先前已经表明, 生物膜中的细胞比它们的浮游相对物3更耐抗生素 10-1000 倍。因此, 需要采取替代战略来克服这一问题。
光动力疗法是一种治疗细菌感染的替代疗法, 它利用适当波长的光线激活光敏。这导致了活性氧种类 (ROS) 的生产, 这是致命的靶细胞通过扰乱细胞壁, 失活酶, 并损害 DNA4。这种多目标特性使得细菌难以对 PDT 治疗产生抗药性。
在以前的报告5、6中, 研究了 PDT 对细菌和真菌生物膜的抗菌作用, 其中包括甲苯胺蓝、孔雀石绿、亚甲基蓝、氯 e6 和卟啉等多种光敏. 7,8,9,10,11,12,13。5-阿拉, 药的实际光敏剂, PpIX, 其特点是其小分子量和快速清除12,14。这些优势给了阿拉 PDT 的主要潜力作为治疗应用。虽然对浮游细菌的影响已经研究了许多组12, 但对细菌生物膜的抗菌作用尚未阐明。同时, 比较以往研究的结果是很难的。其中一个原因是不同的协议被各种组使用。因此, 本协议描述了一种基于我们以前的工作15的一种用于 PDT 系统的体外模型。通过 CFU 计算和 CLSM 活性染色证实了该模型的有效性。

<table><tbody><tr><td>Tryptone Soya Broth (TSB)</td><td>OXOID</td><td>CM0129B</td><td></td></tr><tr><td>Tryptone Soya Agar (TSA)</td><td>OXOID</td><td>CM0131</td><td></td></tr><tr><td>SYTO9</td><td>Thermo Fisher Scientific</td><td>L7012</td><td>The LIVE/DEAD BacLight Bacterial Viability Kits&amp;nbsp;</td></tr><tr><td>Propidium iodide (PI)</td><td>Thermo Fisher Scientific</td><td>L7012</td><td>The LIVE/DEAD BacLight Bacterial Viability Kits&amp;nbsp;</td></tr><tr><td>Pancreatin</td><td>Sigma-Aldrich</td><td>P3292</td><td></td></tr><tr><td>5-aminolevulinic acid (ALA)</td><td>Fudan Zhangjiang Bio-Pharm</td><td>3.1</td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt; strain USA300</td><td>/</td><td>/</td><td>The source of USA 300 references &amp;ldquo;Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6&amp;rdquo;.</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt; clinical strains (C1-C3)</td><td>/</td><td>/</td><td>All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627].</td></tr><tr><td>96-well microplate</td><td>Corning Inc</td><td>3599</td><td>Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile</td></tr><tr><td>Fluorodish</td><td>NEST Biotechnology</td><td>801001</td><td>Glass bottom, Non-pyrogenic</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>Eppendorf microcentrifuge 5417</td><td>Eppendorf</td><td>Z365998 |&amp;nbsp;SIGMA</td><td></td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific</td><td>SHKE4000</td><td>MaxQ 4000 Benchtop Orbital Shakers</td></tr><tr><td>Light emitting diode (LED)</td><td>Wuhan Yage Optic and Electronic Technique CO</td><td>LED-IB</td><td></td></tr><tr><td>Leica TCS SP8 confocal laser-scanning microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica LAS AF software</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>IMARIS software</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm

金黄色葡萄球菌(金黄色葡萄球菌) 是一种常见的人类病原体, 引起化脓性和系统性感染。金黄色葡萄球菌感染很难根除, 这不仅是由于抗药性菌株的出现, 而且是它形成生物膜的能力。近年来, 光动力疗法 (PDT) 已被指出是控制生物膜感染的潜在治疗方法之一。然而, 需要进一步的研究, 以提高我们对其对细菌生物膜的影响的认识, 以及潜在的机制。这篇手稿描述了一个体外模型的 PDT 与 5-氨基乙酰丙酸 (5-阿拉), 一个前兆的实际光敏剂, 原卟啉 IX (PpIX)。简单地, 成熟的金黄色葡萄球菌生物膜与阿拉巴马一起孵化, 然后暴露在光中。随后, 通过共聚焦激光扫描显微镜 (CLSM) 计算菌落形成单元 (CFUs), 并通过活性荧光染色可视化方法对金黄色葡萄球菌生物膜的抗菌作用进行了量化。具有代表性的结果表明, 对金黄色葡萄球菌生物膜具有较强的抗菌作用。该协议很简单, 可用于开发一种体外模型, 用于研究用 ALA PDT 治疗金黄色葡萄球菌生物膜的方法。在未来的研究中, 也可以借鉴其他光敏对不同菌株的光动力分析, 并进行最小的调整。

在 USA300 和三株临床菌株中, 与对照组 (以阿拉巴马为首的、以阿拉为主导的 led 和阿拉巴马为首的 +) 相比, 生物膜中细菌的生存能力下降了 (图 1)。
为证实 CFU 测定的结果, 观察其对金黄色葡萄球菌生物膜的抗菌作用, USA300 生物膜通过 CLSM 与活性染色进行可视化. 存活和死亡细胞分别染色绿色?...

Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

本手稿描述了一种研究 5-氨基乙酰丙酸介导光动力疗法 (ALA)对金黄色葡萄球菌生物膜的抗菌作用的协议。该协议可用于开发一种体外模型来研究细菌生物膜与 PDT 在未来的治疗。

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

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Research

JoVE Journal

Biology

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

7.8K 次观看.  Fudan University. 本手稿描述了一种研究 5-氨基乙酰丙酸介导光动力疗法 (ALA)对金黄色葡萄球菌生物膜的抗菌作用的协议。该协议可用于开发一种体外模型来研究细菌生物膜与 PDT 在未来的治疗。

视频: An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

科研

免疫与感染

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 ...

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 ...

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

生物化学

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 ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

一种体外光动力治疗方法的研究

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

医学

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

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

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

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

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

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

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

<meta charset="utf8"></head><body>用于细胞内 金黄色葡萄球菌 感染研究的优化体内模型

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

<meta charset="utf8"></head><body>金黄色葡萄球菌细胞内感染模型的开发与评价

Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.

金黄色葡萄球菌细胞内感染模型的开发与评价

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

摘要

探索更多视频

此视频中的章节

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

针对相关生物膜

适合与天然化合物库的探索抗生物试验的平台

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子

使用 COMSTAT 软件量化抗菌剂对体外生物膜结构的影响

一种体外光动力治疗方法的研究

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

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

金黄色葡萄球菌细胞内感染模型的开发与评价

金黄色葡萄球菌细胞内感染模型的开发与评价

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

摘要

探索更多视频

此视频中的章节

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

针对相关生物膜

适合与天然化合物库的探索抗生物试验的平台

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

使用 COMSTAT 软件量化抗菌剂对 体外 生物膜结构的影响

一种体外光动力治疗方法的研究

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

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

金黄色葡萄球菌细胞内感染模型的开发与评价

金黄色葡萄球菌细胞内感染模型的开发与评价

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