Name: broth microdilution in vitro screening: an easy and fast method to detect new antifungal compounds
Uploaded: 2018-02-14T15:00:00
Description: Watch this Scientific Journal Video about Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds at JoVE.com

我们感谢海角-巴西, CNPq 巴西, FAP/DF 为财政支持。我们感谢 Hugo Paes 博士修改了手稿。

1. 解决方案和媒体
<ol><li>准备2X 的罗斯威尔公园纪念研究所 (RPMI) 1640 中等, 磷酸盐缓冲盐水 (PBS), Sabouraud 葡萄糖汤和 Sabouraud 葡萄糖琼脂按表 1。</li>
</ol>2. 真菌接种生长条件
<ol><li>将所有真菌菌株储存在35% 甘油-80 摄氏度, 直到需要。</li>
	<li>在每个实验之前执行以下步骤。<ol>
<li>对于白色念珠菌菌株:<ol>
<li>解冻一个股票瓶和转移200µL 到10毫升 Sabouraud 葡萄?...

<ol>
	<li>Armstrong-James, D., Meintjes, G., Brown, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neglected+epidemic:+fungal+infections+in+HIV/AIDS.">A neglected epidemic: fungal infections in HIV/AIDS.</a> Trends Microbiol. 22 (3), 120-127 (2014).</li><li>Romani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+fungal+infections.">Immunity to fungal infections.</a> Nat Rev Immunol. 11 (4), 275-288 (2011).</li><li>Pfaller, 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=Antifungal+drug+resistance:+mechanisms,+epidemiology,+and+consequences+for+treatment.">Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment.</a> Am J Med. 125 (1 Suppl), S3-S13 (2012).</li><li>Hancock, R. 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CLSI document M27-A3. , (2008).</li><li>Guilhelmelli, 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=Activity+of+Scorpion+Venom-Derived+Antifungal+Peptides+against+Planktonic+Cells+of+Candida+spp.+and+Cryptococcus+neoformans+and+Candida+albicans+Biofilms.">Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms.</a> Front Microbiol. 7, 1844 (2016).</li><li>Roongruangsree, U. T., Kjerulf-Jensen, C., Olson, L. W., Lange, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+Tests+for+Thick+Walled+Fungal+Spores+(ex:+Oospores+of+Peronospora+manshurica).">Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica).</a> Journal of Phytopathology. 123 (3), 244-252 (1988).</li><li>Boedijn, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+as+stain+for+fungi.">Trypan blue as stain for fungi.</a> Stain Technol. 31 (3), 115-116 (1956).</li><li>Goihman-Yahr, 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=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71 (2), 73-83 (1980).</li><li>Tati, 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=Histatin+5-spermidine+conjugates+have+enhanced+fungicidal+activity+and+efficacy+as+a+topical+therapeutic+for+oral+candidiasis.">Histatin 5-spermidine conjugates have enhanced fungicidal activity and efficacy as a topical therapeutic for oral candidiasis.</a> Antimicrob Agents Chemother. 58 (2), 756-766 (2014).</li><li>Petrou, M. A., Shanson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Cryptococcus+neoformans+by+the+NCCLS+microdilution+and+Etest+methods+using+five+defined+media.">Susceptibility of Cryptococcus neoformans by the NCCLS microdilution and Etest methods using five defined media.</a> J Antimicrob Chemother. 46 (5), 815-818 (2000).</li><li>Zaragoza, O., 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=Process+analysis+of+variables+for+standardization+of+antifungal+susceptibility+testing+of+nonfermentative+yeasts.">Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts.</a> Antimicrob Agents Chemother. 55 (4), 1563-1570 (2011).</li><li>Rodriguez-Tudela, J. 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=Influence+of+shaking+on+antifungal+susceptibility+testing+of+Cryptococcus+neoformans:+a+comparison+of+the+NCCLS+standard+M27A+medium,+buffered+yeast+nitrogen+base,+and+RPMI-2%+glucose.">Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose.</a> Antimicrob Agents Chemother. 44 (2), 400-404 (2000).</li><li>Beggs, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase+in+relation+to+ketoconazole+and+miconazole+susceptibilities+of+Candida+albicans.">Growth phase in relation to ketoconazole and miconazole susceptibilities of Candida albicans.</a> Antimicrob Agents Chemother. 25 (3), 316-318 (1984).</li><li>Alcouloumre, M. S., Ghannoum, M. A., Ibrahim, A. S., Selsted, M. E., Edwards, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+properties+of+defensin+NP-1+and+activity+against+Cryptococcus+neoformans+in+vitro.">Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro.</a> Antimicrob Agents Chemother. 37 (12), 2628-2632 (1993).</li></ol>

稀释试验可以用少量的化合物分析靶化合物的潜在抗真菌活性, 同时在浓度范围内对其进行测试。因此, 建议将本议定书作为筛选潜在新的抗真菌化合物的第一步。这里提出的协议是基于 M27-A3 协议, 最初旨在帮助选择在诊所的抗真菌疗法, 并可适应各种新的抗真菌化合物。总的来说, 该协议可以集中于提取物或化合物的物理化学特征。例如, 由于介质中的化合物可能阻止其与真菌细胞的相互作用, 如...

真菌感染已成为近几十年来一个重要的医疗问题, 主要是由于免疫机能丧失者的数量增加, 如接受癌症治疗的人和患有艾滋病毒/艾滋病者或移植的器官1,2。然而, 一组非常有限的可用抗真菌药物和越来越多的真菌耐药性报告导致了关于系统性部真菌病3 治疗的主要问题。
新的抗真菌化合物的潜在来源是抗菌肽 (安培), 许多生物体产生的小阳离子肽, 作为其对感染的先天免疫反应的一部分4。然而, 检测这些化合物对真菌病原体的筛选方法并不标准化。不同的程序已经被用来评估安培的抗真菌活性, 有时对于相同的模型微生物5,6,7。在某些协议中, 这些差异和细节的缺乏使化合物的比较复杂化, 并阻碍了重现性。
对新药候选者进行标准化测试的一种方法是遵循在临床环境中确定抗真菌敏感性的指导方针, 如临床和实验室标准研究所 (CLSI) M27-A3 指南。然而, 这些抗真菌敏感性测试是太限制性的, 并没有考虑到不同物种的新陈代谢变化, 因为它们只是建立了一些选择剂。例如, 它们不考虑非发酵酵母的代谢需要。
该协议允许评估潜在的抗真菌化合物的活性, 并在这里实施, 以寻找抗真菌肽。它的基础上的汤稀释敏感性测试从 CLSI M27-A3 指南与修改, 优化筛选新化合物8,9。这些变化允许使用少量的化合物, 温度或初始接种的变化, 和不同的介质, 以获得最佳的预试生长, 同时使用参照抗真菌药物作为控制来标准化结果。这种方法, 利用多井培养板, 使其能够快速和可靠地筛选出大量的化合物。
由于其固有的灵活性, 本议定书可用于不同的化学类化合物和其他微生物, 几乎没有适应。

<table>
	<tbody>
		<tr>
			<td>Media and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium with l-glutamine, without sodium bicarbonate</td>
			<td>Thermo Fisher</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propane sulfonic acid (MOPS) (o que a gente usa tem um s&#243;dio, completa o nome dele please)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use to buffer 2X RPMI medium</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Din&#226;mica</td>
			<td>1528-1</td>
			<td>137 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>J.T.Baker</td>
			<td>3040-01</td>
			<td>2.7 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>V000129</td>
			<td>10 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>60230</td>
			<td>2 mM for Phosphate buffered saline (PBS)</td>
		</tr>
		<tr>
			<td>BD Difco Sabouraud dextrose broth</td>
			<td>BD</td>
			<td>238230</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Sabouraud Dextrose Agar</td>
			<td>BD</td>
			<td>210950</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>V000123</td>
			<td>35% for (solu&#231;&#227;o de estoque? Criopreserva&#231;&#227;o?)</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td></td>
			<td></td>
			<td>Para dilui&#231;&#227;o das drogas na dilui&#231;&#227;o seriada</td>
		</tr>
		<tr>
			<td>Antifungal drugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluconazole</td>
			<td>Sigma-Aldrich</td>
			<td>F8929</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspofungin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1160</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dish petri</td>
			<td>J.Prolab</td>
			<td>0304-5</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Solution Reservoir</td>
			<td>KASVI</td>
			<td>K30-208</td>
			<td>Use to pippet the solutions using the multichannel pippet</td>
		</tr>
		<tr>
			<td>Equipment and other materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Nikon</td>
			<td>E200MV</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugue</td>
			<td>Thermo Fisher</td>
			<td>MegaFuge 16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ethik Technology</td>
			<td>403-3D</td>
			<td>Set to 37&#176; C</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E25</td>
			<td>Set to 37&#176; C, 200 RPM</td>
		</tr>
		<tr>
			<td>Cell counting chamber, Neubauer</td>
			<td>BOECO Germany</td>
			<td>BOE 13</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>HTL</td>
			<td>5123</td>
			<td></td>
		</tr>
	</tbody>
</table>

broth microdilution in vitro screening: an easy and fast method to detect new antifungal compounds

真菌感染已成为近几十年来的重要医学条件, 但现有抗真菌药物的数量有限。在这种情况下, 寻找新的抗真菌药物是必要的。此处报告的协议详细介绍了一种筛选肽的抗真菌性质的方法。它是基于从临床和实验室标准研究所 (CLSI) M27-A3 指导方针的汤稀释敏感性测试, 以适应研究抗菌肽作为潜在的新抗真菌药物。该协议描述了一种功能性的检测方法来评估抗真菌化合物的活性, 并且可以很容易地修改, 以适应任何特定的分子类别的调查。由于检测是用小体积的96井板进行的, 所以大规模的筛分可以在短时间内完成, 特别是在自动化环境下进行。这一过程说明了标准化和可调节的临床协议如何帮助新分子的板凳工作追求, 以改善真菌疾病的治疗。

MIC 被定义为在潜伏期结束时完全抑制可见真菌生长的最低抗菌化合物浓度。由于本议定书的目的是要有一个快速的方法来筛选潜在的抗真菌药物, 任何与透明介质类似的空白井被认为是一个积极的结果, 而任何与浊度类似的负/生长控制井是被认为是负面的。然而, 如果有兴趣知道一个给定的安培是否是抑菌作用或杀菌剂, 从积极的水井的媒体也可以镀到 Sabouraud 葡萄糖琼脂?...

Watch this Scientific Journal Video about Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds at JoVE.com

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

一种简便、适应性强的肉汤稀释法筛选抗真菌化合物和提取物。

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Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

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This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

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Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...