Name: quadruple-checkerboard: a modification of the three-dimensional checkerboard for studying drug combinations
Uploaded: 2021-07-24T14:30:00
Description: Watch this Scientific Journal Video about Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations at JoVE.com

1. 准备步骤
<ol><li>准备穆勒-欣顿汤（MHB），将25克MH汤加入1升蒸馏水中混合。高压灭菌在121°C为2.5小时。然后，在室温下或冰箱中存储自动切割的介质。</li>
	<li>使用四象限条纹法在阿加介质上对有关细菌（大肠杆菌 ESBL）进行亚培养，并在37°C处过夜孵化。<ol>
<li>使用无菌循环，采取一个殖民地，并通过做密切的平行条纹在麦康基阿加板的上半部分传播它。</li>
		<li>使用循环，通过从第?...

<ol>
	<li>Ibezim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+resistance+to+antibiotics.">Microbial resistance to antibiotics.</a> African Journal of Biotechnology. 4, 1606-1611 (2006).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> P &amp; T: A Peer-Reviewed Journal for Formulary Management. 40 (4), 277-283 (2015).</li><li>Alanis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antibiotics:+Are+we+in+the+post-antibiotic+era.">Resistance to antibiotics: Are we in the post-antibiotic era.</a> Archives of Medical Research. 36 (6), 697-705 (2005).</li><li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+at+the+crossroads.">Antibiotics at the crossroads.</a> Nature. 431 (7011), 899-902 (2004).</li><li>Bollenbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+interactions:+mechanisms+and+implications+for+drug+discovery+and+resistance+evolution.">Antimicrobial interactions: mechanisms and implications for drug discovery and resistance evolution.</a> Current Opinion in Microbiology. 27, 1-9 (2015).</li><li>Mehta, K. C., Dargad, R. R., Borade, D. M., Swami, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+antibiotic+resistance+in+common+infectious+diseases:+role+of+antibiotic+combination+therapy.">Burden of antibiotic resistance in common infectious diseases: role of antibiotic combination therapy.</a> Journal of Clinical and Diagnostic Research: JCDR. 8 (6), (2014).</li><li>Chanda, S., Rakholiya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy:+Synergism+between+natural+plant+extracts+and+antibiotics+against+infectious+diseases.">Combination therapy: Synergism between natural plant extracts and antibiotics against infectious diseases.</a> Science against Microbial Pathogens: Communicating Current Research and Technological Advances. , (2011).</li><li>Cottarel, G., Wierzbowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+drugs,+an+emerging+option+for+antibacterial+therapy.">Combination drugs, an emerging option for antibacterial therapy.</a> Trends in Biotechnology. 25 (12), 547-555 (2007).</li><li>Kristiansen, J., Amaral, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+management+of+resistant+infection+with+non-antibiotics.">The potential management of resistant infection with non-antibiotics.</a> The Journal of Antimicrobial Chemotherapy. 40, 319-327 (1997).</li><li>Tamma, P. D., Cosgrove, S. E., Maragakis, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+for+treatment+of+infections+with+gram-negative+bacteria.">Combination therapy for treatment of infections with gram-negative bacteria.</a> Clinical Microbiology Reviews. 25 (3), 450-470 (2012).</li><li>Leekha, S., Terrell, C. L., Edson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+principles+of+antimicrobial+therapy.">General principles of antimicrobial therapy.</a> Mayo Clinic Proceedings. 86 (2), 156-167 (2011).</li><li>Eliopoulos, G. M., Eliopoulos, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+combinations:+Should+they+be+tested.">Antibiotic combinations: Should they be tested.</a> Clinical Microbiology Reviews. 1 (2), 139-156 (1988).</li><li>Doern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+does+2+plus+2+equal+5?+A+review+of+antimicrobial+synergy+testing.">When does 2 plus 2 equal 5? A review of antimicrobial synergy testing.</a> Journal of Clinical Microbiology. 52 (12), 4124-4128 (2014).</li><li>Stein, 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=Three+dimensional+checkerboard+synergy+analysis+of+colistin,+meropenem,+tigecycline+against+multidrug-resistant+clinical+klebsiella+pneumonia+isolates.">Three dimensional checkerboard synergy analysis of colistin, meropenem, tigecycline against multidrug-resistant clinical klebsiella pneumonia isolates.</a> PloS One. 10 (6), 0126479 (2015).</li><li>Langeveld, W. T., Veldhuizen, E. J. A., Burt, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+essential+oil+components+and+antibiotics:+a+review.">Synergy between essential oil components and antibiotics: a review.</a> Critical Reviews in Microbiology. 40 (1), 76-94 (2014).</li><li>Pankey, G., Ashcraft, D., Kahn, H., Ismail, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-kill+assay+and+Etest+evaluation+for+synergy+with+polymyxin+B+and+fluconazole+against+Candida+glabrata.">Time-kill assay and Etest evaluation for synergy with polymyxin B and fluconazole against Candida glabrata.</a> Antimicrobial Agents and Chemotherapy. 58 (10), 5795-5800 (2014).</li><li>Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy,+antagonism,+and+what+the+chequerboard+puts+between+them.">Synergy, antagonism, and what the chequerboard puts between them.</a> The Journal of Antimicrobial Chemotherapy. 52 (1), 1 (2003).</li></ol>

四重棋盘方法类似于棋盘和协议中的三维棋盘。但是，应考虑某些关键步骤，以避免在实验过程中出现错误。
在启动协议之前，请务必对每种药物的MIC进行针对测试分离物的测试，以了解使用药物 1 和药物 2 开始稀释所需的浓度，这些浓度需要在盘子中连续稀释。关于药物 3 和药物 4，还应知道 MIC 计算需要测试的浓度（1/8 MIC、1/4 MIC、1/2 MIC 和 MIC）。有很多方法可以确定MIC?...

由于过度使用和滥用抗生素1，2，耐药性增加，开发治疗细菌感染的新药和制剂的必要性变得至关重要。开发新药等新方法对于克服耐药性危机非常重要。然而，制药业对开发新的抗菌剂不感兴趣。此外，如果开发新药，细菌将继续进化和发展对这些新药的耐药性3，4。因此，耐药性问题将无法解决，因此需要另一种方法，必须考虑和研究，以克服细菌耐药性。
药物组合是治疗细菌感染的一个非常重要的概念，主要是那些由耐多药病原体5，6引起的。它减少了治疗过程，减少了给予的剂量：因此，降低给定药物的毒性，有助于降低耐药性发展速度，并在某种程度上使细菌对附带敏感性5、7、8、9概念中描述的给定药物敏感。
一种药物的耐药性发展需要单一的突变：然而，针对多种途径的药物组合的耐药性发展需要几个独立的突变，这些突变会因这种组合而减慢。使用联合疗法时抵抗力下降的一个例子是10号分枝杆菌对里法姆平的抵抗率下降。另一个例子是Gribble等人所做的一项研究，该研究表明，单独服用皮拉西林的患者中耐药菌株的出现率高于服用卡博西霉素和阿米诺糖苷10组合的患者。研究表明，在进化的细菌中对阿米糖苷的耐药性发展使这些菌株对各种其他药物敏感。β-乳酸类药物阿莫西林与乳糖酶抑制剂克拉武拉尼酸的结合表明，在治疗耐药细菌菌株8方面取得了成功。
减少治疗时间是药物组合产生的一个很好的优势。例如，一种将青霉素或西夫特里亚松与根塔米辛结合2周的疗法，在给予4周11时，将单独给予青霉素或西夫特里亚松相同的疗效。组合药物允许使用剂量较低的药物，这些药物在单独服用时（如子 MIC）无效。硫化物的例子可以给出，其中使用三磺酰平减小，在较低的剂量，产生的毒性是晶体形成或结晶时，使用不溶性磺酰平地全剂量12。
因此，减少剂量和治疗时间最终将减少药物对身体的毒性。开发评估联合药物相互作用的方法的想法非常重要。在一项研究中，结果表明，联合疗法对治疗抗药性抗性阿 西诺菌 和 P.阿鲁吉诺萨8种更为有效。
联合给药 研究药物组合的方法有多种，如棋盘法、消磨曲线法、电子测试法等。棋盘方法可以在一个实验中研究两种药物之间的所有可能组合。此外，它被开发为研究三种药物的组合14。现在，我们扩展这一点，以研究四种药物的组合，主要用于治疗耐多药病原体。
时间杀死曲线检测通常用于测试某种药物的杀菌效果。它也被用来测试药物组合的效果，其中几个药物在特定浓度下结合。该协议要求准备几个无菌管或杯子，在每杯中，我们添加汤，药物的组合，和所需的细菌菌株。经过几个时间点的光密度的孵化和记录，结果与用过的菌株的正常增长率进行比较，看增长率是增、减，还是不变。
电子测试方法通常用于测试最低抑制浓度 （MIC），其中含有有关药物梯度浓度的条状被放在接种板上。它也被用来测试两种药物之间的组合，其中两条条被添加到板垂直的方式相交在他们的MIC13。
根据文献，没有黄金标准来定义和研究协同作用：因此，很难评估哪种方法用于研究组合更好，哪种方法产生更好、更可靠的结果，主要是13。然而，时间杀人检测是劳动密集型的，耗时的，昂贵的15，16，而电子测试方法的开发，以研究两种药物之间的组合只。棋盘可以研究测试的两种药物之间的所有可能组合，这就是为什么这种技术已被选择开发。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330000402</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Citotest</td>
			<td>4330-0013-17</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>corning</td>
			<td>430828</td>
			<td>For drug 3 and 4 preparation</td>
		</tr>
		<tr>
			<td>5 mL polysterene round-bottom Tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td>For 0.5 MacFarland bacterial inoculum preparation</td>
		</tr>
		<tr>
			<td>90mm petri dishes</td>
			<td>JRZ Plastilab</td>
			<td></td>
			<td>As bed for the solutions to be added using the multichannel pipette</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>corning</td>
			<td>3596</td>
			<td>For serial diltuion and combining drugs</td>
		</tr>
		<tr>
			<td>Bactrim 200, 40 mg (Trimethoprim-sulamethoxazole</td>
			<td>By CRNEXI SAS Fontenay-sous-Bois, France</td>
			<td>10177403</td>
			<td>Drug 4</td>
		</tr>
		<tr>
			<td>Ceforane, 1 g (Cefotaxime)</td>
			<td>PHARCO Pharmaceuticals</td>
			<td>24750/2006</td>
			<td>Drug 1</td>
		</tr>
		<tr>
			<td>Densitometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. Coli ESBL strain</td>
			<td>Retreived as a medical strain from the Saint-George Hospital Lebanon</td>
			<td></td>
			<td>Bacterial strain</td>
		</tr>
		<tr>
			<td>Mac Conkey + crystal violet agar</td>
			<td>BIO-RAD</td>
			<td>64169508</td>
			<td>For making agar plates used for subculturing</td>
		</tr>
		<tr>
			<td>Miacin 500 mg/2 mL (Amikacin)</td>
			<td>HIKMA Pharmaceuticals</td>
			<td>2BXMIA56N-AEF</td>
			<td>Drug 2</td>
		</tr>
		<tr>
			<td>Muller-Hinton Broth</td>
			<td>BIO-RAD</td>
			<td>69444</td>
			<td>For making bacterial media</td>
		</tr>
		<tr>
			<td>Multichannel Pipette</td>
			<td>Thermo Scientific</td>
			<td>GJ54761</td>
			<td>For serial dilution and addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Paper Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel pipettes</td>
			<td>Thermo Scientific</td>
			<td>OH19855 HH40868</td>
			<td>For the addition of media, bacteria and drugs</td>
		</tr>
		<tr>
			<td>Tavanic, 500 mg (Levofloxacin)</td>
			<td>sanofi aventis</td>
			<td>221937/2009</td>
			<td>Drug 3</td>
		</tr>
	</tbody>
</table>

quadruple-checkerboard: a modification of the three-dimensional checkerboard for studying drug combinations

药物组合疗法的概念正变得非常重要，主要是随着对药物的抵抗力的急剧增加。四重棋盘也称为 Q-棋盘，旨在最大限度地增加一个实验中四种药物之间可能获得的组合数量，以最大限度地减少与其他协议实现相同结果所需的时间和工作。此协议基于简单的微稀释技术，其中药物被稀释并组合在几个 96 井板中。
在第一组96井板，穆勒-欣顿汤被添加，其次是第一个所需的药物（如Cefotaxime这里）连续稀释它。第一步完成后，另一组96井板用于稀释第二种药物（例如阿米卡奇），通过去除特定量的药物2进行转移，并在第一组含有药物的96井板中放入相应的井中。第三步是将第三种药物（如利沃弗洛沙辛）的所需浓度添加到包含药物1和2组合的初始组的适当板中。第四步是在第一组中将第四种药物（如三聚丙酮-磺酰硫磺醇）的所需浓度添加到适当的盘子中。然后，将准备和添加 大肠杆菌 ESBL细菌。
此方法对于评估所有可能的组合非常重要，并且具有更广泛的可能性，可进一步进行 体内 测试。尽管是一项需要大量关注的累人技术，但结果非常出色，而且节省了时间，许多组合可以在一个实验中进行测试。

图2A 表示通过将塞福塔西姆和阿米卡辛与特定浓度的利沃福沙辛和三氯环己烷-磺胺佐尔结合而获得的结果。我们可以在图的左侧看到图右侧显示药物浓度的四个板。箭头表示增长/无增长界面上的井。彩色油井是包含生长的井。我们注意到，第四板不包含包含组合的象限中的生长。这是因为在这个板块中，我们有利沃弗洛沙辛的MIC，将抑制增长。在这个数字中，我们可以看?...

Watch this Scientific Journal Video about Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations at JoVE.com

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

该协议描述了如何研究在一个实验中四种药物之间可以获得的所有可能的组合。该方法基于标准的96井板微稀释检测和分数抑制浓度（FICs）的计算来评估结果。

四重检查板：研究药物组合的三维棋盘的修改

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