Name: a reference broth microdilution method for dalbavancin in vitro susceptibility testing of bacteria that grow aerobically
Uploaded: 2015-09-09T14:00:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically at JoVE.com

Prior work with regard to susceptibility testing of dalbavancin has been valuable in development of a standardized methods and acknowledgement for this work is given to JMI Laboratories, Robert Rennie and Beth Goldstein.

注:参见CLSI文件M7-A10和M100-S25和/或ISO / FDIS 20776-1为参考肉汤稀释法的药敏2，3，4全部细节的参考方法允许在一些选项。具体到接种物制备和MIC面板生产程序，以实现相同的最终结果。这里详述的方法涉及一种抗微生物剂，达巴万星，和某些步骤代表潜在几种方式参考过程可以做到的。适当的安全防范措施（生物安全2级保持一致），应使用10。用于该视频发布用途的MIC面板格式示于表1中。 1.存储达巴万星粉<ol><li>在接收到诊断级达巴万星的粉末，保存于-20℃在一干燥的环境中以非除霜冰箱。在使用前，该粉末应该平衡到开幕前达到室温。 </li></ol> 2.准备MIC板稀释<ol><li>准备原液达巴万星在纯（纯）DMSO的无菌玻璃或塑料管不高于1600微克/毫升，并在-20制备或商店的同一天使用到-60 度或以下为将来使用非除霜冰箱。考虑到作为设置在与粉末接收的文档达巴万星的效力，称量粉末时（参见公式1，例如800微克/毫升的浆料制备）。 </li><li>稀释库存稀释类似于方案如示于第1列（"源浓度"） 在表2与纯DMSO中无菌玻璃或塑料管。使用一个移液管，用于测量稀释剂和另一个移液管，用于将初始达巴万星股票到第一管。对于后面的每万星股票集中使用新的吸管。 </li><li>准备100X最终MIC面板concent比稀释（中间浓度），用纯DMSO。如示于列表2的 2-4结合的源和DMSO中达到所需的中间体浓度的适当体积（体积要使用将取决于MIC板数目必须作出）。 </li><li>准备0.004％P80:通过添加0.1毫升P80至4.9毫升卫生署2 O.准备2％P80的全新工作原液通过穿过上制备的当天0.22微米过滤器和使用溶液消毒。稀释500 2％P80（例如，0.3毫升的2％P80至149.7毫升CAMHB的）:通过使1制备0.004％P80的稀释剂。 </li><li>进一步稀释在步骤2.2中1中制备的中间体的浓度:100在阳离子调节的Mueller Hinton肉汤（CAMHB），补充有0.004％（体积/体积）聚山梨醇酯-80（P-80）的P-​​80和/或LHB（链球菌）加入在双最终浓度因加入接种物（步骤4.3）将导致一个1:2稀释。 参见表2的列6和7 </st荣&gt;。 </li></ol> 3.准备MIC板<ol><li>免除50微升2.3步准备每万星液进入MIC面板的适当的孔中，包括媒体只在一个井（生长控制好）。多通道移液管，用无菌的提示可以被用于此步骤。 </li><li>马上用板或密封塑料薄膜，地点在塑料袋中，并立即在非除霜冰箱放置在≤-20℃（最好在≤-60℃），直到需要。如果冻结面板的使用，消除密封件和放置在实验室工作台上各个面板15-30分钟（直至以及内容解冻）在进行面板接种前。 </li></ol> 4.接种MIC板，进行纯度和设置菌落总数<ol><li>选择从18-24小时血琼脂或其它非选择性琼脂平板一些众所周知的分离菌落。触摸各菌落的顶部用无菌环或拭子，并转移到1-5毫升CAMHB或s艾琳直到浊度相当于一个0.5麦法兰标。通过视觉比较的0.5麦克法兰或具有光度设备评估浊度。 </li><li>内制备的15-30分钟，稀释接种物1:100在CAMHB（100微升到10ml CAMHB）。对于大多数细菌对抗达巴万星测试，除S.肺炎 ，这种稀释会提供一个最后阱的5×10 5 CFU / ml的浓度（可接受的范围为2-8×10 5 CFU / ml）中。对于S.肺炎 ，基于浊度的比较0.5麦氏细菌浓度通常相当少，因此，稀释的接种1点25（400微升到10ml CAMHB + 10％+ LHB）。 </li><li>内接种物制备后15分钟，转移50微升最终接种物，每孔（有例外的无菌性控制的孔）在第3步中制备的MIC面板使用无菌尖端的多通道移液管可用于本步骤的。 </li><li>执行纯度检查通过转移和使用无菌环到一个非选择性琼脂（例如，胰蛋白酶大豆琼脂，用5％绵羊血）以及从正增长控制扩频1-10微升等分试样。 </li><li>设置菌落计数通过从生长对照孔用单声道吸管和无菌尖端和转让去除10微升至10ml盐水（1:1000稀释）。混合并加入100μl用单声道吸管和无菌尖端转移到一个合适的，非选择性琼脂培养基（例如，胰蛋白酶大豆琼脂，用5％绵羊血）并扩展到整个琼脂表面用无菌环，重复在不同方向的两个倍以保证均匀分布的接种（1:10稀释）的。 </li></ol> 5.孵育MIC板，落计数和纯度板<ol><li>密封每个MIC面板不超过4板或堆叠在一个塑料袋，用塑料胶带或孵化之前紧身塑料盖。或者，将空MIC面板上的T不超过4的MIC板堆的运算，将湿纸巾在一个塑料容器中，放置的MIC板在塑料容器中，并紧密容器安全地与盖子。 </li><li>孵育在环境空气培养箱MIC板在35℃±2℃16-20小时（葡萄球菌和肠球菌）和20-24小时（链球菌）内接种的30分钟。孵育相同 ​​的条件下的菌落计数和纯度板除在一个5％CO 2培养箱温育链球菌。 </li></ol> 6.阅读MIC和落计数板;检查纯度板<ol><li>读将MIC作为完全抑制细菌生长在用肉眼检测出的孔中的最低浓度。 </li><li>指望菌落计数板的殖民地。乘以各菌落稀释因子（1:10,000）（例如，50个菌落是相当于5×10 5 CFU / ml）中。可接受的范围是20-80菌落（2-8×10 5 CFU / ml）和用作Appro公司ximate方针。 </li><li>检查纯度板。如果所有的菌落都类似于在步骤4.1中使用的菌落，然后接种可以认为纯的。如果有存在的任何其它的菌落，然后有潜在的污染物是存在在MIC面板和测试应该重复。 </li></ol> 7.检查质量控制结果。 <ol><li> 请参考表3为CLSI质量控制菌株的预期范围。只报告试验的结果的分离物如果达巴万星MIC结果质量控制生物体（多个）是在预期范围内。 </li><li>如果任何临床S.获得的≥0.25微克/毫升的MIC 金黄色葡萄球菌分离，重复万星MIC检测和/或发送到参考实验室进行验证。 </li></ol>

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桑德拉·麦柯迪是DURATA治疗的雇员;实验室的专家，公司，雇主劳拉Koeth和吉娜费舍尔，由DURATA治疗签约，准备稿件。本文的出版费用由实验室的专家，公司支付

备战万星MIC测试时，下列方法的细节提出了额外的考虑。将MIC面板的设计将取决于待测试的抗微生物剂的数量（即，单独或多个代理达巴万星）和细菌的物种进行测试。达巴万星浓度应包括解释断点预期为至少一个的QC应变所有细菌物种进行测试和稀释的整个范围。抗微生物稀释系列可以组织或者行或列中取决于抗微生物剂和/或隔离/板和接种的方法的数量。达巴万星粉末和培养液的体积应计算，以确保最佳量产生和浪费是最小的。对于本出版物中使用的示例中，卷的基础上编制两次MIC板。阳离子调节的Mueller Hinton肉汤应根据人准备和灭菌ufacturer的指示和媒体的无菌验证。 MIC板可以制成和制备当天使用，或者它们可以通过适当的质量控制生物体和供将来使用冷冻贮存的验证板进行测试。在整个过程中使用无菌技术是重要的。 没有严格的标准验证接种浓度与菌落计数。菌落计数最初可以检查测试和/或周期性地验证在步骤4.2进行稀释步骤每一物种。重要的是，在0.5麦克法兰标准的有效性还检查例行与 E. 大肠杆菌 ATCC 25922使用类似于一个过程到步骤4.5。如果结果不等于1-2×10 8 CFU / ml的，一个新的0.5麦克法兰标准应获得。如果从MIC井菌落计数的可接受范围之外（2-8×10 5 CFU /毫升和MIC结果的临床分离群从第变化Ë预期正态分布（如0.03-0.12微克/毫升为金黄色葡萄球菌 ）和/或MIC结果质量控制分离物（S）的预期范围之外，验证测试是必要的，方法的修改可能需要进行。有人建议，一个验证0.5麦法兰标准来重复菌落计数（多个），如果结果是再次预期结果之外，如在步骤4.2进行的接种物稀释应作相应的调整和MIC重复。需要注意的是即使进行菌落计数因为菌落计数过程使用稀释体积可能不一定检测较低污染微生物的浓度的纯度检查是必要的。 对于MIC试验被认为是有效的，可接受的增长必须发生在生长控制良好。对于大多数的细菌，将针对达巴万星进行测试（即，葡萄球菌，链球菌和肠球菌，生长将被看作是一个按钮（通常&gt; 2毫米）和更少的FRequently，增长可能会出现浑浊的好。不像一些抗菌剂可能会出现拖尾的效果浓度增加，万星端点通常很好的定义。还有为了便于阅读微稀释试验各种观看设备，其可以被用来读取达巴万星含有平板只要有在辨别生长在孔中的能力没有妥协。 在协议中的关键步骤涉及的程序与相关的达巴万星和加成P80的溶解度的部分。重要的是，达巴万星的使用的最大浓度不大于1600微克/毫升，使DMSO正在使肉汤稀释之前用作溶剂和中间体的浓度在DMSO中制备，使DMSO在MIC中的最终浓度面板的孔是不大于1％。 DMSO是吸湿，因此，利用旧的瓶的DMSO是一个的体贴离子关于达巴万星的溶解度。然而，在质量控制株小BMD研究表明这并非是一个重要的变量（见结果部分），它是使用新的或近期开瓶DMSO的好习惯。它也是重要的P80被添加到肉汤中稀释药物制剂的最后步骤，是在0.002％在MIC的最终浓度良好。 包括骨密度过程这里利用以及在达巴万星和P-80的浓度的终浓度2倍和相加接种物用50μl/孔，使得最终体积/孔为100微升制备用50微升/ MIC板。这种方法允许一个MIC面板的格式产生两个S的测试金黄色葡萄球菌和肺炎因为溶解的马血中可加入到接种物。使用100μl/孔并加入到10微升的其它方法制备的MIC板/孔中被描述CLSI和ISO程序。 在万星敏感断点S.金黄色葡萄球菌是≤0.12微克/毫升（FDA药品说明书）。如示于最近的监视，大多数菌株是由达巴万星在0.06微克/毫升15个级别的抑制。随着易感断点接近模态MIC在MIC的方法不同造成的任何变化都会影响敏感率。因为BMD程序达巴万星，包括一些独特的步骤（具体地说，在在MIC中以及制备的库存和中间药物稀释和0.002％P80的使用DMSO，当务之急是，该方法是完全遵循的金黄色葡萄球菌的QC生物体（ATCC 29213）与0.06微克/毫升的很清楚达巴万星MIC模态，一直方法变化的良好指标和应常规用于验证骨密度方法。如果MIC结果与0.03-0.12微克预期范围/毫升，但趋向于任一低或HIG此范围H侧，该方法的进一步的验证是必要的。 加入0.002％P80的被推荐用于所有相关的革兰氏阳性生物体（staphylococcci，肠球菌，和链球菌）。然而，值得注意的是，它已经表明，溶解的马血的作用类似于P-80相对于它的抗结合能力和，加入的P-80，以CAMHB + LHB对达巴万星MIC链球菌结果没有额外的影响。 目前尚没有纸片法进行测试达巴万星和由于骨密度的相关性差和AD的结果，使用AD方法达巴万星的敏感性测试，不推荐12,14。骨密度方法应当用于达巴万星的敏感性的未来的评估而作为金标准的商业药敏试验的发展。

达巴万星的是，于2014年5月由FDA批准用于治疗急性细菌性皮肤和皮肤结构感染引起的革兰氏阳性生物体1的治疗的半合成脂糖肽类。在视频格式详述此方法的主要目标是对临床实验室和研究科学家用于测试的准确和可重复的达巴万星的敏感性的结果和报告提供明确的指导。 是经常在临床实验室进行了对致病菌代理的体外活性评价的抗菌剂MIC商业方法的确定。这里介绍的方法是微量肉汤稀释法所推荐的CLSI和ISO 2,3,4。由于达巴万星的商业敏感性的方法是当前不可用（在本出版物的时间）和纸片和琼脂稀释方法目前不推荐˚F或者万星，参考肉汤稀释方法可用12,14当前选项。如该参考文献的方法不是一个商业和FDA批准的体外诊断方法，临床实验室中使用此过程病人测试必须通过用于实验室开发的测试（LDT）法规要求遵守美国。可以预料，随着达巴万星使用的增加，更多的实验室将有需要测试此化合物。万古霉素测试作为替代测定S.万星敏感性金黄色葡萄球菌已确认5。然而，达巴万星可有效对抗万古霉素中间体S.金黄色葡萄球菌 （VISA），因此，临床医生可以要求万星MIC结果。另外，MIC结果其他细菌物种可以请求。 美国食品药物管理局容易断点万星对S.金黄色葡萄球菌已建立在≤0.12微克/毫升。 FDA指定只有易感性BLE类别由于缺乏对耐药株的数据。其中野生型S.正常MIC分布金黄色葡萄球菌分离株是/毫升之间0.03-0.12微克，0.06微克/毫升的明确模式的MIC。在MIC结果由于方法差A轻微变化，除了在±一个稀释的固有骨密度变化，有可能对敏感率一个显著影响。6类似的脂糖肽类药物，达巴万星药敏试验可以是作为结果有挑战性的溶解度和分子的相对大的尺寸和它的结合特性7,8,9。这里所描述的参考骨密度方法包括所特有的不溶性活性剂和/或脂糖肽类药物两个特定步骤:（1）使用DMSO制备储备溶液和（2）加入0.002％的P-80在最终的MIC面板稀释液。此视频出版物的目的是提供清晰度达巴万星骨密度的方法和到的specificaLLY关注这些关键步骤，以确保精确和可重复的MIC结果。

<table><tbody><tr><td>Dalbavancin</td><td>Metrics, Inc.</td><td>Greenville, NC</td><td>BI-397</td></tr><tr><td>Vancomycin</td><td>Sigma-Aldrich</td><td>St. Louis, MO</td><td>C5793</td></tr><tr><td>Cation Adjusted Mueller Hinton Broth</td><td>Becton Dickinson</td><td>Sparks, MD</td><td>212322</td></tr><tr><td>Lysed horse blood</td><td>Cleveland Scientific</td><td>Bath, OH</td><td></td></tr><tr><td>Dose-it peristaltic pump</td><td>Integra Biosciences</td><td>Hudson, NH</td><td></td></tr><tr><td>Multi-channel pipet (Viaflo II)</td><td>Integra</td><td>Hudson, NH</td><td>4164</td></tr><tr><td>12 channel pipet (Matrix)</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>Single channelpPipets (Ovation 10 &amp;micro;l - 100 &amp;micro;l)</td><td>VistaLab</td><td>Brewster, NY</td><td></td></tr><tr><td>96 well microplate</td><td>Greiner Bio-one</td><td>Monroe, NC</td><td>650161</td></tr><tr><td>50 ml flat cap conical bottom centrifuge tubes</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>16x125 mm disposable glass tubes</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>12x17 mm snap cap, polystyrene tubes</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>16 ml centrifuge tubes</td><td>BioExpress</td><td>Kaysville, UT</td><td>C-3394-2</td></tr><tr><td>Reagent reservoirs</td><td>Integra Biosciences</td><td>Hudson, NH</td><td>4312</td></tr><tr><td>Disposable sterilelLoops (1 &amp;micro;l &amp;amp; 10 &amp;micro;l)</td><td>Biologix</td><td>Lenexa, KA</td><td>65-0001,65-0010</td></tr><tr><td>Tryptic soy agar + 5% sheep blood</td><td>Becton Dickinson</td><td>Sparks, MD</td><td></td></tr><tr><td>Incubator (ambient)</td><td>Sanyo</td><td></td><td></td></tr><tr><td>Incubator (5% CO2)</td><td>Sanyo</td><td></td><td></td></tr><tr><td>Analytical balance&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>Gloves</td><td>Kimberly-Clark</td><td></td><td>52817</td></tr><tr><td>Lab coats</td><td></td><td></td><td></td></tr></tbody></table>

a reference broth microdilution method for dalbavancin in vitro susceptibility testing of bacteria that grow aerobically

药敏试验（AST）被执行以评估对各种细菌的抗微生物剂的体外活性。 AST的结果，这被表示为最低抑菌浓度（MIC）用于研究抗性发展的抗微生物发展和监测，并在临床上用于抗微生物治疗的指导。达巴万星的是，被批准在美国食品和药物管理局（FDA）2014年5月为急性细菌性皮肤和皮肤结构感染引起的革兰氏阳性生物体的治疗的半合成脂糖肽类抗微生物剂。达巴万星过电流抗葡萄球菌疗法的优点是它的半衰期长，其允许每周一次给药。达巴万星具有对金黄色葡萄球菌的活性（包括甲氧西林敏感的金黄色葡萄球菌 [MSSA]和耐甲氧西林金黄色葡萄球菌 [MRSA]），凝固酶阴性葡萄球菌， <em&gt; 肺炎链球菌， 咽颊炎基，β溶血性链球菌和万古霉素敏感肠球菌。类似于其他最近脂糖肽类代理商，CLSI和ISO肉汤易感性试验方法优化包括使用二甲基亚砜（DMSO）作为溶剂的制备储备溶液和聚山梨醇酯80（P80）时，以缓解剂塑料的粘附。在此之前的临床研究和达巴万星的初始开发期间，未进行与使用的P-80的敏感性的研究和MIC结果往往是2-4倍高且类似更高的MIC结果与琼脂稀释易感性的方法获得的。达巴万星首次被列入CLSI肉汤稀释方法表中2005年和2006年修订，以澄清使用DMSO和P-80的。的微量肉汤稀释这里显示（BMD）程序是特定于达巴万星，是按照CLSI和ISO方法，用步骤一步细节和FOC我们的关键步骤增加了清晰度。

标准化MIC方法是必要的，以监测耐药性的发展，各种抗菌监测计划的一个重要目标。达巴万星MIC结果在2011至2013年收集在欧洲和美国分离物&lt;0.25微克/毫升为99.9％ 的金黄色葡萄球菌和链球菌的100％（ 表4）。 为了评估使用新鲜（盖章）DMSO中，万星BMD中等收入国家的QC菌株S.三次测试的重要性黄色葡萄球菌ATCC 29213 和 E. 粪肠球菌 ATCC 29212和重复测试S.肺炎 ATCC 49619，采用（1）的DMSO由密封的安瓿，（2）的DMSO从1升的瓶子是在有效期限内即与先前已使用2年和执行（3）的DMSO来自同一1升的等分试样瓶（样品2），该静置在一个开放的烧杯在使用前72小时。使用具有三个DMSO样品制成的面板达巴万星MIC结果相似和Within可接受的QC范围（ 表5）。 达巴万星活性以下的原液塑料和不加入的P-80的曝光的损失已有案可稽7。在2个额外的研究中，加入P-80的初始和中间储备溶液（表6）和使用玻璃或塑料管的初始和中间储备溶液的制备（表7）没有产生影响达巴万星MIC结果。还有上达巴万星MIC结果没有影响时从三个厂家聚丙烯和聚苯乙烯板用于（表7）。 的溶解的马血中的肉汤培养基链球菌测试的存在已经显示出具有类似表面活性剂的活性，可比到P-80 7,8。对于链球菌的达巴万星参考方法不包括P-80和溶解的马血，然而，不同于葡萄球菌和肠球菌，达巴万星MIC结果链球菌是相似的（在+/- 1稀释）有和没有添加P-80的。 如先前已经提出并在图1中示出，达巴万星琼脂稀释（AD）的MIC通常2-8倍相比，目前的BMD基准12，13，14高。不像骨密度，添加0.002％的P-80不影响AD万星MIC 结果 12。 <img alt="式（1）" src="/files/ftp_upload/53028/53028eq1.jpg" /> 公式1:的 25毫升800微克/毫升万星的解决方案，达巴万星储备液准备准备步骤例 。 <img alt="图1" src="/files/ftp_upload/53028/53028fig1.jpg" /> 达巴万星和万古霉素BMD图1的比较广告。研究比较dalbava结果ncin和万古霉素骨密度和AD MIC结果选择革兰氏阳性细菌分离。平均（A）达巴万星和 （B）万古霉素肉汤稀释和琼脂稀释MIC结果在微克/毫升15 金黄色葡萄球菌 ，8凝固酶阴性葡萄球菌（CNS），5 粪肠球菌和14链球菌（组A，B和C）。 <a href="https://www.jove.com/files/ftp_upload/53028/53028fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td>五</td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr><td>一个</td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL; 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </td><td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td>乙</td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </td><td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td> C </td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL0.12 </td><td> DAL 0.25 </td><td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td> ð </td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </td><td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td> Ë </td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </TD&gt; <td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td> F </td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </td><td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td> G </td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </td><td> DAL 00.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td> H </td><td> DAL 0.002 </td><td> DAL 0.004 </td><td> DAL 0.008 </td><td> DAL 0.015 </td><td>德尔0.03 </td><td> DAL 0.06 </td><td> DAL 0.12 </td><td> DAL 0.25 </td><td> DAL 0.5 </td><td> DAL 1 </td><td> DAL 2 </td><td>波什Ctrl键。 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> DAL </td><td colspan="2">达巴万星</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td&gt; </td><td></td></tr><tr><td></td><td colspan="12">浓度后，50微升/接种也指出在微克/毫升</td></tr></tbody></table> 表1 MIC板格式。达巴万星稀释和阳性对照孔的这个视频出版物中使用的，例如，将96孔板形式示出的位置<table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td>五</td><td> 6 </td><td> 7 </td><td> 8 </td></tr><tr><td>来源浓。 （微克/毫升） </td><td>体积源（毫升） </td><td>体积的DMSO（毫升） </td><td>中级浓。在DMSO（微克/毫升） </td><td>音量中级浓。 （毫升） </td><td>体积CAMHB + 0.004％P80 *（毫升） </td><td> 2X浓。经过1:100稀释于CAMHB（微克/毫升） </td><td>最后一个面板浓。在接种后（微克/毫升） </td></tr><tr><td> 800 </td><td> 1.0 </td><td>1.0 </td><td> 400 </td><td> 0.02 </td><td> 1.98 </td><td> 4 </td><td> 2 </td></tr><tr><td> 400 </td><td> 0.5 </td><td> 0.5 </td><td> 200 </td><td> 0.02 </td><td> 1.98 </td><td> 2 </td><td> 1 </td></tr><tr><td> 400 </td><td> 0.5 </td><td> 1.5 </td><td> 100 </td><td> 0.02 </td><td> 1.98 </td><td> 1 </td><td> 0.5 </td></tr><tr><td> 400 </td><td> 0.5 </td><td> 3.5 </td><td> 50 </td><td> 0.02 </td><td> 1.98 </td><td> 0.5 </td><td> 0.25 </td></tr><tr><td> 50 </td><td> 0.5 </td><td> 0.5 </td><td> 25 </td><td> 0.02 </td><td> 1.98 </td><td> 0.25 </td><td> 0.125 </td></tr><tr><td> 50 </td><td> 0.5 </td><td> 1.5 </td><td> 12.5 </td><td> 0.02 </td><td> 1.98 </td><td> 0.125 </td><td> 0.06 </td></tr><tr><td> 50 </td><td> 0.5 </td><td> 3.5 </td><td> 6.25 </td><td> 0.02 </td><td> 1.98 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td> 6.25 </td><td> 0.5 &lt;/ TD&gt; <td> 0.5 </td><td> 3.13 </td><td> 0.02 </td><td> 1.98 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td> 6.25 </td><td> 0.5 </td><td> 1.5 </td><td> 1.5 </td><td> 0.02 </td><td> 1.98 </td><td> 0.015 </td><td> 0.008 </td></tr><tr><td> 6.25 </td><td> 0.5 </td><td> 3.5 </td><td> 0.8 </td><td> 0.02 </td><td> 1.98 </td><td> 0.008 </td><td> 0.004 </td></tr><tr><td> 0.8 </td><td> 0.5 </td><td> 0.5 </td><td> 0.4 </td><td> 0.02 </td><td> 1.98 </td><td> 0.004 </td><td> 0.002 </td></tr><tr><td colspan="5">该表已经被修改CLSI M100-S25，表8B 3 </td><td></td><td></td><td></td></tr></tbody></table> 可用于肉汤稀释药敏试验表2方案准备不溶于水的抗菌剂稀释液，万星的工作方案和稀释剂的体积为准备最后的MIC的0.002-2微克/毫升达巴万星的解决方案。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>质量控制应变</td><td>预期达巴万星MIC（微克/毫升） </td></tr><tr><td>金黄色葡萄球菌ATCC 29213 </td><td> 0.03-0.12 </td></tr><tr><td>粪肠球菌ATCC 29212 </td><td> 0.03-0.12 </td></tr><tr><td>肺炎链球菌ATCC 49619 </td><td> 0.008-0.03 </td></tr></tbody></table> 表3.预期达巴万星MIC结果的质量控制菌株3，11。CLSI质量控制结果表明菌株一起被临床分离以验证正确的方法进行测试<img alt="表4" src="/files/ftp_upload/53028/53028table4.jpg" /> 表4.来自美国和欧洲的2011至2013年Surveill分布达巴万星MIC结果（数量在每个MIC）ANCE研究15。达巴万星MIC结果从最近的临床分离株的发表的研究报告作为当前达巴万星在体外活性的指标。 <table border="0" cellpadding="0" cellspacing="0"><colgroup></colgroup><tbody><tr><td rowspan="2">隔离＃ </td><td colspan="3">达巴万星MIC（微克/毫升） </td></tr><tr><td>二甲基亚砜1 </td><td>二甲基亚砜2 </td><td> DMSO 3 </td></tr><tr><td rowspan="3"> 金黄色葡萄球菌ATCC 29213 </td><td> 0.12 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 0.12 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td rowspan="3"> 粪肠球菌 ATCC 29212 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td rowspan="2"><em&gt;秒。肺炎链球菌ATCC 49619 </td><td> 0.015 </td><td> 0.015 </td><td> 0.015 </td></tr><tr><td> 0.03 </td><td> 0.015 </td><td> 0.015 </td></tr><tr><td colspan="4">二甲基亚砜1 =未开封的阻燃密封10毫升安瓿</td></tr><tr><td colspan="2">二甲基亚砜2 =常开1L瓶</td><td></td><td></td></tr><tr><td colspan="4"> DMSO 3 =经常打开一瓶1L - 在盖烧杯，坐在板凳上的72小时</td></tr></tbody></table> 表5.达巴万星MIC结果（微克/毫升），使用DMSO股票及中稀释准备从3来源DMSO，万星MIC研究比较，准备了万星股票和中间稀释使用不同DMSO溶剂效应的结果。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2">隔离号码</td><td rowspan="2">复制号码</td><td colspan="2">达巴万星MIC（微克/毫升） </td></tr><tr><td> DALB瓦特/ P80在DMSO </td><td> DALB W / O P80在DMSO </td></tr><tr><td rowspan="4"> 金黄色葡萄球菌ATCC 29213 </td><td> 1 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 2 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 3 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 4 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 金黄色葡萄球菌临床DP80-002SA </td><td> 1 </td><td> 0.03 </td><td> 0.03 </td></tr><tr><td> 金黄色葡萄球菌ATCC 700699 </td><td> 1 </td><td> 0.5 </td><td> 0.5 </td></tr><tr><td rowspan="4"> 肺炎链球菌 ATCC 49619 </td><td> 1 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td> 2 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td> 3 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td> 4 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td>化脓性链球菌临床: </td><td></td><td></td><td></td></tr><tr><td> DP80-004PY </td><td> 1 </td><td> 0.015 </td><td> 0.015 </td></tr><tr><td> DP80-005PY </td><td> 1 </td><td> 0.03 </td><td> 0.03 </td></tr><tr><td> DP80-006PY </td><td> 1 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td> DP80-007PY </td><td> 1 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td> DP80-008PY </td><td> 1 </td><td> 0.03 </td><td> 0.015 </td></tr><tr><td> 无乳链球菌临床: </td><td></td><td></td><td></td></tr><tr><td> DP80-009AG </td><td> 1 </td><td> 0.06 </td><td> 0.12 </td></tr><tr><td> DP80-010AG </td><td> 1 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td> DP80-011AG </td><td> 1 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> DP80-012AG </td><td> 1 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> 停乳链球菌克里尼CAL DP80-013DY </td><td> 1 </td><td> 0.03 </td><td> 0.03 </td></tr><tr><td> 肺炎链球菌临床: </td><td></td><td></td><td></td></tr><tr><td> DP80-014SP </td><td> 1 </td><td> 0.06 </td><td> 0.06 </td></tr><tr><td> DP80-015SP </td><td> 1 </td><td> 0.03 </td><td> 0.03 </td></tr><tr><td> DP80-016SP </td><td> 1 </td><td> 0.03 </td><td> 0.03 </td></tr><tr><td> DP80-017SP </td><td> 1 </td><td> 0.06 </td><td> 0.06 </td></tr></tbody></table> 使用DMSO股票和中间体制备稀释使用和不使用0.002％，表6达巴万星MIC结果P-80，万星MIC的研究，相比增加0.002％的P-80达巴万星股票和中间的稀释作用的结果。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="3">离析</td><td rowspan="3">复制号码</TD&gt; <td colspan="4" rowspan="2">股票稀释管</td><td colspan="4">微孔板（塑料型） </td></tr><tr><td rowspan="2">聚丙烯，格瑞纳</td><td colspan="3">聚苯乙烯</td></tr><tr><td>玻</td><td>塑料</td><td>塑料1小时</td><td>塑料1小时/中级1小时</td><td>格雷纳</td><td>康宁</td><td> NUNC </td></tr><tr><td rowspan="3">金黄色葡萄球菌ATCC 29213 </td><td> 1 </td><td> 0.06 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.06 </td><td> 0.03 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td> 2 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.06 </td></tr><tr><td> 3 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td><td> 0.03 </td></tr><tr><td rowspan="3">粪肠球菌ATCC 29212 </td><tD&gt; 1 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td><td> 0.03 </td><td> 0.03 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td> 2 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td><td> 0.03 </td><td> 0.03 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td> 3 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td><td> 0.06 </td><td> 0.03 </td><td> 0.03 </td><td> 0.06 </td><td> 0.03 </td></tr><tr><td rowspan="2">肺炎链球菌ATCC 49619 </td><td> 1 </td><td> 0.015 </td><td> 0.015 </td><td> 0.015 </td><td> 0.015 </td><td> 0.008 </td><td> 0.008 </td><td> 0.008 </td><td> 0.008 </td></tr><tr><td> 2 </td><td> 0.015 </td><td> 0.015 </td><td> 0.015 </td><td> 0.015 </td><td> 0.008 </td><td> 0.015 </td><td> 0.008 </td><td> 0.008 </td></tr><tr><td colspan="10">在玻璃瓶玻璃=初始库存，立即用于制造中等浓度在塑料试管，立即使用s到做肉汤稀释。 </td></tr><tr><td colspan="10">塑料=初始库存的塑料离心管材质，立即用于制造中等浓度的塑料管，直接用来做肉汤稀释。 </td></tr><tr><td colspan="10">在塑料离心管中制成的塑料1小时=初始库存，让坐在板凳上1小时，然后用来制作中间体的浓度在塑料管中，立即用于制作浓度的肉汤。 </td></tr><tr><td colspan="10">塑料1小时/中级1小时=初始的塑料离心管中取得的股票，让坐在板凳上1小时，然后用来制作中间体的浓度在塑料管，让坐在板凳上1小时，然后用来制作浓度的肉汤。 </td></tr></tbody></table> 使用股票稀释准备表7.达巴万星MIC结果在塑料和玻璃管和MIC板胶种。结果○FA万星MIC研究比较使用塑料或玻璃管准备库存解决方案时，也使用不同的来源和类型的塑料酶标板的作用效果。

Watch this Scientific Journal Video about A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically at JoVE.com

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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Immunology and Infection

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

28.9K 次观看.  Laboratory Specialists, Inc. Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology. 

视频: A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

结核分枝杆菌综合肉汤稀释药敏试验微孔板格式为一线和二线药物

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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免疫与感染

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

木炭琼脂刃天青分析的可视化分枝杆菌的半定量，中等吞吐量枚举

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

喷墨打印机辅助自动跳棋阵列和手动消时法的抗菌协同试验

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

递延生长抑制试验来量化竞争性细菌来源的抗菌剂的影响

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

生物学

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

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

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

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

肉汤稀释体外筛选: 一种简便快速检测新抗真菌化合物的方法

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

通过受激拉曼散射成像对氘掺入单个细菌进行快速抗菌药敏试验

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Source: Zheng, X., et al. <a href="https://app.jove.com/v/55273">System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.

评估测试化合物对肉汤中分枝杆菌的有效性

1. Bacterial Strain and Growth Medium

	Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

Source: Moran, J. C. et al., <a href="https://app.jove.com/v/54437">Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition.</a> J. Vis. Exp.&#160;(2016)
This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.

评估细菌来源的抗菌化合物效果的延迟生长抑制试验

The following method is adapted&#160;to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with ...

A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

Source: Ketelboeter, L. M., et al. <a href="https://app.jove.com/v/53105">Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress.</a> J. Vis. Exp. (2015).
This video demonstrates a microtiter plate-based method for determining the Minimum Inhibitory Concentration (MIC) of test antibiotics against bacteria. Upon adding a suspension of the bacterial pathogen Pseudomonas aeruginosa to a microtiter plate containing serial dilutions of a test antibiotic, and an inhibitor of the production of pyomelanin by the bacteria, the optical density of the wells is measured to compute the minimum inhibitory concentration of the antibiotic, and the impact of the pyomelanin production inhibitor the antibiotic sensitivity of the bacteria.

评估抗生素最低抑制浓度的微量滴定板法

1. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates

	Set up overnight cultures of the strains to be tested in LB (Luria-Bertani) ...

参考微量稀释法达巴万星

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