作者要感谢李章、李贤志、阿龙·欣兹和克莱顿·霍尔对这份手稿的编辑帮助。这种检测最初是在达特茅斯盖塞尔医学院乔治·奥图尔的实验室中开发的。Mah博士实验室的研究得到了加拿大自然科学和工程研究理事会和加拿大囊性纤维化研究理事会的资助。

1. MBC-B
<ol><li>
种植生物膜（改编自奥图尔9）。
	<ol>
<li>在 37 °C 的丰富介质中种植野生型兴趣菌株和突变菌株，持续 16 小时。</li>
		<li>将饱和的隔夜培养物 1:100 稀释到新的耐药性检测介质中。 P. aeruginosa 的标准介质是 M63 最小介质，辅以硫酸镁和精氨酸（见 表 1）。这种介质刺激形成更坚固的生物膜。</li>
		<li>在 96 井微蒂特菜中加入每口井稀释 100μl（见 表 1）。由于这些检测通常以三份执行，因此每个菌株应有 24 口井。</li>
		<li>在 37 °C 下孵化微质板 24 小时。</li>
	</ol>
</li>
	<li>
将预制生物膜暴露在抗生素的浓度梯度下
	<ol>
<li>为 7 口井准备 10 倍稀释系列抗生素。示例:对于抗生素托霉素，井中的最终浓度为 0.4、0.2、0.1、0.05、0.025、0.0125 和 0.006 毫克/毫升 5-8。 从25毫克/毫升的库存中，准备10倍稀释4，2，1，0.5，0.25，0.125和0.06毫克/毫升。留在冰上</li>
		<li>使用多通道移液器取出已用的超自然（包含浮游生物细胞）（见表1）。</li>
		<li>在所有油井中加入 90 微升 M63 （Mg/Arg）。</li>
		<li>每10倍抗生素浓度中加入10微克，以达到所需的最终浓度。为每个复制品和菌株添加 10 μl 水（无抗生素控制）到最终井中。</li>
		<li>在 37 °C 下孵化微质板 24 小时。</li>
	</ol>
</li>
	<li>
刷新媒体，并允许活细胞分离。
	<ol>
<li>使用多通道移除已用的超自然（包含浮游生物细胞）。</li>
		<li>在所有油井中加入 115 微升 M63 （Mg/Arg）。</li>
		<li>在 37 °C 下孵化微质板 24 小时。</li>
	</ol>
</li>
	<li>
活细胞的检测。
	<ol>
<li>标记每个 96 井微蒂特板的两个 LB agar 板。</li>
		<li>通过将爪子浸入 100% 乙醇中并将爪子穿过 Bunsen 燃烧器的明火，对多管设备（见 表 1）进行消毒。重复。让爪子稍微冷却一下。使用多管齐下装置，将浮石培养物的~3 μl（通常保留在爪尖上的量）从微蒂特板的每口井转移到LB agar板的表面。</li>
		<li>在37°C下孵化LB agar板16小时。</li>
		<li>通过眼睛识别细菌生长的切口（图1），确定抗生素的杀菌浓度最低。</li>
	</ol>
</li>
</ol>2. MBC-P
<ol><li>
准备细菌菌株
	<ol>
<li>在 37 °C 的丰富介质中种植野生型兴趣菌株和突变菌株，持续 16 小时。</li>
		<li>将饱和的隔夜培养物 1:100 稀释到新的耐药性检测介质中。P. aeruginosa的标准介质是 M63 最小介质，辅以硫酸镁和精氨酸（见表 1）。</li>
		<li>在 96 井微蒂菜中加入每口井稀释 90μl（见表 1）。由于这些检测通常以三份执行，因此每个菌株应有 24 口井。</li>
	</ol>
</li>
	<li>
将浮石细胞暴露在抗生素的浓度梯度上
	<ol>
<li>为 7 口井准备 10 倍稀释系列抗生素。示例:对于抗生素托霉素，井中的最终浓度为 0.032、0.016、0.008、0.004、0.002、0.001 和 0.0005 毫克/毫升。从25毫克/毫升的库存中，准备稀释0.32、0.16、0.08、0.04、0.02、0.01和0.005毫克/毫升。留在冰上</li>
		<li>每10倍抗生素浓度中加入10微克，以达到所需的最终浓度。为每个复制品和菌株添加 10 μl 水（无抗生素控制）到最终井中。</li>
		<li>在 37 °C 下孵化微质板 24 小时。</li>
	</ol>
</li>
	<li>
活细胞的检测。
	<ol>
<li>标记每个 96 井微蒂特板的两个 LB agar 板。</li>
		<li>通过将爪子浸入 100% 乙醇中并将爪子穿过 Bunsen 燃烧器的明火，对多管设备（见 表 1）进行消毒。重复。让爪子稍微冷却一下。使用多管齐下装置，将浮石培养物的~3 μl（通常保留在爪尖上的量）从微蒂特板的每口井转移到LB agar板的表面。</li>
		<li>在37°C下孵化LB agar板16小时。</li>
		<li>通过眼睛识别细菌生长的切断（图2），确定抗生素的细菌浓度最低。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int. J. Antimicrob. Agents. 35 (4), 322-332 (2010).</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>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., 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=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Zhang, L., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Involvement+of+a+Novel+Efflux+System+in+Biofilm-Specific+Resistance+to+Antibiotics.">The Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics.</a> J. Bacteriol. 190 (13), 4447-4452 (2008).</li><li>Zhang, L., Hinz, A. J., Nadeau, J. P., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+tssC1+Links+Type+VI+Secretion+and+Biofilm-specific+Antibiotic+Resistance.">Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-specific Antibiotic Resistance.</a> J. Bacteriol. 193 (19), 5510-5513 (2011).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biofilm-Specific+Antibiotic+Resistance+Gene,+ndvB,+is+Important+for+Expression+of+Ethanol+Oxidation+Genes+in+Pseudomonas+aeruginosa+Biofilms.">The Biofilm-Specific Antibiotic Resistance Gene, ndvB, is Important for Expression of Ethanol Oxidation Genes in Pseudomonas aeruginosa Biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>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=Microtiter+Dish+Biofilm+Formation+Assay.">Microtiter Dish Biofilm Formation Assay.</a> J. Vis. Exp. (47), e2437 (2011).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+tolerance+of+biofilms+and+persister+cells.">Multidrug tolerance of biofilms and persister cells.</a> Curr. Top. Microbiol. Immunol. 322, 107-131 (2008).</li><li>Merritt, J. H., Kadouri, D. E., 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=Growing+and+Analyzing+Static+Biofilms.">Growing and Analyzing Static Biofilms.</a> Curr. Protoc. Microbiol. 1, 1B.1.1-1B.1.17 (2005).</li><li>Ramey, B. E., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.+Growing+and+analyzing+biofilms+in+fermenters.">Chapter 1. Growing and analyzing biofilms in fermenters.</a> Curr. Protoc. Microbiol. 1, 1B.3.1-1B.3.14 (2005).</li></ol>

提交人宣称她没有相互竞争的经济利益。

浮石细胞中的抗生素耐药性被定义为由于细胞的永久变化（如 突变）而增加抗生素的最低抑制浓度 （MIC）。迄今已查明的生物膜特异性抗性或耐受性机制是生物膜中野生类型基因表达的结果。因此，抗药性的经典定义不适用于生物膜。然而，另一组定义已经提出:耐药性机制阻止抗生素进入其目标，而耐受性机制关闭抗生素的目标10。生物膜特异性抗生素耐药性机制包括这两个定义。在整个协议中使用了"电阻"一词，但必须进行进一步实验，以确定基因是否有助于抵抗或耐受性。
MBC-P 和 MBC-B 检测可能适用于任何生物膜形成细菌菌株和任何杀菌抗菌剂。使这种检测适应其他细菌和其他抗菌剂的两个重要标准是确定导致生物膜形成的条件，并使用野生类型的感兴趣菌株确定抗菌剂的适当浓度范围。适当的生物膜形成条件可以确定使用乔夫生物膜形成协议公布的乔治奥图尔9。为了确定 MBC-B 测定合适的浓度范围，一个很好的经验法则是使用 25 x MIC（最小抑制浓度11）作为范围的中间浓度，并相应地进行调整。对于 MBC-P 检测，使用 MBC-B 测定所标识值的 1/10。目标是定义一系列浓度，使野生型菌株的 MBC-B 与敏感突变菌株之间有明显的差异。因此，该范围的高端应接近野生型菌株的 MBC-B。我们重复这个实验至少三次，每个菌株至少给出9个数据点。
这些检测中最关键的步骤是避免污染。为了防止菌株之间的污染，应在菌株之间留一个空柱。另一个关键步骤是留出足够的时间让多管设备上的金属爪冷却到足够。这可以通过燃烧爪子、定时冷却期和用手快速触摸爪子来测试。
如果接触抗生素后细胞从生物膜中分离是个问题，则可以修改 MBC-B 方法以包括声波步骤。跳过步骤 1.3.3（添加新介质后 24 小时孵化）。相反，在加入115 微量新介质（步骤1.3.2）后，用无菌胶板密封和声波板覆盖96井微晶板的井，将生物膜细胞从井壁中分离出来。使用水浴声波器。必须事先确定从塑料中有效去除生物膜细胞的具体条件（时间、强度）。
MBC-B的变种用于筛选一个库，该库由4，000个插入P.aeruginosa的转子插入突变体进行生物膜特异性抗生素耐药突变体。选择了一种浓度为托布拉霉素（50微克/毫升），不会杀死野生类型的生物膜细胞，但可以识别比野生生物膜敏感3倍的突变体。每口井没有用一种特定的细菌来接种井柱，而是接种了不同的突变体。突变体被留在形成一个生物膜，暴露在50 μ克/毫升的托布拉霉素，离开从布拉霉素暴露恢复，然后检测生存能力。在相同的筛查条件下，对未能存活50 μ克/毫升的抗霉素的突变体进行了重新测试。利用MBC-P和MBC-B协议来确认突变体的对肌霉素敏感表型。
还有其他方法来检测生物膜中的抗生素耐药性， 即 在生物反应器12，13中生长的殖民地生物膜和生物膜。这些方法要费力得多，而且菌株数量有限，很容易被检测。因此，MBC-B在这些其他方法上的优势是，它具有高通量，允许研究人员进行筛选或检测几个菌株，以及几个不同的抗生素浓度一次。
<table border="1"><tbody>
<tr>
<td>试剂的名称</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>评论（可选）</td>
		</tr>
<tr>
<td>1x M63</td>
			<td></td>
			<td></td>
			<td>通过溶解 15 g KH2PO 4、35 g K2HPO4和 10 g （NH4）2SO4在 1 L 水中作为 5x M63 库存做好准备。此库存无需自动切割，可在室温下存储。 稀释 5x 库存 1:5，高压灭菌，冷却，然后添加所需的组件。</td>
		</tr>
<tr>
<td>KH2PO4
</td>
			<td>渔夫</td>
			<td>P285-500</td>
			<td></td>
		</tr>
<tr>
<td>K2HPO4
</td>
			<td>渔夫</td>
			<td>P288-500</td>
			<td></td>
		</tr>
<tr>
<td>（NH4）2SO4
</td>
			<td>西格马</td>
			<td>A5132</td>
			<td></td>
		</tr>
<tr>
<td>硫酸镁</td>
			<td>渔夫</td>
			<td>M63-500</td>
			<td>添加到1mM最终浓度。 准备作为1M股票在水中和高压灭菌器。</td>
		</tr>
<tr>
<td>托布拉米辛</td>
			<td>西格马</td>
			<td></td>
			<td>准备50毫克/毫升库存。阿里报价和存储在-20°C。</td>
		</tr>
<tr>
<td>精氨酸</td>
			<td>西格马</td>
			<td>A5131</td>
			<td>增加 0.4% 的最终浓度。 准备作为20%的库存在水中和过滤器消毒。 这种替代碳/能源可以取代葡萄糖和木糖酸</td>
		</tr>
<tr>
<td>96 井微蒂特板</td>
			<td>康宁</td>
			<td>3595</td>
			<td>无菌，平底，低蒸发</td>
		</tr>
<tr>
<td>特兰弗佩特（多通道移液器）</td>
			<td>品牌科技</td>
			<td>2703610</td>
			<td>8 通道， 20-200 μl</td>
		</tr>
<tr>
<td>多管设备</td>
			<td>丹-卡尔</td>
			<td>MC48</td>
			<td>48 个钳子适合 96 井微蒂板的 1/2</td>
		</tr>
</tbody></table>表1。

抗生素耐药性检测最初是为了检测浮游生物（自由游泳）细菌培养物的耐药性而开发的。由于许多细菌感染涉及生物膜（表面连接细胞），我们有兴趣开发一种检测生物膜特异性抗生素耐药性的方法。然而，大多数抗生素耐药性检测不适合测量生物膜的耐药性。例如，确定最低抑制浓度（MIC）是确定浮石细菌培养物 1抗生素耐药性的黄金标准。这种检测需要将稀释的浮石培养物与稀释系列抗生素混合。 抑制浮石细胞可见生长的抗生素浓度是MIC。由于这种检测依赖于对生长的抑制，根据定义，它不能与生物膜培养物配合使用，这需要检查预生生物膜中细胞的抗生素敏感性。此处描述的 MBC-B 检测结果不是测量生长抑制，而是确定杀死生物膜中已有细胞的抗生素浓度。因此，这种检测旨在模仿已建立的生物膜感染的抗生素治疗，并提供一个更相关的视野 中的细菌抗生素耐药性。
由于生物膜通常比浮石培养物2-4更具抗生素耐药性，因此有必要设计出一种将生物膜的抗生素耐药性与浮石培养物直接相关的方法。因此，这种方法的另一个目标是能够直接比较浮石细胞和生物膜细胞之间的抗生素耐药性水平。此处描述的 MBC-P 和 MBC-B 分析使这一点成为可能，因为细胞是在类似条件下培养的。我们利用这种方法研究了几个对生物膜特异性抗生素耐药性很重要的基因。

<table><tbody><tr><td> &lt;p&gt;1x&amp;nbsp;M63&lt;/p&gt; </td><td></td><td>&lt;p align=&quot;center&quot;&gt;&lt;/p&gt;</td><td> &lt;p&gt;Prepare as a 5x&amp;nbsp;M63 stock by dissolving 15g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 35g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 10g (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&amp;nbsp; Dilute 5x&amp;nbsp;stock 1:5, autoclave, cool, then add the desired components.&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt; </td><td>Fisher</td><td>&lt;p align=&quot;center&quot;&gt;P285-500&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt; </td><td>Fisher</td><td>&lt;p align=&quot;center&quot;&gt;P288-500&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt; </td><td>Sigma</td><td>&lt;p align=&quot;center&quot;&gt;A5132&lt;/p&gt;</td><td> &lt;p&gt;&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Magnesium sulfate&lt;/p&gt; </td><td>Fisher</td><td>&lt;p align=&quot;center&quot;&gt;M63-500&lt;/p&gt;</td><td> &lt;p&gt;Add to 1 mM final concentration.&amp;nbsp; Prepare as a 1 M stock in water and autoclave.&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Tobramycin&lt;/p&gt; </td><td>Sigma</td><td>&lt;p align=&quot;center&quot;&gt;&lt;/p&gt;</td><td> &lt;p&gt;Prepare 50 mg/m&amp;nbsp;stock. Aliquot and store at &lt;span style=&quot;font-size: 11.199999809265137px;&quot;&gt;-&lt;/span&gt;20&amp;deg;C.&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Arginine&lt;/p&gt; </td><td>Sigma</td><td>&lt;p align=&quot;center&quot;&gt;A5131&lt;/p&gt;</td><td> &lt;p&gt;Add to 0.4% final concentration.&amp;nbsp; Prepare as a 20% stock in water and filter sterilize.&amp;nbsp; This alternative carbon/energy source can replace glucose and casamino acids&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;96-well microtiter plates&lt;/p&gt; </td><td>Corning</td><td>&lt;p align=&quot;center&quot;&gt;3595&lt;/p&gt;</td><td> &lt;p&gt;Sterile, flat-bottom, low evaporation&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Tranferpette (multichannel pipette)&lt;/p&gt; </td><td>BrandTech</td><td>&lt;p align=&quot;center&quot;&gt;2703610&lt;/p&gt;</td><td> &lt;p&gt;8-channel, 20-200 &amp;mu;l&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Multiprong device&lt;/p&gt; </td><td>Dan-Kar</td><td>&lt;p align=&quot;center&quot;&gt;MC48&lt;/p&gt;</td><td> &lt;p&gt;48 prongs fit into &amp;frac12; of a 96-well microtiter plate&lt;/p&gt; </td></tr></tbody></table>

establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)

该协议允许浮石和生物膜耐药性之间的直接比较的细菌菌株，可以形成一个生物膜 体外。细菌被接种到96井微质板的井中。在浮游生物检测中，选择的抗菌剂的连续稀释被添加到细菌悬浮剂中。在生物膜检测中，一旦接种，细菌就会在一定的时间内形成生物膜。未连接的细胞从井中去除，媒体补充，并添加选择的抗菌剂的连续稀释。接触抗菌剂后，浮石细胞被检测为生长。对于生物膜检测，媒体刷新了缺乏抗菌剂的新鲜介质，生物膜细胞有留给恢复。生物膜细胞的生存能力在恢复期后被检测。抗菌剂的MBC-P被定义为杀死浮石培养细胞的药物浓度最低。 相比之下，菌株的 MBC-B 通过将预制生物膜暴露在 24 小时内抗菌剂浓度增加的程度来确定。MBC-B 被定义为杀死生物膜中细胞的抗菌剂的最低浓度。

进行了 MBC-P 和 MBC-B 检测，比较了 PA14 野生类型的灵敏度与 PA14 ∆ndvB。托布拉霉素被用作抗生素。结果与步骤 1.4.4（图 1）和步骤 2.3.4（图 2）相对应。PA14 和∆ndvB 接种到 MBC-P 和 MBC-B 三方检测中。在完成 MBC-B 协议的第 1.0-1.4 步和 MBC-P 协议的步骤 2.0-2.3 后，可行单元被镀在 LB agar 板上。细胞左侧显示托布拉霉素（μ克/毫升）的浓度。PA14 的 MBC-B 为 100 μg/ml，∆ndvB 的 MBC-B 为 12.5 μg/ml。PA14 和∆ndvB 突变体的 MBC-P 为 8 μg/ml。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50854/50854fig1highres.jpg" src="/files/ftp_upload/50854/50854fig1.jpg"> 图1。具有代表性的结果来自 Mbc - b 检测与 Pa14 野生类型 vs. Pa14 ∆ndvb和托布拉霉素。值是指托布拉霉素（μg/ml）的最终浓度。
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50854/50854fig2highres.jpg" src="/files/ftp_upload/50854/50854fig2.jpg"> 图2。具有代表性的结果来自 Mbc - p 检测与 Pa14 野生类型 vs. Pa14 ∆ndvb和托布拉霉素。值是指托布拉霉素的最终浓度（μ克/毫升）。

Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

该协议允许使用 96 井微蒂板直接比较浮石和生物膜耐药性，以识别细菌菌株 ，从而在体外 形成生物膜。浮游生物或生物膜细菌暴露在首选抗菌剂的连续稀释中。活力被阿加板块的生长所测定。

建立浮星细胞（MBC-P）和生物膜细胞（MBC-B）抗菌剂的最小杀菌浓度

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

establishing-minimal-bactericidal-concentration-an-antimicrobial

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

19.9K 次观看.  University of Ottawa. 该协议允许使用 96 井微蒂板直接比较浮石和生物膜耐药性，以识别细菌菌株 ，从而在体外 形成生物膜。浮游生物或生物膜细菌暴露在首选抗菌剂的连续稀释中。活力被阿加板块的生长所测定。

视频: Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

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 ...

科研

免疫与感染

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

病原生物膜利用工程定额淬Lactonases防有毒中断

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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 ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

使用等温微计算法确定新天然产物的杀菌或杀菌作用的代谢分析

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

生物工程

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

体外评估纳米颗粒和纳米结构表面的抗菌活性

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

使用PCR板深孔微孔设备生成更大的细菌生物膜生物质

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

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

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

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

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

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

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

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

建立浮星细胞（MBC-P）和生物膜细胞（MBC-B）抗菌剂的最小杀菌浓度

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使用人工痰介质测试抗生素药效试验

病原生物膜利用工程定额淬Lactonases防有毒中断

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