Name: measuring bacterial resistance with e-tests
Uploaded: 2023-04-30T07:10:10.000Z
Duration: 13 min 39 s
Channel: JoVE

K12 Standards

Louisiana Student Standards for Science

High School

Life Sciences

from-molecules-to-organisms-structures-and-processes

From Molecules To Organisms: Structures And Processes

HS-LS1-8

antibiotic-susceptibility-testing-epsilometer-tests-to-determine-mic

Antibiotic Susceptibility Testing: Epsilometer Tests to Determine MIC Values of Two Antibiotics and Evaluate Antibiotic Synergy

1. 最小抑菌浓度测试（E-测试）
<ol>
	<li>实验准备
	<ol>
		<li>佩戴手套和实验服</li>
		<li>使用70%乙醇对工作区域进行消毒，准备好实验台</li>
		<li>准备 Mueller-Hinton 琼脂平板（MHA 平板）</li>
	</ol>
	</li>
	<li>制备 0.5 号麦氏比浊标准液
	<ol>
		<li>制备1%氯化钡（BaCl2）溶液： 
		将1克无水氯化钡（BaCl2）加入100 mL蒸馏水中，充分涡旋混匀。</li>
		<li>制备1%硫酸（H2SO4）溶液： 
		将1 mL浓硫酸（H2SO4）加入99 mL蒸馏水中，充分涡旋混匀。</li>
		<li>制备0.5号麦氏比浊标准液： 
		取50 µL BaCl2溶液加入5 mL 1% H2SO4溶液中，充分涡旋，形成浑浊悬液。</li>
		<li>将0.5号麦氏比浊标准液置于铝箔包裹的试管中，于25°C保存，最长不超过6个月。使用前充分涡旋至均匀。</li>
	</ol>
	</li>
	<li>制备 MHA 平板
	<ol>
		<li>用无菌接种环从血琼脂平板上刮取Streptococcus G群细菌，混入1 mL生理盐水中，涡旋混匀制成菌悬液。</li>
		<li>将菌悬液与0.5号麦氏比浊标准液比较，调整浊度一致，以确保实验中接种量相同。根据需要添加生理盐水或细菌调节浓度。</li>
		<li>使用无菌棉签蘸取菌液，在MHA平板表面均匀涂布。随后采用以下三种方法之一进行实验（1.4–1.6）。</li>
	</ol>
	</li>
	<li>单一抗生素耐药性测试。Streptococcus G群细菌对青霉素G或庆大霉素的耐药性
	<ol>
		<li>将E-test试纸条（青霉素G或庆大霉素）置于MHA平板中央（图1 A,B）。</li>
		<li>在37°C下孵育18–20小时。</li>
		<li>读取结果。最小抑菌浓度（MIC）为抑菌圈与梯度抗生素试纸条交界处的数值（图1 C,D）。</li>
	</ol>
	</li>
</ol>
<img alt="抗生素敏感性，琼脂平板上的E-test方法，微生物生长抑制分析。" class="xfigimg" src="/files/ftp_upload/10512/10512fig1.jpg" data-altupdatedat="1761830439000" data-alt="图1"> 
图1：单个E-test。 将 A) 青霉素G 和 B) 庆大霉素的E-test试纸条放置于涂布有G群链球菌菌落的Mueller Hinton琼脂平板上，在37°C、5% CO2条件下过夜培养前（A 和 B）及培养后（C 和 D）的结果。<a href="https://www.jove.com/files/ftp_upload/10512/10512fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>
<ol start="5">
	<li>协同作用测试交叉法。 Streptococcus G群，对青霉素G和庆大霉素耐药。
	<ol>
		<li>将含有不同抗生素的两根E-test试纸条（例如青霉素G和庆大霉素）以交叉形式放置在接种好的MHA平板上。
		<ol>
			<li>为获得最准确的结果，应尽量使两根试纸条在交叉处呈约90°角，交叉点位于各自最小抑菌浓度（MIC）值刻度线的交点处，该MIC值此前已通过单种抗生素耐药性试验确定（图2 A）。</li>
			<li>注意，一旦试纸条放置于琼脂平板上，便不应再移动，因为部分抗生素可能已被培养基吸收。因此，若角度略有偏差（如85°），或交叉点偏离实际MIC值1-2 mm，也应保持试纸条不动。建议将实验重复三次，以减少此类误差。</li>
		</ol>
		</li>
		<li>在37°C下孵育18-20小时。</li>
		<li>读取结果。最小抑菌浓度（MIC）的测定依据为抑菌圈与每根E-test试纸条上梯度抗生素浓度刻度相交的位置（图2 B）。</li>
		<li>使用部分抑菌浓度（FIC）计算公式（公式1）来判断是否存在协同作用。</li>
	</ol>
	</li>
</ol>
<img alt="使用E-test条带在琼脂平板上进行抗生素敏感性试验，显示生长抑制区。" class="xfigimg" src="/files/ftp_upload/10512/10512fig2.jpg" data-altupdatedat="1761830439000" data-alt="图2"> 
图2：协同作用检测——交叉试验。 青霉素G与庆大霉素对G群链球菌的最低抑菌浓度（MIC）协同抗菌试验结果，分别为37°C、5% CO2条件下培养过夜前（A）和培养过夜后（B）。两种药物各自的MIC值之间形成90°夹角（青霉素G：0.094 µg/mL，庆大霉素：8 µg/mL）。<a href="https://www.jove.com/files/ftp_upload/10512/10512fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>
<ol start="6">
	<li>协同作用测试非交叉法。 Streptococcus G群，对青霉素G和庆大霉素耐药。
	<ol>
		<li>将E-test试纸条置于MHA平板中央（图3 A,D）。</li>
		<li>标记出此前已确定的每条试纸条上的MIC值位置。</li>
		<li>在室温下孵育1小时。</li>
		<li>移除每块MHA平板上的E-test试纸条（图3 B,E）。</li>
		<li>将第二条E-test试纸条（含有另一种抗生素）分别放置在先前移除试纸条的位置，使两条试纸条的MIC值对齐所做标记并保持平行。</li>
		<li>在37°C下孵育18–20小时。</li>
		<li>读取结果。MIC值测定为每个E-test试纸条上抑菌浓度梯度试纸与抑菌圈相交处的数值（图3 C,F）。</li>
		<li>为确定协同作用，使用部分抑菌浓度（FIC）计算公式（公式1）。</li>
	</ol>
	</li>
</ol>
<img alt="在含抗生素纸片的琼脂平板上进行细菌生长实验；分析药物敏感性与耐药性。" class="xfigimg" src="/files/ftp_upload/10512/10512fig3.jpg" data-altupdatedat="1761830439000" data-alt="Figure 3"> 
图3：协同作用检测——非交叉试验。 青霉素G与庆大霉素对G群链球菌的最低抑菌浓度（MIC）协同抗菌试验结果。A) 庆大霉素纸片（中心浓度8 µg/mL）置于G群链球菌表面，B) 移除庆大霉素纸片，C) 联合庆大霉素/青霉素G纸片（中心浓度0.094 µg/mL）置于G群链球菌表面，D) 青霉素G纸片（中心浓度0.094 µg/mL），E) 移除青霉素G纸片，F) 联合青霉素G/庆大霉素纸片（中心浓度8 µg/mL）置于G群链球菌表面。<a href="https://www.jove.com/files/ftp_upload/10512/10512fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>
2. 肉汤试验
<ol>
	<li>设置
	<ol>
		<li>佩戴手套和实验服</li>
		<li>使用70%乙醇对工作区域进行消毒，准备实验台面</li>
		<li>收集含50%裂解马血和20 mg/mL β-NAD的15 mL MH肉汤（MH-F）</li>
	</ol>
	</li>
	<li>（可选）进行E-test [方案1]，以测定固体培养基上的最低抑菌浓度（MIC）
	<ol>
		<li>尽管此步骤为可选，但获得该信息有助于优化实验设计（例如，可根据平板测定的MIC值设定抗生素添加浓度，使其围绕该值），从而提高实验成功的可能性。</li>
	</ol>
	</li>
	<li>制备细菌接种物。 如上所述，细菌浓度可通过OD nm测定或McFarland比浊标准进行估算
	<ol>
		<li>OD600 nm法
		<ol>
			<li>获取已知细菌浓度的细菌悬液</li>
			<li>将菌液用MH-F肉汤稀释，使OD600达到0.003</li>
		</ol>
		</li>
		<li>McFarland比浊法
		<ol>
			<li>在无菌试管中加入15 mL MH-F肉汤</li>
			<li>从平板取细菌接种至MH-F肉汤中，达到McFarland标准浊度。剧烈涡旋混匀后，将溶液倒入无菌培养皿中</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>准备抗生素
	<ol>
		<li>确定所需抗生素浓度
		<ol>
			<li>从E-test中获取MIC值（例如，青霉素G为0.125 µg/mL，庆大霉素为8 µg/mL）</li>
			<li>将琼脂平板MIC值乘以24–27，对应四到七次2倍倍比稀释，所得结果即为抗生素的起始浓度（例如，青霉素G进行七次2倍稀释：0.125 µg/mL × 27 = 16 µg/mL；庆大霉素进行四次2倍稀释：8 µg/mL × 24 = 128 µg/mL）</li>
			<li>将所需起始浓度扩大100倍，以确定抗生素的储备液浓度（例如，青霉素G储备液为1.6 mg/mL，庆大霉素储备液为12.8 mg/mL）</li>
		</ol>
		</li>
		<li>据此配制100倍浓度的抗生素储备液
		<ol>
			<li>将抗生素溶解于10 mL经高压灭菌的水中，并涡旋混匀以制备储备溶液（例如，使用16 mg青霉素G和128 mg庆大霉素制备上述储备液）</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>将细菌加入微孔板孔中
	<ol>
		<li>将含有细菌接种物的200 µL MH-F肉汤分装至96孔微量滴定板前3行的各孔中，每组实验设三个重复。</li>
	</ol>
	</li>
	<li>将抗生素加入微孔板孔中
	<ol>
		<li>向第一列孔（A1、B1、C1）中额外加入200 µL含细菌的MH-F肉汤，使总体积达到400 µL。</li>
		<li>向第一列孔中加入4 µL抗生素储备液。由于样品总体积为400 µL，抗生素将被稀释100倍。</li>
		<li>通过将200 µL菌液/抗生素混合液从A1转移至A2，依次至A11，进行2倍倍比稀释。每次转移后需充分吹打混匀。对其他行重复此操作。</li>
		<li>从第11列孔中移除200 µL液体，使所有孔的最终体积均为200 µL。</li>
		<li>最后一列（A12、B12、C12）不加抗生素，作为空白对照。</li>
	</ol>
	</li>
	<li>确定MIC值
	<ol>
		<li>将96孔微量滴定板在37°C下静置培养24小时。</li>
		<li>MIC值定义为稀释系列中最后一个无可见细菌生长的孔（图4）。然而，该数值的可靠性依赖于初始接种量的准确性。</li>
	</ol>
	</li>
</ol>
<img alt="采用96孔板的微量稀释法；显示青霉素G和庆大霉素抗生素最低抑菌浓度（MIC）的图表。" class="xfigimg" src="/files/ftp_upload/10512/10512fig4.jpg" data-altupdatedat="1761830439000" data-alt="图4"> 
图4：通过肉汤稀释法测定最低抑菌浓度（MIC）。 此处MIC定义为在由澄清变为浑浊前最后一个保持澄清的孔（即无细菌生长）。每行分别为青霉素G的MIC值重复孔和庆大霉素的MIC值重复孔，两者均针对一株G群链球菌（Streptococcus group G）分离株。 A) 实际实验结果，B) 对A中结果的示意图解（灰色 = 无生长；白色 = 有生长）。 <a href="https://www.jove.com/files/ftp_upload/10512/10512fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>
<img alt="显示微量滴定板设置、PBS对照和连续稀释的细菌稀释与涂布示意图" class="xfigimg" src="/files/ftp_upload/10512/10512fig5.jpg" data-altupdatedat="1761830439000" data-alt="图5"> 
图5：用于测定原始细菌浓度的系列稀释示意图。 按照所述方法进行稀释（20 µL样品加入180 µL中，形成10倍系列稀释），然后从A-H各列取10 µL分别涂布于两个独立的血琼脂平板上，如图所示。 <a href="https://www.jove.com/files/ftp_upload/10512/10512fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>
<ol start="8">
	<li>确定初始接种量大小 
	注意：微量肉汤稀释法对抗生素敏感性试验中所用的初始接种量大小极为敏感。若接种量过大，会导致假阳性结果，因为在此比例下添加的抗生素将无法再抑制细菌生长。因此，验证加入微孔板中的细菌数量至关重要。尽管该数据在实验进行时无法获得（由于需要24小时培养），但其可作为对照使用。若加入的细菌数量处于规定浓度范围内，则最小抑菌浓度（MIC）值可信。若接种量过高或过低，则需重复实验。
	<ol>
		<li>对细菌进行系列稀释
		<ol>
			<li>准备一块96孔微孔板，用于稀释原始细菌浓度以确定接种量大小。理想终体积为200 µL，含细菌105-6个。进行稀释时，首先在B-H列的每孔中加入180 µL无菌PBS（每组设三个重复孔，即1-3行）。</li>
			<li>接着，在A列的每孔（三个重复孔，即1-3行）中加入100 µL细菌溶液。</li>
			<li>通过将20 µL细菌液从A列转移至B列并剧烈吹打，进行10倍系列稀释（设三个重复）。依次重复此步骤至H列。</li>
		</ol>
		</li>
		<li>接种稀释后的细菌以测定接种量大小
		<ol>
			<li>根据图5标记血琼脂平板。</li>
			<li>根据图5所示，将10 µL系列稀释液点种至平板上。</li>
			<li>将平板在37°C下培养20–24小时。</li>
		</ol>
		</li>
		<li>确定细菌接种量大小
		<ol>
			<li>计数每个点上菌落数在5–50之间的菌落数量（图6）。</li>
			<li>通过计算三个重复样本的平均值，乘以稀释倍数（例如B组样本为10倍，C组为100倍，D组为1000倍，依此类推），再乘以100（以校正10 µL点种体积），得到接种量（单位：cfu/mL）。若接种量在105-6 cfu/mL范围内，则MIC数据可信。</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="细菌生长实验；琼脂平板，菌落计数分析，A-D区，测试的生长抑制剂。" class="xfigimg" src="/files/ftp_upload/10512/10512fig6.jpg" data-altupdatedat="1761830439000" data-alt="图6"> 
图6：接种量的确定。 按照图5接种的细菌在37°C下培养20-24小时，然后进行计数。D行的菌落数量适中（例如5-50个），适合计数。A组样品未稀释，B组稀释10倍，C组稀释100倍，D组稀释1000倍，每点接种量为10 µL。<a href="https://www.jove.com/files/ftp_upload/10512/10512fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

抗生素耐药性是一个全球性的健康问题。为了确定微生物的耐药机制，检测不同抗生素之间协同作用与拮抗作用的方法至关重要。E-试验法快速、易于重复，可用于研究联合治疗的任何潜在协同效应。也可采用肉汤稀释法来预测杀菌活性。为了深入研究不同微生物的耐药机制，了解抗生素之间的协同与拮抗相互作用至关重要。联合使用抗生素可能是提高治疗效果、应对抗生素耐药性的策略之一。在本实验中，我们测定了青霉素G和庆大霉素对G群链球菌的最低抑菌浓度（MIC）值。同时证实，这两种抗生素未表现出协同作用，因此不推荐作为此类感染的首选治疗方案。

抗生素敏感性试验：采用epsilometer试验测定两种抗生素的最小抑菌浓度（mic）值并评估抗生素协同作用

来源：Anna Bläckberg1, Rolf Lood1 
1 瑞典隆德大学隆德临床科学系感染医学部，生物医学中心，隆德 221 00，瑞典
了解抗生素与细菌之间的相互作用对于理解微生物如何进化出抗生素耐药性至关重要。1928年，亚历山大·Fleming（Alexander Fleming）发现了青霉素，这种抗生素通过干扰细菌细胞壁的再生来发挥其抗菌作用（1）。随后，人们又发现了其他具有不同作用机制的抗生素，包括抑制细菌DNA复制和蛋白质翻译的药物；然而，近年来尚未开发出新的抗生素。当前抗生素的耐药性日益增加，导致一些严重的感染性疾病无法得到有效治疗（2）。本文介绍几种评估细菌群体耐药性的方法。这些方法均有效，且不受所用抗生素作用机制的影响，因为其检测的最终指标是细菌的死亡。抗生素耐药性不仅在医院环境中迅速传播，也在整个社会广泛扩散。为了研究此类耐药现象，已发展出多种检测方法，包括Epsilometer试验（E试验）和肉汤稀释法（3）。
E试验是一种成熟的方法，也是一种经济有效的工具，用于量化最小抑菌浓度（MIC）数据，即能够抑制微生物可见生长的抗菌剂的最低浓度。根据所用的细菌菌株和抗生素种类，MIC值可能在亚微克/毫升至&gt;1000 微克/毫升之间变化（4）。E试验使用一种含有预设抗生素梯度的塑料试纸条进行，该试纸条上印有以微克/毫升为单位的MIC读数刻度。当将其应用于接种后的琼脂平板时，试纸条可直接转移至琼脂基质上。经过培养后，由于细菌生长受到抑制，沿试纸条周围可观察到对称的椭圆形抑菌区。MIC由抑菌区域确定，即椭圆与试纸条交界处的刻度值。另一种常用的测定MIC的方法是微量肉汤稀释法。微量肉汤稀释法将不同浓度的抗菌剂加入含接种细菌的液体培养基中。培养后，MIC定义为能够阻止细菌可见生长的最低抗生素浓度（5）。该方法也是一种定量方法，适用于多种细菌。此方法的缺点包括试剂浓度配制过程中可能出现误差，以及实验所需试剂数量较多。从临床和科研角度来看，测定抗生素耐药性至关重要，下文将讨论并展示这些用于研究耐药性的in vitro方法。
针对特定细菌的耐药性谱可用于优化抗生素治疗方案，以确定患者是否能从联合治疗中获益，而非单一药物治疗。同时使用一种以上抗生素时，必须了解它们之间的相互作用，以及它们是否具有相加、协同或拮抗效应。当抗生素联合作用的效果等于各抗生素在相同剂量下单独使用时的效力之和时，即表现为相加效应。而当抗生素的联合作用强于各药物单独使用时的效果，则表明存在协同作用（6）。采用抗菌药物联合治疗旨在避免抗菌药物耐药性的发生，从而增强单个抗生素的治疗效果（7）。了解拮抗作用同样重要，以防止不必要的抗菌药物联合使用。E-test 方法提供了简便且多种途径来确定不同抗菌药物之间可能存在的协同或拮抗作用。为应对耐药病原体的不断蔓延，了解特定抗生素之间可能存在的协同与拮抗机制至关重要，这有助于提高临床疗效并对抗多重耐药性。
使用E-test测定协同作用可分为两大类方法：交叉法和非交叉法。尽管这两种协同试验均依赖于已知的单个抗生素最低抑菌浓度（MIC）值，但它们在方法学和概念上略有不同。在非交叉协同试验中，首先将待测抗生素组合中的第一种抗生素置于接种了细菌的琼脂平板上。在允许第一条试纸中的抗生素扩散入平板一段时间后（例如1小时），移除该试纸，并将含有第二种抗生素的新试纸放置在与第一条完全相同的位置，确保两个试纸各自的MIC值对齐重叠。随后可按照前述方法分析所产生的抑菌圈，并根据公式1计算协同作用。
公式 1 - 抑制分数浓度（FIC）
<img src="/files/ftp_upload/10512/10512eq1.jpg" alt="抗菌协同分析的FIC指数方程，药物组合效果的计算公式。" data-altupdatedat="1761830439000">
数值 &gt;0.5 表明具有协同作用。
尽管该方法能为检测者提供易于分析的平板，但由于需要更换试纸条，且每次实验需使用两块平板，因此过程较为繁琐且耗时。取而代之的是，常采用交叉试验法。该方法不再将两种不同的E-test试纸条依次叠加放置（移除第一条后再放置第二条），而是将两条试纸条同时以交叉形式（<img align="center" src="/files/ftp_upload/10512/10512eq3.jpg" alt="静力平衡；ΣFx=0，ΣFy=0示意图；力矢量，平衡分析。" data-altupdatedat="1761830439000"> 90°角）放置，使两条试纸条各自的已知最低抑菌浓度（MIC）值构成直角。通过此方法，每次协同作用检测仅需一块平板，操作更简便，因此尽管分析难度略高，仍为首选方法。联合使用抗生素时的新MIC值可通过观察修饰后的抑菌圈来判断，随后根据公式1确定是否存在协同作用。
与使用琼脂平板法相比，微量肉汤法通常更具优势，因其灵活性更高（例如，可以选择超出E-test试纸条浓度范围的特定抗生素浓度）。此外，由于抗生素在液体溶液中分布均匀，不依赖于在固相（琼脂平板）中的解离过程，微量肉汤法被认为更为敏感。在96孔微孔板的孔中接种定量的细菌（106 cfu/mL：细菌浓度可通过OD600 nm测定、浊度标准或对10倍系列稀释的菌液进行涂布平板法进行估计），并在各孔中加入不同稀释度的抗生素。与E-test试纸条法类似，最低抑菌浓度（MIC）被确定为能够抑制细菌可见生长的最低抗生素浓度所对应的孔/位点。
实验目的
<ul>
	<li>以下项目描述了测定青霉素G和庆大霉素MIC值的策略 链球菌 采用两种不同方法对G组进行分组，即E-test和微量肉汤稀释法。对于E-test，使用接种了待测菌的穆勒-希尔顿琼脂平板 链球菌属 G组采用青霉素G和/或庆大霉素梯度条联合使用；而含50%裂解马血的MH肉汤及20 mg/mL β-NAD则与可溶性抗生素联合使用 链球菌 采用微量肉汤法的G组。</li>
</ul>
材料
<ul>
	<li>保存于4°C、&lt;7天内的血琼脂平板上的细菌菌落</li>
	<li>血琼脂平板</li>
	<li>0.5麦氏比浊标准
	<ul>
		<li>1% BaCl2</li>
		<li>1% H2SO4</li>
	</ul>
	</li>
	<li>生理盐水试管（2 mL）</li>
	<li>棉签</li>
	<li>穆勒-欣顿琼脂平板（MHA平板）</li>
	<li>含50%溶血马血和20 mg/mL β-NAD的MH肉汤（MH-F）</li>
	<li>E-test青霉素/庆大霉素（或其他关注的抗生素）（BioMerieux，法国马西莱图瓦勒，瑞典）</li>
	<li>抗生素青霉素/庆大霉素（或其他关注的抗生素，粉末/溶液）</li>
</ul>
注意：不同细菌种类所使用的特定培养基可能有所不同。

E-test 中的 MIC 值 
在图 1中确定的个体 MIC 值分别为：青霉素 G 为 0.094 μg/mL，庆大霉素为 8 μg/mL。在协同试验中，青霉素 G 在交叉与非交叉试验中均显示 MIC 值为 0.064 μg/mL（图 2、3），而庆大霉素在交叉与非交叉试验中的 MIC 值均为 4 μg/mL。需注意，由于两种试验条件下试纸条孵育时间不同，交叉与非交叉试验之间可能出现轻微差异。
协同作用的计算 
FIC 的计算公式为：
<img align="center" src="/files/ftp_upload/10512/10512eq2.jpg" alt="分数加法方程；涉及 0.064/0.094 + ...

抗生素敏感性试验：采用Epsilometer试验测定两种抗生素的最小抑菌浓度（MIC）值并评估抗生素协同作用

来源：Anna Bläckberg1，Rolf Lood1  
1 隆德大学临床科学系，感染医学科，生物医学中心，隆德大学，221 ...

抗生素敏感性是指细菌对抗生素的敏感程度，可通过肉汤稀释法或Epsilometer试验（也称为E试验）进行测定。在肉汤稀释法中，将标准化数量的细菌接种到含有系列抗生素稀释液的培养基中。若细菌对该抗生素敏感，则在较高抗生素浓度下无法生长，但在较低浓度下仍可继续增殖，导致培养基变浑浊。细菌无法存活或增殖的最低抗生素浓度被称为该抗生素对特定细菌的最低抑菌浓度，即 MIC 值。在E试验中，将一条含有预设抗生素浓度梯度的塑料条置于刚涂布有细菌的Mueller-Hinton琼脂（MH-A）培养皿表面。抗生素从试条中扩散至琼脂培养基内，并被细菌吸收。若细菌对该抗生素敏感，则无法增殖并发生死亡，在E试条周围形成清晰的抑菌区域，称为抑菌圈。当抑菌圈边缘与E试条相交处所对应的刻度值，即为该抗生素的最低抑菌浓度（MIC）值。通常联合使用抗生素以防止细菌产生耐药性菌株。这种联合用药常产生协同作用，而非简单的相加效应。协同作用是指两种抗生素联合使用的效果大于各自单独使用效果的总和。然而，只有当联合用药的最低抑菌浓度（MIC）值比单独使用时至少降低两倍，该效应才被认为具有显著意义。这一标准通过计算部分抑菌浓度（FIC）指数来评估。将每种抗生素在联合用药时的MIC与其单独使用时的MIC之比相加，若所得FIC指数小于0.5，则表明存在协同作用。抗生素协同作用可通过两种基于E-test的方法进行测定：非交叉试验或交叉试验。在非交叉试验中，首先将两种不同抗生素（具有预设的MIC值）的E条分别贴于两个独立的平板上。待抗生素在培养基中扩散后，移去原始E条，并将另一种抗生素的E条放置于原位置，使其MIC刻度与先前E条的MIC刻度完全重合。交叉试验是非交叉试验的快速版本，将两种抗生素的E条以十字交叉方式同时放置，使它们的MIC刻度在交叉点处相互成90度角。两种方法经孵育后，分别读取每种抗生素在与另一种抗生素联用时的MIC值，读数位置为抑菌圈边缘与E条边缘相交处。随后计算FIC指数。本视频将演示如何利用E-test和微量肉汤稀释法测定特定抗生素对某种细菌的最小抑菌浓度（MIC）值。您还将学习如何通过交叉试验和非交叉试验来确定两种抗生素之间的协同作用。首先，穿戴适当的个人防护装备，包括实验手套和实验服。接着，使用70%乙醇对工作区域进行消毒。然后，收集15毫升无菌的含50%裂解马血和20毫克/毫升β-烟酰胺的Mueller-Hinton肉汤，以及五到八个Mueller-Hinton琼脂平板。现在，准备0.5麦氏浊度标准液：量取9.95毫升1%硫酸溶液，然后向其中加入50微升1%氯化钡溶液。充分涡旋混匀，形成浑浊悬液。用铝箔覆盖试管并暂时放置备用。接下来，向一个15毫升试管中加入1毫升生理盐水溶液。使用无菌接种环从细菌测试平板上刮取细菌生长物样本（此处为G群链球菌），然后将带有细菌的接种环放入生理盐水中，轻轻搅拌，并充分涡旋振荡试管。接着，将细菌悬液与麦氏比浊标准管并排放置，比较两者的浊度是否一致。根据需要添加适量生理盐水或细菌菌落，直至细菌悬液的浊度与标准管相匹配。达到所需浊度后，将无菌棉签浸入细菌悬液中。为接种MH-A平板，用棉签以“之”字形动作轻柔涂布平板整个表面。随后，在平板底部侧面标记细菌名称及日期。首先，用镊子夹住青霉素G E-test试纸条的边缘，取出试纸条。轻轻将试纸条置于 freshly swabbed MH-A 平板的中央，然后盖上皿盖。在此示例中，同时测试第二种抗生素——庆大霉素。因此，使用第二个平板和庆大霉素E-test试纸条重复上述试纸条放置步骤。为判读E-test结果，先取含有青霉素G E-test试纸条的第一个平板。观察抑菌圈与抗生素试纸条相交的位置，读取试纸条刻度上对应的数值。该数值即为青霉素G的最低抑菌浓度（MIC）值。以相同方法确定庆大霉素的MIC值。首先，将A群链球菌菌株接种于MH-A平板上。在平板底部标记细菌名称、待用的抗生素以及日期。接着，将待测抗生素的E-test试纸条置于平板中央。然后，将第二条试纸条以90度角垂直于第一条试纸条，并定位其MIC标记。在两条试纸条的MIC值交汇处，轻轻将第二条E-test试纸条覆盖于第一条之上。试纸条放置完毕后，切勿移动。随后，将平板在37摄氏度下孵育18至20小时。接种两块MH-A平板，并在每块平板上接种G族链球菌菌株后，将一种抗生素的E-test条放置于其中一块平板表面。随后，如图所示，将另一种抗生素的E-test条放置于第二块平板表面。使用塑料接种环，在每块平板表面标记出各自抗生素的最低抑菌浓度（MIC）值。接着，盖好平板，在室温下孵育1小时。此后，用镊子移除E-test条。然后，取其中一块平板及另一种抗生素的E-test条，将E-test条置于原先第一条E-test条留下的印迹上方，找到E-test条上MIC值与所标记线对齐的位置，并轻轻将E-test条放置于该交叉点处。对第二块平板重复此操作，随后将两块平板置于37摄氏度下孵育18至20小时。首先，获取具有已知细菌浓度的细菌悬液，并用MHF肉汤稀释培养物，使OD600达到0.003。接着，称取16毫克青霉素G和128毫克庆大霉素。将每种称量好的干燥抗生素分别转移至215毫升锥形管中。向每个锥形管中加入10毫升蒸馏水，并通过涡旋充分混匀。用抗生素名称和浓度标记各管。将400微升工作菌液分别加入96孔微孔板三行的第一孔中，每个处理设三个重复。接着，向这三行的各孔中加入200微升含MHF肉汤的工作菌液。然后，进行抗生素的两倍系列稀释：首先向第一孔中加入4微升抗生素母液，使抗生素稀释100倍；随后，从每行的第一孔开始，依次向前移取200微升菌液-抗生素混合液至下一孔，直至倒数第二孔，在每次移液后通过移液器吹打两到三次以确保充分混匀。最后，弃去最后一孔中的200微升菌液-抗生素混合液。为了确定青霉素G的肉汤微量稀释试验结果，首先找到未见明显细菌生长的孔，其特征为无浑浊现象。在这些孔中，识别出抗生素浓度最低的孔，该孔对应的浓度即为青霉素G对受试细菌的最低抑菌浓度（MIC）值。庆大霉素的MIC值可采用相同的实验方法和技术进行测定。为了确定非交叉试验的结果，取第一个含有青霉素G E试纸条的平板。然后确定抑菌圈与抗生素试纸条相交的位置。刻度上对应的数值即为青霉素G与庆大霉素联用时的最低抑菌浓度（MIC）值。在此示例中，联用时的MIC值为0.064微克/毫升。现在，收集第二个平板，该平板含有庆大霉素 E 纸片，并如前所述确定联合用药时的 MIC 值。为了评估联合效应，首先通过将联合用药时的 MIC 值除以青霉素 G 单独使用时的 MIC 值，计算青霉素 G 的部分抑制浓度（FIC）。对庆大霉素重复此过程。然后，使用此处所示的公式计算 FIC 指数。联合用药时 MIC 值降低两倍，可得到小于或等于 0.5 的 FIC 指数值，表明青霉素 G 与庆大霉素之间存在协同作用。在本例中，计算得到的 FIC 值为 1.18，大于 0.5。因此，结果未显示青霉素 G 与庆大霉素对中国台湾链球菌 G 群菌株具有协同作用。为了确定交叉试验的结果，首先确定生长抑制圈与其相应的E条带相交的点。读取每个E试验条带上对应于此相交点的数值。这些数值代表青霉素G和庆大霉素联合使用的最低抑菌浓度（MIC）值。接下来，为了评估该组合的效果，使用此处所示的公式计算FIC指数。在此示例中，计算得到的FIC值为1.18，大于0.5。这意味着青霉素G和庆大霉素对中国链球菌G群菌株不具有协同作用。

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抗生素敏感性测试:Epsilo计测试，以确定两种抗生素的MIC值，并评估抗生素协同效应

measuring bacterial resistance with e-tests

Measuring bacterial resistance with E-tests helps show how strongly an antibiotic can stop growth. The method is used to find the minimal inhibitory concentration, or MIC, which is the lowest antibiotic level that prevents visible bacterial growth. It is a practical way to study resistance in bacteria and compare how different antibiotics perform.

The E-test uses a plastic strip that contains a built-in antibiotic gradient. The strip is placed on an agar plate that has been inoculated with bacteria, and the MIC scale is printed directly on the strip in micrograms per milliliter. After incubation, an ellipse-shaped zone appears where growth is blocked. The MIC is read where that inhibition zone meets the strip.

A similar MIC method is microbroth dilution. In this approach, bacteria are placed in broth with different antibiotic concentrations. After incubation, the MIC is the lowest concentration that still stops visible growth. This method is quantitative and can be used with several bacteria, although it can require many reagents and careful preparation of the concentrations.

Knowing a bacterium’s resistance profile can help guide treatment choices. It can show whether a patient may benefit from one antibiotic or from a combination. When two antibiotics are used together, they may act in an additive, synergistic, or antagonistic way. Additive means the combined effect matches the effect of each drug at equal dose, while synergy means the combined effect is stronger than either drug alone.

E-tests can also be used to look for synergy and antagonism between antimicrobial agents. One method is non-cross testing. In this setup, one antibiotic strip is first placed on the agar plate, allowed to diffuse, and then removed. A second strip is placed in the same position, with the two MIC values aligned, and the result is used to calculate the fractional inhibitory concentration, or FIC. Values above 0.5 show synergy. This method is simple to read, but it takes more time and uses two plates.

A faster option is the cross test. Here, both E-test strips are placed at the same time in a 90-degree cross, with the known MIC values meeting at the angle. Only one plate is needed, so the method saves work, although the result can be a little harder to analyze. The changed inhibition zones are then used to determine whether the antibiotics act synergistically.

A broth-based approach can also be used for combination testing. It gives more flexibility because specific antibiotic concentrations can be chosen outside the limits of an E-test strip. It may also be more sensitive because the antibiotics are evenly distributed in liquid, rather than relying on diffusion through agar. In this method, wells in a 96-well plate are filled with a set number of bacteria and different antibiotic dilutions, and the MIC is the lowest concentration that prevents visible growth.

Measuring Bacterial Resistance with E-Tests

measuring-bacterial-resistance-with-e-tests

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Bacteriophage transduction moves DNA between bacteria by using a phage, or bacterial virus, as the carrier. In this process, a phage can deliver genetic material from one bacterium to another. The method is a form of horizontal gene transfer and is important in both bacterial evolution and laboratory genetics. Phages were first described in the early 1900s, and transduction was named in 1951 by Norton Zinder and Joshua Lederberg. The transcript describes two main phage classes. Lytic phages...

microbiology

该集合展示了微生物调查的关键工具，包括适当的无菌技术和电镀，如何使用选择性培养基和丰富样品，以及混合或纯样品的培养方法。此外，它着眼于识别微生物分离物的常见方法，以及细菌基因操作的程序。

Learn how E-tests and microbroth dilution measure MIC values, and how cross and non-cross setups reveal antibiotic synergy in bacteria.

Measuring Bacterial Resistance with E-Tests | JoVE K12

Georgia Standards of Excellence

9-12

Biology

SB6.e

Anna Bläckberg

Rolf Lood

Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center

Lund University

Concepts

Preparation of Plates and Bacteria

Determining MIC Using E-Test

Synergy Testing: Cross Approach

Synergy Testing: Non-Cross Approach

MIC Determination Using Broth Dilution

Data Analysis: Broth Microdilution

Data Analysis and Results: Synergy Testing

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Measuring Bacterial Resistance with E-Tests

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