作者感谢 DBT（生物技术教学计划）、DBT（BUILDER 计划）和 FIST 的资助。MR 感谢 SHODH 的奖学金。惠普感谢CSIR的奖学金。

铜绿假单胞菌的所有细菌分离株均来自印度巴罗达和斋浦尔的医学微生物学实验室。所有选定的临床分离株均在生物安全柜（BSL2）中处理，并且在实验期间处理细菌分离株时要格外小心。所有试剂/溶液的商业细节均在材料表中提供。
1. 天蓝色铬磺酸铬（CAS）染料和琼脂培养基的制备
<ol><li>用以下组合物制备CAS染料（100mL）：<ol><li>将 60 mg CAS 溶解在 50 mL 蒸馏水（溶液 1）中。</li>
		<li>将 2.7 mg FeCl3·6H2O 溶于 10 mL 10 mM HCl（溶液 2）中。</li>
		<li>将 73 mg 西曲溴铵 （HDTMA） 溶解在 40 mL 蒸馏水（溶液 3）中。</li>
		<li>小心混合溶液 1、溶液 2 和溶液 3。将其存放在玻璃瓶中。</li>
	</ol></li>
	<li>按照以下步骤准备CAS琼脂：<ol><li>将 100 mL MM9 盐溶液加入 750 mL 蒸馏水中。</li>
		<li>溶解32.24g哌嗪-N，N'-双（2-乙磺酸）（PIPES）游离酸。</li>
		<li>加入 15 克琼脂。高压灭菌器并使其冷却。</li>
		<li>向混合物中加入 30 mL 无菌酪氨基酸溶液和 10 mL 无菌 20% 葡萄糖溶液。</li>
		<li>加入 100 mL CAS 染料，混合，并在无菌条件下倒入。 注： 补充文件 1 中提供了 MM9 培养基、酪蛋白氨基酸溶液和 8-羟基喹啉的制备。PIPES缓冲液对pH值敏感。在达到 pH 值 5.6 之前，它不会溶解。确保持续监测 pH 值，因为当 PIPES 开始溶解在水中时，它会进一步降低 pH 值。用相同体积的3%8-羟基喹啉溶解在氯仿中提取Casamino酸。将不混溶液在4°C下放置约20分钟，小心地收集溶液的上相，不要干扰下相。</li>
	</ol></li>
</ol>2. 铁载体生产的定性分析
<ol><li>将OD600nm 设置为0.2，用于 铜绿假单胞菌的24小时培养物。</li>
	<li>使用无菌线环，在CAS琼脂平板上划线细菌培养物。</li>
	<li>在30°C孵育24小时。 注意：蛋白胨水培养基或 0.8% 生理盐水可用于稀释细菌培养物。如果在24小时未观察到细菌生长，则将CAS板孵育48至72小时。</li>
</ol>3. 总铁载体的定量估计
<ol><li>将OD600nm调节至0.25后，在蛋白胨水培养基中重新接种24小时生长的铜绿假单胞菌培养物，并在30°C下孵育48小时。</li>
	<li>48小时后，在室温下以4650× g 离心细菌培养物10分钟。</li>
	<li>离心后，向 96 孔微量滴定板中加入 100 μL 无细胞上清液，并向其中加入 100 μL CAS 染料。</li>
	<li>用铝箔盖住板，在室温下孵育20分钟。</li>
	<li>孵育后，在630nm处进行分光光度读数。</li>
	<li>将总铁载体定量的结果计算为铁载体单位百分比（PSU）。 注意： PSU 的计算公式为：[（Ar - As）/Ar] x 100 其中，Ar = 参比在 630 nm 处的吸光度，As = 样品游离上清液的吸光度。作为参考，应将CAS染料添加到未接种的蛋白胨水培养基中。用6M HCl填充所有玻璃器皿，如试管，烧瓶等2小时，并用蒸馏水冲洗两次，以去除其上的任何铁痕迹。</li>
</ol>4. 吡啶的定量估计
<ol><li>将OD600nm调节至0.25并在30°C孵育48小时后，在蛋白胨水培养基中重新接种24小时生长的铜绿假单胞菌培养物。</li>
	<li>48小时后，在进一步进行之前测量细菌生长的OD600nm 。</li>
	<li>在室温下以4650× g 离心细菌培养物10分钟。</li>
	<li>离心后，向 96 孔微量滴定板中加入 100 μL 无细胞上清液，并向其中加入 100 μL 50 mM Tris-HCl （pH 8.0）。</li>
	<li>在OD405 nm处进行分光光度读数。</li>
</ol>5. 脓气素提取和分光光度法
<ol><li>将OD600nm调整为0.25并在30°C孵育24小时后，在King's B培养基（补充文件1）中重新接种24小时生长的铜绿假单胞菌培养物。</li>
	<li>24小时后，取100mL培养物，并在室温下以4650× g 离心10分钟。</li>
	<li>离心后，向上清液中加入 5 mL 1 M 柠檬酸。用 50 mL 乙酸乙酯提取两次。</li>
	<li>通过注射器过滤器用硫酸镁过滤有机相。将过滤后的有机相储存在-20°C。</li>
	<li>在 320 nm 处获取分光光度读数。 注意：由于 pyochelin 在室温下是一种高度不稳定的化合物，因此请在冰上进行提取过程。使用 50 mL 无菌注射器进行过滤器分离。将棉花放在注射器的尖端，并在上面加入 1 克硫酸镁。将无菌注射器过滤器固定在注射器的尖端，并将滤液收集在无菌管中。</li>
</ol>

铜 绿假单胞 菌分离株铁载体产量的评估

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作者没有什么可透露的。

该方案使研究人员能够从细菌游离上清液中定量铜绿假单胞菌的总铁载体和两种不同的铁载体，即pyoverdine和pyochelin。在CAS琼脂平板测定中，CAS染料和Fe3+离子形成络合物。当细菌产生铁载体时，它们会淬灭 CAS-Fe3+ 复合物中的 Fe3+ 离子，导致细菌生长周围的颜色发生变化。这种变化导致细菌生长周围出现明显的橙色光晕14,15。虽然细菌可以直接在 CAS 琼脂平板上划线，但整体结果可能会受到影响。对大量细菌细胞进行条纹处理可能会在细菌生长周围形成一个宽大的橙色光晕，而使用较少的细菌细胞可能会导致狭窄的透明区域。为了解决这个问题，我们通过调整OD600来划定均匀数量的细菌细胞。挑剔的细菌可能在 24 小时内显示结果，而生长缓慢的细菌可能需要超过 72 小时才能表现出铁载体的产生。
对于总铁载体的定量测定，最初采用经典光谱法，这需要大量的上清液进行铁载体检测，因此不切实际。现在使用带有改进的 CAS 测定法的 96 孔板进行总铁载体的定量。与传统的光谱方法相比，这种方法减少了试剂、细菌上清液的使用、时间和准确性16。其他研究也报告了使用微量滴定板作为一种省时且更有效的方法17。使用微量滴定板定量总铁载体已被证明是一种有效的方法。在这项研究中，选择在无铁培养基中生长的 48 小时龄细菌培养物进行检查。还进行了一项比较研究，以评估不同生长培养基中的铁载体产生，例如 Luria 肉汤、Luria 肉汤（用 3% 8-羟基喹啉提取）、King's B 培养基（用 3% 8-羟基喹啉提取）和蛋白胨水培养基（用 3% 8-羟基喹啉提取）。据观察，蛋白胨水培养基表现出最高的铁载体产生量，因此，它被选择用于进一步的实验。描述不同生长培养基中铁载体产生的图表包含在 补充 图1中。
吡啶可使用荧光光谱读数进行检测和定量，但已知只有吡啶表现出荧光。Pyoverdine 与 Fe3+ 离子复合物不表现出荧光18,19。对于吡啶的检测和定量，选择48小时龄的细菌培养物进行研究。已知铜绿假单胞菌在最初的缺铁条件下产生脓皮素，并在暴露于严重的铁胁迫条件下后转而产生吡咯烷。已经证明吡喃定可以螯合其他金属阳离子，如铝、镓、锰和铬，但只有铁被成功转运到细胞膜19。
与总铁载体和吡喃苜蓿类似，脓脓霉素不能直接从无细胞上清液中定量，必须使用二氯甲烷20 提取。二氯甲烷与无细胞上清液不混溶，并在底部形成一个单独的层。在这项研究中，测试了各种营养培养基，包括 Luria 肉汤、蛋白胨水和酪蛋白胨酸培养基，但从 King's B 培养基中获得了大量的 pyochelin。虽然 Hoegy 等人在他们的方案中使用了 CAA 培养基，但我们用 King's B 培养基对其进行了修改，因为 CAA 培养基不支持细菌生长21。通过用8-羟基喹啉提取营养培养基以去除培养基中的任何铁残留物，从而进一步修改了该方法，从而形成缺铁培养基。此外，还发现孵育时间会显着影响脓蘑菇素的产生。Dumas等人证明， 铜绿假单 胞菌在严格的铁限制条件下从脓脓果素转变为脓脓苷的产生12。在这项研究中，从 24 小时的细菌培养物中提取和检测 pyochelin，因为在 48 小时培养物中未检测到 pyochelin。上清液的体积也会影响表皮脂蛋白的检测，因此，使用100 mL无细胞上清液进行提取。此外，MR1、TL7 和 J3 分离株中 pyochelin 的产生显着更高，这可能是由于细菌菌株尽管具有有效的铁螯合剂22，但无法产生吡啶。
Ji 等人介绍了一种新颖且灵敏的 pyochelin 定量方法，并使用 LC/MS/MS 对其进行了验证。然而，这种方法仅适用于从囊性纤维化患者痰液中分离出 的铜绿假单 胞菌。LC/MS/MS是一种昂贵的设置，对于大多数实验室来说可能负担不起23.Visaggio 等人开发了一种基于生物发光的全细胞生物传感器，可实现快速、灵敏和一步的 pyochelin 定量。然而，该方法不能提供总铁载体和吡替定24 的定量。
铁载体铁摄取系统可能在多重耐药细菌的药物递送中具有抗菌应用，因为它们在细菌存活和毒力中起着至关重要的作用。这种方法可能提供一种对抗多重耐药细菌的新方法25。不能穿透细菌细胞膜的药物可以与铁载体连接，并且产生的Fe-铁载体复合物可以在细菌膜内运输26。

生物体需要铁来执行各种重要功能，例如电子传输和 DNA 复制1。 铜绿假单胞菌是一种革兰氏阴性机会性病原体，已知具有多种毒力因子在宿主中建立感染，其中一种机制是铁载体形成2。在缺铁条件下， 铜绿假单 胞菌会释放出称为铁载体的特殊分子，这些分子会从周围环境中淬灭铁。铁载体在细胞外螯合铁，产生的铁载体复合物被主动转运回细胞3。
已知铜绿假单胞菌产生两种铁载体，pyoverdine 和 pyochelin。已知 Pyoverdine 具有较高的铁螯合亲和力 （1：1），而已知 pyochelin 具有较低的铁螯合亲和力 （2：1）4。脓蘑菇素也被称为次级铁载体，因为它具有较低的铁螯合亲和力5。铜绿假单胞菌 6 中铁载体的产生和调节受到群体感应 （QS） 系统的主动控制。
除铁淬火外，铁载体还参与调节毒力因子，并在生物膜形成中发挥积极作用7。铁载体还发挥着其他关键作用，包括参与细胞信号传导、防御氧化应激和促进微生物群落之间的相互作用8.铁载体通常根据它们螯合铁的特定官能团进行分类。该分类中的三种主要双酸配体是儿茶酚酸盐、羟肟酸盐和α-羟基羧酸盐3。Pyoverdines 是荧光假单胞菌属物种的标志，例如铜绿假单胞菌和荧光假单胞菌 5。它们由与含有 6-12 个氨基酸的寡肽偶联的混合绿色荧光发色团组成。几种非核糖体肽合成酶 （NRP） 参与其合成9.参与吡啶产生和调控的四个基因是 pvdL、pvdI、pvdJ 和 pvdD10。Pyoverdine 还负责哺乳动物的感染和毒力11.铜绿假单胞菌在中度限铁条件下产生脓蘑菇素，而吡脓草定在严格的限铁环境中产生12。参与 pyochelin 生产的两种操纵子是 pchDCBA 和 pchEFGHI13。值得注意的是，在碧蓝蛋白存在下，脓脓苷（儿茶酚酸酯）诱导氧化损伤和炎症，并产生羟基自由基，其对宿主组织有害11。
与微生物测定相比，蓝铝磺酸铬 （CAS） 测定因其全面、高灵敏度和更大的便利性而被广泛采用，微生物测定虽然灵敏，但可能过于特异性14.CAS测定可以在琼脂表面或溶液中进行。它依赖于当三价铁离子从其强烈的蓝色络合物转变为橙色时发生的颜色变化。CAS 比色法定量了 Fe-CAS 表面活性剂三元络合物中铁的消耗。这种特殊的络合物由金属、有机染料和表面活性剂组成，呈蓝色，并在 630 nm 处表现出吸收峰。
本报告提出了一种定性检测铁载体产生的方法，其中可以检测琼脂平板上铁载体的产生。还提供了一种定量估计微量滴定板中总铁载体产生的方法，以及来自 铜绿假单胞菌的两种铁载体pyoverdine和脓脓明的检测和定量分析方法。

<table><tbody><tr><td>Agar Agar, Type I</td><td>HIMEDIA</td><td>GRM666</td><td></td></tr><tr><td>8-Hydroxyquinoline</td><td>Loba Chemie</td><td>4151</td><td></td></tr><tr><td>Casamino Acid</td><td>SRL Chemicals</td><td>68806</td><td></td></tr><tr><td>Cetyltrimethyl Ammonium Bromide (CTAB)</td><td>HIMEDIA</td><td>RM4867-100G</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>1070242521</td><td></td></tr><tr><td>Chrome azurol sulfonate</td><td>HIMEDIA</td><td>RM336-10G</td><td></td></tr><tr><td>Citric acid</td><td>Merck</td><td>100241</td><td></td></tr><tr><td>Dextrose monohydrate</td><td>Merck</td><td>108342</td><td></td></tr><tr><td>Dichloromethane</td><td>Merck</td><td>107020</td><td></td></tr><tr><td>Ferric chloride hexahydrate</td><td>HIMEDIA</td><td>GRM6353</td><td></td></tr><tr><td>Glass Flasks</td><td>Borosil</td><td>5100021</td><td></td></tr><tr><td>Glass Test-tubes</td><td>Borosil</td><td>9820U05</td><td></td></tr><tr><td>Hydrochloric acid</td><td>SDFCL</td><td>20125</td><td></td></tr><tr><td>King&#039;s medium B base</td><td>HIMEDIA</td><td>M1544-500G</td><td></td></tr><tr><td>M9 Minimal Medium Salts</td><td>HIMEDIA</td><td>G013-500G</td><td></td></tr><tr><td>Magnesium Sulphate&amp;nbsp;</td><td>Qualigens</td><td>10034</td><td></td></tr><tr><td>MultiskanGO UV Spectrophotometer</td><td>Thermo Scientific</td><td>51119200</td><td></td></tr><tr><td>Peptone Type I, Bacteriological</td><td>HIMEDIA</td><td>RM667-500G</td><td></td></tr><tr><td>PIPES free acid</td><td>MP Biomedicals</td><td>190257</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>1048731000</td><td></td></tr><tr><td>Proteose peptone</td><td>HIMEDIA</td><td>RM005-500G</td><td></td></tr><tr><td>Shimadzu UV-Vis Spectrophotometer</td><td>Shimadzu</td><td>2072310058</td><td></td></tr><tr><td>Sigma Laborzentrifuge</td><td>Sigma-Aldrich</td><td>3-18K</td><td></td></tr><tr><td>Sodium chloride</td><td>Qualigens</td><td>15915</td><td></td></tr></tbody></table>

qualitative and quantitative analysis of siderophore production from pseudomonas aeruginosa

铜绿假单胞 菌（铜绿假单胞菌）以其产生多种毒力因子以在宿主中建立感染而闻名。其中一种机制是通过铁载体的产生来清除铁。 铜绿假单 胞菌产生两种不同的铁载体：铁螯合亲和力较低的脓脓苷和铁螯合亲和力较高的脓苷。本报告表明，吡啶可以直接从细菌上清液中定量，而脓皮单在定量前需要从上清液中提取。
定性分析铁载体产生的主要方法是蓝铝磺酸铬 （CAS） 琼脂平板测定。在该测定中，Fe3+-染料复合物中释放的 CAS 染料导致颜色从蓝色变为橙色，表明铁载体的产生。为了定量总铁载体，将细菌上清液与微量滴定板中的CAS染料等比例混合，然后在630nm处进行分光光度分析。通过将Pyoverdine与50 mM Tris-HCl等比例混合，直接从细菌上清液中定量，然后进行分光光度法分析。380 nm处的峰证实了pyoverdine的存在。至于Pyochelin，不可能从细菌上清液中直接定量，因此必须首先提取。随后的分光光度分析显示存在 pyochelin，峰值为 313 nm。

在对临床分离株的铁载体进行定量定量之前，对铁载体的产生进行了定性筛选，以确保铁载体的产生。通过在CAS琼脂平板上划线细菌来观察来自临床分离株的铁载体的定性检测。本研究选择了三种临床分离株，即MR1、TL7、J3以及PAO1（参考菌株）。所有三种临床分离株和 PAO1 都显示出铁载体产生的阳性结果，其中蓝色琼脂表面细菌生长周围的透明橙色光晕表明铁载体产生阳性（图 1）。CAS琼脂上的晕形成（图1）粗略估计了临床分离株产生的铁载体。因此，使用 CAS 试剂和液体培养基对铁载体的产生进行了定量估计。
直接从无细胞上清液中进行总铁载体定量。孵育后，观察到颜色变化，其中黄色表示从Fe复合物中去除CAS染料。在这里，使用带有未接种生长培养基的CAS染料作为对照，用于铁载体计算。分离株 MR1 和 TL7 的总铁载体产量没有显著差异，而与 PAO1 相比，J3 的总铁载体产量显著（图 2）。
接下来，直接从游离上清液中进行吡啶定量。吡啶在细胞环境中释放，因此使用游离上清液进行吡啶定量。还通过分光光度计进行了紫外范围扫描，其中380nm处的峰证实了吡喃定的存在（图3A）。分光光度法读数在 405 nm 处采集，结果解释为 OD405/OD600 nm。所有分离株均显示吡非定产生，其中分离株 MR1 和 J3 的吡非定比 PAO1 显著降低，而 TL7 未观察到显着差异（图 3B）。
脓橙素不能直接从无细胞上清液中定量。无法从 1 mL 无细胞上清液中提取 pyochelin，因此从 100 mL 无细胞上清液中提取。用1M柠檬酸酸化。由于脓蘑菇素是一种高度不稳定的化合物，因此提取过程在冰上进行。通过在300至600nm范围内运行UV范围扫描进行脓皮脂蛋白检测，其中OD320nm 处的峰证实了脓皮脂蛋白的存在（图4A）。通过在OD320 nm处获取分光光度读数进行Pyochelin定量。通过除以 OD320/OD600 进行计算。与参考分离株 PAO1 相比，分离株 MR1、TL7 和 J3 显示出明显更高的 pyochelin 产量（图 4B）。
在各种细菌生长培养基中进行比较铁载体的产生，以检查在各种生长培养基中产生的总铁载体的量。选择四种不同的培养基，其中用 3% 8-羟基喹啉提取 Luria 肉汤、King's B 培养基和蛋白胨水，并选择未提取的 Luria 肉汤进行研究。提取的卢里亚肉汤、King's B 培养基和蛋白胨水培养基的铁载体产量无显著差异，而未提取的卢里亚肉汤的铁载体产量存在显著差异。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65980/65980fig1v2.jpg"> 图1：CAS琼脂上产生的铁载体。 将24小时培养物的CAS琼脂平板上的 铜绿假单 胞菌的生长点在CAS琼脂平板上，并在30°C下孵育24小时。铁载体的存在由细菌菌落（PAO1、MR1、TL7 和 J3）周围的橙色光晕表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig1v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65980/65980fig2v2.jpg"> 图2：总铁载体产量。 评估了 PAO1 和三种临床分离株（MR1、TL7 和 J3）的总铁载体产生量。将 100 μL 游离上清液（在蛋白胨水液中生长 24 小时的 PAO1、MR1、TL7 和 J3）加入 96 孔微量滴定板中，然后加入 100 μL CAS 染料。采用单因素方差分析检验，具有统计学意义。误差线表示三个生物学重复之间的±标准偏差 （SD）。*对应 p < 0.05;ns 对应于 p > 0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig2v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65980/65980fig3v2.jpg"> 图3：吡啶生产。铜绿假单胞菌菌株 PAO1 和临床分离株（MR1、TL7 和 J3）在蛋白胨水培养基中生长 24 小时后评估的吡替啶产生。离心细菌培养物，向 96 孔微量滴定板中加入 100 μL 游离上清液。此后，加入 100 μL 50 mM Tris-HCl （pH 8.0）。吡啶检测通过紫外分光光度法在350 nm至600 nm范围内进行。在 380 nm 处进行吡替定生产的光谱分析 （A）。吡替定产量的定量 （B）。误差线表示一式三份实验的标准误差。误差线表示三个生物重复之间的±SD。对应 p < 0.0001，**对应 p < 0.01，ns 对应 p > 0.05，基于单因素方差分析检验。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig3v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65980/65980fig4v2.jpg"> 图 4：脓气素生产。 使用乙酸乙酯和硫酸镁提取 PAO1（用作参考）和临床分离株（MR1、TL7 和 J3）的 Pyochelin 生产。分光光度分析范围为 230 nm 至 400 nm （A）。脓皮脂素的定量 （B）。对每种分离株进行了三次独立实验。误差线表示三个生物重复之间的±SD。对应 于基于 单因素方差分析检验的 p < 0.001。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/65980fig4v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充 图1：各种细菌生长培养基中铁载体的比较。在四种不同的生长培养基中产生总铁载体，其中用 3% 8-羟基喹啉提取 King's B 培养基、蛋白胨水培养基和 Luria 肉汤以去除其中的任何微量铁，并选择 Luria 肉汤（未提取）。通过向培养基中加入等体积的3%8-羟基喹啉进行3%8-羟基喹啉的萃取。误差线表示三个生物重复之间的±SD。**对应 p < 0.001，ns 对应于 p > 0.5 基于单因素方差分析检验。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/Supplemental%20Figure%201.pdf" target="_blank">请点击这里查看此图的较大版本.</a>
补充文件1：用于本研究的培养基配方。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65980/Supplementary%20File%201.docx" target="_blank">请按此浏览档案。</a>

Watch this Scientific Journal Video about Quantifying Siderophores and Pyochelin for Infection Control at JoVE.com

作者聚焦：定量铁载体和绿茶素用于感染控制 

该方案提供来自 铜绿假单胞菌的总铁载体，pyoverdine和脓霉素的定性和定量分析。

铜绿假单胞菌产生铁载体的定性和定量分析

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

铁载体与铁形成复合物，并在铁限制条件下将其转运到细胞中。铁载体是有效的毒力因子，有助于在宿主中建立严重感染，并且已知在调节铜绿假单胞菌的群体感应中发挥作用。我们的研究旨在定量估计假单胞菌中铁载体的产生。铁载体是细胞外分子，因此使用游离上清液定量总铁载体以及吡喃蕊素和脓粒素。由于不能检测到小体积的脓蘑菇素，因此从大量无细胞上清液中提取。最近，LCMS和生物传感器已被开发用于定量化脓皮脂素。LCMS对于小型实验室来说是一种昂贵的设置，而生物传感器只能定量化pyochelin。这种简单且图文并茂的方案使用最少的实验室试剂和仪器，并允许使用更短的时间从无细胞上清液中估计铁载体。

qualitative-quantitative-analysis-siderophore-production-from

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Biology

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

3.8K 次观看.  The Maharaja Sayajirao University of Baroda. 铁载体与铁形成复合物，并在铁限制条件下将其转运到细胞中。铁载体是有效的毒力因子，有助于在宿主中建立严重感染，并且已知在调节铜绿假单胞菌的群体感应中发挥作用。我们的研究旨在定量估计假单胞菌中铁载体的产生。铁载体是细胞外分子，因此使用游离上清液定量总铁载体以及吡喃蕊素和脓粒素。由于不能检测到小体积的脓蘑菇素，因此从大量无细胞上清?...

视频: 作者聚焦：定量铁载体和绿茶素用于感染控制 

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

科研

生物化学

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

从环境样品中筛选高通量的安德森菌: 植物组织、散装土壤和根际土壤

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

环境科学

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

免疫与感染

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

生物工程

铜绿假单胞菌菌株中鼠李糖脂产生的比较分析

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This bacterium produces high levels of biosurfactants known as rhamnolipids (RL), which are molecules with significant biotechnological value but are also associated with virulence traits. In this respect, the detection and quantification of RL may be useful for both biotechnology applications and biomedical research projects. In this article, we demonstrate step-by-step the technique to detect the production of the two forms of RL produced by P. aeruginosa using thin-layer chromatography (TLC): mono-rhamnolipids (mRL), molecules constituted by a dimer of fatty acids (mainly C10-C10) linked to one rhamnose moiety, and di-rhamnolipids (dRL), molecules constituted by a similar fatty acid dimer linked to two rhamnose moieties. Additionally, we present a method to measure the total amount of RL based on the acid hydrolysis of these biosurfactants extracted from a P. aeruginosa culture supernatant and the subsequent detection of the concentration of rhamnose that reacts with orcinol. The combination of both techniques can be used to estimate the approximate concentration of mRL and dRL produced by a specific strain, as exemplified here with the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3).

作者聚焦:了解铜绿假单胞菌中的鼠李糖脂调控

Pseudomonas aeruginosa&#160;produces the rhamnolipid biosurfactants. Thin-layer chromatography detects and determines the proportion of mono- and di-rhamnolipids produced by each strain. Quantification of total rhamnolipids involves assessing rhamnose equivalents present in these biosurfactants extracted from the culture supernatants using the orcinol method.

铜绿假单胞菌产生的单鼠李糖脂和二鼠李糖脂的检测和定量

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is ...

铜绿假单胞菌产生铁载体的定性和定量分析

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此视频中的章节

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

从环境样品中筛选高通量的安德森菌: 植物组织、散装土壤和根际土壤

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

微孔阵列平台中微生物群落发展的组装与跟踪

铜绿假单胞菌产生的单鼠李糖脂和二鼠李糖脂的检测和定量

铜绿假单胞菌产生的单鼠李糖脂和二鼠李糖脂的检测和定量

铜绿假单胞菌产生的单鼠李糖脂和二鼠李糖脂的检测和定量

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

伪多莫纳斯航空感染模型实时评估工具

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression