这项工作得到了传染病和微生物学、公共卫生中心的支持。作者没有收到任何其他资助这项研究。作者感谢 Drs 阿诺特, 内森. 巴赫曼和 Ranjeeta 梅农对整个基因组测序实验的专家建议和帮助。

这项研究不需要道德上的认可。
1.光滑念珠菌的亚文化和接种制备
<ol><li>选择要研究的c. 光滑隔离的面板, 其中还应包括至少一个c. 光滑美式类型区域性集合 (ATCC), 并具有已知的敏感度模式。</li>
	<li>亚文化, 通过触摸一个单一的殖民地使用无菌一次性塑料循环和裸奔到一个 Sabouraud 的葡萄糖琼脂 (不育) 板8。</li>
	<li>在35° c 的情况下孵育24-48 ĥ的纯培养基, 生长良好。</li>
</ol>2. 抗真菌药敏性的测定
<ol><li>使用无菌一次性塑料循环, 从一个新鲜的继C. 光滑隔离板上选取大约1毫米直径的4-5 菌落。重在3毫升的无菌蒸馏水中。用温和的移混合均匀的悬挂。</li>
	<li>使用密度5将单元格密度调整为0.5 麦克法兰, 该值相当于 1 x 106到 10 x 68单元格/mL。</li>
	<li>使用商业化验 (见材料表和试剂) 对所有的C. 光滑进行敏感性测试, 按照制造商的说明进行隔离。根据 CLSI 指南和准备报告 (表 1)8, 解释基于合成的抗真菌药物对菌株的敏感性。</li>
</ol>3. 基因组 DNA 提取测序
<ol><li>重在1.5 毫升的试管中, 用50毫米的 EDTA 在300µL 中, 从一个刚生长的自失能板中 loopful 出菌落。</li>
	<li>在悬浮液中加入40µL zymolyase (10 毫克/毫升), 轻轻吸管5次, 直至悬挂均匀。</li>
	<li>孵育样品在37° c 为 1-2 h 消化细胞壁。室温冷却5分钟。</li>
	<li>离心悬浮在 1.4万x g为2分钟, 然后小心地去除上清。</li>
	<li>提取 dna 提取试剂盒的基因组 dna 的指南 (见材料表)。</li>
	<li>重提取的 DNA 颗粒在50µL 10 毫米的三个缓冲 (pH 7.5-8.5), 而不是洗脱缓冲器提供的套件。</li>
	<li>通过测量 260/280 nm25的光学密度 (O. D) 来检查 DNA 的纯度。</li>
</ol>4. 基因组 DNA 定量
<ol><li>根据制造商的荧光检测指南 (见材料表), 在化验试剂盒中准备一个1x 的三 EDTA (TE) 缓冲器。</li>
	<li>通过在一次性96井板中加入2µL 至98µL 的 1x TE 化验缓冲液 (最终容积100µL, 稀释因子 1:50) 稀释 DNA 样本。此稀释步骤可以手动使用多通道吸管或自动液体处理工作站进行。</li>
	<li>通过稀释荧光检测试剂盒 (表 2) 中提供的 lambda DNA (100 µg/毫升) 来准备标准的范围, 并包括用于测量的样品。</li>
	<li>添加100µL 的荧光染料到100µL 稀释的 DNA 样品和标准的反应。在室温下孵育5分钟, 免受光照。</li>
	<li>根据制造商的指导方针测量所有样品的荧光。</li>
	<li>使用荧光读数绘制标准曲线, 并计算 DNA 样本的原始浓度。</li>
	<li>确定 dna 的体积和10毫米的三个缓冲 (pH 值 8) 来调整 dna 浓度到 0.2 ng/µL。</li>
	<li>使用自动液体处理工作站执行 DNA 稀释。这一步也可以通过手动移实现。</li>
</ol>5. DNA 文库的制备
注意: 库的准备和排序是按照制造商提供的协议和指南 (图 1A) 执行的 (请参阅材料表)。
<ol><li>Tagmentation 与 PCR 扩增
	<ol>
<li>标签一个新的96井硬壳薄墙板。</li>
		<li>添加5µL 的定量输入 DNA 在 0.2 ng/µL (总共 1 ng), 以每个样本井的板块。</li>
		<li>添加10µL 的 tagmentation 缓冲和5µL 的放大缓冲, 每一个井含有 DNA 和轻轻吸管混合。用胶板密封密封板。</li>
		<li>将盘置于热循环中, 并运行以下 PCR 程序:55 ° c 为5分钟, 并保持在10° c。当样品到达10° c 时, 立即进行中和。</li>
		<li>加入5µL 中和缓冲器, 以中和增反应, 轻轻吸管混合。密封板, 室温孵育5分钟。</li>
		<li>在样品中加入15µL 的 PCR mastermix。</li>
		<li>使用一箱索引底漆管的设置 96-井板格式, 使每个样本得到一个独特的组合索引模板的基础上 (表 3)。</li>
		<li>使用表3中模板中提供的顺序, 在索引板机架 (图 1B) 中排列索引底漆管, 并记录该模板上指数的位置。</li>
		<li>将 tagmentation 板与添加的 PCR mastermix 放在指数板架上, 并按顺序排列。</li>
		<li>将索引引物1管置于垂直排列, 并在索引架上的水平排列上索引底漆2管。使用多通道吸管, 小心地为每个样品添加5µL 的索引底漆。</li>
		<li>用新的盖子取代旧的索引管帽, 以避免指数之间的交叉污染。</li>
		<li>密封板使用 96-清楚的板封口和执行第二 PCR 如下:95 ° c 为三十年代, 12 个周期95° c 为十年代, 55 ° c 为三十年代, 72 ° c 为三十年代和72° c 为 5 min。</li>
	</ol></li>
	<li>PCR 清除
	<ol>
<li>将 pcr 产物转移到 pcr 清除, 从 tagmentation 板到深井板 (见材料表)。</li>
		<li>涡流的商业磁珠解决方案 (见材料表) 根据制造商的说明, 并添加30µL 的每个 PCR 产品的深井板。</li>
		<li>摇动在 1800 rpm 2 分钟的微振动筛板, 在室温下孵育, 无需晃动5分钟。</li>
		<li>把盘子放在一个磁性的支架上2分钟, 直到清液清除。</li>
		<li>将上清液小心地丢弃在磁性支架上。</li>
		<li>添加200µL 新鲜准备的80% 乙醇与板的磁性立场。</li>
		<li>在三十年代的磁性支架上孵育板, 小心地除去和丢弃上清, 而不干扰珠子。</li>
		<li>重复洗涤步骤, 让珠子风干15分钟. 如果有的话, 去掉多余的乙醇。</li>
		<li>从磁性支架上取下板, 并在珠子上加52.5 µL 的悬浮缓冲器。</li>
		<li>在 1800 rpm 的微振动筛上摇动盘子2分钟, 在室温下孵育2分钟而不摇晃。</li>
		<li>将盘放在磁性支架上, 使上清液清晰。</li>
		<li>使用多通道吸管, 小心地转移50µL 的上清从清理板到一个新的硬壳板。</li>
	</ol></li>
	<li>库规范化
	<ol>
<li>根据制造商的指导方针, 解冻库规范化试剂 (见材料表)。</li>
		<li>从清理盘转移20µL 到新的深井板。</li>
		<li>加45µL 磁珠悬浮液, 用平板封口机封板。</li>
		<li>在 1800 rpm 的微振动筛上摇动盘子30分钟。这种潜伏期是至关重要的, 不应超过。</li>
		<li>将板放在磁性支架上2分钟, 确认清液已清除 (图 1B)。</li>
		<li>在一个适当的危险废物容器中除去并丢弃上清液, 而该板仍在磁架上。</li>
		<li>从磁性支架上取下盘子, 用45µL 洗涤缓冲器清洗珠子。</li>
		<li>在 1800 rpm 为5分钟的微振动筛上用洗涤缓冲器摇动盘子。</li>
		<li>将板放在磁性支架上2分钟, 当它变清晰时丢弃上清。</li>
		<li>从磁性支架上取下板, 再用洗涤缓冲器重复洗涤。</li>
		<li>从磁性支架上取下板, 并加入30µL 0.1 N 氢氧化钠。</li>
		<li>摇动板与 0.1 N 氢氧化钠在一个微振动筛在 1800 rpm 5 分钟, 并把板上的磁性立场为2分钟或直到液体是明确的。</li>
		<li>将30µL 的洗脱缓冲液添加到新的96井硬壳薄墙最终规范化的库板中。</li>
		<li>将30µL 上清液从正常化板转移到最终规范化的库板, 使最终体积60µL。现在, 这些库已准备就绪, 可以进行排序。</li>
	</ol></li>
	<li>qPCR 法测定 DNA 文库浓度
	<ol>
<li>根据制造商的指南, 解冻 qPCR 套件中提供的 mastermix、底漆和标准 (见材料表)。</li>
		<li>结合 PCR 试剂 mastermix 和 qPCR 试剂盒中提供的底漆, 按照制造商的说明和等分可以存储在-20 ° c。</li>
		<li>为了确定 dna 库的浓度, 通过执行1:100 稀释 (1.5 µL 脱氧核糖核酸库到148.5 µL 三缓冲), 然后1:80 稀释 (2 µL 从1:100 到158µL 三缓冲), 使脱氧核糖核酸图书馆的1/8000 稀释使用10毫米三个缓冲 (pH 8)。</li>
		<li>摇动稀释板在 700 rpm 至少1分钟, 然后离心机在 14000 x g为1分钟。</li>
		<li>通过混合4µL 稀释 dna 库或 dna 标准和16µL mastermix, 制备20µL 的最终 PCR 反应。</li>
		<li>执行 PCR 以下制造商的设置在 thermocycler:95 ° c 为 5 min, 35 周期95° c 为三十年代和60° c 为四十五年代, 并且最后65° c 到95° c 为融解曲线分析。</li>
		<li>从 qPCR thermocycler 中获取样本 DNA 库和标准的 Ct 值。</li>
		<li>从标准的 Ct 值生成标准曲线 (图 2)。定义上和下 QC 范围由±3周期的平均值。例如, 如果平均 Ct 值是13周期, 那么 QC 范围是在16和10周期之间。</li>
		<li>根据标准曲线和 Ct 值确定个体和平均库浓度 (ALC)。</li>
		<li>确定要使用的总计库 (PAL) 的体积, 根据下面给出的计算结果, 考虑到目标库的浓度介于 1.4-1.8 pM 之间。 注意:从 qPCR = ALC 获得的平均图书馆浓度;汇总库 (PAL) = ALC/2;变性 pal (达尔) = pal * 0.666 根据与缓冲混合的达尔的体积, 是将库的浓度添加到流单元中。例如, 如果将库的65µL 添加到835µL 的缓冲区中, 则从该稀释 (Dil 1) 195 添加到1300年的总容量µL: (65/900) * dnPAL = Dil1 Dil1 * (195/1300) = 最终浓度 (应介于 1.4-1.8 pM 之间)</li>
	</ol></li>
</ol>6. 台式排序器中的库池和初始排序
<ol><li>根据制造商的指导方针, 解冻试剂盒。从4° c 的存储中取出一个新的流动单元, 并在测序前至少30分钟将室温带入房间温度。使用前取出缓冲盒和 prechill 测序缓冲器 (见资料表)。</li>
	<li>通过混合5µL 库 (1 nM) 和5µL 0.2 N 氢氧化钠, 准备一个控制库。在室温下进行5分钟的涡旋和孵育, 将控制库变性成单股。</li>
	<li>添加5µL 200 毫米的三盐酸, pH 7 和涡流。添加235µL prechilled 测序缓冲器并轻轻混合。总容量是250µL 与控制图书馆最后的集中在晚上20点。</li>
	<li>通过将每个样本库的5µL 传输到一个单一的 low-bind 1.5 mL 管中, 将每一个采样文库的序列从最终规范化的库板中进行排序。</li>
	<li>添加30µL 的共用库和30µL 的 0.2 N 氢氧化钠, 变性图书馆在另一个低绑定管。</li>
	<li>涡 low-bind 管和孵化5分钟的室温变性图书馆成单股。</li>
	<li>添加30µL 200 毫米的三盐酸, pH 7 到管与变性库中和反应。</li>
	<li>添加65µL 的中和变性图书馆悬浮和835µL 的 pre-chilled 测序缓冲和旋涡混合好。</li>
	<li>在最后的低束缚管结合以下: 195 µL 从中立的变性图书馆, 1.30 µL 的控制库和1103.70 µL 的测序缓冲器。正确混合。</li>
	<li>将最终的库组合 (1300 µL) 加载到试剂盒上的指定位置。</li>
	<li>设置排序运行通过输入项目和样本详细信息在排序器指定的网站遵循的准则。</li>
	<li>按照指导方针开始测序。台式排序器中的负载流单元、带库的试剂盒和缓冲盒。</li>
	<li>记录顺序中使用的所有试剂盒和墨盒的批号。</li>
</ol>7. 从测序网站下载数据
<ol><li>按照制造商在网站上提供的说明下载 FASTQ 文件。</li>
	<li>为一个良好的质量运行检查百分比 Q30 是≥75% 和集群密度是介于 170-280 k/mm2与最佳在 200-210 k/毫米2 (表 4)。</li>
</ol>8. 测序数据分析
<ol><li>将已测序样本的 FASTQ 文件导入数据分析集成软件包 (见资料表)。</li>
	<li>通过使用表5中列出的设置, 在软件中添加功能, 即修剪、映射到参考 (选择参考基因组)、局部重新排列和变体分析 (图 3a), 创建一个排序工作流。</li>
	<li>通过选择单个示例或一批示例 FASTQ 文件并将输出文件保存在指定的示例文件夹中来运行工作流。</li>
	<li>生成序列覆盖率深度、映射区域和基因组中结构变量列表的报告 (图 3B)。</li>
	<li>使用结构变量列表搜索同义单核苷酸多态性 (snp) 的基因赋予抗药性和毒力。</li>
	<li>通过列出 SNP 位置、基因和耐药或易感菌株的数量来准备报告 (表 6)。</li>
</ol>

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作者没有相互竞争的金融利益, 也没有利益冲突的披露。

本研究确定了在光滑中 WGS 引导检测耐药性的可行性、近似时间线和精密度。图书馆准备和测序的周转时间为四天, 报告分析结果1/2 天。这与培养皿和桑格测序中的药敏测定方法相比, 至少有相当数量的样品。大约 30-90 C. 光滑基因组可以根据测序流细胞容量进行测序, 80-100% 测序覆盖率。由于测序是在 in-house WGS 实验室设置, 因此本研究的成本/资源需求相当于桑格测序和探测器分析的?...

念珠菌光滑是一种日益受到重视的病原体, 其重要性作为一个物种, 它对唑以及最近的棘1、2、3进行了抵抗。不同于二倍体的c. 白念珠菌,光滑的单倍体基因组可能允许它获得突变和更容易地发展多重耐药性。两个药物类的共同抵抗也被报告了4。因此, 早期评估的抗真菌药敏性和检测耐药性在C. 光滑是至关重要的正确的, 有针对性的治疗, 以及在抗真菌的情况下, 以限制驱动器的抗菌素耐药性1,5,6. 建立一个有效的工作流程, 快速检测耐药菌株中的抗性生物标志物的验证性突变, 也有助于改善处方决定和临床结局。
抗真菌药易感性通常通过测量最小的抑制浓度 (MIC) 来评估, 这被定义为最低的药物浓度, 从而导致微生物的生长与无药物的生长相比显著降低。控制.临床和实验室标准研究所 (CLSI) 和欧洲抗菌药易感性检测委员会 (EUCAST) 有标准化的易感性测试方法, 以使 MIC 测定更准确和一致的7, 8。然而, 抗真菌 MIC 的效用仍然有限, 特别是对棘, 特别是在实验室比较, 在不同的方法和条件使用9。也有不确定的相关的中等收入与反应的棘治疗和无法区分重量 (或易感) 菌株与那些窝藏 FKS 突变 (棘耐药菌株)10,11。尽管有验证性基因 PCRs 和桑格测序的抗真菌抵抗标志, 结果的实现往往延迟, 由于缺乏同时检测多电阻标记5,12。因此, 在整个基因组 sequencing-based 分析的支持下, 同时检测基因组不同位置的抗性突变, 比目前的方法具有明显的优势。
整个基因组测序 (WGS) 已成功地实施, 以跟踪疾病传播在爆发期间, 以及在细菌和病毒的基因组范围内的风险评估和耐药性测试的方法13。核酸测序技术的最新进展使病原体的全基因组测序 (WGS) 在技术上和经济上都是可行的。DNA 测序提供了重要的优势比其他方法的病原体鉴定和定性在微生物学实验室使用14,15,16。首先, 它提供了一个具有高吞吐量、速度和质量的通用解决方案。测序可以应用于任何微生物, 并允许在地方或区域实验室的规模经济。其次, 它以 "经得起未来考验" 的方式生产数据, 可以在国家和国际两级进行比较。最后, WGS 在医学中的潜在效用已经通过包含参考基因组的公共数据库的快速增长而得到了增强, 这可以与包含其他临床和流行病学元数据的等效数据库相链接17 ,18。
最近的研究表明, WGS 用于鉴定临床分离的念珠菌的抗真菌性标志物的效用。10,19,20. 这主要是由于高通量台式器的可用性, 建立了生物信息学管道, 降低了测序的成本21,22。真菌 WGS 对桑格测序的优势在于, WGS 允许在一次运行中对多个基因组进行测序。此外, WGS 的念珠菌基因组可以识别药物靶点的新突变, 跟踪遗传进化, 以及临床相关序列类型的出现20,22,23。最重要的是, 在固有的多药耐药性的情况下, WGS 可以帮助及早发现治疗选择前的抗性突变22,24。
在这里, 我们研究了 WGS 的可行性筛选的突变相关的耐药性不同类别的抗真菌药物。我们提出了从 end-user 和诊断真菌实验室角度实施 WGS 的方法。我们在这项分析中包括三分离对培养从三独立的临床病例, 其中体外抗棘和 5-嘧啶在经过一段时间的抗真菌治疗后发展。

<table><tbody><tr><td>DensiCHECK Plus</td><td>BioM&amp;eacute;rieux Inc</td><td>K083536</td><td>Densitometer used for McFarland readings</td></tr><tr><td>Sensititre YeastOne</td><td>TREK Diagnostic Systems, Thermo Scientific</td><td>YO10</td><td>Commercial susceptibility assay plate with standard antifungal drugs.</td></tr><tr><td>Fisherbrand Disposable Inoculating Loops and Needles</td><td>Fisher Scientific, Thermo Fisher Scientific</td><td>22-363-605</td><td>Disposable plastic loops can be used directly from package. No flaming required.</td></tr><tr><td>Eppendorf Safe-Lock microcentrifuge tubes</td><td>Sigma Aldrich, Merck</td><td>T2795&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Volume 2.0 mL, natural&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>ZYMOLYASE 20T from Arthrobacter luteus</td><td>MP Biomedicals, LLC</td><td>8320921</td><td>Used for cell wall lysis of fungal isolate before&lt;br/&gt; DNA extraction</td></tr><tr><td>Wizard Genomic DNA Purification Kit&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>A1120</td><td>Does 100 DNA extractions</td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Thermo Fisher Scientific</td><td>P7589</td><td>&amp;nbsp;Picogreen reagent referred to as fluorescent dye in the protocol.&lt;br/&gt; Includes Lambda DNA standard and picogreen reagent for assay.</td></tr><tr><td>Nextera XT DNA Sample Preparation Kit</td><td>Illumina</td><td>FC-131-1096</td><td>Includes Box 1 and Box 2&amp;nbsp; reagents for 96 samples</td></tr><tr><td>Nextera XT Index Kit v2</td><td>Illumina</td><td>FC-131-2001,&lt;br/&gt; FC-131-2002,&lt;br/&gt; FC-131-2003,&lt;br/&gt; FC-131-2004</td><td>Index set A&lt;br/&gt; Index set B&lt;br/&gt; Index set C&lt;br/&gt; Index set D</td></tr><tr><td>NextSeq 500/550 High Output Kit v2&amp;nbsp;</td><td>Illumina</td><td>FC-404-2004</td><td>300 cycles, More than 250 samples per kit</td></tr><tr><td>NextSeq 500 Mid Output v2 Kit</td><td>Illumina</td><td>FC-404-2003</td><td>300 cycles, More than 130 samples per kit</td></tr><tr><td>PhiX Control Kit</td><td>Illumina</td><td>FC-110-3001</td><td>To arrange indices from Index kit in order</td></tr><tr><td>TruSeq Index Plate Fixture Kit</td><td></td><td>FC-130-1005</td><td>&amp;nbsp;2 Fixtures</td></tr><tr><td>KAPA Library Quantification Kit&lt;br/&gt; for Next-Generation Sequencing</td><td>KAPA Biosystems</td><td>KK4824</td><td>Includes premade standards, primers and MasterMix</td></tr><tr><td>Janus NGS Express Liquid handling system&amp;nbsp;</td><td>PerkinElmer</td><td>YJS4NGS</td><td>Used for DNA dilutions during sequencing</td></tr><tr><td>&amp;nbsp;0.8 mL Storage Plate</td><td>Thermo Scientific</td><td>AB0765B</td><td>MIDI Plate for DNA Library cleanup and&lt;br/&gt; normalisation</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>&amp;nbsp;Magnetic beads in solution for library purification</td></tr><tr><td>Magnetic Stand-96</td><td>Thermo Fisher Scientific</td><td>AM10027</td><td>Used for magnetic bead based DNA purification</td></tr><tr><td>OrbiShaker MP&amp;nbsp;</td><td>Benchmark Scientific</td><td>BT1502</td><td>96-well plate shaker with 4 platforms</td></tr><tr><td>Hard Shell PCR Plate</td><td>BioRad</td><td>HSP9601</td><td>Thin Wall, 96 Well</td></tr><tr><td>LightCycler 480 Instrument II&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>5015278001</td><td>Accomodates 96 well plate</td></tr><tr><td>Microseal &#039;B&#039; PCR Plate Sealing Film, adhesive, optical&amp;nbsp;</td><td>BioRad</td><td>&amp;nbsp;MSB1001</td><td>Clear 96-well plate sealers</td></tr><tr><td>CLC Genomics Workbench</td><td>Qiagen</td><td>CLCBio</td><td>Software for data analysis, Version 8</td></tr><tr><td>NextSeq500 instrument</td><td>Illumina&amp;nbsp;</td><td>Illumina&amp;nbsp;</td><td>Benchtop Sequencer used for next generation sequencing</td></tr></tbody></table>

whole genome sequencing of candida glabrata for detection of markers of antifungal drug resistance

光滑念珠菌可以快速获得导致耐药性的突变, 特别是对唑和棘。基因突变的识别是必不可少的, 因为检测到的电阻体外往往与临床失败相关。我们研究了使用全基因组测序 (WGS) 进行基因组分析抗真菌药物耐药性的可行性, 在C. 光滑中。目的是 torecognize 执行 WGS 的推动因素和障碍, 并衡量其有效性。本文概述了关键的质量控制检查点和 WGS 方法的基本组成部分, 以研究与降低抗真菌药易感性相关的遗传标记。它还估计了数据分析的准确性和测试的转过来时间。
通过抗真菌药敏试验测定了12例临床和一 ATCC光滑的表型敏感性。其中包括三个隔离对, 来自三患者, 在药物最小抑制浓度上升。在两对, 每对的第二个隔离开发了抵抗对棘。第三对的第二个分离开发了抵抗对 5-嘧啶。其余组成的易感性和唑耐药菌株。在抗棘、唑和 5-嘧啶抗性的基因中, 单核苷酸多态 (snp) 通过 WGS 的下一代测序, 在耐药菌株中得到证实。识别抗 真菌抗性基因的非同义 snp, 如FKS1、 FKS2、 CgPDR1、CgCDR1和FCY2 。总的来说, 平均98% 的 WGS 读取的C. 光滑孤立映射到参考基因组, 大约有75倍的读深度覆盖率。周转时间和费用与桑格测序相媲美。
总之, 在不需要多重 PCR/DNA 测序反应的情况下, WGS 的C. 光滑是可行的, 可以揭示抗性不同抗真菌药物类的临床重要基因突变。这是一个积极的步骤, 建立 WGS 能力的临床实验室, 同时检测抗真菌耐药性授予替代品。

十三c. 光滑包括c. 光滑ATCC 90030 和12株从临床真菌参考实验室 (分离 CMRL1 到 CMRL12), Westmead 医院, 悉尼进行了研究 (表 1)。其中包括三对分离 CMRL-1/CMRL-2, CMRL-3/CMRL-4 和 CMRL-5/CMRL-6 获得的抗真菌治疗前后没有流行病学联系, 他们之间的24 (表 1)。
采用 CLSI 解释断点对九抗真菌药物进行了测定, 即两性霉素...

Watch this Scientific Journal Video about Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance at JoVE.com

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

这项研究实施了全基因组测序分析的基因突变, 赋予抗真菌药物耐药性的念珠菌光滑。对棘、唑和 5-嘧啶的耐光滑的菌株进行了测序, 以说明方法。分离株的敏感性分布与基因特异性突变模式的存在或缺失相关。

念珠菌光滑全基因组测序检测抗真菌耐药性标志物

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations ...

whole-genome-sequencing-candida-glabrata-for-detection-markers

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

13.6K 次观看.  Westmead Hospital. 这项研究实施了全基因组测序分析的基因突变, 赋予抗真菌药物耐药性的念珠菌光滑。对棘、唑和 5-嘧啶的耐光滑的菌株进行了测序, 以说明方法。分离株的敏感性分布与基因特异性突变模式的存在或缺失相关。

视频: Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

遗传学

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

生物工程

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

医学

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

生物学

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&amp;RUN Method and Data Analysis Workflow

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&amp;RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&amp;RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&amp;RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&amp;RUN data analysis.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow

This protocol describes an experimental method and data analysis workflow for cleavage under targets and release using nuclease (CUT&amp;RUN) in the human fungal pathogen Candida albicans.

白色念珠菌中转录因子-DNA结合相互作用的全基因组分析:全面的CUT&amp;RUN方法和数据分析工作流程

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations ...

分析光滑念珠菌的小集落形成和耐药性

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

作者聚焦:光动力疗法作为在耐药 念珠菌 中诱导娇小菌落用于抗真菌研究的新方法

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

通过 Rose Bengal 介导的光动力疗法诱导念珠菌中的小菌落

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

念珠菌光滑全基因组测序检测抗真菌耐药性标志物

摘要

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此视频中的章节

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

在荧光蛋白融合的一代

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

癌症治疗监测的切伦科夫发光成像（CLI）

条形码酵母图书馆竞争力的基因屏幕

白念珠菌实时细胞外通量实时分析的生物能量学研究

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

白色念珠菌中转录因子-DNA结合相互作用的全基因组分析:全面的CUT&RUN方法和数据分析工作流程

通过 Rose Bengal 介导的光动力疗法诱导念珠菌中的小菌落

念珠菌光滑全基因组测序检测抗真菌耐药性标志物

摘要

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此视频中的章节

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

在荧光蛋白融合的一代

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

癌症治疗监测的切伦科夫发光成像（CLI）

条形码酵母图书馆竞争力的基因屏幕

白念珠菌实时细胞外通量实时分析的生物能量学研究

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

白色念珠菌中转录因子-DNA结合相互作用的全基因组分析:全面的CUT&RUN方法和数据分析工作流程

通过 Rose Bengal 介导的光动力疗法诱导念珠菌中的小菌落

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备