Name: rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry
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Description: Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

<html> 1，血培养液标为"正" <ol><li>从连续监测温育箱中取出信号血培养瓶，并把它变成一个生物安全柜。注意:瓶可以含有有害微生物和普遍预防需要遵循。由于采样传染性气溶胶的危险性，所有抽样程序必须在生物安全II级层流柜中进行。 </li></ol> 2，革兰氏染色准备<ol><li>从信号血培养肉汤按当地机构的协议准备革兰氏染色。注意:当革兰氏阴性生物体的显微鉴定的血培养液处理为每个下面的方法。当革兰氏阳性菌鉴定，基因靶向识别和抗性标记另一种分子生物学方法应用于肉汤（本文中未提及）13。 </li></ol>标记的血培养肉汤电子"&gt; 3。转移到血清分离管<ol><li>轻轻颠倒2-3倍混合血培养瓶。 </li><li>在生物安全柜连接10 ml注射器的安全血液传输设备。 </li><li>附加的血液转移装置至血液培养瓶和撤回将5ml培养液进入注射器。转让吸气公元前清汤倒入血清分离管。 </li></ol>血培养液对4。浓度<ol><li>离心血清分离管中以1250×g离心15分钟，除去大量的红血细胞。 </li><li>吸出物，并用无菌移液管小心离开紧接凝胶/液体界面上方大约1毫升的血沉棕黄层的弃上清。注意:需氧瓶显示的红血细胞的明确分离到管的底部与凝胶/液界面出现白色不透明的颜色。相反，当加到凝胶/液体界面显示为深红色由于裂解红血细胞成分在上清液中余下的悬浮裂解厌氧血培养肉汤。 </li></ol> 5，重复离心洗涤步骤<ol><li>轻轻混合，最后加入1ml的血沉棕黄层的流体上面的凝胶界面，用无菌移液管，然后将整个体积转移到1.5ml的离心管中。离心新管中于288×g离心30秒。 </li><li>使用一次性塑料1毫升移液管插入一个新的1.5 ml微量离心管转移上清液并丢弃管与沉淀。注意:这是在该步骤中小心地避免粒料的转移是至关重要的。这是用于从厌氧公元前瓶子被处理的上清液仍颜料和颗粒并不总是清楚地看到试样尤为重要。 </li></ol>残余细胞6。裂解</p&gt; <ol><li>离心机的样品在18407×g离心1分钟，然后吸出物（和废弃），使用细尖移液管离开尽可能少的残余液体尽可能不破坏沉淀上清液。 </li><li>通过上下吹打重悬沉淀物残留在1ml无菌无DNA酶和RNA酶的游离水。注:有些作者所描述的使用酒精溶液代替无菌水重悬这使得细菌非可行的沉淀。 </li><li>离心悬浮液于18,407×g离心1分钟。 </li></ol>细菌蛋白质的7。提取<ol><li>吸弃上清液，用吸用细尖吸管确保尽可能多的液体尽量除去了。 </li><li>重悬沉淀于10μl甲酸（70％V / V）。注:甲酸可影响身体，如果它被吸入或者如果它与皮肤接触。在准备或操作的解决方案是为recommended员工使用通风柜除了个人防护装备。 </li><li>拌匀使用移液管，以确保均匀的悬浮液。使用移液管尖端的物理破坏的颗粒可能是必要的，以确保更均匀的溶液。 </li></ol> 8，准备MALDI-TOF的目标板<ol><li>接种干净的MALDI-TOF靶板与2μl每个目标点的准备暂停。制备3靶位点的每个样品，以克服失败读取偶然问题。 </li><li>让MALDI-TOF靶板干燥。干燥速率可以增大通过将板放在通风橱中的边缘，以最大化整个板的气流。 </li><li>覆盖各干燥点与1微升的基质溶液（10 mg / ml的α-氰基-4 - 羟基肉桂酸（HCCA），50％乙腈，2.5％三氟乙酸）。注:基质溶液可以影响身体，如果它被吸入或者如果它进入其所含克拉皮肤。在准备或操作的解决方案，建议员工使用通风柜除了个人防护装备。 </li><li>允许的MALDI-TOF靶板上干燥，如上所述的烟橱的边缘。确保均匀制剂填充每个平板上的目标点。 </li></ol> 9。地方MALDI-TOF靶板进入质谱仪<ol><li>将MALDI-TOF目标板进入MALDI-TOF质谱仪。确保板坐在弹簧加载板齐平。注意:请不要将目标板，直到所有的目标点都干。 </li><li>确保橡胶密封件是干净的线头，灰尘和毛发，使可以创建所需的真空条件。 </li><li>关闭MALDI-TOF阶段盖子，然后按下仪器前面的"输入/输出"按钮。盖子现在将锁定并允许创建必要的真空度。 </li></ol> 10，MALDI-TOF MS Spectra收购<ol><li>打开MALDI生物分型和FlexControl软件。 </li><li>内的打字软件请从下拉菜单中选择"新分类"标题为"文件"。将项目命名，然后选择"新建"。选择"下一步"的打字软件内完成设置。 </li><li>在控制软件窗口识别屏幕上的图形目标板。突出显示MALDI-TOF目标点正在使用通过移动鼠标在接种点，同时按住鼠标左键。 </li><li>用鼠标右键单击任意位置上选定的目标位置，然后选择从下拉菜单中选择"添加分析物"。 </li><li>加血培养鉴定的详细信息的"ID"列中为每个目标点，然后点击"下一步"完成时。 </li><li>选择商业"分类学"光谱数据库，然后选择"下一步"。注:Labo的ratories可以利用额外的内部或商业数据库，以提高参考光谱的数目。 </li><li>点击"完成"和MALDI-TOF MS将开始产生光谱。注意:MALDI-TOF设置为按制造商的设定（线性正模式，60赫兹激光频率，20千伏的加速电压，16.7千伏电压IS2，170毫微秒的提取延迟和2,000-20,137 m / z范围）。 </li><li>如果MALDI控制软件无法生成的山峰，人工采集的光谱可以进行（没有描述的方法）。 </li></ol> 11发布分析MALDI-TOF MS分数的解释<ol><li>一旦光谱采集和分析完成后，请在打字的软件旁边的物种鉴定的"+"按钮，展开对每个目标的识别列表。 </li><li>研究了前5名标识，并指出，如果前5名是相似的。不和谐的前5个属，分数≥1.7相同的目标点引发混合肉汤的可能性。注:MALDI-TOF技术具有敏感性差检测混合肉汤。例如，当发酵液包含混合物种高置信度得分可以被识别为混合种中的一种。 </li><li>当得分≥2.0是注意到在目标点的最高匹配，报告该物种。 </li><li>当最高得分上的目标点为≥1.7，但&lt;2.0，只报告了属。如果前5名标识的附加条件是一致的，然后向种的水平。 </li></ol> 12，去除MALDI-TOF靶板<ol><li>当所有的光谱是完全按MALDI-TOF机器上的"IN / OUT"按钮。 </li><li>打开MALDI-TOF靶板插座和移除MALDI-TOF靶板。 </li><li>如果使用的是可重复使用的MALDI-TOF靶板，用酒精清洁所有目标点和MALDI-TOF板储存在室温，一旦它的清洁和干燥。 </li></ol>

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运用MALDI-TOF MS到后离心步骤都与足够的照顾不进行重新混合分离的成分血培养肉汤时，这一点很重要。特别重要的是，除去血培养成分和人类细胞的蛋白质，包括血红蛋白，这可能会产生尖峰与MALDI-TOF光谱干扰。 虽然MS制造商推荐切得分≥2.0的物种和≥1.7，属鉴定，其他报告表明较低的对数得分（范围≥1.4至≥1.6）时，适用于公元前肉汤10,12,14-18可以实现。在临床微?...

由于细菌患者的血流感染（BSI）继续有较高的住院死亡率，范围从6-48％1。适当的经验性抗生素交付促进生存和在严重脓毒症患者的子集，每个小时的延迟，以适当的治疗相关的生存2,3下降。因此，临床实验室的主要目标是快速检测，识别和沟通细菌在血培养存在的，告知临床决策。它已经证明，微生物实验室对抗菌治疗在报告的革兰氏染色4，最近的一次影响最大，观察研究证明，飞行质谱基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）直接在血培养液进行影响超过三分之一BSI引起的革兰阴性菌5订明。 ，6,7微生物的鉴定。该技术是目前公认，并已集成到许多实验室进行快速准确的识别微生物中分离固体培养基上6,8。直接应用MALDI-TOF MS对血培养（BC）的肉汤已经表示"积极"的微生物，因为获得低成本的早期识别微生物的潜在的临床医生和实验室管理人员的上诉。 直接应用MALDI-TOF MS的血培养液的临床应用已被广泛观察到的灵敏度，当与鉴定标准表型文化为基础的方法，以成功识别革兰阴性菌不等的报告相比，限制从47-98.9 ％9-11。在灵敏度的变化可能涉及对BC肉汤组成，初始细菌浓度，变化的样品制备方法，以及在研究人群9中遇到的革兰氏阴性生物体的数组。与这些其他的发布的协议相比，这里提出的方法可避免使用乙醇，氯化铵或附加的（非矩阵）乙腈。其结果是将细菌沉淀将保持存活（直到蛋白提取的点），允许潜在敏感性表型试验方法，可直接应用于这些微生物在液体培养基。此外，所提出的方法已经被证明是便宜的，可靠的，快速，细菌鉴定内的血培养物革兰氏染色结果25分钟内可用的，以最小的手上的时间12。 这种方法是利用甲酸提取一个简单的内部自旋裂解方案直接应用到血培养阳性肉汤识别革兰氏阴性bacteria与MALDI-TOF MS技术。

<table><tbody><tr><td>BACTEC Plus Aerobic/F Medium</td><td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td><td>442192</td><td></td></tr><tr><td>BACTEC Lytic/10 Anaerobic/F Medium</td><td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td><td>442265</td><td></td></tr><tr><td>BACTEC Peds Plus Medium</td><td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td><td>442194</td><td></td></tr><tr><td>Vacutainer - Blood transfer device</td><td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td><td>364880</td><td>Single use sampling device reducing the risk of needlestick injury</td></tr><tr><td>Vacutainer SST 5.0 ml, Advance plus</td><td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td><td>367954</td><td>Serum separating tube</td></tr><tr><td>Syringe 10 ml</td><td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td><td>302143</td><td></td></tr><tr><td>Transfer pipette</td><td>Samco, USA</td><td>222-20S</td><td></td></tr><tr><td>Transfer pipette (fine tipped)</td><td>Samco, USA</td><td>232-20S</td><td></td></tr><tr><td>Microcentrifuge tube (2.0 ml)</td><td>Eppendorf, Hamburg, Germany</td><td>0030.120.094</td><td></td></tr><tr><td>Sterile water (DNAse and RNAse free)</td><td>Life Technologies, Carlsbad, California, USA</td><td>10977-015</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich, St. Louis, Missouri, USA</td><td>F0507</td><td></td></tr><tr><td>Matrix solution&amp;nbsp;</td><td>Bruker Daltonics, Bremen Germany</td><td>285074</td><td>10 mg/ml &amp;alpha;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td></tr><tr><td>Benchtop microflex LT MALDI-TOF MS</td><td>Bruker Daltonics, Bremen Germany</td><td></td><td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td></tr></tbody></table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

临床微生物学实验室的一个重要作用是提供快速鉴定细菌，导致血液感染的。传统的识别需要信号血培养肉汤识别可用的亚文化不仅已经成熟后，在固体琼脂菌落。 MALDI-TOF MS是用于识别大多数临床相关的细菌时，适用于菌落在固体培养基上可靠的，快速的方法。 MALDI-TOF MS直接到血培养液中的应用是一个有吸引力的方法，因为它有潜力加速了细菌的种属鉴定，提高临床管理。然而，要克服的一个重要问题是，在分析前除去包含在血培养标本，如不去除，干扰MS谱，并可能导致判别识别分数不足或低干扰的树脂，蛋白质和血红蛋白。此外，有必要集中硝化细菌IA开发足够的质量谱。该方法描述了浓缩，纯化和提取的革兰氏阴性细菌，允许从一个信号血液培养液中的早期识别细菌。

所生成的MALDI-TOF MS谱进行比较，以光谱的集成参考数据库。对数分数分配给测试分离和参考数据库隔离之间的比赛的信心，用分数≥1.7所需的鉴定可能要属水平（ 图1），≥2.0为可能的识别物种（ 图的建议2）。报告以种的水平时，得分≥1.7和所述第一5标识所匹配的打字软件是一致的（ 图1）12。图3演示了当混合物种的血培养液进行分析?...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

的飞行（MALDI-TOF）质谱（MS），直接向血培养肉汤基质辅助激光解吸/电离飞行时间质谱的应用加快细菌的鉴定。该方法是一种快速和可靠的方法，可直接从血培养肉汤鉴定革兰氏阴性细菌。

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

24.3K 次观看.  Westmead Hospital. 的飞行（MALDI-TOF）质谱（MS），直接向血培养肉汤基质辅助激光解吸/电离飞行时间质谱的应用加快细菌的鉴定。该方法是一种快速和可靠的方法，可直接从血培养肉汤鉴定革兰氏阴性细菌。 

视频: Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

器的子类型<em&gt;空肠弯曲菌</em&gt; SSP。<em&gt; doylei</em&gt;分离株使用基于光谱质谱PhyloProteomics（MSPP）

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

科研

遗传学

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

生物化学

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

免疫与感染

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

化学

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

生物学

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

血培养瓶中的加速药敏试验

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

作者聚焦：通过直接鉴定血培养瓶中的革兰氏阴性菌来提高诊断准确性

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

摘要

探索更多视频

此视频中的章节

器的子类型

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

为快速细菌鉴定和药敏试验的准备血培养微丸

肽聚糖的液相色谱质谱和生物信息学半定量分析

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

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此视频中的章节

器的子类型

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

为快速细菌鉴定和药敏试验的准备血培养微丸

肽聚糖的液相色谱质谱和生物信息学半定量分析

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

使用自动微生物鉴定系统进行直接微生物鉴定，以促进 EUCAST RAST 方法，无需质谱法

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作