自然科学和工程研究理事会（NSERC）的加拿大与Sentinel生物活性纸网络，为这项工作提供了资金。

<ol>
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没有利益冲突的声明。

今天最常见的细菌检测方法是缓慢的（经典的微生物）或（抗体，PCR）技术要求高。因此，我们认为，应配合向速度和简单的检测工具的下一代。为此，我们已经建立了一个RNA裂解和荧光信号核酶可用于开发简单的检测报告通过荧光信号的产生细菌的存在。功能核酶探针，RFD的EC1的，被激活的大肠杆菌的生长过程中产生的CEM在培养基中的大肠杆菌 。由于我们的方法使用细菌的原油外混合物作为检测目标和绕过艰苦的目标提取和扩增步骤，它可以用来设置很简单，“混合和阅读”类型的细菌检测的检测。使用我们的核酶不仅限于基于荧光检测方法。例如，比色法检测使用相同的核酶系统检测Ç设计使用以前报道的方法，利用有机染料结合滚环扩增32，我们预计使用细菌检测脱氧核酶作为一个有吸引力的途径，产生新的细菌生物传感器具有更大的操作简便。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号或型号 </td></tr><tr><td>洋菜</td><td> BioShop加拿大</td><td> AGR003 </td></tr><tr><td>过硫酸铵（APS） </td><td> BioShop加拿大</td><td> AMP001 </td></tr><tr><td>丙烯酰胺/双丙烯酰胺（40％，29:1） </td><td> BioShop加拿大</td><td> ACR004 </td></tr><tr><td>硼酸</td><td> BioShop加拿大</td><td> BOR001 </td></tr><tr><td>溴酚蓝</td><td> Sigma-Aldrich公司</td><td> B8026 </td></tr><tr><td> EDTA的</td><td>电磁学</td><td> EXO539-1 </td></tr><tr><td>盐酸</td><td> Sigma-Aldrich公司</td><td> 38281 </td></tr><tr><td>肝素钠</td><td> bioshop加拿大</td><td> HEP001 </td></tr><tr><td> LB培养基</td><td> Sigma-Aldrich公司</td><td> L3022 </td></tr><tr><td> MgCl 2的 </td><td>电解二氧化锰化学制品</td><td> B10149-34 </td></tr><tr><td>氯化钠</td><td> BioShop加拿大</td><td> SOD002 </td></tr><tr><td>醋酸钠</td><td>电解二氧化锰化学品</td><td> SXO255-1 </td></tr><tr><td>氢氧化钠</td><td>电解二氧化锰化学品</td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShop加拿大</td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShop加拿大</td><td> TEM001 </td></tr><tr><td> Tris碱</td><td> BioShop加拿大</td><td> BST666 </td></tr><tr><td>吐温20 </td><td> Sigma-Aldrich公司</td><td> P9416 </td></tr><tr><td>尿素</td><td> BioShop加拿大</td><td> URE001 </td></tr><tr><td> xylenecyanol法郎</td><td> Sigma-Aldrich公司</td><td> X4126 </td></tr><tr><td> DNA的浓缩</td><td> Thermo Scientific的</td><td>学者的DNA Speed​​Vac离心120 </td></tr><tr><td> millex过滤器单元</td><td> Millipore公司</td><td> SLGP033RS </td></tr><tr><td>凝胶加载提示</tD&gt; <td> Diamed </td><td> TEC200EX - K </td></tr><tr><td> ImageQuant软件</td><td>分子动力学</td><td>版本5.0 </td></tr><tr><td> kimwipes </td><td>金佰利专业</td><td> 34705 </td></tr><tr><td>迷你Vortexer </td><td>厂商VWR </td><td> 58816-121 </td></tr><tr><td>封口膜</td><td>佩希内塑料包装</td><td> PM996 </td></tr><tr><td>培养皿</td><td> Fisher Scientific则</td><td> fisherbrand 08-757-12 </td></tr><tr><td>加stripettor（移液枪） </td><td>康宁</td><td> 07764714 </td></tr><tr><td>石英比色皿</td><td>瓦里安公司</td><td> 66-100216-00 </td></tr><tr><td>沙克尔/孵化器</td><td>新不伦瑞克省科技</td><td>经典系列C24 </td></tr><tr><td>台风扫描器</td><td> GE医疗集团</td><td> 9200变模式</td></tr><tr><td>离心分离</td><td>贝克曼</td><td> Allegra的X22-R的</td></tr><tr><td>紫外分光光度计</td><td> Thermo Scientific的</td><td> GenesysUV 10 </td></tr><tr><td>荧光分光光度计</td><td>瓦里安公司</td><td>卡里的Eclipse </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

挂钩经由食物传染的医院获得性病原体的帐户和百万人死亡，住院以及巨大的经济损失每一年的爆发。预防此类疾病暴发和持续流行的地方不断增加的需求分析方法，可以准确地找出罪魁祸首病原体在最早阶段的影响最小。虽然是大阵的病原体检测的有效方法，他们没有能满足以下五个体现为一种理想的检测方法总理要求：高特异性（检测只利益细菌），灵敏度高（能够检测低作为一个单一的活细菌细胞），时间短，结果（几分钟到几小时），伟大的操作简便（无需冗长的抽样程序和使用专门的设备），和成本效益。例如，传统的微生物方法具有高度特异性，但需要很长一段时间（DAYS到几周）获得一个明确的结果。1聚合酶链反应和抗体为基础的技术，提供更短的等待时间（数小时至数天），但它们需要使用昂贵的试剂和/或设备精良。2-4因此，仍然存在为对科研开发创新的细菌检测方法，提供一个或多个上述要求的改进特性的需求量很大。我们的实验室是研究作为一类新型生物传感应用，包括细菌检测的分子探针的脱氧核酶潜在兴趣。 脱氧核酶（也称为核酶或DNA酶）是人造的单链DNA分子具有催化化学反应的能力。6-8这些分子可以从一个巨大的随机DNA序列池（其中包含多达10个隔离16个单独序列的过程）称为“ 体外选择”OR“的SELEX”（9-16这些特殊的DNA分子系统进化指数富集的配体）。已广泛研究，近年来随着分子生物传感应用工具。6-8 我们的实验室已经建立了隔离的RNA裂解荧光脱氧核酶（RFDS;图1） 在体外选择程序和调查作为分析工具的使用RFDS 17-29 RFDS催化裂解的DNA-RNA嵌合基板在一个单核苷酸。交界处（R）的两侧，是由荧光（F）和（Q）的猝灭。接近的F和Q呈现的uncleaved的基板的最小荧光。然而，卵裂事件，导致分离的F和Q，这是伴随着荧光强度显着增加。 最近，我们开发的RFDS隔离细菌检测的方法。5这些特殊RFDS的分离“点亮“在原油外混合背后由特定类型的细菌在他们的环境，或在他们所培养的媒体（ 图1）离开（CEM）的存在。原油混合使用，绕过繁琐的净化过程，使用外的目标，确定一个合适的目标利益为生物传感器的发展微生物（这可能需要数月或数年才能完成）。意味着化验过程很简单，因为那里是没有需要的步骤，以获取细胞内的目标。 利用上述方法，我们得出一个RFD的切割底物只由大肠杆菌产生的CEM（FS1的。图2A） 大肠杆菌 （CEM - EC）。5这五大肠杆菌检测RFD的，名为RFD的EC1的（ 图2A），被认为是严格CEM - EC，但没有响应从其他细菌的主机（ 图3）商业电子讯息。 在这里，我们预先选择的窗口新台币设立大肠杆菌的关键实验程序， 大肠杆菌检测实验使用RFD的EC1的和有代表性的结果。

Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

Detection of Bacteria Using Fluorogenic DNAzymes

我们最近报道一种新的方法，产生荧光脱氧核酶探针，可用于设立一个简单的“混合和阅读的”细菌检测荧光法。这些特殊的DNA探针，催化裂解1生色修饰DNA-RNA的存在原油外的一个特定的细菌，从而转化为细菌检测荧光信号的产生，产生的混合物（CEM）的嵌合基板。在这份报告中，我们将描述模型的细菌检测采用特定的核酶探针记“RFD的EC1的”为重点的实验程序，<em&gt;大肠埃希氏菌（大肠杆菌）</em&gt;。

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

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JoVE Journal

Immunology and Infection

18.8K 次观看.  McMaster University. 我们最近报道一种新的方法，产生荧光脱氧核酶探针，可用于设立一个简单的“混合和阅读的”细菌检测荧光法。这些特殊的DNA探针，催化裂解1生色修饰DNA-RNA的存在原油外的一个特定的细菌，从而转化为细菌检测荧光信号的产生，产生的混合物（CEM）的嵌合基板。在这份报告中，我们将描述模型的细菌检测采用特定的核酶探针记“RFD的EC1的”为重点的实验程...

视频: Detection of Bacteria Using Fluorogenic DNAzymes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

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

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

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 ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

细菌中无机聚磷酸盐的检测

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

厌氧菌小鼠阴道定植模型

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

通过荧光报告基因定量进行全器官病原体追踪

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

作者聚焦：揭示细菌对组织中抗生素和免疫系统的反应

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染的高分辨率三维全器官断层扫描

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...