この仕事のための資金は、自然科学とカナダの工学研究会（NSERC）とSentinel生理活性紙ネットワークによって提供されました。

<ol>
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利害の衝突が宣言されません。

今日一般的な細菌の検出方法のほとんどは、低速（古典的な微生物）または技術的に（抗体、PCR）を求めています。そこで、検出ツールの次の世代は、スピードとシンプルさに向かって応えるべきだと思います。この目的のために、我々は、RNA切断し、蛍光シグナルの生成を介して細菌の存在を報告するためにシンプルなアッセイを開発するために使用することができ、蛍光シグナルDNAザイムを作成しました。注目のDNAザイムプローブ、RFD-EC1は、E.の成長時に生成CEMによって活性化される培養培地中の大腸菌 。提案手法は、検出の対象として細菌の粗細胞混合物を使用して、面倒なターゲットの抽出と増幅のステップをバイパスしているので、それは細菌検出のためのアッセイの非常に単純な、 &quot;ミックス·アンド·読む&quot;のタイプを設定するために使用することができます。私たちのDNAザイムの使用は、蛍光ベースの検出方法に限定されません。同じDNAザイムシステムアッセイcを使用して、例えば、比色検出有機色素と一緒にローリングサークル増幅を利用し、以前に報告された方法を用いて設計することができます。32我々は、より大きな運用の簡素化と新たな細菌のバイオセンサーを生成するための魅力的な道のような細菌を検出するためのDNAzymesの使用を予見する。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号またはモデル </td></tr><tr><td>寒天</td><td> BioShopカナダ</td><td> AGR003 </td></tr><tr><td>過硫酸アンモニウム（APS） </td><td> BioShopカナダ</td><td> AMP001 </td></tr><tr><td>アクリルアミド/ビスアクリルアミド（40％、29:1） </td><td> BioShopカナダ</td><td> ACR004 </td></tr><tr><td>ホウ酸</td><td> BioShopカナダ</td><td> BOR001 </td></tr><tr><td>ブロモフェノールブルー</td><td> Sigma-Aldrich社</td><td> B8026 </td></tr><tr><td> EDTA </td><td> EMサイエンス</td><td> EXO539-1 </td></tr><tr><td>塩酸</td><td> Sigma-Aldrich社</td><td> 38281 </td></tr><tr><td> HEPES </td><td> Bioshopカナダ</td><td> HEP001 </td></tr><tr><td> LBブロス</td><td> Sigma-Aldrich社</td><td> L3022 </td></tr><tr><td> MgCl 2を </td><td> EMD化学品</td><td> B10149-34 </td></tr><tr><td> NaClを</td><td> BioShopカナダ</td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> EMDケミカルズ</td><td> SXO255-1 </td></tr><tr><td> NaOHで</td><td> EMDケミカルズ</td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShopカナダ</td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShopカナダ</td><td> TEM001 </td></tr><tr><td>トリス塩基</td><td> BioShopカナダ</td><td> BST666 </td></tr><tr><td> Tween 20を</td><td> Sigma-Aldrich社</td><td> P9416 </td></tr><tr><td>尿素</td><td> BioShopカナダ</td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> Sigma-Aldrich社</td><td> X4126 </td></tr><tr><td> DNAコンセントレータ</td><td>サーモフィッシャーサイエンティフィック</td><td> SavantのDNA Speed​​Vac 120 </td></tr><tr><td>マイレクスフィルタユニット</td><td>ミリポア</td><td> SLGP033RS </td></tr><tr><td>ゲルローディングのヒント</tD&gt; <td> Diamed </td><td> TEC200EX-K </td></tr><tr><td> ImageQuantソフトウェア</td><td>分子動力学</td><td>バージョン5.0 </td></tr><tr><td>キムワイプ</td><td>キンバリークラークプロフェッショナル</td><td> 34705 </td></tr><tr><td>ミニボルテックス</td><td> VWR </td><td> 58816-121 </td></tr><tr><td>パラフィルム</td><td> Pechineyプラスチック包装</td><td> PM996 </td></tr><tr><td>ペトリ皿</td><td>フィッシャー·サイエンティフィック</td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettorプラス（ピペットガン） </td><td>コー​​ニング</td><td> 07764714 </td></tr><tr><td>石英キュベット</td><td>バリアン社</td><td> 66-100216-00 </td></tr><tr><td>シェーカー/インキュベーター</td><td>ニューブランズウィックサイエンティフィック</td><td>クラシックシリーズC24 </td></tr><tr><td>台風スキャナ</td><td> GEヘルスケア</td><td> 9200可変モード</td></tr><tr><td>遠心分離</td><td>ベックマン·コールター</td><td>アレグラX22-R </td></tr><tr><td> UV分光光度計</td><td>サーモフィッシャーサイエンティフィック</td><td> GenesysUV 10 </td></tr><tr><td>蛍光分光光度計</td><td>バリアン社</td><td>ケアリーのEclipse </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

発生はそれぞれ毎年死亡と入院の何百万人と同様に巨大な経済的損失のために食品を媒介と院内感染病原体のアカウントにリンクされています。進行中の流行の場所の影響を正確に早い段階で犯人の病原体を識別することができる分析法のためにますます需要のような流行と最小化の防止。高い特異性（興味のある唯一の細菌を検出）、高感度（低として検出することができる：病原体を検出するための効果的な方法の大きな配列があるが、それらのどれも理想的な検出方法のために具現すべて次の5つの最初の要件を満たすことはできません単一のライブ細菌細胞など）を、短時間-結果に（数分から数時間）、偉大な運用の簡素化（長いサンプリング手順や特殊な装置の使用の必要はありません）、および費用対効果。例えば、古典的な微生物学的方法は非常に特異的であるが、長い時間（DAを必要とする週間YS）が1 PCRおよび抗体ベースの技術は短い待機時間（数時間から数日）を提供しています。決定的な結果を取得するために、彼らは高価な試薬および/ ​​または洗練された装置の使用。結果2-4を必要とする、まだある前述の要件の1つまたは複数の特性の改善を提供する革新的な細菌の検出方法を開発に向けた科学研究のための大きな需要。当研究室では、細菌の検出を含むバイオセンシングアプリケーションのための分子プローブの新規クラスとしてDNAzymesの可能性を調べるに興味がある5。 DNAzymes（またdeoxyribozymesまたはDNA酵素として知られている）は人工の化学反応を触媒する機能を備えた一本鎖DNA分子を6-8、これらの分子は、多くの10とを含む広大なランダムシーケンスDNAプール（から単離することができる16個の配列）o &quot;はin vitroでの選択で &quot;として知られるプロセスによってR &quot;SELEX&quot;（指数関数的濃縮によるリガンドの系統的進化）。9月16日これらの特別なDNA分子は、広くバイオセンシングアプリケーションのための分子ツールとして近年検討されている6-8。 我々の研究室ではRNA-切断蛍光DNAzymes（RFDs、図1）を単離するためのインビトロでの選択の手順で確立したと分析のツールとしてRFDsの使用を検討した17から29 RFDsは、単一のリボヌクレオチドでDNA-RNAキメラ基質の切断を触媒する。蛍光体（F）およびクエンチャー（Q）によって挟まれているジャンクショ​​ン（R）。 FとQの近くには未開基板最小限の蛍光をレンダリングします。しかし、切断イベントは、蛍光強度の顕著な増加を伴っているFとQの分離につながる。 最近では、我々は、細菌を検出するためのRFDsを単離する方法を開発しました。5これらの特別なRFDsは&quot;に分離された粗混合物を使用すると、浄化の退屈なプロセスを回避します。自分の環境で、またはそれらが培養されているメディアの中の細菌の特定のタイプ（ 図1）によって残された原油の細胞外液（CEM）の存在下で、 &quot;点灯し、バイオセンサーの開発（完了するまで数ヶ月または数年かかる可能性がある）の対象となる微生物から適切なターゲットを識別します。外のターゲットを使用すると、細胞内のターゲットを取得する手順については、必要はありませんので、検定の手順はシンプルであることを意味します。 のみE.によって生成されたCEMの存在下で、上記のアプローチを使用して、我々は（ 図2A。FS1）は、その基板を切断するRFDを導出した大腸菌 （CEM-EC）。5このE. RFD-EC1（ 図2A）という名前の大腸菌検出RFDは、厳密にCEM-ECへの応答が他の細菌のホスト（ 図3）からのCEMに応答しなくなることが判明した。 ここでは、presaの複数形NT E.を設定するための重要な実験手順RFD-EC1と代表的な結果を用いて大腸菌の検出アッセイ。

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

Detection of Bacteria Using Fluorogenic DNAzymes

我々は最近、単純な細菌検出用蛍光アッセイを &quot;ミックス·アンド·読む&quot;を設定するために適用できる蛍光DNAザイムプローブを生成するための斬新なアプローチが報告されています。これらの特殊なDNAプローブは、それによって蛍光シグナルの生成に細菌検出を翻訳する特定の細菌によって産原油の細胞外液（CEM）の存在下で発色団修飾DNA-RNAキメラ基質の切断を触媒する。本報告では、特定のDNAザイムプローブは、 &quot;RFD-EC1&quot;を表すモデルの細菌の検出のために採用されている重要な実験手順を説明します。<em&gt;大腸菌（Escherichia coli）</em&gt;。

蛍光性DNAzymesを使用した細菌の検出

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

detection-of-bacteria-using-fluorogenic-dnazymes

Research

JoVE Journal

Immunology and Infection

18.8K ビュー.  McMaster University. 我々は最近、単純な細菌検出用蛍光アッセイを "ミックス·アンド·読む"を設定するために適用できる蛍光DNAザイムプローブを生成するための斬新なアプローチが報告されています。これらの特殊なDNAプローブは、それによって蛍光シグナルの生成に細菌検出を翻訳する特定の細菌によって産原油の細胞外液（CEM）の存在下で発色団修飾DNA-RNAキメ?...

ビデオ: 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).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

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

免疫・感染学

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.

オリゴヌクレオチド結合蛍光マイクロスフェアを使用して複雑な臨床及び環境試料中の細菌の多重検出

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.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

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.

16S rRNAのをターゲットジゴキシン標識DNAプローブを用いたパラフィン包埋組織内細菌のその場検出で

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.

分子インプリントを用いたバイオ マーカーの超高感度検出による静電容量センサー

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.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

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

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.

フローサイトメトリーを用いた経月細菌に結合するヒトノロウイルスウイルス様粒子の定量化

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.

嫌気性増殖細菌によるマウス膣コロニー形成モデル

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

Fluorescent Reporter 定量による全臓器病原体追跡(英語)

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.

著者スポットライト:組織における抗生物質と免疫系に対する細菌の応答を解明する

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

微生物感染症の高解像度三次元全臓器断層撮影法

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