この作品は、ビル＆メリンダ·ゲイツ財団（PneuCarriageプロジェクト、グラント52099）とビクトリア州政府の運用インフラストラクチャのサポートプログラムによってサポートされていました。原稿の重要な読書のためのアイリーン·ダン、ジャネット·ストラッチャン、キム·ハレに感謝します。

1。タイピングのための細胞懸濁液を準備<ol><li>無菌使い捨て1μLループを使用して、固体の非選択的なウマ血液寒天培地上に成長させた肺炎球菌の新鮮な一夜純粋培養の小さなスイープを取り、100μlのハートインフュージョン（HI）の培養液に接種する。サスペンションは、単に目に見えて濁ったように見えるはずです。静かに乳化するチューブを攪拌。 注：そのようなブレインハートインフュージョンブロスのような他の媒体も適切であり得る。血清の添加（ 例えば 、最終濃度10％（v / v）のウマ血清）使用前に約1時間37℃でインキュベートして反応を増強するために使用され得る。 </li></ol> 2。細胞懸濁液の密度をチェックするステップ1.1で調製した細胞懸濁液はまた、陰性対照として使用され、タイピング血清を添加する前に細胞密度をチェックすべきである。 <ol><li> 1μlのループを用いて、ガラスヒーターの上に、ステップ1.1で調製した接種材料の液滴を転送するS顕微鏡スライド。 </li><li>ピンセットを使用して、サスペンションに小さな円形のカバーガラスを配置します。 </li><li>位相コントラスト設定を使用して、サスペンション（ 図1A）の密度をチェックするために、400倍の倍率で顕微鏡的に準備を観察します。 <ol><li>接種物（ 図2A）が重すぎる場合は、それを希釈するためのステップ1.1で行われた細胞懸濁液に、よりスープを追加します。優しく再び混ぜる。 </li><li>接種材料が軽すぎる場合には、例えば、顕微鏡視野（ 図2B）に表示&lt;50セルがあると、ステップ1.1で行われた、穏やかに混合細胞懸濁液の準備に多くの細菌を追加します。 注：経験により、細胞懸濁液の密度が揺れ、それだけで目に見えて濁っように見えるべき光にかざすことで判断することができる。 </li></ol></li><li>好適な数の細胞が顕微鏡視野内に表示されるまで繰り返して2.1から2.3を繰り返します。 注意：ガラススライドとcoverslIPSは、鋭利物危険が。使用後は、スライドとカバーガラスを感染性物質で汚染されており、バイオハザードシャープコンテナに配置しなければならない。 </li></ol> 3。タイピング<ol><li>顕微鏡スライドガラス上に（ 例えば 1μLループを使用して）準備された接種物〜1μLを転送します。バイオハザードシャープコンテナにループを捨てる。 </li><li>スライド上に室温抗血清を等量の（1μlの白金耳）を追加し、両方のループとよく下がる混ぜる。 </li><li>ピンセットを使用して、ドロップに触れないように注意しながら、サスペンション上に小さな円形のカバーガラスを配置します。乾燥からスライド上の流体を防止するために、すぐに抗血清を添加した後サスペンションにカバースリップを配置します。 </li><li>顕微鏡を用いて、400倍の倍率で位相差の下でサスペンションを観察します。肺炎球菌細胞のほとんど又は全く「膨潤」は40で見ることができる場合、負の膨張反応が観察され無血清添加（陰性対照）を有する肺炎球菌細胞懸濁液において観察されるものに類似0X倍率。細胞は陰性対照中の細胞と比較した場合、拡大または「膨潤」と表示されたとき、より可視正の膨張反応が観察される。 注：曖昧反応は室温で5〜10分間のインキュベーション後、またはそれ以上の抗血清との反応を繰り返すことにより反応を検査することによって、例えば、さらに調査されるべきである。 </li><li>陽性反応が観察されるまで、個々の抗血清プールの連続した​​テストに肺炎球菌血清型別を行っています。代わりに、プールは血清型のローカル発生率に応じて、「ラウンド」で適用することができる。 注：「ラウンド」でのテストでは、最も一般的な血清型に対する抗体を含むプールは労力とコストを最小限に抑え、最初にテストされていることを保証します。複数のプールを同時にテストもINTERPに役に立つ追加ネガティブコントロールを提供し、retation。特定の「ラウンド」内のすべてのプールが負の場合、プールのその後のラウンドでの分離株をテストします。 </li><li>反応性プール内に含まれる適切な型またはグループ抗血清を用いて分離株をテストします。必要に応じて、さらに血清型を区別する因子抗血清を使用しています。 注意：メーカーから供給される肺炎球菌血清への鍵は、適切なプール、タイプ、グループ、または因子抗血清および（任意の交差反応を含む）の結果の解釈の選択に使用されている。 </li><li>否定的な結果は、すべてのプールで得られている場合は、（91血清型21に対する抗体が含まれている）Omniserum試薬と分離株をテストします。肺炎球菌を試験するときに正の膨張反応が依然として観察されない場合Omniserum試薬と分離する分離株は、ほとんどまたは全くカプセルを発現する、又は血清型に対する抗体がOmniserumに示されていないことをしているため、これはあり得る。後者のカテゴリでは、新たな血清型を含むことができる。 </li> &lt;/ OL&gt;

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著者らは、開示することは何もありません。

この技術における重要なステップは、純粋培養物を培養液に細菌細胞の適切な数の加算と一緒に懸濁液を調製するために使用されたことを確認する。これは、寒天プレート上のコロニー増殖と形態が異なる肺炎球菌分離株の間で変動することに留意すべきである。中には、ループをピックオフし、培養液中でよく乳化していない困難な場合がガサガサコロニーを産生する。細胞懸濁液を調製する場合、この理由のため、細菌のより大きな掃引は必要とされ得る。いずれの場合も、それらの相対的な大きさは抗血清での細胞と比較することができるので、個々の細胞ではなく、凝集体は、陰性対照に加え明らかであることが肝要である。また、あまりにも多くの細菌細胞は、莢膜抗原の過剰膨張反応10を阻害する可能性が偽陰性反応をもたらすことがあります。このため、適切な細胞密度を有する細菌懸濁液（ 図1）を作成することが重要である。 SUS年金はホルマリン濃縮を数滴添加することによって、数ヶ月間保存することができる。これは簡単にバッチテストを可能にし、また、実験室院内感染のリスクを低減スープを殺菌するが、これは毒性、腐食性、発癌性、増感、及び可燃性であるホルマリンを扱うことが安全性リスク、比較検討しなければならないが。さらに、我々はいくつかの分離株は、ホルマリン（データは示さず）の存在下で弱い反応を示すことを観察した。 膨化反応は顕微鏡を用いて調べた。位相コントラストの使用は、カプセルの視認性を向上させます。いくつかの研究室では、反応1,000 Xの倍率14,16の油浸を使用して表示される。さらに、いくつかの研究室は、さらに、陽性反応10、16、22の視認性を向上させるためにカウンター染色（メチレンブルー）を使用する。ここで紹介する方法は、オイル-Iなしで見られるmmersionおよび400X倍率15、17アンダー。この方法は、迅速、簡単で23を読み取ることが比較的容易である。 それは、抗血清およびテスト手順が順番にあることを確認するために良好な内部品質管理手順を維持することが重要である。外部品質保証プログラムへの参加は、多くの場合、有益である。 肺炎球菌の血清型のための膨張反応の主な制限は、必要な抗血清のコストが高いことである。しかしながら、これは、それぞれの反応に用いた抗血清の少量に起因する上記のプロトコルに最小化される。いくつかの他の方法は、肺炎球菌23,24血清型について記載されている。しかし、膨化は金本位制のままと新たな方法の開発が膨張反応23に対して標準化を必要とします。適切な抗血清を用いて、膨張反応はまた、他のキャップを識別し、入力するために利用することができるULE細菌25を製造する。膨張反応は、現在および新興の肺炎球菌血清型の特性だけでなく、ワクチンの有効性とキャリッジと病気に対するワクチン接種の長期的な影響に関する情報を提供する上で役立つ。

肺炎連鎖球菌 （肺炎球菌）は、病気の負担は資源の乏しい国1で主に落下して、毎年5歳未満の推定80万子供の死を担当しています。肺炎球菌疾患は、敗血症、髄膜炎や肺炎などの重篤な2生命を脅かす感染症に急性中耳炎などの局所的な感染症の重症度の範囲である。肺炎球菌の90莢膜血清型3,4に記載されている。現在の肺炎球菌ワクチンは、侵襲性疾患5、6の大部分の原因である血清型に対する保護を提供する。しかし、ワクチンの広範な使用は、鼻咽頭保菌および侵襲性疾患7,8の両方において、nonvaccine血清型によってワクチンに含まれる血清型の交換に関連している。これは疫学のための血清型の重要性を強調して監視と長期的なワクチン影響調査9。 肺炎球菌のタイピングのためのゴールドスタンダードメソッドは、まず1902年にニューフェルドで記述膨張反応として知られている莢膜反応試験、（オーストリアの10を参照）である。一致度の高い研究室間、及び膨張反応および他の血清型別法11-13の間に見出された。膨張反応10、14〜16のいくつかのバリエーションがあります。ここで説明する方法は、カウンタ汚れや油浸レンズの使用を必要としない、むしろ、調製された試料の低下がタイピング血清と混合し、400Xの倍率で直ちに調べた。 膨張反応は、通常、市販の抗血清を用いて行われる。ここで説明する方法は、より費用効果的にする、いくつかの他の方法を図16、図17と比較してより少ない抗血清を使用する。一度ISOL食べたが、肺炎球菌と同定される陽性反応が観察されるまで、それを順次抗血清プールでテストされています。各プールの抗血清は、91肺炎球菌血清型18に対して産生された抗血清の異なる混合物が含まれています。プールが確立されると、反応性のプールに含まれている個々のタイプ及びグループ抗血清を順次個別に試験する。グループ抗血清（ 例えば 、グループ22抗血清は血清型22Fと22Aと反応して）特定のグループ内の全ての血清型と反応するのに対し、タイプの抗血清は、一般的に、単一の血清型（ 例えば 1型抗血清は血清型1と反応する）と反応する。グループ内のいくつかの血清型はさらに因子抗血清（ 例えば血清型22Fが要因22bに反応するが、しない22C）を使用して区別されます。血清を19,20と判定されるまでこの場合、試験は、全ての関連する因子の抗血清を用いて継続される。膨張反応は、単純かつ迅速であり、14リットル任意で行うことができる良質顕微鏡と抗血清の適切なセットを搭載していaboratory。

<table border="0" cellpadding="0" cellspacing="0" width="1305">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Neufeld) antisera</td>
			<td>SSI Diagnostica</td>
			<td></td>
			<td><a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a> 
			Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Omni) antiserum</td>
			<td>SSI Diagnostica</td>
			<td>2438</td>
			<td>Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical Microscope</td>
			<td>Leica Microsystems</td>
			<td>Leica DML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated Disposable Inoculating Loops 1 &#181;l</td>
			<td>Copan</td>
			<td>CD175S01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse Blood Agar (HBA) plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>PP2001</td>
			<td><a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au/</a> 
			Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heart Infusion (HI) broth</td>
			<td>Media Preparation Unit (MPU), The University of Melbourne</td>
			<td>40HIN</td>
			<td>Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass microscope slides 76 mm x 26 mm</td>
			<td>Lomb Scientific</td>
			<td>7101</td>
			<td>Sharps hazard: Dispose in a biohazard sharps container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips</td>
			<td>NeuVitro&#160;</td>
			<td>GG-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">*Excluding the antisera, all other materials can be purchased from alternative suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Plates prepared from defibranated sheep blood are suitable but blood plates prepared from citrated blood or human blood are not</td>
		</tr>
	</tbody>
</table>

capsular serotyping of streptococcus pneumoniae using the quellung reaction

肺炎球菌 （肺炎球菌）の90の異なる莢膜血清型以上あります。だけでなく、肺炎球菌の疫学を理解するためのツールであるとして、莢膜血清型はワクチンの有効性と影響の研究のために有用な情報を提供することができます。膨張反応は肺炎球菌莢膜血清型のゴールドスタンダード法である。この方法は、莢膜多糖に対して向けプールされた抗血清と特異的に肺炎球菌の細胞懸濁液を試験することを含む。抗原抗体反応は、微視的に観察される。細菌細胞懸濁液の1）の調製、スライドガラス上の細胞及び抗血清の2）混合、および3）顕微鏡を用いて膨張反応を読み取る：プロトコルは、3つの主なステップを有している。膨張反応を行うことが合理的に簡単であり、適切な顕微鏡と抗血清が使用可能な場所に適用することができる。

<html>型特異的抗体は、その屈折率の10の変化につながる、肺炎球菌のカプセルに結合するとき、正の膨張反応が起こる。顕微鏡で観察すると、細菌は「腫れ」と14より見やすく表示されます。 図1（a）は、ハワイブロス中で肺炎球菌細胞を示す。型特異的抗血清を細菌懸濁液に添加されると、肺炎球菌（ 図1B）、「膨潤」屈折率がより丸みを帯びた現れる。 ブロスに加え、あまりにも多くの細菌細胞は、細胞の凝集や偽陰性反応になることがあります。「重すぎる」（ 図2A）または「明るすぎる」（ 図2B）であるハワイブロス中肺炎球菌の細胞の接種物2に示す図 。 08fig1highres.jpg &quot;SRC =&quot; / files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; 図1。負と正の膨張反応。血清型9Vの肺炎球菌の準備がNO抗血清（1A）で、グループ9抗血清（1B）で分離し、400倍の倍率で観察した。後者は「腫れ」と一般的に肯定的な膨張反応で見られる細菌細胞の丸めを示しています。 <img alt="図2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> 図2。あまりにも重い肺炎球菌細胞懸濁液（2A）または明るすぎる（2B）の重すぎるし、あまりにも軽い接種材料。準備、400Xの倍率で観察した。

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

膨張反応は肺炎球菌血清型のためのゴールドスタンダード技術です。この技術は、顕微鏡と、特定の肺炎球菌血清を利用し、一般的に世界的なリファレンス·研究所で使用されています。

の莢血清型<em&gt;肺炎球菌</em&gt;膨張反応を使用して、

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

capsular-serotyping-streptococcus-pneumoniae-using-quellung

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K ビュー.  Murdoch Childrens Research Institute. 膨張反応は肺炎球菌血清型のためのゴールドスタンダード技術です。この技術は、顕微鏡と、特定の肺炎球菌血清を利用し、一般的に世界的なリファレンス·研究所で使用されています。 

ビデオ: Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

免疫・感染学

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更<em&gt;肺炎球菌</em&gt;蛍光染料を使用

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

の莢膜血清型<em&gt;肺炎球菌</em&gt;ラテックス凝集による

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

免疫担当げっ歯類で堅牢な肺炎モデルはに対して抗菌有効性を評価するために<i&gt; S。ニューモニエ</i&gt;、<i&gt; H。インフルエンザ</i&gt;、<i&gt; K。ニューモニエ</i&gt;、<em&gt; P。緑膿菌</em&gt;または<em&gt; A。バウマニ</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

感染マウス感染モデルにおける髄膜炎の連鎖球菌性バイオ フィルムの役割の調査のための頭蓋内クモ膜下ルート

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.