Ce travail a été soutenu par la Fondation Bill et Melinda Gates (projet PneuCarriage, Grant 52099) et du Programme opérationnel de soutien de l&#39;infrastructure du gouvernement victorien. Merci à Eileen Dunne, Janet Strachan, et Kim Hare pour la lecture critique du manuscrit.

Une. Préparation de suspension cellulaire pour taper <ol><li> L&#39;utilisation d&#39;un jetable boucle 1 pl stérile, prendre un petit balayage de la nuit fraîche et pure culture de pneumocoques cultivé sur gélose au sang de cheval non sélectif solide et inoculer 100 pi Heart Infusion bouillon (HI). La suspension doit apparaître comme juste visiblement trouble. Agiter doucement le tube pour émulsionner. Remarque: d&#39;autres médias tels que le bouillon Brain Heart Infusion peuvent également convenir. L&#39;addition de sérum (par exemple une concentration finale de 10% (en volume) de sérum / de cheval) et incubation à 37 ° C pendant environ 1 heure avant l&#39;utilisation peut être utilisé pour améliorer la réaction. </li></ol> 2. Contrôle de la densité de suspension cellulaire La suspension de cellules préparée à l&#39;étape 1.1 est également utilisé en tant que témoin négatif, et doit être vérifiée pour la densité des cellules avant d&#39;ajouter les sérums de typage. <ol><li> L&#39;utilisation d&#39;une boucle de 1 ul, transférer une goutte de l&#39;inoculum préparé dans l&#39;étape 1.1 sur un verreglissière de microscope. </li><li> En utilisant une paire de forceps, placer une petite lamelle circulaire sur la suspension. </li><li> Utilisation du réglage de contraste de phase, observer la préparation au microscope sous un grossissement de 400X, pour vérifier la densité de la suspension (figure 1A). <ol><li> Si l&#39;inoculum est trop lourde (Figure 2A), ajouter plus de bouillon à la suspension cellulaire effectuée à l&#39;étape 1.1 de la diluer. Mélanger doucement à nouveau. </li><li> Si l&#39;inoculum est trop clair, par exemple, il ya &lt;50 cellules visibles dans le champ microscopique (figure 2B), ajouter plus de bactéries à la préparation de la suspension cellulaire fait dans l&#39;étape 1.1 et Mélanger doucement. Remarque: Avec l&#39;expérience, la densité de la suspension cellulaire peut être jugé par la secousse et la tenue à la lumière, où il doit apparaître comme juste visiblement trouble. </li></ol></li><li> Répéter les étapes 2.1 à 2.3 jusqu&#39;à ce qu&#39;un nombre adéquat de cellules sont visibles dans le champ microscopique. Remarque: les lames de verre et coverslips présentent un danger d&#39;objets tranchants. Après utilisation, les lames et lamelles sont contaminés avec du matériel infectieux et doivent être éliminés dans un récipient biohazard objets tranchants. </li></ol> 3. Dactylographie <ol><li> Transférer ~ 1 ul de l&#39;inoculum préparé (par exemple en utilisant une boucle de 1 ul) sur la lame de microscope en verre. Jeter la boucle dans le récipient biohazard objets tranchants. </li><li> Ajouter un volume égal (1 pi de anse) de sérum à température ambiante sur la lame et mélanger deux gouttes bien avec la boucle. </li><li> En utilisant une paire de pinces, placer une petite lamelle circulaire sur la suspension, en prenant soin de ne pas toucher la baisse. Placer la lamelle couvre-objet sur la suspension juste après l&#39;addition d&#39;anti-sérums à empêcher le liquide sur la lame de se dessécher. </li><li> En utilisant un microscope, observer la suspension sous contraste de phase avec un grossissement de 400X. Une réaction négative Quellung est observée lorsque peu ou pas de «gonflement» des cellules pneumocoque peut être vu à 400X grossissement, similaire à ce qui est observé dans la suspension de cellules pneumococciques avec des antisérums ajouté aucun (témoin négatif). Une réaction Quellung positif est observé lorsque les cellules apparaissent agrandie ou &quot;gonflé&quot; et la plus visible par rapport aux cellules du contrôle négatif. Remarque: les réactions équivoques doivent être étudiés plus loin, par exemple en examinant la réaction au bout de 5 à 10 min d&#39;incubation à température ambiante, ou en répétant la réaction avec le plus d&#39;antisérum. </li><li> Effectuer sérotypage pneumocoque aux essais successifs de piscines antisérums individuels jusqu&#39;à une réaction positive est observée. Alternativement, des piscines peuvent être appliquées en «cycles» en fonction de l&#39;incidence locale des sérotypes. Remarque: Test en «cycles» veille à ce que les piscines contenant des anticorps pour les sérotypes les plus communs sont testés en premier, en minimisant l&#39;effort et de coût. Essais simultanés de plusieurs piscines fournit également des contrôles négatifs supplémentaires, ce qui peut être utile dans interpretation. Si toutes les piscines dans un «rondes» sont particulièrement négatif, tester l&#39;isolat avec les rondes subséquentes de piscines. </li><li> Testez l&#39;isolat en utilisant le type approprié ou un groupe anti-sérum contenu dans la piscine réactive. Le cas échéant, utilisez facteur antisérums de différencier davantage le sérotype. Remarque: Les clés de la antisérums pneumocoques fournis par le fabricant sont utilisés dans la sélection de pool approprié, le type, le groupe ou le facteur antisérums et l&#39;interprétation des résultats (y compris les réactions croisées). </li><li> Si les résultats négatifs sont obtenus avec toutes les piscines, tester l&#39;isolat avec le réactif Omniserum (contient des anticorps contre 91 sérotypes 21). Si une réaction Quellung positif n&#39;est pas encore observé lors de l&#39;essai d&#39;un pneumocoque isoler avec le réactif Omniserum, c&#39;est peut-être parce que l&#39;isolat exprime peu ou pas de capsule, ou que des anticorps contre le sérotype ne sont pas représentés dans le Omniserum. Cette dernière catégorie peut inclure de nouveaux sérotypes. </li> &lt;/ Ol&gt;

<ol>
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Dis. 4, 144-154 (2004).</li><li>Henrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+newly+recognized+types+of+Streptococcus+pneumoniae.">Six newly recognized types of Streptococcus pneumoniae.</a> J. Clin. Microbiol. 33, 2759-2762 (1995).</li><li>Calix, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical,+genetic,+and+serological+characterization+of+two+capsule+subtypes+among+Streptococcus+pneumoniae+Serotype+20+strains:+discovery+of+a+new+pneumococcal+serotype.">Biochemical, genetic, and serological characterization of two capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: discovery of a new pneumococcal serotype.</a> J. Biol. Chem. 287, 27885-27894 (2012).</li><li>Hausdorff, W. P., Bryant, J., Paradiso, P. R., Siber, G. 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Les auteurs n&#39;ont rien à révéler.

Une étape cruciale de cette technique est d&#39;assurer une culture pure est utilisée pour préparer la suspension en même temps que l&#39;addition d&#39;un nombre approprié de cellules bactériennes dans le bouillon. Il convient de noter que la croissance de la colonie et la morphologie sur des plaques de gélose varie entre différents isolats pneumococciques. Certains produisent des colonies sèches et rugueuses, qui peuvent être difficiles à enlever avec une boucle et ne pas émulsionner bien dans le bouillon. Pour cette raison, une plus grande balayage de bactéries peut être nécessaire lors de la préparation de la suspension cellulaire. Dans tous les cas, il est impératif que les cellules individuelles, et non des agrégats, sont évidentes dans le contrôle négatif de sorte que leur importance relative peut être comparée à des cellules avec des antiserums ajoutés. En outre, un trop grand nombre de cellules bactériennes peuvent conduire à une fausse réaction négative, où un excès d&#39;antigène capsulaire peut inhiber la réaction Quellung 10. Pour cette raison, il est important pour créer une suspension bactérienne avec une densité de cellules appropriée (figure 1). La suspensionpension peut être conservé pendant plusieurs mois par l&#39;ajout de quelques gouttes de formol concentré. Cela permet le contrôle par lot plus facile et aussi stérilise le bouillon de réduire le risque d&#39;infections acquises au laboratoire, bien que ce doit être pesé contre le risque de sécurité de formol manipulation, qui est toxique, corrosif, cancérogène, sensibiliser et inflammable. En outre, nous avons observé que certains isolats montrent les réactions les plus faibles en présence de formol (données non présentées). Quellung réactions sont examinées en utilisant un microscope. L&#39;utilisation de contraste de phase améliore la visibilité de la capsule. Dans certains laboratoires, la réaction est visualisé en utilisant l&#39;huile d&#39;immersion à 1000 grossissement X 14, 16. En outre, certains laboratoires utilisent un contre-colorant (bleu de méthylène) pour améliorer encore la visibilité d&#39;une réaction positive de 10, 16, 22. La méthode présentée ici est considérée sans huile-iMMERSION et sous un grossissement de 400X 15, 17. Cette méthode est rapide, simple et relativement facile à lire 23. Il est important de maintenir de bonnes procédures de contrôle interne de la qualité pour s&#39;assurer que les procédures d&#39;antisérums et d&#39;essai sont dans l&#39;ordre. La participation à un programme d&#39;assurance de qualité externe est souvent bénéfique. Une limitation importante de la réaction Quellung pour le sérotypage des pneumocoques est le coût élevé des antisérums nécessaires. Cependant, ceci est minimisé dans le protocole ci-dessus en raison de la faible quantité d&#39;antisérum utilisé pour chaque réaction. Plusieurs autres procédés ont été décrits pour le sérotypage des pneumocoques 23, 24. Cependant, Quellung reste l&#39;étalon-or et le développement de nouvelles méthodes nécessite normalisation contre la réaction Quellung 23. Utilisant des antisérums échéant, la réaction Quellung peut également être utilisée pour identifier et saisir d&#39;autres bouchonsule bactéries produisant 25. La réaction Quellung contribue à la caractérisation des sérotypes pneumococciques actuelles et émergentes, ainsi que de fournir des informations sur l&#39;efficacité du vaccin et l&#39;impact à long terme de la vaccination sur le transport et la maladie.

Streptococcus pneumoniae (le pneumocoque) est responsable de la mort d&#39;environ 800 000 enfants de moins de cinq ans chaque année, avec le fardeau de la maladie relevant principalement dans les pays pauvres en ressources 1. Les maladies pneumococciques varient en gravité des infections localisées telles que l&#39;otite moyenne aiguë à la vie en danger des infections sévères telles que sepsis, la méningite et la pneumonie 2. Plus de 90 sérotypes capsulaires des pneumocoques ont été décrits 3, 4. Vaccins antipneumococciques actuelles offrent une protection contre les sérotypes qui sont responsables de la majorité de la maladie invasive 5, 6. Cependant, l&#39;utilisation largement répandue de vaccins a été associé avec le remplacement des sérotypes inclus dans le vaccin par des sérotypes inclus dans le vaccin, à la fois dans le transport du nasopharynx et la maladie invasive 7, 8. Cela souligne l&#39;importance de sérotypage pour épidémiologiquesurveillance et l&#39;impact du vaccin études à long terme 9. La méthode de l&#39;étalon-or pour le typage pneumocoque est le test de réaction capsulaire, connu comme la réaction Quellung, d&#39;abord décrit par Neufeld en 1902 (voir autrichienne 10). Un haut degré de concordance a été trouvée entre les laboratoires, et entre la réaction Quellung et d&#39;autres méthodes de sérotypage 11-13. Il existe plusieurs variantes de la réaction Quellung 10, 14-16. Le procédé décrit ici ne nécessite pas l&#39;utilisation d&#39;une contre-coloration ou la lentille à immersion d&#39;huile, mais plutôt, une goutte d&#39;échantillon préparé est mélangé avec le sérum de frappe et immédiatement examinés sous un grossissement de 400X. La réaction est généralement effectuée Quellung disponible dans le commerce en utilisant des antisérums. Le procédé décrit ici utilise moins d&#39;antisérums par rapport à d&#39;autres méthodes 16, 17, ce qui rend plus rentable. Une fois un isolmangé est identifié comme un pneumocoque, il est testé de manière séquentielle avec des pools d&#39;antisérums jusqu&#39;à ce qu&#39;une réaction positive est observée. Chaque sérum de la piscine contient différents mélanges d&#39;antisérums dirigés contre 91 sérotypes pneumococciques 18. Une fois la piscine est établie, le type individuel et de groupe antisérums qui sont inclus dans la piscine réactive sont testés individuellement dans l&#39;ordre. Type antisérums réagit généralement avec un seul sérotype (par exemple de type 1 antisérum réagit avec le sérotype 1), alors que les antisérums de groupes réagissent avec tous les sérotypes dans un groupe particulier (par exemple le Groupe 22 antisérum réagit avec les sérotypes 22F et 22A). Certains sérotypes au sein des groupes se différencient en utilisant le facteur antisérums (par exemple sérotype 22F réagit avec facteur 22b, mais pas 22c). Dans ce cas, l&#39;essai se poursuit avec tous les antisérums facteur pertinent jusqu&#39;à ce sérotype est déterminé 19, 20. La réaction Quellung est simple et rapide 14 et peut être réalisée dans n&#39;importe quel laboratory qui est équipé d&#39;un microscope de bonne qualité et un ensemble approprié d&#39;antisérums.

<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

Il ya plus de 90 différents sérotypes capsulaires de Streptococcus pneumoniae (pneumocoque) l&#39;. En plus d&#39;être un outil pour comprendre l&#39;épidémiologie pneumocoque, sérotypage capsulaire peut fournir des informations utiles pour les études sur l&#39;efficacité du vaccin et d&#39;impact. La réaction Quellung est la méthode de l&#39;étalon-or pour le sérotypage capsulaire pneumococcique. Le procédé consiste à tester une suspension de cellules avec des antiserums réunis pneumococcique et spécifique dirigé contre le polysaccharide capsulaire. Les réactions antigène-anticorps sont observées au microscope. Le protocole comporte trois étapes principales: 1) Préparation d&#39;une suspension de cellules bactériennes, 2) le mélange des cellules et des antiserums sur une lame de verre, et 3) la lecture de la réaction Quellung en utilisant un microscope. La réaction Quellung est relativement simple à réaliser et peut être appliqué partout où un microscope et des antiserums appropriés sont disponibles.

<html> Une réaction positive est observée lorsque Quellung anticorps spécifique d&#39;un type se lie à la capsule du pneumocoque, ce qui conduit à une modification de son indice de réfraction 10. Vu sous un microscope, les bactéries semblent &#39;gonflé&#39; et plus visible 14. Figure 1A montre des cellules pneumocoque dans un bouillon HI. Lorsque l&#39;anti-sérum spécifique de type est ajouté à la suspension bactérienne, les pneumocoques apparaissent «gonflée», de réfraction et plus arrondie (figure 1B). Trop de cellules ajoutées au bouillon de bactéries peuvent entraîner l&#39;agrégation de cellules ou une réaction faussement négative. Montre la figure 2 inoculations de cellules pneumocoque dans un bouillon HI qui sont «trop lourd» (figure 2A) ou «trop léger» (figure 2B). 08fig1highres.jpg &quot;src =&quot; / files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; Figure 1. Réaction Quellung négatif et positif. Préparations de sérotype 9V pneumocoque isoler sans sérum (1A) et avec le groupe 9 antisérum (1B), ont été vu sous un grossissement de 400X. Ce dernier montre le «gonflement» et l&#39;arrondissement des cellules bactériennes généralement vu dans une réaction Quellung positif. <img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> Figure 2. Inoculum trop lourd et trop léger. Préparations de suspensions cellulaires pneumocoque qui sont trop lourds (2A) ou trop léger (2B), lorsqu&#39;on les examine sous un grossissement de 400X.

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

La réaction Quellung est la technique de référence en matière de sérotypage Streptococcus pneumoniae. Cette technique utilise un microscope et antisérums pneumocoque spécifique et est couramment utilisé dans les laboratoires de référence et de recherche dans le monde entier.

Capsulaire sérotypage des<em&gt; Streptococcus pneumoniae</em&gt; Utilisation de la réaction Quellung

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 vues.  Murdoch Childrens Research Institute.  La réaction Quellung est la technique de référence en matière de sérotypage Streptococcus pneumoniae. Cette technique utilise un microscope et antisérums pneumocoque spécifique et est couramment utilisé dans les laboratoires de référence et de recherche dans le monde entier. 

Vidéo: 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.

Utiliser l&#39;imagerie Bioluminescent chargé d&#39;enquêter sur une synergie entre Streptococcus pneumoniae Et virus Influenza A de souriceaux

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

Recherche

Immunologie et infection

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.

Imagerie de cellules vivantes de la<emBacillus subtilis> Et Streptococcus pneumoniae À l&#39;aide automatisée de microscopie time-lapse

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.

Caractérisation des réponses inflammatoires au cours de la colonisation intranasale avec Streptococcus pneumoniae

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.

La surveillance des changements dans la membrane de polarité, Membrane intégrité, et intracellulaires d&#39;ions concentrations dans<em&gt; Streptococcus pneumoniae</em&gt; Utilisation des colorants fluorescents

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.

Capsulaire sérotypage<em&gt; Streptococcus pneumoniae</em&gt; Par agglutination au latex

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.

Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves de<em&gt; Streptococcus iniae</em&gt; Infection

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.

Un modèle murin du Groupe B<em&gt; Streptococcus</em&gt; Vaginal Colonisation

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.

Un modèle de pneumonie robuste en immunocompétents Rongeurs pour évaluer Efficacité antibactérienne contre<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; ou<em&gt; A. baumannii</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.

Intracrânienne subarachnoïdien voie d’Infection pour enquêter sur les rôles des Biofilms de Streptococcus suis en méningite dans un modèle d’Infection de souris

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.