Questo lavoro è stato sostenuto dalla Fondazione Bill e Melinda (progetto PneuCarriage, Grant 52.099) e Operational Support Program infrastrutture del Governo del Victoria. Grazie a Eileen Dunne, Janet Strachan, e Kim Hare per la lettura critica del manoscritto.

1. Preparazione della sospensione cellulare per la tipizzazione <ol><li> Utilizzando un usa e getta loop 1 ml sterile, prendere una piccola distesa di pernottamento pura cultura fresca di pneumococchi cresciuto su un solido agar sangue di cavallo non selettivo e inoculare 100 ml Heart Infusion (HI) brodo. La sospensione dovrebbe apparire solo visibilmente torbido. Agitare delicatamente il tubo per emulsionare. Nota: Altri media come Brain Heart Infusion brodo possono essere idonei. Aggiunta di siero (ad esempio una concentrazione finale di 10% (v / v) di siero di cavallo) ed incubazione a 37 ° C per circa 1 ora prima dell&#39;uso può essere utilizzato per migliorare la reazione. </li></ol> 2. Controllo Densità di sospensione cellulare La sospensione cellulare preparato al punto 1.1 sarà anche utilizzato come controllo negativo, e dovrebbe essere controllato per densità delle cellule prima di aggiungere i sieri digitazione. <ol><li> Utilizzando un ciclo 1 ml, trasferire una goccia d&#39;inoculo preparato nella fase 1.1 su un bicchieres vetrino da microscopio. </li><li> Usando un paio di pinze, mettere un piccolo vetrino circolare sulla sospensione. </li><li> Utilizzando l&#39;impostazione a contrasto di fase, osservare la preparazione al microscopio sotto 400X ingrandimento, per verificare la densità della sospensione (Figura 1A). <ol><li> Se l&#39;inoculo è troppo pesante (Figura 2A), aggiungere altro brodo alla sospensione cellulare fatta allo step 1.1 diluirlo. Mescolare ancora delicatamente. </li><li> Se l&#39;inoculo è troppo leggero, per esempio, ci sono &lt;50 cellule visibile nel campo microscopico (Figura 2B), aggiungere più batteri alla preparazione sospensione cellulare fatta allo step 1.1 e mescolare delicatamente. Nota: Con l&#39;esperienza, la densità della sospensione cellulare può essere giudicata agitando e sollevando alla luce, dove dovrebbe apparire solo visibilmente torbido. </li></ol></li><li> Ripetere i passaggi 2,1-2,3 finché un adeguato numero di cellule è visibile nel campo microscopico. Nota: vetrini e coverslips presentano un rischio taglienti. Dopo l&#39;uso, diapositive e coprioggetto sono contaminati con materiale infetto e devono essere smaltite in un contenitore a rischio biologico taglienti. </li></ol> 3. Dattilografia <ol><li> Trasferire ~ 1 ml di inoculo preparato (per esempio utilizzando un ciclo 1 microlitri) sul vetrino per microscopio. Scartare il loop nel contenitore rischio biologico taglienti. </li><li> Aggiungere un volume equivalente (1 ml un&#39;ansata) di antisiero temperatura ambiente sul vetrino e mescolare entrambe gocce bene con il loop. </li><li> Usando un paio di pinze, mettere un piccolo vetrino circolare sulla sospensione, facendo attenzione a non toccare la goccia. Posizionare il coprioggetto sulla sospensione immediatamente dopo l&#39;aggiunta di antisieri per evitare che il liquido sul vetrino si secchi. </li><li> Utilizzando un microscopio, osservare la sospensione sotto a contrasto di fase con ingrandimento 400X. Una reazione negativa Quellung si osserva quando poco o nessun &#39;gonfiore&#39; delle cellule pneumococco può essere visto a 400X ingrandimento, simile a quello osservato nella sospensione cellulare pneumococcica senza antisieri aggiunto (controllo negativo). Una reazione positiva Quellung si osserva quando le cellule appaiono allargata o &#39;gonfio&#39; e più visibili rispetto alle celle nel controllo negativo. Nota: reazioni ambigui devono essere studiati ulteriormente, ad esempio esaminando la reazione dopo 5-10 minuti di incubazione a temperatura ambiente, o ripetendo la reazione con più antisiero. </li><li> Condurre sierotipizzazione pneumococcica con successiva prova di ogni pool sieri specifici, fino a quando si osserva una reazione positiva. In alternativa, piscine possono essere applicate in «round» a seconda della incidenza locale dei sierotipi. Nota: I test a «turni» assicura che le piscine contenenti anticorpi per i sierotipi più comuni sono testati prima, riducendo al minimo lo sforzo e costo. Sperimentazione contemporanea di diverse piscine fornisce inoltre ulteriori controlli negativi, che può essere utile in interpretation. Se tutte le piscine in un particolare &#39;round&#39; sono negativi, testare l&#39;isolato con le successive serie di piscine. </li><li> Testare l&#39;isolato utilizzando il tipo appropriato o gruppo antisiero contenuta all&#39;interno del pool reattiva. Se del caso, utilizzare il fattore antisieri per differenziare ulteriormente il sierotipo. Nota: Chiavi di sieri pneumococcica forniti dal costruttore sono utilizzati per la selezione del pool appropriato, tipo, di gruppo o fattore di sieri e l&#39;interpretazione dei risultati (comprese le eventuali reazioni incrociate). </li><li> Se i risultati negativi si ottengono con tutte le piscine, testare l&#39;isolato con Omniserum reagente (contiene anticorpi contro 91 sierotipi 21). Se una reazione positiva Quellung non viene ancora osservata nel test di un pneumococcica isolare con il reagente Omniserum, puo essere perché l&#39;isolato esprime poca o nessuna capsula, o che gli anticorpi contro il sierotipo non sono rappresentati nella Omniserum. Quest&#39;ultima categoria può includere nuovi sierotipi. </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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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+serotyping+of+Streptococcus+pneumoniae+in+Europe.">Validation of serotyping of Streptococcus pneumoniae in Europe.</a> Vaccine. 23, 1368-1373 (2005).</li><li>Lovgren, M., 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=Evolution+of+an+international+external+quality+assurance+model+to+support+laboratory+investigation+of+Streptococcus+pneumoniae,+developed+for+the+SIREVA+project+in+Latin+America,+from.">Evolution of an international external quality assurance model to support laboratory investigation of Streptococcus pneumoniae, developed for the SIREVA project in Latin America, from.</a> J. Clin. 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Gli autori non hanno nulla da rivelare.

Un punto critico in questa tecnica è garantire una coltura pura viene utilizzato per preparare la sospensione con l&#39;aggiunta di un adeguato numero di cellule batteriche al brodo. Va notato che la crescita delle colonie e morfologia su piastre di agar varia tra diverse pneumococchi isolati. Alcuni producono colonie secche e ruvide, che possono essere difficili da far fuori con un ciclo e non emulsionare bene nel brodo. Per questo motivo, una grande spazzata di batteri può essere necessaria quando si prepara la sospensione cellulare. In ogni caso, è imperativo che le cellule individuali, e non aggregati, sono evidenti nel controllo negativo quindi la loro dimensione relativa può essere paragonato alle cellule con antisieri aggiunte. Inoltre, troppe cellule batteriche possono causare una falsa reazione negativa, dove un eccesso di antigene capsulare può inibire la reazione Quellung 10. Per questo motivo è importante creare una sospensione batterica con una densità cellulare adatto (Figura 1). Il suspensione può essere conservata per diversi mesi con l&#39;aggiunta di qualche goccia di formalina concentrata. In questo modo le prove per lotto più facile e anche sterilizza il brodo di ridurre il rischio di infezioni acquisite in laboratorio, anche se ciò deve essere valutato rispetto al rischio la sicurezza del trattamento con formalina, che è tossico, corrosivo, cancerogeno, sensibilizzanti, e infiammabili. Inoltre, abbiamo osservato che alcuni isolati mostrano reazioni deboli in presenza di formalina (dati non mostrati). Reazioni Quellung sono esaminati usando un microscopio. L&#39;uso di contrasto di fase aumenta la visibilità della capsula. In alcuni laboratori, la reazione viene visualizzato utilizzando ad immersione in olio a 1000 ingrandimenti X 14, 16. Inoltre, alcuni laboratori hanno una macchia contatore (blu di metilene) per migliorare ulteriormente la visibilità di una reazione positiva 10, 16, 22. Il metodo qui presentato è visto senza olio-immersion e sotto 400X 15, 17. Questo metodo è veloce, semplice e relativamente facile da leggere 23. E &#39;importante mantenere buoni procedure di controllo di qualità interno per garantire le procedure di sieri e di prova sono in ordine. La partecipazione a un programma di controllo esterno della qualità è spesso utile. Una limitazione importante della reazione Quellung sierotipizzazione degli pneumococchi è il costo elevato degli antisieri richiesta. Tuttavia, questo è minimizzato nel protocollo sopra a causa della piccola quantità di antisiero utilizzato per ciascuna reazione. Diversi altri metodi sono stati descritti per la sierotipizzazione pneumococchi 23, 24. Tuttavia, Quellung rimane il gold standard e lo sviluppo di nuovi metodi richiede standardizzazione contro la reazione Quellung 23. Uso appropriato antisieri, la reazione Quellung può anche essere utilizzata per identificare e digitare altri tappiule batteri che producono 25. La reazione Quellung è strumentale nella caratterizzazione dei sierotipi di pneumococco attuali ed emergenti, oltre a fornire informazioni sulla efficacia del vaccino e l&#39;impatto a lungo termine della vaccinazione sul carrello e la malattia.

Streptococcus pneumoniae (pneumococco) è responsabile della morte di circa 800.000 bambini sotto i cinque anni ogni anno, con il peso delle malattie che rientrano prevalentemente nei paesi poveri di risorse 1. Malattie da pneumococco variano in gravità da infezioni localizzate quali otite media acuta a gravi infezioni che minacciano la vita come sepsi, meningite e polmonite 2. Oltre 90 sierotipi capsulari di pneumococchi sono stati descritti 3, 4. Pneumococco attuali forniscono protezione contro i sierotipi che sono responsabili della maggior parte delle malattie invasive 5, 6. Tuttavia, l&#39;uso diffuso di vaccini è stata associata con sostituzione di sierotipi inclusi nel vaccino da sierotipi non vaccinali, sia nel trasporto nasofaringeo e malattie invasive 7, 8. Questo evidenzia l&#39;importanza di sierotipizzazione per epidemiologicala sorveglianza e l&#39;impatto del vaccino studi a lungo termine 9. Il metodo gold standard per la tipizzazione pneumococcica è la prova di reazione capsulare, nota come la reazione Quellung, descritta da Neufeld nel 1902 (vedi austriaco 10). Un alto grado di concordanza trovata tra laboratori, e tra la reazione Quellung e altri metodi sierotipizzazione 11-13. Ci sono diverse varianti della reazione Quellung 10, 14-16. Il metodo qui descritto non richiede l&#39;uso di un colorante contatore o la lente immersione in olio, ma piuttosto una goccia di campione preparato viene miscelato con il siero di battitura e esaminato immediatamente sotto ingrandimento 400X. La reazione Quellung viene di solito eseguita usando appositi antisieri. Il metodo qui descritto utilizza meno antisieri rispetto ad altri metodi 16, 17, rendendolo più conveniente. Una volta che un isolmangiato è identificato come pneumococco, è sequenzialmente testato con piscine sieri finché si osserva una reazione positiva. Ogni antisiero piscina contiene diverse miscele di sieri sollevate contro 91 sierotipi di pneumococco 18. Una volta che la piscina è stabilito, il tipo individuale e di gruppo antisieri che sono inclusi nel pool reattivi sono testati singolarmente in sequenza. Tipo antisieri generalmente reagiscono con un solo sierotipo (ad esempio di tipo 1 antisiero reagisce con sierotipo 1), mentre antisieri del gruppo reagiscono con tutti i sierotipi in un particolare gruppo (ad esempio Gruppo 22 antisiero reagisce con sierotipi 22F e 22A). Alcuni sierotipi all&#39;interno dei gruppi sono ulteriormente differenziati con fattore di antisieri (ad es sierotipo 22F reagisce con fattore 22b, ma non 22c). In questo caso il test è proseguita con tutti fattore rilevante antisieri fino sierotipo è determinata 19, 20. La reazione Quellung è semplice e veloce 14 e può essere eseguita in qualsiasi laboratory che è dotato di un microscopio buona qualità e appropriato insieme di antisieri.

<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

Ci sono oltre 90 diversi sierotipi capsulari di Streptococcus pneumoniae (pneumococco). Oltre ad essere uno strumento per comprendere l&#39;epidemiologia pneumococcica, sierotipizzazione capsulare può fornire informazioni utili per studi di efficacia del vaccino e di impatto. La reazione Quellung è il metodo gold standard per lo pneumococco capsulare sierotipizzazione. Il metodo prevede saggiando una sospensione cellulare pneumococcica con antisieri pool e specifico diretto contro il polisaccaride capsulare. Le reazioni antigene-anticorpo sono osservate al microscopio. Il protocollo ha tre fasi: 1) preparazione di una sospensione cellulare batterica, 2) miscelazione delle cellule e antisieri su un vetrino, e 3) la lettura della reazione Quellung utilizzando un microscopio. La reazione Quellung è ragionevolmente semplice da eseguire e può essere applicata ovunque sono disponibili un microscopio adatto e antisieri.

<html> Una reazione positiva Quellung verifica quando anticorpo specifico del tipo lega alla capsula del pneumococco, portando ad una variazione del suo indice di rifrazione 10. Quando hanno visto al microscopio, i batteri appaiono &#39;gonfio&#39; e più visibile 14. Figura 1A mostra cellule pneumococco in brodo HI. Quando antisiero specifico del tipo viene aggiunto alla sospensione batterica, pneumococchi appaiono &#39;gonfio&#39;, rifrazione e più arrotondato (Figura 1B). Troppi cellule aggiunte al brodo batteriche possono causare aggregazione di cellule o una reazione falso negativo. Figura 2 mostra inoculi di cellule pneumococco in brodo HI pari &#39;troppo pesante&#39; (Figura 2A) o &#39;troppo leggero&#39; (Figura 2B). 08fig1highres.jpg &quot;src =&quot; / files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; Figura 1. Negativo e positivo reazione Quellung. Preparazioni di sierotipo 9V pneumococcica isolare senza antisiero (1A) e con il gruppo 9 antisiero (1B), sono stati visti sotto 400X. Quest&#39;ultimo mostra il &#39;gonfiore&#39; e l&#39;arrotondamento delle cellule batteriche tipicamente osservati in una reazione positiva Quellung. <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> Figura 2. Inoculi troppo pesante e troppo leggero. Preparazioni di sospensioni pneumococco cellulari che sono troppo pesanti (2A) o troppo leggero (2B), se visto sotto 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 reazione Quellung è la tecnica gold standard per sierotipizzazione Streptococcus pneumoniae. Questa tecnica utilizza un microscopio e specifico antisieri pneumococco ed è comunemente usato in riferimento e laboratori di ricerca in tutto il mondo.

Capsulare Sierotipizzazione di<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo della reazione Quellung

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

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Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K Views.  Murdoch Childrens Research Institute.  La reazione Quellung è la tecnica gold standard per sierotipizzazione Streptococcus pneumoniae. Questa tecnica utilizza un microscopio e specifico antisieri pneumococco ed è comunemente usato in riferimento e laboratori di ricerca in tutto il mondo. 

Video: 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.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

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

Ricerca

Immunologia e infezioni

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.

Cellulare Imaging in diretta di Bacillus subtilis E Streptococcus pneumoniae Utilizzando automatico Time-lapse Microscopia

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.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco 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.

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo di tinture fluorescenti

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.

Capsulare Sierotipizzazione di<em&gt; Streptococcus pneumoniae</em&gt; Da agglutinazione al lattice

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.

Imaging non invasiva della risposta immunitaria innata in un modello di Zebrafish larvale<em&gt; Streptococcus iniae</em&gt; Infezione

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 modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

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.

Una polmonite Modello robusto in immunocompetenti roditori per valutare antibatterico efficacia contro<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; o<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.

Intracranica Subarachnoidal itinerario dell'infezione per indagare ruoli di biofilm di Streptococcus suis nella meningite in un modello di infezione del topo

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.

RNA del Virus di febbre rompiossa nel supernatante di coltura delle cellule infettate da reazione a catena della polimerasi quantitativa in tempo reale di misurazione