Name: following in real time the impact of pneumococcal virulence factors in an acute mouse pneumonia model using bioluminescent bacteria
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Forskning i laboratoriet blev støttet af tilskud fra Deutsche Forschungsgemeinschaft (DFG HA 3125/3-2, DFG HA 3125/4-2) og Forbundsministeriet for Uddannelse og Forskning (BMBF) Medicinsk Infektion Genomics (FKZ 0315828A) til SH.

De dyr infektion eksperimenter er beskrevet her, skal udføres i nøje overensstemmelse med lokale og internationale (f.eks European Health Lov af Federation of Laboratory Animal Science Associations (FELASA)) retningslinjer og forordninger for brug af hvirveldyr. Eksperimenterne skal godkendes af den lokale etiske bestyrelse og Institutional Animal Care udvalget. Alle forsøg med S. pneumoniae i laboratoriet eller animalske infektioner er udført i et klasse II biosikkerhed kabinet. <p class="jove...

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	<li>Niederman, M. S., 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=Guidelines+for+the+management+of+adults+with+community-acquired+pneumonia.+Diagnosis,+assessment+of+severity,+antimicrobial+therapy,+and+prevention.">Guidelines for the management of adults with community-acquired pneumonia. Diagnosis, assessment of severity, antimicrobial therapy, and prevention.</a> Am. J. Respir. Crit. 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<html> Alle eksperimenter udført i dyr skal godkendes af de lokale myndigheder og etik provisioner. I in vivo infektion eksperimenter den bakterielle belastning i de forskellige host nicher af inficerede dyr skal bestemmes på forskellige tidspunkter efter infektion. Under disse eksperimentelle forhold har de dyr, der skal ofres, før isolering af bakterier fra blodet, nasopharynx, bronchoalvelar udskylning, eller organer såsom lunger, milt og hjerne. For at beregne antallet af bakterier pr vært niche og vurd...

Luftvejsinfektioner forårsaget af virus eller bakterier er fortsat en af ​​de mest almindelige samfunds-erhvervet eller kliniske problemer på verdensplan forårsager cirka en tredjedel af alle dødsfald på verdensplan. De vigtigste bakteriearter er Haemophilus influenzae og Streptococcus pneumoniae 2. Men disse bakteriearter er normalt fælles bestanddele af den naturlige luftveje flora. Således bakteriel vogn er også vis risiko for invasiv sygdom og afhængig af immunstatus eller forfordele af enkeltpersoner. Den asymptomatisk kolonisering udløses til invasive infektioner. Streptococcus pneumoniae er den førende patogen af community-erhvervet pneumoni (CAP) og en af de mest almindelige årsager til bakteriæmi hos mennesker. Hos raske individer S. pneumoniae (pneumokokker) er ofte asymptomatiske og harmløse kolonisatorer i de øvre luftveje, hvor de konfronteres med nonpathogenic bakterieraf de bosiddende flora, men også med patogener, såsom Haemophilus spp. eller Staphylococcus aureus og den første linje af det menneskelige immunforsvar. Carriage er højest i små børn (37%) og endnu højere i overfyldte daginstitutioner (58%) 3-5. Den yngste befolkning og de ​​ældre, der modtager pneumokokker via aerosol transmission fra luftfartsselskaber og nasopharynx sekreter 6, hører til de højrisikogrupper og vaccination ved hjælp af en af de pneumokok konjugerede vacciner (PCV10 eller PCV13 hos børn og 23-valent polysaccharid PPSV23 hos voksne) anbefales i USA (US) og mange europæiske lande 4. Den PPSV23 dækker serotyper, der er ansvarlige for ~ 90% af de bakteriæmiske pneumokok-sygdomme i USA og Europa, som forhindrer dermed effektivt invasiv pneumokoksygdom sygdomme (IPD) hos voksne, mens PCVs dækker de mest udbredte serotyper i børn. Følgelig IPD grund af vaccine typer (VT) er reducerede men nonvaccine serotyper viser en høj virulens potentiale og antibiotikaresistens er dukket 4,7-12. Nasopharynx som reservoiret er udgangspunktet for pneumokokker at sprede sig til bihulerne eller midterste ører initierende skadelige lokale infektioner. Mere vigtigt, pneumokokker spredes direkte via luftvejene til bronkierne og lungerne resulterer i livstruende CAP 4,13. Lungeinfektioner er ofte ledsaget med væv og ødelæggelse barriere, således at patogenet spredes i blodet og forårsager IPD. Forekomsten af den fælles landbrugspolitik og IPD er højest i immunsvækkede personer eller i de yderste områder af alder 4,13. De omstændigheder, der er ansvarlige for omdannelsen fra en commensal for et patogen med høj virulens er stadig under debat. Imidlertid har foruden ændringer i host modtagelighed og evolutionær tilpasning ledsaget med højere virulens og stigningen i antibiotikaresistenser blevet foreslået at have en afgørende indflydelse på PNEumococcal infektioner 14-16. Patogenet er udstyret med en flerhed af adhæsiner, der medierer intim kontakt til mukosale epitelceller. Efter overvindelsen af den luftvejs slim, er pneumokok vedhæftning til værtsceller lettes via direkte interaktioner af overfladeeksponerede adhesiner med cellulære receptorer og ved at udnytte ekstracellulære matrix komponenter eller serumproteiner som bro molekyler 4,17,18. Som alsidige patogener pneumokoktyper er også udstyret med faktorer involveret i unddragelse af værtens immun forsvarsmekanismer. Desuden har de evnen til at tilpasse til forskellige host miljøer såsom lunge, blod og cerebrospinalvæske (CSF), henholdsvis 5,17,19,20. Virkningen af bakterielle faktorer patogenese og inflammatoriske værtsresponser er undersøgt i eksperimentelle dyremodeller for lungebetændelse, bakteriæmi eller meningitis 21-25. Trods et humant patogen, disse modeller er vill-etableret for at dechifrere pneumokok væv tropisme, virulens mekanismer eller protectivity af pneumokokvaccine kandidater. Den genetiske baggrund af indavlede musestammer bestemmer modtagelighed for pneumokokker. BALB / c-mus intranasalt inficeret med pneumokokker blev fundet at være resistente, mens CBA / Ca-og SJL-mus var mere modtagelige over for pneumokokinfektioner 22. Dette indebærer, at ligner mennesker, den genetiske baggrund og værtsforsvarsmekanismer bestemme udfaldet af infektionen. Derfor er det nødvendigt at opklare modstand loci i genomet af mus mindre modtagelige for pneumokokinfektioner yderligere indsats. Resultaterne har ført til ændringer i in vivo virulens protokoller. I stedet for de indavlede BALB / c mus, der ofte anvendes i fortiden, er de meget modtagelige CD-1/MF1 udavlede musestammer dag ofte bruges til at undersøge effekten af tab af funktion pneumokok virulens eller fitness faktorer 26-28. Desuden er tilgængelighedenaf bioluminescerende pneumokokker og optiske billeddannende teknikker tillader tidstro bioluminescens Bioimaging infektioner. I pneumokokker er indsat den optimerede luxABCDE genkassette (plasmid Paul-A Tn 4001 luxABCDE Km R) i en enkelt integration stedet for kromosomet ved transposon mutagenese. Bioluminiscerende pneumokokker er blevet anvendt til at vurdere dæmpningen af pneumokok mutanter med mangelfuld virulens eller fitness faktorer og deres translokation fra en anatomisk websted til et andet 26,28-31. Her giver vi en protokol for bioimaging af pneumokok infektioner i en murin lungebetændelse eller sepsis model. Forstærkning og formidling af bioluminiscerende pneumokokker i intranasalt eller intraperitonealt inficerede mus kan nemt overvåges over tid ved hjælp af et optisk imaging system, og det samme dyr på forskellige tidspunkter.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Todd Hewitt broth</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">X936.1</td>
			<td style="width:280px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">2363.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Blood agar plates</td>
			<td style="width:280px;">Oxoid, Wesel, Germany</td>
			<td style="width:129px;">PB5039A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">T832.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erythromycin</td>
			<td style="width:280px;">Sigma-Aldrich,Taufkirchen, Germany</td>
			<td style="width:129px;">E6376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">fetal bovine serum (FBS)</td>
			<td style="width:280px;">PAA Laboratories, Coelbe, Germany</td>
			<td style="width:129px;">A11-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CD-1 mice, female</td>
			<td style="width:280px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:129px;">CD1SIFE06W08W</td>
			<td>female CD-1 mice, six to eight weeks old</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamin 500mg, Curamed injection solution</td>
			<td>Schwabe-Curamed, Karlsruhe, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Rompun 2%, injection solution</td>
			<td>Bayer Animal Health, Monheim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Plastipak 1 ml syringes</td>
			<td>Becton Dickinson, Heidelberg, Germany</td>
			<td style="width:129px;">300015</td>
			<td>sterile Luer-Lok&#8482; syringes with needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Gel Loader Tips</td>
			<td style="width:280px;">peqlab</td>
			<td style="width:129px;">81-13790</td>
			<td>M&#181;ltiFlex&#8482; Tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hyaluronidase</td>
			<td style="width:280px;">Sigma-Aldrich</td>
			<td style="width:129px;">H3884-100mg</td>
			<td>Hyaluronidase Type IV-S from Bovine test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Oxygen</td>
			<td style="width:280px;">Air Liquide, D&#252;sseldorf, Germany</td>
			<td style="width:129px;">M1001L50R2A001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Isofluoran</td>
			<td style="width:280px;">Baxter, Unterschlei&#223;heim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pGEM-T Easy</td>
			<td style="width:280px;">Promega, Mannheim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Oligonucleotides </td>
			<td style="width:280px;">Eurofins MWG, Ebersberg, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Qiaprep Spin Midiprep Kit </td>
			<td style="width:280px;">Qiagen, Hilden, Germany</td>
			<td style="width:129px;">27104</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:296px;">PCR DNA purification kit</td>
			<td style="width:280px;">Qiagen, Hilden, Germany</td>
			<td style="width:129px;">28106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:296px;">Equipment</td>
			<td style="width:280px;"> </td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Living Image 4.1 software</td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">XGI-8 Gas Anesthesia System</td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">IVIS Spectrum Imaging System </td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Biophotometer</td>
			<td style="width:280px;">Eppendorf AG, Hamburg, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
	</tbody>
</table>

following in real time the impact of pneumococcal virulence factors in an acute mouse pneumonia model using bioluminescent bacteria

Lungebetændelse er en af ​​de store sundhedsvæsenets problemer i udviklingslandene og de industrialiserede lande og er forbundet med betydelig sygelighed og dødelighed. Trods fremskridt i viden om denne sygdom, tilgængeligheden af intensivafdelinger (ICU), og brugen af potente antimikrobielle stoffer og effektive vacciner, dødeligheden fortsat høje 1.. Streptococcus pneumoniae er den førende patogen af community-erhvervet pneumoni (CAP) og en af ​​de mest almindelige årsager til bakteriæmi hos mennesker. Dette patogen er udstyret med en udrustning af overflade-eksponerede adhæsinerne og virulens faktorer, der bidrager til lungebetændelse og invasiv pneumokoksygdom (IPS). Vurderingen af in vivo rollen af bakterielle fitness eller virulensfaktorer er af allerstørste betydning at trævle S. pneumoniae patogenicitet mekanismer. Murine modeller af lungebetændelse, bakteriæmi og meningitis bliver brugt til at fastslå virkningen af ​​pneumokok-faktorer ved difskellige faser af infektionen. Her beskriver vi en protokol til at overvåge i realtid pneumokok udbredelse i mus efter intranasal eller intraperitoneal infektioner med selvlysende bakterier. Resultaterne viser formering og spredning af pneumokokker i de nedre luftveje og blod, som kan visualiseres og evalueres ved hjælp af et billedbehandlingssystem og den ledsagende analyse software.

Købet og optagelse af methionin er af central betydning for pneumokokker at opretholde fitness i deres vært niche 32,33. Methionin ABC transportør lipoprotein er kodet i D39 ved SPD _ 0151 gen (TIGR4: sp_0149) og navngivet MetQ 32.. Pneumokokker yderligere producere methionin biosyntese enzymer (D39: Spd_0510 - Spd_0511, TIGR4 Sp_0585 - Sp_0586, Mete og MetF). Den manglende methionin i et kemisk defineret medium påvirker væksten af pneumokokker og lignende, manglende...

Watch this Scientific Journal Video about Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria at JoVE.com

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Streptococcus pneumoniae er den førende patogen forårsager alvorlige community-erhvervet pneumoni og ansvarlig for over 2 millioner dødsfald på verdensplan. Kan følges op på virkningen af ​​bakterielle faktorer impliceret i fitness eller virulens i realtid i en akut mus lungebetændelse eller bakteriæmi model ved hjælp af selvlysende bakterier.

Efter i Real Time konsekvenserne af Pneumokok virulensfaktorer i en akut Mouse Lungebetændelse Model Brug Bioluminescent Bakterier

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Research

JoVE Journal

Immunology and Infection

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

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 ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Real-time Live Imaging af T-celle signalering Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Undersøge effekterne af probiotika på Pneumokok Colonization Brug af en<em&gt; In Vitro</em&gt; Adhærenceprøven

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Kapsulær Serotypning af<em&gt; Streptococcus pneumoniae</em&gt; Brug af Quellung Reaction

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

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

En<em&gt; Ex vivo</em&gt; Model af en oligodendrocyt rettet T-celle Angrib akut Brain Skiver

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Anvende en Inducerbar Expression System til Studieinformationen Interferens med bakteriel virulensfaktorer med intracellulær signalering

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Visualisere virkningerne af sputum på Biofilm Development Ved hjælp af en Chambered dækglas Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Evaluering af virulens og patogenese af Aeromonas infektion i en Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Billeddannelse af in situ-interferon gamma produktion i mus milt efter infektion med Listeria monocytogenes

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Generation af in-frame-Gensletnings mutanter i Pseudomonas aeruginosa og testning for virulens dæmpning i en simpel muse model af infektion

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...