Name: intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model
Uploaded: 2018-07-01T13:00:00
Description: Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

Detta arbete stöds av bidrag från nationella nyckel forskning och utveckling Program i Kina [2017YFD0500102]; Nationella naturvetenskapliga grunden för Kina [31572544]; Statliga centrala laboratoriet för veterinärmedicinska etiologisk biologi [SKLVEB2016KFKT005]; Shanghai jordbruk tillämpas teknik utveckling Program, Kina [G2016060201].

Mus infektion experimenten godkändes av den laboratorium djur övervakning kommittén i Jiangsu-provinsen, Kina och utföras i laboratorium djur Center av Nanjing jordbruket universitetet (tillståndsnummer: SYXK (Su) 2017-0007).
1. beredning av bakterier
Obs: S. suis serotyp 2 virulent stam P1/7 isolerades från en diseased gris med meningit19. Stam P1/7 odlades i Todd-Hewitt buljong (THB, formel per liter THB: hjärtat Infusion, 3,1...

<ol>
	<li>Gottschalk, M., Xu, J., Calzas, C., Segura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+suis:+a+new+emerging+or+an+old+neglected+zoonotic+pathogen?.">Streptococcus suis: a new emerging or an old neglected zoonotic pathogen?.</a> Future Microbiology. 5, 371-391 (2010).</li><li>Goyette-Desjardins, G., Auger, J. P., Xu, J., Segura, M., Gottschalk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+suis,+an+important+pig+pathogen+and+emerging+zoonotic+agent-an+update+on+the+worldwide+distribution+based+on+serotyping+and+sequence+typing.">Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing.</a> Emerging Microbes &amp; Infections. 3 (6), e45 (2014).</li><li>Kerdsin, A., 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=Emergence+of+Streptococcus+suis+serotype+9+infection+in+humans.">Emergence of Streptococcus suis serotype 9 infection in humans.</a> Journal of Microbiology, Immunology and Infection. 50 (4), 545-546 (2017).</li><li>Hatrongjit, R., 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=First+human+case+report+of+sepsis+due+to+infection+with+Streptococcus+suis+serotype+31+in+Thailand.">First human case report of sepsis due to infection with Streptococcus suis serotype 31 in Thailand.</a> BMC Infect Diseases. 15, 392 (2015).</li><li>Fittipaldi, N., Segura, M., Grenier, D., Gottschalk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+factors+involved+in+the+pathogenesis+of+the+infection+caused+by+the+swine+pathogen+and+zoonotic+agent+Streptococcus+suis.">Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis.</a> Future Microbiology. 7 (2), 259-279 (2012).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: from the natural environment to infectious diseases.</a> Nature Reviews Microbiology. 2 (2), 95-108 (2004).</li><li>Fuxman Bass, J. I., 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=Extracellular+DNA:+a+major+proinflammatory+component+of+Pseudomonas+aeruginosa+biofilms.">Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms.</a> Journal of Immunology. 184 (11), 6386-6395 (2010).</li><li>Isiaku, A. I., 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=Biofilm+is+associated+with+chronic+streptococcal+meningoencephalitis+in+fish.">Biofilm is associated with chronic streptococcal meningoencephalitis in fish.</a> Microbial Pathogenesis. 102, 59-68 (2017).</li><li>Oggioni, M. R., 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=Switch+from+planktonic+to+sessile+life:+a+major+event+in+pneumococcal+pathogenesis.">Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis.</a> Molecular Microbiology. 61 (5), 1196-1210 (2006).</li><li>Xiao, G., 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=Streptococcus+suis+small+RNA+rss04+contributes+to+the+induction+of+meningitis+by+regulating+capsule+synthesis+and+by+inducing+biofilm+formation+in+a+mouse+infection+model.">Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model.</a> Veterinary Microbiology. 199, 111-119 (2017).</li><li>Dominguez-Punaro, M. C., 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=Streptococcus+suis+serotype+2,+an+important+swine+and+human+pathogen,+induces+strong+systemic+and+cerebral+inflammatory+responses+in+a+mouse+model+of+infection.">Streptococcus suis serotype 2, an important swine and human pathogen, induces strong systemic and cerebral inflammatory responses in a mouse model of infection.</a> Journal of Immunology. 179 (3), 1842-1854 (2007).</li><li>Seitz, 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=A+novel+intranasal+mouse+model+for+mucosal+colonization+by+Streptococcus+suis+serotype+2.">A novel intranasal mouse model for mucosal colonization by Streptococcus suis serotype 2.</a> Journal of Medical Microbiology. 61 (Pt 9), 1311-1318 (2012).</li><li>Busque, P., Higgins, R., Caya, F., Quessy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+of+pigs+against+Streptococcus+suis+serotype+2+infection+using+a+live+avirulent+strain.">Immunization of pigs against Streptococcus suis serotype 2 infection using a live avirulent strain.</a> Canadian Journal of Veterinary Research. 61 (4), 275-279 (1997).</li><li>Williams, A. E., Blakemore, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+Streptococcal+meningitis+following+intravenous+intracisternal+and+natural+routes+of+infection.">Pathology of Streptococcal meningitis following intravenous intracisternal and natural routes of infection.</a> Neuropathology and Applied Neurobiology. 4 (4), 345-356 (1990).</li><li>Dominguez-Punaro, M. C., 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=Severe+cochlear+inflammation+and+vestibular+syndrome+in+an+experimental+model+of+Streptococcus+suis+infection+in+mice.">Severe cochlear inflammation and vestibular syndrome in an experimental model of Streptococcus suis infection in mice.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 31 (9), 2391-2400 (2012).</li><li>Auger, J. P., Fittipaldi, N., Benoit-Biancamano, M. O., Segura, M., Gottschalk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+Studies+of+Different+Sequence+Types+and+Geographical+Origins+of+Streptococcus+suis+Serotype+2+in+a+Mouse+Model+of+Infection.">Virulence Studies of Different Sequence Types and Geographical Origins of Streptococcus suis Serotype 2 in a Mouse Model of Infection.</a> Pathogens. 5 (3), (2016).</li><li>Chiavolini, D., 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=Method+for+inducing+experimental+pneumococcal+meningitis+in+outbred+mice.">Method for inducing experimental pneumococcal meningitis in outbred mice.</a> BMC Microbiology. 4, 36 (2004).</li><li>Madsen, L. W., Svensmark, B., Elvestad, K., Jensen, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Otitis+interna+is+a+frequent+sequela+to+Streptococcus+suis+meningitis+in+pigs.">Otitis interna is a frequent sequela to Streptococcus suis meningitis in pigs.</a> Veterinary Pathology. 38 (2), 190-195 (2001).</li><li>Holden, M. T., 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=Rapid+evolution+of+virulence+and+drug+resistance+in+the+emerging+zoonotic+pathogen+Streptococcus+suis.">Rapid evolution of virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis.</a> PLoS One. 4 (7), e6072 (2009).</li><li>Dominguez-Punaro Mde, L., 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=In+vitro+characterization+of+the+microglial+inflammatory+response+to+Streptococcus+suis,+an+important+emerging+zoonotic+agent+of+meningitis.">In vitro characterization of the microglial inflammatory response to Streptococcus suis, an important emerging zoonotic agent of meningitis.</a> Infection and Immunity. 78 (12), 5074-5085 (2010).</li><li>Hassan, A., 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=Evaluation+of+different+detection+methods+of+biofilm+formation+in+the+clinical+isolates.">Evaluation of different detection methods of biofilm formation in the clinical isolates.</a> Brazilian Journal of Infectious Diseases. 15 (4), 305-311 (2011).</li><li>Vanier, G., 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=New+putative+virulence+factors+of+Streptococcus+suis+involved+in+invasion+of+porcine+brain+microvascular+endothelial+cells.">New putative virulence factors of Streptococcus suis involved in invasion of porcine brain microvascular endothelial cells.</a> Microbial Pathogenesis. 46 (1), 13-20 (2009).</li><li>Takeuchi, D., 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=The+contribution+of+suilysin+to+the+pathogenesis+of+Streptococcus+suis+meningitis.">The contribution of suilysin to the pathogenesis of Streptococcus suis meningitis.</a> Journal of Infectious Diseases. 209 (10), 1509-1519 (2014).</li><li>Tenenbaum, T., 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=Polar+bacterial+invasion+and+translocation+of+Streptococcus+suis+across+the+blood-cerebrospinal+fluid+barrier+in+vitro.">Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.</a> Cellular Microbiology. 11 (2), 323-336 (2009).</li></ol>

Intrakraniell subarachnoidal rutten av infektion som beskrivs här har uppenbara fördelar över andra vägar av infektion. Det gör att utredarna att studera värd-bakterien interaktionen och effekten av bakteriella komponenter på värd immunsvar direkt i hjärnan, som härma bakteriell hänrycka in i det centrala nervsystemet. Denna väg av infektion kan således förlängas för att undersöka mekanismerna av hjärnhinneinflammation som orsakas av andra bakterier. Det kan dessutom också användas för att testa effe...

Streptococcus suis (S. suis) är stora bakteriella patogener av svin i världen, vilket orsakar svåra sjukdomar inklusive hjärnhinneinflammation, lunginflammation, septikemi, endokardit och artrit1. Det är också en framväxande zoonotiskt agens. Hittills har det rapporterats att nio serotyper kan orsaka infektion hos människa, inklusive serotyper 2, 4, 5, 9, 14, 16, 21, 24 och 312,3,4. Människor och svin är meningit en av de största kliniska tecknen på S. suis infektioner. I Vietnam och Thailand är S. suis den största orsaken till meningit hos vuxna5. Mikrobiell biofilmer är mikroorganismer som fäster vid varandra och är koncentrerade på ett gränssnitt; de är viktiga för bakteriell virulens, överlevnad i olika miljöer och antibiotikaresistens5. Biofilmer är oftast omgivna av en extracellulär matrix som generellt innehåller polysackarider, proteiner och DNA6. Det sistnämnda är kunna framkalla värd inflammatoriskt svar och cytokin produktion7. Biofilm bildning har rapporterats vara inblandade i streptokocker meningit i tidigare studier. Biofilmer bidra till Streptococcus agalactiae meningit i tilapia fisk modell och biofilm bildning har avslöjats inom hjärnans vävnader och runt meningeal ytor i vivo genom intraabdominella inympning8. Under meningit, Streptococcus pneumoniae är i en biofilm-liknande tillstånd och bakterier i sådan biofilm stat var effektivare för att framkalla meningit hos en mus infektion modell9. Dessutom i vår tidigare studie, biofilm staten är associerad med S. suis i mus hjärnan bidrar till bakteriell virulens av överlevnad analys10. Direkta bevis för biofilm inblandning i S. suis meningit kräver dock ytterligare utredning.
Djurmodeller av S. suis infektion har utvecklats hos möss med intraperitoneal (IP)11, intranasalt (i.n.)12, intravenös (i.v.)13och intracisternal (i.c.) rutter för infektion14, 15 , 16. i.p., i.n. och i.v. vägar på infektion är dock inte lämplig för att studera rollerna av S. suis ytan komponenter i hjärnhinneinflammation direkt i hjärnan. Dessa inkluderar extracellulär matrix från biofilmer. Även om i.c. inympningen användes för S. suis infektion, har exakt injektionsstället inte beskrivits i dessa papper. Däremot har de stereotaxic koordinaterna för injektionsstället för intrakraniell subarachnoidal inympningen tydligt beskrivits i en tidigare studie17. Detta gjorde det möjligt för enkel igenkänning av inokuleringspunkten och mer förenklade experimentellt protokoll. Dessutom härmar intrakraniell subarachnoidal rutten av infektion bakteriell hänrycka in i det centrala nervsystemet från bihålorna eller mellanörat17, och förhållandet mellan mellanörat och hjärnhinneinflammation orsakad av S. suis har påvisats genom Madsen et al18. Dessutom, genom att tillämpa intrakraniell subarachnoidal rutten av infektion hos möss, vi har visat att S. suis små RNA rss04 bidrar till hjärnhinneinflammation i vår tidigare studie10.
I förevarande studie, intrakraniell subarachnoidal rutten av infektion användes i möss för att undersöka rollerna av biofilmer i S. suis meningit. Möss var infekterade med plankton celler eller biofilm staten celler av S. suis av denna väg för infektion. Histopatologisk analys och ökat mRNA-uttryck av TLR2 och cytokiner från hjärnvävnaden hos möss som injicerats med biofilm staten celler tydligt anges att S. suis biofilm bidrar till hjärnhinneinflammation.

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td height="46">Todd Hewitt Broth(THB)</td>
			<td style="width:225px;">Becton, Dickinson and Company</td>
			<td style="width:115px;">DF0492078</td>
			<td style="width:304px;">Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td style="width:225px;">DSBIO</td>
			<td style="width:115px;">16C0050</td>
			<td style="width:304px;">Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:184px;">Milli-Q Reference Water Purification System</td>
			<td style="width:225px;">Merck KGaA</td>
			<td style="width:115px;">Z00QSVCUS</td>
			<td style="width:304px;">Without Dnase/ Rnase</td>
		</tr>
		<tr height="21">
			<td>NaCl</td>
			<td style="width:225px;">Tianjin Kemiou Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10019318</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>Na2HPO3</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009012-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>KCl</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009017-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="20">
			<td>KH2PO4</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009019-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="24">
			<td>KOH</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009014-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="42">
			<td>Glycerol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10010618</td>
			<td style="width:304px;">Diluted with equal volumu of purified water, autoclave at 121&#176; for 15 min</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">4% paraformaldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">80096675</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">25% Glutaraldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">30092436</td>
			<td style="width:304px;">10-fold diluted with purified water for fixation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ethanol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10009218</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10006818</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spctrophotometre</td>
			<td style="width:225px;">DeNovix Inc.</td>
			<td style="width:115px;">DS-11+</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ultrasound cell crusher</td>
			<td style="width:225px;">NingBo Scientz Biotechnology Co.,Ltd</td>
			<td style="width:115px;">JY96-IIN</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Centrifuge</td>
			<td style="width:225px;">Hitachi Koki Co., Ltd</td>
			<td style="width:115px;">CT15RE</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Refrigerator</td>
			<td style="width:225px;">Aucma Co., Ltd</td>
			<td style="width:115px;">DW-86L500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Scanning electron microscope</td>
			<td>Zeiss</td>
			<td style="width:115px;">EVO-LS10</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastRNA Pro Green Kit</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">#6045-050</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep-24 Instrument</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">116005500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Instrument for PCR</td>
			<td style="width:225px;">SensoQuest GmbH</td>
			<td style="width:115px;">1124310110</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantStudio 6 Flex</td>
			<td style="width:225px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">4485689</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">SYBR Premix Ex Taq II</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR820A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">PrimeScript RT reagent kit with gDNA Eraser</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR047A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Fully Enclosed Tissue Processor</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ASP200S</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Heated Paraffin Embedding Module</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">EG1150H</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Semi-Automated Rotary Microtome</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">RM2245</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Water bath for paraffin sections</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">HI1210</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Autostainer XL</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ST5010</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Agilent 2100</td>
			<td style="width:225px;">Agilent Technologies</td>
			<td style="width:115px;">G2939A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Optical microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:115px;">BX51</td>
			<td style="width:304px;"></td>
		</tr>
	</tbody>
</table>

intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model

Streptococcus suis är inte bara stora bakteriella patogener av svin i hela världen, men också en framväxande zoonotiskt agens. Människor och svin är meningit en viktig manifestation av S. suis infektioner. En lämplig infektion modell är ett viktigt verktyg att förstå mekanismerna av sjukdomar orsakade av patogener. Flera vägar av infektion i möss har utvecklats för att studera patogenesen av S. suis infektion. Intraperitoneal, intranasalt och intravenös vägar på infektion är dock inte lämplig för att studera S. suis ytan komponenter i hjärnhinneinflammation direkt i hjärnan, såsom den extracellular matrisen från biofilmer roller. Även om intracisternal inympning har använts för S. suis infektion, har exakt injektionsstället inte beskrivits. Här, beskrevs intrakraniell subarachnoidal rutten av infektion i en musmodell för att undersöka rollerna av biofilmer i S. suis meningit. S. suis plankton celler eller biofilm staten celler injicerades direkt i subaraknoidalrummet möss genom injektionsstället ligger 3,5 mm rostralt från bregma. Histopatologisk analys och ökat mRNA-uttryck av TLR2 och cytokiner av hjärnvävnad från möss som injicerats med biofilm staten celler anges tydligt att S. suis biofilm spelar slutgiltiga roller i S. suis meningit. Denna väg för infektion har uppenbara fördelar över andra vägar av infektion, möjliggör studiet av värd-bakterien interaktionen. Dessutom tillåter det effekten av bakteriella komponenter på värd immunsvar direkt i hjärnan för att bedömas, och härmar bakteriell hänrycka in i det centrala nervsystemet. Denna väg av infektion kan förlängas för att undersöka mekanismerna av hjärnhinneinflammation som orsakas av andra bakterier. Det kan dessutom också användas för att testa effekten av läkemedel mot bakteriell meningit.

SEM analys utfördes för att undersöka biofilm bildning de experimentella villkor. Som visas i figur 1, finns det en betydande skillnad i biofilm bildning mellan plankton (figur 1A) och biofilm staten celler (figur 1B). SEM analys visade att biofilm bakterier fanns i klumpar och flera lager och de var inneslutet i den extracellulära matrixen, medan plankton bakter...

Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

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

Här beskriver vi intrakraniell subarachnoidal rutten av infektion hos möss att studera roller av biofilmer i Streptococcus suis meningit. Denna infektion modell passar också studerar patogenesen av andra Bakteriell meningit och effekten av nya läkemedel mot bakteriell meningit.

Intrakraniell Subarachnoidal rutten av infektion för undersöker roller av Streptococcus suis biofilmer i hjärnhinneinflammation i en musmodell för infektion

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 ...

This method can help answer key questions about the roles of streptococcus suis'components in many sites, such as the extracellular matrix from its biofilms. The main advantage of this technique is that it enables investigations of bacterial components that affect how the immune responses directly in a brain and mimic bacteria entrance into the central nervous system. Demonstrating the procedure will be Mister Shouming Zhang, a graduate student from my laboratory.To collect strain P1/7 planktonic cells, first collect five milliliters of the cells from mid-lock phase culture. Centrifuge for three minutes at 8000 times G before washing the cells three times in PBS. Re-suspend the cells with five milliliters of 25%glycerol in THB and aliquot into five tubes.Alternatively, to collect strain P1/7 biofilm state cells, take 20 milliliters of an overnight culture and add to 180 milliliters of fresh THB. Equally divide the diluted culture into 10 round culture plates, then incubate the plates at 37 degrees celsius in 5%carbon dioxide for 24 hours. Following incubation, shake the plate gently to re-suspend the bacteria that have not adhered to the plate and avoid re-suspending the sediment.Then, discard the supernatant by aspiration. Add five milliliters of PBS to harvest biofilm state cells. Re-suspend the sediment completely and then transfer the sample to a new tube.Sonicate the re-suspended biofilm state cells as listed in the text protocol. Next, centrifuge the sonicated cells for three minutes at 8000 times G and store the supernatant at minus 80 degrees celsius. The supernatant may contain biofilm components and it can be used to re-suspend the biofilm bacteria before infection.Re-suspend the sediment with 10 milliliters of 25%glycerol in THB before observing the samples by scanning electron microscopy as described in the text protocol. Infect all mice using planktonic cells or biofilm state cells and perform euthanasia as described in the text protocol. Next, extract the total RNA from brain tissue using an RNA extraction kit.For each mouse, use whole brain tissue for extracting RNA. Divide whole brain tissue into five tubes. Take up to 100 milligrams of brain tissue and add one milliliter of lysis solution to each tube containing the lysis matrix provided by the kit.Extract the RNA following the steps listed in the text protocol. Next, perform CDMA synthesis, including elimination of genomic DNA, using a thermocycler with a reverse transcription reagent kit. Add one microgram of RNA to a tube containing two microliters of 5X genomic DNA elimination buffer and one microliter of geneomic DNA elimination enzyme.Add RNAse free water until the volume reaches 10 microliters. Incubate for two minutes at 42 degrees celsius. Add 10 microliters of master mix to the reaction solution and then mix gently.Proceed immediately with the reverse transcription reaction by placing the tube at 37 degrees celsius for 15 minutes, then transfer the reaction to 85 degrees celsius for five seconds. Perform the quantitative real-time PCR analysis using a real-time PCR machine with a cyber quantitative real-time PCR kit. Following the kit instructions, combine the enzyme, primers, 50X ROX reference dye two, and CDMA template, then add RNAse free water until a total volume of 20 microliters is reached.Run each sample in triplicate using the thermal parameters listed in the text protocol and calculate the relative fold-change based on the two minus delta delta CT method. Also perform the observation and scoring of histological sections of the infected brain tissue as detailed in the text protocol. The effect of biofilms on inflammatory response in mirroring brain tissue is shown here.At 12 hours post-infection, the expression of CCL2, IL6, TNF-alpha, and TLR2 from brains of infected mice was significantly higher for biofilm state cells as compared to planktonic cells. Far more severe pathological changes were observed in the meninges and cerebral cortex of mice infected with the biofilm bacteria as compared to mice infected with the planktonic bacteria. These changes included large areas of necrosis and intense inflammatory cell infiltration.However, much less inflammatory cell infiltration was observed in the meninges from mice infected with the planktonic bacteria. Furthermore, there was far more intense inflammatory cell infiltration observed in ventricles from mice infected with biofilm bacteria as compared to mice infected with the planktonic bacteria. As shown here, much less inflammatory cell infiltration was observed in ventricles from mice infected with the planktonic bacteria.While attempting this procedure, it is important to keep in mind that different strains may have different infective doses, so a preliminary experiment is strongly recommended to determine the appropriate infection dose. After watching this video, you should have a good understanding of how to collect biofilm cells and perform intercranial subretinal direct infection in mice.

Research

JoVE Journal

Immunology and Infection

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

Introducing Shear Stress in the Study of Bacterial Adhesion

Introduktion Shear stress i studien av bakteriell vidhäftning

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Encelliga Analys av Bacillus subtilis Biofilmer Använda fluorescensmikroskopi och flödescytometri

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction och 2-D-gelelektrofores Metoder för<em&gt; Burkholderia</em&gt; Arter

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Kapselkontraktur Serotypningen av<em&gt; Streptococcus pneumoniae</em&gt; Av latexagglutination

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

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

Icke-invasiv avbildning av det medfödda immun Response i en Zebrafish Larv Modell av<em&gt; Streptococcus iniae</em&gt; Infektion

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

A Murine Model of Group B Streptococcus Vaginal Colonization

En musmodell av grupp B<em&gt; Streptococcus</em&gt; Vaginal Colonization

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

Influenza A Virus Studies in a Mouse Model of Infection

Influensa A Virus studier på en musmodell av infektion

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Konstruera mutanter i Serotyp 1 Streptococcus pneumoniae stam 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

Antibiotikaeffekttestning i en Ex vivo-modell av Pseudomonas aeruginosa och Staphylococcus aureus Biofilms i cystisk fibros lunga

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

En musmodell för övergången av Streptococcus pneumoniae från kolonisatör till patogen vid viral saminfektion rekapitulerar åldersförvärrad sjukdom

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...