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

Dette arbejde blev støttet af tilskud fra den nationale nøgle forskning og udvikling Program i Kina [2017YFD0500102]; National Natural Science Foundation i Kina [31572544]; den statslige centrale laboratorium af veterinære ætiologisk biologi [SKLVEB2016KFKT005]; Shanghai landbrug anvendt teknologi udvikling Program, Kina [G2016060201].

Musen infektion eksperimenter blev godkendt af det laboratorium dyr overvågning Udvalget af Jiangsu-provinsen, Kina og udført i Laboratory Animal Center af Nanjing Landbohøjskole (autorisationsnummer: SYXK (Su) 2017-0007).
1. forberedelse af bakterier
Bemærk: S. suis serotype 2 ondartet stamme P1/7 blev isoleret fra syge svin med meningitis19. Stamme P1/7 blev dyrket i Todd-Hewitt bouillon (THB, formel pr. liter af THB: hjertet Inf...

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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>

Ruten intrakranielle subarachnoidal af infektion beskrevet her har åbenlyse fordele i forhold til andre ruter af infektion. Det giver mulighed for efterforskerne at studere vært-bakterien interaktion og påvirkning af bakteriel komponenter vært immunrespons direkte i hjernen, som efterligner bakteriel indgangen i det centrale nervesystem. Således, denne rute af infektion kan udvides for at undersøge mekanismerne af meningitis forårsaget af andre bakterier. Det kan desuden også bruges til at teste effekten af læge...

Streptococcus suis (S. suis) er en større bakteriel patogen svin på verdensplan, forårsager alvorlige sygdomme herunder meningitis, lungebetændelse, septikæmi, endocarditis, og gigt1. Det er også en spirende zoonotisk agens. Hidtil er blevet rapporteret, at ni serotyper kan forårsage infektion hos mennesker, herunder serotyper 2, 4, 5, 9, 14, 16, 21, 24 og 312,3,4. Hos mennesker og grise er meningitis en af de store kliniske tegn på S. suis infektioner. I Vietnam og Thailand er S. suis den største årsag til meningitis hos voksne5. Mikrobielle biofilm er mikroorganismer, som holde sig til hinanden og er koncentreret på en grænseflade; de er uundværlige for bakteriel virulens, overlevelse i forskellige miljøer og antibiotikaresistens5. Biofilm er typisk omgivet af en ekstracellulære matrix, der generelt indeholder polysaccharider, proteiner og DNA6. Sidstnævnte er i stand til at fremkalde vært inflammatoriske respons og cytokin produktion7. Biofilmdannelse er blevet rapporteret at være involveret i streptokok meningitis i tidligere undersøgelser. Biofilm bidrager til Streptococcus agalactiae meningitis i en tilapia fisk model og Biofilmdannelse er blevet afsløret i hjernen væv og omkring meningeal overflader i vivo gennem intra-abdominal podning8. Under meningitis, Streptococcus pneumoniae er i en biofilm-lignende tilstand og bakterier i en biofilm situation var mere effektiv i inducerende meningitis i en mus infektion model9. Desuden, i vores tidligere undersøgelse, biofilm staten forbundet med S. suis i mus hjernen bidrager til bakteriel virulens af overlevelse analyse10. Dog kræver direkte beviser for biofilm inddragelse i S. suis meningitis yderligere undersøgelser.
S. suis infektion-dyremodeller er blevet udviklet i mus bruger intraperitoneal (i.p.)11, intranasal (i.n.)12, intravenøs (IV)13, og intracisternal (IC) ruterne af infektion14, 15 , 16. i.p., i.n. og i.v. ruterne af infektion er dog ikke velegnet til at studere roller S. suis overflade komponenter i meningitis direkte i hjernen. Disse omfatter ekstracellulære matrix fra biofilm. Selv om IC inokulation blev brugt til S. suis infektion, er præcise injektionsstedet ikke blevet beskrevet i disse papirer. Derimod stereotaxisk koordinaterne for injektionsstedet til intrakraniel subarachnoidal podning er klart blevet beskrevet i en tidligere undersøgelse17. Dette gav let anerkendelse af punktet, podning og mere forsimplede forsøgsplan. Derudover efterligner ruten intrakranielle subarachnoidal af infektion bakteriel indgangen til centralnervesystemet fra bihulerne eller mellemøret17og forholdet mellem mellemøret og meningitis forårsaget af S. suis er blevet påvist ved Madsen mfl18. Derudover anvender ruten intrakranielle subarachnoidal infektion hos mus, har vi vist, at S. suis små RNA rss04 bidrager til meningitis i vores tidligere undersøgelse10.
I nuværende undersøgelse, intrakraniel subarachnoidal rute af infektion blev brugt i mus til at undersøge roller af biofilm i S. suis meningitis. Mus blev smittet med planktoniske celler eller biofilm stat celler af S. suis ad denne vej af infektion. Histopatologisk analyse og øget mRNA udtryk af TLR2 og cytokiner fra hjernevæv af musene injiceret med biofilm stat celler tydeligt angivet, S. suis biofilm bidrager til meningitis.

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			<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 er ikke kun en stor bakteriel patogen svin på verdensplan, men også en spirende zoonotisk agens. Hos mennesker og grise er meningitis en større manifestation af S. suis infektioner. En egnet infektion model er et vigtigt redskab til at forstå mekanismerne af sygdomme forårsaget af patogener. Flere ruter af infektion i mus er blevet udviklet for at studere patogenesen af S. suis infektion. Ruterne intraperitoneal, intranasal og intravenøs infektion er dog ikke velegnet til at studere roller S. suis overflade komponenter i meningitis direkte i hjernen, såsom den ekstracellulære matrix fra biofilm. Selvom intracisternal podning har været brugt til S. suis infektion, præcise injektionsstedet ikke er blevet beskrevet. Her, blev intrakraniel subarachnoidal rute af infektion beskrevet i en musemodel at undersøge roller af biofilm i S. suis meningitis. S. suis planktoniske celler eller biofilm stat celler blev direkte sprøjtet ind i subaraknoid rummet af mus gennem injektionsstedet beliggende 3,5 mm rostralt fra bregma. Histopatologisk analyse og øget mRNA udtryk af TLR2 og cytokiner af hjernevæv fra musene injiceret med biofilm stat celler tydeligt angivet, S. suis biofilm spiller endelige roller i S. suis meningitis. Denne rute af infektion har åbenlyse fordele i forhold til andre ruter af infektion, giver mulighed for undersøgelse af vært-bakterien interaktionen. Desuden, det tillader bakteriel komponenter indvirkning på værten immunrespons direkte i hjernen til at blive vurderet, og efterligner bakteriel indgangen i det centrale nervesystem. Denne rute af infektion kan udvides for at undersøge mekanismerne af meningitis forårsaget af andre bakterier. Det kan desuden også bruges til at teste effekten af lægemidler mod bakteriel meningitis.

SEM analyse blev udført for at undersøge Biofilmdannelse under forsøgsbetingelserne. Som vist i figur 1, er der en betydelig forskel i Biofilmdannelse mellem planktoniske celler (fig. 1A) og biofilm stat celler (figur 1B). SEM analyse viste, at biofilm bakterier var i klumper og flere lag, og de var indkapslet i den ekstracellulære matrix, mens planktoniske bakte...

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

Her, beskriver vi ruten intrakranielle subarachnoidal af infektion i mus at studere roller af biofilm i Streptococcus suis meningitis. Denne infektion model er også velegnet til at studere patogenesen af andre bakteriel meningitis og effekten af nye lægemidler mod bakteriel meningitis.

Intrakranielle Subarachnoidal rute for infektion for at undersøge roller af Streptococcus suis biofilm i Meningitis i en musemodel 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

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