Name: assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface
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Forfatterne parlamentsarbejdet finansiel støtte fra Flandern Research Foundation til Marta Calatayud Arroyo (CVE postdoc stipendium-12N2815N). Emma Hernandez-Sanabria er postdoc stipendiat understøttes af Flandern Innovation og Entrepreneurship (Agentschap voor Innovatie dør Wetenschap da Technologie, IWT).

1. stammer og kultur betingelser
Bemærk: Syntetisk mundtlige Fællesskabet blev komponeret af stammer ofte er til stede i den mundtlige microbiome3.
<ol><li>Opnå de følgende stammer fra amerikansk Type kultur samling (ATCC): Aggregatibacter tanddannelsesforstyrrelser (ATCC 43718), Fusobacterium nucleatum (ATCC 10953), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedia (ATCC 25611), Strepto...

<ol>
	<li>Sender, R., Fuchs, S., Milo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+estimates+for+the+number+of+human+and+bacteria+cells+in+the+body.">Revised estimates for the number of human and bacteria cells in the body.</a> PLoS Biology. 14, e1002533 (2016).</li><li>Kelly, D., King, T., Aminov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+microbial+colonization+of+the+gut+in+early+life+to+the+development+of+immunity.">Importance of microbial colonization of the gut in early life to the development of immunity.</a> Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis. , 58-69 (2007).</li><li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+normal+bacterial+flora+of+the+oral+cavity.">Defining the normal bacterial flora of the oral cavity.</a> Journal of Clinical Microbiology. 43, 5721-5732 (2005).</li><li>Marsh, P. D., Head, D. A., Devine, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+plaque+as+a+biofilm+and+a+microbial+community-Implications+for+treatment.">Dental plaque as a biofilm and a microbial community-Implications for treatment.</a> Journal of Oral Biosciences. 57, 185-191 (2015).</li><li>Socransky, S. S., Haffajee, A. D., Cugini, M. A., Smith, C., Kent, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+complexes+in+subgingival+plaque.">Microbial complexes in subgingival plaque.</a> Journal of Clinical Periodontology. 25, 134-144 (1998).</li><li>Haffajee, A. 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=Subgingival+microbiota+in+healthy,+well-maintained+elder+and+periodontitis+subjects.">Subgingival microbiota in healthy, well-maintained elder and periodontitis subjects.</a> Journal of Clinical Periodontology. 25, 346-353 (1998).</li><li>Venema, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+metabolites+produced+by+the+colonic+microbiota+as+drivers+for+immunomodulation+in+the+host.">Microbial metabolites produced by the colonic microbiota as drivers for immunomodulation in the host.</a> The FASEB Journal. 27, (2013).</li><li>Marzorati, 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=The+HMI+(TM)+module:+A+new+tool+to+study+the+Host-Microbiota+Interaction+in+the+human+gastrointestinal+tract+in+vitro.">The HMI (TM) module: A new tool to study the Host-Microbiota Interaction in the human gastrointestinal tract in vitro.</a> BMC Microbiology. 14, 133 (2014).</li><li>Tanzer, J. M., Kurasz, A. B., Clive, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+displacement+of+mutans+streptococci+and+inhibition+of+tooth-decay+by+Streptococcus-Salivarius+Tove-R.">Competitive displacement of mutans streptococci and inhibition of tooth-decay by Streptococcus-Salivarius Tove-R.</a> Infection and Immunity. 48, 44-50 (1985).</li><li>Slomka, V., 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=Nutritional+stimulation+of+commensal+oral+bacteria+suppresses+pathogens:+the+prebiotic+concept.">Nutritional stimulation of commensal oral bacteria suppresses pathogens: the prebiotic concept.</a> Journal of Clinical Periodontology. 44, 344-352 (2017).</li><li>Hernandez-Sanabria, E., 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+increased+respiratory+activity+of+selected+oral+bacteria+may+explain+competitive+and+collaborative+interactions+in+the+oral+microbiome.">In vitro increased respiratory activity of selected oral bacteria may explain competitive and collaborative interactions in the oral microbiome.</a> Frontiers in Cellular and Infection Microbiology. 7, 235 (2017).</li><li>Alvarez, G., Gonzalez, M., Isabal, S., Blanc, V., Leon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+quantify+live+and+dead+cells+in+multi-species+oral+biofilm+by+real-time+PCR+with+propidium+monoazide.">Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide.</a> AMB Express. 3, (2013).</li><li>Ehsani, E., 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=Initial+evenness+determines+diversity+and+cell+density+dynamics+in+synthetic+microbial+ecosystems.">Initial evenness determines diversity and cell density dynamics in synthetic microbial ecosystems.</a> Scientific Reports. 8, 340 (2018).</li><li>Hernandez-Sanabria, E., 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=Correlation+of+particular+bacterial+PCR-denaturing+gradient+gel+electrophoresis+patterns+with+bovine+ruminal+fermentation+parameters+and+feed+efficiency+traits.">Correlation of particular bacterial PCR-denaturing gradient gel electrophoresis patterns with bovine ruminal fermentation parameters and feed efficiency traits.</a> Applied and Environmental Microbiology. 76, 6338-6350 (2010).</li><li>Masters, J. R., Stacey, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+medium+and+passaging+cell+lines.">Changing medium and passaging cell lines.</a> Nature Protocols. 2, 2276-2284 (2007).</li><li>Calatayud, M., Velez, D., Devesa, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+inorganic+arsenic+in+intestinal+epithelial+cell+lines.">Metabolism of inorganic arsenic in intestinal epithelial cell lines.</a> Chemical Research in Toxicology. 25, 2402-2411 (2012).</li><li>Minekus, 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+standardised+static+in+vitro+digestion+method+suitable+for+food+-+an+international+consensus.">A standardised static in vitro digestion method suitable for food - an international consensus.</a> Food &amp; Function. 5, 1113-1124 (2014).</li><li>Berney, M., Hammes, F., Bosshard, F., Weilenmann, H. U., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+interpretation+of+bacterial+viability+by+using+the+LIVE/DEAD+BacLight+kit+in+combination+with+flow+cytometry.">Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry.</a> Applied and Environmental Microbiology. 73, 3283-3290 (2007).</li><li>Props, R., Monsieurs, P., Mysara, M., Clement, L., Boon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+biodiversity+of+microbial+communities+by+flow+cytometry.">Measuring the biodiversity of microbial communities by flow cytometry.</a> Methods in Ecology and Evolution. 7, 1376-1385 (2016).</li><li>Yamashita, Y., Takeshita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oral+microbiome+and+human+health.">The oral microbiome and human health.</a> Journal of Oral Science. 59, 201-206 (2017).</li><li>Wade, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oral+microbiome+in+health+and+disease.">The oral microbiome in health and disease.</a> Pharmacological Research. 69, 137-143 (2013).</li><li>Yu, 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+resource+for+cell+line+authentication,+annotation+and+quality+control.">A resource for cell line authentication, annotation and quality control.</a> Nature. 520, 307 (2015).</li><li>Pelaseyed, 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=The+mucus+and+mucins+of+the+goblet+cells+and+enterocytes+provide+the+first+defense+line+of+the+gastrointestinal+tract+and+interact+with+the+immune+system.">The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.</a> Immunological Reviews. 260, 8-20 (2014).</li><li>Bengoa, A. 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=Simulated+gastrointestinal+conditions+increase+adhesion+ability+of+Lactobacillus+paracasei+strains+isolated+from+kefir+to+Caco-2+cells+and+mucin.">Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.</a> Food Research International. 103, 462-467 (2018).</li><li>Kleiveland, C. R., et al., Verhoeckx, K., 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> The Impact of Food Bioactives on Health: in vitro and ex vivo models. , 197-205 (2015).</li><li>Laparra, J. M., Sanz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+in+vitro+models+to+study+bacterial+adhesion+to+the+intestinal+epithelium.">Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium.</a> Letters in Applied Microbiology. 49, 695-701 (2009).</li><li>Gagnon, M., Berner, A. Z., Chervet, N., Chassard, C., Lacroix, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Caco-2,+HT-29+and+the+mucus-secreting+HT29-MTX+intestinal+cell+models+to+investigate+Salmonella+adhesion+and+invasion.">Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion.</a> Journal of Microbiological Methods. 94, 274-279 (2013).</li><li>Gro&#223;kopf, T., Soyer, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+microbial+communities.">Synthetic microbial communities.</a> Current Opinion in Microbiology. 18, 72-77 (2014).</li></ol>

Den mundtlige microbiome er et centralt element i den menneskelige sundhed som for nylig rapporteret af flere forfattere20,21. Tidligere resultater antyder, at indtagelse af spyt, som indeholder store masser af bakterier kan påvirke det mikrobielle økosystem af tyndtarmen, som er en af de vigtigste steder for immun priming. Kombinationen af en statisk øvre gastrointestinal fordøjelsen model med værtsinterface repræsenteret af epitel og slim-secernerende tar...

Mennesker bor sammen med bakterier, som findes på det samme nummer som menneskelige celler1. Derfor er det af afgørende vigtigt at opnå en omfattende forståelse af den menneskelige microbiome. Mundhulen er et unikt miljø, idet den er opdelt i flere mindre levesteder, som således indeholder en lang række bakterier og biofilm i de forskellige steder. Som et åbent økosystem, kan nogle arter i munden være forbigående besøgende. Dog kolonisere visse mikroorganismer snart efter fødslen og form organiseret biofilm2. Disse findes i tænder overfladen ovenfor gingival sprække, subgingival sprække, tunge, slimhindeinfektioner og dental proteser og fyld3. Bakterier kan også være til stede som flocs og planktoniske celler i lumen af tand-kanalen, enten blandet med nekrotisk pulp væv eller opslæmmet i en væske fase.
Der er aktive, sammenhængende cross-talk mellem værtsceller og bosiddende mikrobiota4. Bakterier kommunikere inden for og mellem arter, og kun en lille del af de naturlige kolonisatorer kan overholde væv, mens andre bakterier tillægger disse primære kolonisatorer. F.eks. celle-celle bindingen mellem mikroorganismer er nøglen for at integrere sekundære kolonisatorer i orale biofilm, og opbygge komplekse net af vekselvirkende mikrobielle celler4. Omkring er 70% af bakteriel aggregater i en spyt prøve dannet af Porphyromonas sp., Streptococcus sp., Prevotella sp., Veillonella sp. og uidentificerede Bacteroidetes. F. nucleatum er en mellemliggende kolonisator i subgingival biofilm og aggregater med de sene kolonisatorer P. gingivalis, T. denticola, og Tannerella forsythia, som er impliceret i parodontitis5. Derudover indtager Streptococcus mitis både slimhinde og dental levesteder, mens S. sanguinis og S. gordonii foretrækker at kolonisere tænder3. Således er, S. sanguinis til stede i nedre fortænder og hjørnetænder, mens Actinomyces naeslundii er blevet fundet i øvre anteriors6.
Derudover spiller de indfødte microbiome en rolle i at bevare sundhed2. Hjemmehørende mikrobiota deltager i immun uddannelse og forebyggelse af patogenet ekspansion. Denne kolonisering resistens opstår, fordi de indfødte bakterier kan være bedre tilpasset på knyttet til overflader, og mere effektiv på metabolising de tilgængelige næringsstoffer for vækst. Selvom probiotiske stammer overleve den gastrointestinale passage og forbliver aktive, er persistens på autoktont bakterier slugt en øvre beliggenhed af mave-tarmkanalen ikke blevet fuldt beskrevet. Dermed, vi underkastes en kunstig Fællesskabet, repræsentant for den mundtlige økosystem, simulerede gastrointestinale transit betingelser. Levedygtighed af bakterieceller blev vurderet ved hjælp af en multicompartment model, der ligner tarm epitel. Nuværende gut simulatorer tilbyde passende reproducerbarhed hvad angår analyse af den luminale mikrobielle samfund7. Dog er bakteriel adhæsion og vært-mikrobe interaktion særskilt behandlet, som kombinerer cellelinjer med mikrobielle samfund er udfordrende8. Derimod præsenterer vi en ramme, der giver potentielle mekanistiske forklaring af vellykket kolonisering begivenheder rapporteret på grænsefladen gut. Ja, denne model kan i fællesskab bruges med en statisk gut model til at evaluere virkningen af mikrobielle samfund på vært overflade signalering.

<table>
	<tbody>
		<tr>
			<td>STRAINS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aggregatibacter actinomycetemcomitans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 43718</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusobacterium nucleatum </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 10953</td>
			<td></td>
		</tr>
		<tr>
			<td>Porphyromonas gingivalis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33277</td>
			<td></td>
		</tr>
		<tr>
			<td>Prevotella intermedia </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25611</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mutans </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25175</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sobrinus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 33478</td>
			<td></td>
		</tr>
		<tr>
			<td>Actinomyces viscosus </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15987</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus salivarius&#160; TOVE-R</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus mitis </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49456</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus sanguinis </td>
			<td>BCCM/LMG Bacteria Collection</td>
			<td>LMG 14657</td>
			<td></td>
		</tr>
		<tr>
			<td>Veillonella parvula </td>
			<td>Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures</td>
			<td>DSM 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptococcus gordonii </td>
			<td>American Type Culture Collection</td>
			<td>ATCC 49818</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2 cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>86010202</td>
			<td></td>
		</tr>
		<tr>
			<td>HT29-MTX cells</td>
			<td>European Collection of Authenticated Cell Cultures</td>
			<td>12040401</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS AND CONSUMABLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) broth</td>
			<td>Oxoid</td>
			<td>CM1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar 2</td>
			<td>Oxoid</td>
			<td>CM0055</td>
			<td>Blood Agar medium</td>
		</tr>
		<tr>
			<td>Menadione</td>
			<td>Sigma</td>
			<td>M9429</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma</td>
			<td>H9039</td>
			<td></td>
		</tr>
		<tr>
			<td>5% sterile defibrinated horse blood</td>
			<td>E&#38;O Laboratories Ltd,</td>
			<td>P030</td>
			<td></td>
		</tr>
		<tr>
			<td>InnuPREP PCRpure Kit</td>
			<td>Analytik Jena</td>
			<td>845-KS-5010250</td>
			<td>PCR purification kit</td>
		</tr>
		<tr>
			<td>Big Dye</td>
			<td>Applied Biosystems</td>
			<td>4337454</td>
			<td>Dye for sequencing</td>
		</tr>
		<tr>
			<td>ABI Prism BigDye Terminator v3.1 cycle sequencing kit</td>
			<td>Applied Biosystems</td>
			<td>4337456</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR&#160;Green I</td>
			<td>Invitrogen</td>
			<td>S7585</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 culture flasks uncoated, cell-culture treated, vented, sterile</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T3924-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 
			0.4%, liquid, sterile-filtered</td>
			<td>Sigma-Aldrich</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture media, with GlutaMAX and Pyruvate</td>
			<td>Life technologies</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polyester membrane cell culture inserts</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3450-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach Type II&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Inactivated fetal bovine serum</td>
			<td>Greiner Bio One</td>
			<td>758093</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X 100 for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without calcium, magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce LDH Cytotoxicity Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>88953</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning HTS Transwell-24 well, pore size 0.4 &#181;m</td>
			<td>Corning Costar Corp</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Serva Electrophoresis</td>
			<td>28539010</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber improved</td>
			<td>Carl Roth</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>653118</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer</td>
			<td>MoBio</td>
			<td>13155</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>186-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Flush system</td>
			<td>Custom made</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>InnOva 4080 Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>8261-30-1007</td>
			<td>Shaker for 2.10</td>
		</tr>
		<tr>
			<td>Memmert CO2 incubator</td>
			<td>Memmert GmbH &#38; Co.</td>
			<td>ICO150med</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS (Electrical Resistance System)</td>
			<td>EMD Millipore, Merck KGaA</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore Milli-Q academic, ultra pure water system</td>
			<td>Millipore, Merck KGaA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker (ROCKER 3D basic)</td>
			<td>IKA</td>
			<td>4000000</td>
			<td>Shaker for 6.10</td>
		</tr>
	</tbody>
</table>

assessing the viability of a synthetic bacterial consortium on the in vitro gut host-microbe interface

Samspillet mellem vært og mikrobiota har længe erkendt og udførligt beskrevet. Munden er magen til andre dele af mave-tarmkanalen, som hjemmehørende mikrobiota opstår og forhindrer kolonisering af eksogene bakterier. Ja, mere end 600 arter af bakterier findes i mundhulen, og et enkelt individ kan bære omkring 100 forskellige når som helst. Orale bakterier besidder evnen til at overholde de forskellige nicher i den mundtlige økosystem, således at blive integreret i resident mikrobielle samfund, og fremme vækst og overlevelse. Strømmen af bakterier i tarmen under synke har imidlertid været foreslået at forstyrre balancen i gut mikrobiota. I virkeligheden, flyttet oral administration af P. gingivalis bakteriel sammensætning i ileal mikrofloraen. Vi brugte en syntetisk Fællesskabet som en forenklet gengivelse af den naturlige mundtlige økosystem, for at belyse overlevelse og rentabilitet af orale bakterier udsat for simuleret gastrointestinale transit betingelser. 14 arter blev valgt, udsat for in vitro- spyt, mavens og tarm fordøjelsen processer og præsenteret for en multicompartment celle model Caco-2 og HT29-MTX celler for at simulere gut slimhinde epitel. Denne model tjente til at udrede konsekvenserne af indtagelse bakterier på celler involveret i den enterohepatiske cirkulation. Ved hjælp af syntetiske Fællesskaber giver kontrollerbarhed og reproducerbarhed. Således, denne metodologi kan tilpasses til at vurdere patogen levedygtighed og efterfølgende betændelse-associerede forandringer, kolonisering kapacitet af probiotiske blandinger, og i sidste ende, potentielle bakteriel påvirke præsystemisk cirkulation.

Denne protokol fører til generation af en model velegnet til belyse overlevelse og rentabilitet af orale bakterier udsat for simuleret gastrointestinale transit betingelser. Greverne af intakt celler fra enkelte stammer er ca 108 celler mL-1 før oprettelsen af syntetiske Fællesskabet, mens multispecies mikrokosmos indeholdt over 90% af levedygtige celler under oprettelsen af Fællesskabet ( Figur 1A og 1B</st...

Watch this Scientific Journal Video about Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface at JoVE.com

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut vært-mikrobe interaktioner blev vurderet ved hjælp af en ny tilgang, der kombinerer en syntetisk mundtlige Fællesskabet, in vitro- mave fordøjelse og en model af tyndtarmen epitel. Vi præsenterer en metode, der kan tilpasses til at evaluere celle invasion af patogener og flere arter biofilm, eller selv at teste probiotiske formuleringer overlevelsesevne.

Vurdering af levedygtigheden af en syntetisk bakteriel konsortium på In Vitro Gut Host-mikrobe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Research

JoVE Journal

Biochemistry

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Påvisning af pH-afhængige aktivitet af<em&gt; Escherichia coli</em&gt; Chaperone HdeB<em&gt; In vitro</em&gt; og<em&gt; In vivo</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Brug Stillads Liposomer at Rekonstituer Lipid-proksimal protein-protein interaktioner<em&gt; In vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Oprensning af biotinylerede celleoverfladeproteiner fra<em&gt; Rhipicephalus microplus</em&gt; Epitelceller

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

In Vitro Polymerization of F-actin on Early Endosomes

In Vitro Polymerization of F-actin on Early Endosomes

In Vitro Polymerisering af F-actin på tidlig Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

En simpel metode til High Throughput kemisk Screening i Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Colorimetric Method for Measuring Iron Content in Plants

En kolorimetriske metode til måling af jernindhold i planter

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Toeprinting analyse af oversættelse indledningen kompleks dannelse på pattedyr mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...