Name: a method to assess bacteriocin effects on the gut microbiota of mice
Uploaded: 2017-07-25T15:00:00
Description: Watch this Scientific Journal Video about A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice at JoVE.com

Forfatterne ønsker at takke EEA Grant NILS Videnskab og Bæredygtighed Koordineret Mobilitet for Forskere (referencen 017-ABEL-CM-2013). CB og GP-M. Blev støttet af AGL2015-70487-P tilskud fra det spanske ministerium for økonomi og konkurrenceevne. OCOU og DBD blev støttet af et strategisk stipendiumsprogram for fødevareforskning fra det norske universitets biovidenskab (NMBU) (projekt 1205051025). Vi vil også gerne takke Inmaculada Noguera for hendes hjælp med dyrepleje og prøveudtagning og Jesus Dehesa for hans hjælp med at sikre tilgængeligheden af ​​laboratoriematerialer i dyreanlægget. Vi værdsætter også professor Lars-Gustav Snipen for hans råd om statistik.

Pleje og håndtering skal udføres på en specialiseret dyreplejeproduktion. De procedurer, der er beskrevet her, blev godkendt af den tilsvarende etiske komité for universitetet i Valencia og de lokale myndigheder efter principperne om laboratoriedyrpleje obligatorisk i henhold til EU-lovgivningen og 2010/63 / EU og den spanske regering RD 53/2013 om beskyttelse af dyr Anvendt til videnskabelige formål for at respektere 3R-princippet i dyreforsøg (erstatning, reduktion, forfining). 1. Fros...

<ol>
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Den her beskrevne procedure er blevet brugt til at bestemme, om ændringer i mikrobiota er bundet til helbred eller alder. Forskellige dele af protokollen er vigtige, men blandt dem kan prøveudtagning af fæces, valg af DNA-fragmentet, der sekventeres og analyseres, og udførelse af DNA-ekstraktion og bioinformatisk analyse sikkert være de mest kritiske punkter. Prøveudtagning er afgørende, fordi mus af etiske årsager ikke bør stresses, og fordi det vides at ændre mængden af ​​bakterier i tarmen. Prøver ska...

Bakteriociner er antimikrobielle peptider fremstillet af en bred vifte af bakteriearter 1 , 2 . Disse forbindelser og deres producenter, især LAB, er blevet udforsket og udnyttet verden over i årtier for deres potentielle anvendelser i fødevarebevarelse og medicin 3 . Flere bakteriociner er kendt for at dræbe vigtige patogener, herunder arter af Listeria, Enterococcus, Staphylococcus og Bacillus . Nogle bakteriociner har endda evnen til at modulere immunresponset 4 . Mange bakteriociner har relativt snævre spektra, en egenskab, der er meget værdsat i nogle applikationer. For eksempel kan nogle smalspektrede bakteriociner anvendes til at lede specifik aktivitet mod udvalgte grupper af problematiske bakterier uden stor forstyrrelse på kommensal eller gavnlig flora, der deler samme niche; Dette er især vigtigt i tarmenOnment, hvor mange gavnlige mikrober trives på en interaktiv og dynamisk måde 5 . Bakteriociner er også meget attraktive til profylaktisk eller probiotisk anvendelse, da de kan undertrykke (ud) vækst af patogener, patobioner eller opportunistiske bakterier, der kan ubalancere tarmhomeostasen 6 , 7 . Med hensyn til deres natur og fysisk-kemiske egenskaber er bakteriociner meget forskellige, da de har forskellige strukturer, målspecifikiteter, virkningsmåder mv. De fleste bakteriociner er blevet undersøgt meget detaljeret i in vitro- indstillinger, men meget få er blevet testet i fødevarer Matricer 8 , 9 eller in vivo, såsom i en dyrecut 6 , 10 . In vitro- egenskaberne kan i høj grad afvige, når de vurderes in vivo på grund af kompleksiteten oF tarmmiljøet og også til formodede utilsigtede virkninger på gavnlige bakterier. De fleste probiotika er LAB. De producerer en række andre metabolitter, herunder kortkædede fedtsyrer, som er kendt for at påvirke værtsens fysiologi, samt at demonstrere antimikrobielle egenskaber mod bestemte bakterier. Derfor er det i tilfælde af probiotiske stammer, der producerer bakteriociner, bedst at etablere realistiske analyser, såsom sunde dyr med normal mikrobiota. I den foreliggende undersøgelse giver vi en strategi, der muliggør vurdering af virkningen af ​​forskellige bakteriocinproducerende stammer, hvis bakteriociner har forskellige hæmmende spektre på raske mus. Vores strategi omfatter fodring af mus med isogene ikke-bakteriocinmutanter, som muliggør differentiering af bakteriocin-medierede effekter fra ikke-bakteriocin-medierede effekter. Sekventering 16S rDNA giver mulighed for at følge de dynamiske ændringer af bakteriepopulationen i tarmen. SubsÆkvivalent statistisk analyse bestemmer korrelationer mellem bakteriearter og også mellem bakteriearter og målte fysiologiske parametre ( fx kropsvægt, serumbiokemiske parametre osv. ). Vi mener, at protokollen, der præsenteres i denne undersøgelse, også kan anvendes på andre probiotiske eller præbiotiske anvendelser ud over undersøgelsen af ​​bakteriociner hos levende dyr.

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Balb/c mice (female)</td>
			<td style="width:141px;">Harlan</td>
			<td style="width:183px;"></td>
			<td style="width:287px;">Mice should be 6 - 8 weeks of age</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic Petri dish</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:183px;">101VR20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brain-Heart-Infusion broth</td>
			<td style="width:141px;">Conda</td>
			<td style="width:183px;">1400.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">European Bacteriological Agar</td>
			<td style="width:141px;">Pronadisa</td>
			<td style="width:183px;">1800.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose D1 Low EEO</td>
			<td style="width:141px;">Pronadisa</td>
			<td style="width:183px;">8010.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1XTAE buffer</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:183px;">15558042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MRS broth</td>
			<td style="width:141px;">Difco</td>
			<td style="width:183px;">288130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS tablets</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">P4417-100TAB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scale</td>
			<td style="width:141px;">Mettler Toledo</td>
			<td style="width:183px;">PB602-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sterile forceps</td>
			<td style="width:141px;">Levantina de Laboratorios S.L.</td>
			<td style="width:183px;">260-3710014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:141px;">Eppendorf</td>
			<td style="width:183px;">5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:141px;">Hermle</td>
			<td style="width:183px;">Z383K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:141px;">AppliChem Panreac</td>
			<td style="width:183px;">121659.1211</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Realpure SSS Kit</td>
			<td style="width:141px;">Real Life Science Solutions, Durviz, Spain</td>
			<td style="width:183px;">RBME04 (300 ml)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropanol</td>
			<td style="width:141px;">AppliChem Panreac</td>
			<td style="width:183px;">131090.1611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:141px;">AppliChem Panreac</td>
			<td style="width:183px;">131086.1214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit fluorometer</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:183px;">Q32851</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AMPure XP beads</td>
			<td style="width:141px;">Beckman Coulter Genomics, USA</td>
			<td style="width:183px;">A63881 (60 ml)</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PerfeCta NGS library quantification kit</td>
			<td style="width:141px;">Quanta BioSciences, Maryland, USA</td>
			<td style="width:183px;">733-2300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MiSeq v3 reagent kit</td>
			<td style="width:141px;">Illumina, San Diego, California, USA</td>
			<td style="width:183px;">MS-102-3003</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Primers for 16S rRNA gene amplification</td>
			<td style="width:141px;"></td>
			<td style="width:183px;"></td>
			<td style="width:287px;">Primers contain V3-V4 region of bacterial 16S rRNA gene and Illumina overhang adaptors:5&#8217;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3&#8217; and 5&#39;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nextera XT Index kit</td>
			<td style="width:141px;"></td>
			<td style="width:183px;">FC-131-1002</td>
			<td>Indices and Illumina sequencing adaptors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropestle for 1.5 ml tubes, Eppendorf / Sigma , Ref.</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">Z317314-1PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass beads, 0.1 mm diameter</td>
			<td style="width:141px;">Biospec Products</td>
			<td style="width:183px;">11079-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoSpin Gel and PCR Clean-up Kit</td>
			<td style="width:141px;">Macherey-Nagel</td>
			<td style="width:183px;">740609.25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Omni Bead Ruptor 24</td>
			<td style="width:141px;">Omni International Inc.</td>
			<td style="width:183px;">19-040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mutanolysin</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">M9901-10KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lysozyme</td>
			<td style="width:141px;">Roche</td>
			<td style="width:183px;">10837059001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">proteinase K</td>
			<td style="width:141px;">Roche</td>
			<td style="width:183px;">3115887001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase A</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:183px;">R4875</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to assess bacteriocin effects on the gut microbiota of mice

Meget spændende spørgsmål opstår med vores fremadgående viden om gutmikrobiotasammensætning og forholdet til sundhed, især med hensyn til de faktorer, der bidrager til at opretholde befolkningsbalancen. Der er dog begrænsede tilgængelige metoder til evaluering af disse faktorer. Bakteriociner er antimikrobielle peptider produceret af mange bakterier, der kan give en konkurrencemæssig fordel for fødevareopkøb og / eller niche-etablering. Mange probiotiske mælkesyrebakterier (LAB) -stammer har stort potentiale til at fremme menneskers og dyrs sundhed ved at forhindre vækst af patogener. De kan også bruges til immunomodulation, da de producerer bakteriociner. Imidlertid bestemmes den antagonistiske aktivitet af bakteriociner normalt ved laboratorie bioassays under veldefinerede, men overforenklede betingelser sammenlignet med det komplekse tarmmiljø hos mennesker og dyr, hvor bakterier står over for multifaktoriale påvirkninger fra værten og hundredvis af mikrobielle arter sHaring den samme niche. Dette værk beskriver en komplet og effektiv procedure til vurdering af effekten Af en række bakteriociner med forskellige målspecificiteter i et murinsystem. Ændringer i mikrobiotasammensætningen under bakteriocinbehandlingen overvåges under anvendelse af 16S rDNA-sekvensbestemmelse. Vores tilgang benytter både bakteriocinproducenterne og deres isogene ikke-bakteriocinproducerende mutanter, idet sidstnævnte giver mulighed for at skelne bakteriocinrelateret fra ikke-bakteriocinrelaterede modifikationer af mikrobiota. Fekal DNA-ekstraktionen og 16S rDNA-sekventeringsmetoderne er konsistente og udgør sammen med bioinformatikken en stærk fremgangsmåde til at finde svage ændringer i bakterieprofilerne og etablere korrelationer, hvad angår kolesterol og triglyceridkoncentration mellem bakteriepopulationer og sundhedsmarkører. Vores protokol er generisk og kan således bruges til at studere andre forbindelser eller næringsstoffer med potentialet til at ændre værtsmikrofonenRobiota-sammensætning, enten ved undersøgelse af toksicitet eller gavnlige virkninger.

Produktionen af ​​bakteriociner er blevet betragtet som en positiv probiotisk funktion i LAB, da det antages at forhindre væksten af ​​opportunistiske bakterier og patogener. Formålet med dette arbejde var at vise bakteriocins evne til at modulere tarmmikrobiotapopulationer i en musemodel. Til dette formål blev der udviklet en procedure til sammenligning af effekten af ​​indtaget af bakteriocinproducerende stammer og deres isogene ikke-producerende stammer. Fremgangsmåden...

Watch this Scientific Journal Video about A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice at JoVE.com

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

En metode til vurdering af bakterieocin-virkninger på musens tarmmikrobiotikum

Bakteriociner menes at spille en central rolle i at definere mikrobiell mangfoldighed i forskellige økologiske nicher. Her beskriver vi en effektiv procedure til vurdering af, hvordan bakteriociner påvirker tarmmikrobiotasammensætningen i en dyremodel.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Research

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Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

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Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

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Metode til vurdering af effekter af en række bølgelængder og intensiteter af Rød / nær-infrarødt lys terapi på oxidativt stress<em&gt; In vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

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Mouse models of genetic and acquired risk factors for atrial fibrillation (AF) have proven valuable in investigating the molecular determinants of AF. ...