Name: examination of host phenotypes in gambusia affinis following antibiotic treatment
Uploaded: 2017-02-22T14:00:00
Description: Watch this Scientific Journal Video about Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment at JoVE.com

This project was partially funded by a FAST (Faculty and Student Team) Award to TPP and JMC from EURECA (Center for Enhancing Undergraduate Research Experiences and Creative Activities) at Sam Houston State University.

blev udført Alle eksperimenter dyr under godkendelse af IACUC protokoller, nummereret 14-05-05-1018-3-01, 13-04-29-1018-3-01, og 14-04-17-1018-3-01. 1. Animalske Collection, håndtering og Etisk Care <ol><li> Saml Gambusia affinis fra marken webstedet (identifikation guide på http://www.sms.si.edu/irlspec/Gambusia_affinis.htm) ved hjælp af en lille dip net og sted i 19 L spande. Brug visuel inspektion for at identificere arter. </li><li> Rest fisk til 1 - 2 ud d i en spand med dam vand. Bagefter ov...

<ol>
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Nogle udfordringer kræver en hvileperiode i ren APW efter antibiotisk behandling for stof til at være udtømt i fisk væv. Hvis hvileperioden springes derefter antibiotisk tilstedeværelse kan forvirre resultaterne, især når assay involverer udsættelse for bakterier. For at undersøge effekter fra en ændret microbiome sammensætning uden store ændringer i det samlede antal af mikrober på værten, indledende forsøg overvågning microbiome sammensætning (16S profilering eller hele genomet sekventering) og befolk...

Det er konstateret, at antibiotika kan forstyrre den menneskelige microbiome fører til dysbiosis, hvilket betyder en mikrobielle samfund ubalance. Den mikrobiota kompositoriske ændring efter behandling med antibiotika har vist sig at sænke samfunds mangfoldighed, reducere centrale medlemmer, og ændre samfundet stofskifte, specielt i tarmen 1, 2. Antibiotikum forstyrrelse af tarmen microbiome kan reducere kolonisering modstand mod Clostridium difficile 3, 4 og Salmonella 5. Derudover har afbrydelse af mikrobiota været knyttet til udviklingen af mange syndromer og sygdomme hos mennesker (f.eks antibiotika-associeret enterocolitis, inflammatorisk tarmsygdom, stofskiftesygdomme osv.). Antibiotika er også i vid udstrækning implementeret i landbruget som vækstfremmere ikvæg og fjerkræ produktion 6. Brugen af ​​disse kraftfulde værktøjer er ikke uden sideeffekter, hvilket er tydeligt i den hurtige stigning af antibiotikaresistens, samt virkningerne af et afbrudt microbiome har med sin beboede vært. Mange undersøgelser har vist, at bredspektrede antibiotika forbrug har langvarige konsekvenser for struktur og funktion af mikrobiota, endnu bivirkningerne fra en antibiotika-forstyrret microbiome påvirker vært fysiologi er kun spekulationer, som endnu ikke er understøttet. Samspillet mellem værten, mikrobiota, og antibiotika er langt fra at blive forstået i en kortfattet måde. Derfor er en enkel og mere medgørlig model er en fordel at kaste lys over de meget komplekse pattedyr system. Slimhindeoverflader hos mennesker, herunder tarmen, huser den højeste tæthed og mangfoldighed af mikrober, og også de mest intime mikrobe-vært interaktioner. Den slimhinde hud microbiome af fisk tilbud several fordele som et modelsystem. Den Teleostei (benede fisk) er en af de tidligste slægter at divergerer i Vertebrata betyder, at benfisk har både medfødte og erhvervede immunforsvar, der har co-udviklet et forhold med kommensale bakterielle samfund 7. Fiskeskind deler mange karakteristika med type 1 slimhindeoverflader hos pattedyr, såsom fysiologiske funktioner, Immunity komponenter og arrangement af slim-producerende celler 8. Den eksterne placering af fiskeskind slimhindeoverfladen tilbyder en microbiome let at eksperimentelt manipulere og prøve. Den vestlige mosquitofish, Gambusia affinis (G. affinis), er en model fisk, der har været anvendt i fortiden for at studere parring og toksikologi 9, 10, 11. I betragtning af den lille størrelse, befolkning overflod i naturen som en invasiv art, m<html> inimal pleje omkostninger, og hårdføre natur, har vi udviklet G. affinis som en slimhinde microbiome model. Endvidere Gambusia deler fysiologi for at føde levende unger med viviparous pattedyr, hvilket er usædvanligt i fiskearter. Vi afsluttede den mest omfattende undersøgelse på det tidspunkt, fiskeskind normal mikrobiota hjælp 16S profilering med Gambusia 12. Yderligere arbejde demonstrerede tre negative virkninger på værten efter afbrydelse af huden og tarmen mikrobiota ved hjælp af en bredspektret antibiotikum 13. Fem forskellige effekter blev undersøgt i fisk efter antibiotisk eksponering. Den mest veletablerede vært gavn for microbiome er konkurrencedygtig udelukkelse af patogener. Fiskene patogen Edwardsiella ictaluri vides at forårsage udbrud af enterisk septikæmi i kommercielle havkat gårde 14. E. ictaluri har også vist sig at letalt inficere zebrafiskclass = &quot;xref&quot;&gt; 15, 16 og Gambusia 17. En udfordring med dette patogen fra vandsøjlen kan tjene som et mål for udstødelse. Som en sammenligning med modtagelighed for en individuel patogen, blev overlevelse under eksponering for en høj tæthed af blandede organismer også udført. Afføring og organisk-rige jord blev brugt som almindeligt stødt kilder til mikrobielle samfund. En anden etableret rolle den bakterielle tarm samfund udfører behandler næringsstoffer og energi høst, hvilket påvirker den samlede ernæringsmæssige optagelse for værten. Som en grov måling af ernæring, blev fisk kropsvægt sammenlignet før og efter en måneds blive fodret en standard kost. Antibiotika-behandlede fisk som et gennemsnit tabt vægt, mens kontrol fisk i gennemsnit taget på i vægt i løbet af måneden. Mekanismen for denne mangel på vægtstigning er uklar. En mulig årsag hertil er transittid af fødevarer i tarmen. En GI motiheden metoden blev tilpasset fra zebrafisk (Adam Rich, SUNY Brockport, personlig kommunikation) til at bestemme transittid. Det er endnu ikke blevet fastslået om antibiotika-behandlede fisk har en ændret transittid. En fælles udfordring oplevet i det naturlige miljø ved alle organismer, især fisk, er osmotisk stress. Gambusia har vist sig at hurtigt at tilpasse, når akut understreget i høje koncentrationer af saltholdighed 18. Overraskende fisk med et antibiotikum-ændrede microbiome udviste sænket overlevelse til en høj salt stress. Mekanismen for denne roman fænotype er under efterforskning. En anden almindelig stress på vanddyr, især i akvarier, er giftige former af kvælstof (ammoniak, nitrat og nitrit). Overlevelse mod nitrat var ikke signifikant forskellig mellem antibiotikum-behandlede og kontrol fisk. De metoder, der præsenteres i dette manuskript kan anvendes med Gambusia eller lignende fisk modelorganismer, såsom zebrafisk og Medaka, at måle fænotyper i fisken følgende eksperimentelle manipulation.

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		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Rifampicin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:127px;">557303-1GM</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Nitrate</td>
			<td>Sigma Aldrich</td>
			<td>S5506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:184px;">Fluorescein-labeled 70 kDa anionic dextran</td>
			<td style="width:104px;">ThermoFisher Scientific</td>
			<td>D1823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS tablets</td>
			<td>Calbiochem</td>
			<td>6500-OP</td>
			<td>tablets dissolve in water to make phosphate-buffered saline</td>
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examination of host phenotypes in gambusia affinis following antibiotic treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

En samlet skematisk diagram af eksperimentelle system anvendt til at undersøge fisk host effekter fra antibiotikum eksponering 13 er vist i figur 1A og omfatter teknik til ekstraktion af huden (figur 1B) og tarm (figur 1C) microbiomes fra fisken. Tre dage blev valgt som antibiotiske periode for eksponering, fordi tidligere data viser, at mens den samlede hud dyrkbar antal dråber tidligt i behandlingen, har det v...

Watch this Scientific Journal Video about Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment at JoVE.com

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

Denne undersøgelse involverer fremgangsmåder til at afsløre virkninger på en model fisk vært følgende ændring af huden og tarmen microbiome fællesskaber sammensætning af et antibiotikum.

Undersøgelse af Host fænotyper i<em&gt; Gambusia affinis</em&gt; Efter behandling med antibiotika

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

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Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

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