Name: methodology for the study of horizontal gene transfer in staphylococcus aureus
Uploaded: 2017-03-10T12:30:00
Description: Watch this Scientific Journal Video about Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus at JoVE.com

This work was partly supported by Takeda Science Foundation, Pfizer Academic Contribution and JSPS Postdoctoral Fellowship for Foreign Researchers (FC).

BEMÆRK: stammer og materialer, der anvendes i dette arbejde er anført i tabel 1 og tabel Materialer hhv. I transmissions- eksperimenter blev N315 og COL cfr -positive derivater brugt som donorer af den cfr gen (N315-45 og COL-45). Disse stammer blev tidligere opnået ved konjugation, idet der som donor af en klinisk cfr -positiv Staphyloccocus epidermidis-stamme (ST2), efter standarden konjugation protokollen (se nedenfor). Denne stamme nærede den ...

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<html> Dette arbejde beskriver de tre store metoder til at studere HGT af genetiske determinanter i S. aureus. Selvom transduktion og konjugation er blevet studeret i årtier, blev eksistensen af naturlige transformation først for nylig anerkendt 2. Således er S. aureus udstyret med alle de tre store former for HGT, og teste dem alle er forpligtet til at klarlægge de mulige formidling veje for genetiske determinanter. Målet med dette arbejde er at udarbejde komplette protokol...

Staphylococcus aureus er en kommensal grampositiv bakterie, der naturligt bebor huden og næsehulen af mennesker og dyr. Denne bakterielle arter er den førende årsag til nosokomielle infektioner på hospitaler og sundhedsmiljøerne. Desuden har evnen til at udvikle resistens over for forskellige antimikrobielle forbindelser gjort forvaltningen af ​​infektioner forårsaget af denne bakterie til en global bekymring. To vigtige veje er involveret i spredningen af ​​resistens fænotyper er kendt: den klonale udbredelse af resistente genotyper og udbredelsen af ​​genetiske determinanter blandt den bakterielle pool. I tilfælde af S. aureus, forskellige antibiotiske resistensgener (samt virulensdeterminanter) har vist sig at være forbundet med mobile genetiske elementer (MGEs) 1. Tilstedeværelsen af disse elementer i genomet af S. aureus viser, at erhvervelse og overdragelse af genietisk materiale inden den bakterielle population kunne spille en vigtig rolle for S. aureus tilpasning og evolution. Genetisk materiale kan udveksles gennem tre kendte mekanismer HGT i Gram-positive bakterier: transformation, konjugation og fag transduktion. Transformation indebærer optagelse af frit DNA. At erhverve fremmed DNA, bakterieceller nødt til at udvikle en særlig fysiologisk fase: kompetence scenen. Når denne fase nås, kompetente celler er i stand til at transportere DNA ind i cytoplasmaet, erhverve nye genetiske determinanter. I tilfælde af S. aureus, har eksistensen af naturlig transformation nylig blevet demonstreret 2. I tråd med dette har vores gruppe belyse relevansen af ekspressionen af suk faktor (en kryptisk sekundær transskription sigma faktor) i kompetence udviklingstrin og på hvordan dens konstitutiv ekspression gør S. aureus i stand til reaching kompetence scenen, som giver mulighed for erhvervelse af resistente fænotyper ved naturlig transformation 2. Konjugation er en proces, der involverer overførsel af DNA fra en levende celle (donor) til en anden (recipient). Begge celler skal være i direkte kontakt, således at DNA, der skal udveksles samtidig være beskyttet af særlige strukturer, såsom rør eller porer. Overførsel af DNA ved denne fremgangsmåde kræver konjugative maskiner. I S. aureus, prototypen konjugative plasmid er PGO1, der huser den konjugative operon Traa 3. Phag transduktion indebærer overførsel af DNA fra celle til celle gennem bakteriofaginfektion og indebærer pakning af bakterie-DNA, i stedet for virus-DNA, ind i fag-capsidet. De fleste af S. aureus isolater lysogeniseret af bakteriofager 1. Ved stressbetingelser, kan profager udskæres fra det bakterielle genome og skift til den lytiske cyklus. Det er de tre velkendte mekanismer til DNA transmission i S. aureus. Der er nogle ekstra transfer mekanismer, såsom &quot;pseudo-transformation&quot; 2 og fag-lignende systemer i overførslen af sygdomsfremkaldende evne øer 4. For nylig, en gruppe rapporterede, at &quot;nanorør&quot; er involveret i overførslen af cellulære materialer (herunder plasmid-DNA) mellem naboceller 5, 6, men en opfølgende undersøgelse ikke er mødt fra andre grupper hidtil. Dette arbejde giver den nødvendige metodik til at studere HGT i S. aureus ved at behandle de tre vigtigste transfer veje konjugation, transduktion, og naturlig transformation. De opnåede med disse metoder resultater blev anvendt til at undersøge transmissionen af den cfr gen (chloramphenicol / florfenicol modstand) blandtS. aureus-stammerne 7. Disse tre teknikker er alsidige værktøjer til undersøgelse af MGE transmission i S. aureus.

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			<td height="21" style="height:21px;width:284px;">Tryptic Soy Broth (TSB)&#160;</td>
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			<td height="21" style="height:21px;">Nutrient Broth No. 2</td>
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			<td height="21" style="height:21px;">Sheep blood agar</td>
			<td>Eiken Chemical Co.,Ltd.</td>
			<td>E-MR96</td>
			<td>Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.&#160;</td>
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			<td height="21" style="height:21px;">Cellulose Ester Gridded 0.45 &#956;L HAWG filter</td>
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methodology for the study of horizontal gene transfer in staphylococcus aureus

Et vigtigt træk ved den større opportunistisk humant patogen Staphylococcus aureus er sin ekstraordinære evne til hurtigt udvikler resistens over for antibiotika. Genomiske undersøgelser viser, at S. aureus bærer mange virulens og resistensgener beliggende i mobile genetiske elementer, hvilket antyder, at horisontal genoverførsel (HGT) spiller en kritisk rolle i S. aureus evolution. Men en fuld og detaljeret beskrivelse af metoden til at studere HGT i S. aureus stadig mangler, især med hensyn til naturlig transformation, som for nylig er blevet rapporteret i denne bakterie. Dette arbejde beskriver tre protokoller, som er nyttige til in vitro undersøgelse af HGT i S. aureus konjugation, phag transduktion, og naturlig transformation. Til dette formål, det cfr genet (chloramphenicol / florfenicol modstand), som giver de phenicoler, Lincosamider, Oxazolidinoner, pleuromutiliner, og streptogramin A (PhLOPSA) -resistance fænotype, blev anvendt. Forståelse af de mekanismer, hvorigennem S. aureus overfører genetisk materiale til andre stammer er afgørende for at forstå den hurtige overtagelse af resistens og hjælper med at afklare former for formidling rapporteret i overvågningsprogrammer eller yderligere forudsige spredningen tilstand i fremtiden.

Resultaterne repræsenteret her er tidligere blevet offentliggjort (tilpasset fra henvisning 7 med udgiverens tilladelse). Vi studerede de potentielle overføringsveje af CFR-genet, som forårsager lavt niveau linezolid modstand og ekspressionen af PhLOPSA-resistens fænotype 14, 15 i S. aureus-stammer, ved at undersøge tre mekanismer HGT. <p class="jove_content" fo:keep-together.wit...

Watch this Scientific Journal Video about Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus at JoVE.com

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

Vi beskriver her tre forskellige protokoller for in vitro undersøgelse af konjugation, transduktion, og naturlig transformation i Staphylococcus aureus.

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