Name: biosensor for detection of antibiotic resistant staphylococcus bacteria
Uploaded: 2013-05-08T09:00:00
Description: Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Arbeidet rapporteres her ble støttet med tilskudd fra Auburn University AUDFS og USAF CRADA 07-277-60MDG-01. Synspunktene i denne artikkelen er de av forfatterne, og reflekterer ikke den offisielle politikk eller plasseringen av United States Air Force, Department of Defense, eller den amerikanske regjeringen.

En. Innstilling av Stage <ol><li> Skaff typestammen S. aureus ATCC 12600, S. aureus ATCC 27690 og Bacillus subtilis ATCC 6051. Meticillin-resistente stammer av S. aureus - MRSA1, to MRSA, 5 MRSA, 13 MRSA, 26 MRSA, 34 MRSA, 45 MRSA, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882, The lytisk fag 12600. </li><li> Skaff PBP 2a antistoff konjugert latekskuler. </li><li>...

<ol>
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Det er velkjent at fager kan anvendes som prober for biosensor bakterielle patogener 28. Det er demonstrert i dette arbeidet som phage sammen med PBP 2a-antistoffer kan anvendes for å løse det gamle problemet: rapid diskriminering antibiotika resistente og sensitive stammer. Man fant imidlertid de vanlige umodifiserte stafylokokkale fager er ikke egnet for bakterier deteksjon med QCM-enheter, selv om de binder bakterier. Den fag-hale er så lang at akustiske bølger ikke kan «s...

Meticillin resistente stammer av Staphylococcus aureus har blitt foreslått som en faktor i essensielle infeksjoner og nosokomiale utbrudd 4-8. Vanligste måtene anerkjennelse av meticillin resistens, for eksempel disk diffusjon Oxacillin agar skjermen test, eller kjøttkraft microdilution, er avhengige av skreddersydde kultur vilkår for å forbedre uttrykk for motstand. Endringer omfatter utnyttelse av oksacillin, inkubering ved 30 eller 35 ° C i stedet for 37 ° C, og inkluderingen av NaCl til vekstmediet. Videre, for korrekt deteksjon ved disse typer teknikker, en lang inkubasjonstid på 24 timer i stedet for 16-18 timers er nødvendig. Raske teknikker med passende (&gt; 96%) følsomhet for identifisering av meticillin resistens inkluderer automatiserte microdilution teknikker som Vitek GPS-SA-kort, Rapid ATB Staph system, og Rapid Microscan Panel system som produserer resultater etter 3-11 timer 9-11. The Crystal MRSA-ID-systemet er en hurtig metode basert på erkjennelsen av veksten av S. aureus i nærvær av 2% NaCl og 4 mg av oksacillin per liter med en oksygen-sensitive fluorescenssensorutstyret. Hevdet følsomhet varierer mellom 91 til 100% etter 4 timer med inkubasjon 12-14. Disse fenotypiske metoder er begrenset i sine nøyaktigheter ved virkningen av utbredte stammer som uttrykker heterogen motstand. Derfor er de beste mest aksepterte metoder for anerkjennelse av meticillin resistens PCR eller DNA hybridisering av mecA genet 15. Men denne teknikken krever rensede DNA og er ekstremt følsomme for forskjellige tilsetninger (urenheter), som inkluderer celleavfall 16.. Videre er disse teknikkene trenger lang tid å utføre. Strategier til anerkjennelse av mecA genproduktet, protein PBP 2a, kunne brukes til å bestemme motstand og kan være mer pålitelig i forhold til standard test teknikker 17. Staphylococcus aureus-stammer innbefatter de som har meticillin 1,2,18. I dette arbeidet foreslo vi en ny teknikk i den spesifikke gjenkjenning og påvisning av MRSA, slik som gjenkjennelse av bakteriene sammen med konformasjon av MRSA i sanntid. For dette formålet en S. aureus bakteriofag med et bredt spektrum av verter (inkludert MRSA stammer) i kombinasjon med monoklonalt antistoff mot protein (PBP 2a) har blitt benyttet. PBP 2a er et cellevegg protein og det er årsaken til antibiotikum resistivitet av MRSA. Men PBP 2a antistoff er ikke spesifikke for S. aureus siden noen andre bakterier har antibiotiske proteiner med sekvenslikhet til PBP 2a 19,20. Følgelig i dette arbeidet, S. aureus bakteriofag og antistoffer mot PBP 2a proteinet har blitt brukt. For å være i stand til å utvikle en biosensor for å spesifitisk oppdage og identifisere MRSA en enhet med en to-trinns handling har blitt utnyttet. Den første trinnet brukte en S. aureus bakteriofag monolaget som en sensor probe, mens det andre trinnet anvendes PBP 2a spesifikke antistoffer. Derfor går man vil gjenkjenne S. aureus bakterier, vil som den andre være følsomme for antibiotika-bindende protein. Når signaler mottatt fra to trinnene er positiv, betyr det at spesifikk påvisning av MRSA.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biosensor for detection of antibiotic resistant staphylococcus bacteria

Et strukturelt transformert lytisk bakteriofag ha en bredde i vertsområde av Staphylococcus aureus-stammer og en penicillin-bindende protein (PBP 2a) antistoff konjugert latekskuler har blitt benyttet for å lage en biosensor beregnet for diskriminering av meticillinresistent (MRSA) og følsom (MSSA) S . aureus arter 1,2. Lytisk phages har blitt konvertert til fag-sfæroider ved kontakt med vann-kloroform grensesnitt. Phage sfæroide monolayers har blitt flyttet på en biosensor overflate ved Langmuir-Blodgett (LB) teknikk tre. Den opprettede biosensorer har blitt undersøkt av en kvartskrystall mikrovekt med utskeielser sporing (QCM-D) for å evaluere bakterier-fag-interaksjoner. Bakterier-sfæroide interaksjoner ført til redusert resonans frekvens og en økning i spredning energi for både MRSA og MSSA stammer. Etter bakteriell binding, har disse sensorene er videre utsatt for penicillin-bindende protein antistoff lateks vulstens. Sensorer som analyseres med MRSA svart på PBP 2a antistoffkuler, selv om sensorer inspisert med MSSA ga ingen respons. Denne eksperimentelle skillet bestemmer en entydig skille mellom meticillin resistente og sensitive S. aureus stammer. Like bundet og ubundet bakteriofager undertrykke bakterievekst på overflater og i vann suspensjoner. Når lytisk phages blir alle forvandlet til sfæroider, beholder de sin sterke lytisk aktivitet og viser høy bakteriell fangst evne. Fagen og phage sfæroider kan benyttes for testing og sterilisering av antibiotikaresistente mikroorganismer. Andre anvendelser kan omfatte bruk i bakteriofag terapi og antimikrobielle overflater.

Den fag demonstrert lytisk aktivitet mot alle testede stammer av S. aureus, inkludert MRSA-stammer, som indikert av fag stikkprøve. Plakk størrelser generelt varierte fra 5 til 15 mm. Ingen aktivitet ble funnet mot andre test-kulturer (tabell 1). En normal vekst av S. aureus ATCC 12600 i NZY medium på shaker-inkubator ved 37 ° C er vist i figur 1A (en kurve som er merket med tomme sirkler). Antallet bakterier økte fra 3,2 10 til 6...

Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Lytisk phage biosensorer og antistoffkuler er i stand til å diskriminere mellom meticillinresistent (MRSA) og sensitive Staphylococcus bakterier. De phages ble immobilisert ved en Langmuir-Blodgett metoden på en overflate av en kvartskrystall mikrovekt sensor og jobbet så bredt spekter Staphylococcus sonder. Antistoffkuler gjenkjenne MRSA.

Biosensor for påvisning av antibiotikaresistens resistente Staphylococcus bakterier

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Research

JoVE Journal

Bioengineering

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