Name: detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system
Uploaded: 2018-04-09T12:30:00
Description: Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Riconosciamo Chi-Chung Chen dal dipartimento di ricerca medica, Chi Mei Medical Center (Tainan, Taiwan) per l'aiuto nel topo di infezione e il supporto dal centro laboratorio animale della National Cheng Kung University. Questo lavoro è sostenuto dal Ministro della scienza e tecnologia (MOST) concede (più 104-2321-B-006-019, 105-2321-B-006-011,-005 and106-2321-B-006) a CC.

Attenzione: EHEC O157: H7 è un livello di biosicurezza 2 (BSL-2) agente patogeno secondo i Centers for Disease Control and Prevention (CDC) istruzioni di sicurezza biologica (https://www.cdc.gov/). Di conseguenza, tutte le procedure sperimentali che coinvolgono EHEC devono essere effettuate in una struttura di BSL-2. Indossare guanti e camici da laboratorio quando si effettua l'esperimento. Lavorare in una certificato di biosicurezza (BSC). Disinfettare il banco sperimentale prima e dopo la procedura sperimentale con et...

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J., Gur-Arie, L., Rosenshine, I., Finlay, B. B. <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+and+in+vivo+model+systems+for+studying+enteropathogenic+Escherichia+coli+infections.">In vitro and in vivo model systems for studying enteropathogenic Escherichia coli infections.</a> Cold Spring Harb Perspect Med. 3 (3), a009977 (2013).</li><li>Ritchie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Enterohemorrhagic+Escherichia+coli+Infection.">Animal Models of Enterohemorrhagic Escherichia coli Infection.</a> Microbiol Spectr. 2 (4), EHEC-0022-2013 (2014).</li><li>Chou, T. 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È stato segnalato che EHEC trasformati con il plasmide luciferasi è stato utilizzato per esaminare la localizzazione nel host o espressione genica in vivo8,11,12. Il modello murino dimostrato qui è stato segnalato anche per rilevare i tempi di EHEC colonizzato e localizzazione in host murino8. Tuttavia, forniamo il protocollo di dettaglio di come amministrare l'inoculazione di EHEC ai topi int...

EHEC O157: H7 è un patogeno che causa diarrea1, HS2, HUS3e anche insufficienza renale4 attraverso cibo o acqua contaminato. EHEC è un patogeno enterobacterium e colonizza il tratto gastrointestinale di esseri umani1. Quando EHEC prima aderire all'epitelio intestinale host, iniettano i fattori di colonizzazione nelle cellule ospiti attraverso il sistema di secrezione di III tipo (T3SS) che funziona come una siringa molecolare che induce un fissaggio e cancellando (A/E) lesione successivamente per imporre adesione (colonizzazione)5. Questi geni coinvolti nella formazione della lesione A/E codificati per il locus dell'enterocita (LEE) il effacement patogenicità isola5.
Bioluminescenza è una reazione chimica produzione di luce, in cui luciferasi catalizza la luciferina di substrato per generare luce visibile6. Questo processo enzimatico richiede spesso la presenza di ossigeno o l'adenosina trifosfato (ATP)6. Bioluminescenza imaging (BLI) permette ai ricercatori la visualizzazione e la quantizzazione delle interazioni ospite-patogeno in animali vivi7. BLI possono caratterizzare il ciclo di infezione batterica in animali vivi seguendo i batteri bioluminescenti come migrare e invadere i tessuti differenti7; Questo rivela una progressione dinamica dell'infezione. Inoltre, la carica batterica negli animali è relativo al segnale bioluminescenti8; così, è un comodo indicatore per stimare le condizioni patologiche degli animali da esperimento in modo semplice e diretto.
Il plasmide usato qui contenute l'operone luciferasi, luxCDABE, che è il batterio Photorhabdus luminescens che codifica la propria luciferasi substrato7,9. Trasformando questo plasmide esprimendo la luciferasi in batteri, i processi di colonizzazione e l'infezione possono essere monitorati osservando questi batteri bioluminescenti animali vivi. Nel complesso, BLI e bioluminescenza-identificati batteri permettono ai ricercatori di monitorare i numeri batterici e la posizione, la vitalità batterica con antibiotici/terapia trattamento ed espressione genica batterica infezione/colonizzazione6, 7. sono stati segnalati numerosi batteri patogeni che esprimono l'operone luxCDABE per esaminare la loro espressione di ciclo e/o gene di infezione nell'infezione. Questi batteri, tra cui uropathogenic Escherichia coli10, EHEC8,11,12,13, enteropatogeni e. coli (EPEC)8, Citrobacter Rodentium14,15, Salmonella typhimurium16, Listeria monocytogenes17, Yersinia enterocolitica18,19, e Vibrio cholerae20, sono stati documentati.
Parecchi modelli sperimentali sono stati sviluppati per facilitare lo studio di EHEC colonizzazione in vitro e in vivo21,22,23. Tuttavia, c'è una mancanza di adeguati modelli animali per studiare l'EHEC colonizzazione in vivoe quindi una conseguente scarsità di dettagli. Per facilitare lo studio di EHEC colonizzazione meccanismo in vivo, è prezioso per costruire modelli animali per osservare e quantificare la colonizzazione di EHEC animali vivi in un metodo non-invasivo.
Questo manoscritto descrive un modello di colonizzazione del mouse-EHEC che utilizza un sistema di espressione bioluminescente per monitorare la colonizzazione di EHEC nel tempo in soggiorno ospiti. Topi intragastrically vengono inoculati con bioluminescenza-labeled EHEC e viene rilevato il segnale bioluminescente in topi con un non-invasiva in vivo imaging sistema13. I topi infettati con bioluminescenza-labeled EHEC hanno mostrato significativi segnali bioluminescenti in loro intestino dopo 2 giorni dopo l'infezione, che ha suggerito che tali batteri colonizzarono nell'intestino ospite dopo 2 giorni dopo l'infezione. Ex vivo immagine dati hanno mostrato che questa colonizzazione è specificamente nell'intestino cieco e del colon dei topi. Utilizzando questo modello di mouse-EHEC, la colonizzazione di EHEC bioluminescente può essere rilevata nell'ospite vivente da uno in vivo imaging system, per studiare i meccanismi dettagliati della colonizzazione di batteri enterici, che possono promuovere ulteriore comprensione in Cambiamenti fisiologici e patologici indotti da EHEC.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Shaker incubator</td>
			<td style="width:175px;">YIH DER</td>
			<td style="width:143px;">LM-570R</td>
			<td style="width:167px;">bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Orbital shaking incubator</td>
			<td style="width:175px;">FIRSTEK</td>
			<td style="width:143px;">S300</td>
			<td>bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">pBSL180</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of nptII gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pAKlux2</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of luxCDABE operon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T&#38;A Cloning Kit</td>
			<td>Yeastern Biotech</td>
			<td style="width:143px;">FYC001-20P</td>
			<td>use for TA cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nsi I</td>
			<td style="width:175px;">NEB</td>
			<td>R0127S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sca I</td>
			<td style="width:175px;">NEB</td>
			<td>R0122S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spe I-HF</td>
			<td style="width:175px;">NEB</td>
			<td>R0133S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sma I&#160;</td>
			<td style="width:175px;">NEB</td>
			<td>R0141S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:175px;">NEB</td>
			<td style="width:143px;">M0202S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ex Taq</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">10X Ex Taq Buffer</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP Mixture&#160;</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">PCR machine</td>
			<td style="width:175px;">applied Biosystem&#160;</td>
			<td style="width:143px;">2720 thermal cycler&#160;</td>
			<td>&#160;for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:175px;">SIGMA</td>
			<td style="width:143px;">G5516-1L</td>
			<td>use for bacteria stocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NaCl</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">31434-5KG-R</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tryptone</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1612.00</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Yeast Extract powder</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:143px;">23547-1 KG</td>
			<td>chemical for making LB medium, 5 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Agar</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1802.00</td>
			<td>chemical for making LB agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">kanamycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">K4000-5G</td>
			<td>antibiotics, use for seleciton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">streptomycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">S6501-100G</td>
			<td>antibiotics, eliminate the microbiota in mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">EDL933 competent cell</td>
			<td style="width:175px;">Homemade</td>
			<td style="width:143px;"></td>
			<td>method is on supplemental document&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporator</td>
			<td style="width:175px;">MicroPulser</td>
			<td style="width:143px;"></td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporation Cuvettes</td>
			<td style="width:175px;">Gene Pulser/MicroPulser</td>
			<td style="width:143px;">1652086</td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">High-speed centrifuge</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">Avanti, J-26S XP</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JA25.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JLA10.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td>Beckman Coulter</td>
			<td>REF357003</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td style="width:175px;">Thermo Fisher scientific</td>
			<td style="width:143px;">3141-0500</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">eppendorf biophotometer plus&#160;</td>
			<td>eppendorf</td>
			<td>AG 22331 hamburg</td>
			<td>for measuring the OD600 value of bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">C57BL/6 mice&#160;</td>
			<td colspan="2">Laboratory Animal Center of NCKU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">lab coat, gloves</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>for personnel protection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane&#160;</td>
			<td>Panion &#38; BF Biotech Inc.</td>
			<td style="width:143px;">G-8669</td>
			<td>for mice anesthesia, pharmaceutical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Reusable 22 G ball-tipped feeding needle</td>
			<td style="width:143px;">&#966;0.9 mm X L 50 mm</td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical&#160; scissors&#160;</td>
			<td></td>
			<td style="width:143px;"></td>
			<td>use for mice experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xenogen IVIS 200 imaging system</td>
			<td>Perkin Elmer</td>
			<td>IVIS spectrum</td>
			<td>use for bioluminescent image capture&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Living Image Software</td>
			<td>Perkin Elmer</td>
			<td style="width:143px;">version 4.1</td>
			<td>use for quantifying the image data</td>
		</tr>
	</tbody>
</table>

detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system

Enteroemorragica e. coli (enteroemorragico EHEC) O157: H7, che è un agente patogeno portato dagli alimenti che causesdiarrhea, colite emorragica (HS), e la sindrome uremica emolitica (HUS), colonizzare il tratto intestinale degli esseri umani. Per studiare il meccanismo dettagliato della EHEC colonizzazione in vivo, è essenziale disporre di modelli animali per monitorare e quantificare la colonizzazione di EHEC. Dimostriamo qui un modello di colonizzazione del mouse-EHEC trasformando il plasmide di espressione bioluminescente per EHEC per monitorare e quantificare la colonizzazione di EHEC in soggiorno ospiti. Animali inoculati con bioluminescenza-labeled EHEC mostrano segnali bioluminescenti intensi in topi tramite rilevazione con un non-invasiva in vivo imaging sistema. Dopo 1 e 2 giorni dopo l'infezione, bioluminescenti segnali potrebbero essere ancora presente in animali infettati, che suggerisce che EHEC colonizzare in padroni di casa per almeno 2 giorni. Inoltre dimostriamo che questi EHEC bioluminescenti individuare all'intestino del mouse, specificamente nell'intestino cieco e del colon, da immagini di ex vivo . Questo modello di colonizzazione del mouse-EHEC può servire come strumento per far progredire la conoscenza corrente del meccanismo di colonizzazione di EHEC.

Abbiamo amministrato la bioluminescenza-labeled EHEC (~ 109 cellule batteriche) ai topi C57BL/6 femminili 6 - settimana-vecchi mediante sonda gastrica orale. Dopo la somministrazione orale di EHEC ai topi all'interno di 1h, gli animali sono stati esaminati per segnale bioluminescenti secondo il sistema di imaging in vivo come mostrato nella Figura 7. I risultati hanno mostrato un forte segnale bioluminescente in topi di sonda gastrica con ...

Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Un protocollo dettagliato di un modello murino per enteroemorragica e. coli (enteroemorragico EHEC) colonizzazione tramite bioluminescenza-identificati batteri è presentato. La rilevazione di questi batteri bioluminescenti di un non-invasiva in vivo imaging sistema animali vivi può avanzare la nostra comprensione corrente della colonizzazione di EHEC.

Rilevamento della colonizzazione di Escherichia Coli enteroemorragica in murino Host di sistema Non invasivo In Vivo bioluminescenza

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Research

JoVE Journal

Immunology and Infection

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

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Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

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Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...