Name: screening foodstuffs for class 1 integrons and gene cassettes
Uploaded: 2015-06-19T13:00:00
Description: Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Dankzij Michaela Hall, Larissa Bispo en Gustavo Tavares voor technische bijstand.

Levensmiddelen die rauw worden gegeten of licht gekookt zijn van de meeste zorg voor de gezondheid van de mens. Voorbeelden zijn salade groenten, fruit, schaal- en schelpdieren. 1. Sample Collection <ol><li> Verzamel monsters onder omstandigheden die verontreiniging te minimaliseren, en in afzonderlijke, schone zakken tijdens het transport. Eenmaal verzameld moeten de monsters worden bewaard bij 4 ° C en binnen 24 uur. </li></ol> 2. Verrijkte Cultuur Voorbereiding </...

<ol>
	<li>Bush, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+antibiotic+resistance.">Tackling antibiotic resistance.</a> Nature Rev. Microbiol. 9, 894-896 (2011).</li><li>Davies, J., Davies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+evolution+of+antibiotic+resistance.">Origins and evolution of antibiotic resistance.</a> Microbiol. Mol. Biol. Rev. 74 (3), 417-433 (2010).</li><li>Costa, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+is+ancient.">Antibiotic resistance is ancient.</a> Nature. 477 (7365), 457-461 (2011).</li><li>Gillings, M. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impacts+of+anthropogenic+activity+on+the+ecology+of+class+1+integrons+and+integron-associated+genes+in+the+environment.">Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment.</a> ISME J. 5, 1253-1261 (2011).</li><li>Gerzova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microbiota+composition+and+presence+of+selected+antibiotic+resistance+genes+in+carriage+water+of+ornamental+fish.">Characterization of microbiota composition and presence of selected antibiotic resistance genes in carriage water of ornamental fish.</a> PLoS ONE. 9, e103865 (2014).</li><li>Power, M., Emery, S., Gillings, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Into+the+wild:+dissemination+of+antibiotic+resistance+determinants+via+a+species+recovery+program.">Into the wild: dissemination of antibiotic resistance determinants via a species recovery program.</a> PLoS ONE. 8, e63017 (2013).</li><li>Stokes, H. W., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+flow,+mobile+genetic+elements+and+the+recruitment+of+antibiotic+resistance+genes+into+Gram-negative+pathogens.">Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens.</a> FEMS Microbiol. Rev. 35 (5), 790-819 (2011).</li><li>Moura, A., Oliveira, C., Henriques, I., Smalla, K., Correia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+diversity+of+conjugative+plasmids+in+integron-carrying+bacteria+from+wastewater+environments.">Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments.</a> FEMS Microbiol. Lett. 330 (2), 157-164 (2012).</li><li>Schl&#252;ter, A., Krause, L., Szczepanowski, R., Goesmann, A., P&#252;hler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+and+composition+of+a+plasmid+metagenome+from+a+wastewater+treatment+plant.">Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant.</a> J. Biotech. 136 (1-2), 65-76 (2008).</li><li>Taylor, N. G. H., Verner-Jeffreys, D. W., Baker-Austin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+systems:+maintaining,+mixing+and+mobilising+antimicrobial+resistance.">Aquatic systems: maintaining, mixing and mobilising antimicrobial resistance.</a> TREE. 26 (6), 278-284 (2011).</li><li>Stalder, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+impact+of+hospital+effluent+on+dissemination+of+the+integron+pool.">Quantitative and qualitative impact of hospital effluent on dissemination of the integron pool.</a> ISME J. 8, 768-777 (2014).</li><li>Allen, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Call+of+the+wild:+antibiotic+resistance+genes+in+natural+environments.">Call of the wild: antibiotic resistance genes in natural environments.</a> Nature Rev. Microbiol. 8, 251-259 (2010).</li><li>Wellington, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+natural+environment+in+the+emergence+of+antibiotic+resistance+in+Gram-negative+bacteria.">The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria.</a> Lancet Infect. Dis. 13 (2), 155-165 (2013).</li><li>Gillings, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+a+Tn402-like+class+1+integron+with+a+novel+cassette+array+via+flanking+miniature+inverted-repeat+transposable+element-like+structures.">Mobilization of a Tn402-like class 1 integron with a novel cassette array via flanking miniature inverted-repeat transposable element-like structures.</a> Appl. Env. Microbiol. 75 (18), 6002-6004 (2009).</li><li>Graham, D. W., Collignon, P., Davies, J., Larsson, D. J., Snape, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underappreciated+role+of+regionally+poor+water+quality+on+globally+increasing+antibiotic+resistance.">Underappreciated role of regionally poor water quality on globally increasing antibiotic resistance.</a> Env. Sci. Technol. 48 (20), 11746-11747 (2014).</li><li>Perry, J. A., Westman, E. L., Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistome:+what's+new.">The antibiotic resistome: what's new.</a> Curr. Opinion Microbiol. 21, 45-50 (2014).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J. Vis. Exp. 63, e3998-e3998 (2011).</li><li>Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Extraction+of+PCR-Competent+DNA+from+Recalcitrant+Environmental+Samples.">Rapid Extraction of PCR-Competent DNA from Recalcitrant Environmental Samples.</a> Env. Microbiol. 1096, 17-23 (2014).</li><li>Sajjad, A., Holley, M. P., Labbate, M., Stokes, H., Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+class+1+integron+with+a+complete+Tn402-like+transposition+module.">Preclinical class 1 integron with a complete Tn402-like transposition module.</a> Appl. Env. Microbiol. 77 (1), 335-337 (2011).</li><li>Wright, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+in+the+environment:+A+link+to+the+clinic.">Antibiotic resistance in the environment: A link to the clinic.</a> Curr. Opinion Microbiol. 13 (5), 589-594 (2010).</li><li>Stokes, H., Nesbo, C., Holley, M., Bahl, M., Gillings, M., Boucher, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community.">Class 1 integrons predating the association with Tn402.-like transposition genes are present in a sediment microbial community.</a> Journal of Bacteriology. 188, 5722-5730 (2006).</li><li>Lane, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acid Techniques in Bacterial Systematics. , 115-175 (1991).</li><li>Holmes, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil.">Recombination activity of a distinctive integron-gene cassette system associated with stutzeri. populations in soil.</a> Journal of Bacteriology. 185, 918-928 (2003).</li></ol>

De identificatie van integronen en de bijbehorende gen cassettes is potentieel een belangrijke stap in het voorspellen van de opkomst van nieuwe opportunistische pathogenen, tracking paden voor ziekteverwekkers in de menselijke voedselketen, en het identificeren van nieuwe weerstand en virulentiedeterminanten 8,21,26. Het doel van dit artikel was om een ​​gestroomlijnde aanpak voor het screenen van monsters voor klasse 1 integronen, het karakteriseren van de cassette arrays en het identificeren van de soo...

De ontdekking van antibiotica was een van de grootste wetenschappelijke prestaties van de 20 ste eeuw. Echter, het gebruik en misbruik van antibiotica heeft geleid tot de snelle evolutie van antibiotica resistente bacteriën, en die nu een ernstige bedreiging voor de volksgezondheid in de 21e eeuw poseren. De opkomst van bacteriestammen resistent tegen de meeste behandelingen verhoogt de mogelijkheid we betreden een tijdperk waarin antimicrobiële geneesmiddelen zijn niet langer effectief 1,2. De genetische machines die antibiotica-resistentie is een oud systeem, die dateren van vóór de mens en antibiotische selectie druk door miljoenen jaren 3. Mobiele genetische elementen, zoals plasmiden, transposons, genomische eilanden, integratieve conjugatieve elementen en integronen kan verspreiden antibiotica resistentiegenen (ARG), zowel binnen als tussen bacteriesoorten 4. Hiervan hebben integrons een centrale rol in de verspreiding van ARG gespeeld, ondanksfeit dat ze vertrouwen op plasmiden en transposons voor de mobilisatie en het inbrengen in bacteriële genomen 5. Integronen vastleggen gencassettes via een integron-integrase, en vervolgens tot expressie cassettes middels een integron gecodeerd promoter 6,7 (figuur 1). Integron gen cassettes zijn kleine mobiele elementen bestaande uit enkele open reading frames (ORF), waarvan de producten kan resistentie tegen antibiotica of ontsmettingsmiddelen 8. Klasse 1 integronen zijn de integronen meest hersteld van klinische isolaten 5, waar ze gezamenlijk hebben verworven meer dan 130 verschillende antibioticumresistentiegen cassettes 9. De verspreiding van klasse 1 integronen in-menselijke geassocieerd commensale en pathogene bacteriën genereert afvalstromen mens dat een groot aantal van deze genetische elementen 10 bevatten. Naar schatting 10 19 bacteriën die klasse 1 integronen bevatten, worden vrijgegeven via zuiveringsslib elk jaar in het Verenigd Koninkrijk 11. Het is daarom niet verwonderlijk dat klasse 1 integronen verlenen antibioticaresistenties worden nu gedetecteerd in microbiota van wilde vogels, vissen en andere inheemse dieren 12-14. Vrijgeven integronen terug in het milieu vormt een belangrijke bedreiging voor de volksgezondheid, omdat de verwerving van nieuw gen cassettes en complexe herschikkingen met andere mobiele elementen blijft optreden, met name in zuiveringsinstallaties en andere waterlichamen 15-18. De natuurlijke omgeving wordt dan een vruchtbare werven voor nieuwe weerstand determinanten en opportunistische pathogenen 19,20. Novel-Integron met bacteriën en nieuwe gegeven argumenten terug omcirkelen in de menselijke gemeenschap via besmet water en voedsel 21,22. Bewaking van het milieu ARGs is een belangrijke strategie voor het begrijpen en beheren van resistentie tegen antibiotica in de toekomst 23. In het bijzonder moet aandacht worden besteed aan levensmiddelen die rauw worden gegeten oflichtvaardig gekookt, omdat deze presenteren de grootste bedreiging voor de overdracht van nieuwe mobiele elementen en ziekteverwekkers. In dit protocol, een gestroomlijnde benadering voor het detecteren, identificeren en karakteriseren van klasse 1 integronen en bijbehorende gencassettes in levensmiddelen worden beschreven (Figuur 2). Met een combinatie van kweken en polymerase kettingreactie (PCR), kan integrons snel worden gedetecteerd in complexe bacteriële en individuele isolaten. Werkwijzen voor het identificeren van de soorten bacteriën en de conformatie en identiteit van het Integron geassocieerde gencassettes gegeven. De werkwijze is geschikt voor een breed scala van plantaardige en dierlijke voedingsmiddelen en voorbeelden zijn werkstromen voor elk van deze soorten voedsel.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>GoTaq Colourless Mastermix</td>
			<td>Promega</td>
			<td>M7132</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>RNAse (Ribonuclease A from bovine pancreas)</td>
			<td>Sigma</td>
			<td>R6513-10MG</td>
			<td>Used in all PCRs</td>
		</tr>
		<tr>
			<td>HinFI restriction enzyme</td>
			<td>Promega</td>
			<td>R6201</td>
			<td>Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA</td>
		</tr>
		<tr>
			<td>100 bp ladder</td>
			<td>GE Healthcare</td>
			<td>27400701</td>
			<td>Used as a size standard on all agarose gels</td>
		</tr>
		<tr>
			<td>GelRed DNA stain</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>Guanidinium Thiocyanate</td>
			<td>Life Technologies</td>
			<td>AM9422</td>
			<td>CAUTION: Personal protection must be worn when handling this material</td>
		</tr>
		<tr>
			<td>CLS-TC Solution</td>
			<td>MP Biomedicals</td>
			<td>6540409</td>
			<td>Resuspension solution used at the begining of the genomic DNA extraction</td>
		</tr>
		<tr>
			<td>Lysing Matrix E FastPrep tubes</td>
			<td>MP Biomedicals</td>
			<td>116914500</td>
			<td>Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.</td>
		</tr>
		<tr>
			<td>binding matrix</td>
			<td>MP Biomedicals</td>
			<td>116540408</td>
			<td>Diluted 1:5 with 6M guanidinium thiocyanate and used in the genomic DNA extraction method.</td>
		</tr>
		<tr>
			<td>Fast Prep machine</td>
			<td>MP Biomedicals</td>
			<td>Number of options available</td>
			<td>MP Biomedicals has a number of FastPrep machines available to purchase. Visit http://www.mpbio.com for more information</td>
		</tr>
	</tbody>
</table>

screening foodstuffs for class 1 integrons and gene cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

Screening van gemengde culturen en bacteriële isolaten van intI1 Primerpaar HS463a / HS464 PCR kan worden gebruikt om de aanwezigheid van de klasse 1 integron-integrase gen, intI1 (figuur 1) te detecteren. Deze primer set werkt goed voor het detecteren intI1 in gemengde culturen, en wordt ook gebruikt om bacteriële kolonies geoogst uit platen uitgespreid (figuur 2) te screenen. Positieve isolaten moet een sterke band te genereren bij 471 bp ...

Watch this Scientific Journal Video about Screening Foodstuffs for Class 1 Integrons and Gene Cassettes at JoVE.com

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Screening Levensmiddelen voor klasse 1 integrons en Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

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