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

Thanks to Michaela Hall, Larissa Bispo and Gustavo Tavares for Technical Assistance.

Foodstuffs that are eaten raw or lightly cooked are of most concern for human health. Examples include salad vegetables, fruit, shellfish and crustaceans.
1. Sample Collection
<ol>
	<li>Collect samples under conditions that minimize contamination, and stored in separate, clean bags during transport. Once collected, samples should be stored at 4 &#176;C and processed within 24 hr.</li>
</ol>
2. Enriched Culture Preparation
<ol>
	<li>Fruits and vegetables:
	<ol>
		<li...

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+consequences+of+antibiotic+use+for+the+resistome,+mobilome+and+microbial+pangenome.">Evolutionary consequences of antibiotic use for the resistome, mobilome and microbial pangenome.</a> Front. Microbiol. 4, 4 (2013).</li><li>Gillings, 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+evolution+of+class+1+integrons+and+the+rise+of+antibiotic+resistance.">The evolution of class 1 integrons and the rise of antibiotic resistance.</a> J. Bacteriol. 190 (14), 5095-5100 (2008).</li><li>Brassard, S., Lapointe, J., Roy, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+relative+strength+of+tandem+promoters+for+the+antibiotic-resistance+genes+of+several+integrons.">Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons.</a> Gene. 142 (1), 49-54 (1994).</li><li>Partridge, S. 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=Definition+of+the+attI1+site+of+class+1+integrons.">Definition of the attI1 site of class 1 integrons.</a> Microbiol. 146 (11), 2855-2864 (2000).</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=Integrons:+Past,+Present,+and+Future.">Integrons: Past, Present, and Future.</a> Microbiol. Mol. Biol. Rev. 78 (2), 257-277 (2014).</li><li>Partridge, S. R., Tsafnat, G., Coiera, E., Iredell, J. 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+cassettes+and+cassette+arrays+in+mobile+resistance+integrons.">Gene cassettes and cassette arrays in mobile resistance integrons.</a> FEMS Microbiol. Rev. 33 (4), 757-784 (2009).</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=Using+the+class+1+integron-integrase+gene+as+a+proxy+for+anthropogenic+pollution.">Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution.</a> ISME J. In press, (2014).</li><li>Gaze, W. 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>

The identification of integrons and their associated gene cassettes is potentially a key step in predicting the emergence of new opportunistic pathogens, tracking pathways for pathogens into the human food chain, and identifying new resistance and virulence determinants8,21,26. The aim of this paper was to describe a streamlined approach for screening samples for class 1 integrons, characterizing their cassette arrays and identifying the bacterial species in which they reside. Critical steps in the protocol in...

The discovery of antibiotics was one of the greatest scientific achievements of the 20th century. However, the use and abuse of antibiotics has led to the rapid evolution of antibiotic resistant bacteria, and these now pose a serious threat to public health in the 21st century. The rise of bacterial strains resistant to most treatment options raises the possibility we are entering an era where antimicrobial drugs are no longer effective1,2.
The genetic machinery that confers antibiotic resistance is an ancient system, predating humans and antibiotic selection pressures by millions of years3. Mobile genetic elements, such as plasmids, transposons, genomic islands, integrative conjugative elements and integrons can disseminate antibiotic resistance genes (ARG) both within and between bacterial species4. Of these, integrons have played a central role in the spread of ARG, despite the fact that they rely upon plasmids and transposons for mobilization and insertion into bacterial genomes5. Integrons capture gene cassettes using an integron-integrase, and then express cassettes using an integron encoded promoter6,7 (Figure 1). Integron gene cassettes are small mobile elements consisting of single open reading frames (ORF) whose products can confer resistance to antibiotics or disinfectants8. Class 1 integrons are the integrons most commonly recovered from clinical isolates5, where they have collectively acquired over 130 different antibiotic resistance gene cassettes9.
The spread of class 1 integrons into human-associated commensal and pathogenic bacteria generates human waste streams that contain large numbers of these genetic elements10. An estimated 1019 bacteria that contain class 1 integrons are released via sewage sludge each year in the United Kingdom11. It is therefore not surprising that class 1 integrons conferring antibiotic resistances are now being detected in microbiota of wild birds, fish, and other native wildlife12-14. Releasing integrons back into the environment poses a significant public health threat, since acquisition of new gene cassettes and complex rearrangements with other mobile elements continues to occur, particularly in sewage treatment plants and other water bodies15-18. The natural environment then becomes a fertile recruiting ground for new resistance determinants and opportunistic pathogens19,20. Novel integron-containing bacteria and new ARGs can circle back into the human community through contaminated water and food21,22. Surveillance of environmental ARGs is a key strategy for understanding and managing antibiotic resistance in the future23. In particular, attention should be paid to foodstuffs that are eaten raw or lightly cooked, since these present the greatest threat for transmission of new mobile elements and pathogens.
In this protocol, a streamlined approach for detecting, identifying and characterizing class 1 integrons and their associated gene cassettes in foodstuffs are outlined (Figure 2). Using a combination of culturing and polymerase chain reaction (PCR), integrons can be rapidly detected in complex bacterial communities and individual isolates. Methods for identifying the species of bacteria and the conformation and identity of the integron-associated gene cassettes are given. The method is suitable for a broad range of plant and animal foods, and examples of typical workflows are given for each of these food types.

<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 of mixed cultures and bacterial isolates for intI1
Primer set HS463a/HS464 PCR can be used to detect the presence of the class 1 integron-integrase gene, intI1 (Figure 1). This primer set works well for detecting intI1 in mixed cultures, and is also used to screen bacterial colonies harvested from spread plates (Figure 2). Positive isolates should generate a single strong band at 471 bp using this primer set (Figure 3A...

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.

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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