Name: Screening Foodstuffs for Class 1 Integrons and Gene Cassettes (Video) | JoVE Journal
Uploaded: 2015-06-19T13:00:00.000+00:00
Duration: 9 min 37 s
Description: Learn to detect class 1 integrons and antibiotic resistance gene cassettes in foodstuffs using PCR screening and DNA sequencing. This protocol enables monitoring of resistance genes in raw foods for public health surveillance.

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5 minutes

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>Place approximately 10 g of material in a durable plastic bag. If processing larger material, concentrate on the skin of the fruit or vegetable. Add 90 ml 0.1 M sodium phosphate buffer, pH 7.0 and process in a paddle blender for 30 sec. Alternatively, the material can be manually agitated if a paddle blender is not available.</li>
		<li>Collect the liquid into two 50 ml Falcon tubes. Centrifuge tubes at 4,000 x g for 7 min to pellet bacteria.</li>
		<li>Carefully remove the supernatant and suspend the pelleted bacteria in 30 ml of sterile Luria Bertani (LB) broth.</li>
		<li>Incubate on a shaker O/N, holding one tube at 25 &#176;C and the other at 37 &#176;C. Cultures should be shaken at 200 oscillations per minute (opm). Store cultures at 4 &#176;C until required.</li>
	</ol>
	</li>
	<li>Seafood:
	<ol>
		<li>Dissect out the stomach (oysters) or digestive tract (prawns) using sterile forceps and tweezers and place into a sterile 1.5 ml tube containing 200 &#181;l of 0.1 M sodium phosphate buffer, pH 7.0. Macerate the sample to create a homogenate.</li>
		<li>Dispense 5 ml of LB broth into two sterile 5 ml tubes, and inoculate each with the seafood homogenate.</li>
		<li>Incubate on a shaker O/N, holding one tube at 25 &#176;C and the other 37 &#176;C. Cultures should be shaken at 200 opm. Store cultures at 4 &#176;C until required.</li>
	</ol>
	</li>
</ol>
3. Screening Cultures for Integrons
NOTE: Standard PCR protocols are used throughout this methodology, using buffers supplied with the enzyme, and a final MgCl2 concentration of 2.5 mM. If required, Lorenz (2011)24 has outlined PCR optimization and troubleshooting methods in an earlier issue of this journal.
<ol>
	<li>Boiled DNA preparation:
	<ol>
		<li>Dispense 100 &#181;l of enriched culture into a sterile 0.5 ml PCR tube. Heat the sample to 99 &#176;C for 10 min, using a water bath or heat block. Snap chill on ice for 2 min.</li>
		<li>Microcentrifuge at 14,000 x g for 5 min to pellet cell debris. Return to ice. DNA samples can be frozen at -20 &#176;C until required, but must be thawed on ice.</li>
	</ol>
	</li>
	<li>Screening for class 1 integrons using PCR: 
	NOTE: Mixed cultures from the O/N incubation are screened for class 1 integrons using the HS463a/HS464 PCR protocol (Table 1).
	<ol>
		<li>Thaw all PCR reagents on ice, away from any potential sources of contaminating DNA. Make up 48 &#181;l PCR mastermix per sample, including negative controls (Table 2). Dispense 48 &#181;l of PCR master mix to each tube, using barrier tips.</li>
		<li>Add 2 &#181;l of thawed DNA to individual PCR tubes using barrier tips. Run appropriate program in the PCR thermocycler.</li>
		<li>To assess the results of the amplification, electrophorese 7 &#181;l of PCR product on a 2% agarose gel poured and run in TBE buffer (90 mM TRIS-borate, 2 mM EDTA). Include a molecular weight marker, such as a 100 bp ladder.</li>
		<li>Post stain the gel with a DNA stain, and visualize using a UV transilluminator. Check that there has been no amplification in the negative control. A strong band at around 470 bp for any particular sample indicates that the mixed culture contains bacteria that carry a class 1 integron. Pure colonies will now be isolated from any mixed culture that returned a positive PCR.</li>
	</ol>
	</li>
</ol>
4. Screening of Single Colonies for Class 1 Integrons
<ol>
	<li>Serial dilutions and spread plates: 
	NOTE: To isolate single colonies from the PCR-positive mixed cultures, the mixed culture is serially diluted 10-fold in sodium phosphate buffer, and then plated to recover single colonies.
	<ol>
		<li>Add 1 ml of mixed culture to 9 ml of 0.1 M sodium phosphate buffer, pH 7.0 and mix by inversion. Add 1 ml of the dilution to a further 9 ml of phosphate buffer, and repeat until a serial dilution of 10-8 is reached.</li>
		<li>Spread 100 &#181;l of the 10-4 to 10-8 dilutions onto LB agar, in duplicate. Incubate the plates O/N at the same temperature used for the initial mixed culture. Plates can then be stored at 4 &#176;C until required, however it is best to process the single colonies fairly promptly.</li>
	</ol>
	</li>
	<li>Single colony selection and boiled DNA preparation:
	<ol>
		<li>Pick single colonies from the serial dilution plates, selecting as many different colony types as possible, using criteria such as colony size, shape and color. Use sterile toothpicks to select single colonies from the spread plate.</li>
		<li>Touch the toothpick to the colony, and then transfer into a PCR tube containing 100 &#181;l of sterile water. If large numbers of colonies are to be screened, then colonies can be prepared in a microtiter tray. Spin the toothpick between the fingers to dislodge some of the cells into the water.</li>
		<li>Using the same toothpick, inoculate an LB plate with a streak of the colony. Large numbers of colonies can be stored on labelled plates if each streak is about 1 cm long.&#160;Repeat steps 1 to 3 until you have selected an appropriate number of isolates.</li>
		<li>Incubate the LB plates O/N, at the same temperature as that used for the initial enriched culture. Cultures can then be stored at 4 &#176;C until required.</li>
		<li>For preparing DNA from the bacterial suspensions, follow the steps as outlined in section 3.1. For screening of pure culture lysates, perform the HS463a/HS464 PCR as outlined in section 3.2. Isolates that return a positive PCR will be used for preparation of genomic DNA (section 5), and for further analyses.</li>
	</ol>
	</li>
</ol>
5. Genomic DNA Extraction of Integron-positive Single Colonies Using Bead Beating25
<ol>
	<li>Inoculate 5 ml of LB broth with a sample of the pure culture isolated in section 4.2. Incubate O/N, shaking at the same temperature that was used for the initial enriched culture.</li>
	<li>Pellet cells at 4,000 x g for 7 min, then remove the supernatant. Resuspend the pelleted cells in 1 ml CLS-TC solution in a Lysing Matrix E fast prep tube. Using a bead beating machine, process the sample at 5.5 m/sec for 30 sec.</li>
	<li>Centrifuge tube at 14,000 x g for 5 min and recover 700 &#181;l of supernatant into a sterile 1.5 ml tube.</li>
	<li>Add 700 &#181;l of binding matrix diluted 1:5 with 6 M guanidinium thiocyanate and mix at RT for 5 min. Centrifuge at 14,000 x g for 1 min, discard supernatant.</li>
	<li>Add 800 &#181;l salt ethanol wash (70% ethanol, 0.1 M sodium acetate) and vortex until the pellet is fully resuspended. Wash for 5 min at RT, centrifuge at 14,000 x g for 1 min and discard supernatant. Ensure all supernatant has been removed, using a micropipette if necessary, then allow to air dry for 5 min.</li>
	<li>Resuspend the pellet matrix in 200 &#181;l TE (10 mM Tris-HCl, 1mM EDTA, pH 7.6) by pipetting up and down. Incubate at RT for 3 min. Centrifuge at 14,000 x g for 3 min and transfer 160 &#181;l of the eluted DNA into a sterile tube. Run an aliquot of the genomic DNA extraction on a 2% w/v agarose gel to check the yield and integrity of the DNA extraction. The purified genomic DNA is stored at -20 &#176;C and defrosted on ice when required for PCR testing.</li>
</ol>
6. Diagnostic PCRs and DNA Sequencing
NOTE: The genomic DNA prepared in section 5 will be used for all diagnostic PCRs, and to confirm the positive test for class 1 integrons.
<ol>
	<li>Thaw genomic DNA on ice, briefly vortex the DNA sample and pulse spin at 14,000 x g for 20 sec. Repeat the PCR for the class 1 integron-integrase gene using HS463a/HS464 (Table 1), confirming that the isolate is positive for the 471 bp amplicon (Section 3.2).</li>
	<li>Identification of positive cultures to species level is achieved by analysis of the small subunit rRNA gene (16S rDNA). Amplify the 16S gene using primers f27/r1492 (Table 1). Check the PCR products using electrophoresis. All bacterial targets should generate a 16S amplicon of about 1450 bp.</li>
	<li>Sequence the 16S rRNA gene amplicon to determine species identity, but since there are likely to be multiple positive PCRs from step 6.2, and many of these positives will be the same bacterial species, we will first distinguish different species using restriction digestion of the 16S PCR product.
	<ol>
		<li>Digest an aliquot of the 16S PCR with the restriction enzyme Hinf1. Any enzyme that recognizes 4 bp cleavage sites will do, but Hinf1 generates good diversity from 16S targets, and is a reliable, inexpensive enzyme.</li>
	</ol>
	</li>
	<li>Set up a 30 &#181;l restriction digest mastermix (Table 2) in a sterile tube and add 20 &#181;l of un-purfied 16S PCR product. Incubate in a 37 &#176;C water bath O/N. Check the digest on a 2% agarose gel.</li>
	<li>Score the digestion patterns produced by Hinf1. Isolates with the same 16S restriction profile are likely to be the same species. Rather than sequencing every 16S PCR product, sequence at most three representatives of each Hinf1 restriction pattern.</li>
	<li>Purify the 16S PCR products using a commercial kit. A single stranded exonuclease kit can be used, but column based or precipitation methods also work well to remove unincorporated primers and single stranded DNAs. Sequence the 16S amplicons using primer r910 (Table 1), setting up the reactions as specified by the sequencing facility.</li>
	<li>Use the resulting DNA sequence to interrogate the NCBI DNA database with the blastn function (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>). Greater than 97% nucleotide identity is usually taken as species identity.</li>
</ol>
7. Mapping and Characterization of Integrons and Cassette Arrays
NOTE: Class 1 integrons emanating from human-dominated ecosystems are likely to be the most common integrons in all your samples. These integrons all have a recent single origin, and therefore have a highly conserved DNA sequence5.
<ol>
	<li>Differentiate between a class 1 integrons emanating from human sources and those that occur naturally in environmental samples by performing a PCR with primer set intI1F165/intI1R47610 (Table 1). 
	NOTE: Class 1 integrons from clinical or commensal bacteria will generate a ~300 bp product, while environmental class 1 integrons will not generate any product.</li>
	<li>Characterize the clinical integrons by amplifying the cassette array with primer set HS458/HS459. This PCR will amplify the region between intI1 and the 3&#8217; conserved segment at the terminus of most cassette arrays. Because the number and identity of cassettes is variable, the size of the PCR product will also vary.</li>
	<li>Purify amplicons and sequence the DNA using each of the amplification primers. Use sequences to interrogate DNA databases using the blastn function (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>).</li>
	<li>Ensure naming of cassettes follow standardized nomenclature9, since many data depositions for integron gene cassettes use inconsistent or ambiguous names. Give particular attention to novel gene cassettes, since these may encode new phenotypes, including genes that confer increased transmissibility, pathogenicity or virulence8,21. 
	NOTE: Preclinical forms of the class 1 integron still circulate in natural environments. In particular, those that are linked to active Tn402 transposons are of most interest26. The cassette arrays of these integrons will not amplify with primer set HS458/HS459, but can be amplified with the MRG284/MRG285 primer set (Table 1), generating amplicons of variable size dependent on cassette content. PCR products should be purified and DNA sequenced as outlined in 7.2.</li>
</ol>

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<li>Gaze, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impacts+of+anthropogenic+activity+on+the+ecology+of+class+1+integrons+and+integron-associated+genes+in+the+environment%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+microbiota+composition+and+presence+of+selected+antibiotic+resistance+genes+in+carriage+water+of+ornamental+fish%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Into+the+wild%3A+dissemination+of+antibiotic+resistance+determinants+via+a+species+recovery+program%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((Gene+flow%2C+mobile+genetic+elements+and+the+recruitment+of+antibiotic+resistance+genes+into+Gram-negative+pathogens%5BTitle%5D)+AND+%22FEMS+Microbiol.+Rev%22%5BJournal%5D)">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/pubmed?term=((Broad+diversity+of+conjugative+plasmids+in+integron-carrying+bacteria+from+wastewater+environments%5BTitle%5D)+AND+%22FEMS+Microbiol.+Lett%22%5BJournal%5D)">Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments.</a> FEMS Microbiol. Lett. 330 (2), 157-164 (2012).</li>
<li>Schlüter, A., Krause, L., Szczepanowski, R., Goesmann, A., Pühler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+diversity+and+composition+of+a+plasmid+metagenome+from+a+wastewater+treatment+plant%5BTitle%5D)+AND+%22J.+Biotech%22%5BJournal%5D)">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/pubmed?term=((Aquatic+systems%3A+maintaining%2C+mixing+and+mobilising+antimicrobial+resistance%5BTitle%5D)+AND+%22TREE%22%5BJournal%5D)">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/pubmed?term=((Quantitative+and+qualitative+impact+of+hospital+effluent+on+dissemination+of+the+integron+pool%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">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/pubmed?term=((Call+of+the+wild%3A+antibiotic+resistance+genes+in+natural+environments%5BTitle%5D)+AND+%22Nature+Rev.+Microbiol%22%5BJournal%5D)">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/pubmed?term=((The+role+of+the+natural+environment+in+the+emergence+of+antibiotic+resistance+in+Gram-negative+bacteria%5BTitle%5D)+AND+%22Lancet+Infect.+Dis%22%5BJournal%5D)">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>
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<li>Gillings, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+Extraction+of+PCR-Competent+DNA+from+Recalcitrant+Environmental+Samples%5BTitle%5D)+AND+%22Env.+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Preclinical+class+1+integron+with+a+complete+Tn402-like+transposition+module%5BTitle%5D)+AND+%22Appl.+Env.+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Antibiotic+resistance+in+the+environment%3A+A+link+to+the+clinic%5BTitle%5D)+AND+%22Curr.+Opinion+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Class+1+integrons+predating+the+association+with+Tn402.-like+transposition+genes+are+present+in+a+sediment+microbial+community%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">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>
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<li>Holmes, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombination+activity+of+a+distinctive+integron-gene+cassette+system+associated+with+stutzeri.+populations+in+soil%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">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 authors have nothing relevant to disclose.

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><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 6 M 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 &lt;a href=&quot;http://www.mpbio.com&quot; target=&quot;_blank&quot;&gt;http://www.mpbio.com&lt;/a&gt; for more information</td></tr></tbody></table>

screening foodstuffs for class 1 integrons and gene cassettes

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...

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 ...

The overall goal of this procedure is to provide a streamlined approach for detecting class one inter grounds and their associated gene cassettes from foodstuffs. This is accomplished by first making an enriched bacterial culture from your chosen food item. The second step is to screen the culture for the presence of class one inters using PCR and isolate single colonies from the inter positive mixed culture.After screening the single colonies for the presence of integrons, prepare genomic DNA from the single colonies for diagnostic PCRs to differentiate bacterial species and detect the presence of gene cassettes. The final step is to purify PCR products and submit them for DNA sequence analysis. Ultimately, DNA sequence analysis is used to characterize gene cassettes within a class one inter and identify the species in which they reside.Antibiotic resistance is one of the major problems facing medical treatment in the 21st century. One of the ways that we can manage antibiotic resistance is to detect and trace antibiotic resistance genes in the environment from humans to the environment and back again. This protocol is going to show you how to detect antibiotic resistance.Genes in food stuffs that we might eat raw or with minimal processing. The procedure is going to be demonstrated by Leet Waldron, a postdoc working in my laboratory. Foods that are eaten raw or lightly cooked, such as fruits, vegetables, and shellfish are of the most concern for human health.Only the preparation of enriched cultures from seafood will be demonstrated here. Please refer to the protocol text for the preparation of cultures from fruits and vegetables. To prepare an enriched culture from seafood, first use sterile forceps and tweezers to dissect out the digestive tract from prawns.Place the dissected material into a sterile 1.5 milliliter tube containing 200 microliters of a sodium phosphate buffer. Macerate the sample using a pestle to create a homogenate and vortex. Briefly dispense five milliliters of LB broth into two sterile five milliliter tubes and inoculate each with the seafood homogenate.Shake both tubes overnight at 200 OPM incubating one tube at 25 degrees Celsius and the other at 37 degrees Celsius on the following day. Store the cultures at four degrees Celsius until required to recover single colonies from A PCR positive mixed culture. First serially dilute the culture tenfold in sodium phosphate buffer by adding one milliliter of the mixed culture to nine milliliters of sodium phosphate buffer mixed by vortex.Add one milliliter of the dilution to a further nine milliliters of sodium phosphate buffer and repeat until a serial dilution of 10 to the minus eight is reached plate. The diluted culture spread 100 microliters of the 10 to the minus four to 10 to the minus eight dilution onto LB agar. In duplicate, incubate the plates overnight at the same temperature used for the initial mixed culture on the following day.Single colonies can be selected from the serial dilution plates. For DNA preparation, make sure all plates and sample tubes are clearly labeled prior to starting. Touch a sterile toothpick to a single colony and then transfer into a PCR tube containing 100 microliters of sterile water.Spin the toothpick between the fingers to dislodge some of the cells into the water using the same toothpick, inoculate an LB plate with a streak of each picked colony. Large numbers of colonies can be stored on labeled plates. If each streak is about one centimeter long, close the lid immediately to reduce contamination between samples.Select as many different colony types as possible using criteria such as colony size, shape, and color. Incubate the LB plates overnight at the same temperature as that used for the initial enriched culture. Cultures can then be stored at four degrees Celsius until required to prepare DNA from the bacterial suspensions.Heat the samples to 99 degrees Celsius for 10 minutes using a thermocycler micro centrifuge at 14, 000 times, G for five minutes at room temperature to pellet cell debris return to ice. Subsequently screen the DNA by PCR with primer's HS 4 63 A and HS 4 64. Following the procedures described in the protocol text, it is important to avoid any potential sources of contaminating DNA when performing the PCR to assess the results of the amplification electro referee.Seven microliters of each PCR product on a 2%aros gel poured and run in TBE buffer after post staining the gel with the DNA stain. Visualize the DNA using a UV transluminator Isolates that carry a class one integrin will generate a strong single band at 471 base pairs genomic DNA prepared from isolates that return a positive PCR are used for these analyses. Thaw the genomic DNA on ice and briefly vortex and pulse spin at 14, 000 times G for 20 seconds.The primer sequences and PCR conditions used for diagnostic PCRs are shown in table one in the manuscript. First, repeat the PCR for the class oneone integrase gene to confirm that the isolate is positive for the 471 base pair amplicon. Next, identify the positive cultures to the species level by amplifying the small subunit ribosomal RNA gene 16 S-R-D-N-A.All bacterial targets should generate a 16 S amplicon of about 1, 450 base pairs since many of these positive cultures will be the same. Bacterial species distinguish the different species by restriction digestion of the 16 SPCR product. The restriction enzyme HIF one is recommended since it generates good diversity from 16 s targets and is a reliable, inexpensive enzyme.Set up a 30 microliter restriction digest master mix in a sterile tube and add 20 microliters of un purified 16 SPCR product. Incubate in a 37 degree Celsius water bath overnight on the following day, check the digest on a 2%AROS gel selected 16 SPCR products are then purified using a commercial kit and sent to a sequencing facility for DNA sequencing. Use the resulting DNA sequence to interrogate the N-C-B-I-D-N-A database with the Blast N function greater than 97%nucleotide identity is usually taken as species identity.Mixed cultures antibacterial isolates are screened for the presence of the class one integrase gene by PCR. Using the primers HS 4 63 A and HS 4 64 positive colonies generate a strong single band at 471 base pairs. Genomic, DNA from pure isolates is used for characterization of inter cassette arrays, amplification of cassette arrays from TN 4 0 2 integrons using primers HS 4 58 and HS 4 59 generates variable product sizes dependent on the identity and size of the component cassettes in the array.Identification of these cassettes requires DNA sequencing Environmental Class one integrons are often embedded in bacterial chromosomes. Their consider rays can be amplified by primer's MG 2 84 and MG 2 85 that target the proximal and most distal recombination sites respectively. This PCR also generates variable product sizes dependent on the identity and size of the component cassettes in the array.However, cassettes in environmental arrays are unlikely to encode antibiotic resistance. Bacterial species are identified by amplifying 16 S-R-D-N-A and screening the PCR products by digestion. With HIF one individual species will generate distinctive patterns such the isolates of the same species can be easily identified and confirmed by DNA sequencing.After watching this video, you should now have a good idea of how to isolate Class one ingrams and their antibiotic resistance cassettes from everyday foods.

screening-foodstuffs-for-class-1-integrons-and-gene-cassettes

Research

JoVE Journal

Environment

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Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

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Learn to detect class 1 integrons and antibiotic resistance gene cassettes in foodstuffs using PCR screening and DNA sequencing. This protocol enables monitoring of resistance genes in raw foods for public health surveillance.

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

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