Name: quasi-metagenomic analysis of salmonella from food and environmental samples
Uploaded: 2018-10-25T14:45:00
Description: Watch this Scientific Journal Video about Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples at JoVE.com

The authors would like to thank Mark Harrison and Gwen Hirsch of the University of Georgia for kindly providing the bacterial strain and other support to this study.

1. Sample Preparation
NOTE: Food samples are prepared for pre-enrichment according to Microbiology Laboratory Guidebook (MLG) of U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS)11 and Bacteriological analytical manual (BAM) of U.S. Food and Drug Administration (FDA)12.
<ol>
	<li>Aseptically place a 25 g portion of food sample such as black pepper, chicken breast, ground beef, and alfalfa sprouts or an environmental ...

<ol>
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Y., 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=Quasi-metagenomics+and+realtime+sequencing+aided+detection+and+subtyping+of+Salmonella+enterica+from+food+samples.">Quasi-metagenomics and realtime sequencing aided detection and subtyping of Salmonella enterica from food samples.</a> Applied and Environmental Microbiology. , (2017).</li><li>Hyeon, J. Y., Deng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Salmonella+in+raw+chicken+breast+using+real-time+PCR+combined+with+immunomagnetic+separation+and+whole+genome+amplification.">Rapid detection of Salmonella in raw chicken breast using real-time PCR combined with immunomagnetic separation and whole genome amplification.</a> Food Microbiology. 63, 111-116 (2017).</li><li>Hosono, S., 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=Unbiased+whole-genome+amplification+directly+from+clinical+samples.">Unbiased whole-genome amplification directly from clinical samples.</a> Genome Research. 13 (5), 954-964 (2003).</li><li>Seth-Smith, H. 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=Whole-genome+sequences+of+Chlamydia+trachomatis+directly+from+clinical+samples+without+culture.">Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture.</a> Genome Research. 23 (5), 855-866 (2013).</li><li>Jacobson, A. P., Gill, V. S., Irvin, K. A., Wang, H., Hammack, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+to+prepare+samples+of+leafy+green+vegetables+for+preenrichment+with+the+Bacteriological+Analytical+Manual+Salmonella+culture+method.">Evaluation of methods to prepare samples of leafy green vegetables for preenrichment with the Bacteriological Analytical Manual Salmonella culture method.</a> Journal of Food Protection. 75 (2), 400-404 (2012).</li><li>Jacobson, A. P., Hammack, T. S., Andrews, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+sample+preparation+methods+for+the+isolation+of+Salmonella+from+alfalfa+and+mung+bean+seeds+with+the+Bacteriological+Analytical+Manual's+Salmonella+culture+method.">Evaluation of sample preparation methods for the isolation of Salmonella from alfalfa and mung bean seeds with the Bacteriological Analytical Manual's Salmonella culture method.</a> Journal of AOAC International. 91 (5), 1083-1089 (2008).</li><li>Deng, X., 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=Comparative+analysis+of+subtyping+methods+against+a+whole-genome-sequencing+standard+for+Salmonella+enterica+serotype+Enteritidis.">Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.</a> Journal of Clinical Microbiology. 53 (1), 212-218 (2015).</li><li>. Microbiology Laboratory Guidebook Available from: <a target="_blank" href="https://www.fsis.usda.gov/wps/portal/fsis/topics/science/laboratories-and-procedures/guidebooks-and-methods/microbiology-laboratory-guidebook/microbiology-laboratory-guidebook">https://www.fsis.usda.gov/wps/portal/fsis/topics/science/laboratories-and-procedures/guidebooks-and-methods/microbiology-laboratory-guidebook/microbiology-laboratory-guidebook</a> (2018)</li><li>. Bacteriological Analytical Manual (BAM) Chapter 5: Salmonella Available from: <a target="_blank" href="https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm">https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm</a> (2018)</li><li>Malorny, B., 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=Diagnostic+real-time+PCR+for+detection+of+Salmonella+in+food.">Diagnostic real-time PCR for detection of Salmonella in food.</a> Applied and Environmental Microbiology. 70 (12), 7046-7052 (2004).</li><li>June, G. A., Sherrod, P. S., Hammack, T. S., Amaguana, R. M., Andrews, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+effectiveness+of+selenite+cystine+broth,+tetrathionate+broth,+and+Rappaport-Vassiliadis+medium+for+the+recovery+of+Salmonella+from+raw+flesh+and+other+highly+contaminated+foods:+precollaborative+study.">Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella from raw flesh and other highly contaminated foods: precollaborative study.</a> Journal of AOAC International. 78 (2), 375-380 (1995).</li><li>Hammack, T. S., Amaguana, R. M., June, G. A., Sherrod, P. S., Andrews, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+effectiveness+of+selenite+cystine+broth,+tetrathionate+broth,+and+Rappaport-Vassiliadis+medium+for+the+recovery+of+Salmonella+spp.+from+foods+with+a+low+microbial+load.">Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella spp. from foods with a low microbial load.</a> Journal of Food Protection. 62 (1), 16-21 (1999).</li><li>Wood, D. E., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kraken:+ultrafast+metagenomic+sequence+classification+using+exact+alignments.">Kraken: ultrafast metagenomic sequence classification using exact alignments.</a> Genome Biology. 15 (3), R46 (2014).</li><li>Davis, S., Pettengill, J. B., Luo, Y., Payne, J., Shpuntoff, A., Rand, H., Strain, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFSAN+SNP+Pipeline:+an+automated+method+for+constructing+SNP+matrices+from+next-generation+sequence+data.">CFSAN SNP Pipeline: an automated method for constructing SNP matrices from next-generation sequence data.</a> PeerJ Computer Science. 1 (e20), (2015).</li><li>Zhang, S., 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=Salmonella+serotype+determination+utilizing+high-throughput+genome+sequencing+data.">Salmonella serotype determination utilizing high-throughput genome sequencing data.</a> Journal of Clinical Microbiology. 53 (5), 1685-1692 (2015).</li><li>Rodrigue, S., 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=Whole+genome+amplification+and+de+novo+assembly+of+single+bacterial+cells.">Whole genome amplification and de novo assembly of single bacterial cells.</a> PLoS One. 4 (9), e6864 (2009).</li><li>Ramamurthy, T., Ghosh, A., Pazhani, G. P., Shinoda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Perspectives+on+Viable+but+Non-Culturable+(VBNC)+Pathogenic+Bacteria.">Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria.</a> Front Public Health. 2, 103 (2014).</li></ol>

Because of the often-low abundance and in-homogenous presence of Salmonella in food and environmental samples, culture enrichment before IMS-MDA is still necessary for Salmonella detection and subtyping; it is therefore a critical step of the protocol. To identify optimal conditions to increase the abundance of Salmonella relative to sample background flora, different enrichment media may be evaluated for specific samples. According to MLG and BAM, both selective medium such as RV broth and non...

Metagenomics sequencing theoretically allows concerted detection and subtyping of foodborne pathogens. However, food samples present challenges to the pathogen analysis by direct sequencing of the food microbiome. First, foodborne pathogens are often present at low levels in food samples. Most of the commercially available rapid detection methods still require 8&#8211;48 h culturing to enrich pathogen cells to a detectable level1. Second, many foods contain abundant microflora cells and/or food DNA, making foodborne pathogen DNA a small fraction of food metagenome and an elusive target for detection and subtyping by direct metagenomic sequencing.
Modification of food microbiomes has been reported to allow substantial concentration of foodborne pathogen DNA to facilitate sequencing-based detection of Shiga toxin-producing Escherichia coli2,3 and Salmonella enterica4. Because modified food microbiomes are still mixtures of different microbial species, their sequencing is termed as quasi-metagenomic analysis4. Microbiome modification can be performed by culture enrichment alone2,3 or in combination with immunomagnetic separation (IMS) and multiple displacement amplification (MDA)4,5. IMS can selectively capture pathogen cells from the enrichment culture via antibody-coated magnetic beads. MDA can generate sufficient amounts of genomic DNA for sequencing through the highly efficient &#632;29 DNA polymerase6. IMS-MDA has allowed culture-independent pathogen detection from clinical samples7, and shortening of the culture enrichment for quasi-metagenomic detection and subtyping of Salmonella in food samples4.
The overall goal of this method is to prepare quasi-metagenomic DNA from food samples to allow targeted concentration of Salmonella genomic DNA and subsequent detection and subtyping of the Salmonella contaminant by sequencing. Compared to standard methods for Salmonella detection8,9 and subtyping10, the quasimetagenomic approach can substantially shorten the turnaround time from contaminated food and environmental samples to molecular subtypes of the pathogen by unifying the two typically separated analyses into a single workflow. This method is particularly useful for applications such as foodborne outbreak response and other trace back investigations where robust pathogen subtyping is required in addition to pathogen detection and rapid analytical turnaround is important.

<table>
	<tbody>
		<tr>
			<td>Laboratory blender bag w/filter</td>
			<td>VWR</td>
			<td>10048-886</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered peptone water</td>
			<td>Oxoid Micorbiology Products</td>
			<td>CM0509</td>
			<td></td>
		</tr>
		<tr>
			<td>Rappaport Vassiliadis broth</td>
			<td>Neogen Acumedia</td>
			<td>7730A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20&#160;</td>
			<td>Millipore Sigma</td>
			<td>P9416</td>
			<td>Tween 20</td>
		</tr>
		<tr>
			<td>Stomacher blender</td>
			<td>Seward&#160;</td>
			<td>30010108</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75005194</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Techne Prime</td>
			<td>EW-93945-13</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOne Real-Time Thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>4.76357</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>PCR purification beads; mix well before use; store at 4C</td>
		</tr>
		<tr>
			<td>Nextera XT library prep kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>Store at -80C</td>
		</tr>
		<tr>
			<td>MinIon library prep kit</td>
			<td>Oxford Nanopore</td>
			<td>SQK-LSK108</td>
			<td>Store at -80C</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabead Anti-Salmonella beads</td>
			<td>Applied Biosystems</td>
			<td>71002</td>
			<td>Vortex well prior to use</td>
		</tr>
		<tr>
			<td>Illustra GenomiPhi V2 DNA amplification kit (MDA kit) 
			-Sample buffer 
			-Reaction buffer 
			-Enzyme mix</td>
			<td>GE Healthcare</td>
			<td>25-6600-30</td>
			<td>Store at -80C</td>
		</tr>
		<tr>
			<td>HulaMixer</td>
			<td>Invitrogen</td>
			<td>15920D</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag magnetic rack</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR mastermix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td>Mix well before use; store at 4C</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

quasi-metagenomic analysis of salmonella from food and environmental samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Prior to quasimetagenomic sequencing, the overall quantity and purity of IMS-MDA products can be evaluated by fluorospectrometer (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Enrichment time (h)</td>
			<td>Ct value</td>
			<td>Concentration (ng/ul)</td>
			<td>Purity (260/280)</td>
		</tr>
		<tr>
			<td><stron...

Watch this Scientific Journal Video about Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples at JoVE.com

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

This method can be used to detect Salmonella from food samples and identify the pathogen to the strain level through genetic fingerprinting. The main advantage of this technique is that it combines Salmonella detection and subtyping into a single workflow and substantially shortens the turnaround time from Salmonella-contaminated food sample to fingerprint of the pathogen. To begin, aseptically place a 25 gram sample of food inside a sterile laboratory blender bag with a built-in filter or optionally prepare an environmental swab by aseptically moistening a sponge with enrichment broth, dragging it across a pre-determined surface and placing it inside a blender bag.Using a laboratory blender, thoroughly mix each sample with 225 milliliters of RV enrichment broth. Then incubate the mixture at 42 degrees Celsius for four to 21 hours. After this, collect a 50 milliliter subsample of enrichment broth from the filtered side of the bag.Then centrifuge the subsample at 100 times g for 10 minutes. Carefully transfer the supernatant to a new 50 milliliter centrifuge tube and centrifuge at 3, 000 to 6, 000 times g for 10 minutes. Discard the supernatant and wash the pellet in five milliliters of BPW.Resuspend the pellet in five milliliters of BPW. First, thaw the sample buffer and the reaction buffer from the MDA kit on ice. Place one milliliter of resuspended cells in BPW and 20 microliters of anti-Salmonella beads in a microcentrifuge tube.Place the microcentrifuge tube on a rotating mixer for 30 minutes at room temperature. After this, place the tube on a magnetic stand for three minutes. Invert the magnetic rack several times to concentrate the beads into a pellet.Keeping the tube on the magnetic rack, aspirate and discard the supernatant. Next, add one milliliter of wash buffer to the tube. Remove the tube holder from the rack and invert the tube holder several times to remove any non-specifically binding bacteria from the complex.After the third wash, aspirate the supernatant from the tube and discard it. Then remove the tube from the magnetic rack and centrifuge it for one second. Then place the tube on the magnetic rack for three minutes before removing any residual wash buffer.Resuspend the bead Salmonella complexes in nine microliters of sample buffer and incubate the tube at 95 degrees Celsius for three minutes. After cooling the complexes on ice, add nine microliters of reaction buffer and one microliter of enzyme mix. Incubate the tube in a thermal cycler according to the text protocol and cool the tube on ice.Next, use a fluorospectrometer instrument to measure the DNA concentration and purity of the sample. Store the final products at negative 20 degrees Celsius for future experiments. Prepare 18 microliters of PCR mixture per sample according to the text protocol.Then mix the MDA product by gently pipetting up and down. Add two microliters of the MDA product suspension to the PCR mixture. Using an optimized real-time PCR protocol, run the real-time PCR according to the text protocol.Add buffer solutions and reagents to MDA product as specified in the text protocol and transfer the resulting 40 microliters of the sequencing prepped MDA product to a new plate and add 20 microliters of PCR purification beads to each well. Next, incubate the plate at room temperature for 10 minutes. Then wash the beads with 80%ethanol and dry them for 12 minutes.Resuspend the dried beads in 53 microliters of resuspension buffer and incubate the beads for two minutes at room temperature. Finally, dilute and pool libraries according to the manufacturer's instructions. In this protocol, targeted quantity assessment of Salmonella DNA was performed by real-time PCR.Representative results from two brands of Salmonella-contaminated chicken breast ranged from 701.54 to 945.86 nanograms per microliter indicating that the sample DNA was effectively amplified. While attempting this procedure, it is important to remember to wash and handle IMS beads carefully, keep MDA reagents and products free from contaminants and maintain a clean hood. After its development, this technique paved the way for researchers in the field of food safety and public health to explore rapid and high-resolution tracking of Salmonella contaminants in food samples, product environments and supply chains.Don't forget that working with foodborne pathogens can be extremely hazardous and precautions such as wearing personal protective equipment and following good laboratory practices should always be taken while performing this procedure.

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