Name: enrichment and detection of clostridium perfringens toxinotypes in retail food samples
Uploaded: 2019-10-18T14:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples at JoVE.com

This research did not receive any specific funding from the public, commercial, or not-for profit sectors.

NOTE: C. perfringens is considered a biosafety hazard level 2 (BSL2) organism. Although not all food samples will contain C. perfringens, all cultured samples should be treated as such. All proper precautions and personnel protective equipment (PPE) should be worn at all times. Decontaminate all material prior to disposal.
1. Prepare modified RPM4,15
<ol>
	<li>Combine 30 g/L fluid thioglycolate medium, 60 g/L gelatin, 5 g/L peptone, 5 g/L glucose, 5 g/L potassium phosphate dibasic, 3 g/L yeast extract, 1.5 g/L sodium chloride, 0.5 g/L ferrous sulfate in deionized water until evenly dispersed. We typically make 1 L batches.</li>
	<li>Autoclave at 121 &#176;C for 15 min.</li>
	<li>Allow to cool to approximately 40 &#176;C.</li>
	<li>Once RPM is cool, add D-cycloserine to a final concentration of 440 mg/L. 
	NOTE: Stock concentrations of D-cycloserine dissolved in sterile water at 50 mg/mL may be stored at -20 &#176;C for future use.</li>
	<li>Aseptically transfer 10 mL of RPM to 15 mL conical tubes. RPM can be prepared in batches and stored at 4 &#176;C for up to one month.</li>
	<li>Warm RPM to 37 &#176;C prior to use.</li>
</ol>
2. Sample collection and RPM incubation
<ol>
	<li>Transport food to the laboratory under ambient temperatures within 1 h. Food may be transported in original packaging or sterile containers. If not testing immediately, food may be stored at -20 &#176;C until use. Make note of the type of food and the country of origin according to the packaging label, if available, as well as any other relevant information.</li>
	<li>Remove approximately 1.0&#8211;2.0 g of food and transfer to sterile Petri dish or equivalent. Finely mince with a sterile razor blade or scalpel.</li>
	<li>Inoculate into 10 mL of phosphate buffered saline (PBS) in a 15 mL conical tube. Mix well.
	<ol>
		<li>To select for vegetative cells, transfer 5 mL of the PBS-food mixture into a 15 mL conical tube containing 10 mL of RPM. Secure the lid tightly.</li>
		<li>To select for spores, heat the remaining 5 mL of PBS-food mixture at 85 &#176;C for 15 min and then transfer to a 15 mL conical tube containing 10 mL of RPM. Secure the lid tightly.</li>
	</ol>
	</li>
	<li>Vortex and invert the food-RPM cultures to ensure complete mixing.</li>
	<li>Tightly seal the conical tubes and wrap lids with paraffin film or plastic wrap to ensure an anaerobic environment.</li>
	<li>Incubate overnight (ON) at 37 &#176;C.</li>
	<li>The following morning note any turbidity or fermentation in RPM tubes.</li>
</ol>
3. TSC &#34;sandwich&#34;&#160;plating
<ol>
	<li>Inoculate ON RPM cultures into TSC agar using a &#8220;sandwich&#8221; technique (Figure 2) described below.</li>
	<li>Prepare TSC agar as per the manufacturer&#39;s instructions.
	<ol>
		<li>Prepare the base layer of the TSC plates by transferring 10 mL of molten TSC agar to sterile Petri dishes and allow to solidify. These may be prepared in advanced and stored at 4 &#176;C. Ensure plates are warmed to room temperature prior to use.</li>
		<li>For the top layer of the TSC plate, maintain the remaining molten TSC agar at 40 &#176;C. 
		NOTE: Although agar&#39;s melting point is above 85 &#176;C, molten agar solidifies around 32&#8211;45 &#176;C.</li>
	</ol>
	</li>
	<li>Carefully retrieve the ON RPM cultures and transfer 100 &#181;L to the TSC agar base. Because of fermentation, some cultures may be under pressure. Be sure to wear all appropriate PPE.</li>
	<li>Spread the RPM inoculum using sterilized glass beads or cell spreader.</li>
	<li>Allow RPM to be &#34;absorbed&#34;&#160;into TSC agar for 5&#8211;10 min at room temperature.</li>
	<li>Carefully transfer 20 mL of molten, 40 &#176;C TSC agar to plate using a serological pipette.</li>
	<li>Replace the Petri dish lid and allow TSC agar to completely solidify at room temperature.</li>
	<li>Invert the Petri dish and incubate at 37 &#176;C ON.</li>
	<li>If serial dilutions are desired, perform dilutions of the ON RPM cultures into fresh, pre-warmed RPM prior to inoculation into TSC agar.</li>
</ol>
4. Sub-culturing of sulfite-reducing colonies
<ol>
	<li>The following morning, remove plates from incubator and examine for bacterial growth. Aerobic bacteria may be present on the surface of the agar. Anaerobic bacteria will be found growing embedded within the agar. Sulfite-reducing bacteria will turn surrounding agar black. Possible C. perfringens colonies will be black and embedded in the agar.</li>
	<li>C. perfringens suspected colonies will be positive for sulfite reduction and will appear black, embedded within the agar. Using a sterile, single-use eyedropper, &#34;pluck&#34;&#160;the black colonies from the agar and transfer to 10 mL of fresh RPM in a 15 mL conical tube. It is important that the air from the eyedropper be expelled prior to piercing agar.</li>
	<li>If there is a dense amount of aerobic bacterial growth on the surface of the plate, use a sterile cell scraper to remove colonies from selected areas. Multiple C. perfringens suspected colonies can be sampled from the same TSC plate into separate RPM cultures.</li>
	<li>Tightly secure conical tube lids and wrap in paraffin film or plastic wrap. Incubate ON at 37 &#176;C.</li>
</ol>
5. DNA Extraction
<ol>
	<li>Remove ON RPM cultures and examine for signs of growth including turbidity and fermentation.</li>
	<li>Gently invert RPM culture to disperse any settled bacteria and carefully open. Transfer 1 mL of the culture to a microcentrifuge tube.</li>
	<li>Centrifuge at top speed (approximately 15,000 x g) for 10 min to pellet bacteria.</li>
	<li>Wash pellet with 1 mL of sterile PBS.
	<ol>
		<li>In some circumstances, undigested gelatin will settle on top of pelleted bacteria. To remove gelatin, &#34;fluff&#34;&#160;off gelatin by gently agitating with PBS using a micropipette. This will &#34;fluff-up&#34;&#160;the gelatin while leaving the bacterial pellet intact.</li>
		<li>Remove the PBS-gelatin mixture with gentle aspiration. Re-suspend the remaining bacterial pellet with 1 mL of fresh PBS.</li>
	</ol>
	</li>
	<li>Centrifuge the resuspended bacterial pellet at top speed for 10 min and carefully aspirate the supernatant.</li>
	<li>Immediately perform DNA extraction using a DNA extraction kit and a protocol specifically created for extraction DNA from Gram-positive bacteria (see the Table of Materials).</li>
	<li>Use the extracted DNA immediately or store at -20 &#176;C until PCR analysis.</li>
</ol>
6. Detection of C. perfringens via PCR genotyping
<ol>
	<li>To determine if cultures are positive for C. perfringens toxinotypes, examine extracted DNA for different toxin genes via PCR. 
	NOTE: Because we are most interested in the epsilon toxin producing strains type B and D C. perfringens, we evaluate for the presence of the alpha, beta, and epsilon toxin genes. Primers are listed in Table 2. Primers for 16s ribosomal DNA are used as a DNA extraction control. Additional primers can be used to test for toxinotypes A through G and can be found in published literature2,12,13.</li>
	<li>Use previously extracted C. perfringens DNA as a positive control. This control ensures that all components of the PCR reactions are working. We use DNA extracted from C. perfringens type B (ATCC 3626) as our positive control. Grow ATCC 3636 ON in RPM at 37 &#176;C and extract DNA using the same methods described in steps 5.1&#8211;5.7. Store extracted DNA at -20 &#176;C until use.</li>
	<li>Set PCR conditions as follows: 94.0 &#176;C for 3 min; 94.0 &#176;C for 30 s; 47.9 &#176;C for 30 s, 72.0 &#176;C for 1 min (Repeat steps 2&#8211;4 for 35 cycles); 72.0 &#176;C for 10 min.</li>
	<li>To confirm PCR products, run PCR reactions on a 1.8 g/100 mL agarose gel using standard techniques. Expected PCR product sizes are listed in Table 2. Typical PCR results are displayed in Figure 3.</li>
</ol>

<ol>
	<li>Petit, L., Gibert, M., Popoff, 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=Clostridium+perfringens:+toxinotype+and+genotype.">Clostridium perfringens: toxinotype and genotype.</a> Trends in Microbiology. 7, 104-110 (1999).</li><li>Rood, J. I., 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=Expansion+of+the+Clostridium+perfringens+toxin-based+typing+scheme.">Expansion of the Clostridium perfringens toxin-based typing scheme.</a> Anaerobe. , (2018).</li><li>Murrell, T. G., O'Donoghue, P. J., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+sheep-multiple+sclerosis+connection.">A review of the sheep-multiple sclerosis connection.</a> Medical Hypotheses. 19, 27-39 (1986).</li><li>Rumah, K. R., Linden, J., Fischetti, V. A., Vartanian, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Clostridium+perfringens+type+B+in+an+individual+at+first+clinical+presentation+of+multiple+sclerosis+provides+clues+for+environmental+triggers+of+the+disease.">Isolation of Clostridium perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease.</a> PLoS One. 8, 76359 (2013).</li><li>Wagley, 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=Evidence+of+Clostridium+perfringens+epsilon+toxin+associated+with+multiple+sclerosis.">Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis.</a> Multiple Sclerosis. , (2018).</li><li>Linden, J. 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=Clostridium+perfringens+Epsilon+Toxin+Causes+Selective+Death+of+Mature+Oligodendrocytes+and+Central+Nervous+System+Demyelination.">Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination.</a> MBio. 6, 02513 (2015).</li><li>Popoff, 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=Epsilon+toxin:+a+fascinating+pore-forming+toxin.">Epsilon toxin: a fascinating pore-forming toxin.</a> FEBS Journal. 278, 4602-4615 (2011).</li><li>Lee, C. A., Labbe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Enterotoxin-+and+Epsilon-Positive+Clostridium+perfringens+Spores+in+U.S.+Retail+Spices.">Distribution of Enterotoxin- and Epsilon-Positive Clostridium perfringens Spores in U.S. Retail Spices.</a> Journal of Food Protection. 81, 394-399 (2018).</li><li>Wen, Q., McClane, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+enterotoxigenic+Clostridium+perfringens+type+A+isolates+in+American+retail+foods.">Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods.</a> Applied and Environmental Microbiology. 70, 2685-2691 (2004).</li><li>Cooper, K. K., Bueschel, D. M., Songer, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+Clostridium+perfringens+in+retail+chicken+livers.">Presence of Clostridium perfringens in retail chicken livers.</a> Anaerobe. 21, 67-68 (2013).</li><li>Strong, D. H., Canada, J. C., Griffiths, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Clostridium+perfringens+in+American+foods.">Incidence of Clostridium perfringens in American foods.</a> Applied and Environmental Microbiology. 11, 42-44 (1963).</li><li>Buogo, C., Capaul, S., Hani, H., Frey, J., Nicolet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+Clostridium+perfringens+type+C+enteritis+in+pigs+using+a+DNA+amplification+technique+(PCR).">Diagnosis of Clostridium perfringens type C enteritis in pigs using a DNA amplification technique (PCR).</a> Journal of Veterinary Medicine, Series B. 42, 51-58 (1995).</li><li>Yamagishi, T., Sugitani, K., Tanishima, K., Nakamura, S. <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+test+for+differentiation+of+five+toxin+types+of+Clostridium+perfringens.">Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens.</a> Microbiology and Immunology. 41, 295-299 (1997).</li><li>Johansson, A., Engstrom, B. E., Frey, J., Johansson, K. E., Baverud, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+clostridium+perfringens+during+simulated+transport+and+stability+of+some+plasmid-borne+toxin+genes+under+aerobic+conditions.">Survival of clostridium perfringens during simulated transport and stability of some plasmid-borne toxin genes under aerobic conditions.</a> Acta Veterinaria Scandinavica. 46, 241-247 (2005).</li><li>Erickson, J. E., Deibel, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+medium+for+rapid+screening+and+enumeration+of+Clostridium+perfringens+in+foods.">New medium for rapid screening and enumeration of Clostridium perfringens in foods.</a> Applied and Environmental Microbiology. 36, 567-571 (1978).</li><li>Regan, S. 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=Identification+of+epsilon+toxin-producing+Clostridium+perfringens+strains+in+American+retail+food.">Identification of epsilon toxin-producing Clostridium perfringens strains in American retail food.</a> Anaerobe. 54, 124-127 (2018).</li></ol>

No disclosures.

Here we describe a method to identify C. perfringens prevalence in retail food samples with limited subculturing and without use of an anaerobic chamber system. This method uses a combination of techniques to increase identification of C. perfringens from food samples. By using a modified version of RPM media, we allow for the selective growth of C. perfringens. By sandwiching the inoculated RPM in between layers of TSC agar, we are able to identify and isolate anaerobic, sulfite-reducing bacteria characteristic of C. perfringens. To confirm the presence of C. perfringens, sulfite-reducing colonies are sub-cultured into fresh RPM. The modified version or RPM allows us to easily extract DNA from cultures, enabling PCR confirmation of specific toxin genes. Confirmation of C. perfringens contaminated food samples can be achieved within three days.
In early experiments, food samples were simply inoculated into RPM and DNA extracted from ON cultures. This method resulted in detection of C. perfringens in a limited number of samples (data not shown). Although RPM is selective for C. perfringens growth, it is not exclusive for C. perfringens growth. Other, gram-positive, D-cycolserine resistant bacteria can still grow in RPM. We hypothesized that contamination by other bacterial species may have decreased our detection of C. perfringens strains by decreasing the sensitivity of our PCR analysis in our first ON RPM culture. A critical step in increasing the detection of C. perfringens was the inclusion of the TSC agar &#8220;sandwich&#8221; technique. This allowed us to differentiate and select for anaerobic, sulfite-reducing colonies, characteristic of C. perfringens. A key step in this process is ensuring that the top layer of molten TSC agar is at 40 &#176;C. Although some C. perfringens strains can grow at increased temperatures (46&#8211;48 &#176;C)15,16, addition of molten agar at increased temperatures greatly reduces the amount of cultures recovered, mostly likely due to cell death.
There are several potential limitations to this method. As mentioned previously, neither the RPM nor TSC agar selects or differentiates for C. perfringens exclusively, allowing for growth of other bacterial species present in food samples. This may reduce the sensitivity of the assay to select for C. perfringens only. However, this is a common limitation in almost all culturing techniques. Genotyping confirmation of purified isolates is the best method for definitively identifying C. perfringens and other bacterial species. Another limitation of this study is that we do not test the purified isolates. We purposely did this to limit the amount of subculturing, as repeated subculturing is feared to result in plasmid loss. Because we do not isolate to purity, it is possible that multiple C. perfringens toxinotypes may be present in the same sample or subculture. If researchers wish to obtain purified isolates, standard purification methods can be used on the last RPM culture described in this method; this typically requires the use of anaerobic chambers. Although originally used to isolate C. perfringens from food, this method can be used to identify and isolate C. perfringens from a multitude of sources. Specifically, one such application of this method is to test fecal samples from humans (or animals) that are suspected to be infected with C. perfringens and toxinotype the bacteria to better understand the source of infection.

Clostridium perfringens (C. perfringens) is a Gram positive, anaerobic, spore-forming, rod shaped bacterium that is found ubiquitously in the environment. This species of bacteria carries genes that encode for over 17 toxins and, historically, has been characterized into five toxinotypes (A-E) based on the presence of four different toxin genes: alpha, beta, epsilon, and iota toxin (Table 1)1. Recently, it has been suggested that this typing-scheme needs to be expanded to include types F and G, which harbor the C. perfringens enterotoxin (CPE) and NetB toxin, respectively2. However, more research is needed before this scheming system is formally accepted. While the alpha toxin gene is strictly chromosomally located, the CPE gene can be found both on the chromosome and plasmids. In comparison, the remaining toxins&#39;&#160;genes are found on various differently-sized plasmids. We are particularly interested in the prevalence of C. perfringens types B and D as these strains produce epsilon toxin, an extremely potent, pore-forming toxin, which has been suggested to play a role in triggering multiple sclerosis (MS) in humans3,4,5,6,7. How people become infected or colonized by these strains is unknown. One possible explanation is through consumption of contaminated food products. To help answer this question, we sought to determine the prevalence of different C. perfringens toxinotypes in American food samples.
The presence of C. perfringens toxinotypes in American food samples is understudied and often requires use of anaerobic container systems and numerous sub-culturing steps8,9,10,11. Although numerous sub-culturing steps are needed to obtain purified isolates, this method can lead to loss of plasmids over time12,13,14, possibly affecting the detection of plasmid-borne toxin genes including the epsilon toxin gene. We sought to develop an easy method, with fewer sub-culturing steps, to selectively culture C. perfringens without the use of anaerobic chambers, jars, or bags. Briefly, food samples are inoculated into Rapid Perfringens Media (RPM) overnight (ON), then &#34;sandwiched&#34;&#160;into TSC agar and incubated ON. Colonies suspected to be C. perfringens are then sub-cultured into RPM and incubated again ON. DNA is extracted and PCR performed to determine genotype (Figure 1). We chose to use RPM as it has been demonstrated to increase the recovery of C. perfringens strains from food samples compared to other more standard media15. In addition, RPM was successfully used to isolate an epsilon toxin producing type B strain from an MS patient4. We use a modified version of RPM instead of the original version to allow easy DNA extraction. While this method allows easy identification of toxin genes within samples, it is possible that an individual sample will contain more than one C. perfringens toxinotype. Because our method does not isolate purified strains using multiple rounds of purification, identification of multiple toxinotypes from one sample is not possible. However, standard purification techniques (typically streaking onto TSC plates or blood agar plates) can be applied at the end of our protocol to achieve purified cultures.

<table><tbody><tr><td>D-Cycloserine</td><td>Sigma-Aldrich</td><td>C6880</td><td></td></tr><tr><td>Dextrose</td><td>Sigma Life Science</td><td>D9434-250G</td><td></td></tr><tr><td>Disposable Transfer Pipets</td><td>any brand</td><td></td><td>Select one with slim tip like Thermo Scientific Disposable Transfer Pipets 137116M/EMD</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kits</td><td>Qiagen</td><td>69504</td><td>Note: numerous DNA and plasmid extractions kits were evaluated, this kit gave the most desirable results.</td></tr><tr><td>Dry Incubator</td><td>any brand</td><td></td><td></td></tr><tr><td>Fluid thioglycolate medium</td><td>Remel</td><td>R453452</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma Life Science</td><td>G1890-500G</td><td></td></tr><tr><td>Individual Primers</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Iron (II) sulfate heptahydrate</td><td>Sigma Life Science</td><td>F8633-250 G</td><td></td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich</td><td>L6876</td><td>Needed for DNA extraction, not provided in kit</td></tr><tr><td>microcentrifuge tube</td><td>any brand</td><td></td><td></td></tr><tr><td>parafilm or plastic wrap</td><td>any brand</td><td></td><td></td></tr><tr><td>Peptone from casein and other animal proteins</td><td>Sigma-Aldrich</td><td>70173-100G</td><td></td></tr><tr><td>Perfringens Agar Base (TSC + SFP)</td><td>Oxoid</td><td>CM0587</td><td>Make TSC agar according to instructions</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma-Aldrich</td><td>P2222-100G</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S-7653</td><td></td></tr><tr><td>Sterile Cell Scraper</td><td>any brand</td><td></td><td></td></tr><tr><td>Sterile cell Spreader</td><td>any brand</td><td></td><td></td></tr><tr><td>Sterile petri dishes</td><td>any brand</td><td></td><td></td></tr><tr><td>Supplies and equipment for gel electrophoresis</td><td>any brand</td><td></td><td></td></tr><tr><td>table top centrifuge</td><td>any brand</td><td></td><td></td></tr><tr><td>Taq&amp;nbsp;PCR Master Mix Kit</td><td>Qiagen</td><td>201443</td><td></td></tr><tr><td>Thermocycler for PCR reaction</td><td>any brand</td><td></td><td></td></tr><tr><td>water bath</td><td>any brand</td><td></td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>70161-100G</td><td></td></tr></tbody></table>

enrichment and detection of clostridium perfringens toxinotypes in retail food samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Using this method, 15–20% of our sampled foods test positive for C. perfringens. While most strains are positive for toxinotype A, we have successfully detected both Type B and D in food samples. In a previously published paper we tested a total of 216 food samples purchased from New York retail stores16 (Table 3). These samples included various meat samples (beef, lamb, pork and lamb), poultry samples (chicken and turkey), and seafood samples (cod, salmon, shellfish, snapper, flounder, squid, tilapia, tuna, and various other fishes). Produce and dairy samples were also tested. Of 216 samples, 34 (16%) were positive for C. perfringens. Of the 34 C. perfringens positive samples, 31 samples (91.2%) contained the alpha toxin, one sample (2.9%) contained the alpha, beta and epsilon toxin, and two samples (5.9%) contained the alpha and epsilon toxin.
Interestingly, we also discovered that C. perfringens was more prevalent as vegetative cells compared to spores. Twenty-five samples were compared for the presence of vegetative C. perfringens cells or spores. Spores were selected for by heat shocking samples at 85 °C for 15 min. Of the 25 samples tested, 16% were positive for vegetative C. perfringens strains versus 4% for spores. This indicates that it may be more cost effective to test for only vegetative cells instead of both.
<img alt="Foodborne pathogen detection process, flowchart, DNA extraction, PCR analysis, vegetative cells, spores." class="xfigimg" src="/files/ftp_upload/59931/59931fig01.jpg" data-altupdatedat="1747736610000" data-alt="Figure 1"> 
Figure 1: Overview of procedure. 
Food samples are minced and diluted into sterile PBS. Half of the PBS-food sample is inoculated into RPM to select for vegetative cells. The remaining PBS-food sample is heat shocked at 85 °C for 15 min to select for spores prior to inoculation in RPM. Cultures are incubated ON at 37 °C then plated into TSC agar. TSC agar is incubated ON at 37 °C and black, sulfite-reducing cultures are sub-cultured into fresh RPM. Sub-cultured RPM cultures are incubated ON at 37 °C and DNA is extracted to perform genotyping via PCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59931/59931fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Layered chromatography diagram, showing TSC and RPM layers for separation experiment." class="xfigimg" src="/files/ftp_upload/59931/59931fig02.jpg" data-altupdatedat="1747736610000" data-alt="Figure 2"> 
Figure 2: Schematic of TSC agar sandwich technique. 
RPM media containing the C. perfringens bacteria are plated between two layers of TSC agar in order to promote an anaerobic environment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59931/59931fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Agarose gel electrophoresis diagram; DNA bands; species identification; 750bp to 250bp markers." class="xfigimg" src="/files/ftp_upload/59931/59931fig03.jpg" data-altupdatedat="1747736610000" data-alt="Figure 3"> 
Figure 3: Selected PCR results. 
Example images of the genotyping results of C. perfringens from seven different types of food, and a positive control of C. perfringens type B. A molecular weight ladder (first lane of each gel) was used to approximate the size of PCR results in base pairs (bp). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59931/59931fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Toxinotype</td>
			<td>Alpha</td>
			<td>Beta</td>
			<td>Epsilon</td>
			<td>Iota</td>
			<td>CPE</td>
			<td>NetB</td>
		</tr>
		<tr>
			<td rowspan="5">Established</td>
			<td>A</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>B</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>C</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td>D</td>
			<td>+</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td>E</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td rowspan="2">Proposed</td>
			<td>F</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td>G</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
		</tr>
		<tr>
			<td colspan="8"></td>
		</tr>
		<tr>
			<td colspan="8">+ present 
			- not present 
			+/- may or may not be present</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of C. perfringens genotypes. A chart of the combinations of toxins produced by each C. perfringens toxinotype.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Target</td>
			<td>Primer Pairs</td>
			<td>Expected PCR Product (bp)</td>
		</tr>
		<tr>
			<td>alpha</td>
			<td>F: GCT AAT GTT ACT GCC GTT GA 
			R: CCT CTG ATA CAT CGT GTA AG</td>
			<td>325</td>
		</tr>
		<tr>
			<td>beta</td>
			<td>F: GCG AAT ATG CTG AAT CAT CTA 
			R: GCA GGA ACA TTA GTA TAT CTT C</td>
			<td>196</td>
		</tr>
		<tr>
			<td>epsilon</td>
			<td>F: GCG GTG ATA TCC ATC TAT TC 
			R: CCA CTT ACT TGT CCT ACT AAC</td>
			<td>655</td>
		</tr>
		<tr>
			<td>16s RNA</td>
			<td>F: AGA GTT TGA TCC TGG CTC A 
			R: GGT TAC CTT GTT ACG ACT T</td>
			<td>~1300</td>
		</tr>
	</tbody>
</table>
Table 2: Primers and expected PCR products for selected toxin genes. Primers used in the PCR genotyping step.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3" rowspan="2">Food Type</td>
			<td colspan="2">Type A</td>
			<td colspan="2">Type B</td>
			<td colspan="2">Type C</td>
			<td colspan="2">Type D</td>
			<td colspan="2">C. perf +</td>
		</tr>
		<tr>
			<td colspan="2">alpha toxin positive</td>
			<td colspan="2">alpha, beta, and epsilon toxin positive</td>
			<td colspan="2">alpha and beta toxin positive</td>
			<td colspan="2">alpha and epsilon toxin positive</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>n</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
		</tr>
		<tr>
			<td rowspan="5">Meat</td>
			<td>beef</td>
			<td>38</td>
			<td>8</td>
			<td>21%</td>
			<td>1</td>
			<td>3%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>9</td>
			<td>24%</td>
		</tr>
		<tr>
			<td>lamb</td>
			<td>10</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>pork</td>
			<td>15</td>
			<td>2</td>
			<td>13%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>13%</td>
		</tr>
		<tr>
			<td>mixed</td>
			<td>1</td>
			<td>1</td>
			<td>100%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>64</td>
			<td>11</td>
			<td>17%</td>
			<td>1</td>
			<td>2%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>12</td>
			<td>19%</td>
		</tr>
		<tr>
			<td rowspan="3">Poultry</td>
			<td>chicken</td>
			<td>19</td>
			<td>5</td>
			<td>26%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>5</td>
			<td>26%</td>
		</tr>
		<tr>
			<td>turkey</td>
			<td>7</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>26</td>
			<td>5</td>
			<td>19%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>5</td>
			<td>19%</td>
		</tr>
		<tr>
			<td rowspan="11">Seafood</td>
			<td>cod</td>
			<td>4</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>mixed</td>
			<td>1</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>salmon</td>
			<td>11</td>
			<td>2</td>
			<td>18%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>18%</td>
		</tr>
		<tr>
			<td>shelfish</td>
			<td>32</td>
			<td>1</td>
			<td>3%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>snapper</td>
			<td>4</td>
			<td>3</td>
			<td>75%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>3</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>flounder</td>
			<td>12</td>
			<td>4</td>
			<td>33%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>4</td>
			<td>33%</td>
		</tr>
		<tr>
			<td>squid</td>
			<td>4</td>
			<td>1</td>
			<td>25%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>tilapia</td>
			<td>21</td>
			<td>2</td>
			<td>10%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>10%</td>
			<td>4</td>
			<td>19%</td>
		</tr>
		<tr>
			<td>tuna</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>other</td>
			<td>8</td>
			<td>1</td>
			<td>13%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>13%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>100</td>
			<td>14</td>
			<td>14%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>2%</td>
			<td>16</td>
			<td>16%</td>
		</tr>
		<tr>
			<td rowspan="4">Dairy</td>
			<td>cow</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>goat</td>
			<td>4</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>milk</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>10</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td rowspan="4">Produce</td>
			<td>vegetable</td>
			<td>12</td>
			<td>1</td>
			<td>8%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>8%</td>
		</tr>
		<tr>
			<td>fruit</td>
			<td>1</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>herb</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>16</td>
			<td>1</td>
			<td>6%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>6%</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td>216</td>
			<td>31</td>
			<td>14%</td>
			<td>1</td>
			<td>0.50%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>0.90%</td>
			<td>34</td>
			<td>16%</td>
		</tr>
	</tbody>
</table>
Table 3: Prevalence of different C. perfringens toxinotypes in 216 food samples. An example of the results obtained when using this method to test retail food for C. perfringens. This table has been modified from the previously published manuscript Regan et al.16.

Watch this Scientific Journal Video about Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples at JoVE.com

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...

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19.8K Views.  Weill Cornell Medical College. We hebben een eenvoudige methode ontwikkeld om voedselmonsters te testen op de verschillende C-toxineotypen. We zijn vooral geïnteresseerd in de prevalentie van toxineotypen B en D die het epsilontoxine produceren. Ons protocol maakt eenvoudige verrijking en detectie van de verschillende C perfringens toxinotypen zonder het gebruik van anaerobe kamers of incubators met beperkte sub-culturing.Deze methode kan ook worden gebruikt om bodem, water, fecale monsters en meer te testen. Het a...

Video: Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Immunology and Infection

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.

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 ...

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Bioengineering

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

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