These studies were funded by TECHLAB, Inc.

1. Enumeration of Campylobacter in Contrived Human Fecal Specimens
NOTE: All steps are carried out using sterile technique and materials on a disposable protective sheet within a disinfected laminar flow safety hood.
CAUTION: Live Campylobacter are infectious and can cause illness, including diarrhea. Wear gloves, a lab coat and safety glasses whenever handling bacteria. Do not mouth pipet. Dispose of all material that has contacted bacteria in proper biohazard containers.
<ol>
	<li>Growth of stock culture of bacteria
	<ol>
		<li>Obtain strains of C. jejuni (ATCC-33560) or C. coli (ATCC 33559) (Table of Materials) as dried or frozen cultures and rehydrate or thaw bacteria according to manufacturer&#39;s instructions. Streak the rehydrated bacteria onto a Campylobacter-specific agar plate to start the culture. Incubate the plate 48 h at 37 &#176;C in an anaerobic jar containing a microaerobic atmosphere gas-generating sachet.</li>
		<li>On the following day, prepare 100 mL of brain-heart infusion (BHI) growth broth containing 0.5% trypticase, 0.5% protease peptone, 0.0125% sodium pyruvate, and 0.0125% sodium bisulfite.</li>
		<li>Prereduce the BHI broth by covering the flask loosely and placing it in an anaerobic jar with a sachet that will produce a microaerophilic environment. Allow broth to prereduce overnight at 37 &#176;C. Similarly, prereduce Campylobacter-specific plates to be used for colony counts in steps 1.1.10 and 1.2.2.</li>
		<li>As Campylobacter are sensitive to air, gather all materials before inoculating broth and do not dawdle while handling cultures. When ready to inoculate, add fetal bovine serum (FBS) to prereduced broth to 4% of total volume. Retain 1 mL of prereduced broth to serve as a blank in measurements of optical density at 600 nm (OD600).</li>
		<li>Remove 3 mL of prereduced broth containing FBS and use broth to scrape the starter plate containing the Campylobacter culture. Gently scrape the plate with an inoculating loop, and then transfer the bacterial slurry to a sterile tube.</li>
		<li>Inoculate the 100 mL of prereduced broth with approximately 3 mL of bacterial slurry and incubate with moderate shaking at 115 rpm at 37 &#176;C in an anaerobic jar containing a gas-generating sachet.</li>
		<li>Monitor growth of the bacteria spectrophotometrically by turbidity at OD600. Use the reserved broth as a blank. If the anaerobic jar is opened, replace the gas-generating sachet.</li>
		<li>Stop broth incubation after 48&#8722;72 h or before the OD600 value reaches ~0.4. 
		NOTE: This OD600 typically equates to 107 to 108 CFU/mL. See Table 1 for typical results.</li>
		<li>To establish number of bacteria in pure stock culture, perform eight 10-fold dilution series of 100 &#181;L of broth in 900 &#181;L of dilution buffer (Table of Materials). After the 100 &#181;L of broth has been removed for the first dilution, return flask to anaerobic jar with fresh gas-generating sachet to await use in spiking fecal pool.</li>
		<li>Use sterile plating beads to spread 100 &#181;L of the 10-5 to 10-7 dilutions on duplicate prereduced Campylobacter-specific plates from step 1.1.3. Label plates with dilution used, place them in a second anaerobic jar with gas-generating sachet, and incubate at 37 &#176;C for 48&#8722;72 h. 
		NOTE: See Figure 1 and Figure 2 for dilution scheme and photographs of colonies.</li>
		<li>After growth, choose the plate with between 30&#8722;300 colonies to count. Utilize the counts to determine the CFU/mL of the stock broth culture using Equation 1: 
		 
		CFU/mL in stock = Average # of colonies on chosen (duplicate) analytical plates &#247; (mL plated x dilution of plate) [Equation 1] </li>
	</ol>
	</li>
	<li>Preparation and enumeration of contrived clinical fecal specimens
	<ol>
		<li>Immediately after the plates for analytical counts are prepared in step 1.1.10, make a second set of stock broth dilutions by preparing 10 serial 2-fold dilutions from the stock broth and a Campylobacter-negative fecal pool (NFP). For example, prepare the first dilution by mixing equal volumes of broth and NFP (e.g., 0.1 mL each) and make subsequent dilutions by transferring a designated volume of broth and NFP mixture into a tube with an equal designated volume NFP. Add a control plate with broth containing no Campylobacter added to the fecal pool to help identify non-Campylobacter colonies.
		<ol>
			<li>Make the NFP from de-identified, diarrheal patient surveillance specimens or healthy donor stools that have previously been tested and found to be Campylobacter-negative by methods such as a Campylobacter enzyme immunoassay and by 16S rRNA qPCR.</li>
		</ol>
		</li>
		<li>T-streak 10 &#181;L of each Campylobacter/stool dilution on duplicate prereduced Campylobacter-specific agar plates. Place plates in the anaerobic jar with a gas-generating sachet and incubate at 37 &#176;C for 48 &#177; 2 h.</li>
		<li>Examine the streaked plates visually for colonies resembling those from pure Campylobacter cultures. 
		NOTE: The third quadrant is typically where these will be found. See Figure 1 and Figure 2 for dilution scheme and images of colony size, color and morphology.</li>
		<li>Select multiple Campylobacter-like colonies and Gram stain. Using microscopy with an oil immersion lens, examine a thinly streaked area for gram-negative curved, spiral, or cigar-shaped small bacteria. 
		NOTE: Campylobacter are Gram-negative and require basic fuchsin as a counterstain (instead of the typical safranin) to be visualized accurately. Classic gull-winged bacteria may be seen but are not a requirement. See Figure 2 for representative micrograph.</li>
		<li>If either of the duplicate plates at a specific dilution has 1 or more Campylobacter colonies present, consider that dilution fecal-culture positive.</li>
		<li>Consider the last dilution that contains a visible Campylobacter-like gram-negative colony the limit of culture detection. Use Equation 2 to calculate the CFU/mL of the positive dilution in contrived clinical fecal specimen: 
		 
		CFU/mL in fecal sample = Analytical CFU/mL &#247; Dilution with last positive colony [Equation 2] 
		 
		NOTE: See Table 2 for typical results.</li>
	</ol>
	</li>
</ol>
2. Viability Determination of Campylobacter Stored in Transport Media
<ol>
	<li>Mix 1 mL of Campylobacter broth culture (step 1.1.8) with 1 mL of NFP and prepare 10 duplicate two-fold serial dilutions in NFP. Further dilute each dilution an additional 1:4 in Cary-Blair media, just as a clinical specimen prepared in transport media is treated.</li>
	<li>Store the 20 dilution tubes and a negative control in Cary-Blair medium in capped tubes at 2&#8722;8 &#176;C for 96 h and count colonies from each dilution occurring at time zero and every 24 h. For colony counting, sample the broth:fecal tubes and setup fecal culture for colony counting of each dilution, in duplicate.</li>
	<li>Each day plate 10 &#181;L portions of the fecal dilutions on Campylobacter-selective agar and incubate at 37 &#176;C for 48 h, as described above in steps 1.1.9&#8722;1.2.7.</li>
	<li>Perform a simultaneous analytical plate count of the original bacterial stock (from step 1.1.8 or a freshly grown broth stock) as described above (steps 1.1.9&#8722;1.1.11). Calculate the CFU/mL of the original bacterial stock (Equation 1) to calculate the concentration of bacteria in the transport media fecal sample and its dilutions (Equation 2).</li>
</ol>
3. Non-culture Assays for Verifying Culture Results
<ol>
	<li>Use an enzyme immunoassay (EIA) that gives minimal false positive results12 and perform according to package insert instructions to verify culture results.</li>
	<li>Use a molecular assay that can detect the 16S rRNA gene or other gene of a broad range of Campylobacter species13. Confirm that the molecular assay reacts with species such as C. upsaliensis or C. lari that grow poorly on standard antibiotic-containing agar14. Follow manufacturer&#39;s instructions for extraction of DNA from fecal samples and performing the test. 
	NOTE: Bidirectional DNA sequencing of the 16S amplicon can be used to confirm the species of Campylobacter in a positive specimen. Species-specific PCR (see Table 3 for target genes) can also be used to identify species present in discrepant or positive specimens15.</li>
</ol>

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	<li>. Annual Summaries of Foodborne Outbreaks Available from: <a target="_blank" href="https://www.cdc.gov/fdoss/annual-reports/index.html">https://www.cdc.gov/fdoss/annual-reports/index.html</a> (2018)</li><li>Kaakoush, N. O., Casta&#241;o-Rodr&#237;guez, N., Mitchell, H. M., Man, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Epidemiology+of+Campylobacter+Infection.">Global Epidemiology of Campylobacter Infection.</a> Clinical Microbiology Reviews. 28 (3), 687-720 (2015).</li><li>Pitkanen, T. a., H, M. L., Rose, J. B., Jimenez-Cisneros, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Global Water Pathogen Project. , (2017).</li><li>Fitzgerald, C., 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=Multicenter+Evaluation+of+Clinical+Diagnostic+Methods+for+Detection+and+Isolation+of+Campylobacter+spp.+from+Stool.">Multicenter Evaluation of Clinical Diagnostic Methods for Detection and Isolation of Campylobacter spp. from Stool.</a> Journal of Clinical Microbiology. 54 (5), 1209-1215 (2016).</li><li>Kirkpatrick, B. D., Tribble, D. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+upsaliensis:+Another+Pathogen+for+Consideration+in+the+United+States.">Campylobacter upsaliensis: Another Pathogen for Consideration in the United States.</a> Clinical Infectious Diseases. 34 (11), 59-60 (2002).</li><li>Bullman, S., O'Leary, J., Corcoran, D., Sleator, R., Lucey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular-based+detection+of+non-culturable+and+emerging+campylobacteria+in+patients+preseting+with+gastroenteritis.">Molecular-based detection of non-culturable and emerging campylobacteria in patients preseting with gastroenteritis.</a> Epidemiology and Infection. 140, 684-688 (2012).</li><li>Giltner, C. L., Saeki, S., Bobenchik, A. M., Humphries, R. M. <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+Campylobacter+Antigen+by+Enzyme+Immunoassay+Leads+to+Increased+Positivity+Rates.">Rapid Detection of Campylobacter Antigen by Enzyme Immunoassay Leads to Increased Positivity Rates.</a> Journal of Clinical Microbiology. 51 (2), 618-620 (2013).</li><li>M'ikanatha, N. 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=Culturing+stool+specimens+for+Campylobacter+spp.,+Pennsylvania,+USA.">Culturing stool specimens for Campylobacter spp., Pennsylvania, USA.</a> Emerging Infectious Disease. 18, 484-487 (2012).</li><li>Al Amri, A., Senok, A. C., Ismaeel, A. Y., Al-Mahmeed, A. E., Botta, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+PCR+for+direct+identification+of+Campylobacter+spp.+in+human+and+chicken+stools.">Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools.</a> Journal of Medical Microbiology. 56 (10), 1350-1355 (2007).</li><li>Buss, J. E., 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=Campylobacter+culture+fails+to+correctly+detect+Campylobacter+in+30%+of+positive+patient+stool+specimens+compared+to+non-cultural+methods.">Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 38, 1087-1093 (2019).</li><li>Wohlwend, N., Tiermann, S., Risch, L., Risch, M., Bodmer, T. <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+a+Multiplex+Real-Time+PCR+Assay+for+Detecting+Major+Bacterial+Enteric+Pathogens+in+Fecal+Specimens:+Intestinal+Inflammation+and+Bacterial+Load+Are+Correlated+in+Campylobacter+Infections.">Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections.</a> Journal of Clinical Microbiology. 54 (9), 2262-2266 (2016).</li><li>Couturier, B. A., Hale, D. C., Couturier, 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=Association+of+Campylobacter+upsaliensis+with+Persistent+Bloody+Diarrhea.">Association of Campylobacter upsaliensis with Persistent Bloody Diarrhea.</a> Journal of Clinical Microbiology. 50 (11), 3792-3794 (2012).</li><li>Chaban, B., Musil, K. M., Himsworth, C. G., Hill, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cpn60-Based+Real-Time+Quantitative+PCR+Assays+for+the+Detection+of+14+Campylobacter+Species+and+Application+to+Screening+of+Canine+Fecal+Samples.">Development of cpn60-Based Real-Time Quantitative PCR Assays for the Detection of 14 Campylobacter Species and Application to Screening of Canine Fecal Samples.</a> Applied and Environmental Microbiology. 75, 3055-3061 (2009).</li><li>Allos, B., Blaser, M. J., Mandell, G. L., Bennett, J. E., Dolin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, 7th ed. , 2793-2802 (2009).</li><li>Anderson, N. W., Buchan, B. W., Ledeboer, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+BD+MAX+Enteric+Bacterial+Panel+to+Routine+Culture+Methods+for+Detection+of+Campylobacter,+Enterohemorrhagic+Escherichia+coli+(O157),+Salmonella,+and+Shigella+Isolates+in+Preserved+Stool+Specimens.">Comparison of the BD MAX Enteric Bacterial Panel to Routine Culture Methods for Detection of Campylobacter, Enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella Isolates in Preserved Stool Specimens.</a> Journal of Clinical Microbiology. 52 (4), 1222-1224 (2014).</li><li>Wasfy, M., Oyofo, B., Elgindy, A., Churilla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+preservation+media+for+storage+of+stool+samples.">Comparison of preservation media for storage of stool samples.</a> Journal of Clinical Microbiology. 33 (8), 2176-2178 (1995).</li><li>Bess&#232;de, E., Delcamp, A., Sifre, E., Buissonniere, A., M&#233;graud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Methods+for+Detection+of+Campylobacters+in+Stool+Samples+in+Comparison+to+Culture.">New Methods for Detection of Campylobacters in Stool Samples in Comparison to Culture.</a> Journal of Clinical Microbiology. 49 (3), 941-944 (2011).</li><li>Bess&#232;de, E., 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=Evaluation+of+the+Diagnostic+Accuracy+of+Two+Immunochromatographic+Tests+Detecting+Campylobacter+in+Stools+and+Their+Role+in+Campylobacter+Infection+Diagnosis.">Evaluation of the Diagnostic Accuracy of Two Immunochromatographic Tests Detecting Campylobacter in Stools and Their Role in Campylobacter Infection Diagnosis.</a> Journal of Clinical Microbiology. 56 (4), (2018).</li><li>Shane, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+Diseases+Society+of+America+Clinical+Practice+Guidelines+for+the+Diagnosis+and+Management+of+Infectious+Diarrhea.">Infectious Diseases Society of America Clinical Practice Guidelines for the Diagnosis and Management of Infectious Diarrhea.</a> Clinical Infectious Diseases. 65 (12), 1963-1973 (2017).</li><li>Toledo, Z., Simaluiza, R. 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The authors are employees of TECHLAB, Inc. that produces the QUIK CHEK&#8482; kit used as a comparator in this article.

The culture methods described here are built on simple, widely used techniques and materials available in most laboratories10. It is the combination of analytical and contrived samples that provide new information of a clinically relevant detection threshold for fecal cultures. Additionally, the adjudication of culture results with 5 separate assays strengthens the conclusions that Campylobacter fecal culture mis-identifies a significant portion of patient specimens. The EIA and molecular assays are useful as controls because they are each based on a different principle (antigen interaction with antibody vs. DNA amplification) and, importantly, do not rely on viability of bacteria. Note that the EIA assay used for these studies is well-validated and has been shown to agree fully with 4 molecular tests12.
Culture of Campylobacter spp. is particularly troublesome, with sensitivity reported to range from 60&#8722;76%19,20, and as evident from its ~30% rate of failure to detect true-positive specimens here. Personnel can expect that control EIA and molecular tests will frequently produce positive results when culture data are negative.
The most critical step in the protocol is the identification of pin-point colonies among competing fecal flora. It is not unusual, as dilutions near the detection threshold, to have alternating zero and non-zero colony count estimates (e.g., 2, 0, 1, 0, 0). It is important to recognize that culture thresholds will be a range of concentrations, not a specific CFU/mL. Nevertheless, the estimate of ~1 x 106 CFU/mL feces as a lower limit for culture detection compares well with reports that infected humans shed 106 to 109 Campylobacter per gram feces21. Changes in antibiotics or agar plates and variations inevitable in individual fecal specimens will undoubtedly change threshold values. This protocol should enable improvements in growth media.
This first information on a limit for culture detection makes it possible to set clinically relevant thresholds for diagnostic tests, and lays the microbiological foundation which is needed to address unstudied issues of non-symptomatic carriage22,23 by Campylobacter, or if bacterial load correlates with symptoms or serious sequelae.

The United States Centers for Disease Control (CDC) recently published that the Foodborne Diseases Active Surveillance Network (FoodNet) surveillance program reported 9,723 cases of laboratory-diagnosed Campylobacter infections in 20181. This represents a 12% increase in Campylobacter case reports over 2015&#8722;20171. Worldwide, Campylobacter spp. are among the most common bacterial intestinal infections2. Nevertheless, the numbers of Campylobacter-based intestinal illnesses that occur each year are suspected to be underreported3. This underestimation is predictable because most patients can recover with only moderate discomfort and no medical treatment. However, for patients with more severe symptoms or who are at higher risk for serious disease, and who then seek medical care, stool culture is the most common method for assessing whether Campylobacter is the pathogen that is causing their distress4.
For Campylobacter spp., stool culture is particularly troublesome. The most common pathogenic organisms, C. jejuni, C. coli, C. upsaliensis, and C. lari, are microaerophilic5. This means that the bacteria will die at random, unknown rates once exposed to air. The time between specimen collection and culture setup thus becomes an uncontrolled variable in the ability to detect viable Campylobacter spp. by culture.
For direct culture of fecal specimens, the slow growth of Campylobacter is also a problem. Campylobacter colonies are very small even after 48 h of incubation and can easily be covered by competing organisms in the fecal matrix. Plates that contain antibiotics to which most strains of C. jejuni and C. coli are resistant are widely used, as the antibiotics inhibit growth of many (but not all) competing fecal bacteria, allowing better visualization of Campylobacter colonies6. However, other Campylobacter species such as C. lari and C. upsaliensis are sensitive to some of these antibiotics, and either grow poorly or not at all. This contributes to the underreporting of Campylobacter infections from these antibiotic-sensitive species7.
There is a third reason why a culture for Campylobacter may be inaccurate. The bacteria, when stressed, may remain viable but can become &#34;non-culturable&#34;8. This by definition means that the culture will not detect the bacteria present in the sample. How often this occurs is not known8.
Given these potential issues with culture, we used multiple comparison reference methods so that faulty culture results did not make a single comparator assay appear inaccurate9. The culture methods used (e.g., Campylobacter-selective plates, transport medium, gas-generating sachets) were chosen because they are used widely in clinical laboratories for stool specimen culture10.
The culture protocols described here were developed because the lowest number of Campylobacter jejuni that could be detected by culture in human stool was not known. Although estimates have been published for numbers of colony forming units (CFU) present in poultry feces11, these results cannot be equated to human stool, as Campylobacter spp. are commensals in chickens, and do not cause diarrhea. This fundamental information is needed to establish the numbers of Campylobacter bacteria that will produce symptoms of gastroenteritis in humans and to compare virulence between strains or species.

<table><tbody><tr><td>Anaerobic 3.5L Jar</td><td>Thermo Fisher</td><td>HP0031A</td><td></td></tr><tr><td>AnaeroGRO Campylobacter Selective Agar</td><td>Hardy Diagnostics</td><td>AG701</td><td></td></tr><tr><td>Bacto Brain Heart Infusion</td><td>BD Biosciences</td><td>237500</td><td></td></tr><tr><td>Bacto Protease Peptone</td><td>Life Technologies Corp</td><td>211684</td><td></td></tr><tr><td>Basic Fuchsin</td><td>Fisher Scientific</td><td>B12544</td><td></td></tr><tr><td>BBL Trypticase Peptone</td><td>Life Technologies Corp</td><td>211921</td><td></td></tr><tr><td>C. coli Type strain</td><td>ATCC</td><td>33559</td><td></td></tr><tr><td>C. jejuni Type strain</td><td>ATCC</td><td>33560</td><td></td></tr><tr><td>CampyGen gas generating system sachet</td><td>Thermo Fisher</td><td>CN0025A</td><td></td></tr><tr><td>Campylobacter QUIK CHEK</td><td>TechLab, Inc.</td><td>T5047 / T31025</td><td></td></tr><tr><td>Cary-Blair transport medium</td><td>Fisher Scientific</td><td>23-005-47</td><td></td></tr><tr><td>Coli Roller Sterile plating beads</td><td>Millipore Sigma</td><td>71013</td><td></td></tr><tr><td>Dilution Buffer</td><td>Anaerobe Systems</td><td>AS-908</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech-Bio, Inc</td><td>SFBM30</td><td></td></tr><tr><td>Sodium bisulfite</td><td>Sigma-Aldrich</td><td>243973</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>Spectrophotometer cuvettes</td><td>USA Scientific</td><td>9090-0460</td><td></td></tr></tbody></table>

culture methods to determine the limit of detection and survival in transport media of campylobacter jejuni in human fecal specimens

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

Identifying Campylobacter spp. colonies among competing fecal flora requires keen eyesight and considerable judgement. The lowest number of colonies that can be detected by culture has not been studied, although specimens from patients have been estimated to harbor 106&#8722;109 CFU/mL16,17. However, patient samples cannot be used quantitatively as there is no independent method to establish accurate bacterial numbers. To overcome this limitation, two simultaneous measurements are made with one bacterial stock. One test is used for visual detection of Campylobacter colonies from serial dilutions of the stock bacteria in a fecal matrix, simulating clinical specimens; the other is used analytically to quantify the CFU/mL present in the bacterial stock culture used for spiking (Figure 2A).
The detection thresholds for Campylobacter will not be defined values. This is to be expected because each fecal matrix is complex and unique, and growth of bacteria is variable. A key parameter for success is identifying the pinpoint size colonies amongst the competing fecal flora. A representative plate of spiked stool culture is shown in Figure 2C and Figure 2D. The negative control plate without added Campylobacter is important to help identify other fecal flora. Gram staining many candidates also trains the eye to distinguish the correct glossy colonies and the intermediate pink color of fuchsin-stained gram-negative bacteria and confirms the morphology of the bacteria in the selected colonies (Figure 2B). Seven independent experiments were performed, using 5 C. jejuni and 2 C. coli broths, and gave thresholds that overlapped and spanned from 0.3&#8722;5 x 106 CFU/mL. See Table 2 for typical data. The detection limits averaged 2 x 106 for C. jejuni and 1.2 x 106 CFU/mL for C. coli. This indicates that culture can likely detect ~1&#8722;2 x 106 C. jejuni or C. coli per gram of fecal specimen on standard antibiotic-containing Campylobacter-specific agar used by many clinical laboratories. There are multiple specialized agars with different antibiotics that may give different thresholds for colony detection. The methods described here should encourage more quantitative and comparative studies to improve the accuracy of culture and broaden the versatility of new media. For example, 152 colonies were counted on the first 10-5 plate and 144 colonies on the second 10-5 plate. The average between the two plates is 148 colonies. The plates were inoculated with 0.1 mL (100 &#181;L) of 10-5 dilution, which by Equation 1 equates to 148 x 106 (14.8 x 107) CFU/mL in the pure culture stock. When the fecal dilutions were made, the culture was spiked into negative fecal pool at a 1:1 ratio. Therefore, by Equation 2, the first point (plate &#34;a&#34;) on the fecal curve corresponds to 14.8 x 107 divided by 2 and equals 7.4 x 107 CFU/mL. This &#34;a&#34; tube is used to make 9 additional dilutions. In Figure 1, the last dilution with one visible gram-negative colony with Campylobacter-like morphology is on plate &#34;g&#34;. This equates to 1.1 x 106 CFU/mL for the fecal culture threshold of detection in this example.
Even though sustained viability is key to culture&#39;s accuracy, retention of viability of Campylobacter spp. during handling and shipment of specimens from patients to clinics to reference labs is problematic. Typical storage is to refrigerate specimens in ordinary capped tubes with air exposure and with no special atmosphere. Specimens in transport media (also known as preserved samples) are thought to have better survival, but there are few reports that provide quantitative data18.
The combination of analytical and contrived sample methods shown above was used again to obtain viability and survival time estimates of C. jejuni in transport media. A bacterial stock broth was used to prepare ten duplicate 2-fold to 1024-fold sample dilutions in fecal matrix. The initial broth was found by the analytical counts to have a concentration of 4.8 x 107 CFU/mL. On plates made on day 0, C. jejuni was detected (2 days later) on the plate streaked with the 32-fold dilution, equivalent to 1.5 x 106 CFU/mL. However, on the plates made after refrigerating the Cary Blair fecal sample for 24 hours, only the 2-fold dilution (equivalent to 2.4 x 107 CFU/mL) grew visible colonies. No further loss of viability was seen out to 96 hours, when the study was stopped. This loss of viability equates to a 16-fold (94%) loss of culturable organisms in less than 24 hours and indicates that, even with refrigeration, stool in Cary Blair medium with less than 107 CFU/mL C. jejuni may be missed by culture.
In contrast to the results of culture, the EIA detected the presence of C. jejuni at the 256-fold dilution at the initial time point and throughout the 4 day testing period. The C. jejuni detection threshold for this EIA using spiked fecal samples is 8.4 x 104 CFU/mL. This threshold is below that of fecal culture and allows more sensitive and stable detection of C. jejuni.
To test the ability of culture to detect Campylobacter spp. in an actual clinical setting, 1,552 clinical stool specimens were characterized by 6 procedures: fecal culture, a new immunoassay for Campylobacter spp., and 4 molecular methods. All samples were prospectively collected and initially classified by conventional culture at 3 laboratories in the United States, and then cross-checked by EIA. Any culture-positive or EIA/culture-discrepant specimens were then screened by the molecular methods12. Specimens were assigned a true-positive or true-negative status based on the results of the 5 non-culture methods. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60457/60457fig1a.jpg" /> 
Figure 1: Scheme for simultaneous preparation of analytical and spiked fecal samples. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60457/60457fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60457/60457fig2a.jpg" /> 
Figure 2: Identification of C. jejuni colonies from pure and fecal cultures. (A) Photograph of C. jejuni colonies from pure bacterial culture after 72 hours incubation. (B) Gram stain of C. jejuni from pure bacterial culture, oil-immersion 400x magnification. (C) Photograph of C. jejuni-positive spiked fecal culture after 48 h incubation. (D) Enlarged area in box in (C), 10x magnification. White arrows indicate pin-point size gram-negative C. jejuni colonies. The black arrowhead indicates a colony that is slightly larger, gram-positive, and not C. jejuni. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60457/60457fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Cultures</td>
			<td>OD600 @ T0</td>
			<td>OD600 @ T Final1</td>
			<td>Final CFU/mL</td>
		</tr>
		<tr>
			<td>C. jejuni</td>
			<td>0.146</td>
			<td>0.321</td>
			<td>1.28 x 107</td>
		</tr>
		<tr>
			<td>C. coli</td>
			<td>0.245</td>
			<td>0.508</td>
			<td>4.50 x 108</td>
		</tr>
	</tbody>
</table>
Table 1: Typical growth and CFU/mL of C. jejuni and C. coli stocks.1 C. jejuni culture was stopped after 48 h of incubation. C. coli culture was stopped after 54 h of incubation.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Dilution tube for spiked fecal sample</td>
			<td>Number of Campylobacter-like colonies</td>
			<td>Number of Gram-negative colonies1</td>
			<td>Culture positive?</td>
			<td>Calculated CFU/mL of spiked sample</td>
		</tr>
		<tr>
			<td rowspan="10">C. jejuni (1.28 x 108 CFU/mL stock)</td>
			<td>(2-fold) a</td>
			<td>Dense</td>
			<td>nd2</td>
			<td>yes</td>
			<td>6.40 x 107</td>
		</tr>
		<tr>
			<td>(4-fold) b</td>
			<td>20+</td>
			<td>nd</td>
			<td>yes</td>
			<td>3.20 x 107</td>
		</tr>
		<tr>
			<td>(8-fold) c</td>
			<td rowspan="3">4-10</td>
			<td>nd</td>
			<td>yes</td>
			<td>1.60 x 107</td>
		</tr>
		<tr>
			<td>(16-fold) d</td>
			<td>nd</td>
			<td>yes</td>
			<td>8.00 x 106</td>
		</tr>
		<tr>
			<td>(32-fold) e</td>
			<td>nd</td>
			<td>yes</td>
			<td>4.00 x 106</td>
		</tr>
		<tr>
			<td>(64-fold) f</td>
			<td rowspan="4">1-3</td>
			<td>1 of 2</td>
			<td>yes</td>
			<td>2.00 x 106</td>
		</tr>
		<tr>
			<td>(128-fold) g</td>
			<td>2 of 3</td>
			<td>yes</td>
			<td>1.00 x 106</td>
		</tr>
		<tr>
			<td>3(256-fold) h</td>
			<td>1 of 2</td>
			<td>yes</td>
			<td>5.00 x 105</td>
		</tr>
		<tr>
			<td>(512-fold) i</td>
			<td>0 of 1</td>
			<td>no</td>
			<td>2.50 x 105</td>
		</tr>
		<tr>
			<td>(1024-fold) j</td>
			<td>0</td>
			<td>nd</td>
			<td>no</td>
			<td>NFP</td>
		</tr>
		<tr>
			<td rowspan="10">C. coli (4.50 x 108 CFU/mL stock)</td>
			<td>(2-fold) a</td>
			<td rowspan="2">Dense</td>
			<td>nd</td>
			<td>yes</td>
			<td>2.25 x 108</td>
		</tr>
		<tr>
			<td>(4-fold) b</td>
			<td>nd</td>
			<td>yes</td>
			<td>1.13 x 108</td>
		</tr>
		<tr>
			<td>(8-fold) c</td>
			<td>50+</td>
			<td>nd</td>
			<td>yes</td>
			<td>5.63 x 107</td>
		</tr>
		<tr>
			<td>(16-fold) d</td>
			<td>30+</td>
			<td>nd</td>
			<td>yes</td>
			<td>2.81 x 107</td>
		</tr>
		<tr>
			<td>(32-fold) e</td>
			<td>10+</td>
			<td>nd</td>
			<td>yes</td>
			<td>1.41 x 107</td>
		</tr>
		<tr>
			<td>(64-fold) f</td>
			<td rowspan="2">3-8</td>
			<td>nd</td>
			<td>yes</td>
			<td>7.03 x 106</td>
		</tr>
		<tr>
			<td>(128-fold) g</td>
			<td>nd</td>
			<td>yes</td>
			<td>3.52 x 106</td>
		</tr>
		<tr>
			<td>3(256-fold) h</td>
			<td rowspan="2">1-3</td>
			<td>1 of 3</td>
			<td>yes</td>
			<td>1.76 x 106</td>
		</tr>
		<tr>
			<td>(512-fold) i</td>
			<td>0 of 1</td>
			<td>no</td>
			<td>8.79 x 105</td>
		</tr>
		<tr>
			<td>(1024-fold) j</td>
			<td>0</td>
			<td>nd</td>
			<td>no</td>
			<td>NFP</td>
		</tr>
	</tbody>
</table>
Table 2: Typical numbers of colonies on plates of spiked fecal samples.1 Gram negative colonies among Campylobacter-like colonies, 2nd = not determined, 3Data in bold type indicates last positive dilution.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Gene target</td>
		</tr>
		<tr>
			<td>C. jejuni</td>
			<td>hipO</td>
		</tr>
		<tr>
			<td>C. coli</td>
			<td>cadF</td>
		</tr>
		<tr>
			<td>C. upsaliensis</td>
			<td>cpn60</td>
		</tr>
		<tr>
			<td>C. lari</td>
			<td>cpn60</td>
		</tr>
		<tr>
			<td>C. helveticus</td>
			<td>cpn60</td>
		</tr>
		<tr>
			<td>C. fetus</td>
			<td>cpn60</td>
		</tr>
		<tr>
			<td>C. hyointestinalis</td>
			<td>cpn60</td>
		</tr>
		<tr>
			<td>C. concisus</td>
			<td>cpn60</td>
		</tr>
	</tbody>
</table>
Table 3: Genes useful for detection of individual Campylobacter species qPCR.

Watch this Scientific Journal Video about Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens at JoVE.com

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Although stool culture for Campylobacter is imprecise, it is still considered the gold standard for identification. Methods to determine the limit of detection and survival in transport media of C. jejuni in human stool are described and compared with a new immunoassay with better accuracy.

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician ...

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Immunology and Infection

11.4K Views.  TECHLAB, Inc.. Although stool culture for Campylobacter is imprecise, it is still considered the gold standard for identification. Methods to determine the limit of detection and survival in transport media of C. jejuni in human stool are described and compared with a new immunoassay with better accuracy.

Video: Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Environment

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 ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

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 a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

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 High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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. ...

Biology

Enhanced Campylobacter Isolation from Meat

Detection and Isolation of Campylobacter spp. from Raw Meat

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 &#181;m and 0.65 &#181;m pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 &#181;m pore-size filter compared to the 0.45 &#181;m pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Author Spotlight: Development of an Enhanced Protocol for Rapid and Accurate Isolation of Campylobacter from Food Products

Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Despite establishments enacting measures to reduce the prevalence throughout their facilities, contaminated products consistently reach consumers. The technique developed over the past twelve years addresses limitations of existing methods for isolating and detecting Campylobacter spp. from raw meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and ...

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Summary

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Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

Detection of Bacteria Using Fluorogenic DNAzymes

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

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

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Colorimetric Detection of Bacteria Using Litmus Test

Detection and Isolation of Campylobacter spp. from Raw Meat

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias