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Article Type: Original Article
Evaluation of Rapid Screening Techniques for Detection of Salmonella spp. from Produce Samples after Pre-enrichment According to FDA BAM and a Short Secondary Enrichment
Abbreviated Running Headline: Screening of Salmonella in Produce
K. J. Yoshitomi1, K.C. Jinneman1, P.A. Orlandi2, S.D. Weagant3, R. Zapata4, and W.M. Fedio4*
1
Food and Drug Administration, Bothell, WA 98021; 2 Food and Drug Administration,
Rockville, MD 20857; 3 Weagant Consulting, Poulsbo, WA 98370; 4 New Mexico State University, Food Safety Laboratory, Las Cruces, NM 88003
* Corresponding author Willis Fedio New Mexico State University MSC 3BF
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12422 This article is protected by copyright. All rights reserved.
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P.O. Box 30003 Las Cruces, NM 88003-8003 Phone: 575-646-7345 Fax: 575-646-7344 E-mail: [email protected]
SIGNIFICANCE AND IMPACT OF THE STUDY: In the event of a foodborne disease outbreak, rapid identification and characterization of the pathogen is essential to prevent the spread of disease and reduce the number of illnesses. This study reports the utility of an abbreviated secondary enrichment for the isolation of Salmonella in artificially contaminated fresh produce at very low levels. In addition, incorporation of rapid, easy-to-use lateral flow devices to screen enrichments can provide a low cost (equipment and highly trained personnel), high return (rapid identification of contaminated food) investment in the timely pathogen screening of fresh produce.
ABSTRACT: Conventional detection of Salmonella from foods involves enrichment and isolation on selective media which can significantly lengthen time to result. The objective of this study was to evaluate the utility of an accelerated plating procedure and the use of rapid screening devices for Salmonella detection. Fresh produce was inoculated with Salmonella at ~2.5, ~7.5 and ~25 cfu sample-1. After 24 h pre-enrichment, subcultures
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were made into TT and RV broths and further incubated at 42°C for an additional 7 and 24 h. Enrichments were streaked for isolation of Salmonella as well as tested by rapid screening methods. The seven hour accelerated plating procedure worked well from 4/6 to 6/6 in all produce samples inoculated at the lowest level. Both the RapidChek and Neogen Reveal tests worked as well as the VIDAS-SLM after 24 h secondary enrichment, but failed to detect the pathogen after 7 h selective enrichment in romaine lettuce and tomatoes, while fractional detection was observed in cilantro and jalapenos. Both devices detected Salmonella on cantaloupe at the lowest level of inoculation. An abbreviated selective enrichment procedure worked well to accelerate the isolation of colonies of Salmonella from contaminated samples providing isolates for further characterization one day earlier than standard analysis.
Keywords: Salmonella, Produce, Rapid Screening
INTRODUCTION Fresh produce is increasingly found to be involved in foodborne disease outbreaks (Sivapalasingam et al. 2004; Lynch et al. 2009; Kozak et al. 2013). Examination of outbreak associated illnesses from 1998-2008 estimated that as many as 46% of illnesses were attributed to produce (Painter et al. 2013). Salmonella has been associated with outbreaks involving alfalfa sprouts, lettuce and tomatoes as well as other produce (Anon 2009; Behravesh et al. 2012; Gajraj et al. 2012; Bennett et al. 2014) and is a pathogen of concern in these commodities. Rapid and reliable screening methods for detecting bacterial pathogens in produce samples would allow for more timely testing of food samples and potentially quicker response to foodborne outbreaks.
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Rapid screening methods for foodborne pathogens can help reduce the amount of time and labor needed for detecting pathogens. A wide range of rapid methods are available for detection of pathogenic bacteria. However, many rapid screening methods such as automated immunoassays and real-time PCR procedures require investment in expensive equipment or require trained/experienced personnel to perform the analysis. Although potentially more costly, molecular or immuno-based screening assays can offer greater sensitivity in screening food samples, in addition to being more rapid than standard cultural techniques. The use of lateral flow devices developed for detection of foodborne pathogens offers an economical method to screen food enrichments. The Reveal for Salmonella Complete System (Neogen Corporation, Lansing, MI, USA) along with RV medium provides for rapid recovery of Salmonella spp. in foods, allowing detection and presumptive identification of the target organism in 20-28 h. The enriched sample is placed into a Salmonella Reveal device with specific antibodies against Salmonella. If a line develops only in the control zone, the sample is negative for Salmonella spp. If lines are present in the control and test zones, the sample is considered positive for salmonellae (Neogen Corp. 2008). The RapidChek® Salmonella system (Romer Labs, Union, MO, USA) uses a similar mechanism to rapidly detect Salmonella. The RapidChek® lateral flow strip contains a proprietary panel of anti-Salmonella antibodies engineered to enhance the overall performance of the method. The complete test utilizes a proprietary enrichment base supplemented with phage as well as a secondary enrichment media to selectively grow Salmonella to be detected by the test strip. Upon immersion into the enrichment media and uptake via capillary action, positive results are attained in a similar manner with presence of a red line in the control and test location (Romer Labs, 2007).
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The VIDAS (Vitek Immuno Diagnostic Assay System, bioMerieux, Askim, Sweden) automated instrument uses technology that is adaptable to a wide range of assays, and combines an enzyme immunoassay method with a final fluorescence reading to obtain a test result. This technology is known as ELFA (Enzyme-Linked Fluorescent Assay) which uses alkaline phosphatase to hydrolyze a substrate to a fluorescent product which is proportional to the abundance of pathogen present in the sample, (bioMerieux 2009). The VIDAS® SLM Assay incorporates the same primary enrichment and secondary selective enrichment as described in the FDA Bacteriological Analytical Manual (BAM), followed by enrichment in M-Broth prior to inoculation of test strip and incubation on the VIDAS instrument.
This study was designed to evaluate the utility of an accelerated plating procedure and rapid screening techniques for Salmonella detection and isolation using FDA BAM ( Andrews et al. 2014) enrichment procedures. Selective plating of 7h enrichments (TT and RV) could potentially be used to obtain isolates earlier than with the traditional, cultural method, allowing quicker confirmation of presumptive isolates and further characterization. In addition, simple, easy-to-use lateral flow devices were evaluated at the 7 h and 24 h secondary enrichment time points to determine if such devices could be used in this manner. Although, it should be emphasized that these devices were used following standard BAM pre-enrichment and abbreviated secondary enrichment and not the manufacturer’s specific enrichment. Examining the applicability of such devices as used in this study could eliminate the need for parallel enrichment schemes and provide a potentially useful test that is simple, rapid and relatively low in cost.
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RESULTS AND DISCUSSION Plating of the secondary selective enrichment after 7 h, as opposed to 24 h, performed well to accelerate cultural isolation of Salmonella from contaminated produce samples as shown in Table 1 (7 h) and Table 2 (24 h) under the BAM column heading. For Romaine lettuce artificially contaminated with S. Typhimurium ATCC 14028, plating 7 h selective enrichment broths resulted in confirmed salmonellae in 4/6 low inoculum samples (2.4 cfu per 375g sample), while none of the rapid devices gave positive results. For cilantro contaminated with S. Anatum ATCC 9270, fractional recovery or detection was observed at the medium level of inoculation (5.6 sample-1) with 5/6, 3/6, and 2/6 positive for BAM, RapidChek, and Reveal TT, respectively, with all replicates positive at the higher level (15 cfu sample-1). Fractional detection (2/6) was observed with the Reveal RV treatment at these high levels and none were detected at the two lower levels of inoculation. Tomato samples were artificially inoculated with Salmonella Montevideo ATCC 8387. Similar to the Romaine lettuce samples, Salmonella was fractionally isolated from the low (5/6 at 2.8 cfu g-1) as well as medium levels (5/6 at 8.6 cfu g-1), but not detected by any of the devices after 7 h selective enrichment. All isolates were recovered by BAM plating procedures at the high inoculation level (28.4 cfu g-1). S. Heidelberg ATCC 8326 was successfully isolated from inoculated jalapeño peppers at all levels as low as 2.7 cfu sample-1. However, fractional detection by lateral flow devices was achieved at medium and lower levels with 4/6 samples positive, except for the Reveal RV treatment having 6/6 positive. Cantaloupe artificially contaminated with S. Panama was positive when screened by both RapidChek and Reveal devices and culturally recovered by direct plating after 7 h selective enrichment at all inoculum levels, as low as 1.6 cfu sample-1.
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Direct plating of the secondary enrichment after 7 h was successful in recovery for most matrix/strain combinations. In most cases, 5/6 samples or greater were positive for all levels. The response of the lateral flow devices as employed at this time point was more variable. Romaine lettuce and tomatoes resulted in no positive screening results, while cilantro and jalapenos were 88% successful across all lateral flow tests at the highest level of inoculum, with fractional detection at the lower levels. Detection of S. Panama in cantaloupes was 100% successful by devices for all treatment combinations. After 24 h enrichment, all of the BAM samples, all of the VIDAS–SLM screening tests and all of the lateral flow devices were positive for all levels of inoculum for jalapenos and cantaloupe. This was not surprising given the high detection rates after 7 h for these foods. Detection rates by devices were markedly improved for romaine lettuce, cilantro and tomatoes compared to the 7 h time point. There was no difference (p < 0.05) in the detection rates between BAM, VIDAS SLM and devices for romaine lettuce and cilantro after 24 h, showing a dramatic increase over the 7 h rates of detection. Improvement in detection rates after 24 h was also observed for tomatoes for the Reveal RV treatment with 6/6 positive as low as 2.8 cfu sample-1. However, the RapidChek® (0/6 at all levels) and Reveal TT (3/6 at highest level and 0/6 at others) detection rates remained significantly lower than BAM plating results, similar to the 7 h detection rates for the tomato sample matrix. Both devices were tested on TT broths, suggesting that perhaps Salmonella did not grow as well in TT broth from this particular matrix. Except for this result, all lateral flow devices performed to the same level of sensitivity as the BAM and VIDAS-SLM methods after 24 h in secondary enrichment. Both the RapidChek® and Neogen Reveal® tests worked as well as the VIDASSLM after 24h secondary enrichment for most strains tested, but did not reliably detect the pathogen after 7 h selective enrichment in most of the artificially contaminated
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samples. It was possible to recover Salmonella isolates from artificially contaminated fresh produce at levels ranging from 1.6 to 28.4 cfu sample-1 with a 7 h abbreviated secondary enrichment, depending upon the commodity/strain. The slightly better sensitivity observed with the direct plating after 7 h was most likely attributed to increased recovery of low numbers of Salmonella from the plating media. The reported sensitivity of lateral flow devices are approximately 107 cfu mL-1 (Seo et al. 2003), a level at which Salmonella numbers may have not been reached either through matrix effects or competition from other organisms. In the current study, we found the sensitivity of the Salmonella RapidChek® devices to range from 107-108 cfu mL-1. Although the devices were not used as recommended by the manufacturer, the utility of having the option to use such device in standard protocol can prove valuable in certain circumstances. Detection or lack thereof, by devices was not dependent upon which secondary enrichment media was tested, and both devices performed very similarly across all produce matrices at 7 h. After 24 h, the devices performed as well as the standard protocol (except for the RapidChek® and Reveal TT tomato treatment). Tomatoes did not have any positive detection values by devices at 7 h, and at 24 h only the RV enriched samples were positive by devices. Use of lateral flow devices for detection of Salmonella has shown similar sensitivity with reference methods after secondary enrichment (Hoerner et al. 2011; Torlak et al. 2012). This study suggests that the lateral flow devices have similar sensitivity as standard plating methods after 24 h. The sensitivity of the devices were variable after a shortened 7 h enrichment in secondary media, but can be of value if a screening result was necessary one day sooner. In addition, plating of the 7 h enrichment proved as sensitive as the full 24 h, suggesting isolates can be obtained for further characterization one day sooner than conventional methods. Introducing rapid, simple to use tests such as lateral flow devices at key points during standard analytical methods for Salmonella and abbreviating a secondary
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enrichment by plating sooner can potentially speed time to a confirmed result. This can be especially valuable when considering timely response to foodborne outbreaks involving fresh produce.
MATERIALS AND METHODS Microorganisms The following strains were used in the study: Salmonella Typhimurium ATCC 14028, Salmonella Anatum ATCC 9270, Salmonella Montevideo ATCC 8387, Salmonella Heidelberg ATCC 8326 and Salmonella Panama S4RB39. S. Panama S4RB39 was obtained from the Salmonella Genetic Stock Center, University of Calgary, Calgary, AB. The strains were maintained in 20% glycerol/peptone broth medium at -70°C. Preparation of Inoculum Frozen stock cultures of the microorganisms were streaked onto Tryptic Soy Agar with 0.6% Yeast Extract (TSAYE; BD, Sparks, MD, USA) plates and incubated at 37°C for 20 h. A well-separated colony from the plate was transferred to 5 mL of Brain Heart Infusion broth (BHI; BD, Sparks, MD, USA) and incubated at 37°C for 18-20 h without shaking. Ten-fold serial dilutions of the enriched sample were prepared in Butterfields Phosphate Buffer (BPB) to obtain low, medium and high inoculum levels. Inoculum Levels Produce (Romaine lettuce, cilantro, jalapeno peppers, tomatoes and cantaloupe) was artificially contaminated with the various Salmonella species at low (~2.5 cfu sample-1), medium (~7.5 cfu sample-1) and high (~25 cfu sample-1) levels. The inoculum levels were
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determined by plate counts of dilutions of the initial inoculum. Inoculum was not aged in this particular study. Uninoculated controls were also prepared with produce samples which only received BPB. Experimental parameters Six replicates at each inoculum level were tested per food matrix, as well as six uninoculated controls. Samples were pre-enriched in sterile plastic bags (12” x 18”, NASCO, Ft. Atkinson, WI, USA) using the appropriate standard enrichment broth for the product being tested. Enrichment broths used were as follows: 375 g portions of Romaine lettuce were enriched in 3375 mL of lactose broth (LB); 454 g jalapeño peppers in 1.5x volume of lactose broth; 375 g cilantro in 3375 mL tryptic soy broth (TSB); each cantaloupe (approximately 1300 g) in 1.5x volume of Universal preenrichment broth (UP) until floating, and 454 g tomatoes in 1.0x volume of UP. Enrichment broths were incubated at 35°C. After 24 h, Enrichments were subcultured using 1.0 mL and 0.1 mL aliquots from the broths to 10 mL portions of Tetrathionate (TT) broth and Rappaport-Vassiliadis (RV) medium, respectively. For the accelerated method, the TT and RV broths were incubated at 42°C for 7 h, and then screened with Neogen devices. RapidChek device was used only to screen TT broths. Regardless of results from device, enrichments were streaked onto Hektoen Enteric (HE; Difco, BD, Sparks, MD, USA), Xylose Lysine Desoxycholate (XLD; Difco, BD, Sparks, MD, USA) and Bismuth Sulfite (BS; Difco, BD, Sparks, MD, USA) plates for isolation of Salmonella. The BS, HE, and XLD agars were incubated for 24 h at 35°C. Isolates were screened on Triple Sugar Iron (TSI; Difco, BD, Sparks, MD, USA) and Lysine Iron Agar (LIA; Difco, BD, Sparks, MD, USA) slants and confirmed with biochemical tests and by serology as described in the BAM.
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Incubation of the selective enrichments (TT and RV) was continued for an additional 17 h (24 h total) at 42°C as described in BAM for high background foods and used for conventional cultural isolation and confirmation. Similar to the 7 h time point, Neogen devices were used on both RV and TT broths while RapidChek was only used on the 24 h TT broths. Samples were further streaked to HE, BS, and XLD plates and were incubated for 24 ± 2 h at 35 ± 2°C. Identification of isolates was then performed as described in the BAM. M-broth (10 mL) was inoculated from the 24 h TT and RV broths (1 mL) and used for VIDAS-SLM analyses and incubated at 41-42°C for 6-8 h (AOAC International 2004). The AOAC VIDAS SLM Salmonella screening assay was followed in order to determine if the sample “Can Not be Ruled Out” (CROs) for the presence of Salmonella or is considered negative for the presence of Salmonella. If a CRO result was obtained from the VIDAS SLM assay, typical isolates from the selective agars (HE, BS, XLD) were picked to triple sugar iron (TSI) and lysine iron (LIA), as outlined in the BAM. The TSI/LIA slants were incubated for 24 h at 35°C. One or more presumptive-positive TSI cultures were picked to TSAYE plates for purification, serology and API 20E.
Statistical Analysis Chi square, χ2, analysis of the data was performed to determine differences between detection by lateral flow devices and cultural isolation by BAM plating procedure (Bird et al. 2001). χ2 = [(a-b)-1]2/(a + b) where a = samples positive by lateral flow devices and negative by BAM plating, and b = samples negative by lateral flow devices and positive by BAM plating. Significant differences between treatments at p > 0.05 exist for calculated χ2 values greater than 3.84.
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ACKNOWLEDGEMENTS: The authors wish to acknowledge Jorge Beall for technical support in accomplishing this work.
Conflict of Interest The authors have no conflict of interest to declare.
Disclaimer: The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.
REFERENCES Andrews, W.H., Jacobson, A. and Hammack, T. (2014) FDA Bacteriological Analytical Manual (BAM) Online, Chapter 5 Salmonella. Anon. (2009) Outbreak of Salmonella serotype Saintpaul infections associated with eating alfalfa sprouts - United States, 2009. MMWR Morb Mortal Wkly Rep 58, 500-503. AOAC International (2004) AOAC Official Method 2004.03. Salmonella in Foods. Gaithersburg, MD. Behravesh, C.B., Blaney, D., Medus, C., Bidol, S.A., Phan, Q., Soliva, S., Daly, E.R., Smith, K., Miller, B., Taylor, T., Jr., Nguyen, T., Perry, C., Hill, T.A., Fogg, N., Kleiza, A., Moorhead, D., AlKhaldi, S., Braden, C. and Lynch, M.F. (2012) Multistate outbreak of Salmonella serotype Typhimurium infections associated with consumption of restaurant tomatoes, USA, 2006: hypothesis generation through case exposures in multiple restaurant clusters. Epidemiol Infect 140, 2053-2061.
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Bennett, S.D., Littrell, K.W., Hill, T.A., Mahovic, M. and Behravesh, C.B. (2014) Multistate foodborne disease outbreaks associated with raw tomatoes, United States, 1990-2010: a recurring public health problem. Epidemiol Infect 1-8. bioMerieux (2009) VIDAS Salmonella (SLM). Bird, C.B., Hoerner, R.J. and Restaino, L. (2001) Comparison of the Reveal 20-hour method and the BAM culture method for the detection of Escherichia coli O157:H7 in selected foods and environmental swabs: collaborative study. J AOAC Int 84, 737-751. Gajraj, R., Pooransingh, S., Hawker, J.I. and Olowokure, B. (2012) Multiple outbreaks of Salmonella braenderup associated with consumption of iceberg lettuce. Int J Environ Health Res 22, 150-155. Hoerner, R., Feldpausch, J., Gray, R.L., Curry, S., Islam, Z., Goldy, T., Klein, F., Tadese, T., Rice, J. and Mozola, M. (2011) Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801. J AOAC Int 94, 14671480. Kozak, G.K., MacDonald, D., Landry, L. and Farber, J.M. (2013) Foodborne outbreaks in Canada linked to produce: 2001 through 2009. J Food Prot 76, 173-183. Lynch, M.F., Tauxe, R.V. and Hedberg, C.W. (2009) The growing burden of foodborne outbreaks due to contaminated fresh produce: risks and opportunities. Epidemiol Infect 137, 307-315. Neogen Corp. (2008) Neogen Reveal Microbial Screening Test Kit for Salmonella.
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Painter, J.A., Hoekstra, R.M., Ayers, T., Tauxe, R.V., Braden, C.R., Angulo, F.J. and Griffin, P.M. (2013) Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998-2008. Emerg Infect Dis 19, 407-415. Romer Labs, I. (2007) RapidCheck Salmonella Test Kit. Seo, K.H., Holt, P.S., Stone, H.D. and Gast, R.K. (2003) Simple and rapid methods for detecting Salmonella enteritidis in raw eggs. Int J Food Microbiol 87, 139-144. Sivapalasingam, S., Friedman, C.R., Cohen, L. and Tauxe, R.V. (2004) Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J Food Prot 67, 2342-2353. Torlak, E., Akan, I.M. and Inal, M. (2012) Evaluation of RapidChek Select for the screening of Salmonella in meat and meat products. J Microbiol Methods 90, 217-219.
Table 1. Comparison of BAM, RapidChek and Reveal (TT and RV) test results from produce items (Romaine lettuce, cilantro, jalapenos, tomatoes and cantaloupe) artificially contaminated with various Salmonella strains after 7 h in secondary enrichment. Results (number positive/total) Produce Item/Species
Inoculum Sample
-1
cfu sample
RapidChek BAM
(TT)
Reveal (TT)
Reveal (RV)
Romaine lettuce S. Typhimurium ATCC 14028
Control
None
0/6
0/6
0/6
0/6
Low
2.4
4/6
0/6
0/6
0/6
Medium
7.1
6/6
0/6
0/6
0/6
High
21.4
6/6
0/6
0/6
0/6
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Cilantro Control
None
0/6
0/6
0/6
0/6
Low
1.9
5/6
2/6
1/6
0/6
Medium
5.6
5/6
3/6
2/6
0/6
High
15
6/6
6/6
6/6
2/6
Control
None
0/6
0/6
0/6
0/6
Low
2.8
5/6
0/6
0/6
0/6
Medium
8.6
5/6
0/6
0/6
0/6
High
28.4
6/6
0/6
0/6
0/6
Control
None
0/6
0/6
0/6
0/6
Low
2.7
6/6
4/6
4/6
6/6
Medium
7.7
6/6
4/6
4/6
4/6
High
27
5/5*
5/5
5/5
5/5
Control
None
0/6
0/6
0/6
0/6
S. Panama
Low
1.6
6/6
6/6
6/6
6/6
S4R B39
Medium
4.8
6/6
6/6
6/6
6/6
High
16
6/6
6/6
6/6
6/6
S. Anatum ATCC 9270
Tomatoes
S. Montevideo ATCC 8387
Jalapenos
S. Heidelberg ATCC 8326
Cantaloupe
*Sample lost due to lab error Bold = Chi square (χ2) value where χ2 > 3.84, indicating a significant difference (p < 0.05) between positive rate of lateral flow devices compared to BAM plating of enrichments.
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Table 2. Comparison of BAM, RapidChek, Reveal (TT and RV) and VIDAS Salmonella test results from produce items (Romaine lettuce, cilantro, jalapenos, tomatoes and cantaloupe) artificially contaminated with various Salmonella strains after 24 h in secondary enrichment. Results (number positive/total) Produce Item/Species
Sample
Inoculum -1
cfu sample
RapidChek BAM (TT)
Reveal (TT)
Reveal (RV)
VIDAS (M-broth)
Romaine lettuce
S. Typhimurium ATCC 14028
Control
None
0/6
0/6
0/6
0/6
0/6
Low
2.4
4/6
4/6
4/6
5/6
4/6
Medium
7.1
6/6
6/6
6/6
6/6
6/6
High
21.4
6/6
6/6
6/6
6/6
6/6
Control
None
0/6
0/6
0/6
0/6
0/6
Low
1.9
5/6
5/6
5/6
5/6
5/6
Medium
5.6
5/6
5/6
5/6
5/6
5/6
High
15
6/6
6/6
6/6
6/6
6/6
Control
None
0/6
0/6
0/6
0/6
0/6
Low
2.8
6/6
0/6
0/6
6/6
6/6
Medium
8.6
6/6
0/6
0/6
6/6
6/6
High
28.4
6/6
0/6
3/6
6/6
6/6
Control
None
0/6
0/6
0/6
0/6
0/6
Low
2.7
6/6
6/6
6/6
6/6
6/6
Medium
7.7
6/6
6/6
6/6
6/6
6/6
High
27
5/5*
5/5
5/5
5/5
5/5
Cilantro
S. Anatum ATCC 9270
Tomatoes
S. Montevideo ATCC 8387
Jalapenos
S. Heidelberg ATCC 8326
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Cantaloupe Control
None
0/6
0/6
0/6
0/6
0/6
S. Panama
Low
1.6
6/6
6/6
6/6
6/6
6/6
S4R B39
Medium
4.8
6/6
6/6
6/6
6/6
6/6
High
16
6/6
6/6
6/6
6/6
6/6
* Sample lost due to lab error Bold = Chi square (χ2) value where χ2 > 3.84, indicating a significant difference (p < 0.05) between positive rate of lateral flow devices compared to BAM plating of enrichments.
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Article Type: Original Article
Evaluation of Rapid Screening Techniques for Detection of Salmonella spp. from Produce Samples after Pre-enrichment According to FDA BAM and a Short Secondary Enrichment
Abbreviated Running Headline: Screening of Salmonella in Produce
K. J. Yoshitomi1, K.C. Jinneman1, P.A. Orlandi2, S.D. Weagant3, R. Zapata4, and W.M. Fedio4*
1
Food and Drug Administration, Bothell, WA 98021; 2 Food and Drug Administration,
Rockville, MD 20857; 3 Weagant Consulting, Poulsbo, WA 98370; 4 New Mexico State University, Food Safety Laboratory, Las Cruces, NM 88003
* Corresponding author Willis Fedio New Mexico State University MSC 3BF
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12422 This article is protected by copyright. All rights reserved.
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P.O. Box 30003 Las Cruces, NM 88003-8003 Phone: 575-646-7345 Fax: 575-646-7344 E-mail: [email protected]
SIGNIFICANCE AND IMPACT OF THE STUDY: In the event of a foodborne disease outbreak, rapid identification and characterization of the pathogen is essential to prevent the spread of disease and reduce the number of illnesses. This study reports the utility of an abbreviated secondary enrichment for the isolation of Salmonella in artificially contaminated fresh produce at very low levels. In addition, incorporation of rapid, easy-to-use lateral flow devices to screen enrichments can provide a low cost (equipment and highly trained personnel), high return (rapid identification of contaminated food) investment in the timely pathogen screening of fresh produce.
ABSTRACT: Conventional detection of Salmonella from foods involves enrichment and isolation on selective media which can significantly lengthen time to result. The objective of this study was to evaluate the utility of an accelerated plating procedure and the use of rapid screening devices for Salmonella detection. Fresh produce was inoculated with Salmonella at ~2.5, ~7.5 and ~25 cfu sample-1. After 24 h pre-enrichment, subcultures
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were made into TT and RV broths and further incubated at 42°C for an additional 7 and 24 h. Enrichments were streaked for isolation of Salmonella as well as tested by rapid screening methods. The seven hour accelerated plating procedure worked well from 4/6 to 6/6 in all produce samples inoculated at the lowest level. Both the RapidChek and Neogen Reveal tests worked as well as the VIDAS-SLM after 24 h secondary enrichment, but failed to detect the pathogen after 7 h selective enrichment in romaine lettuce and tomatoes, while fractional detection was observed in cilantro and jalapenos. Both devices detected Salmonella on cantaloupe at the lowest level of inoculation. An abbreviated selective enrichment procedure worked well to accelerate the isolation of colonies of Salmonella from contaminated samples providing isolates for further characterization one day earlier than standard analysis.
Keywords: Salmonella, Produce, Rapid Screening
INTRODUCTION Fresh produce is increasingly found to be involved in foodborne disease outbreaks (Sivapalasingam et al. 2004; Lynch et al. 2009; Kozak et al. 2013). Examination of outbreak associated illnesses from 1998-2008 estimated that as many as 46% of illnesses were attributed to produce (Painter et al. 2013). Salmonella has been associated with outbreaks involving alfalfa sprouts, lettuce and tomatoes as well as other produce (Anon 2009; Behravesh et al. 2012; Gajraj et al. 2012; Bennett et al. 2014) and is a pathogen of concern in these commodities. Rapid and reliable screening methods for detecting bacterial pathogens in produce samples would allow for more timely testing of food samples and potentially quicker response to foodborne outbreaks.
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Rapid screening methods for foodborne pathogens can help reduce the amount of time and labor needed for detecting pathogens. A wide range of rapid methods are available for detection of pathogenic bacteria. However, many rapid screening methods such as automated immunoassays and real-time PCR procedures require investment in expensive equipment or require trained/experienced personnel to perform the analysis. Although potentially more costly, molecular or immuno-based screening assays can offer greater sensitivity in screening food samples, in addition to being more rapid than standard cultural techniques. The use of lateral flow devices developed for detection of foodborne pathogens offers an economical method to screen food enrichments. The Reveal for Salmonella Complete System (Neogen Corporation, Lansing, MI, USA) along with RV medium provides for rapid recovery of Salmonella spp. in foods, allowing detection and presumptive identification of the target organism in 20-28 h. The enriched sample is placed into a Salmonella Reveal device with specific antibodies against Salmonella. If a line develops only in the control zone, the sample is negative for Salmonella spp. If lines are present in the control and test zones, the sample is considered positive for salmonellae (Neogen Corp. 2008). The RapidChek® Salmonella system (Romer Labs, Union, MO, USA) uses a similar mechanism to rapidly detect Salmonella. The RapidChek® lateral flow strip contains a proprietary panel of anti-Salmonella antibodies engineered to enhance the overall performance of the method. The complete test utilizes a proprietary enrichment base supplemented with phage as well as a secondary enrichment media to selectively grow Salmonella to be detected by the test strip. Upon immersion into the enrichment media and uptake via capillary action, positive results are attained in a similar manner with presence of a red line in the control and test location (Romer Labs, 2007).
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The VIDAS (Vitek Immuno Diagnostic Assay System, bioMerieux, Askim, Sweden) automated instrument uses technology that is adaptable to a wide range of assays, and combines an enzyme immunoassay method with a final fluorescence reading to obtain a test result. This technology is known as ELFA (Enzyme-Linked Fluorescent Assay) which uses alkaline phosphatase to hydrolyze a substrate to a fluorescent product which is proportional to the abundance of pathogen present in the sample, (bioMerieux 2009). The VIDAS® SLM Assay incorporates the same primary enrichment and secondary selective enrichment as described in the FDA Bacteriological Analytical Manual (BAM), followed by enrichment in M-Broth prior to inoculation of test strip and incubation on the VIDAS instrument.
This study was designed to evaluate the utility of an accelerated plating procedure and rapid screening techniques for Salmonella detection and isolation using FDA BAM ( Andrews et al. 2014) enrichment procedures. Selective plating of 7h enrichments (TT and RV) could potentially be used to obtain isolates earlier than with the traditional, cultural method, allowing quicker confirmation of presumptive isolates and further characterization. In addition, simple, easy-to-use lateral flow devices were evaluated at the 7 h and 24 h secondary enrichment time points to determine if such devices could be used in this manner. Although, it should be emphasized that these devices were used following standard BAM pre-enrichment and abbreviated secondary enrichment and not the manufacturer’s specific enrichment. Examining the applicability of such devices as used in this study could eliminate the need for parallel enrichment schemes and provide a potentially useful test that is simple, rapid and relatively low in cost.
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RESULTS AND DISCUSSION Plating of the secondary selective enrichment after 7 h, as opposed to 24 h, performed well to accelerate cultural isolation of Salmonella from contaminated produce samples as shown in Table 1 (7 h) and Table 2 (24 h) under the BAM column heading. For Romaine lettuce artificially contaminated with S. Typhimurium ATCC 14028, plating 7 h selective enrichment broths resulted in confirmed salmonellae in 4/6 low inoculum samples (2.4 cfu per 375g sample), while none of the rapid devices gave positive results. For cilantro contaminated with S. Anatum ATCC 9270, fractional recovery or detection was observed at the medium level of inoculation (5.6 sample-1) with 5/6, 3/6, and 2/6 positive for BAM, RapidChek, and Reveal TT, respectively, with all replicates positive at the higher level (15 cfu sample-1). Fractional detection (2/6) was observed with the Reveal RV treatment at these high levels and none were detected at the two lower levels of inoculation. Tomato samples were artificially inoculated with Salmonella Montevideo ATCC 8387. Similar to the Romaine lettuce samples, Salmonella was fractionally isolated from the low (5/6 at 2.8 cfu g-1) as well as medium levels (5/6 at 8.6 cfu g-1), but not detected by any of the devices after 7 h selective enrichment. All isolates were recovered by BAM plating procedures at the high inoculation level (28.4 cfu g-1). S. Heidelberg ATCC 8326 was successfully isolated from inoculated jalapeño peppers at all levels as low as 2.7 cfu sample-1. However, fractional detection by lateral flow devices was achieved at medium and lower levels with 4/6 samples positive, except for the Reveal RV treatment having 6/6 positive. Cantaloupe artificially contaminated with S. Panama was positive when screened by both RapidChek and Reveal devices and culturally recovered by direct plating after 7 h selective enrichment at all inoculum levels, as low as 1.6 cfu sample-1.
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Direct plating of the secondary enrichment after 7 h was successful in recovery for most matrix/strain combinations. In most cases, 5/6 samples or greater were positive for all levels. The response of the lateral flow devices as employed at this time point was more variable. Romaine lettuce and tomatoes resulted in no positive screening results, while cilantro and jalapenos were 88% successful across all lateral flow tests at the highest level of inoculum, with fractional detection at the lower levels. Detection of S. Panama in cantaloupes was 100% successful by devices for all treatment combinations. After 24 h enrichment, all of the BAM samples, all of the VIDAS–SLM screening tests and all of the lateral flow devices were positive for all levels of inoculum for jalapenos and cantaloupe. This was not surprising given the high detection rates after 7 h for these foods. Detection rates by devices were markedly improved for romaine lettuce, cilantro and tomatoes compared to the 7 h time point. There was no difference (p < 0.05) in the detection rates between BAM, VIDAS SLM and devices for romaine lettuce and cilantro after 24 h, showing a dramatic increase over the 7 h rates of detection. Improvement in detection rates after 24 h was also observed for tomatoes for the Reveal RV treatment with 6/6 positive as low as 2.8 cfu sample-1. However, the RapidChek® (0/6 at all levels) and Reveal TT (3/6 at highest level and 0/6 at others) detection rates remained significantly lower than BAM plating results, similar to the 7 h detection rates for the tomato sample matrix. Both devices were tested on TT broths, suggesting that perhaps Salmonella did not grow as well in TT broth from this particular matrix. Except for this result, all lateral flow devices performed to the same level of sensitivity as the BAM and VIDAS-SLM methods after 24 h in secondary enrichment. Both the RapidChek® and Neogen Reveal® tests worked as well as the VIDASSLM after 24h secondary enrichment for most strains tested, but did not reliably detect the pathogen after 7 h selective enrichment in most of the artificially contaminated
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samples. It was possible to recover Salmonella isolates from artificially contaminated fresh produce at levels ranging from 1.6 to 28.4 cfu sample-1 with a 7 h abbreviated secondary enrichment, depending upon the commodity/strain. The slightly better sensitivity observed with the direct plating after 7 h was most likely attributed to increased recovery of low numbers of Salmonella from the plating media. The reported sensitivity of lateral flow devices are approximately 107 cfu mL-1 (Seo et al. 2003), a level at which Salmonella numbers may have not been reached either through matrix effects or competition from other organisms. In the current study, we found the sensitivity of the Salmonella RapidChek® devices to range from 107-108 cfu mL-1. Although the devices were not used as recommended by the manufacturer, the utility of having the option to use such device in standard protocol can prove valuable in certain circumstances. Detection or lack thereof, by devices was not dependent upon which secondary enrichment media was tested, and both devices performed very similarly across all produce matrices at 7 h. After 24 h, the devices performed as well as the standard protocol (except for the RapidChek® and Reveal TT tomato treatment). Tomatoes did not have any positive detection values by devices at 7 h, and at 24 h only the RV enriched samples were positive by devices. Use of lateral flow devices for detection of Salmonella has shown similar sensitivity with reference methods after secondary enrichment (Hoerner et al. 2011; Torlak et al. 2012). This study suggests that the lateral flow devices have similar sensitivity as standard plating methods after 24 h. The sensitivity of the devices were variable after a shortened 7 h enrichment in secondary media, but can be of value if a screening result was necessary one day sooner. In addition, plating of the 7 h enrichment proved as sensitive as the full 24 h, suggesting isolates can be obtained for further characterization one day sooner than conventional methods. Introducing rapid, simple to use tests such as lateral flow devices at key points during standard analytical methods for Salmonella and abbreviating a secondary
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enrichment by plating sooner can potentially speed time to a confirmed result. This can be especially valuable when considering timely response to foodborne outbreaks involving fresh produce.
MATERIALS AND METHODS Microorganisms The following strains were used in the study: Salmonella Typhimurium ATCC 14028, Salmonella Anatum ATCC 9270, Salmonella Montevideo ATCC 8387, Salmonella Heidelberg ATCC 8326 and Salmonella Panama S4RB39. S. Panama S4RB39 was obtained from the Salmonella Genetic Stock Center, University of Calgary, Calgary, AB. The strains were maintained in 20% glycerol/peptone broth medium at -70°C. Preparation of Inoculum Frozen stock cultures of the microorganisms were streaked onto Tryptic Soy Agar with 0.6% Yeast Extract (TSAYE; BD, Sparks, MD, USA) plates and incubated at 37°C for 20 h. A well-separated colony from the plate was transferred to 5 mL of Brain Heart Infusion broth (BHI; BD, Sparks, MD, USA) and incubated at 37°C for 18-20 h without shaking. Ten-fold serial dilutions of the enriched sample were prepared in Butterfields Phosphate Buffer (BPB) to obtain low, medium and high inoculum levels. Inoculum Levels Produce (Romaine lettuce, cilantro, jalapeno peppers, tomatoes and cantaloupe) was artificially contaminated with the various Salmonella species at low (~2.5 cfu sample-1), medium (~7.5 cfu sample-1) and high (~25 cfu sample-1) levels. The inoculum levels were
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determined by plate counts of dilutions of the initial inoculum. Inoculum was not aged in this particular study. Uninoculated controls were also prepared with produce samples which only received BPB. Experimental parameters Six replicates at each inoculum level were tested per food matrix, as well as six uninoculated controls. Samples were pre-enriched in sterile plastic bags (12” x 18”, NASCO, Ft. Atkinson, WI, USA) using the appropriate standard enrichment broth for the product being tested. Enrichment broths used were as follows: 375 g portions of Romaine lettuce were enriched in 3375 mL of lactose broth (LB); 454 g jalapeño peppers in 1.5x volume of lactose broth; 375 g cilantro in 3375 mL tryptic soy broth (TSB); each cantaloupe (approximately 1300 g) in 1.5x volume of Universal preenrichment broth (UP) until floating, and 454 g tomatoes in 1.0x volume of UP. Enrichment broths were incubated at 35°C. After 24 h, Enrichments were subcultured using 1.0 mL and 0.1 mL aliquots from the broths to 10 mL portions of Tetrathionate (TT) broth and Rappaport-Vassiliadis (RV) medium, respectively. For the accelerated method, the TT and RV broths were incubated at 42°C for 7 h, and then screened with Neogen devices. RapidChek device was used only to screen TT broths. Regardless of results from device, enrichments were streaked onto Hektoen Enteric (HE; Difco, BD, Sparks, MD, USA), Xylose Lysine Desoxycholate (XLD; Difco, BD, Sparks, MD, USA) and Bismuth Sulfite (BS; Difco, BD, Sparks, MD, USA) plates for isolation of Salmonella. The BS, HE, and XLD agars were incubated for 24 h at 35°C. Isolates were screened on Triple Sugar Iron (TSI; Difco, BD, Sparks, MD, USA) and Lysine Iron Agar (LIA; Difco, BD, Sparks, MD, USA) slants and confirmed with biochemical tests and by serology as described in the BAM.
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Incubation of the selective enrichments (TT and RV) was continued for an additional 17 h (24 h total) at 42°C as described in BAM for high background foods and used for conventional cultural isolation and confirmation. Similar to the 7 h time point, Neogen devices were used on both RV and TT broths while RapidChek was only used on the 24 h TT broths. Samples were further streaked to HE, BS, and XLD plates and were incubated for 24 ± 2 h at 35 ± 2°C. Identification of isolates was then performed as described in the BAM. M-broth (10 mL) was inoculated from the 24 h TT and RV broths (1 mL) and used for VIDAS-SLM analyses and incubated at 41-42°C for 6-8 h (AOAC International 2004). The AOAC VIDAS SLM Salmonella screening assay was followed in order to determine if the sample “Can Not be Ruled Out” (CROs) for the presence of Salmonella or is considered negative for the presence of Salmonella. If a CRO result was obtained from the VIDAS SLM assay, typical isolates from the selective agars (HE, BS, XLD) were picked to triple sugar iron (TSI) and lysine iron (LIA), as outlined in the BAM. The TSI/LIA slants were incubated for 24 h at 35°C. One or more presumptive-positive TSI cultures were picked to TSAYE plates for purification, serology and API 20E.
Statistical Analysis Chi square, χ2, analysis of the data was performed to determine differences between detection by lateral flow devices and cultural isolation by BAM plating procedure (Bird et al. 2001). χ2 = [(a-b)-1]2/(a + b) where a = samples positive by lateral flow devices and negative by BAM plating, and b = samples negative by lateral flow devices and positive by BAM plating. Significant differences between treatments at p > 0.05 exist for calculated χ2 values greater than 3.84.
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ACKNOWLEDGEMENTS: The authors wish to acknowledge Jorge Beall for technical support in accomplishing this work.
Conflict of Interest The authors have no conflict of interest to declare.
Disclaimer: The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.
REFERENCES Andrews, W.H., Jacobson, A. and Hammack, T. (2014) FDA Bacteriological Analytical Manual (BAM) Online, Chapter 5 Salmonella. Anon. (2009) Outbreak of Salmonella serotype Saintpaul infections associated with eating alfalfa sprouts - United States, 2009. MMWR Morb Mortal Wkly Rep 58, 500-503. AOAC International (2004) AOAC Official Method 2004.03. Salmonella in Foods. Gaithersburg, MD. Behravesh, C.B., Blaney, D., Medus, C., Bidol, S.A., Phan, Q., Soliva, S., Daly, E.R., Smith, K., Miller, B., Taylor, T., Jr., Nguyen, T., Perry, C., Hill, T.A., Fogg, N., Kleiza, A., Moorhead, D., AlKhaldi, S., Braden, C. and Lynch, M.F. (2012) Multistate outbreak of Salmonella serotype Typhimurium infections associated with consumption of restaurant tomatoes, USA, 2006: hypothesis generation through case exposures in multiple restaurant clusters. Epidemiol Infect 140, 2053-2061.
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Bennett, S.D., Littrell, K.W., Hill, T.A., Mahovic, M. and Behravesh, C.B. (2014) Multistate foodborne disease outbreaks associated with raw tomatoes, United States, 1990-2010: a recurring public health problem. Epidemiol Infect 1-8. bioMerieux (2009) VIDAS Salmonella (SLM). Bird, C.B., Hoerner, R.J. and Restaino, L. (2001) Comparison of the Reveal 20-hour method and the BAM culture method for the detection of Escherichia coli O157:H7 in selected foods and environmental swabs: collaborative study. J AOAC Int 84, 737-751. Gajraj, R., Pooransingh, S., Hawker, J.I. and Olowokure, B. (2012) Multiple outbreaks of Salmonella braenderup associated with consumption of iceberg lettuce. Int J Environ Health Res 22, 150-155. Hoerner, R., Feldpausch, J., Gray, R.L., Curry, S., Islam, Z., Goldy, T., Klein, F., Tadese, T., Rice, J. and Mozola, M. (2011) Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801. J AOAC Int 94, 14671480. Kozak, G.K., MacDonald, D., Landry, L. and Farber, J.M. (2013) Foodborne outbreaks in Canada linked to produce: 2001 through 2009. J Food Prot 76, 173-183. Lynch, M.F., Tauxe, R.V. and Hedberg, C.W. (2009) The growing burden of foodborne outbreaks due to contaminated fresh produce: risks and opportunities. Epidemiol Infect 137, 307-315. Neogen Corp. (2008) Neogen Reveal Microbial Screening Test Kit for Salmonella.
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Painter, J.A., Hoekstra, R.M., Ayers, T., Tauxe, R.V., Braden, C.R., Angulo, F.J. and Griffin, P.M. (2013) Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998-2008. Emerg Infect Dis 19, 407-415. Romer Labs, I. (2007) RapidCheck Salmonella Test Kit. Seo, K.H., Holt, P.S., Stone, H.D. and Gast, R.K. (2003) Simple and rapid methods for detecting Salmonella enteritidis in raw eggs. Int J Food Microbiol 87, 139-144. Sivapalasingam, S., Friedman, C.R., Cohen, L. and Tauxe, R.V. (2004) Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J Food Prot 67, 2342-2353. Torlak, E., Akan, I.M. and Inal, M. (2012) Evaluation of RapidChek Select for the screening of Salmonella in meat and meat products. J Microbiol Methods 90, 217-219.
Table 1. Comparison of BAM, RapidChek and Reveal (TT and RV) test results from produce items (Romaine lettuce, cilantro, jalapenos, tomatoes and cantaloupe) artificially contaminated with various Salmonella strains after 7 h in secondary enrichment. Results (number positive/total) Produce Item/Species
Inoculum Sample
-1
cfu sample
RapidChek BAM
(TT)
Reveal (TT)
Reveal (RV)
Romaine lettuce S. Typhimurium ATCC 14028
Control
None
0/6
0/6
0/6
0/6
Low
2.4
4/6
0/6
0/6
0/6
Medium
7.1
6/6
0/6
0/6
0/6
High
21.4
6/6
0/6
0/6
0/6
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Cilantro Control
None
0/6
0/6
0/6
0/6
Low
1.9
5/6
2/6
1/6
0/6
Medium
5.6
5/6
3/6
2/6
0/6
High
15
6/6
6/6
6/6
2/6
Control
None
0/6
0/6
0/6
0/6
Low
2.8
5/6
0/6
0/6
0/6
Medium
8.6
5/6
0/6
0/6
0/6
High
28.4
6/6
0/6
0/6
0/6
Control
None
0/6
0/6
0/6
0/6
Low
2.7
6/6
4/6
4/6
6/6
Medium
7.7
6/6
4/6
4/6
4/6
High
27
5/5*
5/5
5/5
5/5
Control
None
0/6
0/6
0/6
0/6
S. Panama
Low
1.6
6/6
6/6
6/6
6/6
S4R B39
Medium
4.8
6/6
6/6
6/6
6/6
High
16
6/6
6/6
6/6
6/6
S. Anatum ATCC 9270
Tomatoes
S. Montevideo ATCC 8387
Jalapenos
S. Heidelberg ATCC 8326
Cantaloupe
*Sample lost due to lab error Bold = Chi square (χ2) value where χ2 > 3.84, indicating a significant difference (p < 0.05) between positive rate of lateral flow devices compared to BAM plating of enrichments.
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Table 2. Comparison of BAM, RapidChek, Reveal (TT and RV) and VIDAS Salmonella test results from produce items (Romaine lettuce, cilantro, jalapenos, tomatoes and cantaloupe) artificially contaminated with various Salmonella strains after 24 h in secondary enrichment. Results (number positive/total) Produce Item/Species
Sample
Inoculum -1
cfu sample
RapidChek BAM (TT)
Reveal (TT)
Reveal (RV)
VIDAS (M-broth)
Romaine lettuce
S. Typhimurium ATCC 14028
Control
None
0/6
0/6
0/6
0/6
0/6
Low
2.4
4/6
4/6
4/6
5/6
4/6
Medium
7.1
6/6
6/6
6/6
6/6
6/6
High
21.4
6/6
6/6
6/6
6/6
6/6
Control
None
0/6
0/6
0/6
0/6
0/6
Low
1.9
5/6
5/6
5/6
5/6
5/6
Medium
5.6
5/6
5/6
5/6
5/6
5/6
High
15
6/6
6/6
6/6
6/6
6/6
Control
None
0/6
0/6
0/6
0/6
0/6
Low
2.8
6/6
0/6
0/6
6/6
6/6
Medium
8.6
6/6
0/6
0/6
6/6
6/6
High
28.4
6/6
0/6
3/6
6/6
6/6
Control
None
0/6
0/6
0/6
0/6
0/6
Low
2.7
6/6
6/6
6/6
6/6
6/6
Medium
7.7
6/6
6/6
6/6
6/6
6/6
High
27
5/5*
5/5
5/5
5/5
5/5
Cilantro
S. Anatum ATCC 9270
Tomatoes
S. Montevideo ATCC 8387
Jalapenos
S. Heidelberg ATCC 8326
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Cantaloupe Control
None
0/6
0/6
0/6
0/6
0/6
S. Panama
Low
1.6
6/6
6/6
6/6
6/6
6/6
S4R B39
Medium
4.8
6/6
6/6
6/6
6/6
6/6
High
16
6/6
6/6
6/6
6/6
6/6
* Sample lost due to lab error Bold = Chi square (χ2) value where χ2 > 3.84, indicating a significant difference (p < 0.05) between positive rate of lateral flow devices compared to BAM plating of enrichments.
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