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DeCory, Thomas R., Richard A. Durst, Scott J. Zimmerman, Linda A. Garringer, Gary Paluca, Heleen H. DeCory, and Richard A. Montagna. "Development of an Immunomagnetic Bead-Immunoliposome Fluorescence Assay for Rapid Detection of Escherichia coli O157:H7 in Aqueous Samples and Comparison of the Assay with a Standard Microbiological Method." Applied and Environmental Microbiology 71, no. 4 (April 2005): 1856–64. http://dx.doi.org/10.1128/aem.71.4.1856-1864.2005.
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ABSTRACT The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-β-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.
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BLAIS, BURTON W., RONALD A. BOOTH, LUCILLE PHILLIPPE, SITHIAN PANDIAN, and HIROSHI YAMAZAKI. "Polymacron™ Enzyme Immunoassay System for Detection of Escherichia coli O157 Inoculated into Foods†." Journal of Food Protection 60, no. 2 (February 1, 1997): 98–101. http://dx.doi.org/10.4315/0362-028x-60.2.98.
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A rapid and simple enzyme immunoassay system was developed for detection of the food-borne pathogen Escherichia coli O157. This system is based on the use of anti-E. coli O157 antibody-coated Polymacron™, an inexpensive macroporous polyester fabric, as a high-surface-area immunoadsorbent for the rapid capture and subsequent immunoenzymatic detection of E. coli O157 antigens extracted from test samples by heating at 100°C for 10 min in the presence of sodium cholate. A dot blot format was used which facilitated the assay of multiple samples. The method was specific for all strains tested bearing the O157 and related antigens (e.g., group N Salmonella), giving positive reactions in the assay of pure cultures of 29 E. coli O157:H7 strains, one E. coli O157:H12, one E. coli O157:NM (nonmotile) and one Salmonella urbana strain, but not in the assay of a variety of other gram-negative and gram-positive bacteria. The method permitted the detection of ca. 400 E. coli O157:H7 CFU in a 5-μl spot applied to Polymacron™, and of as few as 0.4 CFU of E. coli O157:147 cells per g inoculated into ground beef samples, which were assayed after overnight enrichment.
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HUANG, SHIU W., and TSUNG C. CHANG. "Specific Identification of Escherichia coli O157 by an Immunostick Method Using Commercially Available Antibodies." Journal of Food Protection 59, no. 6 (June 1, 1996): 670–73. http://dx.doi.org/10.4315/0362-028x-59.6.670.
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A sandwich enzyme-immunoassay performed on plastic sticks was developed to specifically identify Escherichia coli O157. Colonies of test bacteria grown on tryptic soy agar were suspended in phosphate-buffered saline, heated in a 100°C water bath for 15 min, and incubated with the plastic sticks coated with a commercial preparation of anti-E. coli O157 antibodies at 37°C for 1.5 h. After incubation, the same antibodies labeled with peroxidase were used to produce the signal of antigen-antibody reaction. For 35 strains of E. coli O157 (among them 34 were E. coli O157:H7) tested, all produced strong reactions by the immunoassay. For 162 strains of E. coli with somatic antigens other than O157 and 38 strains of other genera tested, only one strain (Salmonella bietri) produced a false-positive reaction. The specificity and sensitivity of the immunostick assay were 100% (35/35) and 99.5% (199/200), respectively. The detection limit of the assay for E. coli O157:H7 (CCRC 15991) was about 105 CFU/ml. The method, which can be carried out within 3 h, is useful for rapid identification of suspect E. coli O157 isolated on selective media.
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FRATAMICO, PINA M., and LORI K. BAGI. "Comparison of Methods for Detection and Isolation of Cold- and Freeze-Stressed Escherichia coli O157:H7 in Raw Ground Beef†." Journal of Food Protection 70, no. 7 (July 1, 2007): 1663–69. http://dx.doi.org/10.4315/0362-028x-70.7.1663.
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A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at ≤0.5 and ≤2 CFU/g, and samples were then enriched immediately or were stored at 4°C for 72 h or at −20°C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42°C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35°C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42°C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35°C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+nat35°C, 3.7 times more likely with an initial inoculum of ≤2.0 CFU/g than with ≤0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.
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AYDIN, MUHSIN, GENE P. D. HERZIG, KWANG CHEOL JEONG, SAMANTHA DUNIGAN, PARTH SHAH, and SOOHYOUN AHN. "Rapid and Sensitive Detection of Escherichia coli O157:H7 in Milk and Ground Beef Using Magnetic Bead–Based Immunoassay Coupled with Tyramide Signal Amplification." Journal of Food Protection 77, no. 1 (January 1, 2014): 100–105. http://dx.doi.org/10.4315/0362-028x.jfp-13-274.
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Escherichia coli O157:H7 is a major foodborne pathogen that has posed serious problems for food safety and public health. Recent outbreaks and recalls associated with various foods contaminated by E. coli O157:H7 clearly indicate its deleterious effect on food safety. A rapid and sensitive detection assay is needed for this harmful organism to prevent foodborne illnesses and control outbreaks in a timely manner. We developed a magnetic bead–based immunoassay for detection of E. coli O157:H7 (the most well-known Shiga toxigenic E. coli strain) with a 96-well microplate as an assay platform. Immunomagnetic separation (IMS) and tyramide signal amplification were coupled to the assay to increase its sensitivity and specificity. This immunoassay was able to detect E. coli O157:H7 in pure culture with a detection limit of 50 CFU/ml in less than 3 h without an enrichment step. The detection limit was decreased 10-fold to 5 CFU/ml with addition of a 3-h enrichment step. When this assay was tested with other nontarget foodborne pathogens and common enteric bacteria, no cross-reactivity was found. When tested with artificially contaminated ground beef and milk samples, the assay sensitivity decreased two- to fivefold, with detection limits of 250 and 100 CFU/ml, respectively, probably because of the food matrix effect. The assay results also were compared with those of a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an ELISA coupled with IMS; the developed assay was 25 times and 4 times more sensitive than the standard ELISA and the IMS-ELISA, respectively. Tyramide signal amplification combined with IMS can improve sensitivity and specificity for detection of E. coli O157:H7. The developed assay could be easily adapted for other foodborne pathogens and will contribute to improved food safety and public health.
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Ingram, David T., Chinta M. Lamichhane, David M. Rollins, Lewis E. Carr, Edward T. Mallinson, and Sam W. Joseph. "Development of a Colony Lift Immunoassay To Facilitate Rapid Detection and Quantification of Escherichia coli O157:H7 from Agar Plates and Filter Monitor Membranes." Clinical Diagnostic Laboratory Immunology 5, no. 4 (July 1, 1998): 567–73. http://dx.doi.org/10.1128/cdli.5.4.567-573.1998.
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ABSTRACT E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 ± 1°C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3′,5,5′-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling.
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Feldsine, Philip T., Shannon T. Green, Andrew H. Lienau, and David E. Kerr. "Comparative Validation Study to Demonstrate the Equivalence of a Minor Modification to AOAC Methods 996.09, VIP® for EHEC and 996.10, Assurance EIA® EHEC with the Reference Culture Method for the Detection of Escherichia coli O157:H7 in Beef." Journal of AOAC INTERNATIONAL 88, no. 4 (July 1, 2005): 1193–96. http://dx.doi.org/10.1093/jaoac/88.4.1193.
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Abstract The Visual Immunoprecipitate Assay (VIP®) method for the detection of enterohemorrhagic Escherichia coli O157:H7 (VIP for EHEC) and Assurance® Enzyme Immunoassay (EIA) method for the detection of EHEC (EHEC EIA) are AOAC INTERNATIONAL Official Methods 996.09 and 996.10, respectively. A minor modification to the enrichment medium used in both methods has been developed. This modification, the BioControl modified EHEC medium™ (BioControl mEHEC™) provides a more cost-effective procedure with performance equivalent to that of the cultural method for detection of E. coli O157:H7 in beef.
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CALICCHIA, MELISSA L., E. L. PARKER, S. GAMBREL-LENARZ, and RICHARD R. MATNER. "Use of the Petrifilm™ Test Kit-HEC Direct Blot Enzyme Immunoassay Method without Swab Pre-Enrichment to Screen for Low Levels of Escherichia coli O157 : H7 on Beef Carcasses by Surface Swabbing." Journal of Food Protection 60, no. 7 (July 1, 1997): 870–73. http://dx.doi.org/10.4315/0362-028x-60.7.870.
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A total of 750 beef carcasses were assayed to detect E. coli O157:H7 by surface swabbing. Each swab represented a 200-cm2 composite surface area. Escherichia coli O157:H7 was not detected in any of the carcass samples. Assays were conducted from carcass swab suspensions by direct plating on Petrifilm™ E. coli Count plates followed by an E. coli O157:H7 assay using the Petrifilm™ Test Kit-HEC direct blot enzyme immunoassay without sample pre-enrichment, and by enrichment using a modified U.S. Department of Agriculture (USDA) Petrifilm™ E. coli O157:H7 procedure. Additionally, beef slabs were inoculated with E. coli O157 :H7 to verify that recovery by the direct swab technique was at least as efficient as enrichment of excised tissue samples, which is the usual USDA method. Escherichia coli O157:H7 inoculation of 4,32, and 180 CFU/25 cm2 were designated low, medium, and high surface contamination levels, respectively. The percentage of recovery of E. coli O157:H7 using the direct swab technique without sample pre-enrichment was 60, 80, and 100% for low, medium, and high surface inoculation levels, respectively. In comparison, recovery from 25-g enrichments of ground excised samples from beef slabs containing 1.4,9.6, and 50 CFU of E. coli O157:H7 was 0, 20, and 73%, respectively. Similarly, the percent recovery of this organism from nonground excised samples containing 56 CFU/25 g enrichment was 68%, versus 100% by direct swab technique without sample pre-enrichment from beef containing 190 CFU/25 cm2 Direct surface swab tests with the 3M Petrifilm™ Test Kit-HEC without sample pre-enrichment proved to be a sensitive, nondestructive, alternate means of evaluating beef carcasses for the presence of E. coli O157:H7.
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Patiño-Burbano, Rocio Esperanza, Ana Karina Carrascal, Jorge Luis Parra-Arango, and José Luis Rodríguez-Bautista. "Assessment of a multiplex detection method for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in cow milk." Universitas Scientiarum 24, no. 1 (April 8, 2019): 277–94. http://dx.doi.org/10.11144/javeriana.sc24-1.aoam.
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Raw cow milk is considered one of the most important vehicles for pathogenic bacteria like Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. These three bacteria are responsible for foodborne diseases. Routine microbiological methods to detect these microorganisms in cow milk can be complicated and time consuming. The aim of this work was to evaluate a method to simultaneously detect Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in experimentally contaminated cow milk. The assessed method combined a standard microbiological culture step, using a pre-enrichment medium that favors the growth of the three focal microorganisms: SEL broth, followed by a single PCR assay. A total of 43 interference bacterial strains were used to evaluate the method’s specificity. The detection rate for the microbiological method with standard culture media was 10 UFC/mL, and that of the PCR detection, following pre-enrichment in SEL broth, was 10 UFC/mL for S. enterica and L. monocytogenes and between 1 and 5 UFC/mL for E. coli O157:H7. The PCR method showed specificity for the reference strains. Simultaneous detection by multiple PCR using SEL broth was successful for the detection of S. enterica, E. coli O157:H7, and L. monocytogenes in samples of experimentally contaminated cow milk, featuring both a high detection rate and a high specificity. This approach promises to be a feasible routine procedure when testing milk samples in industry and public health control setups.
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HUSSEIN, HUSSEIN S., LAURIE M. BOLLINGER, and MARK R. HALL. "Growth and Enrichment Medium for Detection and Isolation of Shiga Toxin–Producing Escherichia coli in Cattle Feces." Journal of Food Protection 71, no. 5 (May 1, 2008): 927–33. http://dx.doi.org/10.4315/0362-028x-71.5.927.
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Detection methods of Shiga toxin–producing Escherichia coli (STEC) in cattle feces varied in using enrichment media containing different antibiotic combinations. To examine efficacy of a new detection method for STEC, three O157:H7 (ATCC 43889, 43890, and 43895) and 41 non-O157:H7 (members of the O1, O15, O26, O86, O103, O111, O125, O127, O128, O136, O146, O153, O158, O165, O166, and O169 serogroups) isolates were tested. These isolates were grown in tryptic soy broth for 6 h, and their concentrations were determined before inoculation of tubes containing 1 g of cattle feces (sterile [experiment 1; evaluating growth] and fresh [experiment 2; evaluating enrichment]) to simulate the high and low levels of STEC shedding by cattle (105 versus 102 CFU/g feces, respectively). Eight STEC isolates (the three O157:H7 and five non-O157:H7 selected at random) were tested at a very low level (10 CFU/g feces). The feces were incubated in 50 ml of brain heart infusion broth containing potassium tellurite, novobiocin, and vancomycin (2.5, 20, and 40 mg/liter, respectively) and cefixime (50 μg/liter) at 37°C for 12 h and tested for STEC (VTEC [verotoxin-producing E. coli]–Screen assay [agglutination immunoassay]). Potential STEC isolates were recovered, characterized biochemically, serotyped, and tested for toxin production using Vero (African green monkey kidney) cell toxicity assay and agglutination immunoassay. In both experiments, all the STEC isolates used for fecal inoculation were recovered at the concentrations tested. Our medium supported growth of and enrichment for a wide range of STEC isolates.
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Wang, Jiapeng, and Siu-Tung Yau. "Ultrasensitive and rapid detection of Escherichia coli O157:H7 in beef juice using immunoassay based on field-effect enzymatic detection." Anal. Methods 6, no. 14 (2014): 5387–91. http://dx.doi.org/10.1039/c4ay00593g.
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Gilmour, Matthew W., Linda Chui, Theodore Chiu, Dobryan M. Tracz, Kathryn Hagedorn, Lorelee Tschetter, Helen Tabor, Lai King Ng, and Marie Louie. "Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods." Journal of Medical Microbiology 58, no. 7 (July 1, 2009): 905–11. http://dx.doi.org/10.1099/jmm.0.007732-0.
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The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.
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NYQUIST-BATTIE, CYNTHIA, LAURA E. FRANK, DEANNA LUND, and DANIEL V. LIM. "Optimization of a Fluorescence Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Escherichia coli O157:H7 in Apple Juice." Journal of Food Protection 67, no. 12 (December 1, 2004): 2756–59. http://dx.doi.org/10.4315/0362-028x-67.12.2756.
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Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 104 to 106 CFU/ml, whereas that for spiked phosphate-buffered saline was 106 to 108 CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.
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Gareau, Mélanie G., Nathan K. Ho, Dirk Brenner, Andrew J. Sousa, Lionel LeBourhis, Tak W. Mak, Stephen E. Girardin, Dana J. Philpott, and Philip M. Sherman. "Enterohaemorrhagic, but not enteropathogenic, Escherichia coli infection of epithelial cells disrupts signalling responses to tumour necrosis factor-alpha." Microbiology 157, no. 10 (October 1, 2011): 2963–73. http://dx.doi.org/10.1099/mic.0.051094-0.
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Enterohaemorrhagic Escherichia coli (EHEC), serotype O157 : H7 is a non-invasive, pathogenic bacterium that employs a type III secretion system (T3SS) to inject effector proteins into infected cells. In this study, we demonstrate that EHEC blocks tumour necrosis factor-alpha (TNFα)-induced NF-κB signalling in infected epithelial cells. HEK293T and INT407 epithelial cells were challenged with EHEC prior to stimulation with TNFα. Using complementary techniques, stimulation with TNFα caused activation of NF-κB, as determined by luciferase reporter assay (increase in gene expression), Western blotting (phosphorylation of IκBα), immunofluorescence (p65 nuclear translocation) and immunoassay (CXCL-8 secretion), and each was blocked by EHEC O157 : H7 infection. In contrast, subversion of host cell signalling was not observed following exposure to either enteropathogenic E. coli, strain E2348/69 (O127 : H6) or the laboratory E. coli strain HB101. Heat-killed EHEC had no effect on NF-κB activation by TNFα. Inhibition was mediated, at least in part, by Shiga toxins and by the O157 plasmid, but not by the T3SS or flagellin, as demonstrated by using isogenic mutant strains. These findings indicate the potential for developing novel therapeutic targets to interrupt the infectious process.
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Li, Baoguang, and Jin-Qiang Chen. "Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker." Applied and Environmental Microbiology 78, no. 15 (May 25, 2012): 5297–304. http://dx.doi.org/10.1128/aem.00794-12.
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ABSTRACTThe goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viableEscherichia coliO157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection ofE. coliO157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific forE. coliO157:H7 strains (n= 298). Using this assay, we can detect amounts of genomic DNA ofE. coliO157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)–real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 107dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viableE. coliO157:H7 cells with an 8-h enrichment. In conclusion, this PMA–real-time PCR assay offers a sensitive and specific means to selectively detect viableE. coliO157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viableE. coliO157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.
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Qin, Xuan, Eileen J. Klein, Emmanouil Galanakis, Anita A. Thomas, Jennifer R. Stapp, Shannon Rich, Anne Marie Buccat, and Phillip I. Tarr. "Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples." Journal of Clinical Microbiology 53, no. 7 (April 29, 2015): 2148–53. http://dx.doi.org/10.1128/jcm.00115-15.
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Timely accurate diagnosis of Shiga toxin-producingEscherichia coli(STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targetingstx1,stx2, andrfbEO157with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagicE. coli[EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC.E. coliO157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 usingrfbEO157, and LD-PCR results prompted successful recovery ofE. coliO157 (n= 25) and non-O157 STEC (n= 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and thatE. coliO157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.
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DeMARCO, DANIEL R., and DANIEL V. LIM. "Detection of Escherichia coli O157:H7 in 10- and 25-Gram Ground Beef Samples with an Evanescent-Wave Biosensor with Silica and Polystyrene Waveguides." Journal of Food Protection 65, no. 4 (April 1, 2002): 596–602. http://dx.doi.org/10.4315/0362-028x-65.4.596.
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A portable evanescent-wave fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded 10- and 25-g ground beef samples. The biosensor works by launching light from a 635-nm laser diode into specially designed optical fiber probes, generating an evanescent field that extends approximately 1,000 nm from the fiber surface. Fluorescent molecules within the evanescent field are excited, and a portion of their emission recouples into the fiber probe. The return path emission is transported by an optical fiber to a photodiode within the biosensor that detects and quantifies the fluorescent signal. A sandwich immunoassay was performed on the fiber probes with cyanine 5 dye–labeled polyclonal anti–E. coli O157:H7 antibodies for generation of the specific fluorescent signal. Biotin-streptavidin interactions were used to attach polyclonal antiE. coli O157:H7 antibodies to the surface of the fiber probe. A centrifugation method was developed to obtain samples suitable for biosensor analysis from 10- and 25-g ground beef samples. The assay was shown to be sensitive and repeatable. One hundred percent correct identification of positive samples was demonstrated at 9.0 × 103 CFU/g for 25-g ground beef samples with silica waveguides and at 5.2 × 102 CFU/g for 10-g ground beef samples with polystyrene waveguides. The reaction was highly specific. No false positives were observed for 10-g ground beef samples not spiked with the pathogen. In addition, when samples were spiked with high concentrations of a variety of non–E. coli O157:H7 organisms, no false positives were observed. The method was rapid, with results being obtained within 25 min of sample processing.
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HEUVELINK, ANNET E., JOHANNA T. M. ZWARTKRUIS.NAHUIS, and ENNE DE BOER. "Evaluation of Media and Test Kits for the Detection and Isolation of Escherichia coli O157 from Minced Beef." Journal of Food Protection 60, no. 7 (July 1, 1997): 817–24. http://dx.doi.org/10.4315/0362-028x-60.7.817.
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This study has evaluated the efficacy of selective enrichment and plating media used for the isolation of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157, by examining pure bacterial cultures. In addition, the performance of a variety of commercial test kits for the detection of E. coli O157 strains inoculated into minced beef was compared, using the Ampcor E. coli O157:H7 Kit, 3M Petrifilm™ Test Kit-HEC, Dynabeads anti-E coli O157, EHEC-TEK™, and the Tecra E. coli O157 visual immunoassay. The commercial Verotox F test for the determination of the VT type of VTEC isolates was compared with a polymerase chain reaction (PCR) assay for VT-coding genes. Modified E. coli broth containing novobiocin (mEC + n) and sorbitol MacConkey agar supplemented with cefixime and tellurite (CT-SMAC) were the most efficacious media for selective enrichment and isolation, respectively. After enrichment of the inoculated samples, all kits tested could detect less than one O157 VTEC cell per g of minced beef. While the results of the immunoassays need to be confirmed by isolating the organisms, the use of the immunomagnetic separation technique directly yields isolates. The results of the Verotox F test were consistent with PCR results. A sensitive and cost-effective method for the isolation of O157 VTEC from minced beef in food industry and epidemiological studies involving large numbers of samples is the following: enrichment in mEC + n at 37°C for 6 to 8 h with shaking at 100 rpm, followed by immunomagnetic separation using Dynabeads anti-E. coli O157 and spread plating of the concentrated target cells onto CT-SMAC. The Verotox F test can be used to determine whether the isolates produce VTl and/or VT2.
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JUNG, YANGJIN, ANNA C. S. PORTO-FETT, BRADLEY A. SHOYER, LAURA E. SHANE, ELIZABETH HENRY, MANUELA OSORIA, and JOHN B. LUCHANSKY. "Survey of Intact and Nonintact Raw Pork Collected at Retail Stores in the Mid-Atlantic Region of the United States for the Seven Regulated Serogroups of Shiga Toxin–Producing Escherichia coli." Journal of Food Protection 82, no. 11 (October 10, 2019): 1844–50. http://dx.doi.org/10.4315/0362-028x.jfp-19-192.
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ABSTRACT A total of 514 raw pork samples (395 ground or nonintact and 119 intact samples) were purchased at retail stores in Pennsylvania, Delaware, and New Jersey between July and December 2017. All raw pork samples were screened for serogroup O26, O45, O103, O111, O121, O145, or O157:H7 cells of Shiga toxin–producing Escherichia coli (STEC-7) using standard microbiological and molecular methods. In short, 21 (5.3%) of the 395 ground or nonintact pork samples and 3 (3.4%) of the 119 intact pork samples tested positive via the BAX system real-time PCR assay for the stx and eae virulence genes and for the somatic O antigens for at least one of the STEC-7 serogroups. However, none of these 24 presumptive-positive pork samples subsequently yielded a viable isolate of STEC displaying a STEC-7 serogroup-specific surface antigen in combination with the stx and eae genes. These data suggest that cells of STEC serogroups O26, O45, O103, O111, O121, O145, or O157:H7 are not common in retail raw pork samples in the mid-Atlantic region of the United States.
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WANG, HAIYAN, ERIN BOYLE, and JEFF FARBER. "Rapid and Specific Enzyme Immunoassay on Hydrophobic Grid Membrane Filter for Detection and Enumeration of Thermophilic Campylobacter spp. from Milk and Chicken Rinses." Journal of Food Protection 63, no. 4 (April 1, 2000): 489–94. http://dx.doi.org/10.4315/0362-028x-63.4.489.
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Six commercially available anti-Campylobacter antibodies were examined for their applicability in an enzyme immuno-assay on hydrophobic grid membrane filters, both for the detection and enumeration of Campylobacter spp. When a panel of nine Campylobacter (seven Campylobacter jejuni and two Campylobacter coli) and eight non-Campylobacter strains were used in a dot-blot format enzyme immunoassay to test the specificity of these antibodies, only one polyclonal antibody (Biodesign) detected all Campylobacter strains. Escherichia coli O157:H7 produced weak nonspecific signals due to endogenous peroxidase activity. The specificity of this Biodesign antibody was further tested against 30 more Campylobacter strains and more than 600 non-Campylobacter strains on hydrophobic grid membrane filters grown on modified Campylobacter agar with charcoal and deoxycholate, a Campylobacter selective medium. All the Campylobacter strains were detected, whereas only two (Acinetobacter calcoaceticus, Salmonella Minnesota) of the approximately 130 non-Campylobacter strains, which grew on modified Campylobacter agar with charcoal and deoxycholate, gave false-positive signals. This simple, rapid, and specific enzyme immunoassay also detected Campylobacter spp. from inoculated milk and chicken rinses and naturally contaminated chicken rinses.
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ZHAO, TONG, and MICHAEL P. DOYLE. "Evaluation of Universal Preenrichment Broth for Growth of Heat-Injured Pathogens." Journal of Food Protection 64, no. 11 (November 1, 2001): 1751–55. http://dx.doi.org/10.4315/0362-028x-64.11.1751.
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Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis, and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2°C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37°C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (>103 CFU/ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37°C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation—but not at 6 h—sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.
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KIM, KEUN Y., P. MICHAEL DAVIDSON, and HEE J. CHUNG. "Antibacterial Activity in Extracts of Camellia japonica L. Petals and Its Application to a Model Food System." Journal of Food Protection 64, no. 8 (August 1, 2001): 1255–60. http://dx.doi.org/10.4315/0362-028x-64.8.1255.
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The potential presence of naturally occurring antimicrobials in petals of Camellia japonica L., a member of the tea family, was investigated against foodborne pathogens in microbiological media and food. Petals of the camellia flower (C. japonica L.) were extracted with methanol and fractionated into basic, acidic, and neutral fractions. The acidic fraction (equivalent to 1.0 g of raw sample per disk) produced an inhibitory zone of 14 to 19 mm (diameter) in a disk assay against the pathogens Salmonella Typhimurium DT104, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on agar plates. Silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, and preparative purification by high-pressure liquid chromatography were used to purify compounds in the fraction. The mass spectrum of the antibacterial compound isolated had a molecular ion (M+) of m/z 116 and showed good conformity with the spectrum of fumaric acid (HOOC-CH=CH-COOH). An aqueous extract from the petals of C. japonica L. had an inhibitory effect on growth of all pathogens at 37°C in microbiological media by increasing the lag phase. None of the microorganisms was inhibited completely. Milk was used as a model food system. Aqueous extract at a concentration of 100 mg/ml was bacteriostatic against all the foodborne pathogens in the milk stored at 25°C for up to 4 days.
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BEIZA, ALBERTO A., ZAHRA H. MOHAMMAD, and SUJATA A. SIRSAT. "Persistence of Foodborne Pathogens on Farmers' Market Fomites." Journal of Food Protection 84, no. 7 (February 26, 2021): 1169–75. http://dx.doi.org/10.4315/jfp-20-406.
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ABSTRACT The number of farmers' markets registered by the U.S. Department of Agriculture has seen a significant increase, jumping from 1,755 in 1994 to 8,771 in 2019. Although previous studies have investigated the microbial profile of produce obtained from farmers' markets, literature exploring microbial (bacteria and virus) persistence on a variety of different farmers' market fomites over a 2-month period is limited. The objective of the current study was to investigate the persistence of key foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and MS2 bacteriophage) on these fomites by using a microbiological viability assay. The MS2 bacteriophage was quantified by using a host E. coli strain, and PFU were identified. A repeated measures analysis of variance was used to compare the persistence rates of foodborne pathogens on cardboard, plastic, tablecloth, molded pulp fiber, and wicker baskets used to store, transport, and display produce at farmers' markets. In general, molded pulp fiber, plastic, and wicker surface materials supported the persistence of foodborne pathogens the most, with Salmonella and S. aureus demonstrating the highest log concentrations over the longest period of time. Additionally, E. coli strains also persisted for a significant period of time on all fomites, with the exception of tablecloth. The results suggest that foodborne pathogens on these fomites pose a high risk of cross-contamination, particularly if the fomites cannot be washed, rinsed, and sanitized effectively (e.g., cardboard). The results highlight the need to avoid using porous, single-use storage containers, such as cardboard, molded pulp fiber, and wicker containers for extended periods of time and suggest the use of easily cleanable materials, such as plastic containers. HIGHLIGHTS
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Han, Lu, Kaidi Wang, Lina Ma, Pascal Delaquis, Susan Bach, Jinsong Feng, and Xiaonan Lu. "Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment." Applied and Environmental Microbiology 86, no. 7 (January 31, 2020). http://dx.doi.org/10.1128/aem.02566-19.
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ABSTRACT Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the “viable but nonculturable” (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.