TY - JOUR
AU - Choudary, Prabhakara V.
AB - Abstract The immuno-polymerase chain reaction (PCR) approaches facilitate rapid (8 h) detection of Escherichia coli O157:H7 in contaminated dairy products and ground beef samples with detection sensitivities approaching 1 colony forming unit (cfu) g−1 ml−1. However, no PCR products were obtained when the method was applied to identify E. coli O157:H7 in tainted apple juice. Enzyme-linked immuno-assay (ELISA) results suggested non-specific binding of endogenous polyphenols (ubiquitous in plant products) to antibodies present on the surface of the immunobeads, making the latter unavailable for capturing the target bacteria. Treatment of the test sample, prior to IMS, with a synthetic fining agent, polyvinylpyrrolidone, restored the full function and sensitivity of the immuno-PCR. The study demonstrates the suitability of the improved method as a generic strategy for rapid screening of fruit juices and plant produce for E. coli O157:H7. Enterohemorrhagic Escherichia coli, Food poisoning, Gastroenteritis, Hazard analysis critical control point, Immunomagnetic separation, PCR, Polyphenol, Polyvinylpyrrolidone 1 Introduction The enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an emerging foodborne microbial pathogen of growing concern worldwide [1]. EHEC has originally been discovered in association with tainted ground beef and dairy products [2]. But, in the past few years, EHEC contamination has spread to previously unsuspected food items, including beverages — fruit juices, in particular [3]. The EHEC outbreak in 1996 in the western US and Canada, that culminated in the death of a 16-month-old Colorado girl and sickened at least 66 people, has resulted from drinking commercial non-pasteurized apple juice tainted with EHEC [4]. This led to the first ever criminal conviction of the manufacturer of the contaminated food item; in this case, Odwalla of Half Moon Bay, CA, to pay a $1.5 million criminal penalty for admitted failure to take adequate steps to prevent the contamination. Japan has experienced a severe EHEC epidemic in 1996 with over 6000 school children falling seriously ill, after eating EHEC-contaminated radish sprouts [5]. The 1997 outbreak, that sickened at least 60 people in Michigan and 48 in Virginia, has been traced back to EHEC-contaminated alfalfa sprouts, extending the microbial safety concerns to pre-packaged fresh salads. The infections breaking out in the USA in 1998 from EHEC-tainted potato salad, public pool water and the vegetables, point to the widening diversity of foods this pathogen is associating with. Considering the most common source of EHEC contamination, i.e. the soiling with cattle feces and manure, it is not surprising that vegetables, fruits and their products are contaminated, just as the cattle and their products. Therefore, there is a clear need for rapid methods that can reliably identify EHEC in fruit juices and fresh produce so that effective methods of pathogen reduction can be devised. The antibody-based immunomagnetic separation/capture (IMS) and/or immunoassay [6] and the nucleic acid-based polymerase chain reaction (PCR) [7] have been playing an increasingly prominent role in the new technologies being developed for the detection of foodborne microbial pathogens. IMS and PCR, individually or in combination, have been applied successfully for rapid and sensitive detection of EHEC and other pathogens in a variety of meats and the dairy foods [8]. However, there have been few published methods, tailored for the analysis of fruit juices, fruits and vegetables for potential pathogens. We found the immuno-PCR approaches including those developed recently in our laboratory for beef and dairy products [9] to be unsuitable for direct application to screen fruit juices for EHEC contamination. Since the IMS-PCR method works well for the analysis of beef and dairy foods, we reasoned that the failure encountered in applying it to plant produce/fruit juices should be due to inhibition of the method by components selective to plant products. Examination of fruit juices and fresh produce extracts in an ELISA test for inhibition of each of the steps of our enrichment-IMS-PCR method validated our hypothesis. Furthermore, adsorption of endogenous polyphenols with a synthetic fining agent, polyvinylpyrrolidone (PVP), removed the observed interference. We report here the inhibition of IMS-PCR by fresh produce and fruit juices, describe the adsorption step preceding IMS, and demonstrate extended use of the immuno-PCR method for rapid detection of EHEC in fruit juices. 2 Materials and methods 2.1 Materials Reagent grade PVP was obtained from Merck KgaA (Darmstadt, Germany), polyvinylpolypyrrolidone (Polyclar PVPP) from Cellulo (Fresno, CA), and polyethylene glycol (PEG) 6000 and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Pre-packaged pasteurized apple juice (Gold medal, Martinelli, Watsonville, CA), grape juice (Libby's Juicy juice, Nestle USA, Glendale, CA), peach juice (Safeway, Oakland, CA), green tea (Good Earth Teas, Santa Cruz, CA), slabs of milk chocolate (Hershey Foods, Hershey, PA), fresh leaves of kale and spinach, fresh tomatoes, dried apricots (Mediterranean, TownHouse, Safeway Stores, Oakland, CA), roasted coffee beans, wheat flour (Pillsbury, Cedar Rapids, IA) and red wine (Red Sangria, Carlo Rossi Vineyards, Modesto, CA) were purchased from a local grocery. Betel nut was procured from Rathnam Nut Powder, Puttur, AP, India. 2.2 Bacterial strain and culture conditions The E. coli O157:H7 DECA 4B was a generous gift from Kimberly Seebart and Richard Wilson of the Escherichia coli Reference Center, Pennsylvania State University, University Park, PA. It was grown overnight at 37°C in 50 ml tryptic soy broth (TSB; Difco, Detroit, MI) in a 100-ml Erlenmeyer flask. Plate counts (cfu ml−1) of EHEC inocula were determined by spread plating serial dilutions (in the range of 102 to 106) of overnight cultures. Each dilution (100 µl) was plated on MacConkey sorbitol agar (SMAC; Difco, Detroit, MI) [10] containing cefimide (0.05 mg l−1) and potassium tellurite (2.5 mg l−1) (SMACCT) [11], and colonies on the plates following overnight incubation at 37°C were enumerated. 2.3 Sample preparation Leaf extracts of kale and spinach were each prepared, by homogenizing fresh leaves (~10 g) for 3–5 min at top speed in a blender (Osterizer Pulsematic 12, Sunbeam Oster, Milwaukee, WI) and extracting with cold distilled water (10 ml). One—gram samples each of betel nut, coffee beans, milk chocolate and green tea were separately extracted for 5 min at 25°C with 2 g of sterile acid-washed glass beads (0.45 mm mesh size, Braun Melsungen, Germany) in 20 ml water, using a porcelain mortar and pestle. The homogenates were clarified by centrifugation (Model J2-21 M/E, Beckman Coulter, Fullerton, CA) at 5000 rpm for 5 min at 4°C using a JS 7.5 rotor. The clarified extracts, without any exogenous additions, such as antioxidants, were maintained on ice for the same-day use, or were stored frozen at −20°C for future use. Wheat flour was extracted as above, with 2 vols. of water, and red wine was used directly from the source container. 2.4 Spiking of fruit juices and produce extracts with EHEC The fruit juices and produce extracts (9 ml) were each inoculated with 1 ml overnight culture of EHEC, vortex mixed to a homogeneous suspension and held on laboratory bench until the particulate matter settled down. Each spiked sample was then diluted serially to give 1.5×105 to 0.1 cfu ml−1. 2.5 ELISA procedure Microplates (96-well, Maxisorp, Nalge Nunc International, Rochester, NY) in triplicates were coated overnight at room temperature with mouse IgG (0–200 ng) in 100 µl coating buffer (15 mM Na2CO3, 35 mM NaHCO3 and 3 mM NaN3, pH 9.6). The coated plates were blocked with 200 µl Tris buffer-saline-Tween-20 (TBST: 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20), containing 2% bovine serum albumin (BSA) and were incubated at room temperature for 2 h. The blocking agent was then replaced with test sample (100 µl). The samples included apple, apricot, blueberry, grape, peach, or tomato juice, the extract of kale leaves, spinach leaves, betel nut, coffee beans, green tea, milk chocolate, wheat flour, or red wine. The plates, incubated for 2 h at room temperature, were washed 5× with TBST and incubated for 2 h at room temperature with goat anti-mouse IgG (H+L)-horseradish peroxidase (HRP) conjugate (Immuno-Select, Life Science Technologies, Gaithersburg, MD). The plates were again washed 4× with TBST, and the HRP activity of the bound antibody was assayed for 10 min at 37°C, using the substrates: O-phenylene diamine (OPD) (30 mg) and H2O2 (30 µl), dissolved in 75 ml phosphate-citrate buffer (0.1 M citric acid, 0.2 M Na2HPO4, pH 5.0). The reaction was terminated by the addition of 1 M H2SO4 (100 µl), and the plates were read at 492 nm, using the ‘UVmax kinetic microplate reader’ (Molecular Devices, Menlo Park, CA). All samples were normalized against a sample blank, before reading the control and test samples. The results were analyzed using the software package, ‘Softmax’ (Molecular Devices, Menlo Park, CA). 2.6 Culture enrichment The spiked apple juice (5 ml) was aseptically added to sterile TSB (95 ml) in a 250-ml Erlenmeyer flask and incubated at 37°C for 4 h. To test the effect of pH on the performance of IMS-PCR, TSB, stabilized with 5.0 mM phosphate buffer, pH 8.0 (TSBP), was used. 2.7 Adsorption with fining agents Stock solutions of fining agents (PVP, PVPP, PEG, BSA or free glycine) were prepared by dissolving 20 mg powder in 1 ml water and filter-sterilizing. The stock solutions (1.0 ml) were each mixed with the enriched culture (5 ml) in a 15-ml Falcon tube (Becton Dickinson, Franklin Lakes, NJ) and incubated at 37°C for 25 min with shaking at 120 rpm on a gyratory shaker (Model G24, New Brunswick Scientific, Edison, NJ). At the end of the adsorption step, 1-ml portions of the treated samples were used directly in the IMS step. 2.8 IMS Twenty microliters of magnetic beads coated with anti-E. coli O157 antibody (Dynabeads Anti-E. coli 0157, Dynal, Lake Success, NY) were added to each of the PVP-treated cultures (1 ml each) in 1.5-ml microfuge tubes and incubated at room temperature for 20 min with agitation at 75 rpm, on a vortex mixer (Genie2, Fisher Scientific, Pittsburgh, PA). The EHEC-bound immunobeads were held in place with the aid of a magnetic particle concentrator (Promega, Madison, WI) and washed 3× with PBS (0.01 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl) containing 0.05% (w/v) Tween-20, according to the manufacturer's instructions. Direct amplification (skipping the IMS step) of the Stx genes off the bacterial pellet from a 1-ml portion of the enriched culture served as negative control. 2.9 Amplification and detection of PCR products PCR amplifications were performed as in [8] using the following primer set: 5′-ATACAGAG(AG)G(GA)ATTTCGT-3′ (forward) and 5′-TGATGATG(AG)CAATTCAGTTAT-3′ (reverse), adapted from Paton et al. [12]. These primers, with a G+C content of 34% and a Tm of 56°C, are specific for the plasmid-borne Stx1 and Stx2 genes of EHEC [13] and produce a 215/212 bp amplicon [14]. They were synthesized by the UC Davis Protein Structure Laboratory, using an ABI Automatic DNA synthesizer Model 380A (Applied Biosystems, Foster city, CA). The PCR cocktail (100 µl) contained: 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.15% (v/v) Triton X-100, 1.8 mM MgCl2, 0.2 mM each of the deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Piscataway, NJ), 1.5 U Taq DNA Polymerase (Promega, Madison, WI) and 0.5 µM each of the primers. It was added to EHEC-bound immunobeads in 0.5 ml PCR tubes (Fisher Scientific, Pittsburgh, PA) and overlaid with sterile mineral oil (50 µl). PCR amplifications were performed using a thermocycler (Model PTC 150, M.J. Research, Watertown, MA). A cell lysis-cum-initial denaturation step at 95°C for 5 min was followed by 35 cycles of denaturation at 92°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. Ten microliters of each PCR reaction were electrophoresed for 20 min at 100 V in a horizontal agarose (1.5% w/v) slab in 1×Tris-acetate-EDTA buffer, pH 8.0, containing ethidium bromide (0.05%, w/v) [15], using a mini-gel electrophoresis unit (Mupid-2, Cosmo Bio, Tokyo, Japan). HaeIII-digested PhiX DNA (Life Technologies, Gaithersburg, MD) served as Mr size marker. DNA bands in the gel were visualized on a UV transilluminator and photographed using the Polaroid camera system (Photo/PrepI, Fotodyne, Hartland, WI). 3 Results 3.1 Fruit juices lower the pH of the enrichment culture and inhibit IMS and PCR The method typically comprises of: culture enrichment of total bacterial population of the test sample (4 h±10 min), immunocapture of target bacteria (20 min), PCR amplification of the signature sequences of the pathogen (110 min), and gel electrophoretic identification of the PCR amplicons (30 min), with a 70-min preparation time for assembling various reactions and steps, including the polyphenol adsorption step newly introduced here. In the absence of the adsorption step, addition of even 1 µl of apple juice to the IMS step or to the PCR mixture completely abolished amplification of the Stx sequences (data not shown). Direct enrichment of EHEC-spiked fruit juice and plant produce samples resulted in considerable lowering of the pH of the culture medium by the end of 4 h, and yielded no amplification products, following immuno-PCR. However, maintaining the pH constant at 7.0 by buffering the medium with phosphates did not circumvent the inhibition. 3.2 Plant products interfere with antigen binding activity of the antibody The possible mode and the magnitude of inhibition of IMS by fruit juices and produce extracts were investigated using sandwich ELISA test, by monitoring the decrease in the total binding activity of an anti-mouse antibody to mouse IgG in the presence of plant products. The antibody binding activity was determined (colorimetrically) as a function of the catalytic activity of the reporter enzyme, HRP, conjugated to the anti-mouse antibody used as tracer in the ELISA. The binding activity of the antibody was completely inhibited by all the fruit juices and produce extracts tested (Fig. 1). Milk chocolate (Fig. 1) and red wine (data not shown) also inhibited antigen binding activity of the antibody. Exogenously added (0.25 mg ml−1) condensed tannins (proanthocyanidins, with catechin, epicatechin, epicatechin gallate and epigallocatechin as major constituent units) extracted from wine grapes inhibited the antibody binding activity completely in ELISA (data not shown). Figure 1 View largeDownload slide Effect of fruit juices and produce extracts on antigen-binding activity of the antibody. Test food samples (apple, apricot, peach, blueberry, tomato or grape juice, the extract of spinach leaves, kale leaves, betel nut, coffee beans, green tea or milk chocolate (100 µl each)) were separately added to microplates, coated with mouse IgG (0–200 ng) and blocked with 2% BSA in TBST. The plates were incubated for 2 h at room temperature, washed 5× with TBST and re-incubated with HRP-conjugate of goat anti-mouse IgG (H+L). Plates were again washed 4× with TBST, and developed for 10 min at 37°C with OPD (30 mg) and H2O2 (30 µl). The reaction was terminated, and the plates were read at 492 nm. The A492 readings, corrected against a sample blank, were plotted against the IgG concentration (ng protein well−1). The extent of reduction in the A492 values (HRP activity) directly corresponds to the degree of inhibition caused by the test samples on antigen (mouse IgG)-binding activity of the tracking antibody(−HRP). Open diamonds, positive control (a complete ELISA reaction with none of the fruit juices, extracts or other test samples added); filled diamonds, milk chocolate; filled squares, grape juice; open circles, apple juice; open triangles, peach juice; open squares, spinach extract; filled triangles, apricot extract; filled circles, negative control (ELISA reaction minus tracking antibody and without any test sample). Figure 1 View largeDownload slide Effect of fruit juices and produce extracts on antigen-binding activity of the antibody. Test food samples (apple, apricot, peach, blueberry, tomato or grape juice, the extract of spinach leaves, kale leaves, betel nut, coffee beans, green tea or milk chocolate (100 µl each)) were separately added to microplates, coated with mouse IgG (0–200 ng) and blocked with 2% BSA in TBST. The plates were incubated for 2 h at room temperature, washed 5× with TBST and re-incubated with HRP-conjugate of goat anti-mouse IgG (H+L). Plates were again washed 4× with TBST, and developed for 10 min at 37°C with OPD (30 mg) and H2O2 (30 µl). The reaction was terminated, and the plates were read at 492 nm. The A492 readings, corrected against a sample blank, were plotted against the IgG concentration (ng protein well−1). The extent of reduction in the A492 values (HRP activity) directly corresponds to the degree of inhibition caused by the test samples on antigen (mouse IgG)-binding activity of the tracking antibody(−HRP). Open diamonds, positive control (a complete ELISA reaction with none of the fruit juices, extracts or other test samples added); filled diamonds, milk chocolate; filled squares, grape juice; open circles, apple juice; open triangles, peach juice; open squares, spinach extract; filled triangles, apricot extract; filled circles, negative control (ELISA reaction minus tracking antibody and without any test sample). 3.3 Adsorption with PVP alleviates polyphenol-mediated inhibition of IMS-PCR Fig. 2 shows the efficacy of PVP treatment in enabling the method to detect various levels of EHEC contamination of spiked apple juice. EHEC was tested in the range of 1 and 1.5×105 cfu sample−1 (Fig. 2, lanes 5–10). EHEC was detected to 1 cfu in the PVP-treated sample (Fig. 2, lane 5), while the negative control containing no template DNA or EHEC (Fig. 2, lane 3) and untreated samples yielded no amplification products (data not shown). All the fruit juices tested, produce extracts and other polyphenol-rich foods spiked with EHEC, upon treatment with PVP, each yielded a 215/212-bp amplicon in immuno-PCR, as shown in Fig. 3. Among other adsorption agents tested, PVPP and PEG, unlike BSA and free glycine, countered the inhibition as effectively as PVP (data not shown). Figure 2 View largeDownload slide Agarose (1.5%, w/v) gel electrophoresis of PCR products obtained from spiked fruit juice samples containing EHEC in the range of 0.1 to 1.5×105 cfu sample−1. Lanes 1 and 11, DNA size markers (HaeIII digest of PhiX 174 RF DNA (bp, from top to bottom, 1353, 1078, 872, 603, 310, 281/271, 234, 194, 118, 72)); lane 2, positive control (EHEC, 1.5×103 cfu sample−1); lane 3, negative control (without template DNA or cells); lanes 4–10, apple juice (10 µl) spiked with different sizes of EHEC inocula (0.1, 1, 10, 1.5×102, 1.5×103, 1.5×104 or 1.5×105 cfu sample−1, respectively). The spiked samples were subjected to enrichment, PVP-treatment, immunocapture and amplification, as described in the text. The appearance of a 215/212-bp DNA band (shown by an arrow to the left of the gel), corresponding to the Stx1/Stx2 amplicon, is indicative of contamination with EHEC and detection by the method described. Figure 2 View largeDownload slide Agarose (1.5%, w/v) gel electrophoresis of PCR products obtained from spiked fruit juice samples containing EHEC in the range of 0.1 to 1.5×105 cfu sample−1. Lanes 1 and 11, DNA size markers (HaeIII digest of PhiX 174 RF DNA (bp, from top to bottom, 1353, 1078, 872, 603, 310, 281/271, 234, 194, 118, 72)); lane 2, positive control (EHEC, 1.5×103 cfu sample−1); lane 3, negative control (without template DNA or cells); lanes 4–10, apple juice (10 µl) spiked with different sizes of EHEC inocula (0.1, 1, 10, 1.5×102, 1.5×103, 1.5×104 or 1.5×105 cfu sample−1, respectively). The spiked samples were subjected to enrichment, PVP-treatment, immunocapture and amplification, as described in the text. The appearance of a 215/212-bp DNA band (shown by an arrow to the left of the gel), corresponding to the Stx1/Stx2 amplicon, is indicative of contamination with EHEC and detection by the method described. Figure 3 View largeDownload slide Agarose (1.5%, w/v) gel electrophoresis of PCR products from different fruit juices and extracts of fresh produce spiked with EHEC. The spiked juice/extract samples were diluted to 1.5×103 cfu sample−1, and enriched for 4 h, followed by PVP treatment and IMS-PCR. A 215/212-bp band (shown by an arrow to the left of the gel) corresponds to the Stx1/Stx2 amplicon and indicates the presence of EHEC in the test sample. Lane 1, positive control (EHEC culture, 1.5×103 cfu sample−1); lane 2, negative control (no cells or template DNA); lane 3, red wine; lanes 4–10, extracts (lane 4, wheat flour; lane 5, betel nut; lane 6, green tea; lane 7, coffee bean; lane 8, chocolate; lane 9, spinach leaf; lane 10, kale leaf); lanes 11–16, juices (lane 11, tomato; lane 12, blue berry; lane 13, peach; lane 14, wine grape; lane 15, apricot; lane 16, apple); lane 17, DNA size markers (HaeIII digest of PhiX 174 RF DNA (bp, from top to bottom, 1353, 1078, 872, 603, 310, 281/271, 234, 194, 118, 72)). Figure 3 View largeDownload slide Agarose (1.5%, w/v) gel electrophoresis of PCR products from different fruit juices and extracts of fresh produce spiked with EHEC. The spiked juice/extract samples were diluted to 1.5×103 cfu sample−1, and enriched for 4 h, followed by PVP treatment and IMS-PCR. A 215/212-bp band (shown by an arrow to the left of the gel) corresponds to the Stx1/Stx2 amplicon and indicates the presence of EHEC in the test sample. Lane 1, positive control (EHEC culture, 1.5×103 cfu sample−1); lane 2, negative control (no cells or template DNA); lane 3, red wine; lanes 4–10, extracts (lane 4, wheat flour; lane 5, betel nut; lane 6, green tea; lane 7, coffee bean; lane 8, chocolate; lane 9, spinach leaf; lane 10, kale leaf); lanes 11–16, juices (lane 11, tomato; lane 12, blue berry; lane 13, peach; lane 14, wine grape; lane 15, apricot; lane 16, apple); lane 17, DNA size markers (HaeIII digest of PhiX 174 RF DNA (bp, from top to bottom, 1353, 1078, 872, 603, 310, 281/271, 234, 194, 118, 72)). 4 Discussion We have recently reported a modular immuno-PCR approach for the detection of EHEC in dairy products in a total assay time of 8 h [9]. The application of the method has successfully been extended to the rapid screening of ground beef samples spiked with EHEC as well as to etiological confirmation of contaminated hamburgers and beef patties from earlier EHEC outbreaks [8]. However, our attempts to apply the method for screening the samples of Odwalla unpasteurized apple juice associated with the 1996 EHEC outbreak [4] were unsuccessful (Gooding and Choudary, unpublished results). Others have reported similar experience with apple cider [16]. The inhibition could affect any of the modular steps: pre-enrichment, IMS, and/or PCR. To identify the specific step(s) inhibited by fresh produce and fruit juices, we systematically examined the latter's influence on each individual step of the method. Since there is evidence that low pH is more injurious to bacteria at room temperature than at very cold or freezing temperatures [17, 18], we first considered whether the acid pH was affecting the bacteria and masking their identification. Steady-state maintenance of the pH of the enrichment broth around 7.0, at variance with an earlier report [16], failed to prevent the inhibition of immunocapture of the pathogen, thus ruling out such a possibility. The finding, however, was not surprising, considering the extreme acid-tolerance of EHEC, especially after exposure to warm temperatures around 48°C [19], enabling it to survive in apple juice for over 30 days [20], and that it inhabits and replicates in human intestine. Other considerations, such as diluting the sample or repeated washing, also did not prove useful. Fruit juices contain significant levels of polyphenols (catechins, flavenols, flavones, flavanones, cinnamates, benzoic acid, vitamin E, large flavenols, anthocyanins, etc.), in addition to polysaccharides, peptides (rich in acidic amino acids) and free amino acids [21]. It is known from our experiments that polysaccharides, peptides and free amino acids have no adverse effect on IMS-PCR. However, polyphenols act as common chemical defenses of plants against herbivores, by denaturing and decreasing the nutritional value of proteins to the feeding herbivores. Siebert and coworkers [22] have proposed that polyphenols in the plant products, such as juices and proteins, reversibly combine to form high molecular weight soluble complexes. Tagashira et al. [23] have hypothesized that neutral phenolics block bacterial adherence. Recently, Howell et al. [24] have identified proanthocyanidins as the active ingredients of cranberries, known for long to inhibit the adherence of uro-pathogenic E. coli to mucosal cell surfaces, the initial step in bacterial pathogenesis. Results reported here demonstrate that polyphenols (condensed tannins, flavenols, flavones, flavanones, cinnamates, benzoic acid, vitamin E, large flavenols, anthocyanins, etc.) occurring in a large gamut of fruits, including berries of the Ericaceae family as well in fresh produce inhibit binding between EHEC-specific antibody and EHEC cells. All these observations, together with the strategies deployed by plants using polyphenols as defensive agents, strongly suggest that polyphenols, either as chemically pure composites or as natural ingredients of plant products, could conceivably be used in preventive treatment of bacterial infections, including EHEC, in humans as well as in animals. Our ELISA results suggest that polyphenols, by binding non-specifically to the anti-E. coli O157 antibodies, deprive the immunobeads of their ability to capture target bacteria. Even though fruit juices vary widely in their polyphenol composition [18], the synergy among different members of the polyphenol-pool endogenous to plant products seems to contribute to the observed strong inhibitory effect of fruit juices on IMS. The complete blocking of antibody binding activity in ELISA by purified composite polyphenols (or mixtures) (e.g. condensed tannins (0.25 mg ml−1, w/v) and partial blocking by simple polyphenols alone (e.g. tannic acid, 0.25 mg ml−1, w/v) (data not shown) support this hypothesis. Our objective being to establish a direct cause-and-effect relationship between the polyphenolic content of the test samples and the interference with immuno-PCR, we tested several groups of food items, including milk chocolate and red wine, known to be rich in polyphenols. We included fruits and vegetables in the test, since each fruit or vegetable that enters a processing facility is potentially a carrier of pathogens, especially where the skin or peel is in intimate contact with the end product (e.g. juice). Including apples and tomatoes, all the samples tested inhibited antibody activity completely. We have not attempted to establish quantitative relationships between polyphenol content of the fruit/vegetable/food sample and inhibition of immuno-PCR, because even as small a volume as 1 µl of apple juice was strongly inhibitory (data not shown). The complete inhibition of both IMS and PCR by purified preparations of condensed tannins provides a direct proof for our hypothesis that polyphenols are responsible for the inhibitory effect of fruit juices and fresh produce extracts on immuno-PCR. By pre-treating the sample with PVP prior to IMS, we were able to abolish the inhibition and restore the detection sensitivity of the method to 1 cfu of EHEC (in 1 ml) of fruit juice or fresh produce extract. Comparison of different fining agents indicated PVPP and PEG also to be equally efficient in facilitating capture of EHEC cells from spiked fruit juices, by adsorbing the polyphenols and thus preventing inhibition of the IMS-PCR. However, because of relatively limited water-solubility of PVPP, and the tendency of PEG to precipitate proteins, we used PVP in all our experiments. Free glycine (1%, w/v), prominent in the digestive juices of herbivorous insects as an antagonist of the protein denaturing activity of host plant polyphenols [25], was ineffective in our method (data not shown). In summary, by incorporating the simple chemical adsorption step as described here, we were able to circumvent the non-specific binding activity of polyphenols and adapt the IMS-PCR method for application to fruit juices, fresh produce and other foods rich in polyphenols. It has several ramifications for effective strategies to ensure microbial safety of fruit juices, organically grown and minimally processed fresh produce and other plant products as well as foods rich in polyphenols, foods that were not easily amenable to such scrutiny until now. By providing a simple and reliable way of screening large numbers and varieties of fruit juices and other plant-derived foods for potential or actual contamination with EHEC, our method will help food processors as well as organic farms in developing and maintaining safe manufacturing standards and practices, such as HACCP (hazard analysis critical control point) plans. It should also prove useful to the government in meeting the new challenges, by allowing them to pinpoint the source of contaminants, e.g. the orchard workers with unhygienic practices during processing, cattle wastes contaminating the fruit or vegetables that had fallen to the ground, etc. Using that information, the regulatory agencies, in their effort to minimize the pathogen risk at various levels of production, processing and retail distribution, will be able to prosecute individuals and firms flouting the laws and jeopardizing food supplies. Finally, this method will serve as a model for detection of other food pathogens in a variety of foods, including those derived from plants. Acknowledgements We thank Lisa Argento and Raul Calderon for technical assistance, Sharon Shoemaker and Jennie Hunter-Cevera for advice and support, Kimberly Seebart and Richard Wilson for the reference EHEC strain, Jennifer Donovan and Andrew Waterhouse for generous help with the analysis of polyphenols and for helpful discussions, and Christopher Gooding for carrying out initial experiments with spiked juices and with naturally contaminated apple juice samples from the 1996 outbreak. The contents of this paper are subject to a patent application (UC 99-030-1). References [1] Qadri S.M. Kayali S. ( 1998) Enterohemorrhagic Escherichia coli: a dangerous food-borne pathogen. Postgrad. Med.  103, 179– 180, 185–187. Google Scholar CrossRef Search ADS PubMed  [2] Riley L.W. Remis R.S. Helgerson S.D. McGee H.H. Wells G.J. Davis B.R. Hebert R.J. Olcott E.S. Johnson L.M. Hargrett N.T. Blake P.A. Cohen M.L. ( 1983) Hemorrhagic colitis associated with a rare Escherichia coli serotype. New Engl. 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TI - Adsorption of endogenous polyphenols relieves the inhibition by fruit juices and fresh produce of immuno-PCR detection of Escherichia coli 0157:H7
JF - Journal of the Endocrine Society
DO - 10.1111/j.1574-695X.1999.tb01241.x
DA - 1999-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/adsorption-of-endogenous-polyphenols-relieves-the-inhibition-by-fruit-kVjBQlnbB6
SP - 213
EP - 220
VL - 23
IS - 3
DP - DeepDyve
ER -