Comparison of an immunochromatographic method and the TaqMan� E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

Numerous field studies have revealed that irrigation water can contaminate the surface of plants; however, the occurrence of pathogen internalization is unclear. This study was conducted to determine the sites of Escherichia coli O157:H7 contamination and its survival when the bacteria were applied through spray irrigation water to either field-grown spinach or lettuce. To differentiate internalized and surface populations, leaves were treated with a surface disinfectant wash before the tissue was ground for analysis of E. coli O157:H7 by direct plate count or enrichment culture. Irrigation water containing E. coli O157:H7 at 102, 104, or 106 CFU/ml was applied to spinach 48 and 69 days after transplantation of seedlings into fields. E. coli O157:H7 was initially detected after application on the surface of plants dosed at 104 CFU/ml (4 of 20 samples) and both on the surface (17 of 20 samples) and internally (5 of 20 samples) of plants dosed at 106 CFU/ml. Seven days postspraying, all spinach leaves tested negative for surface or internal contamination. In a subsequent study, irrigation water containing E. coli O157:H7 at 108 CFU/ml was sprayed onto either the abaxial (lower) or adaxial (upper) side of leaves of field-grown lettuce under sunny or shaded conditions. E. coli O157:H7 was detectable on the leaf surface 27 days postspraying, but survival was higher on leaves sprayed on the abaxial side than on leaves sprayed on the adaxial side. Internalization of E. coli O157:H7 into lettuce leaves also occurred with greater persistence in leaves sprayed on the abaxial side (up to 14 days) than in leaves sprayed on the adaxial side (2 days).

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Infrequent Internalization of Escherichia coli O157:H7 into Field-Grown Leafy Greens

Journal of Food Protection ◽

10.4315/0362-028x-73.3.500 ◽

2010 ◽

Vol 73 (3) ◽

pp. 500-506 ◽

Cited By ~ 67

Author(s):

MARILYN C. ERICKSON ◽

CATHY C. WEBB ◽

JUAN CARLOS DIAZ-PEREZ ◽

SHARAD C. PHATAK ◽

JOHN J. SILVOY ◽

...

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Field Studies ◽

Escherichia Coli O157 ◽

E Coli ◽

Leafy Greens ◽

Contaminated Irrigation Water ◽

Green Fluorescent ◽

Field Plots ◽

Spinach Leaves

Several sources of contamination of fresh produce by Escherichia coli O157:H7 (O157) have been identified and include contaminated irrigation water and improperly composted animal waste; however, field studies evaluating the potential for internalization of O157 into leafy greens from these sources have not been conducted. Irrigation water inoculated with green fluorescent plasmid–labeled Shiga toxin–negative strains (50 ml of 102, 104, or 106 CFU of O157 per ml) was applied to soil at the base of spinach plants of different maturities in one field trial. In a second trial, contaminated compost (1.8 kg of 103 or 105 CFU of O157 per g) was applied to field plots (0.25 by 3.0 m) prior to transplantation of spinach, lettuce, or parsley plants. E. coli O157:H7 persisted in the soil up to harvest (day 76 posttransplantation) following application of contaminated irrigation water; however, internalized O157 was not detected in any spinach leaves or in roots exposed to O157 during the early or late growing season. Internalized O157 was detected in root samples collected 7 days after plants were contaminated in mid-season, with 5 of 30 samples testing positive for O157 by enrichment; however, O157 was not detected by enrichment in surface-disinfected roots on days 14 or 22. Roots and leaves from transplanted spinach, lettuce, and parsley did not internalize O157 for up to 50 days in the second trial. These results indicate that internalization of O157 via plant roots in the field is rare and when it does occur, O157 does not persist 7 days later.

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Persistence of Enterohemorrhagic Escherichia coli O157:H7 in Soil and on Leaf Lettuce and Parsley Grown in Fields Treated with Contaminated Manure Composts or Irrigation Water

Journal of Food Protection ◽

10.4315/0362-028x-67.7.1365 ◽

2004 ◽

Vol 67 (7) ◽

pp. 1365-1370 ◽

Cited By ~ 298

Author(s):

MAHBUB ISLAM ◽

MICHAEL P. DOYLE ◽

SHARAD C. PHATAK ◽

PATRICIA MILLNER ◽

XIUPING JIANG

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Poultry Manure ◽

Dairy Manure ◽

Enterohemorrhagic Escherichia Coli ◽

Avirulent Strain ◽

Escherichia Coli O157 ◽

E Coli ◽

Manure Compost ◽

Contaminated Irrigation Water

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 infections associated with lettuce and other leaf crops have occurred with increasing frequency in recent years. Contaminated manure and polluted irrigation water are probable vehicles for the pathogen in many outbreaks. In this study, the occurrence and persistence of E. coli O157:H7 in soil fertilized with contaminated poultry or bovine manure composts or treated with contaminated irrigation water and on lettuce and parsley grown on these soils under natural environmental conditions was determined. Twenty-five plots, each 1.8 by 4.6 m, were used for each crop, with five treatments (one without compost, three with each of the three composts, and one without compost but treated with contaminated water) and five replication plots for each treatment. Three different types of compost, PM-5 (poultry manure compost), 338 (dairy manure compost), and NVIRO-4 (alkaline-stabilized dairy manure compost), and irrigation water were inoculated with an avirulent strain of E. coli O157:H7. Pathogen concentrations were 107 CFU/g of compost and 105 CFU/ml of water. Contaminated compost was applied to soil in the field as a strip at 4.5 metric tons per hectare on the day before lettuce and parsley seedlings were transplanted in late October 2002. Contaminated irrigation water was applied only once on the plants as a treatment in five plots for each crop at the rate of 2 liters per plot 3 weeks after the seedlings were transplanted. E. coli O157:H7 persisted for 154 to 217 days in soils amended with contaminated composts and was detected on lettuce and parsley for up to 77 and 177 days, respectively, after seedlings were planted. Very little difference was observed in E. coli O157:H7 persistence based on compost type alone. E. coli O157:H7 persisted longer (by >60 days) in soil covered with parsley plants than in soil from lettuce plots, which were bare after lettuce was harvested. In all cases, E. coli O157:H7 in soil, regardless of source or crop type, persisted for >5 months after application of contaminated compost or irrigation water.

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Transmission of Escherichia coli O157:H7 to Internal Tissues and Its Survival on Flowering Heads of Wheat

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-298 ◽

2015 ◽

Vol 78 (3) ◽

pp. 518-524 ◽

Cited By ~ 11

Author(s):

BISMARCK MARTINEZ ◽

JAYNE STRATTON ◽

ANDRÉIA BIANCHINI ◽

STEPHEN WEGULO ◽

GLEN WEAVER

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Escherichia Coli O157 ◽

Human Pathogen ◽

Contamination Source ◽

Wheat Seedlings ◽

E Coli ◽

Uremic Syndrome ◽

Low Levels ◽

Fourth Experiment

Escherichia coli O157:H7 is a human pathogen that can cause bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. E. coli O157:H7 illnesses are mainly associated with undercooked beef; however, in recent years, outbreaks have been linked to fresh produce, such as spinach, lettuce, and sprouts. In 2009, flour was implicated as the contamination source in an outbreak involving consumption of raw cookie dough that resulted in 77 illnesses. The objectives of this research were to determine (i) whether E. coli O157:H7 could be translocated into the internal tissues of wheat (Triticum aestivum) seedlings from contaminated seed, soil, or irrigation water and (ii) whether the bacterium could survive on flowering wheat heads. The levels of contamination of kanamycin-resistant E. coli O157:H7 strains in seed, soil, and irrigation water were 6.88 log CFU/g, 6.60 log CFU/g, and 6.76 log CFU/ml, respectively. One hundred plants per treatment were sown in pot trays with 50 g of autoclaved soil or purposely contaminated soil, watered every day with 5 ml of water, and harvested 9 days postinoculation. In a fourth experiment, flowering wheat heads were spray inoculated with water containing 4.19 log CFU/ml E. coli O157:H7 and analyzed for survival after 15 days, near the harvest period. To detect low levels of internalization, enrichment procedures were performed and Biotecon real-time PCR detection assays were used to determine the presence of E. coli O157:H7 in the wheat, using a Roche Applied Science LightCycler 2.0 instrument. The results showed that internalization was possible using contaminated seed, soil, and irrigation water in wheat seedlings, with internalization rates of 2, 5, and 10%, respectively. Even though the rates were low, to our knowledge this is the first study to demonstrate the ability of this strain to reach the phylloplane in wheat. In the head contamination experiment, all samples tested positive, showing the ability of E. coli O157:H7 to survive on the wheat head.

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Fate ofEscherichia coliO157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

Canadian Journal of Microbiology ◽

10.1139/w04-097 ◽

2004 ◽

Vol 50 (12) ◽

pp. 1007-1014 ◽

Cited By ~ 55

Author(s):

A Mark Ibekwe ◽

Pamela M Watt ◽

Peter J Shouse ◽

Catherine M Grieve

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Irrigation Water ◽

Real Time Pcr ◽

The United States ◽

Large Animal ◽

Escherichia Coli O157 ◽

Pcr Assay ◽

E Coli ◽

Rhizosphere Soils

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 × 103in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.Key words: real-time PCR, Escherichia coli O157:H7, irrigation, survival, quantification.

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Internalization of Escherichia coli O157:H7 following Spraying of Cut Shoots When Leafy Greens Are Regrown for a Second Crop

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-217 ◽

2013 ◽

Vol 76 (12) ◽

pp. 2052-2056 ◽

Cited By ~ 11

Author(s):

MARILYN C. ERICKSON ◽

CATHY C. WEBB ◽

JUAN CARLOS DÍAZ-PÉREZ ◽

LINDSEY E. DAVEY ◽

ALISON S. PAYTON ◽

...

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Tissue Repair ◽

Contamination Level ◽

Contaminated Water ◽

Escherichia Coli O157 ◽

E Coli ◽

Leafy Greens ◽

Spraying Plants ◽

High Level

Both spinach and lettuce were grown to harvest, cut, and then regrown after spraying the cut shoots with irrigation water contaminated with Escherichia coli O157:H7. Plant tissue was collected on the day of spraying and again 2 and 14 days later for analysis of total and internalized E. coli O157:H7 populations. Internalization of E. coli O157:H7 occurred on the day of spraying, and larger populations were internalized as the level in the spray increased. Tissue repair was slow and insufficient to prevent infiltration of E. coli O157:H7; internalized E. coli O157:H7 in shoots cut 5 days prior to exposure to E. coli O157:H7–contaminated water were not significantly different from levels in shoots cut on the same day of spraying with contaminated water (P > 0.05). Two days after spraying plants with a high level of E. coli O157:H7 (7.3 log CFU/ml), levels of internalized E. coli O157:H7 decreased by ca. 2.6 and 1.3 log CFU/g in Tyee and Bordeaux spinach, respectively, whereas populations of internalized E. coli O157:H7 decreased very little (ca. 0.4 log CFU/g) in lettuce plants that had been sprayed either on the same day as cutting or 1 day after cutting. When cut plants were sprayed with irrigation water at a lower contamination level (4.5 log CFU/ml), internalized E. coli O157:H7 was not detected in either spinach or lettuce plants 2 days later and therefore would not likely be of concern when the crop was harvested.

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Persistence of Escherichia coli O157:H7 on Lettuce Plants following Spray Irrigation with Contaminated Water

Journal of Food Protection ◽

10.4315/0362-028x-66.12.2198 ◽

2003 ◽

Vol 66 (12) ◽

pp. 2198-2202 ◽

Cited By ~ 113

Author(s):

ETHAN B. SOLOMON ◽

HOAN-JEN PANG ◽

KARL R. MATTHEWS

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

Sampling Period ◽

Repeated Exposure ◽

Human Consumption ◽

Contaminated Water ◽

Escherichia Coli O157 ◽

Plant Leaves ◽

E Coli ◽

Spray Irrigation

Irrigation water collected at farms growing crops for human consumption was artificially contaminated with E. coli O157: H7 and used to irrigate lettuce plants. Plants in a growth chamber were spray irrigated either once or intermittently with water contaminated with 102 or 104 CFU of E. coli O157:H7 per ml and were then sampled over a 30-day period. Only plants exposed to 102 CFU/ml on day 1 did not harbor the pathogen at the end of the sampling period. All other treatments resulted in contaminated plants at harvest. Plants irrigated with 104 CFU/ml contained high levels (up to 5 log CFU/g) of the pathogen at harvest. The results obtained in this study underscore the assertion that spray irrigation (the application of water directly to plant leaves) is linked to the contamination of crops and suggest that repeated exposure increases the E. coli O157:H7 level on the plant.

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Survey of Retail Alfalfa Sprouts and Mushrooms for the Presence of Escherichia coli O157:H7, Salmonella, and Listeria with BAX, and Evaluation of this Polymerase Chain Reaction–Based System with Experimentally Contaminated Samples†

Journal of Food Protection ◽

10.4315/0362-028x-66.2.182 ◽

2003 ◽

Vol 66 (2) ◽

pp. 182-187 ◽

Cited By ~ 22

Author(s):

CHRISTINE M. STRAPP ◽

ADRIENNE E. H. SHEARER ◽

ROLF D. JOERGER

Keyword(s):

Escherichia Coli ◽

Detection Rate ◽

Salmonella Enteritidis ◽

Detection System ◽

Escherichia Coli O157 ◽

Chain Reaction ◽

E Coli ◽

Polymerase Chain ◽

Alfalfa Sprouts ◽

Alfalfa Sprout

BAX, a polymerase chain reaction (PCR)–based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocytogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.

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Evaluation of an Attachment Assay on Lettuce Leaves with Temperature- and Starvation-Stressed Escherichia coli O157:H7 MB3885

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-332 ◽

2014 ◽

Vol 77 (4) ◽

pp. 549-557 ◽

Cited By ~ 8

Author(s):

INGE VAN der LINDEN ◽

BART COTTYN ◽

MIEKE UYTTENDAELE ◽

GEERTRUI VLAEMYNCK ◽

MARTINE MAES ◽

...

Keyword(s):

Escherichia Coli ◽

Quantitative Pcr ◽

Irrigation Water ◽

Escherichia Coli O157 ◽

E Coli ◽

Overhead Irrigation ◽

Plating Method ◽

Plate Counts ◽

Stressed Cells ◽

Butterhead Lettuce

Attachment of enteric pathogens such as Escherichia coli O157:H7 to fresh produce is a crucial first step for contamination to occur, and irrigation water (IW) is considered a potentially important preharvest introduction route. In a natural situation, E. coli O157:H7 may be present in the irrigation water for some time and may, therefore, be starved. Most research, however, is performed with freshly cultured strains. The aim of this study was to examine the behavior of E. coli O157:H7 MB3885 under starvation stress in water used for overhead irrigation in the greenhouse and the consequence on its subsequent ability to attach to butterhead lettuce leaves. E. coli O157:H7 MB3885 was starvation stressed by introducing it at ± 7.5 log CFU/ml into phosphate-buffered saline (PBS), sterile distilled water (SDW), or IW. The suspensions were stored at 4 or 20°C and were used after 0, 2, and 6 days for an attachment assay on butterhead lettuce. E. coli O157:H7 MB3885 levels were determined by plating method and live and dead quantitative PCR technique. A decrease in plate counts, an indicator of stress, was observed for most of the conditions, whereas a die-off, as revealed by the live and dead quantitative PCR data, was only observed in IW stored at 20°C. Overall, stress appeared to be highest in IW and lowest in PBS. The stressed cells were still able to recover, even at 4°C, and to attach to the lettuce. Furthermore, our results show that standard laboratory solutions such as PBS and SDW may not be the best to simulate stressed cells in IW, in which the bacteria may behave significantly differently.

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A Simple Method for the Direct Detection of Salmonella and Escherichia coli O157:H7 from Raw Alfalfa Sprouts and Spent Irrigation Water Using PCR†‡

Journal of Food Protection ◽

10.4315/0362-028x-68.11.2256 ◽

2005 ◽

Vol 68 (11) ◽

pp. 2256-2263 ◽

Cited By ~ 20

Author(s):

LYNETTE M. JOHNSTON ◽

DRISS ELHANAFI ◽

MARYANNE DRAKE ◽

LEE-ANN JAYKUS

Keyword(s):

Escherichia Coli ◽

Irrigation Water ◽

High Speed ◽

Direct Detection ◽

Pcr Amplification ◽

Escherichia Coli O157 ◽

Simple Method ◽

E Coli ◽

Serovar Typhimurium ◽

Alfalfa Sprouts

The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10−1 to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10−1 CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.

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