Heat and Drought Stress during Growth of Lettuce (Lactuca sativa L.) Does Not Promote Internalization of Escherichia coli O157:H7

2009 ◽  
Vol 72 (12) ◽  
pp. 2471-2475 ◽  
Author(s):  
GUODONG ZHANG ◽  
LI MA ◽  
LARRY R. BEUCHAT ◽  
MARILYN C. ERICKSON ◽  
VANESSA H. PHELAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Stress ◽  
Fluorescent Protein ◽  
Escherichia Coli O157 ◽  
Romaine Lettuce ◽  
E Coli ◽  
Leaf Surfaces ◽  
Green Fluorescent ◽  
And Control ◽  
Photoperiod Control

Studies were done to determine the effect of heat stress on internalization of Escherichia coli O157:H7 in lettuce subjected to different watering practices during growth. Iceberg and romaine lettuce were grown in sandy soil in an environmental chamber at 23°C during the day and 7°C at night, with a 12-h photoperiod. Thirty days after transplanting seedlings, potting soil was inoculated with a five-strain mixture of green fluorescent protein–labeled E. coli O157:H7 at populations of 4 and 6 log CFU/g of soil. Lettuce plants were exposed to one of two temperature stress regimes: 36°C during the day and 15°C at night for 2 days, or 32°C during the day and 15°C at night for 3 days, both with a 12-h photoperiod. Control plants were held at 23°C during the day and 7°C at night for 3 days. Plants were either watered daily or not watered during the heat stress and control treatments. E. coli O157:H7 was detected by enrichment in all inoculated soil and rhizosphere samples from plants grown in inoculated soil. Less E. coli O157:H7 was detected in inoculated heat-stressed soil than in control soil. From inoculated pots, all leaf surfaces and macerated leaves that had been surface sanitized were negative for E. coli O157:H7. All surface-sanitized macerated roots from control samples and from 143 of 144 samples of inoculated samples were negative for E. coli O157:H7. Heat stress during growth of lettuce did not promote or enhance internalization of E. coli O157:H7, regardless of the moisture content in the soil.

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

Transfer of Escherichia coli O157:H7 to Romaine Lettuce due to Contact Water from Melting Ice

2008 ◽  
Vol 71 (2) ◽  
pp. 252-256 ◽  
Author(s):  
JIN KYUNG KIM ◽  
MARK A. HARRISON
Keyword(s):  
Escherichia Coli ◽  
Tap Water ◽  
Sampling Site ◽  
Contaminated Water ◽  
Escherichia Coli O157 ◽  
Romaine Lettuce ◽  
E Coli ◽  
Ice Melt ◽  
Leaf Surfaces ◽  
Shipping Containers

Ice can be used to chill romaine lettuce and maintain relative humidity during transportation. Escherichia coli O157:H7 may contaminate water used for ice. The objective of this study was to determine the potential for E. coli O157:H7 contamination of romaine lettuce from either ice contaminated with the pathogen or by transfer from lettuce surfaces via melting ice. In experiment 1, lettuce was spot inoculated with E. coli O157:H7 and chilled with ice prepared from uncontaminated tap water. In experiment 2, water inoculated with this pathogen was frozen and used to ice lettuce. Three heads of lettuce were stacked in each container and stored at 4 or 20°C. After the ice melted, E. coli O157:H7 attachment to and recovery from the lettuce leaves were determined. For experiment 1, the population of E. coli O157:H7 attached to inoculated sites averaged 3.8 and 5.5 CFU/cm2 at 4 and 20°C, respectively. Most of the uninoculated sites became contaminated with the pathogen due to ice melt. For experiment 2, 3.5 to 3.8 log CFU E. coli O157:H7 per cm2 was attached to the top leaf on the first head. After rinsing with chlorinated water (200 μg/ml), E. coli O157:H7 remained on the surface of the top head (1.8 to 2.0 log CFU/cm2). There was no difference in numbers of E. coli O157:H7 recovered from each sampling site at 4 and 20°C. Results show that E. coli O157:H7 can be transferred onto other produce layers in shipping containers from melted ice made of contaminated water and from contaminated to uncontaminated leaf surfaces.

Impact of Preinoculation Culture Conditions on the Behavior of Escherichia coli O157:H7 Inoculated onto Romaine Lettuce (Lactuca sativa) Plants and Cut Leaf Surfaces

2009 ◽  
Vol 72 (7) ◽  
pp. 1553-1559 ◽  
Author(s):  
CHRISTOPHER G. THEOFEL ◽  
LINDA J. HARRIS
Keyword(s):  
Escherichia Coli ◽  
High Standard ◽  
Culture Conditions ◽  
Escherichia Coli O157 ◽  
Late Stationary Phase ◽  
Romaine Lettuce ◽  
Preparation Methods ◽  
E Coli ◽  
Leaf Surfaces ◽  
Inoculum Preparation

Inoculum preparation methods can impact growth or survival of organisms inoculated into foods, thus complicating direct comparison of results among studies. The objective of this study was to evaluate preinoculation culture preparation for impact on Escherichia coli O157:H7 inoculated onto leaves of romaine lettuce plants and cut leaf surfaces. E. coli O157:H7 was grown quiescently or shaken at 15, 25, or 37°C to different growth phases in tryptic soy or M9 minimal salts broth or agar. Cells were harvested, washed, and suspended in 0.1% peptone, Milli Q water, or well water and refrigerated for 0 or 18 h. Prepared inoculum was spotted onto cut romaine lettuce (10 μl; 3 × 104 CFU/10 g) or onto romaine lettuce plants (20 μl; 3 × 106 CFU per leaf). Cut lettuce was sealed in 100-cm2 bags (made from a commercial polymer film) and incubated at 5 or 20°C. Lettuce plants were held at 23°C for 24 h. For all tested conditions, levels of E. coli O157:H7 increased at 20°Concut lettuce and decreased on cut lettuce stored at 5°C or on leaves of lettuce plants. At 20°C, preinoculation culture conditions had little impact on growth of E. coli O157:H7 on cut lettuce. However, survival at 5°C was significantly better (P < 0.05) for cultures grown at 15 or 37°C in minimal medium and to late stationary phase. Impact of preinoculation handling on survival on lettuce plants was less clear due to relatively high standard deviations observed among samples.

Efficacy of Commercial Produce Sanitizers against Nontoxigenic Escherichia coli O157:H7 during Processing of Iceberg Lettuce in a Pilot-Scale Leafy Green Processing Line

2013 ◽  
Vol 76 (11) ◽  
pp. 1838-1845 ◽  
Author(s):  
GORDON R. DAVIDSON ◽  
ANNEMARIE L. BUCHHOLZ ◽  
ELLIOT T. RYSER
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Pilot Scale ◽  
Wash Water ◽  
Escherichia Coli O157 ◽  
Cross Contamination ◽  
E Coli ◽  
Iceberg Lettuce ◽  
Processing Line ◽  
Green Fluorescent

Chemical sanitizers are routinely used during commercial flume washing of fresh-cut leafy greens to minimize cross-contamination from the water. This study assessed the efficacy of five commercial sanitizer treatments against Escherichia coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line. Iceberg lettuce (5.4 kg) was inoculated to contain 106 CFU/g of a four-strain cocktail of nontoxigenic, green fluorescent protein–labeled, ampicillin-resistant E. coli O157:H7 and processed after 1 h of draining at ~22°C. Lettuce was shredded using a commercial slicer, step-conveyed to a flume tank, washed for 90 s using six different treatments (water alone, 50 ppm of peroxyacetic acid, 50 ppm of mixed peracid, or 50 ppm of available chlorine either alone or acidified to pH 6.5 with citric acid [CA] or T-128), and then dried using a shaker table and centrifugal dryer. Various product (25-g) and water (50-ml) samples collected during processing along with equipment surface samples (100 cm2) from the flume tank, shaker table, and centrifugal dryer were homogenized in neutralizing buffer and plated on tryptic soy agar. During and after iceberg lettuce processing, none of the sanitizers were significantly more effective (P ≤ 0.05) than water alone at reducing E. coli O157:H7 populations on lettuce, with reductions ranging from 0.75 to 1.4 log CFU/g. Regardless of the sanitizer treatment used, the centrifugal dryer surfaces yielded E. coli O157:H7 populations of 3.49 to 4.98 log CFU/100 cm2. Chlorine, chlorine plus CA, and chlorine plus T-128 were generally more effective (P ≤ 0.05) than the other treatments, with reductions of 3.79, 5.47, and 5.37 log CFU/ml after 90 s of processing, respectively. This indicates that chlorine-based sanitizers will likely prevent wash water containing low organic loads from becoming a vehicle for cross-contamination.

Validation of Apple Cider Pasteurization Treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes

2001 ◽  
Vol 64 (11) ◽  
pp. 1679-1689 ◽  
Author(s):  
PEGGY P. MAK ◽  
BARBARA H. INGHAM ◽  
STEVEN C. INGHAM
Keyword(s):  
Escherichia Coli ◽  
New York ◽  
Listeria Monocytogenes ◽  
Fluorescent Protein ◽  
Escherichia Coli O157 ◽  
Salmonella Spp ◽  
Apple Cider ◽  
E Coli ◽  
Log Reduction ◽  
Green Fluorescent

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and ∘Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380–94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778, CDC F2833, and CDC HO662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14°Brix was heated under conditions ranging from 60°C for 14 s to 71.1°C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1°C for 14 s. Lower temperatures, or less time at 68.1°C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6°C for 14 s for Salmonella spp. L. monocytogenes survived 68.1°C for 14 s, but survivors died in cider within 24 h at 4°C. Laboratory results were validated with a surrogate E. coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1°C for 14 s (Wisconsin recommendations) and at 71.1°C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1°C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.

Thermal Inactivation of Escherichia coli O157:H7 in Cow Manure Compost

2003 ◽  
Vol 66 (10) ◽  
pp. 1771-1777 ◽  
Author(s):  
XIUPING JIANG ◽  
JENNIE MORGAN ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Thermal Inactivation ◽  
Fluorescent Protein ◽  
Escherichia Coli O157 ◽  
Cow Manure ◽  
E Coli ◽  
Manure Compost ◽  
D Values ◽  
Green Fluorescent

Rates of inactivation of a five-strain mixture of green fluorescent protein–labeled Escherichia coli O157:H7 in autoclaved and unautoclaved commercial cow manure compost with a moisture content of ca. 38% were determined at temperatures of 50, 55, 60, 65, and 70°C. Trypticase soy agar with ampicillin was determined to be the best medium for the enumeration of heat-injured and uninjured cells of green fluorescent protein–labeled E. coli O157:H7. The results obtained in this study revealed that in autoclaved compost, E. coli O157:H7 reductions of ca. 4 log CFU/g occurred within 8 h, 3 h, 15 min, 2 min, and <1 min at 50, 55, 60, 65, and 70°C, respectively. At 65 and 70°C, considerably less time was required to kill the pathogen in unautoclaved compost than in autoclaved compost. Decimal reduction times (D-values) for autoclaved compost at 50, 55, 60, 65, and 70°C were 137, 50.3, 4.1, 1.8, and 0.93 min, respectively, and D-values for unautoclaved compost at 50, 55, and 60°C were 135, 35.4, and 3.9 min, respectively. Considerable tailing was observed for inactivation curves, especially at 60, 65, and 70°C. These results are useful for identifying composting conditions that will reduce the risk of the transmission of E. coli O157:H7 to foods produced in the presence of animal fecal waste.

Effects of Plant Maturity and Growth Media Bacterial Inoculum Level on the Surface Contamination and Internalization of Escherichia coli O157:H7 in Growing Spinach Leaves

2009 ◽  
Vol 72 (11) ◽  
pp. 2313-2320 ◽  
Author(s):  
SHUAIHUA PU ◽  
JOHN C. BEAULIEU ◽  
WITOON PRINYAWIWATKUL ◽  
BEILEI GE
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
The United States ◽  
Surface Contamination ◽  
Growth Media ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Spinach Plant ◽  
Green Fluorescent

The incidence of foodborne outbreaks linked to fresh produce has increased in the United States. Particularly noteworthy was the 2006 Escherichia coli O157:H7 outbreak associated with prepackaged baby spinach. This study aimed to determine whether E. coli O157:H7 would be present in the aerial leaf tissue of a growing spinach plant when introduced at various plant maturities and different inoculum levels in a greenhouse setting. Spinach seeds of a commercial variety were sown in 8-in. (20.32-cm) pots. After seed germination, two levels (103 and 107 CFU/ml) of an E. coli O157:H7 green fluorescent protein–expressing strain were introduced into the plant growth media weekly for a total of five times. Inoculated spinach plants were examined weekly for the presence of E. coli O157:H7 on leaves and in surrounding growth media. Among 120 spinach plant samples examined for internal leaf contamination, only one yielded a positive result. Surface leaf contamination occurred occasionally and clustered between 3 and 5 weeks of age, but not among leaves younger than 3 weeks of age. On the other hand, when inoculated at the 107 CFU/ml level, the E. coli O157:H7 green fluorescent protein strain survived the entire cultivation period, although with gradually reduced levels. The experiments demonstrated that internalization of E. coli O157:H7 of growing spinach plant leaves under greenhouse conditions was a rare event, but surface contamination did occur, primarily when the plants reached 3 weeks of age. The study provided important data to further assess the association between spinach age and potential contamination of E. coli O157:H7.

Efficacy of Antimicrobial Agents in Lettuce Leaf Processing Water for Control of Escherichia coli O157:H7

2009 ◽  
Vol 72 (7) ◽  
pp. 1392-1397 ◽  
Author(s):  
GUODONG ZHANG ◽  
LI MA ◽  
VANESSA H. PHELAN ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Organic Load ◽  
Escherichia Coli O157 ◽  
Peroxyacetic Acid ◽  
E Coli ◽  
Processing Water ◽  
Green Fluorescent ◽  
The Impact

The objectives of this research were to study transfer and control of Escherichia coli O157:H7 during simultaneous washing of inoculated and uninoculated lettuce pieces and to determine the efficacy of antimicrobial agents (peroxyacetic acid, mixed peracid, and sodium hypochlorite) on reducing the transfer of E. coli O157:H7 through processing water with or without organic load. Lettuce leaf pieces (5 by 5 cm) were inoculated with a five-strain mixture of green fluorescent protein–labeled E. coli O157:H7 at 5.6 log CFU per piece. One inoculated lettuce piece was added to five uninoculated leaves during washing. Peroxyacetic acid and mixed peracid were tested at 10, 20, and 30 ppm, and chlorine was tested at 30 and 50 ppm. No organic load (liquefied lettuce leaves) and 10% organic load in processing water were compared. Without organic load, peroxyacetic acid at 30 ppm, mixed peracid at 10, 20, and 30 ppm, and chlorine at 30 and 50 ppm all significantly reduced E. coli O157: H7 in processing water by 1.83, 1.73, 1.50, 1.83, 1.34, and 1.83 log CFU/ml, respectively, compared with washing with water alone. These antimicrobials at all concentrations tested also significantly reduced transfer of the bacteria from an inoculated leaf to uninoculated leaves in the processing water by 0.96 to 2.57 log CFU per piece. A 10% organic load in the processing water reduced efficacy of antimicrobial agents. In this contaminated water, peroxyacetic acid at 10 and 20 ppm and chlorine at 30 ppm produced effects not significantly different from those of water alone. Therefore, it is important to understand the impact of organic load when validating the effectiveness of antimicrobial treatments.

Quantitative Transfer of Escherichia coli O157:H7 to Equipment during Small-Scale Production of Fresh-Cut Leafy Greens

2012 ◽  
Vol 75 (7) ◽  
pp. 1184-1197 ◽  
Author(s):  
ANNEMARIE L. BUCHHOLZ ◽  
GORDON R. DAVIDSON ◽  
BRADLEY P. MARKS ◽  
EWEN C. D. TODD ◽  
ELLIOT T. RYSER
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Small Scale ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Leafy Greens ◽  
Iceberg Lettuce ◽  
Fresh Cut ◽  
Green Fluorescent

Postharvest contamination and subsequent spread of Escherichia coli O157:H7 can occur during shredding, conveying, fluming, and dewatering of fresh-cut leafy greens. This study quantified E. coli O157:H7 transfer from leafy greens to equipment surfaces during simulated small-scale commercial processing. Three to five batches (22.7 kg) of baby spinach, iceberg lettuce, and romaine lettuce were dip inoculated with a four-strain cocktail of avirulent, green fluorescent protein–labeled, ampicillin-resistant E. coli O157:H7 to contain ∼106, 104, and 102 CFU/g, and then were processed after 1 h of draining at ∼23°C or 24 h of storage at 4°C. Lettuce was shredded using an Urschel TransSlicer at two different blade and belt speeds to obtain normal (5 by 5 cm) and more finely shredded (0.5 by 5 cm) lettuce. Thereafter, the lettuce was step conveyed to a flume tank and was washed and then dried using a shaker table and centrifugal dryer. Product (25-g) and water (40-ml) samples were collected at various points during processing. After processing, product contact surfaces (100 cm2) on the shredder (n =14), conveyer (n =8), flume tank (n =11), shaker table (n =9), and centrifugal dryer (n =8) were sampled using one-ply composite tissues. Sample homogenates diluted in phosphate or neutralizing buffer were plated, with or without prior 0.45-μm membrane filtration, on Trypticase soy agar containing 0.6% yeast extract supplemented with 100 ppm of ampicillin to quantify green fluorescent protein–labeled E. coli O157:H7 under UV light. During leafy green processing, ∼90% of the E. coli O157:H7 inoculum transferred to the wash water. After processing, E. coli O157:H7 populations were highest on the conveyor and shredder (P < 0.05), followed by the centrifugal dryer, flume tank, and shaker table, with ∼29% of the remaining product inoculum lost during centrifugal drying. Overall, less (P < 0.05) of the inoculum remained on the product after centrifugally drying iceberg lettuce that was held for 1 h (8.13%) as opposed to 24 h (42.18%) before processing, with shred size not affecting the rate of E. coli O157:H7 transfer.

Location of Escherichia coli O157:H7 on and in Apples as Affected by Bruising, Washing, and Rubbing

2001 ◽  
Vol 64 (9) ◽  
pp. 1328-1333 ◽  
Author(s):  
STEPHEN J. KENNEY ◽  
SCOTT L. BURNETT ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Skin Surface ◽  
Confocal Scanning Laser Microscopy ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Log Reduction ◽  
Peptone Water ◽  
Confocal Scanning Laser ◽  
Green Fluorescent

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 μm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 m below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

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