Transfer of Escherichia coli O157:H7 to romaine lettuce due to contact water from melting ice

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 degrees 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 degrees 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 microg/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 degrees 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.     

Comparison of methods for detection and isolation of cold- and freeze-stressed Escherichia coli O157:H7 in raw ground beef

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 < or =0.5 and < or =2 CFU/g, and samples were then enriched immediately or were stored at 4 degrees C for 72 h or at -20 degrees 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 degrees C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35 degrees 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 degrees C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35 degrees 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+n at 35 degrees C, 3.7 times more likely with an initial inoculum of < or = 2.0 CFU/g than with < or = 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.     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

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 degrees 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 degrees 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 (> 10(3) 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 degrees 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.     

Survival of enterohemorrhagic Escherichia coli O157:H7 in bovine feces applied to lettuce and the effectiveness of chlorinated water as a disinfectant.

Bovine feces are a potential vehicle for transmitting enterohemorrhagic Escherichia coli O157:H7 to humans. A study was undertaken to determine survival characteristics of E. coli O157:H7 on iceberg lettuce using 0.1% peptone water and bovine feces as carriers for inocula. Four levels of inoculum, ranging from 10(0) to 10(5) CFU of E. coli O157:H7 per g of lettuce, were applied. Populations surviving on lettuce stored at 4 degrees C were monitored for up to 15 days. Regardless of the type of carrier, viable cells of E. coli O157:H7 were detected on lettuce after 15 days, even when the initial inoculum was 10(0) to 10(1) CFU/g. Spray treatments of lettuce with 200 ppm chlorine solution or deionized water were equally effective in killing or removing E. coli O157:H7 from lettuce. Holding lettuce for 5 min after spray treatment was not more effective in reducing populations than holding for 1 min before rinsing with water. Prevention of contamination of lettuce with bovine feces that may harbor E. coli O157:H7 as well as other infectious microorganisms is essential to minimizing the risk of illness. The development of sanitizers more efficacious than chlorine for the removal of pathogens from raw fruits and vegetable is needed.     

Effect of repeated irrigation with water containing varying levels of total organic carbon on the persistence of Escherichia coli O157:H7 on baby spinach

The California lettuce and leafy greens industry has adopted the Leafy Greens Marketing Agreement (LGMA), which allows for 126 most-probable-number (MPN) Escherichia coli per 100 ml in irrigation water. Repeat irrigation of baby spinach plants with water containing E. coli O157:H7 and different levels of total organic carbon (TOC) was used to determine the epiphytic survival of E. coli O157:H7. Three irrigation treatments (0 ppm of TOC, 12 or 15 ppm of TOC, and 120 or 150 ppm of TOC) were prepared with bovine manure containing E. coli O157:H7 at either low (0 to 1 log CFU/100 ml) or high (5 to 6 log CFU/100 ml) populations, and sprayed onto baby spinach plants in growth chambers by using a fine-mist airbrush. MPN and direct plating techniques were used to determine the E. coli O157:H7 populations on the aerial plant tissue. Plants irrigated with high E. coli O157:H7 populations, regardless of TOC levels, showed a 3-log reduction within the first 24 h. Low levels of E. coli O157:H7 were observed for up to 16 days on all TOC treatments, ranging from 76.4 MPN per plant (day 1) to 0.40 MPN per plant (day 16). No viable cells were detected on spinach tissue 24 h after irrigation with water containing fewer than 126 CFU/100 ml E. coli O157:H7. Under growth chamber conditions in this study, E. coli O157:H7 populations in irrigation water that complies with the LGMA standards will not persist for more than 24 h when applied onto foliar surfaces of spinach plants.     

Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 10(2) to 10(9) CFU/ml for E. coli O157:H7, 10(3) to 10(9) CFU/ml for Salmonella, and 10(1) to 10(8) CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 10(5) CFU/g for E. coli O157:H7, 10(3) CFU/g for Salmonella, and 10(4) CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 10(3) CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli 0157:H7, Salmonella, and Shigella in food.     

Effects of using reduced volumes of nonselective enrichment medium in methods for the detection of Escherichia coli O157:H7 from raw beef

Recent work from our laboratory revealed that tryptic soy broth (TSB) was a superior enrichment medium for use in test-and-hold Escherichia coli O157:H7 methods at levels down to a ratio of three volumes of medium to one volume of sample. Lower ratios were examined for their effect on results obtained from culture isolation, the BAX E. coli O157:H7 MP assay, and the Assurance GDS E. coli O157:H7 assay. Ground beef and boneless beef trim were inoculated with a high level (170 CFU/65 g of ground beef and 43 CFU/65 g of trim) and a low level (17 CFU/65 g of ground beef and 4 CFU/65 g of trim) of E. coli O157:H7 and enriched in 3, 1, 0.5, and 0 volumes of TSB. The volume of TSB used did not affect E. coli O157:H7 detection by culture isolation, Assurance GDS detection in ground beef or trim, or the BAX MP assay detection in ground beef. However, BAX MP assay detection of E. coli O157:H7 in beef trim was 50, 42, and 33% positive when enrichment volumes of 0.5x, 1x, and 3x, respectively, were used. Optimum results with all methods were obtained using 1 volume of TSB. We concluded that detection test results can be considered valid as long as enrichment medium is used, even when it is less than the specified 3 or 10 volumes.     

Rapid detection of Escherichia coli O157:H7 inoculated in ground beef,chicken carcass,and lettuce samples with an immunomagnetic chemiluminescence fiber-optic biosensor

A biosensor was evaluated with regard to its usefulness in the rapid detection of Escherichia coli O157:H7 inoculated in ground beef, chicken carcass, and romaine lettuce samples. The biosensor consisted of a chemiluminescence reaction cell, a fiber-optic light guide, and a luminometer linked to a personal computer in conjunction with immunomagnetic separation. The samples inoculated with E. coli O157:H7 were first centrifuged and suspended in buffered peptone water and then incubated with anti-E. coli O157 antibody-coated magnetic beads and horseradish peroxidase (HRP)-labeled anti-E. coli O157 antibodies to form antibody-coated bead-bacterium-HRP-labeled antibody sandwich complexes. Finally, the sandwich complexes were separated from the samples in a magnetic field and reacted with luminol in the reaction cell. The number of E. coli O157:H7 cells was determined by collecting the HRP-catalyzed chemiluminescence signal from the bead surface through a fiber-optic light guide and measuring the signal with a luminometer. The chemiluminescence biosensor was specific for E. coli O157:H7 in samples containing other bacteria, including Salmonella Typhimurium, Campylobacter jejuni, and Listeria monocytogenes. The chemiluminescence signal was linear on a log scale from 10(2) to 10(5) CFU of E. coli O157:H7 per ml in samples. Detection could be completed within 1.5 h without any enrichment. The detection limits for ground beef, chicken carcass, and lettuce samples were 3.2 x 10(2), 4.4 x 10(2), and 5.5 x 10(2) CFU of E. coli O157:H7 per ml, respectively.     

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.     

Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds.

Isolating Escherichia coli O157:H7 from batches of alfalfa seeds used to produce sprouts implicated in human illness has been difficult, perhaps due to nonhomogenous and very low-level contamination and inaccessibility of the pathogen entrapped in protected areas of the seed coat. We evaluated the effectiveness of various treatments in releasing E. coli O157:H7 from seeds. The influence of homogenization (blending or stomaching for 1 or 2 min), rinsing method (shaking for 5 min), soaking time (0. 1, 3, 6, or 15 h), soaking temperature (4 or 21 degrees C), and the addition of surfactants (0.1%, 0.5%, or 1.0% Tween 80 or Span 20) to rinse water was determined. Blending or stomaching for 1 or 2 min, and soaking for 1 h or longer at 4 or 21 degrees C, respectively, resulted in maximum release of E. coli O157:H7 from seeds. Soaking seeds at 37 degrees C for 15 h increased cell populations of E. coli O157:H7 by approximately 3.6 log10 CFU/g, likely due to bacterial growth. The maximum number of cells released from seeds by rinse water containing 1.0% Span 20 was at 21 degrees C, whereas at 37 degrees C, 0.1% or 0.5% Tween 80 was more effective. Detection of E. coli O157:H7 on seeds stored at 37 degrees C for up to 13 weeks and on sprouts derived from these seeds was compared. E. coli O157:H7 inoculated on seeds at 2.0 log10 CFU/g was detected after storage of seeds for up to 8 weeks at 37 degrees C and in sprouts produced from the seeds. The pathogen was not detected on seeds stored for 13 weeks at 37 degrees C and was not isolated from sprouts produced from these seeds. Identifying seed treatment methods that enhance removal of E. coli O157:H7 from alfalfa seeds can aid the isolation and enumeration of the pathogen on seeds. With a combination of optimal conditions for detecting the pathogen, i.e. soaking seeds for 1 h and pummeling seeds for 1 min, an enrichment step in modified tryptic soy broth (TSB), and the use of immunomagnetic beads for separation of E. coli O157:H7 cells, E. coli O157:H7 was detected in alfalfa seeds incubated at 37 degrees C for up to 8 weeks as effectively as in sprouts produced from the seeds.     

Optimization of rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes by PCR and application to field test

For rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes, simple methods for sample preparation and PCR were established and applied to a field test. To improve specificity, primer sets LP43-LP44 and C(+)-D(-) were selected for E. coli O157:H7 and L. monocytogenes, respectively. Through centrifugation and partial heat treatment after enrichment, E. coli O157:H7 and L. monocytogenes were detected at 1 initial CFU without genomic DNA extraction in the culture and with artificially inoculated food samples including milk, chicken, ham, and pork. Based on the optimized PCR method, a feasibility test was carried out using randomly collected field samples. To remove false positives and false negatives, a PCR method using several primer sets, including the optimized primer set, and a standard culture method were used. With the PCR detection and standard culture methods, two pork samples were positive for L. monocytogenes after enrichment, indications that the PCR assay could be effectively used for rapid, sensitive, and species-specific detection of foodborne pathogens.     

Influence of inoculation method, spot inoculation site, and inoculation size on the efficacy of acidic electrolyzed water against pathogens on lettuce

The influence of bacterial inoculation methods on the efficacy of sanitizers against pathogens was examined. Dip and spot inoculation methods were employed in this study to evaluate the effectiveness of acidic electrolyzed water (AcEW) and chlorinated water (200 ppm free available chlorine) against Escherichia coli O157:H7 and Salmonella spp. Ten pieces of lettuce leaf (5 by 5 cm) were inoculated by each method then immersed in 1.5 liters of AcEW, chlorinated water, or sterile distilled water for 1 min with agitation (150 rpm) at room temperature. The outer (abaxial) and inner (adaxial) surfaces of the lettuce leaf were distinguished in the spot inoculation. Initial inoculated pathogen population was in the range 7.3 to 7.8 log CFU/g. Treatment with AcEW and chlorinated water resulted in a 1 log CFU/g or less reduction of E. coli O157:H7 and Salmonella populations inoculated with the dip method. Spot inoculation of the inner surface of the lettuce leaf with AcEW and chlorinated water reduced the number of E. coli O157:H7 and Salmonella by approximately 2.7 and 2.5 log CFU/g, respectively. Spot inoculation of the outer surface of the lettuce leaf with both sanitizers resulted in approximately 4.6 and 4.4 log CFU/g reductions of E. coli O157:H7 and Salmonella, respectively. The influence of inoculation population size was also examined. Each sanitizer could not completely eliminate the pathogens when E. coli O157:H7 and Salmonella cells inoculated on the lettuce were of low population size (10(3) to 10(4) CFU/g), regardless of the inoculation technique.     

A simple method for the direct detection of Salmonella and Escherichia coli O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR

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.     

Evaluation of a polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.     

Survival and growth of Escherichia coli O157:H7 inoculated onto cut lettuce before or after heating in chlorinated water, followed by storage at 5 or 15 degrees C

This study determined the effects of mild heat and chlorine treatment followed by storage for up to 18 days at 5 degrees C or 7 days at 15 degrees C on the survival and growth of Escherichia coli O157:H7 inoculated onto fresh-cut iceberg lettuce. The efficacy of treatment with 20 ppm chlorine in killing the pathogen on lettuce at 50 degrees C was determined. Treatment of lettuce with 20 ppm chlorine at either 20 or 50 degrees C did not result in significantly greater reductions in populations of E. coli O157:H7 compared to respective treatments in water without chlorine. The pathogen steadily decreased in viability on treated lettuce throughout subsequent storage at 5 degrees C for 18 days. The population increased by 2.3 to 3.2 log10 CFU/g within 2 days, then continued to increase at a slower rate through 7 days of storage at 15 degrees C. At 4 and 7 days, significantly (alpha = 0.05) higher populations were reached on lettuce that had been treated at 50 degrees C, compared to respective samples that had been treated at 20 degrees C, regardless of the presence of 20 ppm chlorine in the treatment water. Treatment of lettuce with 20 ppm chlorine at 50 or 20 degrees C before or after inoculation with E. coli O157:H7 did not have a marked influence on behavior of the pathogen during subsequent storage at 5 or 15 degrees C.     

Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach

Escherichia coli O157:H7 contaminated spinach has recently caused several outbreaks of human illness in the USA and Canada. However, to date, there has been no study demonstrating an effective way to eliminate E. coli O157:H7 in spinach. Therefore, this study was conducted to investigate the effect of chemical sanitizers alone or in combination with packaging methods such as vacuum and modified atmosphere packaging (MAP) on inactivating E. coli O157:H7 in spinach during storage time. Spinach inoculated with E. coli O157:H7 was packaged in four different methods (air, vacuum, N(2) gas, and CO(2) gas packaging) following treatment with water, 100 ppm chlorine dioxide, or 100 ppm sodium hypochlorite for 5 min at room temperature and stored at 7+/-2 degrees C. Treatment with water did not significantly reduce levels of E. coli O157:H7 in spinach. However, treatment with chlorine dioxide and sodium hypochlorite significantly decreased levels of E. coli O157:H7 by 2.6 and 1.1 log(10)CFU/g, respectively. Levels of E. coli O157:H7 in samples packaged in air following treatments grew during storage time, whereas levels were maintained in samples packaged in other packaging methods (vacuum, N(2) gas, and CO(2) gas packaging). Therefore there were significant differences (about 3-4 log) of E. coli O157:H7 populations between samples packed in air and other packaging methods following treatment with chemical sanitizers after 7 days storage. These results suggest that the combination of treatment with chlorine dioxide and packaging methods such as vacuum and MAP may be useful for improving the microbial safety of spinach against E. coli O157:H7 during storage.     

Modified immunoliposome sandwich assay for the detection of Escherichia coli O157:H7 in apple cider

Detection of Escherichia coli O157:H7 in fruit juices such as apple cider is necessary for diagnosis of infection and epidemiological investigations. However, inhibitors in the apple cider, such as endogenous polyphenols and acids, often decrease the sensitivity of PCR assays and immunoassays, thus routinely requiring laborious cell separation steps to increase the sensitivity. In the current study, polyethylene glycol (PEG)-derivatized liposomes encapsulating sulforhodamine B were tagged with anti-E. coli O157:H7 antibodies and used in an immunoliposome sandwich assay for the detection of E. coli O157:H7 in apple cider. Even without prior separation, this assay can detect E. coli O157:H7 in apple cider samples inoculated with as few as 1 CFU/ml after an 8-h enrichment period. The lower limit of detection in pure cultures without enrichment was 7 x 10(3) CFU/ml (280 CFU/40-microl sample). PEGylated immunoliposomes are suitable as an analytical reagent for the detection of E. coli O157:H7 in fruit juices containing polyphenols.     

Efficacy of acidic electrolyzed water ice for pathogen control on lettuce

Acidic electrolyzed water (AcEW) was used as frozen AcEW (AcEW-ice) for inactivation of Listeria monocytogenes and Escherichia coli O157:H7 on lettuce. AcEW-ice was prepared from AcEW with 20, 50, 100, and 200 ppm of available chlorine by freezing at -40 degrees C and generated 30, 70, 150, and 240 ppm of chlorine gas (Cl2), respectively. The AcEW-ice was placed into styrene-foam containers with lettuce samples at 20 degrees C for 24 h. Although AcEW-ice generating 30 ppm Cl2 had no effect on L. monocytogenes cell counts, AcEW-ice generating 70 to 240 ppm of Cl2 significantly (P < 0.05) reduced L. monocytogenes by ca. 1.5 log CFU/g. E. coli O157:H7 cell counts were reduced by 1.0 log CFU/g with AcEW-ice generating 30 ppm of Cl2. AcEW-ice generating 70 and 150 ppm of Cl2 reduced E. coli O157:H7 by 2.0 log CFU/g. Further significant reduction of E. coli O157:H7 (2.5 log CFU/g) was demonstrated by treatment with AcEW-ice generating 240 ppm of Cl2. However, treatment with AcEW-ice generating 240 ppm of Cl2 resulted in a physiological disorder resembling leaf burn. AcEW-ice that generated less than 150 ppm of Cl2 had no effect on the surface color of the lettuce. AcEW-ice, regardless of the concentration of the emission of Cl2, had no effect on the ascorbic acid content in the lettuce. The weight ratio of lettuce to AcEW-ice required was determined to be over 1:10. The bactericidal effect of AcEW-ice appeared within the first 2 h. The use of AcEW-ice provides simultaneously for low temperature storage and inactivation of bacteria.     

Penetration of Escherichia coli O157:H7 into lettuce as influenced by modified atmosphere and temperature.

The effects of temperature and atmospheric oxygen concentration on the respiration rate of iceberg lettuce and Escherichia coli O157:H7 cells attachment to and penetration into damaged lettuce tissues were evaluated. Respiration rate of lettuce decreased as the temperature was reduced from 37 to 10 degrees C. Reducing the temperature further to 4 degrees C did not affect the respiration rate of lettuce. Respiration rate was also reduced by lowering the atmospheric oxygen concentration. Lettuce was submerged in E. coli O157:H7 inoculum at 4, 10, 22, or 37 degrees C under 21 or 2.7% oxygen. Attachment and penetration of E. coli O157:H7 were not related to the respiration rate. The greatest numbers of E. coli O157:H7 cells attached to damaged lettuce tissues at 22 degrees C at both oxygen concentrations. More cells were attached under 21% oxygen than under 2.7% oxygen at each temperature, but this difference was small. Penetration of E. coli O157:H7 into lettuce tissue was determined by immunostaining with a fluorescein isothiocyanate-labeled antibody. Under 21% oxygen, E. coli O157:H7 cells showed greatest penetration when lettuce was held at 4 degrees C, compared to 10, 22. or 37 degrees C, and were detected at an average of 101 microm below the surfaces of cut tissues. However, under 2.7% oxygen, there were no differences in degree of penetration among four incubation temperatures. The degree of E. coli O157:H7 penetration into lettuce tissue at 4 or 22 degrees C was greater under 21% oxygen than under 2.7% oxygen; however, no difference was observed at 37 degrees C. Conditions that promote pathogen penetration into tissue could decrease the effectiveness of decontamination treatments.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.