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.     

Effect of irrigation method on transmission to and persistence of Escherichia coli O157:H7 on lettuce

In this study, the transmission of Escherichia coli O157:H7 to lettuce plants through spray and surface irrigation was demonstrated. For all treatments combined, the number of plants testing positive following a single exposure to E. coli O157: H7 through spray irrigation (29 of 32 plants) was larger than the number testing positive following surface irrigation (6 of 32 plants). E. coli O157:H7 persisted on 9 of 11 plants for 20 days following spray irrigation with contaminated water. Immersion of harvested lettuce heads for 1 min in a 200 ppm chlorine solution did not eliminate all E. coli O157:H7 cells. The results of this study suggest that regardless of the irrigation method used, crops can become contaminated; therefore, the irrigation of food crops with water of unknown microbial quality should be avoided.     

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

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 10(7) CFU/g of compost and 10(5) 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.     

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.     

Presence and survival of Escherichia coli O157:H7 on lettuce leaves and in soil treated with contaminated compost and irrigation water

Escherichia coli O157:H7 outbreaks associated with produce consumption have brought attention to contaminated compost manure, and polluted irrigation water as potential sources of pathogens for the contamination of these crops. The aim of this study was to determine the potential transfer of E. coli O157:H7 from soil fertilized with contaminated compost or irrigated with contaminated water to edible parts of lettuce together with its persistence in soil under field conditions in two different seasons (fall and spring). Moreover, its survival on lettuce sprinkled with contaminated irrigation water was evaluated, as well as the prevalence of aerobic mesophilic, Enterobacteriaceae and Pseudomonadaceae in control lettuce samples. Four treatments, contaminated compost, surface and sprinkle irrigation with contaminated water and uninoculated pots, were used in this work. Contaminated compost was applied to soil in the pots before lettuce was transplanted and contaminated irrigation water was applied twice and three times on the plants after the seedlings were transplanted, for sprinkle and surface irrigation, respectively. E. coli O157:H7 survived in soil samples for 9 weeks at levels, 4.50 log cfu gdw(-1) (dw, dry weight) in fall and 1.50 log cfu gdw(-1) in spring. The pathogen survives better in fall, indicating an important influence of environmental factors. E. coli O157:H7 population in lettuce leaves after sprinkle irrigation was very high (between 10(3) and 10(6) cfu g(-1)), but decreased to undetectable levels at field conditions. There was also transfer of E. coli O157:H7 from soil contaminated with compost or irrigated with contaminated water to lettuce leaves, mainly to the outer ones. The mean counts for aerobic mesophilic, Enterobacteriaceae and Pseudomonadaceae populations were also influenced by environmental conditions; higher levels were observed under fall conditions than in spring conditions. Contamination of lettuce plants in the field can occur through both contaminated composted manure and irrigation water and persist for several months.     

Efficacy of a peroxyacetic acid formulation as an antimicrobial intervention to reduce levels of inoculated Escherichia coli O157:H7 on external carcass surfaces of hot-boned beef and veal

The efficacy of a peroxyacetic acid formulation (POAA) at reducing Escherichia coli O157:H7 contamination on external carcass surfaces of hot-boned beef and veal with a commercial spray apparatus was determined. Hot-boned external carcass surfaces were inoculated with either a high dose (10(6) CFU/cm2) in fresh bovine feces or with a low dose (10(3) CFU/cm2) in diluent of laboratory-cultured E. coli O157:H7. Treatments included a water wash, a POAA (180 ppm) wash, or a water plus POAA wash. Samples were extracted from the external carcass surface with a cork borer to determine the numbers of viable E. coli O157:H7 remaining on the carcass surface after treatment. Although a water wash alone resulted in a 1.25 (94.4%) and a 1.31 (95.1%) mean log reduction on veal and beef inoculated with a high dose of E. coli O157:H7, the POAA treatment resulted in a substantially greater mean log reduction of 3.56 and 3.59 (>99.9%). The water wash only resulted in a 33.9% reduction on veal and 62.8% on beef inoculated with a low dose of E. coli O157:H7, whereas POAA treatment greatly improved pathogen reduction to 98.9 and 97.4% on veal and beef, respectively. The combination of a water wash followed by a POAA treatment resulted in a similar E. coli O157:H7 reduction to that achieved by POAA treatment alone. In conclusion, POAA treatment significantly reduced viable E. coli O157:H7 numbers on experimentally contaminated beef and veal carcasses, which justifies its use as a chemical intervention for the removal of this human pathogen.     

Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.     

The survival of Escherichia coli O157:H7 in the presence of Penicillium expansum and Glomerella cingulata in wounds on apple surfaces

The survival of Escherichia coli O157:H7 in the presence of one of two plant pathogens, Penicillium expansum and Glomerella cingulata, in wounds on apples was observed during 14 days storage at room temperature (RT) and at 4 degrees C. The aim of this work was to determine if changes in apple physiology caused by the proliferation of fungal decay organisms would foster the survival of E. coli O157:H7. Trials were performed where (A) plant pathogens (4 log10 spores) were added to apple wounds 4 days before the wounds were inoculated with E. coli O157:H7 (3 log10 CFU g(-1) apple) (both RT and 4 degrees C storage), (B) plant pathogens and E. coli O157:H7 were added on the same day (both RT and 4 degrees C storage), and (C) E. coli O157:H7 was added 2 days (RT storage) and 4 days (4 degrees C storage) before plant pathogens. In all trials E. coli O157:H7 levels generally declined to <1 log10 at 4 degrees C storage, and in the presence of P. expansum at 4 degrees C or RT. However, in the presence of G. cingulata at RT E. coli O157:H7 numbers increased from 3.18 to 4.03 log10 CFU g(-1) in the apple wound during trial A, from 3.26 to 6.31 log10 CFU g(-1) during trial B, and from 3.22 to 6.81 log10 CFU g(-1) during trial C. This effect is probably a consequence of the attendant rise in pH from 4.1 to approximately 6.8, observed with the proliferation of G. cingulata rot. Control apples (inoculated with E. coli O157:H7 only) were contaminated with opportunistic decay organisms at RT during trials A and B, leading to E. coli O157:H7 death. However, E. coli O157:H7 in control apples in trial C, where no contamination occurred, increased from 3.22 to 5.97 log10 CFU g(-1). The fact that E. coli O157:H7 can proliferate in areas of decay and/or injury on fruit highlights the hazards associated with the use of such fruit in the production of unpasteurized juice.     

Spread of bacterial pathogens during preparation of freshly squeezed orange juice

To study the potential of three bacterial pathogens to cross-contaminate orange juice during extraction, normal operation conditions during juice preparation at food service establishments were simulated. The spread of Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes from inoculated oranges to work surfaces and to the final product was determined. The transference of these three bacterial pathogens to orange juice made from uninoculated oranges with the use of contaminated utensils was also studied. Fresh oranges were inoculated with a marker strain of rifampicin-resistant Salmonella Typhimurium, E. coli O157:H7, or L. monocytogenes. Final pathogen levels in juice were compared as a function of the use of electric or mechanical juice extractors to squeeze orange juice from inoculated oranges. Pathogen populations on different contact surfaces during orange juice extraction were determined on sulfite-phenol red-rifampicin plates for Salmonella Typhimurium and E. coli O157:H7 and on tryptic soy agar supplemented with 0.1 g of rifampicin per liter for L. monocytogenes. After inoculation, the average pathogen counts for the orange rind surface were 2.3 log10 CFU/cm2 for Salmonella Typhimurium, 3.6 log10 CFU/cm2 for E. coli O157:H7, and 4.4 log10 CFU/cm2 for L. monocytogenes. This contamination was spread over all utensils used in orange juice squeezing. Mean pathogen counts for the cutting board, the knife, and the extractor ranged from -0.3 to 2.1 log10 CFU/cm2, and the juice contained 1.0 log10 CFU of Salmonella Typhimurium per ml, 2.3 log10 CFU of E. coli O157:H7 per ml, and 2.7 log10 CFU of L. monocytogenes per ml. Contact with contaminated surfaces resulted in the presence of all pathogens in orange juice made from uninoculated oranges. These results give emphasis to the importance of fresh oranges as a source of pathogens in orange juice.     

Survival of Salmonella typhimurium and Escherichia coli O157:H7 in cheese brines

Survival of Salmonella typhimurium and Escherichia coli O157:H7 was studied in model brines and brine from three cheese plants. Three strain mixtures of S. typhimurium and E. coli O157:H7 (10(6) CFU/ml) were inoculated separately into 23% model brine with or without added pasteurized whey (2%) and as a combined inoculum into the commercial brines. The model brines were incubated at 8 and 15 degrees C for 28 days, and the commercial brines at 4 and 13 degrees C for 35 days. Populations of both pathogens in the model brine + whey decreased slowly over 28 days (1.0-2.0 log CFU/ml) with greater survival at 8 degrees C than at 15 degrees C. Corresponding decreases in model brine without whey were 1.9-3.0 log CFU/ml, with greater survival at 8 degrees C than at 15 degrees C. Both S. typhimurium and E. coli O157:H7 survived significantly better (P < 0.05) at 4 degrees C than at 13 degrees C in two of the commercial brines. The survival of each pathogen in the commercial brines at 13 degrees C was significantly influenced by brine pH. Both pathogen populations decreased most rapidly in commercial brines during the first week of storage (2.5-4.0 and 2.3-2.8 log CFU/ml for S. typhimurium and E. coli O157:H7, respectively) with significant recovery (ca. 0.5 log CFU/ml increase) often occurring in the second week of storage. Counts changed little thereafter. Overall, E. coli O157:H7 survived better than S. typhimurium, with differences of 0.1-1.2 log CFU/ml between the two pathogens. Results of this study show that cheese brine could support the survival of contaminating S. typhimurium and E. coli O157:H7 for several weeks under typical brining conditions.     

Sensitive detection of Escherichia coli O157:H7 by conventional plating techniques

The direct detection and estimation of concentration of Escherichia coli O157:H7 down to 1 CFU/g of cheese was achieved by conventional plating techniques. Cheese was manufactured with unpasteurized milk inoculated with E. coli O157: H7 at 34 +/- 3 CFU/ml. The numbers of E. coli O157:H7 were monitored during cheese ripening by plating on sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) and on CT-O157:H7 ID medium. Using the pour plate method, E. coli O157:H7 colonies could easily be distinguished from non-O157:H7 colonies on CT-O157:H7 ID medium but not on CT-SMAC. Higher numbers of E. coli O157:H7 were detectable with O157:H7 ID medium. Latex agglutination and PCR were used to confirm the identification of typical E. coli O157:H7 colonies, and nontypical colonies as not being E. coli O157:H7. As few as 1 CFU/g of cheese could be detected. E. coli O157:H7 also was detected in deliberately contaminated milk at concentrations as low as 4 CFU/10 ml.     

Inactivation of Escherichia coli O157: H7 and Listeria monocytogenes by PR-26, a synthetic antibacterial peptide.

This study reports the antibacterial effect of PR-26, a synthetic peptide derived from the first 26 amino acid sequence of PR-39, an antimicrobial peptide isolated from porcine neutrophils. A three-strain mixture of Escherichia coli O157:H7 or Listeria monocytogenes of approximately 10(8) CFU was inoculated to a final concentration of 10(7) CFU/ml in 1% peptone water (pH 7.0), containing 50 or 75 microg/ml of PR-26, and incubated at 37 degrees C for 0, 6, 12, and 24 h; at 24 degrees C for 0, 12, 24, and 36 h; or at 10 or 4 degrees C for 0, 24, 72, and 120 h. Control samples included 1% peptone water inoculated with each pathogen mixture but containing no PR-26. The surviving population of each pathogen at each sampling time was determined by plating on tryptic soy agar with incubation at 37 degrees C for 24 h. At 37 degrees C, PR-26 decreased E. coli O157:H7 and L. monocytogenes populations by >5.0 log CFU/ml at 12 h, with complete inactivation at 24 h. At 24 degrees C, PR-26 reduced E. coli O157:H7 and L. monocytogenes by approximately 3.5, 4.0, and 4.5 log CFU/ml at the end of 12-, 24-, and 36-h incubations, respectively. At 4 and 10 degrees C, the inhibitory effect of PR-26 on E. coli O157:H7 and L. monocytogenes was significantly lower (P < 0.05) than that at 37 and 24 degrees C: a 2- to 3-log CFU/ml reduction was observed at 120-h incubation. Results indicate that PR-26 could potentially be used as an antimicrobial agent, but applications in appropriate foods need to be validated.     

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.     

Behavior of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses manufactured from raw milk

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.     

Effect of xylitol on adhesion of Salmonella Typhimurium and Escherichia coli O157:H7 to beef carcass surfaces

Effects of 10% xylitol (a five-carbon sugar alcohol) on adhesion of Escherichia coli O157:H7 and Salmonella Typhimurium to meat surfaces were examined with three approaches. First, beef outside round was inoculated with rifampin-resistant E. coli O157:H7 and Salmonella Typhimurium dispersed in xylitol or peptone solution. Samples were rinsed with water or not rinsed in a 2 x 2 factorial arrangement. No interaction existed between inoculum and rinsing treatments (P > 0.84). Incubation in xylitol had minimal impact on pathogen adhesion (P > 0.76); however, rinsing reduced pathogen cell counts (P < 0.01). Second, meat samples were treated with water, xylitol, or no rinse; inoculated with pathogens dispersed in peptone solution (8.6 log CFU/ml for each pathogen); and then treated with water, xylitol, or no rinse in a 3 x 3 factorial arrangement. No interactions were observed (P > 0.50). Postinoculation rinsing reduced pathogen loads (P < 0.01) without difference between water and xylitol (P > 0.64). Third, carcass surfaces inoculated with pathogens (5.5 log CFU/cm2) were treated with 35 degrees C water wash, 2.5% L-lactic acid spray, 10% xylitol spray, lactic acid plus xylitol, or hot water plus xylitol. Lactic acid treatments reduced Salmonella Typhimurium at 0 h (P < 0.01) and 24 h (P < 0.02). Hot water treatments tended to reduce Salmonella Typhimurium at 0 h (P < 0.07). Xylitol did not reduce pathogens (P > 0.62) or increase effectiveness of other treatments. Xylitol does not influence E. coli O157:H7 and Salmonella Typhimurium adhesion to meat surfaces.     

The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk.

The objective of this study was to develop a fluorescent bacteriophage assay (FBA) for the detection of E. coli O157:H7 in ground beef and raw milk. The FBA is a two step assay that combines immunomagnetic separation, to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E. coli O157:H7 cells. When used in conjunction with flow cytometry, the FBA was able to detect 2.2 CFU/g of artificially contaminated ground beef following a 6 h enrichment. Between 10(1) and 10(2) CFU/ml of artificially contaminated raw milk were detectable after a 10 h enrichment step. The results show that the FBA is potentially useful as a rapid technique for the preliminary detection of E. coli O157:H7 in food.     

Fate of Escherichia coli O157:H7 during silage fermentation

The survival characteristics of Escherichia coli O157:H7 in silage derived from contaminated grass were investigated. The survival of other enteric bacteria was also investigated to determine if E. coli O157:H7 demonstrates enhanced acid tolerance in comparison. Samples of chopped grass were treated as follows: (i) no additive (control); (ii) inoculation with E. coli O157:H7 to a final concentration of log10 4.0 CFU g(-1); (iii) addition of an 85% solution of formic acid at 3.0 ml kg(-1) grass; and (iv) addition of both E. coli O157:H7 and formic acid, at the above concentrations. Treated 6-kg grass samples were packed into laboratory silos, sealed, and stored at 15 degrees C for up to 180 days. Individual replicate silos were removed from storage periodically and subjected to microbiological and chemical analyses. Chemical analyses of the silage samples indicated that lactic acid-dominant fermentations, with a rapid drop in pH, occurred. Numbers of enteric bacteria decreased from log10 7.0 to 8.0 CFU g(-1) to undetectable levels within 19 days' storage. E. coli O157:H7 did not survive the silage fermentation process, with numbers declining from approximately log10 4.0 CFU g(-1) to undetectable levels within 19 days of ensiling. The pattern of decline in numbers of E. coli O157:H7 was the same as that for the enteric bacteria, indicating that under the conditions tested, the acid tolerance of E. coli O157:H7 was not significantly different from the acid tolerance of other enteric bacteria. This study found that E. coli O157:H7 did not survive a good silage fermentation process, indicating that properly ensiled grass that is correctly stored is unlikely to be a vector for the transmission of the pathogen among cattle.     

Fate of Escherichia coli O157:H7, Salmonella typhimurium DT 104, and Listeria monocytogenes in fresh meat decontamination fluids at 4 and 10 degrees C.

Bacterial pathogens may colonize meat plants and increase food safety risks following survival, stress hardening, or proliferation in meat decontamination fluids (washings). The objective of this study was to evaluate the ability of Escherichia coli O157:H7, Salmonella Typhimurium DT 104, and Listeria monocytogenes to survive or grow in spray-washing fluids from fresh beef top rounds sprayed with water (10 or 85 degrees C) or acid solutions (2% lactic or acetic acid, 55 degrees C) during storage of the washings at 4 or 10 degrees C in air to simulate plant conditions. Inoculated Salmonella Typhimurium DT 104 (5.4 +/- 0.1 log CFU/ml) died off in lactate (pH 2.4 +/- 0.1) and acetate (pH 3.1 +/- 0.2) washings by 2 days at either storage temperature. In contrast, inoculated E. coli O157:H7 (5.2 +/- 0.1 log CFU/ml) and L. monocytogenes (5.4 +/- 0.1 log CFU/ml) survived in lactate washings for at least 2 days and in acetate washings for at least 7 and 4 days, respectively; their survival was better in acidic washings stored at 4 degrees C than at 10 degrees C. All inoculated pathogens survived in nonacid (pH > 6.0) washings, but their fate was different. E. coli O157:H7 did not grow at either temperature in water washings, whereas Salmonella Typhimurium DT 104 failed to multiply at 4 degrees C but increased by approximately 2 logs at 10 degrees C. L. monocytogenes multiplied (0.6 to 1.3 logs) at both temperatures in water washings. These results indicated that bacterial pathogens may survive for several days in acidic, and proliferate in water, washings of meat, serving as potential cross-contamination sources, if pathogen niches are established in the plant. The responses of surviving pathogens in meat decontamination waste fluids to acid or other stresses need to be addressed to better evaluate potential food safety risks.     

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.     

Biological control of postharvest decays of apple can prevent growth of Escherichia coli O157:H7 in apple wounds

Fresh cells of the antagonist Pseudomonas syringae at 2.4 x 10(8) CFU/ml inoculated into wounds of 'Golden Delicious' apple prevented Escherichia coli O157:H7 (concentrations ranging from 2.4 x 10(5) to 2.4 x 10(7) CFU/ml) from growing in the wounds. This occurred when the two microorganisms were co-inoculated or inoculation with E. coli O157:H7 was conducted 1 or 2 days after inoculation with the antagonist. In similar tests, application of the commercial formulation of this antagonist prevented the growth of E. coli O157:H7 in wounds when inoculated 1 or 2 days after application of the antagonist. Populations of E. coli O157:H7 in wounds treated with water (control) before inoculation with this pathogen increased approximately 2 log units during the first 48 h after inoculation. These results indicate that biocontrol agents developed for controlling storage decays of fruits may have the additional benefit of preventing the growth of foodborne pathogens in freshly wounded tissue of intact and fresh-cut fruits.