Efficacy of Acetic Acid or Chitosan for Reducing the Prevalence of Salmonella- and Escherichia coli O157:H7-Contaminated Leafy Green Plants in Field Systems.

Outbreaks associated with fresh-cut leafy greens continue to occur despite efforts to implement horticultural practices that minimize introduction of enteric pathogens to the crop. The experimental trials in this study were designed to examine the efficacy of an acetic acid (AA)- and chitosan-based spray treatment, applied 1 day prior to harvest, for reducing the prevalence of Escherichia coli O157:H7 (O157) and Salmonella in field-grown leafy greens contaminated at levels detectable only through enrichment culture. Responses to the treatment solution were variable and depended on the type of leafy green (leafy lettuce, spinach, or cabbage), cultivar, pathogen, and AA concentration (0.3 to 0.7%). No significant differences in E. coli O157 prevalence were found for untreated and treated cabbage heads and spinach plants (P > 0.05). In contrast, treatment significantly affected Salmonella on 'Bravo F1' green cabbage and '7-Green' spinach (P < 0.05), with odds ratios of 2.2 and 3.3 for finding the pathogen on untreated versus treated greens, respectively. Salmonella was also 7.1 times more likely to be found on an untreated lettuce plant than on a lettuce plant sprayed with a 0.7% AA treatment solution (95% confidence interval [CI], 4.1 to 12.2; P < 0.0001). In studies addressing the efficacy of chitosan (0.1 or 0.3%), this chemical failed to reduce the prevalence of either pathogen on lettuce (P > 0.05). Similarly, spraying with 0.3% AA did not affect the prevalence of Salmonella on lettuce plants (P > 0.05); however, treatment solutions with 0.4% AA reduced the likelihood of detecting Salmonella in treated versus untreated plants by 6.6 times (95% CI, 2.1 to 20.9; P = 0.0007). After the lettuce was harvested and hand washed, consumers failed to distinguish either visually or organoleptically between untreated lettuce and lettuce sprayed with an acetic acid solution (P > 0.05). These results indicate that acetic acid could be used to reduce the microbiological risk of preharvest leafy greens.

Exploratory Study of the Application of Smoke Aerosols to Manure-Based Composting Materials To Reduce Prevalence of Salmonella.

During the early stages of aerobic composting, heat is generated and when the materials are self-insulating, extended exposure of pathogens to this heat source will lead to significant reduction, if not elimination, of the pathogens. However, when insufficient heat is applied to the composting materials, pathogens may survive. Under those conditions if the compost had contained material of animal origin or food waste, it would be considered untreated and would not be allowed in fields growing crops that may be consumed raw. However, alternative treatment processes are allowed, provided they are validated to meet the microbial standards stipulated in the Produce Safety final rule of the Food Safety Modernization Act and that the physical parameters of the process are documented to ensure that the conditions under which the process was validated have been met. Hence, this exploratory study was undertaken in a laboratory setting to determine the potential for application of aerosolized smoke to inactivate Salmonella in manure-based compost. Smoke generated from wood chips (oak or pecan) and introduced to the headspace of contaminated cow manure compost (≤3 log CFU/g) in sealed containers (35 g per container) resulted in no Salmonella detected by enrichment culture in 100% (0 of 14) of the samples after 18 to 48 h of exposure, whereas Salmonella in control samples remained at initial levels over the same time period. Shorter exposure times (6 h) to the smoke aerosols were less effective (11 of 24 samples positive by enrichment culture), and additional flushes with the wood smoke during this time failed to decrease the prevalence of contamination. Smoke aerosols generated from waste agricultural materials and held in containers with Salmonella-contaminated compost for 18 h significantly reduced the prevalence of the pathogen in samples compared with control samples (P < 0.05). The odds of not finding Salmonella in smoke-exposed compost were 14 (pine needles and rice hulls), 23 (cocoa hulls, orange rind, and peanut hulls), and 28 (sunflower hulls) times greater compared with samples not exposed to smoke. Many other variables remain to be examined (e.g., compost composition, compost maturity, and anaerobic conditions) to determine whether this approach could be universally applied to manure-based compost. Validation under field conditions will be required and may entail use of this approach in combination with suboptimal thermal conditions (<55°C).

Salmonella and Enterohemorrhagic Escherichia coli Serogroups O45, O121, O145 in Wheat Flour: Effects of Long-Term Storage and Thermal Treatments.

Salmonella and enterohemorrhagic Escherichia coli (EHEC) are of serious concern in wheat flour and its related products but little is known on their survival and thermal death kinetics. This study was undertaken to determine their long-term viability and thermal inactivation kinetics in flour. Inoculation was performed using mixtures of EHEC serogroups O45, O121, O145 and Salmonella followed by storage at room temperature (23°C) or 35°C (for Salmonella). Plate counting on tryptic soy agar (TSA) and enrichment were used to assess long-term survival. For thermal studies, wheat flour samples were heated at 55, 60, 65, and 70°C and cell counts of EHEC and Salmonella were determined by plating. The δ-values were calculated using the Weibull model. At room temperature, EHEC serovars and Salmonella were quantifiable for 84 and 112 days, and were detectable for the duration of the experiment after 168 and 365 days, respectively. The δ-values were 2.0, 5.54, and 9.3 days, for EHEC O121, O45, and O145, respectively, and 9.7 days for Salmonella. However, the only significant difference among all values was the δ-value for Salmonella and serogroup O121 (p ≤ 0.05). At 35°C, Salmonella counts declined to unquantifiable levels after a week and were not detected upon enrichment after 98 days. Heat treatment of inoculated wheat flour at 55, 60, 65, and 70°C resulted in δ-value ranges of 20.0-42.9, 4.9-10.0, 2.4-3.2, and 0.2-1.6 min, respectively, for EHEC. The δ-values for Salmonella at those temperatures were 152.2, 40.8, 17.9, and 17.4 min, respectively. The δ-values obtained for Salmonella at each temperature were significantly longer than for EHEC (p ≤ 0.05). Weibull model was a good fit to describe the thermal death kinetics of Salmonella and EHEC O45, O121 and O145 in wheat flour. HIGHLIGHTS -EHEC and Salmonella can survive for extended periods of time in wheat flour.-Long-term storage inactivation curves of EHEC and Salmonella were similar.-EHEC was more sensitive to heat than Salmonella.-Weibull model was a good fit to describe thermal death kinetics of EHEC and Salmonella.-Flour storage at 35°C may be a feasible method for microbial reduction.

Survival of Salmonella enterica and Escherichia coli O157:H7 Sprayed onto the Foliage of Field-Grown Cabbage Plants.

To reduce the number of cabbage pathogen outbreaks, it is essential to understand the fate of enteric pathogens that contaminate plants in the field. To assist in that effort, two independent trials were conducted with a red cultivar (cv. Red Dynasty) and a green cultivar (cv. Bravo F1) of field-grown cabbage ( Brassica oleracea var. capitata). In the first trial, plants with small heads were sprayed with an inoculum containing both attenuated Salmonella enterica Typhimurium and Escherichia coli O157:H7 (5.0 log CFU/mL). Initial pathogen levels (ca. 3.9 log CFU per head), determined through plate count enumeration (limit of detection was 1.3 log CFU/g), dropped precipitously such that 2 days later, they could not be detected by enrichment culture in 22 to 35% of the heads. However, subsequent declines were at a slower rate; no differences were observed between red and green cabbage heads ( P > 0.05), and heads were still positive for the pathogens 22 days after being sprayed with the inoculum. As a result, the logistic model revealed that for every 2 days contaminated cabbage heads remained in the field, the probability of finding a positive sample decreased by a factor of 1.1 (95% confidence interval from 1.0 to 1.2, P = 0.0022) and 1.2 (95% confidence interval from 1.0 to 1.4, P ≤ 0.0001) for Salmonella and E. coli O157:H7, respectively. In the second trial occurring 2 weeks later, plants with medium red or green cabbage heads were sprayed with an inoculum at a dose of 3.5 log CFU/mL. A similar decay in prevalence over time occurred for green cabbage as in trial 1; however, pathogen decline in red cabbage was less in trial 2 than in trial 1. The extended persistence of pathogens in cabbage heads exhibited in both trials infers that harvest of contaminated cabbage destined for raw consumption is risky. Additional field studies are necessary to determine whether similar pathogen fates occur in other regions or climates and to clarify the effect of the maturity of red cabbage on pathogen inactivation.

Survival and internalization of Salmonella and Escherichia coli O157:H7 sprayed onto different cabbage cultivars during cultivation in growth chambers.

BACKGROUND: Cabbage may become contaminated with enteric pathogens during cultivation. Using multiple cabbage cultivars at two maturity stages (small plants or plants with small heads) in growth chamber studies, the fate (internalization or surface survival) of Salmonella and Escherichia coli O157:H7 (0157) were examined in conjunction with any potential relationships to the plant's antimicrobial content. RESULTS: Internalized Salmonella was detected in cabbage within 24 h with prevalence ranging from 62% (16 of 26) for the 'Super Red 80' cultivar to 92% (24 of 26) for the 'Red Dynasty' cultivar. Surface survival of pathogens on small cabbage plants over nine days was significantly affected by cultivar with both pathogens surviving the most on the 'Farao' cultivar and Salmonella and O157 surviving the least on the 'Super Red 80' and 'Capture' cultivars, respectively (P < 0.05). Survival of O157 was slightly higher on cabbage heads for O157 than small plants suggesting that the maturity stage may affect this pathogen's fate. An inverse relationship existed between antimicrobial levels and the pathogen's surface survival on cabbage heads (P < 0.05). CONCLUSIONS: The fate of pathogens varied with the cabbage cultivar in growth chamber studies highlighting the potential to explore cultivar in field studies to reduce the risk of microbiological contamination in this crop. © 2019 Society of Chemical Industry.

Pre-harvest internalization and surface survival of Salmonella and Escherichia coli O157:H7 sprayed onto different lettuce cultivars under field and growth chamber conditions.

Plant genotype has been advocated to have an important role in the fate of enteric pathogens residing in lettuce foliage. This study was therefore undertaken under the premise that different pathogen responses could occur in lettuce cultivars with cultivar selection being one of several hurdles in an overall strategy for controlling foodborne pathogens on field-grown produce. Up to eight lettuce cultivars ('Gabriella', 'Green Star', 'Muir', 'New Red Fire', 'Coastal Star', 'Starfighter', 'Tropicana', and 'Two Star') were examined in these experiments in which the plants were subjected to spray contamination of their foliage with pathogens. In an experiment that addressed internalization of Salmonella, cultivar was determined to be a significant variable (P < 0.05) with 'Gabriella' and 'Muir' being the least and most likely to exhibit internalization of this pathogen, respectively. Furthermore, antimicrobials (total phenols and antioxidant capacity chemicals) could be part of the plant's defenses to resist internalization as there was an inverse relationship between the prevalence of internalization at 1 h and the levels of these antimicrobials (r = -0.75 to -0.80, P = 0.0312 to 0.0165). Internalized cells appeared to be transient residents in that across all cultivars, plants sampled 1 h after being sprayed were 3.5 times more likely to be positive for Salmonella than plants analyzed 24 h after spraying (95% CI from 1.5 to 8.2, P = 0.0035). The fate of surface-resident Salmonella and Escherichia coli O157:H7 was addressed in subsequent growth chamber and field experiments. In the growth chamber study, no effect of cultivar was manifested on the fate of either pathogen when plants were sampled up to 12 days after spray contamination of their foliage. However, in the field study, five days after spraying the plants, Salmonella contamination was significantly affected by cultivar (P < 0.05) and the following order of prevalence of contamination was observed: 'Muir' < 'Gabriella' < 'Green Star' = 'New Red Fire' < 'Coastal Star'. Nine days after spray contamination of plants in the field, no effect of cultivar was exhibited due primarily to the low prevalence of contamination observed for Salmonella (8 of 300 plant samples positive by enrichment culture) and E. coli O157 (4 of 300 plant samples positive by enrichment culture). Given the narrow window of time during which cultivar differences were documented, it is unlikely that cultivar selection could serve as a viable option for reducing the microbiological risk associated with lettuce.

Disposition of Salmonella and Escherichia coli O157:H7 following Spraying of Contaminated Water on Cucumber Fruit and Flowers in the Field.

Cucumbers are frequently consumed raw and have been implicated in several recent foodborne outbreaks. Because this item may become contaminated at the farm, it is vital to explore the fate of attenuated Salmonella Typhimurium or Escherichia coli O157:H7 sprayed onto foliage, flowers, and fruit in fields and determine whether pre- or postcontamination spray interventions could minimize contamination. After spraying cucumber plants with contaminated irrigation water (3.8 log CFU/mL of Salmonella Typhimurium and E. coli O157:H7), 60 to 78% of cucumber fruit were not contaminated because the plant's canopy likely prevented many of the underlying fruit from being exposed to the water. Subsequent exposure of contaminated cucumber plants to a simulated shower event did not appear to dislodge pathogens from contaminated foliage onto the fruit, nor did it appear to consistently wash either pathogen from the fruit. Spraying flowers and attached ovaries directly with a pathogen inoculum (4.6 log CFU/mL) initially led to 100% and 65 to 90% contamination, respectively. Within 3 days, 30 to 40% of the flowers were still contaminated; however, contamination of ovaries was minimal (≤10%), suggesting it was unlikely that internalization occurred through the flower to the ovary with these pathogen strains. In another study, both pathogens were found on a withered flower but not on the fruit to which the flower was attached, suggesting that this contaminated flower could serve as a source of cross-contamination in a storage bin if harvested with the fruit. Because pre- and postcontamination acetic acid-based spray treatments failed to reduce pathogen prevalence, the probability that fruit initially contaminated at 1.3 to 2.8 log CFU of Salmonella Typhimurium or E. coli O157:H7 per cucumber would be positive by enrichment culture decreased by a factor of 1.6 and 1.9 for Salmonella Typhimurium and E. coli O157:H7, respectively, for every day the fruit was held in the field ( P ≤ 0.0001). Hence, to reduce the prevalence of Salmonella Typhimurium on cucumbers below 5%, more than 1 week would be required.

Inactivation of Escherichia coli O157:H7 and Salmonella during washing of contaminated gloves in levulinic acid and sodium dodecyl sulfate solutions.

Field workers often wear gloves harvesting ready-to-eat produce; however, fields are not sterile environments and gloves may become contaminated numerous times during a working shift. This study explored the potential for inactivation of Escherichia coli O157:H7 and Salmonella when contaminated gloves were washed in levulinic acid (LV) and sodium dodecyl sulfate (SDS) solutions. Washing nitrile gloves with increasing concentrations of LV above 1.0% led to a decreased prevalence of glove contamination by Salmonella (P = 0.0000). A higher level of prevalence occurred for solid agar-cultured pathogens than liquid broth-cultured pathogens after nitrile gloves were washed in LV/SDS (P = 0.0000). Pathogens residing on latex gloves were more likely to be completely inactivated by washing in 0.5% LV/0.1% SDS solutions than nitrile or Canners gloves that exhibited inconsistent responses dependent on the pathogen strain. However, drying after washing nitrile gloves in 0.5% LV/0.1% SDS led to additional pathogen inactivation (P = 0.0394). Pathogen transfer from gloves to produce was implied as the pathogen prevalence on cantaloupe rind handled by LV/SDS-washed gloves was not statistically different from the prevalence on gloves (P = 0.7141). Hence, the risk of produce contamination may still exist but would be reduced by washing gloves in LV/SDS.

Inactivation of Escherichia coli O157:H7 and Salmonella deposited on gloves in a liquid state and subjected to drying conditions.

Gloves are worn by workers harvesting ready-to-eat produce as a deterrent for contaminating the produce with enteric pathogens that may reside on their hands. As fields are not sterile environments, the probability for gloves to become contaminated still exists and therefore it is critical to understand the conditions that affect the survival of pathogens on gloves. Both Escherichia coli O157:H7 and Salmonella deposited on glove surfaces in a liquid state survived longer when the pathogen had been suspended in lettuce sap than when suspended in water. Despite this protection, pathogens deposited on clean single-use gloves were more likely to survive during drying than pathogens deposited on dirty gloves (a film of lettuce sap had been applied to the surface prior to pathogen application and soil had been ground into the gloves). Survival of both E. coli O157:H7 and Salmonella was biphasic with the greatest losses occurring during the first hour of drying followed by much slower losses in the ensuing hours. Pathogens grown in rich media (tryptic soy broth) versus minimal media (M9) as well as those cultured on solid agar versus liquid broth were also more likely to be resistant to desiccation when deposited onto gloves. Although survival of E. coli O157:H7 on nitrile gloves was in general greater than it was on latex gloves, the relative survival of Salmonella on the two glove types was inconsistent. Due to these inconsistencies, no one glove type is considered better than another in reducing the risk for contamination with enteric pathogens. In addition, the extended survival of what are generally referred to as stress-resistant pathogens suggests that gloves either be changed frequently during the day or washed in a disinfectant to reduce the risk of glove contamination that could otherwise contaminate product handled with the contaminated gloves.