Agricultural Mitigation Strategies to Reduce the Impact of Romaine Lettuce Contamination.

BACKGROUND: Leafy greens, particularly romaine lettuce, are often associated with outbreaks due to their susceptibility to contamination from various environmental sources. This study aimed to evaluate the presence of E. coli, Salmonella, copper, nickel, zinc, and manganese in irrigation water, lettuce leaves, and agricultural soil in the Litani River Basin (LRB), Lebanon. METHOD: Samples were collected from five demonstration plots employing different agricultural practices. Heavy metal concentrations were determined using atomic absorption spectrometry, while E. coli and Salmonella testing were conducted through conventional culturing techniques. The impact of E. coli contamination on seed germination and the interaction effects between E. coli and heavy metals were also examined. The study also compared the effectiveness of various irrigation systems in reducing bacterial contamination. RESULTS: The results demonstrated that contamination levels varied significantly across the plots and irrigation types. This variation underscores the necessity of site-specific mitigation strategies to enhance food safety. Our findings highlight the importance of selecting appropriate irrigation methods and implementing tailored agricultural practices to minimize the risk of contamination. CONCLUSION: This research provides valuable insights for optimizing agricultural practices in the LRB to ensure food safety and environmental sustainability.

Upcycling Romaine lettuce outer leaves by infrared blanching and hot air drying.

Romaine lettuce outer leaves, as opposed to the more commonly marketed heart, are typically discarded and present an opportunity for upcycling as dried powders. Duquesne Romaine lettuce was evaluated to quantify and compare quality attributes of fresh outer and heart leaves, dried powders following hot air drying, and dried powders following an infrared (IR) blanching pretreatment before drying. Attributes measured for fresh leaves included moisture, water activity (Aw), color, total soluble phenolics (TSP), and antioxidant capacity (AC). Drying kinetics and time/energy saving through IR blanching were evaluated. Attributes measured for dried powders included moisture, Aw, color, true density, water vapor isotherms, TSP, AC, cadmium (Cd) content, and pesticide residues. TSP, AC, Cd, and pesticide residues were higher, whereas moisture content and Aw were lower in fresh outer versus heart leaves. Hot air drying reduced TSP and AC to 63.6% and 35.2% of fresh values, respectively, whereas IR blanching further reduced TSP and AC to 37.3% and 25.4% in outer leave powders. On the other hand, TSP and AC increased 237% and 151%, respectively, for unblanched heart powders. Higher increase of TSP than AC in heart leaf powder may indicate synthesis of phenolic compounds activated by abiotic stresses such as cutting and high temperatures at the initial drying stage. IR blanching resulted in significant time/energy savings for drying of outer leaves. Microbial loads were substantially reduced during drying, although microbial population on outer leaves were more resistant. Safe to eat outer leaf Romaine lettuce powders can be produced, assuming appropriate agricultural practices.

Combined treatment of pulsed light and nisin-organic acid based antimicrobial wash for inactivation of Escherichia coli O157:H7 in Romaine lettuce, reduction of microbial loads, and retention of quality.

Microbial safety of fresh produce continues to be a major concern. Novel antimicrobial methods are needed to minimize the risk of contamination. This study investigated the antimicrobial efficacy of pulsed light (PL), a novel nisin-organic acid based antimicrobial wash (AW) and the synergy thereof in inactivating E. coli O157:H7 on Romaine lettuce. Treatment effects on background microbiota and produce quality during storage at 4 °C for 7 days was also investigated. A bacterial cocktail containing three outbreak strains of E. coli O157:H7 was used as inoculum. Lettuce leaves were spot inoculated on the surface before treating with PL (1-60 s), AW (2 min) or combinations of PL with AW. PL treatment for 10 s, equivalent to fluence dose of 10.5 J/cm2, was optimal and resulted in 2.3 log CFU/g reduction of E. coli O157:H7, while a 2 min AW treatment, provided a comparable pathogen reduction of 2.2 log CFU/g. Two possible treatment sequences of PL and AW combinations were investigated. For PL-AW combination, inoculated lettuce leaves were initially exposed to optimum PL dose followed by 2 min AW treatment, whereas for AW-PL combination, inoculated lettuce were subjected to 2 min AW treatment prior to 10 s PL treatment. Both combination treatments (PL-AW and AW-PL) resulted in synergistic inactivation as E. coli cells were not detectable after treatment, indicating >5 log pathogen reductions. Combination treatments significantly (P < 0.05) reduced spoilage microbial populations on Romaine lettuce and also hindered their growth in storage for 7 days. The firmness and visual quality appearance of lettuce were not significantly (P > 0.05) influenced due to combination treatments. Overall, the results reveal that PL and AW combination treatments can be implemented as a novel approach to enhance microbial safety, quality and shelf life of Romaine lettuce.

Effect of Various LED Light Qualities, Including Wide Red Spectrum-LED, on the Growth and Quality of Mini Red Romaine Lettuce (cv. Breen).

Recently, LEDs with various light qualities have been used in closed plant factories, and they are known to have different effects on the growth and quality of crops. Therefore, this study was conducted to investigate the change in growth and quality in mini red romaine lettuce using LEDs with various light qualities. Wide red spectrum (WRS)-LEDs, blue (B)-LEDs, blue + red (BR)-LEDs, red (R)-LEDs, and white (W)-LEDs were used as the artificial light sources. Regarding growth, the R-LED treatment showed the most positive effect, but the leaf shape was not normal and the Hunter b* value was not suitable because it was higher than that of the other treatments. The Hunter a*, SPAD, and NDVI values of the B- and BR-LED treatments were effective, but this was not the case for those of the R- and W-LED treatments. The anthocyanin reflectance index 1 (ARI1) was 20 times higher in the B-LED treatment than in the R-LED treatment, and the ascorbic acid content was the highest in the WRS-LED treatment. In the sensory evaluation, bitterness and sweetness showed opposite tendencies. Regarding the overall preference, the BR-LED treatment received the highest score. Correlation analysis showed that the bitterness was closely correlated with the anthocyanin content and leaf color. Taken together, BR-LEDs provided a good top fresh weight, dark red leaves, and high anthocyanin and ascorbic acid contents, with the highest overall preference; therefore, BR-LEDs were the most suitable for the cultivation of mini red romaine lettuce.

Comparative microbial analyses of hydroponic versus in-soil grown Romaine lettuce obtained at retail.

The overarching goal of this study was to assess the microbiological profile of hydroponically grown Romaine lettuce and in-soil Romaine lettuce (organic and conventional). Thirty-six samples of hydroponic lettuce, seventy-two samples organic lettuce (thirty-six bagged lettuce and thirty-six non-bagged lettuce), and thirty-six conventionally grown lettuce was purchased from retail stores. A portion of each sample was analyzed for aerobic bacteria (APC), coliforms and E. coli, and yeasts and molds (YM). Another portion of each sample was enriched for Salmonella, E. coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus, and confirmed with RT-PCR. No statistical differences were found in the microbial profile (P > 0.05) between the different farming practices. The APC, coliforms, E. coli, and YM counts were similar across bagged samples. The results demonstrated that APC and E. coli were significantly higher (P<0.05) in organic non-bagged samples compared to other practices. Salmonella and L. monocytogenes were detected in some organically and conventionally grown lettuce samples but were only detected in 3 hydroponically grown lettuce samples. This study indicated that hydroponically grown lettuce obtained at retail may have food safety risks similar to organic and conventional systems. These findings highlight the need for food safety training and educational programs.

The Microbial Quality of Commercial Chopped Romaine Lettuce Before and After the "Use By" Date.

In the United States, due to the limited information about the microbial quality and safety of fresh produce after the labeled open dates, unnecessary discarding of fresh produce in good conditions and food loss have been caused. The aim of this study was to address this knowledge gap and evaluate the microbial quality of commercial chopped Romaine lettuce (RL) on the "Use By" dates (UBD) and 5 days after the "Use By" dates (UBD5). The microbial quality was evaluated using culture-dependent and culture-independent methods. Three brands of RL samples, from early and late harvest seasons, were purchased from local grocery stores and stored at 4°C until 5 days after their UBD. On the UBD and UBD5, bagged lettuce was opened, homogenized, diluted, and plated onto plate count agar and anaerobic agar to obtain total aerobic plate counts (APC) and total anaerobic plate counts (AnPC). For the culture-independent method, DNA was extracted from each sample homogenate and used for 16S rRNA gene sequencing. The culture-dependent results showed that there was no significant change in APC or AnPC between UBD and UBD5 samples. The APC and AnPC ranged from 5.71 ± 0.74 to 7.89 ± 0.10 Log CFU/g and 1.75 ± 0.08 to 7.32 ± 0.61 Log CFU/g, respectively. No significant difference in alpha diversity, based on observed features and Shannon index values, was detected between UBD and UBD5 samples using 16S rRNA sequencing. Similarly, no difference was observed in beta diversity based on the Jaccard distance matrixes and the weighted Unifrac distance matrixes. Taxonomic analysis revealed 128 genera in all RL samples. The top five genera were Pseudomonas (with relative abundance ranging from 16.47 to 92.72%), Serratia (0-52.35%), Weissella (0-42.42%), Pantoea (0.17-21.33%), and Lactococcus (0-24.30%). The differential abundance analysis based on the ANCOM test showed that no bacteria were detected to have significantly differential abundance in RL between UBD and UBD5. In summary, both the culture-dependent and culture-independent results showed that there was no significant difference in the microbial quality of RL before and shortly after the UBD.

Understanding inactivation of Listeria monocytogenes and Escherichia coli O157:H7 inoculated on romaine lettuce by emulsified thyme essential oil.

Effects of thyme essential oil (TEO) emulsion (TEE) with cationic charge formulated using cetylpyridinium chloride (CPC) on attachment strength and inactivation of Listeria monocytogenes and Escherichia coli O157:H7 on romaine lettuce surface were examined in this study. Regardless of the inoculation time (2 h and 24 h), pathogen attachment was stronger on the adaxial surface of the romaine lettuce than on the abaxial surface because of the lower roughness of the former. Moreover, attachment strength increased with increasing inoculation time. TEE washing had the strongest inhibitory effect on pathogen attachment at 2 h when compared with that of TEO, CPC, and sodium hypochlorite (SH), demonstrating a 3.32 and 2.53 log-reduction in the size of the L. monocytogenes and E. coli O157:H7 populations, respectively, compared to the control samples. Additionally, the TEE washing effects were maintained even after inoculation for 24 h, and it decreased attachment to adaxial surface of the samples. These results indicate that TEE could be a good alternative to SH in improving the microbiological safety of romaine lettuce.

Application of bacteriophages EP75 and EP335 efficiently reduces viable cell counts of Escherichia coli O157 on beef and vegetables.

Shiga toxin producing Escherichia coli (STEC) are common etiological agents of food borne illnesses and outbreaks, most often caused by consuming contaminated beef products, followed by raw vegetables and dairy products. Patients infected with E. coli O157 are more likely hospitalized than patients infected with non-O157 STEC, making E. coli O157 an important target for microbiological interventions. We show that a cocktail of bacteriophages EP75 and EP335 effectively reduces E. coli O157 on beef, romaine lettuce, spinach, and zucchini. Treatment of contaminated beef with either 2 × 107 or 1 × 108 PFU/cm2 of bacteriophage cocktail EP75/EP335 resulted in reductions of 0.8-1.1 log10 CFU/cm2 and 0.9-1.3 log10 CFU/cm2, respectively (P < 0.0001). Similarly, bacteriophage treatments of contaminated romaine lettuce, zucchini, or spinach showed significant (P < 0.05) E. coli O157 reductions of 0.7-1.9 log10 CFU/cm2 (2 × 107 PFU/cm2), and 1.4-2.4 log10 CFU/cm2 (1 × 108 PFU/cm2). An E. coli O157 reduction of 0.9 log10 and 2.0 log10 was observed already 30 min after phage application of 1 × 108 PFU/cm2 on beef and romaine lettuce, respectively. These data show that bacteriophages EP75 and EP335 can be effectively used as a processing aid on beef and vegetables, and thereby can aid industry to reduce the risk of E. coli O157 food poisoning.

Aqueous chlorine dioxide generated with organic acids have higher antimicrobial efficacy than those generated with inorganic acids.

Chlorine dioxide (ClO2) is commonly generated by mixing sodium chlorite and acid. This study aimed to evaluate how acid affects the release kinetics and antimicrobial property of ClO2. Solutions made with weak acids released ClO2 more slowly and had higher stability than those made with hydrochloric acid. Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes were treated with 1, 2.5, and 5 ppm ClO2 for 3 or 5 min. Lettuce inoculated with the pathogenic bacteria were treated with 2.5 and 5 ppm ClO2 for 5 min. The effects of peptone load at 0.01% and 0.02% on the antimicrobial efficacy of ClO2 were investigated in S. Typhimurium cell suspensions. The contribution of acids alone at the pH of the ClO2 solutions to bacterial reduction was also evaluated. The 2.5 ppm ClO2 solutions made with citric acid, lactic acid, and malic acid showed higher reductions in all three bacteria than ClO2 made with hydrochloric acid and sodium bisulfate. The 5 ppm ClO2 solutions produced with organic acids reduced populations of all bacterial strains from 7 log CFU/mL to undetectable level in 3 min, except S. Typhimurium treated by ClO2 produced with lactic acid. On inoculated Romaine lettuce model, 5 ppm ClO2 produced with lactic acid and malic acid resulted in the highest reduction of E. coli O157:H7, S. Typhimurium, and L. monocytogenes of approximately 1.4, 1.7, and 2.4 log CFU/g, respectively. The antimicrobial efficacy of ClO2 made with HCl and NaHSO4 were affected by 0.01% and 0.02% peptone load, respectively. Food-grade organic acids produced aqueous ClO2 solutions with stronger antimicrobial properties than inorganic acids. The acids alone at the pH of ClO2 did not show significant bacterial reductions.

Loading ferric lignin on polyethylene film and its influence on arsenic-polluted soil and growth of romaine lettuce plant.

This work developed a composite (Pe-FeLs) which loaded ferric lignin on polyethylene film (PE film) by chemical modification and physico-chemically characterized by Microscope, FESEM with elemental mapping analysis, and XRD. Microscope pictures showed that chemical modification did not destroy the appearance of PE film. The FESEM images of Pe-FeLs showed the well-distributed clusters could be clearly seen and most of the particles were spherical morphology. Elemental mapping of individual element on Pe-FeLs clearly indicated the existing of iron. The XRD pattern showed the amorphous hydroxides of iron on Pe-FeLs. In arsenic solution, the total arsenic adsorption capacity of Pe-FeLs was much higher than that of ferric lignin and PE, which showed Pe-FeLs had the ability to adsorb arsenic. For making Pe-FeLs work well in the soil, a Pe-FeLs system was set up with plastic grid plate, PE film with holes, Pe-FeLs, PE film, and plastic grid plate from the upper to bottom in order. With applying Pe-FeLs system under the soil, arsenic was significantly reduced by 25.5 ~ 53.4% in heavily, moderately, and lower arsenic-polluted soils, the biomass of the romaine lettuce increased and arsenic accumulation in the romaine lettuce decreased.

Persistence of Escherichia coli in the microbiomes of red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) - does seed sanitization matter?

BACKGROUND: Seed sanitization via chemical processes removes/reduces microbes from the external surfaces of the seed and thereby could have an impact on the plants' health or productivity. To determine the impact of seed sanitization on the plants' microbiome and pathogen persistence, sanitized and unsanitized seeds from two leafy green crops, red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) were exposed to Escherichia coli and grown in controlled environment growth chambers simulating environmental conditions aboard the International Space Station. Plants were harvested at four intervals from 7 days post-germination to maturity. The bacterial communities of leaf and root were investigated using the 16S rRNA sequencing while quantitative polymerase chain reaction (qPCR) and heterotrophic plate counts were used to reveal the persistence of E. coli. RESULT: E. coli was detectable for longer periods of time in plants from sanitized versus unsanitized seeds and was identified in root tissue more frequently than in leaf tissue. 16S rRNA sequencing showed dynamic changes in the abundance of members of the phylum Proteobacteria, Firmicutes, and Bacteroidetes in leaf and root samples of both leafy crops. We observed minimal changes in the microbial diversity of lettuce or mizuna leaf tissue with time or between sanitized and unsanitized seeds. Beta-diversity showed that time had more of an influence on all samples versus the E. coli treatment. CONCLUSION: Our results indicated that the seed surface sanitization, a current requirement for sending seeds to space, could influence the microbiome. Insight into the changes in the crop microbiomes could lead to healthier plants and safer food supplementation.

A quasimetagenomics method for concerted detection and subtyping of Salmonella enterica and E. coli O157:H7 from romaine lettuce.

Quasimetagenomics refers to the sequencing of a modified food microbiome to facilitate combined detection and subtyping of targeted pathogens in a single workflow. Through quasimetagenomic sequencing, pathogens are detected and subtyped in a shortened time frame compared to traditional culture enrichment and whole genome sequencing-based analyses. While this method was previously used to detect and subtype Salmonella enterica from chicken, iceberg lettuce, and black pepper, it has not been applied to investigate multiple pathogens in one workflow. A quasimetagenomic method to concertedly detect and subtype Salmonella enterica and Escherichia coli O157:H7 from artificially contaminated romaine lettuce in a single workflow was developed. All quasimetagenomic samples with initial target pathogen inoculum levels of ~1 CFU/g were detected and serotyped after co-enrichment of the two pathogens for 12 h. Single nucleotide polymorphism typing was achievable for some initial pathogen inoculum levels as low as ~0.1 CFU/g. Our results suggest that this method can be used for concerted detection and subtyping of multiple bacterial pathogens from romaine lettuce even at low contamination levels.

Outbreaks of Escherichia coli O157:H7 Infections Linked to Romaine Lettuce in Canada from 2008 to 2018: An Analysis of Food Safety Context.

ABSTRACT: Foodborne diseases are a major cause of illness in Canada. One of the main pathogens causing cases and outbreaks of foodborne illness in Canada is Escherichia coli O157:H7. From 2008 to 2018, 11 outbreaks of E. coli O157:H7 infection in Canada were linked to leafy greens, including 7 (63.6%) linked to romaine lettuce, 2 (18.2%) linked to iceberg lettuce, and 2 (18.2%) linked to other or unspecified types of leafy greens. The consumption of lettuce in Canada, the behavior of E. coli O157:H7 on lettuce leaves, and the production practices used for romaine and iceberg lettuce do not seem to explain why a higher number of outbreaks of E. coli O157:H7 infection were linked to romaine than to iceberg lettuce. However, the difference in the shape of iceberg and romaine lettuce heads could be an important factor. Among the seven outbreaks linked to romaine lettuce in Canada between 2008 and 2018, an eastern distribution of cases was observed. Cases from western provinces were reported only twice. The consumption of romaine and iceberg lettuce by the Canadian population does not seem to explain the eastern distribution of cases observed, but the commercial distribution, travel distances, and the storage practices used for lettuce may be important factors. In the past 10 years, the majority of the outbreaks of E. coli O157:H7 infection linked to romaine lettuce occurred during the spring (March to June) and fall (September to December). The timing of these outbreaks may be explained by the availability of lettuce in Canada, the growing region transition periods in the United States, and the seasonality in the prevalence of E. coli O157:H7. The consumption of romaine lettuce by the Canadian population does not explain the timing of the outbreaks observed.

Conditions at the time of inoculation influence survival of attenuated Escherichia coli O157:H7 on field-inoculated lettuce.

The impact of plant development, environmental conditions at the time of inoculation, and inoculum concentration on survival of attenuated BSL1 Escherichia coli O157:H7 strain ATCC 700728 on field-grown romaine lettuce was evaluated over 3 years. E. coli 700728 was inoculated onto 4- and 6-week-old romaine lettuce plants in the Salinas Valley, CA, at night or the next morning with either low (5 log) or high (7 log) cell numbers per plant to simulate a single aqueous contamination event. At night, when leaf wetness and humidity levels were high, E. coli cell numbers declined by 0.5 log CFU/plant over the first 8-10 h. When applied in the morning, E. coli populations declined up to 2 log CFU/plant within 2 h. However, similar numbers of E. coli were retrieved from lettuce plants at 2 and 7 days. E. coli cell numbers per plant were significantly lower (P < 0.05) 7 days after application onto 4-week-old compared to 6-week-old plants. E. coli 700728 could be recovered by plating or enrichment from a greater proportion of plants for longer times when inoculated at high compared with low initial concentrations and after inoculation of 6-week-old plants compared with 4-week-old plants, even at the low initial inoculum. A contamination event near harvest or when leaf wetness and humidity levels are high may enhance survivability, even when low numbers of E. coli are introduced.

Shiga Toxin-Producing Escherichia coli Infections Associated With Romaine Lettuce-United States, 2018.

BACKGROUND: Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. The Centers for Disease Control and Prevention (CDC) queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation. METHODS: A case was defined as an infection between 13 March and 22 August 2018 with 1 of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole-genome sequencing. We conducted epidemiologic and traceback investigations to identify illness subclusters and common sources. A US Food and Drug Administration-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination. RESULTS: We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and 5 died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty subclusters were identified. Product traceback from subcluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157. CONCLUSIONS: We report on the largest multistate leafy greens-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.

Characterization of a Histo-Blood Group Antigen-like Substance in Romaine Lettuce That Contributes to Human Norovirus Attachment.

Human noroviruses (HuNoVs) are among the main pathogens causing acute nonbacterial gastroenteritis. Histo-blood group antigens (HBGAs) are widely accepted receptors for HuNoV specific binding. HBGA-like substances in produce are also considered as the critical ligands for capture of HuNoVs. However, the composition of viral ligands from food substrates remains unknown. In this study, an oligosaccharide (H2N2F2) was captured and isolated from romaine lettuce extract by a bacterial surface display system. Using electrospray ionization mass spectrometry and tandem mass spectrometry, it was shown that H2N2F2 was most likely to be a chimera of type A, H, and Lewis a HBGAs. The composition was consistent with our ELISA results using a panel of monoclonal antibodies against HBGAs. Our results revealed a possible interaction mechanism between HuNoVs and romaine lettuce. Better understanding of the interaction of HuNoVs with easily contaminated produce will ultimately aid in the control of and reduction in disease outbreaks.

Experimental In-Field Transfer and Survival of Escherichia coli from Animal Feces to Romaine Lettuce in Salinas Valley, California.

This randomized controlled trial characterized the transfer of E. coli from animal feces and/or furrow water onto adjacent heads of lettuce during foliar irrigation, and the subsequent survival of bacteria on the adaxial surface of lettuce leaves. Two experiments were conducted in Salinas Valley, California: (1) to quantify the transfer of indicator E. coli from chicken and rabbit fecal deposits placed in furrows to surrounding lettuce heads on raised beds, and (2) to quantify the survival of inoculated E. coli on Romaine lettuce over 10 days. E. coli was recovered from 97% (174/180) of lettuce heads to a maximal distance of 162.56 cm (5.33 ft) from feces. Distance from sprinklers to feces, cumulative foliar irrigation, and lettuce being located downwind of the fecal deposit were positively associated, while distance from fecal deposit to lettuce was negatively associated with E. coli transference. E. coli exhibited decimal reduction times of 2.2 and 2.5 days when applied on the adaxial surface of leaves within a chicken or rabbit fecal slurry, respectively. Foliar irrigation can transfer E. coli from feces located in a furrow onto adjacent heads of lettuce, likely due to the kinetic energy of irrigation droplets impacting the fecal surface and/or impacting furrow water contaminated with feces, with the magnitude of E. coli enumerated per head of lettuce influenced by the distance between lettuce and the fecal deposit, cumulative application of foliar irrigation, wind aspect of lettuce relative to feces, and time since final irrigation. Extending the time period between foliar irrigation and harvest, along with a 152.4 cm (5 ft) no-harvest buffer zone when animal fecal material is present, may substantially reduce the level of bacterial contamination on harvested lettuce.

Characterization of conditions for bacteria-human norovirus capsid P protein complex (BPC) binding to and removal from Romaine lettuce extract.

Norovirus is a very contagious virus that causes acute gastroenteritis. Contaminated produce is a main vehicle for dissemination of human noroviruses (HuNoVs). As HuNoVs could bind to bacteria effectively, it is highly possible that produce could be contaminated by bacteria-HuNoVs complex. In this study, we used a bacterial-surface-display system to express genogroup I (GI) or genogroup II (GII) HuNoV capsid protein (P protein) on the surface of bacteria. The bacteria-P protein complex (BPC) was used to characterize the conditions for binding to Romaine lettuce extract and removal of the bound BPCs. We demonstrated both GI and GII BPCs could bind to extract from leaf (LE) and vein (VE) effectively. Carbohydrates in LE and VE were involved in GI BPCs binding, and both carbohydrates and proteins were involved in GII BPCs binding. Saliva from both type A and O secretors could completely block binding of both BPCs to LE and VE. Saliva from type B secretors only partially blocked binding of GII but not GI BPCs to LE and VE. However, LE- and VE-bound BPCs could not be reversely removed by washing solution containing free HBGAs from saliva. The binding of GI BPCs to LE and VE was enhanced when pH was below pI (6.1) of GI and reduced when pH was above pI of GI (p < 0.05). The optimal binding for GII BPCs to LE and VE occurred at pI (6.4) of GII. All LE- or VE-bound BPCs could be reversely removed by washing with low (3.0-5.0) or high (9.0-10.0) pH buffer. The effect of ionic strength (NaCl and MgCl2, from zero to 100 g/L) on binding of BPCs to LE and VE was tested. The optimal ionic strength for binding of BPCs to LE and VE was 10.0 g/L (GI) and 5.0 g/L (GII) for NaCl, and 5.0 g/L for MgCl2. LE- and VE-bound BPCs could be reversely removed by washing with high ionic solutions. All LE- or VE- bound BPCs could be released when washed with NaCl concentrations of above 75.0 g/L (GI) and 25.0 g/L (GII), or with MgCl2 concentrations of above 75.0 g/L (GI) and 50.0 g/L (GII). Binding of BPCs to LE and VE was inhibited in the presence of Tween-80 (nonionic surfactant) as low as 0.05% (v/v). All LE- and VE-bound BPCs could be reversed by Tween-80 concentrations over 0.1% (v/v). The study provided important parameters for BPCs binding to and removal from lettuce extract.

Environmental inactivation and irrigation-mediated regrowth of Escherichia coli O157:H7 on romaine lettuce when inoculated in a fecal slurry matrix.

Field trials were conducted in July-August and October 2012 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, California. One-half milliliter of rabbit, chicken, or pig fecal slurry, containing an average of 4.05 × 107 CFU E. coli O157:H7 (C0), was inoculated onto the upper (adaxial) surface of a lower leaf on 288 heads of lettuce per trial immediately following a 2.5 h irrigation event. To estimate the bacterial inactivation rate as a function of time, fecal matrix, irrigation and seasonal climate effects, sets of lettuce heads (n = 28) were sampled each day over 10 days and the concentration of E. coli O157:H7 (Ct) determined. E. coli O157:H7 was detected on 100% of heads during the 10-day duration, with concentrations ranging from ≤340 MPN/head (∼5-log reduction) to >3.45 × 1012 MPN/head (∼5-log growth). Relative to C0, on day 10 (Ct = 12) we observed an overall 2.6-log and 3.2-log mean reduction of E. coli O157:H7 in July and October, respectively. However, we observed relative maximum concentrations due to bacterial growth on day 6 (maximum Ct = 8) apparently stimulated by foliar irrigation on day 5. From this maximum there was a mean 5.3-log and 5.1-log reduction by day 10 (Ct = 12) for the July and October trials, respectively. This study provides insight into the inactivation and growth kinetics of E. coli O157:H7 on romaine lettuce leaves under natural field conditions. This study provides evidence that harvesting within 24 h post irrigation has the potential to increase the concentration of E. coli O157:H7 contamination, if present on heads of romaine lettuce; foliar irrigation can temporarily stimulate substantial regrowth of E. coli O157:H7.

Romaine Lettuce/Skullcap Mixture Improves Sleep Behavior in Vertebrate Models.

The aim of this study is to investigate the effects of romaine lettuce leaves extract (RE), skullcap root extract (SE) and their mixture on sleep behaviors in vertebrate models. HPLC analysis showed that RE contains lactucopicrin (0.02±0.01 mg/g extract), chlorogenic acid (4.05±0.03 mg/g extract), caffeic acid (2.38±0.03 mg/g extract), and chicoric acid (7.02±0.32 mg/g extract) as main phenolic compounds, while SE includes baicalin (99.4±0.5 mg/g extract), baicalein (8.28±0.21 mg/g extract), and wogonin (3.09±0.32 mg/g extract). The mixture of RE (100 mg/g extract) and SE (40 mg/g extract) increased total sleep time by 50.9% compared with the control in pentobarbital-induced sleep model. In electroencephalography (EEG) analysis, RE/SE mixture significantly increased Non-Rapid Eye Movement (NREM), in which delta wave was enhanced by around 40% compared with normal control, leading to the increase of sleep time. In caffeine-induced wake model, RE/SE mixture greatly decreased (53%) caffeine-induced wake time, showing a similar level to normal control. In addition, caffeine-induced decreased of NREM and delta wave effectively increased with RE/SE mixture; NREM and delta wave increased by 85% and 108%, respectively. Furthermore, RE/SE mixture was shown to bind to a gamma-aminobutyric acid type A (GABAA)-benzodiazepine (BZD) receptor stronger than RE or SE single extract. Taken together, RE/SE mixture effectively improved sleep behavior with the increase of NREM via GABAA-BZD receptor binding. RE/SE mixture can be used as an herbal agent for sleep disorders.