Corrigendum: Evasion of Plant Innate Defense Response by Salmonella on Lettuce.

[This corrects the article DOI: 10.3389/fmicb.2020.00500.].

Coordination of SARS-CoV-2 wastewater and clinical testing of university students demonstrates the importance of sampling duration and collection time.

Wastewater surveillance has been a useful tool complementing clinical testing during the COVID-19 pandemic. However, transitioning surveillance approaches to small populations, such as dormitories and assisted living facilities poses challenges including difficulties with sample collection and processing. Recently, the need for reliable and timely data has coincided with the need for precise local forecasting of the trajectory of COVID-19. This study compared wastewater and clinical data from the University of Delaware (Fall 2020 and Spring 2021 semesters), and evaluated wastewater collection practices for enhanced virus detection sensitivity. Fecal shedding of SARS-CoV-2 is known to occur in infected individuals. However, shedding concentrations and duration has been shown to vary. Therefore, three shedding periods (14, 21, and 30 days) were presumed and included for analysis of wastewater data. SARS-CoV-2 levels detected in wastewater correlated with clinical virus detection when a positive clinical test result was preceded by fecal shedding of 21 days (p< 0.05) and 30 days (p < 0.05), but not with new cases (p = 0.09) or 14 days of shedding (p = 0.17). Discretely collected wastewater samples were compared with 24-hour composite samples collected at the same site. The discrete samples (n = 99) were composited examining the influence of sampling duration and time of day on SARS-CoV-2 detection. SARS-CoV-2 detection varied among dormitory complexes and sampling durations of 3-hour, 12-hour, and 24-hour (controls). Collection times frequently showing high detection values were between the hours of 03:00 to 05:00 and 23:00 to 08:00. In each of these times of day 33% of samples (3/9) were significantly higher (p < 0.05) than the control sample. The remainder (6/9) of the collection times (3-hour and 12-hour) were not different (p > 0.05) from the control. This study provides additional framework for continued methodology development for microbiological wastewater surveillance as the COVID-19 pandemic progresses and in preparation for future epidemiological efforts.

Zero-Valent Iron Filtration Reduces Microbial Contaminants in Irrigation Water and Transfer to Raw Agricultural Commodities.

Groundwater depletion is a critical agricultural irrigation issue, which can be mitigated by supplementation with water of higher microbiological risk, including surface and reclaimed waters, to support irrigation needs in the United States. Zero-valent iron (ZVI) filtration may be an affordable and effective treatment for reducing pathogen contamination during crop irrigation. This study was performed to determine the effects of ZVI filtration on the removal and persistence of Escherichia coli, and pepper mild mottle virus (PMMoV) in irrigation water. Water was inoculated with E. coli TVS 353, filtered through a ZVI filtration unit, and used to irrigate cucurbit and cruciferous crops. Water (n = 168), leaf (n = 40), and soil (n = 24) samples were collected, the E. coli were enumerated, and die-off intervals were calculated for bacteria in irrigation water. Variable reduction of PMMoV was observed, however E. coli levels were consistently and significantly (p < 0.05) reduced in the filtered (9.59 lnMPN/mL), compared to unfiltered (13.13 lnMPN/mL) water. The die-off intervals of the remaining bacteria were significantly shorter in the filtered (-1.50 lnMPN/day), as compared to the unfiltered (-0.48 lnMPN/day) water. E. coli transfer to crop leaves and soils was significantly reduced (p < 0.05), as expected. The reduction of E. coli in irrigation water and its transfer to crops, by ZVI filtration is indicative of its potential to reduce pathogens in produce pre-harvest environments.

Temporal and Agricultural Factors Influence Escherichia coli Survival in Soil and Transfer to Cucumbers.

Biological soil amendments of animal origin (BSAAO) increase nutrient levels in soils to support the production of fruits and vegetables. BSAAOs may introduce or extend the survival of bacterial pathogens which can be transferred to fruits and vegetables to cause foodborne illness. Escherichia coli survival over 120 days in soil plots (3 m2) covered with (mulched) or without plastic mulch (not mulched), amended with either poultry litter, composted poultry litter, heat-treated poultry pellets, or chemical fertilizer, and transfer to cucumbers in 2 years (2018 and 2019) were evaluated. Plots were inoculated with E. coli (8.5 log CFU/m2) and planted with cucumber seedlings (Supremo). The number of days needed to reduce E. coli levels by 4 log CFU (dpi4log) was determined using a sigmoidal decline model. Random forest regression and one-way analysis of variance (ANOVA; P < 0.05) identified predictors (soil properties, nutrients, and weather factors) of dpi4log of E. coli and transfer to cucumbers. The combination of year, amendment, and mulch (25.0% increase in the mean square error [IncMSE]) and year (9.75% IncMSE) were the most prominent predictors of dpi4log and transfer to cucumbers, respectively. Nitrate levels at 30 days and soil moisture at 40 days were also impactful predictors of dpi4log. Differing rainfall amounts in 2018 (24.9 in.) and 2019 (12.6 in.) affected E. coli survival in soils and transfer to cucumbers. Salmonella spp. were recovered sporadically from various plots but were not recovered from cucumbers in either year. Greater transfer of E. coli to cucumbers was also shown to be partially dependent on dpi4log of E. coli in plots containing BSAAO.IMPORTANCE Poultry litter and other biological soil amendments are commonly used fertilizers in fruit and vegetable production and can introduce enteric pathogens such as Escherichia coli O157:H7 or Salmonella previously associated with outbreaks of illness linked to contaminated produce. E. coli survival duration in soils covered with plastic mulch or uncovered and containing poultry litter or heat-treated poultry litter pellets were evaluated. Nitrate levels on day 30 and moisture content in soils on day 40 on specific days were good predictors of E. coli survival in soils; however, the combination of year, amendment, and mulch type was a better predictor. Different cumulative rainfall totals from year to year most likely affected the transfer of E. coli from soils to cucumbers and survival durations in soil. E. coli survival in soils can be extended by the addition of several poultry litter-based soil amendments commonly used in organic production of fruits and vegetables and is highly dependent on temporal variation in rainfall.

Evasion of Plant Innate Defense Response by Salmonella on Lettuce.

To establish host association, the innate immune system, which is one of the first lines of defense against infectious disease, must be circumvented. Plants encounter enteric foodborne bacterial pathogens under both pre- and post-harvest conditions. Human enteric foodborne pathogens can use plants as temporary hosts. This unique interaction may result in recalls and illness outbreaks associated with raw agricultural commodities. The purpose of this study was to determine if Salmonella enterica Typhimurium applied to lettuce leaves can suppress the innate stomatal defense in lettuce and utilization of UD1022 as a biocontrol against this ingression. Lettuce leaves were spot inoculated with S. Typhimurium wild type and its mutants. Bacterial culture and confocal microscopy analysis of stomatal apertures were used to support findings of differences in S. Typhimurium mutants compared to wild type. The persistence and internalization of these strains on lettuce was compared over a 7-day trial. S. Typhimurium may bypass the innate stomatal closure defense response in lettuce. Interestingly, a few key T3SS components in S. Typhimurium were involved in overriding stomatal defense response in lettuce for ingression. We also show that the T3SS in S. Typhimurium plays a critical role in persistence of S. Typhimurium in planta. Salmonella populations were significantly reduced in all UD1022 groups by day 7 with the exception of fliB and invA mutants. Salmonella internalization was not detected in plants after UD1022 treatment and had significantly higher stomatal closure rates (aperture width = 2.34 µm) by day 1 compared to controls (8.5 µm). S. Typhimurium SPI1 and SPI2 mutants showed inability to reopen stomates in lettuce suggesting the involvement of key T3SS components in suppression of innate response in plants. These findings impact issues of contamination related to plant performance and innate defense responses for plants.

Effectiveness of Bacteriophages Against Biofilm-Forming Shiga-Toxigenic Escherichia coli on Leafy Greens and Cucumbers.

Background: Shiga-toxigenic Escherichia coli (STEC) have caused several produce-associated outbreaks, making it challenging to control these pathogens. Bacteriophages could serve as effective biocontrol. Materials and Methods: Spinach, lettuce, and cucumbers, inoculated with STEC (O157, O26, O45, O103, O111, O121, O145), were treated with lytic bacteriophages and stored at 4°C for 3 days. Surviving STEC were enumerated and observed under scanning electron microscope (SEM), and data analyzed using one-way analysis of variance (ANOVA) (p < 0.05). Results: Bacteriophage treatments significantly reduced STEC populations, compared with the control (p < 0.05). On spinach and romaine, STEC O26, O45, and O103 were reduced to undetectable levels and STEC O157, O111, O121, and O145 by ∼2 logs CFU/cm2. Multiserotype phage cocktail reduced STEC on leafy greens by 1.4 CFU/cm2 and on cucumbers by 1.7 logs CFU/cucumber. Clusters of STEC cells, surrounded by extracellular matrix, were observed under SEM of positive control, whereas phage-treated produce surface showed fewer cells, with cellular damage. Conclusions: Bacteriophages could be utilized as biocontrol against STEC on fresh produce.