Rain splash-mediated dispersal of Escherichia coli from fecal deposits to field-grown lettuce in the mid- and south Atlantic U.S. regions is affected by mulch type.

Introduction: Wildlife feces can contaminate vegetables when enteric bacteria are released by rain and splashed onto crops. Regulations require growers to identify and not harvest produce that is likely contaminated, but U.S. federal standards do not define dimensions for no-harvest zones. Moreover, mulching, used to retain soil moisture and maximize crop yield may impact rain-mediated bacterial dispersal from feces. Methods: To assess Escherichia coli dissemination from a fecal point source to lettuce grown on various mulches, lettuce cv. 'Magenta' was transplanted into raised beds with plastic, biodegradable plastic, straw, or left uncovered at field sites in Maryland and Georgia. Eleven days post-transplant, 10 g of rabbit manure spiked with ~8 log CFU g-1 E. coli were deposited in each bed. One day following natural or simulated rain events, lettuce was sampled along 1.5 m transects on either side of fecal deposits. Lettuce-associated E. coli was semi-quantified with an MPN assay and dependence on fecal age (stale or fresh), lettuce age (baby leaf or mature head), distance from point source, mulch and post-rain days were statistically evaluated. Results: Distance (p<0.001), fecal age (p<0.001) and mulch (p<0.01) were factors for E. coli transfer from point source to lettuce. The highest and lowest E. coli estimates were measured from lettuce grown on biodegradable plastic and straw, respectively, with a 2-log MPN difference (p<0.001). Mulch and distance were also significant factors in E. coli recovery 3 days post-rain (both p<0.001), where plastic mulches differed from bare ground and straw (p<0.01). For all treatments, fewer E. coli were retrieved from lettuce at 0.3 m, 3 days post-rain compared to 1 day (p<0.001). Fitting the data to a Weibull Model predicated that a 7-log reduction in E. coli from fecal levels would be achieved at 1.2-1.4 m from the point source on plastic mulches, 0.75 m on bare soil (p<0.05) and 0.43 m on straw (p<0.01). Discussion: Straw and bare ground limited rain-mediated E. coli dispersal from feces to lettuce compared to plastic mulches. Fecal age was negatively associated with E. coli dispersal. These findings can inform harvesting recommendations for measures related to animal intrusion in vegetable production areas.

Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

Cultivar was more influential than bacterial strain and other experimental factors in recovery of Escherichia coli O157:H7 populations from inoculated live Romaine lettuce plants.

The varied choice of bacterial strain, plant cultivar, and method used to inoculate, retrieve, and enumerate Escherichia coli O157:H7 from live plants could affect comparability among studies evaluating lettuce-enterobacterial interactions. Cultivar, bacterial strain, incubation time, leaf side inoculated, and sample processing method were assessed for their influence in recovering and quantifying E. coli O157:H7 from live Romaine lettuce. Cultivar exerted the strongest effect on E. coli O157:H7 counts, which held up even when cultivar was considered in interactions with other factors. Recovery from the popularly grown green Romaine "Rio Bravo" was higher than from the red variety "Outredgeous." Other modulating variables were incubation time, strain, and leaf side inoculated. Sample processing method was not significant. Incubation for 24 hours post-lettuce inoculation yielded greater counts than 48 hours, but was affected by lettuce cultivar, bacterial strain, and leaf side inoculated. Higher counts obtained for strain EDL933 compared to a lettuce outbreak strain 2705C emphasized the importance of selecting relevant strains for the system being studied. Inoculating the abaxial side of leaves gave higher counts than adaxial surface inoculation, although this factor interacted with strain and incubation period. Our findings highlight the importance of studying interactions between appropriate bacterial strains and plant cultivars for more relevant research results, and of standardizing inoculation and incubation procedures. The strong effect of cultivar exerted on the E. coli O157:H7-lettuce association supports the need to start reporting cultivar information for illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.IMPORTANCEThe contamination of Romaine lettuce with Escherichia coli O157:H7 has been linked to multiple foodborne disease outbreaks, but variability in the methods used to evaluate E. coli O157:H7 association with live lettuce plants complicates the comparability of different studies. In this study, various experimental variables and sample processing methods for recovering and quantifying E. coli O157:H7 from live Romaine lettuce were assessed. Cultivar was found to exert the strongest influence on E. coli O157:H7 retrieval from lettuce. Other modulating factors were bacterial incubation time on plants, strain, and leaf side inoculated, while sample processing method had no impact. Our findings highlight the importance of selecting relevant cultivars and strains, and of standardizing inoculation and incubation procedures, in these types of assessments. Moreover, results support the need to start reporting cultivars implicated in foodborne illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.

Detection of Salmonella enterica and Listeria monocytogenes in alternative irrigation water by culture and qPCR-based methods in the Mid-Atlantic U.S.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results. IMPORTANCE: Detecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence of Salmonella enterica and Listeria monocytogenes in irrigation water samples examined in this study.

Soil Microclimate and Persistence of Foodborne Pathogens Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Newport in Soil Affected by Mulch Type.

Mulching is a common agricultural practice that benefits crop production through soil moisture retention, weed suppression, and soil temperature regulation. However, little is known about the effect of mulch on foodborne pathogens present in soil. In this study, the influence of polyethylene plastic, biodegradable corn-based plastic, paper, and straw mulches on Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Newport populations in soil was investigated. Silt loam soil in troughs was inoculated with a cocktail of the pathogens and covered with mulch or left bare, then incubated for 21 days, during which bacteria were enumerated and environmental parameters monitored. Bacterial counts declined in all treatments over time (p < 0.001) but persisted at 21 days at 0.8-0.95 log CFU/g. Pathogens also declined as a factor of mulch cover (p < 0.01). An exponential decay with asymptote model fit to the data revealed slower rates of decline in soil under mulches for all pathogens (p < 0.05) relative to bare soil. Compared to the average for all treatments, rates of decay in bare soil were 0.60 (p < 0.001), 0.45 (p < 0.05), and 0.63 (p < 0.001) log CFU/g/d for E. coli O157:H7, L. monocytogenes, and Salmonella, respectively. Linear multiple regression revealed that soil hydrological parameters were positively correlated (p < 0.05) with bacterial counts, while day soil temperatures were negatively correlated (p < 0.001), suggesting that higher day temperatures and lower moisture content of bare soil contributed to the faster decline of pathogens compared to mulched soil. A microcosm experiment using field soil from lettuce cultivation suggested no influence of prior mulch treatment on pathogens. In summary, pathogen decline in soil was modified by the soil microclimate created under mulch covers, but the effect appeared was restricted to the time of soil cover. Slower decline rates of pathogens in mulched soil may pose a risk for contamination of fresh market produce crops.

Temporal Stability of Salmonella enterica and Listeria monocytogenes in Surface Waters Used for Irrigation in the Mid-Atlantic United States.

Enteric bacterial pathogen levels can influence the suitability of irrigation water sources for fruits and vegetables. We hypothesize that stable spatial patterns of Salmonella enterica and Listeria monocytogenes levels may exist across surface water sources in the Mid-Atlantic U.S. Water samples were collected at four streams and two pond sites in the mid-Atlantic U.S. over 2 years, biweekly during the fruit and vegetable growing seasons, and once a month during nongrowing seasons. Two stream sites and one pond site had significantly different mean concentrations in growing and nongrowing seasons. Stable spatial patterns were determined for relative differences between the site concentrations and average concentration of both pathogens across the study area. Mean relative differences were significantly different from zero at four of the six sites for S. enterica and three of six sites for L. monocytogenes. There was a similarity between the mean relative difference distribution between sites over growing season, nongrowing season, and the entire observation period. Mean relative differences were determined for temperature, oxidation-reduction potential, specific electrical conductance, pH, dissolved oxygen, turbidity, and cumulative rainfall. A moderate-to-strong Spearman correlation (rs > 0.657) was found between spatial patterns of S. enterica and 7-day rainfall, and between relative difference patterns of L. monocytogenes and temperature (rs = 0.885) and dissolved oxygen (rs = -0.885). Persistence in ranking sampling sites by the concentrations of the two pathogens was also observed. Finding spatially stable patterns in pathogen concentrations highlights spatiotemporal dynamics of these microorganisms across the study area can facilitate the design of an effective microbial water quality monitoring program for surface irrigation water.

Natural variation and drought-induced differences in metabolite profiles of red oak-leaf and Romaine lettuce play a role in modulating the interaction with Salmonella enterica.

Nutrients on produce surfaces are vital for successful enteric pathogen colonisation. In this study, we investigated natural variation in metabolite profiles of Romaine 'Parris Island Cos' and red oak-leaf lettuce 'Mascara' under regular and restricted watering conditions. We also investigated the impact of plant drought stress on the Salmonella - lettuce association. Salmonella Newport and Typhimurium were able to persist at higher levels on regularly watered Romaine than red oak-leaf lettuce. Drought treatment to lettuce impaired epiphytic Salmonella association, with S. Newport and Typhimurium being differentially affected. A higher log reduction of both serotypes was measured on drought-subjected red oak-leaf lettuce plants than controls, but S. Typhimurium was unaffected on water deficit-treated Romaine lettuce (p < 0.05). To assess Salmonella interaction with leaf surface metabolites, leaf washes collected from both cultivars were inoculated and found to be able to support S. Newport growth, with higher levels of Salmonella retrieved from Romaine washes (p < 0.05). The lag phase of S. Newport in washes from water restricted red oak-leaf lettuce was prolonged in relation to regularly-watered controls (p < 0.05). Untargeted plant metabolite profiling using electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) revealed natural variation between Romaine and red oak-leaf lettuce profiles for leaf tissue and leaf washes. Metabolite profile shifts were detected in both lettuce types in response to drought stress, but more unique peaks were detected in red oak-leaf than Romaine lettuce after drought treatment. Variation between the two cultivars was in part attributed to naturally higher levels of flavonoids and anthocyanins in red oak-leaf lettuce compared to Romaine. Moreover, red oak-leaf, but not Romaine lettuce, responded to drought by inducing the accumulation of proline, phenolics, flavonoids and anthocyanins. Drought stress, therefore, enhanced the functional food properties of red oak-leaf lettuce. Salmonella growth dynamics in lettuce leaf washes suggested that natural variation and drought-induced changes in metabolite profiles in lettuce could partly explain the differential susceptibility of various lettuce types to Salmonella, although the primary or secondary metabolites mediating this effect remain unknown. Regulated mild water stress should be investigated as an approach to lower Salmonella contamination risk in suitable lettuce cultivars, while simultaneously boosting the health beneficial quality of lettuce.

Growth and inactivation of Listeria monocytogenes in sterile extracts of fruits and vegetables: Impact of the intrinsic factors pH, sugar and organic acid content.

Intrinsic characteristics of fresh produce, such as pH, water activity, acid content and nutrient availability are critical factors in determining the survival and growth of Listeria monocytogenes (Lm). In this study, sterile fresh produce juice was used to analyze Lm growth potential among 14 different commodities and to identify physicochemical characteristics in those juices that affect Lm growth. Significant growth of Lm was observed in juices with pH ≥5.6 and low acidity (0.04-0.07 % titratable acidity (TA)) (cantaloupe, carrot, celery, green pepper, parsley, and romaine lettuce), slight reduction of Lm was observed in juices with pH 4.1 (tomato) and pH 3.9 (mango), and no Lm counts were recovered from juices with pH ≤3.8 and high acidity (0.28-1.17 % TA) (apple, blueberry, grape, peach, and pineapple). Although these acidic fruit juices possessed a high sugar content, the pH and acidity of produce juice seemed to be the primary determinants for Lm growth. The neutralization of acidic juices (i.e., Fuji and Gala apple, blueberry, grape, mango, pineapple, peach, and tomato) enabled Lm growth at 37 °C in all juices except for Gala apple and peach. Strong decline in Lm populations in Gala apple, grape and peach juices might be linked to sensitivity to organic acids, such as malic acid. Furthermore, Lm populations significantly decreased in pH-neutral (7.6) cauliflower juice, suggesting that potential antilisterial substances may play a role in Lm decline in cauliflower juice.

Tomato Cultivar Nyagous Fruit Surface Metabolite Changes during Ripening Affect Salmonella Newport.

ABSTRACT: Tomatoes are a valuable crop consumed year-round. Ripe fruit is picked for local sale, whereas tomatoes intended for transit may be harvested at late mature green or breaker stages when fruit firmness preserves quality. In this study, we evaluated Solanum lycopersicum cv. BHN602 association with three Salmonella serotypes and S. lycopersicum cv. Nyagous with Salmonella Newport using fruit at two ripeness stages. Counts of Salmonella Javiana and Typhimurium were higher from red ripe fruit surfaces of BHN602, and counts of Salmonella Newport were higher from ripe Nyagous fruit than from mature green fruit (P < 0.05). Aqueous fruit washes containing fruit surface compounds collected from ripe Nyagous fruit supported more Salmonella Newport growth than green fruit washes (P < 0.05). Growth curve analysis showed that between 2 and 6 h, Salmonella Newport grew at a rate of 0.25 log CFU/h in red fruit wash compared with 0.17 log CFU/h in green fruit wash (P < 0.05). The parallel trend in Salmonella interaction between fruit and wash suggested that surface metabolite differences between unripe and ripe fruit affect Salmonella dynamics. Untargeted phytochemical profiling of tomato fruit surface washes with gas chromatography time-of-flight mass spectrometry showed that ripe fruit had threefold-lower amino acid and fourfold-higher sugar (fructose, glucose, and xylose) levels than green fruit. Green fruit had higher levels of lauric, palmitic, margaric, and arachidic acids, whereas red fruit had more capric acid. The phenolics ferulic, chlorogenic, and vanillic acid, as well as tyrosol, also decreased with ripening. Although limitations of this study preclude conclusions on how specific compounds affect Salmonella, our study highlights the complexity of the plant niche for foodborne pathogens and the importance of understanding the metabolite landscape Salmonella encounters on fresh produce. Fruit surface phytochemical profiling generated testable hypotheses for future studies exploring the differential Salmonella interactions with tomato varieties and fruit at various ripeness stages.

Developmentally related and drought-induced shifts in the kale metabolome limited Salmonella enterica association, providing novel insights to enhance food safety.

Plants influence epiphytic bacterial associations but Salmonella enterica colonizes crop plants commensally, raising the possibility of human foodborne illness, but the factors that mediate human pathogen-plant associations remain understudied. We evaluated whether any changes in leaf tissue and surface metabolomes with kale (Brassica oleracea Acephala group) development and in response to drought modulated Salmonella leaf association. Untargeted phytochemical profiling (including primary and secondary metabolites) of kale leaf tissue extracts and leaf surface washes revealed distinct metabolite profiles that shifted with plant development. Metabolomes of juvenile plants also diverged in response to drought stress, an effect not noted in mature kale. Restricted watering in juvenile plants led to up-accumulation of 45 compounds in leaf tissue and 21 in leaf wash and the appearance of several unique peaks, with concomitant increases in phytochemical measurements. The antioxidant capacity and total flavonoid content were higher in mature than juvenile, regularly watered plant leaf extracts. Drought also elicited flavonoids and glucosinolates in juvenile plants. In mature plants, drought did not induce further prominent changes. Regularly watered juvenile kale provided a favorable substrate for inoculated Salmonella but the ability to support Salmonella declined with age and with drought stress. Salmonella growth was impaired in mature or water-stressed plant washes compared to controls and positive correlations were detected between Salmonella counts on leaves and in leaf washes. Moreover, Salmonella counts were inversely correlated with total flavonoids and phenolics in kale tissues from juvenile plants and regularly watered plants. Future studies should assess how changes in primary and secondary metabolites on the kale plant surface can modulate the Salmonella association. Regulated water restriction could be a strategy in controlled agriculture, with the dual purpose of enhancing health beneficial quality and food safety, especially when harvested at the baby kale stage.

Extended Spectrum ß-Lactamase Activity and Cephalosporin Resistance in Escherichia coli from U.S. Mid-Atlantic Surface and Reclaimed Water.

Phylogenetic distribution and extended spectrum ß-lactamase (ESBL) activity of Escherichia coli recovered from surface and reclaimed water in the mid-Atlantic U.S. were evaluated. Among 488 isolates, phylogroups B1 and A were the most and least prevalent, respectively. Water type, but not season, affected phylogroup distribution. The likelihood of detecting group A isolates was higher in reclaimed than pond (P < 0.01), freshwater river (P < 0.01) or brackish river (P < 0.05) water. Homogeneity in group distribution was lowest in pond water, where group B1 comprised 50% of isolates. Only 16 (3.3%) isolates exhibited phenotypic resistance to one or more cephalosporins tested and only four had ESBL activity, representing groups B1, B2 isolates, and D. Phylogroup was a factor in antimicrobial resistance (P < 0.05), with group A (8.7%) and D (1.6%) exhibiting the highest and lowest rates. Resistance to cefoxitin was the most prevalent. Multi- versus single drug resistance was affected by phylogroup (P < 0.05) and more likely in groups D and B1 than A which carried resistance to cefoxitin only. The most detected ß-lactam resistance genes were blaCMY-2 and blaTEM. Water type was a factor for blaCTX-M gene detection (P < 0.05). Phenotypic resistance to cefotaxime, ceftriaxone, cefuroxime and ceftazidime, and genetic determinants for ESBL-mediated resistance were found predominantly in B2 and D isolates from rivers and reclaimed water. Overall, ESBL activity and cephalosporin resistance in reclaimed and surface water isolates were low. Integrating data on ESBL activity and ß-lactam resistance among E. coli populations can inform decisions on safety of irrigation water sources and One Health. IMPORTANCE Extended spectrum ß-lactamase (ESBL) producing bacteria, that are resistant to a broad range of antimicrobial agents, are spreading in the environment but data remain scarce. ESBL-producing Escherichia coli infections in the community are on the rise. This work was conducted to assess presence of ESBL-producing E. coli in water that could be used for irrigation of fresh produce. The study provides the most extensive evaluation of ESBL-producing E. coli in surface and reclaimed water in the mid-Atlantic United States. The prevalence of ESBL producers was low and phenotypic resistance to cephalosporins (types of ß-lactam antibiotics) was affected by season but not water type. Data on antimicrobial resistance among E. coli populations in water can inform decisions on safety of irrigation water sources and One Health.

Effects of season and water type on the distribution and antimicrobial resistance of Enterococcus faecalis and Ent. faecium from surface and reclaimed water.

AIMS: To evaluate the safety of irrigation water sources based on phenotypic antimicrobial resistance (AMR) in Enterococcus spp., a potential environmental reservoir for AMR determinants. METHODS AND RESULTS: Eleven sites representing fresh and brackish water rivers, ponds and reclaimed water, were sampled over 2 years. Samples (n = 333) yielded 198 unique isolates of Ent. faecalis and Ent. faecium which were tested for antimicrobial susceptibility by microbroth dilution. Species distribution was influenced by water type and season. Enterococcus faecalis was more likely found in freshwater rivers and in summer, and Ent. faecium in reclaimed water and in spring. Only 11% of isolates were pansusceptible, while 48.5% and 26.3% were single (SDR) and multidrug resistant (MDR), respectively. MDR was more likely detected in Ent. faecium than Ent. faecalis. Winter isolates were more likely than summer isolates to exhibit MDR than SDR. CONCLUSIONS: Enterococcus faecalis and Ent. faecium in surface and reclaimed water exhibited diverse phenotypic AMR and a low-level resistance to clinically important antimicrobials such as ampicillin, vancomycin and linezolid. SIGNIFICANCE AND IMPACT OF THE STUDY: Single and multidrug resistance in E. faecalis and E. faecium varied by season but not water type. Antimicrobial resistance prevalence can assist decisions on the safety of irrigation water sources for fresh produce crops.

Impact of irrigation water type and sampling frequency on Microbial Water Quality Profiles required for compliance with U.S. Food Safety Modernization Act Produce Safety Rule standards.

The U.S. Food Safety Modernization Act (FSMA) Produce Safety Rule (PSR) requires that farmers generate a Microbial Water Quality Profile (MWQP) from 20 samples per agricultural water source, taken over 2-4 years and five annual samples thereafter. Farmers must use the MWQP to ascertain a geometric mean (GM) of ≤126 CFU/100 mL and statistical threshold value (STV) of ≤410 CFU/100 mL of generic Escherichia coli. Farmers are responsible for collecting samples and paying for testing, incurring a financial and time burden. To determine if testing frequency can be reduced without compromising accuracy, water samples (n = 279) were collected from twelve sites in the U.S. Mid-Atlantic region from 2016 to 2018 comprising tidal brackish river, non-tidal fresh river, pond, vegetable processing, and reclaimed water. The GM and STV were calculated for all sites and water types using all samples, and for multiple sub-samples of <20 from each site and water type. A Monte Carlo simulation was used to determine the proportion of sub-sample sizes that yielded the same determination as the entire sample size of PSR standard compliance. Four sites, two pond and two reclaimed water sites, complied with PSR GM and STV requirements when using the entire sample set. When a water source's calculated GM and STV using the entire sample set hovered close to the PSR thresholds, sub-sample sizes approached the recommended 20 samples to reach a congruent compliance determination. However, 99% agreement was obtained with a sub-sample of five when the absolute difference between the GM and STV from total samples and the PSR thresholds was ≥2.6 and 4.5 log CFU/100 mL E. coli, respectively. These findings suggest that under certain conditions the MWQP may be generated with well below 20 samples, reducing the economic burden on farmers while still maintaining a representative MWQP.

Levels of Salmonella enterica and Listeria monocytogenes in Alternative Irrigation Water Vary Based on Water Source on the Eastern Shore of Maryland.

Irrigation water sources have been shown to harbor foodborne pathogens and could contribute to the outbreak of foodborne illness related to consumption of contaminated produce. Determining the probability of and the degree to which these irrigation water sources contain these pathogens is paramount. The purpose of this study was to determine the prevalence of Salmonella enterica and Listeria monocytogenes in alternative irrigation water sources. Water samples (n = 188) were collected over 2 years (2016 to 2018) from 2 reclaimed water plants, 3 nontidal freshwater rivers, and 1 tidal brackish river on Maryland's Eastern Shore (ESM). Samples were collected by filtration using modified Moore swabs (MMS) and analyzed by culture methods. Pathogen levels were quantified using a modified most probable number (MPN) procedure with three different volumes (10 liters, 1 liter, and 0.1 liter). Overall, 65% (122/188) and 40% (76/188) of water samples were positive for S. enterica and L. monocytogenes, respectively. For both pathogens, MPN values ranged from 0.015 to 11 MPN/liter. Pathogen levels (MPN/liter) were significantly (P < 0.05) greater for the nontidal freshwater river sites and the tidal brackish river site than the reclaimed water sites. L. monocytogenes levels in water varied based on season. Detection of S. enterica was more likely with 10-liter filtration compared to 0.1-liter filtration. The physicochemical factors measured attributed only 6.4% of the constrained variance to the levels of both pathogens. This study shows clear variations in S. enterica and L. monocytogenes levels in irrigation water sources on ESM. IMPORTANCE In the last several decades, Maryland's Eastern Shore has seen significant declines in groundwater levels. While this area is not currently experiencing drought conditions or water scarcity, this research represents a proactive approach. Efforts, to investigate the levels of pathogenic bacteria and the microbial quality of alternative irrigation water are important for sustainable irrigation practices into the future. This research will be used to determine the suitability of alternative irrigation water sources for use in fresh produce irrigation to conserve groundwater.

Aeromonas spp. diversity in U.S. mid-Atlantic surface and reclaimed water, seasonal dynamics, virulence gene patterns and attachment to lettuce.

Aeromonas, a ubiquitous taxon in water environments, is emerging as a foodborne pathogen of concern that remains understudied and under-reported. We evaluated the distribution of 331 Aeromonas spp. isolates collected from irrigation water over one year and characterised their virulence profile, attachment and ability to persist on lettuce. Water sources included non-tidal and tidal river, farm pond and reclaimed water. Twenty Aeromonas species were identified; A. veronii, A. hydrophila and A. jandaei predominated in all water types and seasons, comprising ~63% of isolates. Species distribution was most affected by water type. The highest and lowest diversity were detected in river and pond water, respectively. A. hydrophila and A. veronii ranked highest in frequency in fresh river and reclaimed water, while A. jandaei ranked first in pond water. Only two isolates carried all five virulence genes tested, while 46% of A. hydrophila (n = 50), 54% of A. veronii (n = 61) and 50% of A. jandaei (n = 32) isolates harboured multiple enterotoxin genes. Detection of alt and ast genes was more likely in summer collections, while ast detection was less likely in tidal brackish river and pond water isolates. Season was a factor in attachment to polystyrene, being strongest in spring isolates. The gene flaA was associated with strong attachment and was more likely to be detected in non-tidal fresh river isolates. A. hydrophila and A. jandaei isolates persisted on lettuce leaves for 24 h, but populations dwindled over 120 h, while loosely and strongly attached cells of A. veronii isolates persisted for 120 h. This study provides comprehensive data on Aeromonas species distribution and environmental traits. The associations revealed among diversity, water type, season, virulence factors and phyllosphere attachment capacity can inform agricultural water standards in novel ways. Moreover, understanding Aeromonas-plant interactions is an important step in advancing food safety of fruit and vegetables.

Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters.

Enteric viruses (EVs) are the largest contributors to foodborne illnesses and outbreaks globally. Their ability to persist in the environment, coupled with the challenges experienced in environmental monitoring, creates a critical aperture through which agricultural crops may become contaminated. This study involved a 17-month investigation of select human EVs and viral indicators in nontraditional irrigation water sources (surface and reclaimed waters) in the Mid-Atlantic region of the United States. Real-time quantitative PCR was used for detection of Aichi virus, hepatitis A virus, and norovirus genotypes I and II (GI and GII, respectively). Pepper mild mottle virus (PMMoV), a common viral indicator of human fecal contamination, was also evaluated, along with atmospheric (air and water temperature, cloud cover, and precipitation 24 h, 7 days, and 14 days prior to sample collection) and physicochemical (dissolved oxygen, pH, salinity, and turbidity) data, to determine whether there were any associations between EVs and measured parameters. EVs were detected more frequently in reclaimed waters (32% [n = 22]) than in surface waters (4% [n = 49]), similar to PMMoV detection frequency in surface (33% [n = 42]) and reclaimed (67% [n = 21]) waters. Our data show a significant correlation between EV and PMMoV (R2 = 0.628, P < 0.05) detection levels in reclaimed water samples but not in surface water samples (R2 = 0.476, P = 0.78). Water salinity significantly affected the detection of both EVs and PMMoV (P < 0.05), as demonstrated by logistic regression analyses. These results provide relevant insights into the extent and degree of association between human (pathogenic) EVs and water quality data in Mid-Atlantic surface and reclaimed waters, as potential sources for agricultural irrigation. IMPORTANCE Microbiological analysis of agricultural waters is fundamental to ensure microbial food safety. The highly variable nature of nontraditional sources of irrigation water makes them particularly difficult to test for the presence of viruses. Multiple characteristics influence viral persistence in a water source, as well as affecting the recovery and detection methods that are employed. Testing for a suite of viruses in water samples is often too costly and labor-intensive, making identification of suitable indicators for viral pathogen contamination necessary. The results from this study address two critical data gaps, namely, EV prevalence in surface and reclaimed waters of the Mid-Atlantic region of the United States and subsequent evaluation of physicochemical and atmospheric parameters used to inform the potential for the use of indicators of viral contamination.

Stress response, amino acid biosynthesis and pathogenesis genes expressed in Salmonella enterica colonizing tomato shoot and root surfaces.

Salmonella enterica can colonize all parts of the tomato plant. Tomatoes have been frequently implicated in salmonellosis outbreaks. In agricultural settings, Salmonella must overcome stress, nutritional and competition barriers to become established on plant surfaces. Knowledge of the genetic mechanisms underlying Salmonella-plant associations is limited, especially when growing epiphytically. A genome-wide transcriptomic analysis of Salmonella Typhimurium (SeT) was conducted with RNA-Seq to elucidate strategies for epiphytic growth on live, intact tomato shoot and root surfaces. Six plasmid-encoded and 123 chromosomal genes were significantly (using Benjamini-Hochberg adjusted p-values) up-regulated; 54 and 110 detected in SeT on shoots and roots, respectively, with 35 common to both. Key signals included NsrR regulon genes needed to mitigate nitrosative stress, oxidative stress genes and host adaptation genes, including environmental stress, heat shock and acid-inducible genes. Several amino acid biosynthesis genes and genes indicative of sulphur metabolism and anaerobic respiration were up-regulated. Some Type III secretion system (T3SS) effector protein genes and their chaperones from pathogenicity island-2 were expressed mostly in SeT on roots. Gene expression in SeT was validated against SeT and also the tomato outbreak strain Salmonella Newport with a high correlation (R 2 = 0.813 and 0.874, respectively; both p < 0.001). Oxidative and nitrosative stress response genes, T3SS2 genes and amino acid biosynthesis may be needed for Salmonella to successfully colonize tomato shoot and root surfaces.

Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, Enterococcus spp., and Aeromonas spp. in Alternative Irrigation Water Sources: a CONSERVE Study.

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting.IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.

Breeding Crops for Enhanced Food Safety.

An increasing global population demands a continuous supply of nutritious and safe food. Edible products can be contaminated with biological (e.g., bacteria, virus, protozoa), chemical (e.g., heavy metals, mycotoxins), and physical hazards during production, storage, transport, processing, and/or meal preparation. The substantial impact of foodborne disease outbreaks on public health and the economy has led to multidisciplinary research aimed to understand the biology underlying the different contamination processes and how to mitigate food hazards. Here we review the knowledge, opportunities, and challenges of plant breeding as a tool to enhance the food safety of plant-based food products. First, we discuss the significant effect of plant genotypic and phenotypic variation in the contamination of plants by heavy metals, mycotoxin-producing fungi, and human pathogenic bacteria. In addition, we discuss the various factors (i.e., temperature, relative humidity, soil, microbiota, cultural practices, and plant developmental stage) that can influence the interaction between plant genetic diversity and contaminant. This exposes the necessity of a multidisciplinary approach to understand plant genotype × environment × microbe × management interactions. Moreover, we show that the numerous possibilities of crop/hazard combinations make the definition and identification of high-risk pairs, such as Salmonella-tomato and Escherichia coli-lettuce, imperative for breeding programs geared toward improving microbial safety of produce. Finally, we discuss research on developing effective assays and approaches for selecting desirable breeding germplasm. Overall, it is recognized that although breeding programs for some human pathogen/toxin systems are ongoing (e.g., Fusarium in wheat), it would be premature to start breeding when targets and testing systems are not well defined. Nevertheless, current research is paving the way toward this goal and this review highlights advances in the field and critical points for the success of this initiative that were discussed during the Breeding Crops for Enhanced Food Safety workshop held 5-6 June 2019 at University of California, Davis.