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Outbreaks of foodborne illnesses linked to fresh fruits and vegetables have been key drivers behind a wide breadth of research aiming to fill data gaps in our understanding of the total ecology of agricultural water sources such as ponds and wells and the relationship of this ecology to foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. Both S. enterica and L. monocytogenes can persist in irrigation water and have been linked to produce contamination events. Data describing the abundance of these organisms in specific agricultural water sources are valuable to guide water treatment measures. Here, we profiled the culture independent water microbiota of four farm ponds and wells correlated with microbiological recovery of S. enterica (prevalence: pond, 19.4%; well, 3.3%), L. monocytogenes (pond, 27.1%; well, 4.2%) and fecal indicator testing. Correlation between abiotic factors, including water parameters (temperature, pH, conductivity, dissolved oxygen percentage, oxidation reduction potential, and turbidity) and weather (temperature and rainfall), and foodborne pathogens were also evaluated. Although abiotic factors did not correlate with recovery of S. enterica or L. monocytogenes (p > 0.05), fecal indicators were positively correlated with incidence of S. enterica in well water. Bacterial taxa such as Sphingomonadaceae and Hymenobacter were positively correlated with the prevalence and population of S. enterica, and recovery of L. monocytogenes was positively correlated with the abundance of Rhizobacter and Comamonadaceae (p < 0.03). These data will support evolving mitigation strategies to reduce the risk of produce contamination by foodborne pathogens through irrigation.

Outbreaks of foodborne illnesses linked to fresh fruits and vegetables have been key drivers behind a wide breadth of research aiming to fill data gaps in our understanding of the total ecology of agricultural water sources such as ponds and wells and the relationship of this ecology to foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. Both S. enterica and L. monocytogenes can persist in irrigation water and have been linked to produce contamination events. Data describing the abundance of these organisms in specific agricultural water sources are valuable to guide water treatment measures. Here, we profiled the culture independent water microbiota of four farm ponds and wells correlated with microbiological recovery of S. enterica (prevalence: pond, 19.4%; well, 3.3%), L. monocytogenes (pond, 27.1%; well, 4.2%) and fecal indicator testing. Correlation between abiotic factors, including water parameters (temperature, pH, conductivity, dissolved oxygen percentage, oxidation reduction potential, and turbidity) and weather (temperature and rainfall), and foodborne pathogens were also evaluated. Although abiotic factors did not correlate with recovery of S. enterica or L. monocytogenes (p > 0.05), fecal indicators were positively correlated with incidence of S. enterica in well water. Bacterial taxa such as Sphingomonadaceae and Hymenobacter were positively correlated with the prevalence and population of S. enterica, and recovery of L. monocytogenes was positively correlated with the abundance of Rhizobacter and Comamonadaceae (p < 0.03). These data will support evolving mitigation strategies to reduce the risk of produce contamination by foodborne pathogens through irrigation.

Fresh produce, as a known or suspected source of multiple foodbome outbreaks, harbors large populations of diverse microorganisms, which are partially released into wash water during processing. However, the dynamics of bacterial communities in wash water during produce processing is poorly understood. In this study, we investigated the effect of chlorine (FC) and peracetic acid (PAA) on the microbiome dynamics in spinach and romaine lettuce rinse water. Treatments with increasing concentrations of sanitizers resulted in convergence of distinct microbiomes. The resultant sanitizer resistant microbiome showed dominant presence by Bacillus sp., Arthrobacter psychrolactophilus, Cupriavidus sp., and Ralstonia sp. Most of the FC and PAA resistant bacteria isolated from spinach and lettuce rinse water after sanitation were gram positive spore forming species including Bacillus, Paenibacillus, and Brewbacillus spp., while several PAA resistant Pseudomonas spp. were also isolated from lettuce rinse water. Inoculation of foodbome pathogens altered the microbiome shift in spinach rinse water under PAA treatment, but not in lettuce rinse water or FC treated samples. These inoculated foodbome pathogens were not isolated among the sanitizer resistant strains.

Indigenous bacterial populations in fresh-cut produce processing facilities can have a profound effect on the survival and proliferation of inadvertently contaminating foodborne pathogens. In this study, environmental samples were collected from a variety of Zone 3 sites in a processing plant before and after daily routine sanitation. Viable mesophilic aerobic bacteria population was evaluated using both culturing method and quantitative real-time PCR (qPCR) after propidium monoazide treatment. Zone 3 surface microbiota were analyzed using 16S rRNA gene amplicon sequencing with the Qiime2 bioinformatic pipeline. Over 8000 bacterial species across 4 major phyla were identified in Zone 3 microbiomes in the processing facility. Overall, effective bacterial reduction was observed at the sampling sites on the production floor, while sanitation effect on peripheral surfaces was less evident. Effective sanitation resulted in both quantitative and qualitive shifts of Zone 3 microbiota. Several species were highly abundant at multiple sample sites for both winter and summer samplings. Based on the spatial and temporal distribution of the most abundant species, a Zone 3 core microbiome in the processing facility was tentatively described to included Cupriavidus sp., Pseudomonas sp., Ralstonia sp., Arthrobacter psychrolactophdus, Pseudomonas vemnii, Stenotrophomonas sp., and an unknown species of the family Enterobacteriaceae.

Indigenous bacterial populations in fresh-cut produce processing facilities can have a profound effect on the survival and proliferation of inadvertently contaminating foodborne pathogens. In this study, environmental samples were collected from a variety of Zone 3 sites in a processing plant before and after daily routine sanitation. Viable mesophilic aerobic bacteria population was evaluated using both culturing method and quantitative real-time PCR (qPCR) after propidium monoazide treatment. Zone 3 surface microbiota were analyzed using 16S rRNA gene amplicon sequencing with the Qiime2 bioinformatic pipeline. Over 8000 bacterial species across 4 major phyla were identified in Zone 3 microbiomes in the processing facility. Overall, effective bacterial reduction was observed at the sampling sites on the production floor, while sanitation effect on peripheral surfaces was less evident. Effective sanitation resulted in both quantitative and qualitive shifts of Zone 3 microbiota. Several species were highly abundant at multiple sample sites for both winter and summer samplings. Based on the spatial and temporal distribution of the most abundant species, a Zone 3 core microbiome in the processing facility was tentatively described to included Cupriavidus sp., Pseudomonas sp., Ralstonia sp., Arthrobacter psychrolactophdus, Pseudomonas vemnii, Stenotrophomonas sp., and an unknown species of the family Enterobacteriaceae.

Describing baseline microbiota associated with agricultural commodities in the field is an important step towards improving our understanding of a wide range of important objectives from plant pathology and horticultural sustainability, to food safety. Environmental pressures on plants (wind, dust, drought, water, temperature) vary by geography and characterizing the impact of these variable pressures on phyllosphere microbiota will contribute to improved stewardship of fresh produce for both plant and human health. A higher resolution understanding of the incidence of human pathogens on food plants and co-occurring phytobiota using metagenomic approaches (metagenome tracking) may contribute to improved source attribution and risk assessment in cases where human pathogens become introduced to agro-ecologies. Between 1990 and 2007, as many as 1990 culture-confirmed Salmonella illnesses were linked to tomatoes from as many as 12 multistate outbreaks (Bell et al., 2012; Bell et al., 2015; Bennett et al., 2014; CDC, 2004; CDC, 2007; Greene et al., 2005a; Gruszynski et al., 2014). When possible, source attribution for these incidents revealed a biogeographic trend, most events were associated with eastern growing regions. To improve our understanding of potential biogeographically linked trends in contamination of tomatoes by Salmonella, we profiled microbiota from the surfaces of tomatoes from Virginia, Maryland, North Carolina and California. Bacterial profiles from California tomatoes were completely different than those of Maryland, Virginia and North Carolina (which were highly similar to each other). A statistically significant enrichment of Firmicutes taxa was observed in California phytobiota compared to the three eastern states. Rhizobiaceae, Sphingobacteriaceae and Xanthobacteraceae were the most abundant bacterial families associated with tomatoes grown in eastern states. These baseline metagenomic profiles of phyllosphere microbiota may contribute to improved

Describing baseline microbiota associated with agricultural commodities in the field is an important step towards improving our understanding of a wide range of important objectives from plant pathology and horticultural sustainability, to food safety. Environmental pressures on plants (wind, dust, drought, water, temperature) vary by geography and characterizing the impact of these variable pressures on phyllosphere microbiota will contribute to improved stewardship of fresh produce for both plant and human health. A higher resolution understanding of the incidence of human pathogens on food plants and co-occurring phytobiota using metagenomic approaches (metagenome tracking) may contribute to improved source attribution and risk assessment in cases where human pathogens become introduced to agro-ecologies. Between 1990 and 2007, as many as 1990 culture-confirmed Salmonella illnesses were linked to tomatoes from as many as 12 multistate outbreaks (Bell et al., 2012; Bell et al., 2015; Bennett et al., 2014; CDC, 2004; CDC, 2007; Greene et al., 2005a; Gruszynski et al., 2014). When possible, source attribution for these incidents revealed a biogeographic trend, most events were associated with eastern growing regions. To improve our understanding of potential biogeographically linked trends in contamination of tomatoes by Salmonella, we profiled microbiota from the surfaces of tomatoes from Virginia, Maryland, North Carolina and California. Bacterial profiles from California tomatoes were completely different than those of Maryland, Virginia and North Carolina (which were highly similar to each other). A statistically significant enrichment of Firmicutes taxa was observed in California phytobiota compared to the three eastern states. Rhizobiaceae, Sphingobacteriaceae and Xanthobacteraceae were the most abundant bacterial families associated with tomatoes grown in eastern states. These baseline metagenomic profiles of phyllosphere microbiota may contribute to improved

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012-2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech's Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate - TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (< 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newpo

Background Research to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop. Results DNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe “native” bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes. Conclusions Distinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Domina

The ability to detect a specific organism from a complex environment is vitally important to many fields of public health, including food safety. For example, tomatoes have been implicated numerous times as vehicles of foodborne outbreaks due to strains of Salmonella but few studies have ever recovered Salmonella from a tomato phyllosphere environment. Precision of culturing techniques that target agents associated with outbreaks depend on numerous factors. One important factor to better understand is which species co-enrich during enrichment procedures and how microbial dynamics may impede or enhance detection of target pathogens. We used a shotgun sequence approach to describe taxa associated with samples pre-enrichment and throughout the enrichment steps of the Bacteriological Analytical Manual's (BAM) protocol for detection of Salmonella from environmental tomato samples. Recent work has shown that during efforts to enrich Salmonella (Proteobacteria) from tomato field samples, Firmicute genera are also co-enriched and at least one co-enriching Firmicute genus (Paenibacillus sp.) can inhibit and even kills strains of Salmonella. Here we provide a baseline description of microflora that co-culture during detection efforts and the utility of a bioinformatic approach to detect specific taxa from metagenomic sequence data. We observed that uncultured samples clustered together with distinct taxonomic profiles relative to the three cultured treatments (Universal Pre-enrichment broth (UPB), Tetrathionate (TT), and Rappaport-Vassiliadis (RV)). There was little consistency among samples exposed to the same culturing medias, suggesting significant microbial differences in starting matrices or stochasticity associated with enrichment processes. Interestingly, Paenibacillus sp. (Salmonella inhibitor) was significantly enriched from uncultured to cultured (UPB) samples. Also of interest was the sequence based identification of a number of sequences as Salmonella despite indi

Background Research to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop. Results DNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe “native” bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes. Conclusions Distinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Domina

The ability to detect a specific organism from a complex environment is vitally important to many fields of public health, including food safety. For example, tomatoes have been implicated numerous times as vehicles of foodborne outbreaks due to strains of Salmonella but few studies have ever recovered Salmonella from a tomato phyllosphere environment. Precision of culturing techniques that target agents associated with outbreaks depend on numerous factors. One important factor to better understand is which species co-enrich during enrichment procedures and how microbial dynamics may impede or enhance detection of target pathogens. We used a shotgun sequence approach to describe taxa associated with samples pre-enrichment and throughout the enrichment steps of the Bacteriological Analytical Manual's (BAM) protocol for detection of Salmonella from environmental tomato samples. Recent work has shown that during efforts to enrich Salmonella (Proteobacteria) from tomato field samples, Firmicute genera are also co-enriched and at least one co-enriching Firmicute genus (Paenibacillus sp.) can inhibit and even kills strains of Salmonella. Here we provide a baseline description of microflora that co-culture during detection efforts and the utility of a bioinformatic approach to detect specific taxa from metagenomic sequence data. We observed that uncultured samples clustered together with distinct taxonomic profiles relative to the three cultured treatments (Universal Pre-enrichment broth (UPB), Tetrathionate (TT), and Rappaport-Vassiliadis (RV)). There was little consistency among samples exposed to the same culturing medias, suggesting significant microbial differences in starting matrices or stochasticity associated with enrichment processes. Interestingly, Paenibacillus sp. (Salmonella inhibitor) was significantly enriched from uncultured to cultured (UPB) samples. Also of interest was the sequence based identification of a number of sequences as Salmonella despite indi

Background: Tomato (Solanum lycopersicum) consumption has been one of the most common causes of produceassociated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454- pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production. Results: The two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Pantoea and Enterobacter were the most abundant. Conclusions: Despite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards t