Agricultural water is an important source of foodborne pathogens on produce farms. Managing water-associated risks does not lend itself to one-size-fits-all approaches due to the heterogeneous nature of freshwater environments. To improve our ability to develop location-specific risk management practices, a study was conducted in two produce-growing regions to (i) characterize the relationship between Escherichia coli levels and pathogen presence in agricultural water, and (ii) identify environmental factors associated with pathogen detection. Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds Ratio [OR] = 2.18), and eaeA-stx codetection (OR = 6.49) was significantly greater for MS compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also showed that eaeA-stx codetection in AZ (OR = 50.2) and NY (OR = 18.4), and Salmonella detection in AZ (OR = 4.4) were significantly associated with E. coli levels, while Salmonella detection in NY was not. Random forest analysis indicated that interactions between environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of pathogen detection. Our findings suggest that (i) environmental heterogeneity, including interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not be a suitable indicator of food safety risks. Instead, targeted methods that utilize environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict when there is a high or low risk of surface water being contaminated by pathogens) are needed to assess and mitigate the food safety risks associated with preharvest water use. By identifying environmental factors associated with an increased likelihood of detecting pathogens in agricultural

19 20 21 22 23 produce farms. Managing water-associated risks does not lend itself to one-size-fits-all 24 approaches due to the heterogeneous nature of freshwater environments, and because 25 environmental conditions affect the likelihood of pathogen contamination and the relationship 26 between indicator organism levels (e.g., E. coli) and pathogen presence. To improve our ability 27 to develop location-specific risk management practices, a study was conducted in two produce-28 growing regions to (i) characterize the relationship between E. coli levels and pathogen presence 29 in agricultural water, and (ii) identify environmental factors associated with pathogen detection. 30Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 31 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds 32 Ratio [OR]=2.18), and eaeA-stx codetection (OR=6.49) was significantly greater for MS 33 compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also 34 showed that eaeA-stx codetection in AZ (OR=50.2) and NY (OR=18.4), and Salmonella 35 detection in AZ (OR=4.4) were significantly associated with E. coli levels, while Salmonella 36 detection in NY was not. Random forest analysis indicated that interactions between 37 environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of 38 pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of 39 pathogen detection. Our findings suggest that (i) environmental heterogeneity, including 40 interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not 41 be a suitable indicator of the food safety risks. Instead, targeted methods that utilize 42 environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict 43 when there is a high or low risk of surface water being contaminated by pathogens) are needed to 44 assess and mitigate the food safety risks associated with preharvest water use. By identifying 45 environmental factors associated with an increased likelihood of detecting pathogens in 46 agricultural water, this study provides information that (i) can be used to assess when pathogen 47 contamination of agricultural water is likely to occur, and (ii) facilitate development of targeted 48 interventions for individual water sources, providing an alternative to existing one-size-fits-all 49 approaches. 50 51 52 3 3Preharvest surface water use for produce production (e.g., irrigation, fertigation, pesticide 53 application, dust abatement) has repeatedly been identified as a factor associated with an 54

Although single nucleotide polymorphisms (SNPs) in folate-mediated pathways predict susceptibility to choline deficiency during severe choline deprivation, it is unknown if effects persist at recommended intakes. Thus, we used stable isotope liquid chromatography-mass spectrometry (LC-MS) methodology to examine the impact of candidate SNPs on choline metabolism in a long-term, randomized, controlled feeding trial among pregnant, lactating, and nonpregnant (NP) women consuming 480 or 930 mg/d choline (22% as choline-d, with d indicating a deuterated trimethyl amine group) and meeting folate-intake recommendations. Variants impairing folate metabolism, methylenetetrahydrofolate reductase (MTHFR) rs1801133, methionine synthase (MTR) rs1805087 [wild-type (WT)], MTR reductase (MTRR) rs1801394, and methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) rs2236225, influenced choline dynamics, frequently through interactions with reproductive state and choline intake, with fewer genotypic alterations observed among pregnant women. Women with these variants partitioned more dietary choline toward phosphatidylcholine (PC) biosynthesis via the cytidine diphosphate (CDP)-choline pathway at the expense of betaine synthesis even when use of betaine as a methyl donor was increased. Choline intakes of 930 mg/d restored partitioning of dietary choline between betaine and CDP-PC among NP (MTHFR rs1801133 and MTR rs1805087 WT) and lactating (MTHFD1 rs2236225) women with risk genotypes. Overall, our findings indicate that loss-of-function variants in folate-metabolizing enzymes strain cellular PC production, possibly via impaired folate-dependent phosphatidylethanolamine-N-methyltransferase (PEMT)-PC synthesis, and suggest that women with these risk genotypes may benefit from choline intakes exceeding current recommendations.-Ganz, A. B., Shields, K., Fomin, V. G., Lopez, Y. S., Mohan, S., Lovesky, J., Chuang, J. C., Ganti, A., Carrier, B., Yan, J., Taeswuan, S., Cohen, V. V., Swersky, C. C., Stover, J. A., Vitiello, G. A., Malysheva, O. V., Mudrak, E., Caudill, M. A. Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.