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In the proposed work, we will perform field and laboratory studies to: <oL> <LI> Assess the relative mobility of E. coli and Salmonella isolated from different positions along hydrological flow paths of individual farms (i.e. in manure or wastewater, storm-event surface runoff and tile drains or groundwater); <LI>Examine the roles of extracellular structures (e.g. fimbrae, curli, flagella), cell membrane properties (hydrophily vs. hydrophobicity) and other mechanisms (propensity for biofilm formation under various flow and solution chemistry conditions) on observed mobility and pore interface localization; <LI>Identify genetic markers that correlate with the phenotypic features that affect bacterial mobility; <LI> Use this understanding to guide the design of new (or alteration of existing) on-farm Best Management Practices (BMPs) to minimize potential pathogen transport.
NON-TECHNICAL SUMMARY: Water quality concerns have led to increased focus on the environmental transport and fate of pathogenic microorganisms, including viruses, bacteria, and protozoa. Pathogenics are of colloidal sizes and can thus freely move in hydrologic flowpaths through soil, and pose risks to public health once they enter surface water bodies or groundwater wells. Given the regional preponderance of dairy farms, the close proximity of farms and non-farm populations, the extensive spreading of manures, and the close hydrologic connection of many fields to water resources, conducting in this research in the Northeast US is of particular importance. In addition to posing a direct risk to water supplies, transport may also play an important role in transmission between farms or perpetuating infections within herds. The overall goal of this project is to determine if E. coli and Salmonella isolated from hydrologic flow paths leading to nearby water resources are more mobile than those isolated from surface sources. Bacteria isolated from sources (manure, bedding, wastewater etc.) will be compared with those isolated from drainage tile flow, surface water flow and groundwater through soil on dairy farms, and then assessed for mobility under controlled laboratory conditions. We will identify the microbiological and physicochemical characteristics that correlate with mobility and determine potential best management practices (BMPs) to control highly mobile microorganisms. We will then seek to apply this understanding by designing or altering on-farm BMPs to minimize potential pathogen transport from infected dairy farms to nearby water resources. BMPs can be structural (such as vegetated barriers or changing tile outflow configurations) and/or managerial (such as the timing and placement of manure applications). We will convene an on-campus cross-disciplinary workshop to present our findings and to brainstorm potential implications for BMPs and for monitoring efforts. <P>APPROACH: 1) Investigate in-field mobility on farms by determining relative populations in sources (manure. wastewater, barnyard runoff) vs. potential target water resources (tile flow, groundwater, nearby surface waters). 2) For selected strains, use controlled laboratory experiments to further document differential mobilities in soil (using slab chambers) and over soil (using vegetated and non-vegetated runoff flumes). 3) Image selected serotypes from flow experiments to determine extracellular structures presence. 4) Use expertise in visible and confocal laser microscopy to monitor flow cell mobility and biofilm formation as affected by various flow (slow vs. rapid flow) and solution chemistry (high vs. low dissolved organic carbon concentrations) regimes. 5) Quantify cell hydrophobicity and the production levels of known biofilm phenotype determinants such as BapA, curli, fimbriae, flagella, and cellulose, by real-world isolates. 6) Determine relationship between mobility and known biofilm phenotype determinants. 7) Use DNA sequencing and Polymerase Chain Reaction to compare difference between the genetic context of known biofilm phenotype determinants for isolates with differing mobility. 8) Determine the predictive value of genotypic markers of mobility for estimating the transport of uncharacterized bacterial isolates in laboratory experiments. 9) Evaluate the utility of genotypic markers of mobility for detecting pathogen transport using culture-based and culture-independent PCR analysis of total DNA extracted from farm samples. 10) Use in-field and laboratory findings to determine conditions under which mobility is minimized. 11) Design and evaluate the efficacy of remedial best management practices to minimize transport.