An official website of the United States government.
The long-term goal of the research is to come to an improved mechanistic understanding of produce contamination by pathogens. We will achieve this by using artificial leaf surfaces as experimentally amenable arenas to pick apart the complexity of pathogen attachment and activity in an environment that is characterized by substantial physical, chemical, and biological heterogeneity at the micrometer scale. We will employ newly developed tools for quantifying the activity and fate of individual bacteria, and determine whether they have merit over methods of interrogation that ignore single-cell variation, for identifying factors contributing to survival of pathogenic E. coli on leafy greens. For this 4-year project, we have formulated the following supporting objectives: Generate and compare data from single-cell and population-level analyses of the establishment of E. coli on real and artificial leaf surfaces; Assess the role of attachment, dispersal, nutrient availability, and bacterial community structure as major contributing factors to E. coli survival on leaf surfaces; Develop next-generation artificial leaf surfaces as experimental tools with improved control over surface properties and delivery of chemicals and biologicals.
<p>NON-TECHNICAL SUMMARY:<br/> Attachment of foodborne pathogens to the surfaces of fresh produce is a complex process. It involves interactions between many physical, chemical, and biological factors, the individual impacts of which are hard to resolve without an experimental reduction in complexity. We propose the use of artificial leaf surfaces to explore the attachment, survival, and reproduction of pathogenic Escherichia coli in an environment that mimics the micrometer-scale surface heterogeneity of leafy greens. Lettuce leaves will serve as master surfaces to cast positive replicas using the organosilicon material poly-dimethylsiloxane (PDMS). These replicas are indistinguishable from real leaves in terms of their physical environment, i.e. surface topography, and they can be manipulated to pose different challenges to invading E. coli in terms of the chemical and
biological environment, for example the availability of nutrients and the structure and function of surface-associated microbiota. We will employ newly developed bioreporter tools for quantifying the activity and fate of individual E. coli cells in these environments, and determine whether these tools have merit over methods of interrogation that ignore variation in the single-cell experience. We commit to the development of improved PDMS-based artificial leaves, which will feature greater experimental control over surface properties and environmental heterogeneity. The project will yield quantitative data that can be used in models that simulate (cross-)contamination of lettuce and other produce. By wide dissemination of casting protocols and bioreporter tools, we hope to contribute beyond our own experiments towards an improved mechanistic understanding of the foliar contamination by
human pathogens.<p>
APPROACH: <br/>We will use several E.coli versions of new or established bioreporter systems to quantify the bacterial experience of real and artificial leaf surfaces.For example, the CUSPER bioreporter involves the dilution of green fluorescent protein (GFP) from dividing bacterial cells in the absence of de novo synthesis, which allows us to quantify the reproductive success of individual bacteria based on reverse interpretation of single-cell GFP content (CUSPER in reverse reads REPSUC, for REProductive SUCcess). This bioreporter represents an ideal tool for assessing the impact of environmental heterogeneity and chance events on individual survivorship. We will exploit a recently established rapid and cost-effective method for the reproduction of leaf surfaces using polydimethylsiloxane (PDMS). PDMS is a commonly used material in soft lithography and can achieve
accurate reproduction of reliefs with nanometer resolution.</p>