A microfluidic approach to hierarchical 3D leafy green-mimic structures for studying attachment and colonization of Escherichia coli O157:H7 under fluid shear stress

Pathogenic bacteria can attach and infiltrate onto fresh produce, leading to foodborne disease outbreaks. Pathogens might use hierarchical stalks, crevices and natural openings on the leafy greens to survive washing. In this study, we assessed the 3D configurations and distributions of Escherichia coli O157:H7 colonization on features of leaf surfaces under fluid shear stress. The colonization of green fluorescent protein (GFP)-tagged E. coli was characterized using multiphoton fluorescence microscopy and compared under static (incubated for 36 hrs at 37°C) and shear flow conditions (750 nl/min for 36 hrs at 37°C). For microfabricated features to mimic the trichomes, stomata, and crevices, we demonstrate that natural wax-coated polydimethylsiloxane (PDMS) retains similar topographies to abaxial surface of leafy greens.
For trichomes, E. coli under shear flow aligned its colonization parallel to the direction of flow, while its colonization without flow had no preferential alignment. Depending on size and spacing of the trichome arrays, different bacterial colonization patterns grow radially from initial microcolonies to form in the shape of an arc due to surface hydrophobicity and fluid shear with a nutrient-rich medium. For crevice and stomata arrays, enhanced microcolonies of E. coli were seen at the edges of individual microstructures under shear flow. These colonies existed predominantly in the near-wall side of channels rather than the center stream, due to lower shear at these locations. The results indicate that bacterial translations and colonization in response to shear flow can affect fresh produce contamination during irrigation and washing events.