Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv) gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibrio bacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a β-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species. ■ MATERIALS AND METHODSBacterial Strains, Plasmids and Culturing Conditions. The strains used in this study were Escherichia coli strains MG1655 and DH5α, and Pseudomonas aeruginosa KCOM 1465, which was obtained

This paper describes a system to study how small physical perturbations can affect bacterial community behavior in unexpected ways through modulation of diffusion and convective transport of chemical communication molecules and resources. A culture environment that mimics the chemically open characteristic of natural bacterial habitats but with user-defined spatiotemporal control of bacteria microcolonies is realized through use of an aqueous two phase system (ATPS). The ATPS is formulated with nontoxic dextran (DEX) and poly(ethylene glycol) (PEG) dissolved in cell culture media. DEX-phase droplets formed within a bulk PEG-phase stably confine the bacteria within it while small molecules diffuse relatively freely. Bacteria-containing DEX droplets can also be magnetically relocated, without loss of its bacterial content, when DEX-conjugated magnetic particles are included. We found that decreasing the distance between quorum-sensing (QS)-coupled microcolonies increased green fluorescent protein (GFP) expression due to increased inter-colony chemical communication but with upper limits. Periodic relocation of the chemical signal receiver colony, however, increased GFP expression beyond these typical bounds predicted by quorum sensing concepts alone by maintaining inter-colony chemical communication while also relieving the colony of short-range resource depletion effects. Computer simulations suggest that such increased productive output in response to periodic nonlethal physical perturbations is a common feature of chemically activated interactive cell systems where there is also a short-range inhibitory effect. In addition to providing insights on the effect of bacteria relocation, the magnetic ATPS droplet manipulation capability should be broadly useful for bioanalyses applications where selective partitioning at the microscale in fully aqueous conditions is needed.