Aqueous Two-Phase System-Derived Biofilms for Bacterial Interaction Studies

Abstract

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

Patterning Bacterial Communities on Epithelial Cells

“…Two of these formulations were used before for printing mammalian cells [14] while the third was a more viscous formulation used in a recent study for patterning bacterial cells and biofilms on polystyrene or PDMS surfaces [13]. MCF 10a cell monolayers were covered with the DMEM/F12-infused polyethylene glycol (PEG) rich phase of each of the three formulations.…”

“…In a previous study done by our group [13], we showed that the final size of the DEX spot and, hence, the community formed can be controlled by adjusting the initial volume of the droplet. Here, in this study, this same principle was expanded upon and applied to develop isolated bacterial communities on an epithelial cell layer (Figure 3).…”

“…In a separate study, we expanded this technique to develop localized and patterned biofilms and subsequently used these biofilms to study interactions between different bacterial strains [13]. In this paper, the benefits of ATPS confinement are extended to applications where bacteria are patterned within a restricted aqueous space over epithelial cells for prolonged periods, i.e., 24 h, without release of the bacteria into the surrounding growth media.…”

Effect of Microstructure on Population Growth Parameters of Escherichia coli in Gelatin-Dextran Systems

“…Also, most studies are performed in systems not resembling food products. Clostridium tetani, Lactobacillus rhamnosus, and E. coli W3110, ML308, and MG1655 were shown to have a preference for the dextran phase in polyethylene glycol-dextran systems (40)(41)(42)(43). Also, in polyvinylpyrrolidone-dextran systems, Lactobacillus rhamnosus and Enterococcus faecium M74 were found preferentially in the dextran phase (42,44).…”

Bioprinting Using Aqueous Two-Phase System

“…To this end, Yaguchi et al patterned two separate populations of bacteria directly over each other using ATPS bioprinting [35]. Using a PEG/DEX ATPS system, Escherichia coli suspended in DEX were patterned over PDMS substrate and formed a biofilm over 24 h (Fig.…”