(311d) Deconvolute Bacterial Responses to Surface Nanotopography and Surface Chemistry Using Orthogonally Engineered Biointerfaces

Reducing bacterial attachment to abiotic surfaces have shown great promise in battling adverse biofilm formation, which has immense importance across various human activities ranging from healthcare to food safety. Nevertheless, despite decades of research and discovery of many successful examples of antibiofouling/antibiofilm surfaces, it is still unclear on a fundamental level how surface properties (e.g., surface topography and chemistry), individually and combinatorially, affect attachment and other bacterial behavior.

One prominent challenge lies in that all surfaces exhibit both topography and chemistry simultaneously to the bacteria they interface with, and that modification of surface topography often inadvertently results in changes in surface chemistry, and vice versa. Furthermore, bacterial cells have been shown to sense and respond to stochastic roughness as small as 100 nm, yet their response to sub-50-nm, well-defined nanotopography is largely unexplored due to low accessibility to sub-50-nm nanofabrication. Finally, surface adherent bacteria undergo dynamic sensing of surface properties, which informs their “decision” to adopt a sessile lifestyle and turn on the biofilm formation sequence – a process underexplored in common biofilm assays. Therefore, the ability to differentiate the decision states of the bacteria during their initial interaction with the surfaces will enrich our understanding of how surface properties affect initial attachment.

Objective

Here, we aim to deconvolute bacterial responses to surface nanotopography and surface chemistry by independently varying these two key surface properties, enabled by orthogonal nanoengineering.

Methods & Results

Orthogonal Nanoengineering and Characterization. First, nanoporous anodic aluminum oxide (AAO) surfaces with highly uniform small (25-nm) and large (100-nm) pores were generated through anodization in oxalic acid. A flat aluminum oxide surface was also prepared as a control. Then, half of these topographically-defined surfaces were further subjected to surface chemistry modification by depositing an ultrathin (< 10 nm) poly(4-vinylpyridine-co-divinylbenzene) via initiated chemical vapor deposition (iCVD). The thin film was further rendered zwitterionic (ZW) via exposure to 1,3-propane sultone. As shown in Figure 1A & 1B, the top surfaces and cross-sections of each of the three topographies were well-preserved after surface chemistry modification, and surface chemistry was consistent across surface topographies, as confirmed by FTIR and XPS (data not shown) – hence, orthogonal nanoengineering was achieved.

Probing Bacterial Responses to Surface Properties. Next, we introduced a fluorescence-based, quantitative bacterial response assay. A Pseudomonas aeruginosa strain, PAO1/pCdrA::gfp(ASV), was chosen because it produces a GFP reporter in proportion to the intracellular concentration of cyclic-di-GMP (c-di-GMP), which is a second messenger that regulates bacterial planktonic-to-sessile transition. Thus, more green fluorescence indicates higher c-di-GMP, and probably a cell committed to turning on the pro-biofilm genes. Besides, the PAO1 strain was also stained with DAPI and propidium iodide to reveal the total number of surface-bound cells and cell wall damage, respectively. Together, these fluorescence lables enabled real-time tracking of multiple bacterial responses to the aforementioned surface properties in situ, as shown by a proof-of-principle experiment conducted on flat aluminum oxide surfaces (Figure 1C). Quantitative analysis on such images not only extracted percentage of cells that was alive versus dead, but also provided rich information on the percentage and distribution of cells entering the biofilm sequence, which are likely to become the hotspot for microcolony development. Preliminary experiments conducted on topographically-varied but chemically-identical aluminum oxide surfaces suggest that 25-nm pore surfaces reduce overall attachment whereas 100-nm pore surfaces lead higher cell death (data not shown). Interestingly, both nanostructured surfaces appear to discourage c-di-GMP production. Clearly, these preliminary results require follow-up experiments to confirm.

Ongoing investigations are focused on further isolating the respective effects of surface topography and chemistry individually, as well as understanding the combinatorial effects of the two. Cell tracking with time-lapse images is also underway to investigate the dynamic nature of bacteria-surface interactions as a function of these two surface properties.