# Bacteria Pattern Spontaneously on Periodic Nanostructure Arrays

Entry by Emily Redston, AP 225, Fall 2011

Work in progress

## Reference

Bacteria Pattern Spontaneously on Periodic Nanostructure Array by A. I. Hochbaum, J. Aizenberg. Nano Lett. 10, 3717-3721 (2010)

## Introduction

Bacterial biofilms naturally form on many surfaces, usually at the solid-liquid or liquid-air interface. Biofilms are composed of many cells embedded within a polymeric organic matrix. While biofilm formation is a concern for many industries, they are especially harmful in the medical community, where they cause extensive damage by triggering the human immune response, releasing harmful endotoxins and exotoxins, and clogging indwelling catheters. Hospital-acquired, or nosocomial, infections affect roughly 10% of patients in the United States, and they are responsible for nearly 100,000 deaths. These infections are difficult to treat because the biofilm protects the cells from antibiotic attack. Developing biomedical materials that are resistant to biofilm formation has been a hot topic in research since it would significantly reduce the rate of nosocomial infections and the costs associated with treating them.

In this regard, many people have attempted to use surface chemistry to prevent biofilm formation. Unfortunately, persistently bacteria-resistant materials are difficult to achieve using surface chemistry alone. Even if the bacteria are unable to attach to a substrate directly, nonspecific adsorption of proteins or secreted surfactants to the surface eventually masks the underlying chemical functionality.

On the other hand, the effects of topographical features on bacterial adhesion and subsequent biofilm formation are poorly understood. However, recent studies have shown that the behavior of mammalian cells can be manipulated using only spatial and mechanical clues. Biofilms contain a diversity of microbial phenotypes and form spatial patterns through cooperative organization at the macroscopic and microscopic level. They develop anisotropically in response to surrounding environmental factors. Topographical features can influence the arrangement and the resulting behavior of cells on surfaces. Some bacteria rely on physical interactions between neighboring cells for communication. Therefore, disrupting the natural packing arrangement of cells within biofilms may influence some of the cooperative functions of these microbial populations. Following this train of thought, in this paper, the authors present a very exciting, alternative approach to preventing biofilm formation. They show that periodic arrays of high-aspect-ratio nanostructures can direct the large-scale spontaneous patterning behavior of bacteria.

## Sample Preparation

To study the effects of substrate topography on bacterial ordering and biofilm development, nanostructured substrates were fabricated with dimensions on the order of bacterial cells. Arrays of high-aspect ratio nanometer-scale polymer posts were made using a fast replication molding technique. Using this method, many identical substrates with varying dimensional parameters, such as nanopost diameter, hieght, pitch, and array symmetry, were made so the authors could conduct systematic investigations of bacterial growth on structure surfaces.

The authors focused primarily on the bacteria Pseudomonas aeruginosa, which is a human opportunistic pathogen and one of the most common nosocomial infections in the lining of catheters and the lungs of cystic fibrosis patients. Pseudomonas aeruginosa (strain PA14) was grown on submerged polymer replicas with a gradient post pitch, from 4 down to 0.9 $\mu$m.

## Results

Figure 1. Comparison of P. aeruginosa adhesion on structured and unstructured regions of the growth substrates. (A) Fluorescence microscopy shows the localized effect of substrate topography on bacterial adhesion as compared to flat surfaces. The image shows the interface between a structured and unstructured region on the same substrate. The interface between the flat (upper) and structured (lower) areas is abrupt, as is the transition from ordered packing to random microcolony aggregates, which lack long-range cell order. The cells were stained with SYTOX green nucleic acid stain. (B,C) Cross-sectional SEM images of PA14 cultured on flat and periodically structured epoxy surfaces, respectively, showing the stark difference in attachment morphology. The aligned cells in (C) are false-colored to highlight their orientation. Scale bars are 10 μm in (A) and 1 μm in (B) and (C).
Figure 2. P. aeruginosa assembled on nanopost arrays. Fluorescence microscopy images of assembled bacteria on a post pitch gradient substrate at 2.2 (A), 0.9 (B), and 0.7 μm (C) spacing between posts show the different packing configurations of rodlike bacteria within the periodic arrays. (D) FFTs of these and intermediate post spacing regions elucidate the ordering of cells on varying topographies. The FFT farthest to the left is from a flat substrate for comparison. The rest of the FFTs are from large area images of bacteria adhered to regions with decreasing post spacing (labeled under each FFT) from left to right. They all show positional ordering peaks corresponding to the [01] and [10] directions of the post array, indicating the preferential attachment and the subsequent registration of the bacterial layer with the posts. Scale bar in (A) is 5 μm and applies to (B) and (C).

As can be seen in Figure 1, as opposed to the random packing and three-dimensional growth of biofilms on flat substrates, bacteria grown on these post substrates spontaneously assemble into patterns dictated by the underlying array symmetry. The fluorescence image in Figure 1a shows the interface between a flat region (upper) and one of patterned posts (lower) on the same substrate. The difference in ordering during biofilm formation is apparent, and the abrupt change at the interface suggests a localized response to topographical features rather than an induced cooperative behavior. The SEM images in Figure 1b and 1c show cross-sectional views of the different bacterial conformations in a biofilm grown on a flat substrate (Figure 1b) versus the extreme ordering case where cells are oriented normal to the substrate (Figure 1c). As is evident from the micrographs, the bacteria exhibit a preference for adhering to the posts even when different conformations are possible. This behavior was observed on post substrates irrespective of surface chemistry.

The authors also found that the spontaneous patterning of bacteria within the post arrays is extremely sensitive to the spacing between adjacent posts. Fluorescence microscopy images (Figure 2a−c), and the corresponding fast Fourier transforms (FFTs, Figure 2d), show the range of ordering achieved within the arrays. The FFTs contain both peaks associated with positional ordering of the bacteria and shape variations of the diffuse central spot, indicative of orientational order. The bacterial assembly is more pronounced as the spacing of the posts approaches the characteristic size of the cell. On regions of the sample where the nearest neighbor post spacing is larger than the length of the cell, bacteria adheres to the substrate randomly (Figure 2a). The FFT from these areas shows no orientational order, akin to growth on flat unstructured surfaces and only faint positional ordering peaks, indicative of the preference of the cells to adhere at points where the posts meet the substrate.

As the spacing between neighboring posts approaches the length of the rodlike P. aeruginosa (roughly 1.2−1.5 μm), bacteria begin to adhere to the substrate following the post array. Cells bridging nearest neighbor post positions (attached parallel to the substrate and perpendicular to each other) are aligned with the [10] and [01] directions of the post lattice. The FFTs in Figure 2d show this transition as the post pitch decreases. The central spot of the FFTs extends toward the [10] and [01] ordering peaks, indicative of the preferential alignment of the cells on the substrate. As the post spacing decreases further across the substrate to about 0.8 μm, the bacteria align themselves along the length of the posts, normal to the substrate. Since the cells are oriented along the imaging axis of the microscope, the bacteria appear as dots (Figure 2c), as opposed to rods (Figure 2b), arranged in a square array. The FFT marks this transition with the loss of orientational order in the central spot, since the cells are radially symmetric in this configuration in plan view. The positional ordering peaks are retained due to the persistent association of the cells with the posts. Throughout the transition from disordered to ordered adhesion, these positional ordering peaks move further from the center of the FFT, consistent with the decreasing lattice spacing of the posts. All the cells assembled within the arrays tend to pack with the same configuration at a given post spacing. In areas of the substrate between these regions, the bacteria pack in a mixture of the two flanking ordering phases.

The distinct packing arrangements of bacteria on periodic nanostructures are a result of a preferential association between the bacterial cells and the substrate. The authors believe that the ordering occurs because the bacteria tend to maximize their contact area with the surface, where the posts act as topographical extensions of the substrate. This simplistic model predicts the packing arrangements of bacteria observed by SEM and fluorescence microscopy. The authors also did the same experiment with other bacteria, and they found that the patterned assembly is a general phenomenon, occurring in examples of both gram-positive and gram-negative bacteria, even in the absence of pili or flagella.

## Conclusion

The authors showed that when the characteristic dimensions of confined spaces approach those of bacterial cells, their interactions with the surface changes significantly. These interactions are general and apply to a variety of bacterial species. Tuning the periodicity of structures within the relevant cellular scale leads to distinctive differences in bacterial assembly. In this manner, they demonstrated the ability to direct cell patterning over large areas on a microscopic level. These substrates could provide a novel route to engineer ordered or disordered biofilm structures for a variety of applications ranging from microbial-resistant surfaces that interfere with the natural packing arrangement of cells within biofilms to those that promote specific biofilm functions such as in remediation or bioelectrical systems. Their results have huge implications for the future direction of biofilm research. The effects of topographical features on biofilm formation is still in its infancy, but this paper shows that it has a lot of potential to be an extremely rewarding area of research.