Bacterial strains used in this study were the P. aeruginosa strain PAK and the Proteus vulgaris strain ATCC 13315. P. aeruginosa PAK was maintained on 1.5% LBA [10 g/L tryptone (Oxoid), 5 g/L yeast extract (Oxoid), 5 g/L NaCl, 1.5% agar (Oxoid)] and Pr. vulgaris ATCC 13315 was maintained on low salt 1.5% LBA (2.5 g/L NaCl) at 37°C. P. aeruginosa interstitial biofilms were cultured on nutrient media [4 g/L tryptone (Oxoid), 2 g/L yeast extract (Oxoid), 2 g/L NaCl, 1 g/L MgSO₄.7H₂O] solidified with 8 g/L gellan gum (MP Biomedicals; twitching motility gellan gum; TMGG). Pr. vulgaris interstitial biofilms were grown on LB solidified with 8 g/L gellan gum (Luria Bertani gellan gum; LBGG).

The micro-fabricated transverse furrow design consists of a series of lateral furrows repeating down the length of one half of a 22 × 40 mm surface, the other half being left smooth, i.e., without micro-fabricated features. This design was patterned to a photoresist coated silicon wafer (negative NLOF 2020 photoresist) using photolithography. Excess photoresist was removed by cleaning with O₂ plasma. Aluminum of thicknesses 0.5 and 1 μm were deposited by thermal evaporation on the silicon wafer coated with the patterned photoresist furrows. The silicon wafer was then submerged in N-methly-2-pyrrlidone (NMP) at 80°C to remove the remaining photoresist and the accompanying aluminum deposits, leaving behind the furrow template as a negative mold on the silicon wafer (Supplementary Figure 1).

Positive molds were then cast into thin polydimethylsiloxane (PDMS) films, ~1 mm in height by mixing Sylgard 184 PDMS (Dow Corning) in a 1:10 ratio of curing agent to base and curing for 12 h at 65°C. Three PDMS films were made for each furrow parameter template. Furrow parameters will be described as width × depth × inter-furrow distance.

A modified interstitial biofilm assay (Turnbull and Whitchurch, 2014) was used as follows. The PDMS template was inoculated, rather than the nutrient media, by dotting a pipette tip coated in an inoculum from an overnight plate culture, 1 mm from the start of the micro-fabricated furrow design. This PDMS template was then used in replacement of the glass coverslip in the interstitial biofilm assay (Figure 1A). Interstitial biofilms of P. aeruginosa and Pr. vulgaris were grown at 37°C for 24 and 16 h, respectively. The 3 PDMS sets for each furrow parameter template were each repeated across 3 separate days (n = 9) for P. aeruginosa interstitial biofilms and 4 separate days (n = 12) for Pr. vulgaris interstitial biofilms, due to increased variability of the extent of Pr. vulgaris biofilm expansion compared to that of P. aeruginosa.

Low magnification phase-contrast microscopy images were taken across the length of the biofilm, starting at the leading edge of the biofilm within the micro-fabricated furrows right across to the leading edge on the opposite smooth side, making sure to include the point of inoculation. The slide was also scanned using an Epson Perfection V37 scanner (Epson) to obtain a macroscopic view of the biofilm. High magnification time-lapse microscopy was performed on each side of the biofilm across 30 min with a capture rate of one frame every 2 s to compare the biofilm expansion within the micro-fabricated furrows to that on the smooth side.

The low magnification images were stitched within FIJI using the automated stitching plugin Grid/Collection stitching (Preibisch et al., 2009; Schindelin et al., 2012). The line tool in FIJI was used to measure the length from the inoculation point to the leading edge of the biofilm on both the furrows and smooth side. The distance from the inoculation point to the start of the furrows was also measured and this distanced subtracted from both biofilm lengths to account for any variation of this distance between experiments. These adjusted lengths were then used in subsequent analysis, unless specified. The expansion rate was calculated to account for these adjusted lengths and therefore represent the expansion rate across the time the leading edge was within the micro-fabricated furrows. To calculate the percentage reduction of interstitial biofilm expansion the difference in biofilm length on the smooth and furrow side was calculated and divided by the length of the biofilm on the smooth side. This was then expressed as a percentage by multiplying by 100.

Data are presented as mean ± SD. One-way ANOVA with a Tukey's post-hoc test and Student's t-test was used to compare data sets. Analyses were performed and data presented using GraphPad Prism v.6 (Graphpad Software). Statistical significance was determined using a p < 0.05.

Micro-patterned surfaces were designed to exploit the stigmergic furrow-following behavior used by P. aeruginosa to self-organize during active biofilm expansion (Gloag et al., 2013a,b). Our aim was to determine if a series of parallel micro-fabricated furrows would be effective at inhibiting biofilm expansion. A series of transverse furrows were micro-fabricated on the surface of one half of a thin slab of PDMS, with the opposite half left smooth (Figures 1A,B). The dimensions of the micro-fabricated furrows were based on the dimensions of the furrows underlying P. aeruginosa biofilms (Gloag et al., 2013a,b). We have previously identified that furrows beneath the leading rafts and the lattice network were ~20 and 10 μm wide, respectively (Gloag et al., 2013a). Therefore, we generated micro-fabricated furrows with widths of 5, 10, 20, 50, and 100 μm to both mimic the dimensions of biofilm furrows and to identify an optimal range of inhibition. We had also determined that the depths of the biofilm furrows were on average 0.2 μm, with 0.5 μm the greatest depth measured (Gloag et al., 2013b). As we were attempting to maximize the confinement of cells within the micro-fabricated furrows during biofilm expansion we assayed 0.5 μm deep furrows to mimic the biofilm furrows as well as 1 μm depths to encompass the full height of a P. aeruginosa cell, which we have previously found to be an average height of 0.68 μm (Gloag et al., 2013b). Furrow dimensions deeper than 1 μm were not tested as we wanted to avoid cells becoming rapidly motile via swimming motility which was likely to occur in the deeper volume of liquid that would pool in the furrows. Therefore the furrow dimensions assayed were widths of 5, 10, 20, 50 and 100 μm, each at depths of 0.5 and 1 μm. Inter-furrow distances of 10, 15, and 20 μm were examined. The furrow dimensions are referred throughout the text as width × depth × inter-furrow distances.

To test the effects of different furrow dimensions on P. aeruginosa biofilm expansion we modified our interstitial biofilm assay in which the biofilm is cultured at the interstitial space between solidified nutrient media and a glass coverslip (Turnbull and Whitchurch, 2014), by replacing the glass coverslip with a thin slab of micro-patterned PDMS (Figures 1A,B). We initially utilized an inter-furrow distance of 20 μm with the furrow depth and width varying between the micro-patterned PDMS templates. We found that all micro-patterned surfaces tested caused a reduction in the net expansion of P. aeruginosa biofilms relative to the unpatterned control. We observed extensive expansion from the inoculation point on the smooth side and comparatively limited expansion in the presence of the micro-fabricated furrows (Figures 1B,C). Macroscopically, P. aeruginosa interstitial biofilms were observed on the smooth side as an extensive semi-circle radiating outward from the inoculation point (Figures 2A, 3A), whereas on the furrow side the biofilms were thin and narrow, extending across the width of the PDMS template (Figures 2A, 3A). This was reflected in a significant reduction in the net expansion rate of P. aeruginosa biofilms across the micro-fabricated furrows, compared to the smooth side, for all furrow parameters tested (Figures 2B, 3B; p < 0.0001). For the 1 μm deep furrows a decrease in furrow width correlated to a greater reduction in biofilm expansion, that was observed macroscopically as a narrower biofilm in the presence of the furrows (Figure 3A; p < 0.0001). This trend was not apparent for the 0.5 μm deep furrows, where the inhibition of the 5 μm wide furrows was similar to that of the 20 and 50 μm wide furrows (Figure 2B). Furthermore, for the 5 μm wide furrows, the 1 μm depth inhibited biofilm expansion to a significantly greater extent compared to the furrows of 0.5 μm depth (71.0 ± 2.7 vs. 54.0 ± 3.6%, respectively; Figures 2B, 3B; p < 0.0001). For the other furrow widths tested there was a similar reduction at the two depths, with the exception of the 50 μm wide furrows, which showed a greater reduction with the 1 μm depth compared to 0.5 μm (p < 0.0001). Together these observations indicated that the 1 μm deep micro-fabricated furrows were more effective than 0.5 μm depth furrows at inhibiting biofilm expansion, presumably due to this depth encompassing the full height of the cells and confining the cell movements more effectively. As such, all subsequent experiments used 1 μm deep furrows.

In an attempt to further increase the inhibition of biofilm expansion over that observed for the 5 μm wide furrows, inter-furrow distances of 10 and 15 μm were also examined (Figure 3). While the 5 × 1 × 10 μm furrows (width × depth × inter-furrow spacing) showed the greatest reduction to P. aeruginosa biofilm expansion (75.0 ± 4.3%; Figure 3B), which was also observed macroscopically (Figure 3A), statistically the different inter-furrow distances had no effect, with the reduction being relatively similar across the different furrow dimensions (5 × 1 × 10 μm: 75.0 ± 4.3%, 5 × 1 × 15 μm: 73.0 ± 3.4%, and 5 × 1 × 20 μm: 71.0 ± 2.7%, respectively; Figure 3B). The reduction on P. aeruginosa biofilm expansion by the 5 × 1 × 10 μm furrows was significant in comparison to that of the 10 × 1 × 20 μm furrows (Figure 3B; p < 0.05), and overall the reductions where similar for the 5 and 10 μm wide furrows, with these displaying a significant reduction in comparison to furrows with widths of 20, 50, or 100 μm (Figure 3B; p < 0.0001). These observations indicate that a micro-patterned PDMS surface containing a series of parallel furrows is an effective means of reducing the overall rate of P. aeruginosa biofilm expansion.

To observe the interaction of P. aeruginosa cells with the micro-patterned surfaces during biofilm expansion, we used time-lapse phase-contrast microscopy to observe cell movements as they migrated across either smooth PDMS or PDMS micro-patterned with a series of 5 × 1 × 10 μm furrows (Supplementary Video 1). The P. aeruginosa interstitial biofilm on the smooth side migrated rapidly across the field of view (Supplementary Video 1). In contrast, in the presence of the furrows, progression of the P. aeruginosa biofilm was significantly impeded with cells largely confined to the micro-fabricated furrows (Supplementary Video 1).

Our analyses have also revealed that in order to escape the furrow and traverse across the inter-furrow regions, P. aeruginosa cells at the leading edge re-orient and organize into vanguard rafts (Supplementary Video 1). This process is similar to what has been previously observed during twitching motility-mediated biofilm expansion of P. aeruginosa where it has been noted that leading-edge raft assemblages are required for migration into new territories (Semmler et al., 1999; Gloag et al., 2013b).

Our time-series of P. aeruginosa twitching motility across micro-patterned PDMS also showed that as these leading edge rafts entered narrow furrows with widths of 5 or 10 μm, which showed the greatest inhibition of biofilm expansion, the aggregates of cells immediately disassembled and the cells altered their direction of motion to migrate along the length of the furrow (Figure 4A and Supplementary Video 1). These cells also appeared to preferentially migrate along the furrow edge, rather than migrating along the middle of the channel (Figure 4B and Supplementary Video 1). Furthermore, these cells appeared to migrate independently of neighboring cells, in some cases migrating as well isolated single cells (Figure 4C and Supplementary Video 1). Individual cell motility is rarely observed during twitching motility-mediated interstitial biofilm expansion of P. aeruginosa (Semmler et al., 1999). The observation that individual P. aeruginosa cells preferentially migrate along the furrow edge is reminiscent of the behavior previously reported for Neisseria gonorrhoeae diplococci which were observed to migrate via twitching motility along the edges of the channels etched in PDMS surfaces (Meel et al., 2012). It was proposed that the channel walls provided additional surfaces for the type IV pili of the N. gonorrhoeae to bind to facilitate twitching motility (Meel et al., 2012). Similarly, the type IV pili of P. aeruginosa may bind to the PDMS furrow edges in our micro-fabricated system, thereby enabling their individual movements and accounting for the preferential alignment of cells along these borders (Figure 4 and Supplementary Video 1).

In contrast, when observing biofilm expansion in the presence of the wider micro-fabricated furrows, we noted that for furrows with widths 20 μm or greater, the leading raft assemblages were maintained after entering the furrow (Figure 4D). As these wider furrows were less inhibitory to overall biofilm expansion (Figures 2, 3) we predict that the maintenance of these leading raft assemblages within the wider furrows facilitates a more rapid escape from the furrow, accounting for the greater net expansion across the PDMS surfaces micro-patterned with wider furrows (Figure 3B). In contrast, within the narrower furrows of 5 or 10 μm wide in which the leading rafts dispersed upon entering the furrow, the cells need to re-coordinate their collective behaviors in order to successfully re-organize into the aggregates required to escape from the furrow and traverse into new territory. It appears therefore that the narrower (5 and 10 μm) furrows are more efficient at disrupting bacterial collective behaviors causing the aggregates to disperse and guide the individual cells to migrate along the longitudinal direction of the furrow.

We also examined the ability of 1 μm deep micro-fabricated furrows to inhibit biofilm expansion by Pr. vulgaris, another important CAUTI pathogen whose biofilms actively expand along catheter surfaces using flagella-mediated swarming motility (Fluit et al., 2001). We found that, similar to P. aeruginosa, in the presence of the micro-fabricated furrows the interstitial biofilms of Pr. vulgaris were narrow and extended across the width of the template when viewed macroscopically. This was in contrast to the biofilms cultured on the smooth side where they were observed as a semi-circle displaying a concentric circle pattern (Figure 5A). The concentric circle patterning is typical of the swarming motility of Proteus spp. and forms as a result of continuous rounds of cellular differentiation into filamentous hyper-flagellated swarmer cells, which mediate surface colonization via swarming motility, followed by consolidation via differentiation back into the normal rod cell, which are largely non-motile (Harshey, 1994; Rauprich et al., 1996). Due to these continuous rounds of differentiation cycles, there was variation in the measured expansion rates of the Pr. vulgaris biofilms, particularly on the smooth side (Figure 5B) depending on whether the cells had entered a motility or consolidation cycle by the end of the time course. We found that wider furrows were not as effective at controlling the collective behavior of Pr. vulgaris during biofilm expansion (Figure 5B) and as was observed for P. aeruginosa, narrow furrow widths correlated with a greater reduction to biofilm expansion (Figure 5B; p < 0.0001 for 5 × 1 × 10, 5 × 1 × 20, and 10 × 1 × 20 μm; p < 0.01 for 5 × 1 × 15 μm), with the 5 μm wide furrows displaying the greatest reduction (68.6 ± 7.9%; Figure 6).

To observe the interaction of Pr. vulgaris cells within the micro-fabricated furrows, phase-contrast time-lapse microscopy was performed using PDMS patterned with 5 × 1 × 10 μm furrow series (Supplementary Videos 2, 3). On the smooth side it was observed that the biofilm migrated rapidly via swarming motility. However, in the presence of the micro-fabricated furrows the biofilm advancement was impeded. We noted that as the leading front of the Pr. vulgaris swarm entered the furrow, the cells appeared to dissociate from the swarm front and change their direction of motion to migrate along the length of the furrow (Figure 5C and Supplementary Videos 2, 3). It therefore appears that the micro-patterned surface disrupts the cohesive swarm front and subsequently controls and guides the movements of individual cells to migrate along the furrows.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.