Microtopographic Patterns Affect Escherichia coli Biofilm Formation

The microfabrication via photolithography and soft lithography were carried out by following the procedures reported previously with slight modificati...
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Microtopographic Patterns Affect Escherichia coli Biofilm Formation on Poly(dimethylsiloxane) Surfaces Shuyu Hou,||,†,‡ Huan Gu,||,†,‡ Cassandra Smith,† and Dacheng Ren*,†,‡,§,^ †

Department of Biomedical and Chemical Engineering, ‡Syracuse Biomaterials Institute, §Department of Civil and Environmental Engineering, and ^Department of Biology, Syracuse University, Syracuse, New York 13244, United States

bS Supporting Information ABSTRACT: Biofilms are involved in 80% of human bacterial infections and are up to 1000 times more tolerant to antibiotics than their planktonic counterparts. To better understand the mechanism of bacteria-surface interactions, polydimethylsiloxane (PDMS) surfaces with microtopographic patterns were tested to study the effects of surface topography on bacterial adhesion and biofilm formation. The patterned PDMS surfaces were prepared by transferring complementary surface topography from a silicon wafer etched via photolithography to introduce 10 μm tall square-shape features. The dimension of protruding square features and the distance between adjacent features were systematically varied. Escherichia coli RP437/pRSH103 (with constitutive expression of red fluorescent protein) was found to preferentially attach and form biofilms in valleys between protruding features even when the dimension of plateaus (top of the square features) is considerably larger than valleys. In addition, significant adhesion of E. coli on plateaus was only observed when the plateaus were bigger than 20 μm  20 μm for face-up patterns and 40 μm  40 μm for face-down patterns. This finding suggests that a threshold dimension may be essential for biofilm formation on flat surfaces without physical confinement.

’ INTRODUCTION Biofilms are sessile microbial communities that cause persistent biofouling and biocorrosion in industrial settings and chronic infections in humans.1,2 Biofilm formation is a dynamic process involving significant changes in gene and protein expression. These changes are highly sensitive to environmental conditions such as surface chemistry and surface topography (roughness and configuration).3,4 Surface roughness (Ra) is used for two-dimensional characterization of a surface, which is normally described as the arithmetic mean distance between the peak and valley.5 Roughness has been well documented to influence bacterial adhesion.6 For example, rough surfaces were found to promote dental plaque formation and maturation.7 Taylor et al.8 also reported that increase in surface roughness between 0.04 μm < Ra < 1.24 μm significantly enhances the adhesion of Pseudomonas aeruginosa and Staphylococcus epidermidis on polymethyl methacrylate (PMMA); while surfaces with roughness between 1.86 μm < Ra < 7.89 μm are more resistant to bacterial adhesion than flat surfaces. In comparison, An et al.9 reported that the roughness (0.44 μm < Ra < 1.25 μm) of titanium surface has no effect on the adhesion of S. epidermidis. Although these studies provide valuable information about surface topography and cell adhesion, these experiments involved different materials and Ra only describes the average roughness across the entire surface in two dimensions. To better describe the morphological characters of a substrate in three dimensions, a well-defined surface configuration is r 2011 American Chemical Society

needed.6 It is well-documented that surface topography significantly influences mammalian cell behaviors including cell adhesion, proliferation and differentiation.10-12 For example, Jin et al.10 reported that nanogrooved surface enhances the adhesion and gene expression of madine darby canine kidney cells. More recently, Schulte et al.11 found that poly(ethylene glycol) (PEG) surfaces with μm scale topographic patterns (lines or posts) significantly enhance fibroblast cell adhesion, while the flat PEG surface is intrinsically nonadhesive. In addition, Schumacher et al.13 evaluated several microtographic patterns (e.g., ridges, pillars, triangles/pillars, and ribs) with 2 μm spacing, which were found to significantly reduce the settlement of zoospores of the green alga Ulva compared to smooth PDMS surfaces. Compared to adhesion of eukaryotic cells, bacterial adhesion and biofilm formation on microtopographic patterns have not been well investigated. It was found that increase in irregularity of a surface promotes bacterial adhesion and biofilm formation.14 In this study, we used systematically varied patterns of PDMS to study the effects of surface microtopography on bacterial adhesion and biofilm formation. The dimension of protruding features and the distance between adjacent features were systematically varied to investigate microbe-surface interactions. Different from previous studies focusing on patterns with sizes comparable to bacterial cells, the topographic patterns used in this study are larger than bacterial cells in every dimension. Such patterns allow us to study the effects of Received: May 5, 2010 Published: February 14, 2011 2686

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surface topography on the development of multicellular structures. PDMS is used in this study because it is well-known for good biocompatibility and stability in pharmaceutical and medical applications.15,16 Thus, understanding biofilm formation on PDMS will be helpful not only for understanding microbe-surface interactions in general but also for development of more effective methods for biofilm control through surface engineering.

’ MATERIALS AND METHODS Bacterial Strains and Growth Medium. Escherichia coli RP437 [thr-1(Am) leuB6 his-4 metF159(Am) eda-50 rpsL1356 thi-1 ara-14 mtl1 xyl-5 tonA31 tsx-78 lacY1 F-],17 one of the best model E. coli strains for biofilm study18-22 and E. coli RP437 ΔmotB/pRSH103, an isogenic nonmotile mutant were used in this work. To visualize biofilms using fluorescence microscopy, E. coli RP437 and E. coli RP437 ΔmotB was labeled with constitutively expressed red fluorescent protein by transformation of the vector pRSH103 constructed previously.19,20 E. coli RP437/pRSH103 and E. coli RP437 ΔmotB/pRSH103 were routinely grown at 37 C in Luria-Bertani (LB) medium23 containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl supplemented with 10 μg/mL tetracycline (to maintain the plasmid). Preparation of PDMS Surfaces with Microtopographic Patterns. The microfabrication via photolithography and soft lithography were carried out by following the procedures reported previously with slight modifications.24-27 Briefly, to prepare PDMS surfaces with microtopographic patterns, a silicon master with complementary patterns (10 μm deep square holes) was fabricated via photolithography using the equipment at the Cornell NanoScale Science & Technology Facility (Cornell University, Ithaca, NY). The configuration of the patterns was designed using the software L-Edit (Tanner Research, Monrovia, CA) to create systematically varied side length of protruding square features (2, 5, 10, 15, 20, 30, 40, 50, or 100 μm) and distance between adjacent features (5, 10, 15, or 20 μm). The depth of all square holes was set to be 10 μm. The schematic illustration of the fabrication process is shown in Figure S1A, Supporting Information. Briefly, a photomask was made using a photomask writer to transfer the pattern design, which was then transferred to a silicon wafer with photoresistant coating via precisely controlled UV exposure through the photomask, followed by an etching process to obtain approximately 10 μm deep features (measured with Tencor P10 Profilometer). Finally, the master was coated with (tridecafluoro-1,1,2,2,-tetrahydrooctyl)trichlorosilane (FOTS) to prevent the disks from sticking to each other. Soft lithography was performed to fabricate the PDMS surfaces with microtopographic patterns. The process is described in Figure S1B. Briefly, elastomer base and curing agent (mass ratio of 10:1) from SYLGARD184 Silicone Elastomer Kit (Dow Corning Corporation, Midland, MI) were thoroughly mixed and degassed for 30 min. The mixture was then carefully poured on a silicon master with patterns described above and covered with a glass cover slide to obtain a flat surface. The sandwich was cured at 60 C for 24 h to polymerize and incubated at room temperature for 24 h to obtain full physical properties. Then the PDMS surface (Figure 1) with the desired patterns was carefully peeled off and stored at room temperature until use. To verify the dimension of patterns, the PDMS surfaces were analyzed using AFM Veeco Icon at Cornell NanoScale Science & Technology Facility. Biofilm Formation and Image Analysis. To investigate E. coli biofilm formation on microtopographic patterns, the patterned PDMS surfaces were sterilized by soaking in 200 proof ethanol for 20 min and dried under vacuum. Each surface was then transferred to a Petri dish (face up) containing 20 mL LB medium supplemented with 10 μg/mL tetracycline, which was then inoculated with an overnight culture of E. coli RP437/pRSH103 to an optical density at 600 nm (OD600) of 0.05 and incubated at 37 C without shaking for 24 h. To study bacterial

Figure 1. Schematic representation of microtopographic patterns on PDMS surfaces used in this study. Key: L, side length of the square patterns (2, 5, 10, 15, 20, 30, 40, 50, or 100 μm); D, distance between adjacent protruding features (5, 10, 15, or 20 μm); H, height of the microtopographic features (10 μm). adhesion and biofilm formation on face-down pattern surfaces, the sterilized PDMS patterns were put upside down with each end supported by a piece of microscope glass slide in a Petri dish containing 20 mL LB medium supplemented with 10 μg/mL tetracycline. The Petri dishes were then inoculated with an overnight culture of E. coli RP437/ pRSH103 for biofilm development as described above. In addition to the wild-type strain, the motility mutant E. coli RP437 ΔmotB/pRSH103 was also tested for its biofilm formation on 100 μm  100 μm microtopographic patterns. To test the effects of shaking, the adhesion and biofilm formation of the wild-type strain were studied on 10 μm  10 μm, 40 μm  40 μm, and 100 μm  100 μm face-up microtographic patterns by following the procedure described above except that the cultures were incubated with shaking at 50 rpm. After incubation, the surfaces were gently washed 3 times by dipping in 0.85% NaCl buffer (change to clean buffer for each dipping step) and visualized using an Axio Imager M1 fluorescence microscope (Carl Zeiss Inc., Berlin, Germany). The surface coverage of biofilms was calculated using COMSTAT.28 At least five spots were randomly picked and imaged from each surface. Each pattern was tested with at least duplicate surfaces and consistent results were obtained.

’ RESULTS AND DISCUSSION To get insight into the effects of surface topography on bacterial adhesion and biofilm formation, the PDMS surfaces with systematically varied patterns were prepared. The dimension of PDMS patterns was verified using AFM Veeco Icon (Figure S2, Supporting Information). The surfaces were then challenged with E. coli RP437/pRSH103 for 24 h and the biofilms were imaged using fluorescence microscopy to visualize E. coli cells constitutively expressing red fluorescent protein (DsRed-Express). First, the surfaces were incubated face up. The representative fluorescence images are shown in Figure 2. The height (H) of protruding square features was set to be 10 μm for all the samples and the side length of square patterns (L) was varied as 2, 5, 10, 15, 20, 30, 40, 50, or 100 μm. The distance (D) between adjacent patterns was 5, 10, 15, or 20 μm. As shown in Figure 2, the majority of the biofilm cells attached in the valleys (the base between protruding features) regardless of the side length of square features and distance between adjacent features. Take 20 μm  20 μm patterns with 5 μm spacing as an example; the surface coverage of biofilms formed in the valleys was 26.9 ( 13.3%; while that formed on the plateaus of protruding patterns was only 6.2 ( 0.8% (t test, P < 0.01). Consistently, mammalian cells were found to adhere and align along the grooves (1 μm deep and 0.5-10 μm wide).29 Callow et al.30 also reported that zoospores selectively settled on the grooved floor of the surfaces with protruding ridges and pillars. Using various patterns of PDMS, it was demonstrated that a repulsive force is generated when microorganisms approach the top of topographic patterns if their sizes are comparable.31 Different from previous studies 2687

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Figure 2. Representative fluorescence images of E. coli RP437/pRSH103 biofilms on PDMS surfaces with systematically varied microtopography (D = 10 μm) (A1-A6) and on smooth PDMS surface (B). Bar =10 μm.

Figure 3. Three-dimensional view of biofilm formation on a PDMS surface with 100 μm (L)  100 μm (L)  10 μm (H) patterns and 20 μm spacing (D).

Figure 4. The surface coverage (mean ( one standard error) of E. coli RP437/pRSH103 biofilms formed on top of face-up protruding patterns. The side length of square features (L) tested was 5, 10, 15, 20, 30, 40, 50, or 100 μm. The surface coverage was calculated using COMSTAT software.28

focusing on patterns with sizes comparable to or smaller than cells, we tested larger distance (D = 5, 10, 15, and 20 μm) between adjacent patterns compared to the average size of bacterial cells (1-5 μm) and various sizes of patterns (L = 2, 5, 10, 15, 20, 30, 40, 50, and 100 μm) in order to systematically study bacterial cell-cell interaction and formation of multicellular structures. To compare with biofilm formation on patterned surfaces, E. coli RP437/pRSH103 was also grown on smooth PDMS surfaces (without any microtopographic patterns). As shown in Figure 2, the biofilm surface coverage in the valleys is significantly larger than that on smooth surfaces of the same material (PDMS). For example, the surface coverage of biofilms on smooth PDMS surfaces was 11.6 ( 1.0%, while the surface coverage of biofilms grown in the valleys of PDMS with 20 μm  20 μm patterns and 5 μm spacing was 26.9 ( 13.3% (t-test, P < 0.05). The 3-D view of representative E. coli biofilm formed on PDMS surface with 100 μm  100 μm protruding square features and 20 μm spacing is shown in Figure 3. These data suggest that the microtopographic patterns facilitate the cells that settled in the valleys to interact closely and form denser biofilms. More interestingly, there appears to be a threshold for the dimension of protruding features to allow significant bacterial adhesion on top of the features. As shown in Figure 2, E. coli formed significantly more biofilms per cm2 when the protruding square features were 20 μm  20 μm or larger, which indicates that there might be a critical dimension of surface area for establishing a stable biofilm on flat surface without three-dimensional confinement (more images are shown in Figure S3, Supporting Information). The surface coverage of E. coli biofilms formed on the plateaus was calculated using COMSTAT software and shown in Figure 4. Two-way ANOVA adjusted by the Tukey-Kramer test was used to analyze the data. No statistically significant changes in average surface coverage was observed when side length (L) increased from 5 to 15 μm (p > 0.05). However, it increased dramatically when L increased to 20 μm

(p < 0.0001). Further increase in L did not lead to significant change in surface coverage, which is close to that on smooth PDMS surfaces (Figure 4, p > 0.05). This trend was found for any given D tested, except that the coverage at L = 20 μm was smaller than that at L = 100 μm and smooth PDMS for D = 5 μm patterns (p < 0.05). Thus, the threshold surface area for E. coli biofilm formation on face-up surfaces appears to be 20 μm  20 μm under the condition of this study. In addition to the effects of L, the average surface coverage also appeared to increase slightly with the interpattern distance (D) on the patterns with L = 20, 30, 40, or 50 μm; e.g., the average surface coverage at D = 15 or 20 μm is significantly higher than that for D = 5 μm (p < 0.05) (Figure 4). The dominating role of pattern size on biofilm formation and the existence of a threshold (20 μm  20 μm) deserve attention. Although elongated cells are found in bacterial biofilms,18 e.g., those longer than 10 μm (probably swarming cells) in Figure 2B, the majority of cells in our study were 5.3 ( 1.3 μm. Thus, the critical surface area for biofilm formation, 20 μm  20 μm, is significantly larger than the size of bacterial cells in this study. To understand if the higher cell density in valleys was because the cells on plateaus were pulled down by gravity or because bacteria cells can actively choose the positions to attach, we also tested the patterned surfaces for E. coli adhesion and biofilm formation by incubating the patterns upside down (face-down patterns). As expected, the surface coverage of E. coli RP437/pRSH103 cells on the face-down PDMS surfaces was significantly less than that on faceup surfaces. For example, the surface coverage on flat PDMS was 11.6 ( 1.0% and 2.6 ( 0.3% on face-up and face-down surfaces, respectively. Consistent with the results of face-up patterns, it was found that E. coli RP437/pRSH103 cells still preferred to attach to the valleys than the top of protruding patterns and a threshold dimension still existed for significant adhesion on the top of protruding patterns (see Figure 5 and Figure S4, Supporting Information, for representative images). However, the threshold appeared to be 40 μm  40 μm (Figure 6) for cell adhesion and biofilm formation 2688

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Figure 5. Representative fluorescence images of E. coliRP437/pRSH103 biofilms on face-down PDMS surfaces with systematically varied microtopography (D = 10 μm) (A1-A6) and on face-down smooth PDMS surface (B). Bar =10 μm.

Figure 6. Surface coverage (mean ( one standard error) of E. coliRP437/pRSH103 biofilms formed on top of inverted protruding patterns. The side length of square features (L) tested was 5, 10, 15, 20, 30, 40, 50, or 100 μm. The surface coverage was calculated using COMSTAT software.28

on the patterns that were incubated upside down, bigger than that of face-up patterns (20 μm  20 μm). This finding indicates that gravity does facilitate cell adhesion but the threshold dimensions are intrinsic factors for biofilm formation, rather than simply the result of gravity. The adhesion and biofilm formation were also found to be sensitive to medium flow. For example, in the presence of 50 rpm shaking, E. coli RP437/pRSH103 was found to barely attach to the PDMS surfaces, either in the valleys or on top of the protruding patterns (data not shown). To further corroborate the results that the adhesion on the face-up patterns is not simply due to gravity-driven settlement, we also tested the adhesion of an isogenic nonmotile mutant, E. coli RP437 ΔmotB/pRSH103, on both face-up and face-down PDMS surfaces with 100 μm  100 μm microtopographic patterns. It was found that motility is important for adhesion; e.g., the average surface coverage on all face-up 100 μm  100 μm patterns tested was 3.2 ( 0.5% by the mutant (Figure 7) and 10.2 ( 1.7% by the wild-type strain (Figure 4). The defects of this mutant in adhesion was also observed for the patterns incubated upside down since the mutant cells could barely attach to either the valleys or the top of protruding features. These data confirm that motility is critical for bacterial adhesion,32 and the cells on surfaces shown in Figure 2 are indeed attached cells rather than due to gravity-driven settlement. The existence of threshold dimensions shown in Figure 4 and Figure 6 suggests that cell-cell interaction is essential for biofilm formation, which is consistent with enhanced biofilm formation in valleys. Recently, we reported that bacterial adhesion and biofilm formation on flat surfaces can be confined in specific patterns by tailoring surface chemistry with self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups18,33 and E. coli biofilm formation responds linearly to linear gradients of surface bioinertness.34 We also noticed

Figure 7. Surface coverage (mean ( one standard error) of E. coli ΔmotB/pRSH103 biofilms formed on top of inverted protruding patterns. The side length of square features (L) tested was 100 μm and the distance between features was 5, 10, 15, or 20 μm. The flat PDMS surface was also included for comparison. The surface coverage was calculated using COMSTAT software.28

that E. coli biofilms have more interactions between cell clusters when the patterns are larger than 20 μm  20 μm (Hou et al., unpublished). Further study is necessary to understand the mechanism of such thresholds. The increase in surface coverage with interpattern distance (D) for face-up patterns is also interesting. Collectively, the findings from this study suggest that bacterial cells are able to differentiate plateaus and valleys and actively choose adhesion sites on a surface with topography. Such selective adhesion can lead to uneven distribution of bacterial cells on a surface, which may contribute to the heterogeneity of biofilm structure and associated variation in gene expression.3,4 Thus, the patterned surfaces are useful tools to obtain biofilms with different cell densities and of different dimensions. These surfaces can be applied to study the functions of key genes and cell-cell interaction in biofilm formation. We are currently investigating the expression of genes for quorum sensing, chemotaxis and motility in bacteria-surface interactions using these microtopographically patterned surfaces. It is also interesting to study the structure and content of biofilm matrix on different patterns. Better understanding of the effects of topography on biofilm formation and bacterial gene expression may help develop more effective antifouling surfaces.

’ CONCLUSIONS PDMS surfaces with various microtopographic patterns were successfully prepared using photolithography and soft lithography. By applying these well-defined surfaces, it was demonstrated that E. coli preferentially adhered and formed biofilms in the 2689

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Langmuir valleys between protruding square features regardless of the dimension of the features (2-100 μm tested) and spacing between adjacent features (5-20 μm tested), suggesting that microtopographic confinement promotes bacterial adhesion and biofilm formation. In addition, significant biofilm formation on protruding square features was only observed when the plateaus were 20 μm  20 μm or larger for the face-up patterns and 40 μm  40 μm or larger for the face-down patterns. These threshold dimensions are significantly larger than E. coli cells, indicating that cell-cell interaction is essential for the formation of multicellular structure of biofilms. Compared to the wild-type strain, the motility mutant can barely attach and form biofilm on face-down PDMS surfaces and the surface coverage was significantly lower than that of the wild-type E. coli on face-up patterns, which indicates that motility is important to cell-cell interaction and bacterial adhesion on PDMS.

’ ASSOCIATED CONTENT

bS

Supporting Information. Figures showing the scheme of microfabrication of polydimethylsiloxane (PDMS) surfaces with microtopographic patterns and representative images of PDMS patterns and biofilms. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: (315) 443-4409. Fax: (315) 443-9175. E-mail: [email protected]. )

Author Contributions

These authors contributed equally.

’ ACKNOWLEDGMENT The authors thank the U.S. National Science Foundation (NSF-CMMI, Grant No. 0826288) for financial support. We are grateful to Dr. John S. Parkinson at the University of Utah for providing the strain E. coli RP437 and its ΔmotB mutant, Dr. Arne Heydorn at the Technical University of Denmark for providing the COMSTAT software and Dr. Yan-Yeung Luk at Syracuse University for suggestions on preparation of PDMS surfaces. We thank the Cornell NanoScale Science & Technology Facility for the access to photolithography facilities. Technical assistance from Ms. Jiachuan Pan (Syracuse University) and Jiejing Qiu (Welch Allyn, Inc) is also appreciated. ’ REFERENCES (1) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2004, 2, 95–108. (2) Fux, C. A.; Stoodley, P.; Hall-Stoodley, L.; Costerton, J. W. Bacterial biofilms: a diagnostic and therapeutic challenge. Expert. Rev. Anti. Infect. Ther. 2003, 1, 667–683. (3) Bollen, C. M.; Lambrechts, P.; Quirynen, M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. Dent. Mater. 1997, 13, 258–269. (4) Wim, T.; nele, W. A.; Isabelle, S.; Marc, Q. Effect of material characteristics and/or surface topography on biofilm development. Clin. Oral Implants Res. 2006, 17, 68–81. (5) Gadelmawla, E. S.; Koura, M. M.; Maksoud, T. M. A.; Elewa, I. M.; Soliman, H. H. Roughness parameters. J. Mater. Proc. Technol. 2002, 123, 133–145.

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’ NOTE ADDED AFTER ASAP PUBLICATION This article was published ASAP on February 14, 2011. The second paragraph of the Introduction section has been modified. The correct version was published on February 18, 2011.

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