Article pubs.acs.org/journal/abseba
Colloidal Crystals Delay Formation of Early Stage Bacterial Biofilms Mehdi Kargar,† Yow-Ren Chang,§ Hamoun Khalili Hoseinabad,† Amy Pruden,‡ and William A. Ducker*,§ †
Department of Mechanical Engineering, ‡Via Department of Civil and Environmental Engineering, and §Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia United States S Supporting Information *
ABSTRACT: The objective of this work was to examine whether close-packed spheres of polystyrene (colloidal crystals) could be used to delay the development of biofilms. We examined early stage biofilm formation of Pseudomonas aeruginosa after 2 days on a flat sheet of polystyrene and on the same solid coated in polystyrene spheres of 450 or 1500 nm diameter. All materials were coated in fetal bovine serum to enable comparison of the effects of different surface curvature while maintaining constant surface chemistry. After 2 days, fluorescence imaging showed that the volume of bacterial colonies was much smaller on the 1500 nm colloidal crystals than on the flat film. In addition, electron microscopy showed that the area covered by structures containing more than one layer of bacteria was significantly reduced on both the 450 and 1500 nm colloidal crystals compared to the flat sheet. This provides proof of concept of biofilm inhibition of a pathogen by a simple nonchemical coating that may find future application in reducing the incidence of infections. Even though the density of adhered bacteria on 450 and 1500 nm was similar after 1 day, biofilm formation after 2 days was delayed more on the 1500 nm spheres than on the 450 nm spheres. We also observed that bacteria have preferred adsorption sites on the 1500 nm colloidal crystals and that cell bodies were often separated. This leads us to hypothesize that the greater spacing between favorable sites on the 1500 nm colloidal crystal hindered the early stage biofilm formation by separation of cell bodies. KEYWORDS: biofilm, colloidal crystal, spheres, adsorption, Pseudomonas aeruginosa
1. INTRODUCTION A biofilm is collection of cells, usually in a polymeric matrix, that microorganisms tend to form on surfaces.1 From a medical perspective, a biofilm is troubling because it may not respond to the host immune system or to treatments by antibiotics.2 Compared to planktonic (suspended) cells, the cells in a biofilm can have altered gene expression and metabolism and may be specialized within the biofilm.1 Biofilm formation typically involves a series of stages that begins with adsorption of macromolecules to an interface, which subsequently facilitates reversible adsorption of planktonic cells followed by longer term adsorption under favorable conditions. Subsequently, the cells typically form small clusters, which result from cell division, further recruitment of planktonic cells, or surface migration. The small clusters can grow into colonies and finally evolve into 3D biofilm structures (see Figure S1). The objective of the current work was to examine whether the addition of a layer of close-packed spherical particles (a colloidal crystal) to a flat solid could delay the early stages of biofilm formation. Prior work has shown that topographical modification of surfaces can affect subsequent interactions with bacteria3−17 and other microorganisms.18−23 For example, Chung et al.4 showed that patterns of approximately rectangular ridges (Sharklet surfaces) in polydimethylsiloxane (PDMS) delayed formation of Staphylococcus aureus biofilms. The proposed mechanism was that “the protruded features of the topographical surface provided a physical obstacle to deter the © XXXX American Chemical Society
expansion of small clusters of bacteria present in the recesses into micro-colonies.”4 The Sharklet surface was also shown to reduce the number of adsorbed Mycobacterium abscessus.24 Likewise, Xu and Siedlecki14 observed that biofilm formation by S. aureus or Staphylococcus epidermidis was delayed on pillars of poly(urethane urea) compared to flat surfaces They attributed their observation to the potential effects of the hydrodynamic shear forces present in their experiments. Xu and Siedlecki14 hypothesized that if texturizing the surfaces reduces the available contact area between the cells and the solid surface, then texturizing eases cell removal and delays biofilm formation. Ivanova et al.25 explored a different use of topography: to penetrate and kill bacteria. They showed that the sharp spikes on cicada wings could kill Pseudomonas aeruginosa cells. An important recent development showed that oil-infused PDMS reduced bacterial adhesion and biofilm formation.26 The oil forms a slippery layer on the surface that is thought to inhibit adhesion. To advance antimicrobial material design, it is vital to understand the mechanisms by which surfaces may repel bacteria or prevent biofilm formation. Biofilm formation of P. aeruginosa on flat surfaces has been widely studied;27−29 however, prior work considering the interaction of P. aeruginosa Received: March 23, 2016 Accepted: May 8, 2016
A
DOI: 10.1021/acsbiomaterials.6b00163 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Biofilms are the most important bacterial structure for medical infection control,1 and it is known that the initial adsorption of bacteria is not necessarily correlated with the eventual extent of biofilm formation.33 So, in this study, we extended our previous work to the determine the effect of colloidal crystals on the early stages of biofilm formation. Our broad goals were to identify a way to minimize biofilm formation and to understand the mechanisms by which this might be achieved. Here, as in our prior work, the effect of topography was isolated from surface chemistry first by using a set of PS samples that all have a very similar composition, as confirmed by X-ray photoelectron spectroscopy (XPS), and second, by coating all surfaces with fetal bovine serum (FBS), which renders the surface hydrophilic.11 Thus, the bacteria interacted with the same model coating, regardless of topography. The findings provide new insight into how materials can effectively be designed to repel early stage bacterial biofilm formation, thus providing possible alternatives to chemical antimicrobials and antibiotics.
with topographically engineered surfaces has focused on adhesion and early stage colony formation, where there are only small clusters of cells in two dimensions,7,8,10−12 rather than biofilm formation. In this work, we consider the hypothesis that a coating of close-packed spheres with dimensions similar to the bacterium “body” (i.e., bacterial cell excluding appendages such as flagella or pili) will delay biofilm formation. In previous work,11 we investigated the effect of colloidal crystals on adhesion of P. aeruginosa. P. aeruginosa is a Gramnegative, motile, rod-shaped bacterium that can act as an opportunistic pathogen.31 Surfaces that inhibit biofilm formation are of great interest for P. aeruginosa infection control, especially given how problematic such biofilms have become in hospitals and that they have a tendency to be multiantibiotic resistant.32 In particular, we investigated hexagonally packed spheres because spheres have the same nonzero curvature in all directions at every point so each particle presents a single convex curvature to the bacteria. The hypothesis was that the bacteria would have more difficulty attaching to the curved surface, which was based on prior work by Kargar et al.12 which was in turn based on theories of the adhesion of vesicles to solids.30 In that prior work,11 we examined bacterial adhesion on a flat sheet of polystyrene (PS) as well as on a range of polystyrene spheres (diameter 220−1500 nm) that spanned the diameter of P. aeruginosa. We showed that coating a flat plate with colloidal crystals (a) reduces the number of P. aeruginosa adhering per unit area of the underlying plate, (b) reduces the number of bacterial clusters per unit area after 1 day, and (c) causes selective adsorption to the 2-fold sites (see Figure 1) in
2. MATERIALS AND METHODS 2.1. Chemicals. Chemicals were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise stated. The following chemicals were received as a powder and mixed with deionized (DI) water (18.2 MΩ cm at 25 °C): tryptic soy broth (TSB, 30 g/L), tryptic soy agar (TSA, 40 g/L), phosphate buffered saline (PBS, 9.88 g/L) powder. USP grade 200 proof ethanol (Decon Laboratories, King of Prussia, PA), 10% EM grade glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA), sodium dodecyl sulfate (SDS) (Sigma-Aldrich, St. Louis, MO), and Hyclone FBS (Sigma-Aldrich) were used as received. Colloidal crystals were fabricated from polystyrene (PS) spheres (Bangs Laboratory, Fishers, IN, or Polysciences Inc., Warrington, PA) and the PS flat sheets were purchased from Goodfellow (Coraopolis, PA). PS particles had nominal diameters of 500 and 1500 nm but electron microscopy showed that particles from Bangs Laboratory had diameters of 450 and 1550 nm and that particles from Polysciences had diameters of 470 and 1420 nm. For simplicity, we refer to the smaller particles as 450 nm and the larger particles as 1500 nm. The particles are supplied as aqueous suspensions. To remove the suspending liquid, and as a first rinse of the particles, particle suspensions were aliquoted into 1 mL centrifuge tubes and spun down at 6,500 g for 10 min. The supernatant was decanted and the particles were resuspended with 1 mL of 1:1 v/v deionized water and ethanol mixture. Centrifugation and decantation was repeated 5 times. Fluorescent microscopy used the L7007 Live/Dead (a mixture of propidium iodide and SYTO9 nucleic acid) assay kit (Invitrogen, Grand Island, NY) or only propidium iodide (Life Technologies Corporation, Carlsbad, CA). 2.2. Fabrication and Characterization of Colloidal Crystals. Colloidal crystals were fabricated using a modification of the methods of Lu and Zhou,34 as described previously.11 Briefly, the spheres were assembled into the colloidal crystals at the solution−air interface. Then the colloidal crystals were deposited onto a PS sheet by drawing the sheet out of the solution and into the air. To increase the binding strength, both between particles and between the particles and the PS sheet, the resulting structure was heated close to the glass transition temperature of PS (95 °C for 110 min for particles from Bangs Laboratories or 95 °C for 5 h for particles from Polysciences Inc.). The quality of the colloidal crystal film was confirmed using scanning electron microscopy (SEM) and laser diffraction patterns. A combination of atomic force microscopy and SEM was used to confirm that the flat PS sheet was smooth, that is, there were no defects within the size range of the spheres. The measured root mean squared (rms) roughness was 1.5 nm over an area of 625 μm2. The final PS structure, whether a flat sheet or a flat sheet coated in annealed particles, is referred to as a “substrate”. Our previous work showed that all substrate types were chemically very similar.11 The
Figure 1. (A) Schematic of the colloidal crystal geometry showing the nomenclature used here for surface sites on the crystal: the crown (top of sphere) and the various confined spaces (2-fold site, 3-fold site, and groove). (B) Schematic of bacteria clarifying the terms “side” and “pole” of bacteria as used in this paper. (C, D) Schematic cross section showing the approximate size of the bacterial diameter compared to the particle radius. The diameter of the bacteria in our two day experiments is 390 nm with a standard error of 10 nm.
the crystal. This decrease in number occurred even though the particle coating increased the total surface area compared to the underlying plate. However, we found that that more curved particles were not better than less curved particles at reducing the number of bacteria, so the original hypothesis was not sufficient to explain the results. The reason may be that when the particles are much smaller than the bacterium, the bacterium attaches to multiple particles. The formation of the colloidal crystal produces also some spaces where a bacterium can sit between two or more spheres, for example, the 2-fold, 3fold, and groove sites, as shown in Figure 1a. B
DOI: 10.1021/acsbiomaterials.6b00163 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering term “sample” refers to a 1.5 × 0.5 mm2 rectangular pieces of substrate used in the biological assays. 2.3. Flow-through Biofilm Formation Assay. The samples were cleaned and sterilized as described previously.11 Each sample was washed in deionized (DI) water for 1 h, rinsed in 100% ethanol, sterilized in 70% ethanol, washed in sterile DI water, then coated in FBS by immersion overnight at 37 °C, and finally rinsed in sterile water. Thus, all samples, regardless of topography, had a layer of FBS overlying chemically similar PS (as determined by XPS.) The adsorption of FBS changed the water contact angle from 90° to
