Bactericidal Surfaces
Natural Bactericidal Surfaces: Mechanical Rupture of Pseudomonas aeruginosa Cells by Cicada Wings Elena P. Ivanova,* Jafar Hasan, Hayden K. Webb, Vi Khanh Truong, Gregory S. Watson, Jolanta A. Watson, Vladimir A. Baulin, Sergey Pogodin, James Y. Wang, Mark J. Tobin, Christian Löbbe, and Russell J. Crawford Numerous insects have evolved to possess superhydrophobic surfaces, which are thought to limit bacterial contamination through a self-cleaning action. While investigating the adhesion of Pseudomonas aeruginosa on the wings of a species of cicada (Psaltoda claripennis), we discovered that the wing surfaces were actually deadly to the cells. Electron microscopy showed nanopillars on the surface penetrating the cells, which was confirmed to be lethal through viability experiments. The effect was very fast, with individual bacterial cells killed within approximately 3 min. The bactericidal effect is primarily based on the physical surface structure; significant alteration of chemistry through gold coating of the wing failed to inhibit cell death. The cicada wings were able to maintain a clean surface by continuous cleansing through bactericidal action, rather than repelling bacterial cells. Surfaces that inhibit bacterial
Prof. E. P. Ivanova, J. Hasan, H. K. Webb, V. K. Truong, Prof. R. J. Crawford Faculty of Life and Social Sciences Swinburne University of Technology P.O. Box 218, Hawthorn, 3122, Australia E-mail:
[email protected] Dr. G. S. Watson, Dr. J. A. Watson School of Pharmacy and Molecular Sciences and Centre for Biodiscovery and Molecular Development of Therapeutics James Cook University Townsville, 4811, Australia Dr. V. A. Baulin ICREA, 23 Passeig Lluis Companys Barcelona, 08010, Spain Dr. V. A. Baulin, S. Pogodin Departament d’Enginyeria Quimica Universitat Rovira i Virgili 26 Avenida dels Paisos Catalans, Tarragona, 43007, Spain Dr. J. Y. Wang Faculty of Engineering and Industrial Sciences Swinburne University of Technology P.O. Box 218, Hawthorn, 3122, Australia Dr. M. J. Tobin Australian Synchrotron 800 Blackburn Road, Clayton, 3168, Australia C. Löbbe SciTech, 72-74 Chifley Drive, Preston, 3072, Australia DOI: 10.1002/smll.201200528 small 2012, 8, No. 16, 2489–2494
contamination via their physical structure represent a novel area of research for the development of antibacterial surfaces. Due to the nature of their environments, many insects have a requirement to minimize their contamination by foreign particles in order to retain functionality.[1,2] These foreign particles may be dust or dirt, or bacterial cells that may seek to colonize and infect the insect. As such, these insects have evolved various strategies or mechanisms for coping with contamination.[3,4] Several have evolved to possess superhydrophobic surfaces, particularly on their wings, which not only enable them to remain dry and minimize weight, but also bestow a self-cleaning effect.[5–8] Adhesion of water droplets that contact the surface is so low that they easily slide and roll across the surface, sweeping off contaminating particles such as dust.[9] It is thought that the same mechanism may also be responsible for the ability of these insects to limit bacterial contamination, i.e., that there may be a direct relationship between self-cleaning and antibiofouling.[10,11] Antibiofouling, commonly referred to simply as antifouling, is the property possessed by some materials which prevent the adhesion and subsequent build-up of biological matter on their surfaces.[12–16] Much work has been conducted on the development of artificial antifouling surfaces for applications in medicine and industry.[15,17–19] Naturally occurring surfaces are a rich source of inspiration for this purpose,[20–22] for example lotus leaves (Nelumbo nucifera),[23] shark skin (e.g., the Mako shark, Isurus oxyrinchus),[24] and the feet of geckos (fam. Gekkonidae),[25] along with the superhydrophobic surfaces of insect wings such as cicadae,[20,26,27] dragonflies[28] and butterflies.[29,30] Superhydrophobicity, and subsequently a self-cleaning ability, arises primarily based on the surface structure; superhydrophobic surfaces contain hierarchical topographical features.[10,31–33] In this research, we have investigated the antibiofouling ability of the surfaces of cicada (Psaltoda claripennis) wings. Cicada wings are generally highly hydrophobic and possess excellent self-cleaning properties.[27,34] While it is thought that there may be a direct relationship between self-cleaning and antibiofouling, it has not yet been properly explored. To address this, the initial adhesion behavior of Pseudomonas aeruginosa ATCC 9027 cells on cicada wing surfaces was assessed and characterized, in order to determine the propensity of the wings for resisting bacterial contamination.
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Our investigations into the interaction of Pseudomonas aeruginosa cells with the cicada wings revealed that the wing surface was actually not particularly effective at repelling the bacteria, as many cells were able to adhere to the wing surface. However the cells’ morphology was significantly altered when in contact with the cicada wing surface; the wing nanopillars appeared to penetrate all of the cells that had attached to the surface (Figure 2a, SI Figure S2). Cellular components appeared to be spreading outwards beneath the cell between the nanopillars (Figure 2b). Subsequent viability analysis of these cells revealed that these penetrated cells were in fact dead (Figure 2c, SI Figure S3). The bactericidal effect of the wing surface on Pseudomonas aeruginosa cells was monitored after discrete incubation time intervals over a total period of 1 h (intervals of 1, 5, 10, 20, 30, 40, 50, and Figure 1. Characterization of the cicada wing surface. a) Photograph of cicada, Psaltoda claripennis. b) Structural physiology of the forewing of Psaltoda claripennis, with major veins 60 min) using confocal laser scanning indicated. c) Scanning electron micrograph of the upper surface of the forewing, at 25 000× microscopy (CLSM) and scanning electron magnification. The surface consists of an array of nanoscale pillars, with approximately hexagonal microscopy (SEM) to provide insight into spacing. Scale bar is 2 μm. d) A water droplet contacting the wing surface. When the droplet the kinetics of the cell death (Figure 2c). impacts the wing, it is repelled by the superhydrophobic surface, causing the droplet to bounce These imaging experiments demonstrated several times (an animation of this can be found in SI movie 1). The self-cleaning property of that the nanopillars began to penetrate the surface is also evident, as the droplet can be seen sliding across the surface after it ceases the cells immediately upon cell attachbouncing. e) Wettability map of the wing surface, constructed from repeated measurement of ment, killing most of them within 5 min. static water contact angle in an array of positions. Contact angles were measured at 10 μm intervals, and were found to range between 147° and 172°. The color scale indicates the contact An ‘attachment/killing cycle’ with a period of approximately 20 min became evident, angle (in °). in which the surface appeared to become saturated with cells, which were then The interactions between the bacteria and the superhy- ruptured and dispersed before another group of cells could drophobic wings were characterized in order to elucidate attach (Figure 2c). The damaged and ruptured cells were the nature of the relationship between self-cleaning and dispersed by both sinking down and spreading between the antibiofouling. nanopillars, and/or by detaching from the surface in the form The wings of cicada Psaltoda claripennis are character- of cellular debris (SI movie 2). The rate at which the cells ized by a series of longitudinal veins, cross veins and the areas sank down between the nanopillars was traced using point enclosed by these regions, known as cells (Figure 1a,b).[27] The force microscopy experiments. An atomic force microscopy major chemical components of the wing are protein, chitin (AFM) tip was positioned on top of a single cell (attached to and cuticular waxes, as inferred from infrared microspec- the wing surface) such that a constant force was maintained troscopy analysis performed at the Australian Synchrotron between the tip and the cell (Figure 3a). By recording the (Supporting Information (SI), Figure S1), which is in good piezo movement required to maintain a constant force over agreement with previous literature.[35] The ventral and dorsal time, we found that the cells would slowly move downwards sides of both the fore and hind wings are covered with a peri- approximately 200 nm into the wing surface, before a sudden odic topography consisting of hexagonal arrays of spherically short downward displacement indicating the point of rupture capped, conical, nanoscale pillars (Figure 1c).[36] The height, of the cell (Figure 3b). This rupture point occurred approxispacing, and diameter of the nanopillars vary between spe- mately 3 min after application of the tip. cies; however for Psaltoda claripennis they are 200 nm tall, In order to determine whether the surface chemistry 100 nm in diameter at the base, 60 nm in diameter at the cap, of the cicada wing influences the lethality of the surface to and spaced 170 nm apart from center to center (Figure 1c). Pseudomonas aeruginosa, cicada wings were coated with a The spatial homogeneity of the wettability of the wing sur- 10 nm-thick film of gold using magnetron sputtering. This face was assessed by measuring water droplet contact angles coating substantially decreased the hydrophobicity of the in a grid pattern, before generating a map which was then wing surface, with the water contact angle of the wing dropcolored according to contact angle (Figure 1d,e, SI movie 1). ping from 158.8° to 105.5° (Figure 4a). The surface coating The average water contact angles measured on the wing was did not however visibly change the wing surface topography 158.8°. (Figure 4b,c), and comparative AFM roughness analysis of
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Figure 2. Bactericidal effect of cicada wing surface on Pseudomonas aeruginosa ATCC 9027. a) Pseudomonas aeruginosa cells on the surface of a cicada wing. Cells are clearly penetrated by the nanopillar structures on the wing surface. Scale bar = 1 μm. b) SEM image of a Pseudomonas aeruginosa cell sinking between the nanopillars on the cicada wing surface at 65 000× magnification at an angle of 53 degrees. A section at the front has been excavated using focused ion beam SEM (FIB-SEM) to enable visualization of a cross section of the cell. Scale bar = 200 nm. c) CLSM images and scanning electron micrographs of Pseudomonas aeruginosa cells adhering to the surface of cicada wing. Live cells in CLSM images are stained with SYTO 9 indicated in green, while dead cells are stained with propidium iodide, indicated in red. Yellow cells are an indication of binding of both fluorescent dyes, which is also indicative of dead cells as propidium iodide is unable to stain healthy cells. Cells appear to sink lower as they are killed; live and dead cells in fluorescence images are largely on separate focal planes. Sinking of the cells between the nanopillars can be seen in SI movie 2. Scale bars in all fluorescence images are 5 μm. Electron micrographs clearly show the nanopillars on the wing surface penetrating many cells. SEM scale bars are 2 μm, inset scale bars are 1 μm.
the native and gold coated wing surfaces confirmed that the surface roughness profiles of the two surfaces were virtually identical (Figure 4d). Despite the appreciable change in the surface chemistry of the wing, the bactericidal effect of the gold-coated wing was preserved (Figure 4e), demonstrating that the physical surface structure of the wing, rather than the surface chemistry, is primarily responsible for the lethality of the cicada wing surface for Pseudomonas aeruginosa cells. To the authors knowledge, no previous literature exists describing a direct link between self-cleaning and antibiofouling. Numerous works have been carried out to fabricate superhydrophobic, self-cleaning structures,[37–41] and many of these studies have equated self-cleaning with antifouling; however the link has not been shown. This may be due to some ambiguity in terms. The word antifouling suggests a similar phenomenon to self-cleaning, i.e., remaining clean by preventing any material from ‘fouling’ a surface. However the term is typically used to specifically describe the property of preventing biological accumulation.[13,15,42] Further confusion arises when authors describe a biocidal surface as antifouling.[43] We propose the use of the term antibiofouling when referring to prevention of biological buildup, and recommend that its definition be limited to the property of prevention or limitation of the settlement of biological material. small 2012, 8, No. 16, 2489–2494
Surfaces that limit microbial growth through biocidal action should be referred to as such. The cicada wing surfaces presented here are a case in point. Despite possessing excellent self-cleaning properties, the wing surfaces were in fact quite poor at limiting the degree of attachment of Pseudomonas aeruginosa cells. A relatively high density of cells could be seen in both SEM and CLSM experiments (Figure 2). Instead, the cicada wings were able to maintain a clean surface through very effective bactericidal mechanisms; any Pseudomonas aeruginosa cell that adhered to the surface was killed within just a few minutes. Research into antimicrobial compounds and materials is of vital importance in combating infectious diseases.[44–46] The discovery of antibiotics was an important milestone in medical science; however since the emergence of antibiotic resistance among pathogenic bacteria, the benefits of antibiotics have been largely mitigated.[47] More recently, the production of antimicrobial surfaces has been developed as a new strategy for limiting the spread of infections.[43,48,49] The common approach for fabricating these antimicrobial surfaces has been to functionalize or coat the surface with a substance known to kill the target organisms, such as silver[50] or polycationic compounds.[48,51,52] The problem with developing antimicrobial surfaces based on chemical killing mechanisms is that there exists the same potential for resistance as in
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Figure 3. ‘Sinking’ of a Pseudomonas aeruginosa cell on a cicada wing traced using point force spectroscopy. a) An AFM tip was positioned on top of a Pseudomonas aeruginosa cell in contact with the wing surface, to trace the downwards movement. The piezo movement required to maintain constant force between the cell and the tip was recorded over time. b) The tip was lowered approximately 200 nm over about 220 s, before a sharp short drop indicating the point at which the cell ruptured.
conventional antibiotic treatments. Cicada wings, however, kill bacteria via a mechanism that is purely based on the surface structure, and as such represents an entirely novel approach to the production of antibacterial surfaces. Similar surface topographies have been fabricated over relatively large areas using a variety of techinques such as anodisation, lithographic moulding, micelle lithography and self-assembly,[53–56] however as of yet the range of suitable materials is somewhat limited. This is the first reported example of a naturally existing surface with a physical structure that exhibits such effective bactericidal properties. Cicada wing nanopillars are extremely effective at killing Pseudomonas aeruginosa cells; the wing surface was able to kill individual cells within approximately 3 min. This bactericidal ability of the wing surface is primarily a physico-mechanical effect, as it is retained when the surface chemistry is substantially altered. While many superhydrophobic surfaces do inhibit the attachment of bacterial cells, we have also shown that there is no direct relationship between superhydrophobicity and antibiofouling. The cicada wings do not actually possess any appreciable antibiofouling ability with respect to Pseudomonas aeruginosa; however the net result is somewhat similar in that the bacteria are prevented from proliferating on the surface. Surfaces such as cicada wings that are capable of killing bacteria that attach
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Figure 4. Role of wing surface structure on nanopillar effect on Pseudomonas aeruginosa. a) Water contact angles on cicada wings decreased from 158° on uncoated wings (left) to 105° after coating with gold (right). b) Line profiles of surface topography of uncoated wing and c) gold coated wing, as well as d) surface roughness analysis showed that the gold coating had little effect on surface topography; the only roughness parameter to change significantly was skewness. e) Nanopillar penetration of Pseudomonas aeruginosa on the cicada wing without gold coating (left), and with gold coating (right) show that surface chemistry is not important in the bactericidal effect of cicada wings, but rather it is topography that is the dominant factor.
to them based primarily on their surface structure alone are a completely novel area of research. These surfaces have enormous potential for application in the production of antibacterial materials, and are an alternative to traditional, chemical-based methods.
Experimental Section Insect Wing Preparation: Cicada (Psaltoda claripennis) specimens were collected from the greater Brisbane parkland areas (typically on flora such as eucalypts). All cell regions of the dorsal and ventral sides of the wings possess a homogeneous nanostructuring.[36] All experiments were performed on or near the cell
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regions of M & M21 on the dorsal side of the forewing as shown in Figure 1b, for consistency. Wing sections of approximately 0.5 cm × 0.5 cm were excised using a scalpel or scissors attached by double-sided adhesive tape onto circular cover slips. They were then briefly rinsed with MilliQ H2O (resistivity of 18.2 MΩ cm−1, Millipore, USA) and finally dried using 99.99% purity nitrogen gas.[57] Bacterial Strains, Growth, and Sample Preparation: Pseudomonas aeruginosa ATCC 9027 was used in this study, obtained from American Type Culture Collection (ATCC, USA). Prior to each experiment, bacterial cultures were refreshed from stocks on nutrient agar (Oxoid, U.K.). For cell attachment experiments, fresh bacterial suspensions were prepared for each strain grown overnight at 37 °C in 5 mL of nutrient broth (Oxoid) with shaking at 120 rpm. Bacterial cells were collected at the logarithmic stage of growth, and the suspensions adjusted to OD600 = 0.3, as described elsewhere.[32] The mounted insect wings were immersed in 5 mL of the bacterial suspension, and incubated for 18 h, except for kinetics experiments, in which Pseudomonas aeruginosa was incubated for various discrete time intervals (1, 5, 10, 20, 30, 40, 50, and 60 min). SEM: High-resolution SEM images of cicada wings with adhering bacteria were taken using a field-emission SEM (FESEM; ZEISS SUPRA 40 VP, Germany) at 3 kV under 5000×, 15 000×, 35 000×, and 70 000× magnification. Wing samples with adsorbed bacteria were coated with thin gold films using a Dynavac CS300 according to the procedure developed previously,[58,59] before viewing with the microscope. Wing excavation was performed using a FEI Helios NanoLab 600 focused-ion beam scanning electron microscope, with a 30 kV Ga+ ion beam at a current of 9.7 pA, and under a vacuum of 1 × 10−3 Pa. Cicada Wing Surface Wettability Maps: Water contact angles were measured on the cicada wings using the sessile drop method.[60–63] The contact angle measurements were carried out in air using an FTA1000c equipped with a nanodispenser (First Ten Ångstroms, Inc., USA). Droplet volumes were 1.0 μL. The contact angles were evaluated by recording 50 images over 2 s with a Pelcomodel PCHM 575-4 camera and measuring contact angles after the droplet had rested on the surface for 2 s. An array of 100 measurements was taken at 10 μm intervals over 100 μm × 100 μm. The contact angles were entered into a table and imported into Avizo (Standard v6.3, Visualization Sciences Group, France) to generate color maps. Selected frames were also imported into Avizo as times series to create movie files (Figure 1d,e, SI movie 1). CLSM: CLSM was employed to visualize the proportions of live cells and dead cells using LIVE/DEAD Bac Light Bacterial Viability Kit, L7012, which contains a mixture of SYTO 9 and propidium iodide fluorescent dyes (Molecular Probes, Invitrogen, USA). SYTO 9 permeates all cells, binding to nucleic acids and fluorescing green when excited by a 485 nm wavelength laser. Propidium iodide only enters cells with significant membrane damage, which are considered to be dead, and binds to nucleic acids with a higher affinity than SYTO 9. Bacterial suspensions were stained according to the manufacturer’s protocol, and imaged using a Fluoview FV10i inverted microscope (Olympus, Japan). Gold Coating of Cicada Wings: The cicada wings were coated with a 10 nm layer of gold using a Kurt J Lesker CMS-18 magnetron sputtering deposition system (USA). The thickness of the film was modulated by controlling the process parameters, such as small 2012, 8, No. 16, 2489–2494
argon gas pressure, DC power and the duration of sputtering, as described elsewhere.[64] The 10 nm gold film was deposited under the direct current mode at room temperature, an argon gas pressure of 4 mTorr and a power of 150 kW, with a base pressure of the chamber below 5 × 10−8 Torr prior to introducing the argon gas. AFM: AFM scans were performed using an Innova microscope (Veeco, Bruker, USA) in tapping mode. Phosphorus doped silicon probes (MPP-31120-10, Veeco, Bruker) with a spring constant of 0.9 N/m, tip radius of curvature of 8 nm and a resonance frequency of ∼20 kHz were utilized for surface imaging. Scanning was carried out perpendicular to the axis of the cantilever at 1 Hz. Scans were performed on at least ten areas of each of five samples. The resulting topographical data were processed with first order horizontal and vertical leveling before performing roughness analysis. Determination of the surface roughness parameters was carried out using the instrument software (SPM Lab Analysis v.7.11, Veeco, Bruker). The roughness parameters included the average roughness (Ra), root-mean-square (rms) roughness (Rq), maximum height difference (Rmax), skewness (Rskw), and kurtosis (Rkur). Processed data files were exported to Avizo, and 3D surface profiles were generated (Figure S2, SI). Point force spectroscopy experiments were carried out on a JPK Instruments NanoWizard II AFM in buffered solution at room temperature with a Mikromash CSC37 cantilever; spring constant 0.35 N/m. The tip was positioned on top of a cell in contact with the wing surface, and the piezo movement required to maintain constant force between the cell and the tip monitored.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This research was undertaken in part on the Infrared Microspectroscopy beamline 2BM1 at the Australian Synchrotron, Victoria, Australia, and at the Melbourne Centre for Nanofabrication, which is the Victorian node of the Australian National Fabrication Facility, an initiative partly funded by the Commonwealth of Australia and the Victorian Government. This study was supported in part by Advanced Manufacturing Co-operative Research Centre (AMCRC). V.K.T. and H.K.W. are recipients of Swinburne University Postgraduate Research Award (SUPRA) award.
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Received: March 6, 2012 Published online: June 4, 2012
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