Nano-structured surfaces control bacterial attachment Natasa Mitik-Dineva1, James Wang2, Paul R. Stoddart2, Russell J. Crawford1 and Elena P. Ivanova1 1
Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, 3122, Australia Email:
[email protected] 2 Centre for Atom Optics and Ultrafast Spectroscopy, Swinburne University of Technology, Hawthorn, 3122, Australia bacterial strains belonging to three different taxonomic groups, Pseudomonas aeruginosa ATCC 9027 [19], Pseudoalteromonas issachenkonii KMM 3549T [20] and Vibrio fischeri DSM 507T [21;22] onto two types of glass surface; “as-received” and modified.
Abstract— Surface roughness is known to play a significant role in the cell-surface attachment process, particularly when the surface irregularities are of a dimension that is comparable to the bacterial size and hence provide shelter from unfavorable environmental factors. To explore the influence of nano-scale surface roughness on bacterial attachment this study utilized asreceived and chemically treated glass surfaces as substrata for bacterial adsorption. Surface modification via chemical etching resulted in a 70% decrease in the nano-scale roughness of the glass surface with no alteration of its chemical composition. We have observed that bacteria belonging to three different taxa, while adhering to the modified surface, exhibited similar attachment tendencies to the un-modified substratum, however the number of attached cells increased threefold. The increase in extent of attachment was also associated with bacterial morphologic and metabolic changes. The results obtained suggest that nano-scale surface roughness might strongly influence bacterial attachment.
II.
A. Glass surface physico-chemical characteristics Modification of the “as-received’ glass surface was achieved by chemical etching. Namely, the surfaces of standard glass microscope slides (7105-PPA premium glass slides, Livingstone International) were treated with a buffered solution of hydrofluoric acid (BHF) to achieve a nanometer-scale variation in surface roughness [23]. After the treatment all slides were thoroughly washed with deionized water and stored in 96% alcohol (Aldrich). The chemical composition of the glass surface before and after exposure to BHF was analyzed by means of X-ray fluorescence spectroscopy (XRF) and X-ray photoelectron spectroscopy (XPS). We have further determined the surface wettability by contact angle measurements using an FTA200 (First Ten Ångstroms Inc.) instrument using the embedded needle method [24;25]. Apart from the surface chemical characteristics, surface morphology was also determined. The roughness parameters of both glass surfaces were quantitatively analyzed using a Solver P7LS, NT-MDT microscope.
Keywords- bacterial attchment; glass; nano-scale roughness
I.
INTRODUCTION
The interest in surface-attached bacteria originated decades ago, when it was first discovered that bacterial presence and subsequent growth can result in number of positive and/or negative effects on both the substrate to which the attachment has taken place, and its utilisation. Even though this process has been widely studied over the years, bacterial adhesion to surfaces remains poorly understood mostly because of its complex dependence on factors relating to both the substratum and bacterial cell surface properties [1-9]. A number of theoretical studies (DLVO, the thermodynamic theory) undertaken over the past few decades have revealed some of the basic physico-chemical aspects of this process. For instance, we now accept that the surface charge and hydrophilicity of the substratum are inversely correlated to the number of cells that will attach. On the other hand, very little is known about the effects of the surface roughness on the extent of cell attachment. According to one common scenario of
B. Bacterial surface characteristics Bacterial surface properties such as the surface wettability and charge were also determined. Cell surface hydrophobicity was evaluated from contact angle measurements on lawns of bacteria using the sessile drop method. Bacterial cells (OD600 nm = 0.4) in a PBS buffer were deposited on cellulose acetate membrane filters (Sartorius, 0.2 µm).The wet filters were air dried at ambient temperature (ca. 20ºC) for approximately 30–40 min until a “plateau state” was reached [35-37]. The overall net surface charges of the three strains used in this study were also obtained as described elsewhere [27;28].
bacterial adhesion, the adsorption process is initiated in surface irregularities that serve as microenvironments where bacteria are sheltered from unfavorable environmental factors. The effects of surface roughness have been studied over a wide range of physical scales [10-18], but it has never been shown that surface roughness on a scale smaller than the cell itself might be a major driver in the initial course of bacterial attachment. The study presented here is an investigation of the impact of nanometrescale surface roughness on the attachment strategy of three
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METHODOLOGY
C. Bacterial attachment – sample preparation and imaging Prior to each experiment, a fresh bacterial suspension of OD600 nm =0.2 was prepared from P. issachenkonii and V. fischeri cells grown in marine broth (Difco) and P. aeruginosa cells grown in nutrient broth (Oxoid) at room temperature (~22°C) for 24 h. A portion of 3–5 mL of bacterial suspension was poured into sterile Petri dishes where the glass slides (one glass slide per Petri dish)
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ICONN 2008
were completely immersed and left for 12 h of incubation at room temperature (~22°C). After incubation all of the slides were washed with deionized water and left to dry. This approach allowed the experiments for bacterial attachment to be performed under identical conditions for each half of each microscope slide. For AFM, all slides were imaged only after a couple of hours drying at ambient temperature, which prevented cells from completely drying out. A FeSEM–ZEISS SUPRA 40VP was used to obtain high-resolution images of the bacterial cells. Just before imaging, all slides were gold coated in order to achieve better conductivity. Concanavalin A 488 dye (Molecular Probes Inc.) was used to label the extra-cellular polysaccharide (EPS) produced by the bacteria while attaching to both glass surfaces. After incubation all samples were washed with sterilized nanopure water and carefully stored until being analysed at room temperature (~22°C). The confocal scanning laser microscope (CSLM) Olympus Fluoview FV1000 Spectroscopic Confocal System was used. Excitation and emission wavelengths for concanavalin A are 495 and 519nm, respectively.
III.
The typical topographic images shown in Fig. 1 indicate that the modified glass surface appears smoother and without the relatively prominent high protrusions of the as-received sample. The results indicate that no significant differences in chemical composition and wettability could be detected between the two surfaces, but they also confirm that the treatment with BHF has modified the topographical properties of the glass surface. In the absence of any other apparent significant differences between the two surfaces, it would appear that this striking nanoscale topographical change is the main point of distinction between the samples. Apart from the substratum surface properties, the hydrophobicity, surface charge and EPS production of the three bacterial strains have also been determined. The results presented in Table 2 show that all bacterial strains exhibited negative net surface charge, with P. aeruginosa being the most negatively charged bacterium. These data are in accordance with previously reported for related species [30]. As for the surface wettability, cells of both P. aeruginosa and P. issachenkonii appeared to be less hydrophobic than cells of V. fischeri.
RESULTS AND DISCUSSION
Table 2. Bacterial surface characteristics.
According to the XRF analysis the most abundant chemical components in both samples were SiO2, Na2O, CaO, MgO and Al2O3. The percentage of all detected components (20 in total) in both glass structures was almost identical, with the exception of fluorine, which was found to be present at a level of 0.37 atomic percent (at%) in the modified glass, compared to 0.24 at% in the as-received material. This is consistent with the fact that HF was the main component of the etching solution. [29] The XPS results of high-resolution region spectra (data presented in the Biophysical Journal [29]) indicate that Si and O were, as expected, predominantly present on both glass surfaces. Analysis of the C1s high-resolution spectra was consistent with the presence of hydrocarbons (C-C, C-H), carbon singly bonded to oxygen or nitrogen (C-O, C-N), carbon doubly bonded to oxygen (C=O) and carbonate species (CO3). Although less carbon was detected on the surface of the etched glass compared to native glass, which may indicate removal of organic contaminants, the relative concentration of the various organic species measured by XPS is similar to that of the native glass. The hydrophilicity, surface charge and the surface roughness before and after the exposure to BHF are presented in Table 1 and Figure 1.
Strain
Contact angle, (water)
Surface charge EPM (µs-1 V cm-1)
Zeta (mV)
potential
P. aeruginosa P. issachenkonii
43.3 ± 8 51.9 ± 3
-1.1 ± 0.1 -2.0 ± 0.2
-14.4 ± 0.7 -35.3 ± 0.2
V. fischeri
83.2 ± 5
-2.7 ± 0.7
-34.9 ± 0.9
After determining the basic physico-chemical characteristics of both bacteria and substrata, the attachment behavior of all three strains was observed on both glass surfaces. An initial inspection of the bacterial attachment revealed striking differences in the bacterial response to the two surfaces. As-received glass surface
Table 1. Surface wettability and roughness parameters for the asreceived and the etched glass
Modified glass surface
a
b
c
d
e
f
Surface roughness (nm)
Sample
Contact angle (water)
Ra
Rq
Rmax
Rz
As-received Modified
44.8±0.1° 41.6±0.1°
2.1 1.3
2.8 1.6
27.8 16.2
12.2 4.8
Figure 2. Typical SEM images representing the attached P. aeruginosa (a, b), P. issachenkonii (c, d) and V. fischeri (e, f) cells on the as-received and etched glass surface after 12 h incubation. Scale bar on all images represents 10 µm.
Figure 1. Typical AFM images of as-received (A) and modified (B) glass surfaces. Scanned areas approximately 5 x 5 µm2
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The analysis of cell dimensions indicated that the morphology of the cells attached to the modified glass surface was significantly different than those attached to the ‘as-received’ glass. It appears that not only the cell morphology has been changed, since an increased production of EPS was also observed. Morphological differences in the cells attached to both surfaces after a 12 h incubation can be traced in high magnification scanning electron microscope images (Figure 2). This was further confirmed by the AFM analysis, as shown in Figure 3. The number of the attached cells on the modified glass surfaces was also considerably increased (Table 4). The calculations of the number of the cells have estimated errors of approximately 10% due to local variability in the coverage.
substances during the process of adhesion was observed using CSLM. The attachment pattern of bacterial cells on two regions of glass surfaces after 24 h of incubation is presented in Figure 4. As –received glass
Table 3. Bacterial cell dimensions and the overall number of attached cells on glass surfaces after 12 h incubation
P. aeruginosa P. issachenkon V. fischeri
Number of attached cells, (cells mm-2 As-received Modified 103 000 184 500 36 000 110 000 98 000 131 900
As-received
Cell dimensions (µm) As-received 2.4x1.8x0.25 2.0x1.0x0.14 1.5x0.7x0.18
Modified glass
a
b
c
d
e
f
Modified 2.1x1.1x0.17 2.9x1.3x0.17 1.6x0.8x0.25
Modified
a
b
c
e
Figure 4. Typical CSLM images representing the EPS production of P. aeruginosa (a, b), P. issachenkonii (c, d) and V. fischeri (e, f) cells while attaching to the ‘as-received’ and modified glass surface. Arrows indicate EPS. Scale bar represents 2 µm on image c, d, e and f; and 10 µm on image a and b. It appears that the cells attached to the modified glass surfaces began to form a multilayer structure and produced greater quantities of EPS as confirmed on the CSLM images. As concanavalin A specifically binds to α-mannopyranosyl and α−glucopyranosyl residues, it can be implied that these sugars are components of EPS found on the cell surfaces. However, the granular EPS observed by AFM on the etched glass in Figure 3 were only detected on image b and d, suggesting a distinct chemical composition for this type of EPS produced by V. fischeri cells. This observation is an indication that changes in the nanoscale surface roughness might induce bacterial cells to produce different types of EPS during the attachment process. It has repeatedly been reported that bacteria produce different EPS depending on the environmental conditions (e.g. [31]), but we are not aware of nanoscale surface topography being implicated in this process before. Identification of the chemical composition of the EPS produced by P. issachenkonii cells on both types of glass surface remains a challenging task due to the small amounts of material available for analysis.
d
f
Figure 3. Typical AFM images representing the P. aeruginosa (a, b), P. issachenkonii (c, d) and V. fischeri (e, f) cell morphology on the as-received and the modified glass surface after 12 h incubation. Arrows indicate EPS. The variations in the cell morphology may be a result of dissimilar strategies being employed by the bacteria to better attach and sustain their existence on the glass surfaces. It is most likely that the difference in surface roughness provoked the production of different quantities of EPS, which led to changes in cell morphology. In general, cells on the ‘as-received’ glass surface appeared smaller and flatter than cells on the modified glass surface. Apart from the EPS detected on top of the cells, different amounts of these materials were located on the modified surface (Figure 3, image (b), (c) and (f)) in close proximity to the attached cells. This is indicative of the surface modification strategy utilized by all three bacterial strains to better sustain their existence on this surface. The production of extracellular
IV.
CONCLUSIONS
The results presented here suggest that under similar conditions, bacterial adhesion to glass is significantly influenced by nanoscale changes in the surface roughness and topography. It is evident that the influence of surface roughness on bacterial adhesion is far more important than was previously believed, and should therefore be considered as a parameter of primary interest alongside other well-recognized factors. The bacterial response to these surfaces of different architecture resulted in a significant change in the cellular metabolic activity evident by the
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characteristic cell morphologies and production of extracellular polymeric substances. The suggestion that bacteria may be susceptible to nanoscale surface roughness casts serious doubt on the conventional wisdom that smoother surfaces represent a more repellent environment to bacteria. The effect of nanoscale surface roughness on bacterial adhesion has important implications in a range of applications, for example in the designing of surfaces for various biomedical applications.
ACKNOWLEDGMENT We thank Hans Brinkies, Swinburne University of Technology, Faculty of Engineering and Industrial Sciences, for his assistance with the scanning electron microscopy; Daniel White, Swinburne University of Technology, Centre for Atom Optics and Ultrafast Spectroscopy for his assistance in etching the glass surfaces, Grant van Reissen, LaTrobe University, for the XPS analysis, and Steve Peacock, CSIRO Minerals, for the XRF analysis.
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