Microscale Order Affects the Early Stages of Biofilm Formation

11206

Langmuir 2007, 23, 11206-11210

Nano/Microscale Order Affects the Early Stages of Biofilm Formation on Metal Surfaces C. Dı´az,† P. L. Schilardi,† R. C. Salvarezza,† and M. Ferna´ndez Lorenzo de Mele*,†,‡ Instituto de InVestigaciones Fisicoquı´micas Teo´ ricas y Aplicadas (INIFTA), Facultad de Ciencias Exactas, UniVersidad Nacional de La Plata-CONICET, Sucursal 4 Casilla de Correo 16, (1900) La Plata, Argentina, and Facultad de Ingenierı´a, UniVersidad Nacional de La Plata, La Plata, Argentina ReceiVed March 6, 2007. In Final Form: July 19, 2007 The adhesion of Pseudomonas fluorescens was studied on nano/microengineered surfaces. Results show that these bacteria formed well-defined aggregates on randomly oriented nanosized granular gold substrates. These aggregates consist of aligned ensembles of bacteria, with some of them strongly elongated. This kind of biological structure was not found on ordered engineered surfaces because bacterial alignment and cell-to-cell sticking were hindered. Importantly, differences in cell morphology, length, orientation, and flagellation were observed between bacteria attached on the ordered nano/microstructures and the randomly ordered surfaces. The implications of the results are related to the design of engineered surfaces to enhance (nanostructured filters) or inhibit (medical implants and industrial biofouling) bacterial colonization on the surfaces and to the biocontrol of soil ecosystems.

Introduction Bacterial cells colonize biotic and abiotic surfaces. Living as a group forming biofilms has several advantages over single cells’ way of life: optimization of growth and survival; better access to nutrients; and higher protection against the environment. Detailed investigation of bacterial adhesion, involved in the developmental process from single cells scattered on a surface to complex multicellular biofilms, is crucial to elaborate strategies to control biofilm development. Bacteria can remain localized, move out to colonize larger areas, or form compact three-dimensional (3D) colonies to await more suitable conditions.1 They show several types of motility on a surface: twitching, swarming, gliding, and sliding/spreading. Pseudomonas comprises a diverse group of bacteria found ubiquitously in heterogeneous environments. They are frequently used as model species for studying adhesion and motility of bacteria.2-14 The investigations made with Pseudomonas fluorescens (P. fluorescens) are particularly interesting because * To whom correspondence should be addressed. Telephone: +54 221 4257430/7291. E-mail: [email protected]. † Instituto de Investigaciones Fisicoquı´micas Teo ´ ricas y Aplicadas (INIFTA). ‡ Facultad de Ingenierı´a. (1) Harshey, R. M. Annu. ReV. Microbiol. 2003, 57, 249-273. (2) Doyle, T. B.; Hawkins, A. C.; McCarter, L. J. Bacteriol. 2004, 186, 63416350. (3) Ko¨hler, T.; Kocjancic Curty, L.; Barja, F.; van Belden, C.; Peche`re, J.-C. J. Bacteriol. 2000, 182, 5990-5996. (4) Klausen, M.; Aaes-Jorgensen, A.; Molin, S.; Tolker-Nielsen, T. Mol. Microbiol. 2003, 50, 61-68. (5) Landry, R. M.; An, D.; Hupp, J. T.; Singh, P. K.; Parsek, M. R. Mol. Microbiol. 2006, 59, 142-151. (6) Allesen-Holm, M.; Barken, K. B.; Yang, L.; Klausen, M.; Webb, J. S.; Kielleberg, S.; Molin, S.; Givskov, M.; Tolker-Nielsen, R. Mol. Microbiol. 2006, 59, 1114-1128. (7) Hsueh, P.-R.; Teng, L.-J.; Pan H.-J.; Chen, Y.-C.; Sun, C.-C.; Ho, S.-W.; Luh, K.-T. J. Clin. Microbiol. 1998, 36, 2914-2917. (8) Pappas, G.; Karavasilis, V.; Christoul Tsianos, E. V. Scand. J. Infect. Dis. 2006, 38, 68-70. (9) Kocoglu, M. E.; Bayram, A.; Balci, I. J. Microbiol. 2005, 43, 257-259. (10) Osawa, K.; Nakajima, M.; Kataoka, N.; Arakawa, S.; Kamidono, S. J. Infect. Chemother. 2002, 8, 353-357. (11) O’Toole, G. A.; Kolter, R. Mol. Microbiol. 1998, 30, 295-304. (12) Lequette, Y.; Greenberg, E. P. J. Bacteriol. 2005, 187, 37-44. (13) Kirisits, M. J.; Parsek, M. R. Cell. Microbiol. 2006, 8, 1841-1849. (14) Ramsey, M. M.; Whiteley, M. Mol. Microbiol. 2004, 53, 1075-1087.

they are involved in both detrimental and useful processes. P. fluorescens has been implicated in urinary tract and bloodstream infections as well as procedure-related infections in hospitalized patients.7-10 They are also associated with food contamination. Conversely, the activity of P. fluorescens as a biocontrol species is beneficial in soil ecosystems.15 Surface properties such as chemical composition, wettability, and roughness are important factors in relation to bacterial adhesion and motility. Even though metals are important components in several medical and industrial systems which are susceptible to microbial attachment,16-20 the majority of the evaluations of biofilm development have been made on agar, glass, alumina, and polycarbonate.21-24 It has been demonstrated that the use of nanophase materials is beneficial in environmental and medical applications.25,26 Bacterial distribution and removal are closely related to the size of the microfeatures of the surface.21,23-25,27 However, to the best of our knowledge, the influence of the surface nano/ microstructures on the developmental process of isolated cells attached to complex multicellular biofilms, which involves cell migration, has not been analyzed. The aim of this paper is to elucidate the role of different structural factors of surfaces on the early stages of bacterial (15) DeFlaun, M. F.; Tanzer, A. S.; McAteer, A. L.; Marshall, B.; Levy, S. T. Appl. EnViron. Microbiol. 1900, 56, 112-119. (16) Klug, D.; Wallet, F.; Kacet, S.; Courcol, R. J. Clin. Microbiol. 2003, 4, 3348-3350. (17) Petrini, P.; Arciola, C. R.; Pezzali, I.; Bozzini, S.; Montanaro, L.; Tanzi, M. C.; Speziale, P.; Visai, L. Int. J. Artif. Organs 2006, 29, 434-442. (18) Tunney, M. M.; Dunne, N.; Einarsson, G.; McDowell, A.; Kerr, A.; Patrick, S. J. Orthop. Res. 2007, 25, 2-10. (19) Ferna´ndez Lorenzo de Mele, M.; Cortizo, M. C. Biofouling 2000, 14, 306-315. (20) Viera, M. R.; Guiamet, P. S.; Ferna´ndez Lorenzo de Mele, M.; Videla, H. A. Corros. ReV. 1999, 18, 205-220. (21) Li, X.; Liu, T.; Chen, Y. Biochem. Eng. J. 2004, 22, 11-17. (22) Toutain, C.; Zegans, M. E.; O’Toole, A. J. Bacteriol. 2005, 187, 771777. (23) Whitehead, K. A.; Colligon, J. S.; Verran, J. Int. Biodeterior. Biodegrad. 2004, 54, 143-151. (24) Whitehead, K. A.; Rogers, D.; Colligon, J. S.; Wright, C.; Verran, J. Colloids Surf., B 2006, 51, 44-53. (25) Webster, T. J.; Tong, Z.; Liu, J.; Banks, M. K. Nanotechnology 2005, 16, S449-S457. (26) Cai, K.; Bossert, J.; Jandt, K. Colloids Surf., B 2006, 49, 136-144. (27) Scheuerman, T. R.; Camper, A. K.; Hamilton, M. A. J. Colloid Interface Sci. 1998, 208, 23-33.

10.1021/la700650q CCC: $37.00 © 2007 American Chemical Society Published on Web 09/20/2007

Biofilm Formation on Metal Surfaces

Langmuir, Vol. 23, No. 22, 2007 11207

adhesion. In particular, we have accomplished a nano/microscale visualization and characterization of P. fluorescens adhesion on random structured surfaces and on engineered gold surfaces with sub-microstructures at the early stages of biofilm formation. Gold has been selected for this study because it is chemically inert and biocompatible.28,29 On the other hand, we have used the bacteria P. fluorescens due to its implications in industrial and medical systems. Comparisons of the cellular morphology, flagellar behavior, orientation, alignment, and arrangement of the cells attached on smooth and nano/microstructured gold surfaces were made through atomic force microscopy (AFM) observations. This technique allows the study of multicellular bacterial communities with morphological details on the extracellular ultrastructures at an extremely high resolution.30 We have found that substrate order in the nano/microscale largely affects early stages of bacterial adhesion and distribution, hindering the formation of ordered aggregates. We also found that the surface structures can alter the shape and size of isolated bacteria and aggregates, the cell-to-cell sticking process, the interaction between aggregates, and also the orientation of cellular substructures involved in motion. Experimental Section Three gold substrates with different submicrometer surface structures were used. The first substrate is vapor deposited gold (S1) prepared by physical vapor deposition of a 200 nm gold layer on a glass substrate covered by a thin Cr layer (Gold Arrandee, Germany). The second substrate (S2) is microstructured gold prepared by combining physical vapor deposition (PVD) with molding and replication techniques as described elsewere.31 The third substrate (S3) was also prepared by gold PVD and molding techniques.32 The surface exhibits a random nanometer-sized structure consisting of 50-100 nm grains as shown in Figure 1a I and the cross section analysis (Figure 1a II). The grains introduce a typical wavelength in the order of 100-200 nm. Typical values of the root-mean-square roughness (w) measured on 10 µm2 AFM images result in w ) 2-3 nm. The second substrate (S2) is microstructured gold prepared by combining PVD with molding and replication techniques.31 The substrate consists of a grid of 550 nm wide Au rows separated by 750 nm wide and 120 nm deep channels as can be seen from the AFM image (Figure 1b I). Therefore, the wavelength and amplitude are 1.3 µm and 120 nm, respectively (Figure 1b II). In this case, the w value (from 10 µm2 AFM images) is 40 nm. The size of the trenches fits very well with the bacteria width. The third substrate (S3), also prepared by gold PVD and molding techniques,32 exhibits two rippled structures oriented in the same direction (Figure 1c I and II). The larger structure has a wavelength and amplitude of 250 and 20 nm, respectively (Figure 1c III). The smaller structure consists of ripples of 40 nm in width and 4 nm in height separated by 8 nm channels (Figure 1c IV). The typical w value measured on 10 µm2 AFM images for this substrate is 8 nm. In this case, the larger channels cannot fit bacteria but the 40 nm sized ripples fit the size of motion organelles such as flagella. These substrates were immersed in P. fluorescens cultures. Bacteria were maintained in Cetrimide agar at 28 °C. The inoculum was prepared by suspending a Cetrimide agar slant (24 h old) in 2 mL of sterile nutrient medium. Afterward, the inoculum was poured into (28) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027-1030. (29) Cortizo, M. C.; Ferna´ndez Lorenzo de Mele, M.; Cortizo, A. M. Biol. Trace Elem. Res. 2004, 100, 151-168. (30) Pelling, A. E.; Li, Y.; Shi, W.; Gimzewski, J. K. Proc. Nat. Acad. Sci. U.S.A. 2005, 102, 6484-6489. (31) Azzaroni, O.; Fonticelli, M.; Benı´tez, G.; Schilardi, P. L.; Gago, R.; Caretti, I.; Va´zquez, L.; Salvarezza, R. C. AdV. Mater. 2004, 16, 405-409. (32) Dos Santos Claro, P. C.; Fonticelli, M.; Benı´tez, G.; Azzaroni, O.; Schilardi, P. L.; Luque, N. B.; Leiva, E.; Salvarezza, R. C. Nanotechnology 2006, 17, 3428-3435.

Figure 1. (a) Vapor deposited gold substrate (S1): (I) 10 × 10 µm2 AFM image and (II) cross section. (b) Microstructured gold substrate (S2): (I) 10 × 10 µm2 AFM image and (II) cross section. (c) Nanostructured gold substrate (S3): (I) 10 × 10 µm2 AFM image; (II) 1 × 1 µm2 AFM image; (III) cross section showing the large wavelength; and (IV) power spectral density performed on (c, I). The arrows indicate the large (black) and smaller (gray) wavelengths. an Erlenmeyer flask containing 300 mL of the nutrient broth medium and kept on a rotary shaker for 3 h at 28 °C. After 24 h of growth, the different substrates were placed into the culture so that a bacterial biofilm could be formed on them. The samples were removed after 30 min and then carefully rinsed with sterile distilled water and dried in air. Note that 30 min is an appropriate exposure period to observe early stages of bacterial adhesion and colonization because in our experimental system longer immersion times (2 h) result in compact bacterial aggregates.33 To measure the length of planktonic bacteria, a drop of the culture medium was poured on a slide glass

11208 Langmuir, Vol. 23, No. 22, 2007

Dı´az et al.

Figure 2. (a) 75 × 75 µm2 AFM image (high pass filtered) of P. fluorescens attached to vapor deposited gold surfaces. (b) 10 × 10 µm2 AFM images showing the aligned structure of a typical bacterial raftlike aggregate. Curly flagella around the aggregate are clearly visible. The arrows indicate elongated bacteria. and dried in air. All the samples were imaged by AFM (Nanoscope IIIa, Digital Instruments) operating in contact mode using silicon nitride tips. The hydrophobicity of the surfaces was determined by measurement of the water contact angle from digitalized pictures employing the sessile drop method and using drop shape analysis with a Java plug-in for the ImageJ software.34

Results and Discussion Figure 2 shows AFM images of the S1 surface after 30 min of exposure to the 24 h old culture medium. Groups of cells forming large aggregates are observed in the AFM images. Bacteria are aligned along their axes, in close contact to neighboring cells. This configuration is similar to that previously described for rafts35 of Proteous mirabilis1,36,37 and P. aeruginosa3 involved in swarming and gliding at the agar/air interface. Elongation of some of the aligned cells can be observed in Figure 2 in agreement with earlier reports.3 Some of them are about 4 µm long (see arrows on Figure 2b), twice the length of single cells (1.99 ( 0.12 µm) measured on other places of the sample. In Figure 2a, spearhead ramifications that emerge at various points of the central bacterial line, which were also described for Proteous mirabilis on agar, can be seen. Figure 2b shows flagella that form curls at the border of the raftlike groups. Similar polar flagella and lateral flagella have been shown previously for Pseudomonas bacteria3,15 but at different interfaces. The production of lateral flagella is thought to increase the forces holding the bacteria on the substrate during migration, prior to irreversible attachment. Bacterial groups formed on the sub-micropatterned gold (S2) with parallel trenches are quite different from those on the randomly nanostructured S1 surface. While bacterial cells form densely packed aggregates with well-defined limits on the S1 surface (Figure 2), the aggregates exhibit an open structure and undefined limits on the S2 surface (Figure 3a). These results clearly indicate that the bacterial arrangement is influenced by the substrate. It could be argued that the bacterial ensembles found on S1 could have been formed in the broth. However, if this were the case, they should be observed irrespective of the surface structure. On the S2 surface, many of the cells are not in lateral contact with the neighboring bacteria, as was the case (33) Diaz, C.; Cortizo, M. C.; Schilardi, P. L.; Gomez de Saravia, S.; Ferna´ndez Lorenzo de Mele, M. Mater. Res. 2007, 10, 11-14. (34) Stalder, A. F.; Kulik, G.; Sage, D.; Barbieri, L.; Hoffmann, P. Colloids Surf., A 286, 1-3, 92-103. (35) Jones, B. V.; Young, R.; Mahenthiralingam, E.; Stickler, D. J. Infect. Immun. 2004, 72, 3941-3950. (36) Fraser, G. M.; Hughes, C. Curr. Opin. Microbiol. 1999, 2, 630-635. (37) Kirov, S. M. FEMS Microbiol. Lett. 2003, 224, 151-159.

Figure 3. (a) 75 × 75 µm2 AFM image (high pass filtered) of P. fluorescens aggregates onto microstructured gold (S2). (b) 10 × 10 µm2 AFM image showing short isolated bacteria in the trenches. (c) AFM image showing how longer bacteria lie across the trenches (see the black arrow). (d) AFM image showing details of a typical aggregate. Note that the aggregate was not able to achieve the dense and aligned structure shown in Figure 2b for S1 substrates. Several flagella which link bacteria can be distinguished in (b), (c), and (d) (see the white arrow).

on S1. Interestingly, 76% of the isolated cells attached on S2 are fitted into the trenches and aligned with them (Figure 3b). These cells are shorter than those lying across the surface pattern (Figure 3c). In fact, some bacteria in the trenches are so shortened that they are close to a cocci shape. The evaluation of the length of a single attached bacterium showed that on S2 it was 1.44 ( 0.12.µm, while, as mentioned previously, on S1 it was 1.99 ( 0.12 µm. The size of the planktonic bacteria was 1.861 ( 0.603 µm in agreement with literature data.38 This value is significantly (p < 0.01) longer than the length of individual bacteria attached on the trenches of S2, but it is not significantly different from those attached on S1. The elongation of some of the cells out of the trenches (see the arrow of Figure 3c) could be a consequence of the bacterial stretching used to facilitate contact with the neighboring cells that are fitted in the trenches. These results point out that the environment induces marked changes in bacterial physiological behavior and shape. Accordingly, it was reported that bacteria growing on agar plates experience a significantly altered physiology, a presence of lateral flagella, and changes in shape compared to bacteria growing in broth.39,40 Importantly, the present results showed for the first time the influence of the surface nano/microstructure of solid substrates on flagellar shapes. The AFM image in Figure 3c also shows with particular detail that the small cells trapped in the trenches are stuck to the elongated cells that lie across the trenches and seem to be pulled out of the channels. This can be inferred considering that, instead of being parallel to the trenches, the short cells stuck to the long cells and formed small angles with them and some short cells have half of their bodies in the trench and the other half out of it. (38) Ito, T.; Miyaji, T.; Nakagawa, T.; Tomizuka, N. Biosci., Biotechnol., Biochem. 2007, 71, 366-370. (39) Wang, Q.; Suzuki, A.; Mariconda, S.; Porwollik, S.; Harshey, R. M. EMBO J. 2005, 24, 2034-2042. (40) Gavı´n, R.; Rabaan, A. A.; Merino, S.; Toma´s, J. M.; Gryllos, I.; Shaw, J. G. Mol. Microbiol. 2002, 43, 383-397.

Biofilm Formation on Metal Surfaces

Langmuir, Vol. 23, No. 22, 2007 11209

Figure 5. AFM image (high pass filtered) of P. fluorescens cells on microstructured gold (S2). The arrows indicate the extracellular polymeric substances produced by the bacteria. Flagella connecting the cells can also be seen.

Figure 4. (a) 75 × 75 µm2 AFM image (high pass filtered) of P. fluorescens attached to a gold surface with nanoripples (S3). The image shows cell aggregates with an open structure. (b) 10 × 10 µm2 AFM image (high pass filtered) showing that the aggregate does not have the closely packed and aligned structure shown in Figure 2. (c) 4 × 4 µm2 AFM image (high pass filtered) of P. fluorescens attached to a gold surface showing flagella around the aggregate. (d) Detail of the flagella and the substrate nanoripples (40 nm in size).

Interestingly, the polar flagella form linkages between isolated bacteria and other bacteria (Figure 3b) or groups of bacteria (Figure 3c and d). They are considered sensors of external conditions,39 able to modulate not only their own biogenesis but also other physiological functions. This linkage process connecting groups of bacteria can also be seen in the micrographs reported by Ko¨hler3 for P. aeruginosa on “swarm” agar (0.5% agar) but without any comment or interpretation by the authors. It seems that flagella are useful for cell-to-cell (Figure 3b) and cell-to-group contacts (Figure 3c and d). The distribution of cells on the right side of Figure 3c and d illustrates that the sticking and alignment processes are more difficult for S2 than for S1. The arrangement of cells that are on the patterned surface of S2 is different from the aligned raftlike structure shown in Figure 2. Many bacteria and small groups of bacteria are neither in complete contact with their neighbors nor aligned with them. For longer immersion times, 3D aggregates were found on S2. Figure 4a and b shows the groups of bacteria that are colonizing the rippled nanostructured gold (S3). All the cells lie across the ripples because the width and depth of the features are very small (Figure 1c) in relation to the bacterial dimensions, and they cannot fit into them. The arrangement of bacteria on S3 shows that only groups of three or four cells are aligned and these groups join to other groups that show different arrangement and direction. Even with the small dimension of the two wavelengths that characterize the surface structure, the formation of the aligned aggregates is partially inhibited. This suggests that cellular substructures such as flagella may “feel” the substrate features when their sizes fit. A preferential alignment of bacteria perpendicular to the trenches was noticed. Notwithstanding that, raftlike assembles on S3 (Figures 4a and b) seem to form easier than those in the case of S2, where many of the bacteria are in the trenches and need to climb the 120 nm height of the trench wall to get out of it (Figure 3c). The presence of a solid barrier at S2 and the stagnant experimental conditions may cause different concentration

gradients of autoinducers41 around the bacterial bodies, according to the sub-microstructure of the surface. This may stimulate some kind of differentiation in the bacteria (shorter and thinner cells), making them better suited to attach to a particular roughness or topography. We now discuss the present results in the context of substrate structure. Significant differences are observed in the formation of raftlike aggregates on the S1 and S2 substrates. There are two main differences when the S1 and S2 structures are compared: roughness and order. S2 is an ordered substrate with a wavelength in the order of bacteria size and is markedly rougher (dominated by the pattern amplitude) than S1. In principle, the inhibition of the raftlike ensembles formation could be related to the roughness and the wavelength present in the S2 surfaces. However, S3, which also inhibits the formation of ordered ensembles, exhibits a roughness similar to that of S1 and a wavelength and amplitude significantly smaller than those of S2. However, in this case, the flagella orientation seems to be influenced by the 40 nm wavelength, as their sizes fit very well. Therefore, we propose that the formation of arranged aggregates of cells is not only affected by roughness but also by the order at the nano/microscale. The alignment and sticking of bacteria in raftlike ensembles seem to be dependent on the surface order. The physicochemical surface properties of the solid material play a major role in the adhesion process.42 Bacteria in aqueous suspension are almost always negatively charged. The surface charge of bacteria is influenced by environmental properties (pH and ionic strength), bacterial age, and bacterial surface structure, among others.43 Generally, bacteria with hydrophobic properties such as those of Pseudomonas prefer hydrophobic material surfaces. The measurements of the hydrophobicity of the three gold surfaces assayed showed that S2 was the most hydrophobic. This may induce stronger interactions between Pseudomonas fluorescens and the surface, which could make migration more difficult. However, it must be taken into account that bacteria can mask the original solid surface hydrophobicity and charge by means of exopolysaccharides (EPS) and biosurfactant production.41 The formation of EPS can be seen under and around the cells in Figure 5 (see arrows). The EPS “cushion” seems to be produced to feel the trenches facilitating the bacterial climb up. Present results show that even though bacterial differentiation proceeds on nano/micro-ordered structures, the alignment and sticking processes are hindered by the structured surface. Although (41) Eberl, E.; Molin, S.; Givskov, M. J. Bacteriol. 1999, 181, 1703-1712. (42) Gottenbos, B.; Busscher, H. J.; van der Mei, H. C. J. Mater. Sci. Mater. Med. 2002, 13, 717-722. (43) Katsikogianni, M.; Missirlis, Y. Eur. Cell. Mater. 2004, 8, 37-57.

11210 Langmuir, Vol. 23, No. 22, 2007

raftlike structures were first thought to be confined to nutrient rich agar surfaces,1 our research work demonstrated that they can also be formed on random nanostructured surfaces immersed in a nutrient rich liquid environment. The raftlike structure, particular flagellar behavior, and cellular differentiation found in this work could be compatible with swarming or gliding motility.44-47 Further investigation will be carried out to confirm this conjecture. AFM proves to be a particularly suitable tool to analyze at high resolution the initial stages of bacterial attachment. It shows in detail the flagella locations, the morphological changes of the bacteria, and the distribution of the cells in the sub-microstructured and nanostructured surfaces. To the best of our knowledge, our (44) Lindum, P. W.; Anthoni, C. C.; Eberl, L.; Molin, S.; Givskov, M. J. Bacteriol. 1998, 180, 6384-6388. (45) Senesi, S.; Celandroni, F.; Salvetti, S.; Beecher, D. J.; Wong, A. C. L.; Ghelardi, E. Microbiology 2002, 148, 1785-1794. (46) Senesi, S.; Ghelardi, E.; Celandroni, F.; Salvetti, S.; Parisio, E.; Galizzi, A. J. Bacteriol. 2004, 186, 1158-1164. (47) Calvio, C.; Celandroni, F.; Ghelardi, E.; Amati, G.; Salvetti, S.; Ceciliani, F.; Senesi, S. J. Bacteriol. 2005, 187, 5356-5366.

Dı´az et al.

report presents the first analysis of the influence of submicrostructures on raftlike ensembles and bacterial shape which could be accomplished due to the advantages of AFM. Our results have important implications as both fundamental contributions and biotechnological applications. One of these implications is related to the design of engineered surfaces to enhance (nanostructured filters) or inhibit (medical implants) bacterial adhesion on surfaces. Another application is associated with the development of strategies to improve the eradication of bacteria through the use of biocides in industrial (biofouling removal) or medical (infection treatments with antibiotics) environments. Finally, our results have environmental implications related to the biocontrol of soil ecosystems in which bacterial biofilm formation is involved. Acknowledgment. We are grateful to ANPCyT (PICT 0211111, PICT 05-33225, PICT 05-32906, PAE 22771), UNLP (Projects 11/X425 and 11/I095), and CONICET (PIP 6075) for financial support of this work. LA700650Q