Single-Cell Force Spectroscopy of Bacteria Enabled by Naturally

Article pubs.acs.org/Langmuir

Single-Cell Force Spectroscopy of Bacteria Enabled by Naturally Derived Proteins Guanghong Zeng,† Torsten Müller,§ and Rikke L. Meyer*,†,‡ †

Interdisciplinary Nanoscience Center (iNANO), Faculty of Science and Technology, Aarhus University, Aarhus 8000, Denmark Department of Bioscience, Faculty of Science and Technology, Aarhus University, Aarhus 8000, Denmark § JPK Instruments AG, Bouchéstrasse 12, Berlin 12435, Germany ‡

ABSTRACT: Bringing the study of bacterial adhesion down to a single-cell level is critical for understanding the molecular mechanisms involved in initial bacterial attachment. We have developed a simple and versatile method for making single-cell bacterial probes to study the adhesion of single bacterial cells by atomic force microscopy (AFM). A single-cell probe was made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated with a commercial cell adhesive Cell-Tak. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, and mica). Attachment to the cantilever was stable during the AFM force measurements that were conducted for 2 h, and viability was confirmed by Live/Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and contact time. The single-cell probe offers control of cell immobilization and thus holds advantages over the commonly used multicell probes with which random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single-cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding of the mechanisms of bacterial attachment.

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INTRODUCTION Attachment of bacteria to surfaces is the initial step in the formation of biofilm, which is the main source of microbial contamination during food processing and is also the cause of infections associated with medical implants. Understanding the mechanism of bacterial adhesion is critical for developing new strategies for preventing unintended bacterial colonization and biofilm formation.1−5 The adhesion of bacteria involves unspecific interactions like van der Waals, electrostatic, and acid−base interactions and also specific interactions between, e.g., lectins and sugar moieties of glycosylated proteins.6 Depending on the species of the bacteria, their physiological state, and the property of the surface, different mechanisms may be employed. Diverse biomacromolecules, including polysaccharides, proteins, and extracellular DNA, play a role in assisting attachment to the surface.7 Cell appendages such as flagella and pili or fimbriae also function as adhesins. While most approaches study the adhesion of bacteria by looking at the collective behavior of millions of cells, it is necessary to go down to a single-cell level to elucidate the molecular mechanism of bacterial adhesion, as cell-to-cell and cell− surface heterogeneity can be taken into account.6,8,9 Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) has been used to study and quantify cell−cell and cell−surface interactions of mammalian cells quantitatively.10,11 Via this technique, a single cell is attached to an AFM cantilever that then approaches the surface of another © 2014 American Chemical Society

cell or to measure the adhesion force. A high level of precision in force measurement, versatility in combining optical techniques, and, most importantly, the ability to operate under physiological conditions have made it possible to quantify interactions of living cells in their native environment. The application of this technique to bacterial cells, however, has been hampered by the lack of techniques for effectively immobilizing bacterial cells onto cantilevers. Several approaches of attaching bacterial cells to AFM cantilevers have been reported, such as chemical fixation and adsorption to a positively charged coating,12 gluing by a commercial adhesive,13 adsorption to a hydrophobic coating,14,15 and positively charged coating.16−19 However, these methods usually rely on immobilization by insertion of a cantilever into a bacterial cell suspension, resulting in immobilization of multiple cells at random locations on the cantilever. Some methods affect cell viability because of the use of toxic chemicals, and others were not sufficiently robust to allow firm immobilization of a wide range of bacteria. A need to develop a reliable and easy way of making single-cell probes therefore remains. Recently, a novel method using the mussel-inspired polydopamine as an adhesive to make single-cell probes with a micromanipulator was reported,20 demonstrating the feasibility Received: December 9, 2013 Revised: February 23, 2014 Published: March 23, 2014 4019

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Figure 1. Illustration of the process of making a single-cell bacterial probe. be picked up. To make a single-cell bacterial probe, a single bacterial cell was identified under the optical microscope, and the apex of the Cell-Tak-coated cantilever was navigated above the cell and engaged at a set point of 1 nN. Five minutes later, the cantilever was retracted with the cell now immobilized to the cantilever. Surface Preparation and Characterization. Fresh glass was made by cleaning glass coverslips by sequential sonication for 5 min in acetone, ethanol, and deionized water. Hydrophilic glass was prepared via a 10 min UV ozone treatment of fresh glass. Mica was cleaved with scotch tape before being used. The static water contact angle was measured on Drop Shape Analysis System DSA100. The rms (rootmean-square) surface roughness was measured by AFM (NanoWizard II, JPK Instruments) tapping mode imaging in air on a 5 μm square on the surface, using OMCL-AC160TS (Olympus) cantilevers. AFM Force Measurements with Bacterial Probes. Force measurements were conducted in PBS. The sensitivity of tipless cantilevers was calibrated on glass before immobilization of bacterial cells, and the spring constant was obtained by thermal tuning. After attachment of a bacteria cell, the cantilever was moved to approach the surface, and force curves were captured at the 1 nN set point with a contact time from 0 to 30 s. For each contact time, 30 force curves were obtained. A control experiment indicated that adhesion forces were not dependent on the order of measurements with different contact times (data not shown). For each strain, adhesion force measurements were repeated with three independent cultures. Force curves were analyzed by data processing software from JPK Instruments. Peaks in the retraction part of the force curves were defined as adhesion peaks. The adhesion force was determined by the peak with the maximal adhesion force, and the rupture length was defined by the length from the contact point to the rupture point of the last adhesion peak. The standard deviation is shown for the adhesion force and rupture length at certain contact times. Statistical analysis between two sets of adhesion forces or rupture lengths was performed with Analysis Toolpack in Excel 2010, using a two-sample t test assuming unequal variance. Viability Assay of the Immobilized Bacteria. The viability of the cell on the tipless cantilever was tested after force measurement by Live/Dead Baclight staining kit, which is based on cell membrane permeability. Fluorescence images were captured on a Zeiss Axiovert 200 M epifluorescence microscope with a 40× objective lens using Zeiss filter sets 10 and 43.

of using a universal wet adhesive to make bacterial probes. A significant improvement to this method, which combined polydopamine coating and colloid probes, allowed better control of the position of the cells and therefore cell−substrate contact.21−23 We have previously demonstrated the effective immobilization of bacterial cells for AFM imaging in liquid by using Cell-Tak, a commercial wet cell adhesive, composed of naturally derived polyphenolic proteins. It was demonstrated that Cell-Tak allowed robust immobilization of viable bacterial cells with no apparent disturbance of cell function.24 The adhesiveness of Cell-Tak has also been utilized to detach marine bacteria from the substrate for the quantification of attachment strength.25 We here report a simple approach for making single-cell bacterial probes for a wide range of bacteria, using a combined AFM and optical microscope to transfer a single cell to a Cell-Tak-coated tipless cantilever. The strong adhesiveness of Cell-Tak allows attachment of a single cell in a controlled manner, which can be applied to a wide range of bacterial cells, independent of their shapes, sizes, and surface properties. It guarantees hours of stability and cell viability for further manipulation for force spectroscopy.

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EXPERIMENTAL SECTION

Preparation of Bacterial Probes. Staphylococcus xylosus DSM 20266 and Staphylococcus epidermidis DSM 20044 were grown in 1 and 3% tryptic soy broth (TSB), respectively, and Pseudomonas f luorescens AH126 and Escherichia coli DSM 429 were grown in Luria broth (LB). Bacteria were first streaked on agar plates and stored at 4 °C for up to 4 weeks before they were inoculated into growth medium and incubated overnight to form precultures. A preculture was diluted to 1% in fresh medium and incubated for 16 h while being shaken at 180 rpm to obtain a sample culture. All strains were cultured at 37 °C, except for P. f luorescens, which was cultured at 30 °C. Bacterial cells were harvested by centrifugation at 5000 rpm for 5 min, washed three times with phosphate-buffered saline (PBS), and diluted in PBS to an optical density at 600 nm (OD600) of 0.1. To immobilize bacterial cells, 50 μL was placed on a hydrophilic glass coverslip, which had been pretreated with a UV ozone cleaner (ProCleaner, BioForce Nanosciences) for 10 min immediately beforehand. After 2 min incubation, the glass coverslip was gently washed with PBS to remove unattached cells and mounted on the combined AFM and optical microscope (JPK Nanowizard II/Zeiss Axiovert 200M) with a drop of PBS to protect bacterial cells from drying out. A tipless silicon cantilever (CSC12/Tipless/NoAl, MikroMasch) was cleaned by UV ozone for 10 min, before it was submerged in a droplet of 2 μL of Cell-Tak dissolved in 57 μL of NaHCO3. Polymerization of the adhesive was initiated by addition of 1 μL of 1 M NaOH. The cantilever was allowed to incubate for 20 min, washed with Milli-Q water, and mounted on the AFM microscope. Cell-Tak coating has to be conducted immediately before use, as overnight storage of coated cantilevers in a 4 °C refrigerator caused the cantilevers to lose adhesiveness to an extent that bacterial cells cannot

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RESULTS AND DISCUSSION An Easy and Versatile Method of Making Single-Cell Bacterial Probes. Single-cell bacterial probes were successfully made from all four bacterial strains tested in this study. The process is illustrated in Figure 1. Most of the experiments were successful on first attempts, while control experiments with unmodified cantilevers failed to pick up cells from the surface with repeated attempts. In some cases, the UV-cleaned cantilevers were exposed to the air for >2 h before Cell-Tak modification, and the subsequent pickup of bacterial cells failed. It is well-known that UV-cleaned silicon surfaces lose hydrophilicity in air over time. Therefore, it is important that 4020

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silicon cantilevers remain hydrophilic to make Cell-Tak modification and subsequent pickup successful. A single bacterial cell suitable to be picked up was located under the 40× objective lens before the cantilever was engaged. It was important to dilute the bacterial culture before immobilization to the hydrophilic coverslip to ensure the availability of single cells, and for rod-shaped cells, it was also convenient to find a cell in the right alignment, i.e., perpendicular to the direction of the cantilever. This orientation would secure good contact along the cell surface in subsequent force measurements. It is noteworthy that hydrophilic treatment of glass coverslips is critical to make bacterial probes. Attempts to pick up cells from untreated fresh coverslips failed, probably because of the firm attachment of bacterial cells on fresh glass, as will be demonstrated in adhesion force measurements in the following section. The successful immobilization of both Gram-positive (S. epidermidis and S. xylosus) and Gram-negative (E. coli and P. f luorescens) bacteria with highly different cell shapes and surface chemistries demonstrates that Cell-Tak is a versatile adhesive for bacteria. Controlling the position of the immobilized cell on the cantilever is crucial for ensuring contact between the cell and the surface during force measurements. We therefore evaluated the maximal distance of the cell from the edge of the cantilever, which would allow appropriate contact between the cell and a surface during the approach of the single-cell probe (Figure 2a). The tilt angle of the cantilever is 10°, and D can be assumed to be a typical bacterial cell size of 1 μm; therefore, the longest allowed distance to the end of the cantilever from the center of the cell is calculated as D × tan−1(θ) = 1 μm × tan−1(10°) = 5.7 μm. Appropriate contact can thus be expected when the cell is positioned within a distance of 5.7 times the cell diameter from the end of the cantilever. Elastic deformation of the cells during force measurement will decrease the cell diameter slightly, but this is estimated to be only approximately 20 nm at the 1 nN set point according to the analysis of force curves in this study. In practice, the immobilization process was visualized by optical microscopy, and bacterial cells were easily immobilized within 2 μm of the cantilever edge (Figure 2c,d). Considerations of the positioning of the cell toward the cantilever edge can be overcome by using colloid probes to pick up bacterial cells.21−23 While this method makes it easier to position the cells by simply aligning them in the center of the colloids, it requires an extra step to attach the colloids to the cantilevers, and the attached colloids may hinder the visualization of the immobilization process under the microscope with transmission light. Bacterial Probes Are Stable and Viable after Being Manipulated for 2 h. We wondered if a Cell-Tak-modified cantilever provided firm attachment of the immobilized cells on the cantilevers during subsequent manipulation. The stability of single-cell probes was tested by repeated force measurement to confirm the reproducibility of the results over time, and subsequent optical visualization and Live/Dead staining of the cell probes to confirm viability. An S. xylosus single-cell probe was submitted to more than 360 force curves on fresh glass, hydrophilic glass, and mica, with contact times of 0, 2, 5, and 10 s. At the end of the measurements, the experiment was repeated, and similar adhesion forces were obtained under the same conditions in each of the two experiments (data not shown). Each experiment took approximately 2 h, and the probe was thus stable for at least 4 h. The location and viability of immobilized cells were confirmed at the end of force curve

Figure 2. Illustrations and optical images of single-cell bacterial probes. (a) Illustration of a single-cell probe that barely touches the surface when the cantilever approaches the surface. (b) Illustration of a typical single-cell probe in contact with the surface. Bright field and fluorescence images of (c and e) the S. epidermidis probe and (d and f) the P. f luorescens probe after force measurements for 2 h followed by Live/Dead staining. The edge of the downward-facing side of the cantilever in the bright field images is denoted with an arrow.

measurements. Two examples are shown in Figure 2. After acquisition of 360 force curves over 2 h, bacterial cells remained on the apex of the cantilevers and showed no indication of compromised viability. The stability of the cell probes was conferred by the strong adhesiveness of Cell-Tak, which provided an adhesion force much stronger than the interaction force between the bacterial cells and the surfaces during subsequent force measurements. Adhesion Force Curves of Single-Cell Probes of Four Bacteria on Three Surfaces. One of the advantages of singlecell probes is that it allows measurement of adhesion of a single cell on many surfaces. We measured adhesion of single-cell probes of four bacterial strains on three different surfaces: fresh 4021

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Figure 3. Representative retraction force curves of S. epidermidis, S. xylosus, P. f luorescens, and E. coli single-cell probes and control probes (Cell-Takcoated cantilevers) on three surfaces after contact for 10 s.

glass, hydrophilic glass, and mica. Typical retraction force curves after contact between the cell and surface for 10 s are presented in Figure 3. It is noteworthy that adhesion on different surfaces was measured with the same probe, therefore allowing comparison of different surfaces in a most reliable way. Force curves of the closely related S. epidermidis and S. xylosus were similar, indicating that the two species could use similar attachment mechanisms. Retraction force curves of staphylococci on all surfaces were characterized by a major adhesion

peak with a number of overlapping minor peaks. Furthermore, most of them end with a more or less independent peak at the final rupture. These results indicate that a large number of relatively short adhesins are located close to the cell surface, which altogether contribute to the large adhesion peak, and long adhesins extend out as long as 1500 nm, which contribute to the peak at the rupture position. The length of these long adhesins is striking, compared to the size of the bacteria (approximately 1000 nm). A possible explanation could be that 4022

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length were observed when moving from fresh glass to hydrophilic glass and then to mica (p < 0.001, except for the rupture length between fresh glass and hydrophilic glass, where no significant difference was found), and the number of adhesion peaks was severely reduced on mica compared to the number on glass surfaces. In general, hydrophobic surfaces have fewer surface-bound water molecules. As removal of surface water molecules is required to allow the approach of a cell surface and establishment of attachment by bacterial adhesins, it is not surprising that the less hydrophilic fresh glass is the most adhesive surface. In the case of P. f luorescens, adhesion profiles were not significantly different between fresh and hydrophilic glass surfaces, while mica still showed a significantly weaker adhesion force and a significantly shorter rupture length. While these results confirm previously reported trends of the adhesion pattern on hydrophilic and hydrophobic glass30 and mica,31 they illustrated the versatility and robustness of single-cell force spectroscopy in studying the adhesion of a single cell toward different abiotic surfaces. More insights could be gained by looking into the adhesion of S. epidermidis and P. f luorescens after different surface contact times (Figure 4). Adhesion forces of S. epidermidis increased as the contact time increased from 0 to 10 s on all surfaces. In the case of P. fluorescens, adhesion forces increased as the contact time increased from 0 to 2 s and showed no further increase after prolonged contact with fresh and hydrophilic glass. This observation together with the result that P. f luorescens shows no difference in adhesion on the two surfaces suggests that P. f luorescens uses adhesins that establish attachment on glass much faster, and they are not dependent on surface hydrophobicity in the range explored. As opposed to adhesion to glass, the adhesion of P. f luorescens to mica was close to zero, and no adhesion peaks were observed. Mica is a surface very different from hydrophilic glass despite the fact that both are very hydrophilic. All bacterial strains show much weaker adhesion forces and slower bond strengthening on mica than on glass. This is attributed to the ability of mica to bind water molecules more strongly, making it difficult for bacterial adhesins to establish acid−base interactions by removing surface-bound water molecules. For both strains, although the adhesion force increased as the contact time was extended, the final rupture length was unaffected when extending the contact time beyond 2 s (Figure 4). If the rupture length had changed as the contact time was increased, it would have indicated a sequential involvement of adhesins of different lengths during the bond strengthening. As this was not the case, we suggest that bond strengthening does not involve a sequence of different adhesins, but rather an increasing number of adhesins contributing to the interaction. Advantage of Single-Cell Probes. A potentially simpler way of making bacterial probes is to attach multiple cells on the same cantilever by immersing the cantilever in a cell suspension, assuming that only one cell close to the tip of the cantilever is in contact with the surface during force measurements. This might not always be the case, as demonstrated in Figure 5. A multicell probe was made by incubating a Cell-Tak-coated cantilever in an S. xylosus suspension for 5 min. In the bright field image, it looks like a single cell (denoted with an arrow) is most likely the only one that interacts with the surface, but it is revealed in the fluorescence image that a dead cell is right next to the cell and another cell is closer to the tip of the cantilever. The multicell probe has much weaker adhesion forces than its single-cell

unfolding and stretching of polymers might be involved in the retraction, as the sawtoothlike pattern in the force curves is quite similar to the protein unfolding and stretching of bacterial pili.27,28 Staphylococci have been reported to use a number of different adhesins, such as polysaccharide intercellular adhesin (PIA), biofilm-associated protein (Bap), accumulation-associated protein (Aap), teichoic acids, and extracellular DNA (eDNA).2 We cannot speculate about the contribution of each of these, but this could be studied by comparing force curves of the wild-type strain with those of knockout mutants lacking specific adhesins. The adhesion profile of P. fluorescens was very different from those of the two staphylococci. While adhesion forces were much weaker, typical retraction force curves are composed of consecutive adhesion peaks, ending with a relatively high peak at the rupture point. The rupture length was even greater than that of staphylococci, reaching 2000 nm in some curves. P. f luorescens is a motile strain with flagella and fimbriae,29 which might contribute to the long interaction distance between the cell and surface. E. coli showed a weak adhesion force on fresh glass and almost no adhesion on hydrophilic glass and mica. E. coli was the only strain among the bacteria tested in this study that does not form a biofilm (data not shown). The low adhesiveness of this strain is thus in agreement with its phenotype. Control experiments with Cell-Tak-modified cantilevers showed distinctive adhesion behavior on all surfaces compared to that of bacterial probes (Figure 3). Adhesion forces were strong on all surfaces (∼15 nN on fresh glass and ∼8 nN on hydrophilic glass and mica), confirming the adhesive nature of Cell-Tak coatings. Force curves were highly reproducible among repetitive measurements, and all of them were composed of single peaks, with rupture lengths much shorter than those of bacterial probes. These results agree well with the fact that Cell-Tak coatings do not have the complex polymers extending from the surface and therefore do not produce force curves with multiple adhesion peaks with long rupture lengths as seen for bacterial cells. The combination of a strong adhesion force, a single peak adhesion profile, and a short rupture length can be used to identify the adhesion of Cell-Tak coatings. When the adhesion of Cell-Tak coatings appears during the measurement of a bacterial probe, the immobilized cell either has detached or has dislocated, and therefore, the bacterial probe should be discarded. The hydrophobicity and surface roughness of the three surfaces were characterized by water contact angle and AFM imaging. All three surfaces were hydrophilic, and the mica and UV-treated glass were so hydrophilic that water contact angles could not be measured (Table 1). A special feature of mica is its flatness, with a rms roughness as low as 0.11 nm. From the force curves of the two staphylococci on three surfaces, significant decreases in both the adhesion force and the rupture Table 1. Water Contact Angles and AFM-Measured RootMean-Square (rms) Roughnesses of the Three Surfaces Used in This Study water contact angle (deg) rms roughness (nm)

fresh glass

hydrophilic glass

mica

44.3 ± 1.1 0.36

N/Aa 0.23

N/Aa 0.11

a

Water spread on the surface so fast that a sessile droplet could not be formed. 4023

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Figure 4. Adhesion forces and final rupture lengths of S. epidermidis and P. f luorescens on three surfaces after contact times of 0−10 s. Data were analyzed from 90 force curves recorded with three bacterial probes from independent cultures.

more attempts, we propose that single-cell probes offer better control for choosing which cell to immobilize and how and where it should be located on the cantilever, which leads to more reproducible results.

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CONCLUSIONS In this paper, a simple and versatile method for making a singlecell bacterial probe for AFM force spectroscopy was developed. A single bacterial cell was picked up by a Cell-Tak-coated tipless AFM cantilever with the aid of a combined AFM and optical microscope. The single-cell bacterial probes were stable after AFM manipulation for at least 2 h, and bacterial cells were still viable. The adhesion of four bacterial strains on three surfaces was investigated by single-cell force spectroscopy using the single-cell probes. S. xylosus and S. epidermidis showed much stronger adhesion forces than P. f luorescens, and E. coli showed almost no adhesion to any surface. Both adhesion force and rupture length were significantly smaller on mica than on glass. Staphylococci adhere stronger on fresh glass than on hydrophilic glass, while the weaker adhesion by P. f luorescens was similar on both types of glass. The bond strengthening effect of S. epidermidis on glass was observed up to 10 s, while that of P. f luorescens stabilized within 2 s. Compared to a multicell probe, single-cell probes have better control of cell immobilization, resulting in more reproducible results in force measurements. This method provides a general platform for investigating bacterial interactions on the single-cell level by AFM.

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Figure 5. Bright field images, Live/Dead fluorescence images, and typical retraction force curves on hydrophilic glass of a single-cell probe (a, c, and e, respectively) and a multicell probe (b, d, and f, respectively) of S. xylosus.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +45 8715 6739. Fax: +45 8942 2722. Notes

counterparts. It is therefore likely that the dead cell is mainly involved in the observed interaction, leading to a reduced adhesion force. In fact, the force curve of the multicell probe resembled those of dead cell probes made accidentally by using a large force set point during pickup (data not shown). While it is still possible that a better multicell probe could be made by

The authors declare that two months’ salary for post doc G.Z. was donated by JPK Instruments. JPK Instruments (T.M.) made an intellectual contribution to the project, but JPK Instruments has not made financial gains from the research and has not affected the communication of the research results. 4024

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(20) Kang, S.; Elimelech, M. Bioinspired Single Bacterial Cell Force Spectroscopy. Langmuir 2009, 25, 9656−9659. (21) Herman, P.; El-Kirat-Chatel, S.; Beaussart, A.; Geoghegan, J. A.; Vanzieleghem, T.; Foster, T. J.; Hols, P.; Mahillon, J.; Dufrene, Y. F. Forces Driving the Attachment of Staphylococcus epidermidis to Fibrinogen-Coated Surfaces. Langmuir 2013, 29, 13018−13022. (22) Beaussart, A.; Herman, P.; El-Kirat-Chatel, S.; Lipke, P.; Kucharíková, S.; Van Dijck, P.; Dufrêne, Y. Single-Cell Force Spectroscopy of the Medically Important Staphylococcus epidermidisCandida albicans Interaction. Nanoscale 2013, 5, 10894−10900. (23) Beaussart, A.; El-Kirat-Chatel, S.; Herman, P.; Alsteens, D.; Mahillon, J.; Hols, P.; Dufrene, Y. F. Single-Cell Force Spectroscopy of Probiotic Bacteria. Biophys. J. 2013, 104, 1886−1892. (24) Meyer, R. L.; Zhou, X. F.; Tang, L. N.; Arpanaei, A.; Kingshott, P.; Besenbacher, F. Immobilisation of Living Bacteria for AFM Imaging under Physiological Conditions. Ultramicroscopy 2010, 110, 1349−1357. (25) Friedrichs, J.; Zieris, A.; Prokoph, S.; Werner, C. Quantifying the Effect of Covalently Immobilized Enzymes on Biofilm Formation by Atomic Force Microscopy-Based Single-Cell Force Spectroscopy. Macromol. Rapid Commun. 2012, 33, 1453−1458. (26) Gram, L.; Wedellneergaard, C.; Huss, H. H. The Bacteriology of Fresh and Spoiling Lake Victorian Nile Perch (Lates niloticus). Int. J. Food Microbiol. 1990, 10, 303−316. (27) Tripathi, P.; Beaussart, A.; Alsteens, D.; Dupres, V.; Claes, I.; von Ossowski, I.; de Vos, W. M.; Palva, A.; Lebeer, S.; Vanderleyden, J.; Dufrene, Y. F. Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG. ACS Nano 2013, 7, 3685−3697. (28) Alsteens, D.; Beaussart, A.; El-Kirat-Chatel, S.; Sullan, R. M. A.; Dufrêne, Y. F. Atomic Force Microscopy: A New Look at Pathogens. PLoS Pathog. 2013, 9, e1003516. (29) Vesper, S. J. Production of Pili (Fimbriae) by Pseudomonas fluorescens and Correlation with Attachment to Corn Roots. Appl. Environ. Microbiol. 1987, 53, 1397−1405. (30) Das, T.; Sharma, P. K.; Krom, B. P.; van der Mei, H. C.; Busscher, H. J. Role of Edna on the Adhesion Forces between Streptococcus mutans and Substratum Surfaces: Influence of Ionic Strength and Substratum Hydrophobicity. Langmuir 2011, 27, 10113− 10118. (31) Xu, H.; Murdaugh, A. E.; Chen, W.; Aidala, K. E.; Ferguson, M. A.; Spain, E. M.; Nunez, M. E. Characterizing Pilus-Mediated Adhesion of Biofilm-Forming E. coli to Chemically Diverse Surfaces Using Atomic Force Microscopy. Langmuir 2013, 29, 3000−3011.

ACKNOWLEDGMENTS This work was supported by The Danish Council for Independent Research (Sapere Aude career program) (Grant 11-105250) and by JPK Instruments AG (Berlin, Germany).

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REFERENCES

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dx.doi.org/10.1021/la404673q | Langmuir 2014, 30, 4019−4025