Influence of biomaterial nanotopography on the adhesive and elastic properties of Staphylococcus aureus cells

Abstract

Despite the well-known beneficial effects of biomaterial nanopatterning on host tissue integration, the influence of controlled nanoscale topography on bacterial colonisation and infection remains unknown. Therefore, the aim of the present study was to determine the nanoscale effect of surface nanopatterning on biomaterial colonisation by S. aureus, utilising AFM nanomechanics and single-cell force spectroscopy (SCFS). Nanoindentation of S. aureus bound to planar (PL) and nanopatterned (SQ) polycarbonate (PC) surfaces suggested two distinct areas of mechanical properties, consistent with a central bacterial cell surrounded by a capsullar component. Nevertheless, no differences in elastic moduli were found between bacteria bound to PL and SQ, suggesting a minor role of nanopatterning in bacterial cell elasticity. Furthermore, SCFS demonstrated increased adhesion forces and work between S. aureus and SQ surfaces at 0 s and 1 s contact times. Although WLC modelling showed similarities in contour lengths for attachment to both surfaces, Poisson analysis suggests increased short-range forces for the S. aureus–SQ interactions. In the case of S. aureus–PL, long-range forces were found to not only be dominant but also repulsive in nature, which may help explain the reduced adhesion forces observed during AFM probing. In conclusion, although surface nanopatterning does not significantly influence the elasticity of attached bacterial cells, it was found to promote the early-adhesion of S. aureus cells to the biomaterial surface.

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1. Introduction

Biomaterials are currently being employed in modern medicine for the augmentation or replacement of missing or diseased tissues.^1,2 More specifically, they are used in a wide range of medical applications such as catheters, artificial heart valve replacements, and orthopaedic and dental implants.³ In recent years, many surface modifications have been incorporated into the design of biomaterials in the hope of improving their biological activity and host tissue integration.^3,4 Amongst these improvements, controlled nanopatterning of the biomaterial surface has been shown to directly influence human stem cell proliferation and differentiation, giving it an important advantage compared to uncontrolled topographies and planar surfaces.^5,6 Despite these positive effects, the effect of biomaterial nanopatterning on bacterial surface colonisation remains unknown.⁷

After implantation, many bacterial strains have demonstrated an increased capacity of adhering to the surface of biomaterials and artificial implants.⁸ As implant surface infection has been repeatedly shown to be detrimental for biomaterial-host tissue integration and can lead to complications as severe as replacement surgery, there is current focus on understanding the process of bacterial adhesion to biomaterial surfaces.⁹ Amongst others, Staphylococcus aureus has shown an increased likelihood to colonise and infect implants in humans and its presence is related to negative clinical prognosis.^10–12 S. aureus is a facultative anaerobic and coagulase-positive Gram positive cocci,¹³ with the ability to cause disease in humans and animals ranging from bone infection to pneumonia and septicaemia.¹⁴ Some of the many virulence factors of S. aureus are the presence of capsule and surface proteins, which promote adhesion to biotic and abiotic surfaces and aid in the process of biofilm formation.¹⁵ In recent years, S. aureus has demonstrated increased antibiotic resistance and as a result, novel antibacterial and anti-adhesive approaches are currently being explored in hopes of developing new strategies against biomaterial surface infection.^16–18

Atomic force microscopy (AFM) has proven to be a valuable tool for the in vitro characterisation of cellular and sub-cellular mechanics.¹⁹ Particularly for microbiology, it allows the possibility to study the nanomechanics of living bacterial cells in buffer conditions without the need for prior sample preparation.²⁰ Using this methodology, it is possible to assess the elastic properties of surface-bound bacteria by indenting the bacterial surface with an AFM cantilever.²¹ Furthermore, by employing approaches such as single-cell force spectroscopy (SCFS), bacterial adhesion to biological and non-biological surfaces can be studied in the nano- and pico-meter ranges.^22–24

Several studies have used AFM to probe the nanomechanics of S. aureus and its adhesion to substrates and other cells.^25–27 However, little is known regarding the influence that nanopatterning exerts on S. aureus adhesion and early-colonisation of the implant surface. As a result, the aim of this study was to determine the nanoscale effect of surface nanopatterning on S. aureus biomaterial colonisation by utilising AFM nanoindentation and single-cell force spectroscopy (SCFS) techniques.

2. Experimental

2.1. Polycarbonate surface characterisation

Two distinct engineered polycarbonate (PC) surfaces were employed throughout the study. Nanopatterned PC surfaces, consisting of 120 nm pits with 300 nm centre–centre separation in a square arrangement (SQ), were obtained with a previously reported protocol.⁵ A planar PC surface (PL) was employed as a smooth control.

Previous to any measurements, nanopatterned surfaces were prepared and cleaned by sonication in dH₂O for 5 min, washed with 70% ethanol and dried under N₂ airflow. Characterisation of surface topography was obtained by AFM imaging (Dimension 3100, Bruker, Santa Barbara, USA) employing intermittent contact mode in air, utilising MSNL-10 (Bruker, USA) cantilevers with a scanning rate of 1.0 Hz. Average surface roughness (R_a) was determined using height images obtained during AFM scanning (n = 3), and processed using proprietary NanoScope Analysis 1.5 software (Bruker, USA). For bacterial experiments, surfaces were placed in a UV-chamber and sterilised with a 20 min cycle (BR-506, UVC Light Products, UK). Surface hydrophilicity was determined after surface sterilisation, by contact angle measurements with deionised water (dH₂O) utilising a Cam 200 Optical Contact Angle Meter (Biolin Scientific, Germany). A single 5 μl droplet was applied to the surfaces, and the average angle of contact over 10 seconds was measured and recorded (n = 3).

2.2. Bacterial cultures

Stocks of S. aureus (strain 8325-4) were maintained at −80 °C in 85% glycerol/15% tryto-soy broth (TSB, Oxoid Ltd, UK) medium. For experiments, S. aureus were grown in TSB for 16 h at 37 °C and aeration until stationary phase. Subsequently, 100 μl of bacterial suspension was diluted 10-fold in phosphate-buffer saline (PBS 1×, Lonza, Belgium) and harvested at 5000 rpm for 1 min (Eppendorf 5417R, UK). Resulting pellets were re-suspended in 1 mL PBS and utilised immediately for experiments.

2.3. Sample preparation for scanning electron microscopy/focused ion beam (SEM-FIB) milling and imaging

To obtain images of S. aureus colonisation of PC surfaces, a 500 μl droplet of bacterial suspension was incubated on each surface for 10 min, rinsed with PBS to remove unattached cells, and fixed immediately with 4% glutaraldehyde. Samples were then dehydrated with 10 min serial washes in 50, 70, 90 and 100% ethanol, and sputter coated with gold. Imaging was carried out with an XB1540 (Carl Zeiss, Germany) SEM-FIB system with an acceleration voltage of 10 kV at magnifications of 50 000× and 100 000×. FIB milling was carried out with a 30 kV:20 mA gallium beam probe, by tilting the sample 54° and performing serial linear millings on S. aureus cells until exposing the bacterial–surface interface.

2.4. Sample preparation for AFM

2.4.1. AFM imaging and bacterial nanomechanics. To attach single S. aureus cells onto substrates for imaging and nanomechanics, a 20 μl droplet of bacterial suspension was deposited onto each PC surface and incubated for 10 min. For imaging, samples were washed after incubation with dH₂O and softly dried under N₂ airflow. For nanoindentation, samples were washed with PBS to remove unattached cells, and re-suspended with 100 μl of TRIS buffer (Sigma-Aldrich, UK). For all nanomechanic experiments, bacterial cells were maintained submerged in TRIS buffer throughout experimentation.

Both imaging and force–volume mapping of the bacterial surface of S. aureus were obtained by employing a JPK Nanowizard system (JPK Instruments, Germany) mounted on an inverted optical microscope (Olympus IX71, Olympus, Japan). Imaging was carried out with a NCS35 cantilever (MikroMasch, USA) in intermittent contact mode in air, tuned at ∼110 kHz. Set point and gain values were adjusted in situ during scanning for image optimisation. Images were obtained at 512 × 512 pixels with an average scanning rate of 0.5 Hz.

For force–volume mapping, MSNL-10 cantilevers with a spring constant of ∼0.1 N m⁻¹ were used. After locating an isolated attached bacterium, force-curves were obtained (constant speed of 2 μm s⁻¹ and a loading force of 3 nN) at random points of the cell centre and perimeter of each bacterium. Six independent S. aureus cells were indented and analysed per surface. As minimal adhesion between the cantilever and bacterial cell was recorded and an indentation depth of ∼50 nm was obtained, Young's modulus (YM) was determined from the extension curve by applying the Hertzian model as previously described in the literature:²⁸

where F is force, E is the Young's modulus, R is the radius of the indenter, δ is indentation distance and v is the Poisson's ratio of the indented sample (considered as 0.5).

2.4.2. Single-cell force spectroscopy (SCFS). For SCFS experiments, customised colloidal probes were fabricated in order to immobilise S. aureus cells by utilising a protocol previously published.²⁶ Briefly, the end of a tipless cantilever (NP-O10, Bruker, Santa Barbara, USA) was brought into contact with a thin layer of UV-curable glue (Loctite, UK) for 10 s. Subsequently, the glue-coated tip was approached to a ∼10 μm silica microsphere (Whitehouse Scientific, UK) with a maximum loading force of 0.5 nN for 3 min. Upon retraction, effective attachment of the silica bead was observed by optical microscopy. Functionalised cantilevers were then UV-cured for 10 min, and correct placing of the microsphere was assessed by SEM imaging (Philips XL30 FEG SEM, FEI, Eindhoven, Netherlands). As a next step, functionalised cantilevers were coated for 1 h with a solution of 4 mg ml⁻¹ dopamine hydrochloride (poly-DOPA) in 10 mM TRIS buffer (pH 8.0), washed with dH₂O and dried with N₂. All cantilevers were calibrated using thermal tuning (∼0.3 N m⁻¹ spring constants), and stored at 4 °C until AFM experiments.

To functionalise colloidal probes with living S. aureus cells, cantilevers were mounted onto the AFM and submerged into a 20 μl droplet of bacterial suspension. The probe was then brought into contact with an isolated cell, with a loading force of 0.5 nN for ∼3 min until attachment was observed. Cantilevers were then retracted, transferred above the PC surface and submerged in TRIS buffer being careful not to dehydrate the S. aureus probe. Experiments were carried out with a loading force of 0.5 nN, a constant speed of 2 μm s⁻¹, and surface delay times of 0 s and 1 s. Each of the S. aureus functionalised probes were utilised only for a single experiment and discarded thereafter. Four independent S. aureus probes were utilised for each surface (totalling 8 probes).

2.5. Data analysis

All obtained images and force curves were analysed using the JPK Data Processing Software v.5.1.8 (JPK Instruments, Germany). For nanomechanics, histograms and median (Mdn) values were obtained for each surface and contact time, and significance was determined by applying the non-parametric Mann–Whitney test (p < 0.05). From SCFS force curves, maximum adhesion force (nN) between the bacterial cell and cantilever was obtained as the lowest negative value for force during the retraction phase, and overall adhesion work (aJ) was obtained by integrating the area under the retraction curve. Unbinding peaks observed during retraction were fitted with the worm-like chain (WLC) model as previously described assuming a persistent length of 0.36 nm.²⁹ Finally, a Poisson analysis of S. aureus–PC unbinding was performed employing a previously published approach, to decouple adhesion into short-range (F_SR) and long-range (F_LR) forces.³⁰

3. Results and discussion

3.1. Characterisation of bacterial adhesion onto PC surfaces

AFM imaging of PC substrates demonstrated different topographies for the PL and SQ surfaces (Fig. 1). SQ exhibited a very distinct patterning, with clearly defined nanopits at regular intervals consistently throughout the surface, which contrasted strongly with the smooth topography observed for PL. AFM surface cross sections of SQ further showed rounded nanopits with an average diameter of 99 ± 6 nm (n = 20) and a depth of ∼70 nm. Also, R_a measurements showed an increased surface roughness of 13.7 ± 0.8 nm for SQ surfaces compared to 0.4 ± 0.0 nm for PL ones. No difference was found in surface wettability, with values for PL and SQ at 80.6 ± 2.7° and 80.3 ± 1.5° respectively. However these values do indicate that PC surfaces, irrespective of patterning, are slightly hydrophilic which has been previously shown to favour staphylococcal attachment.³¹

Fig. 1 Characterisation of planar (PL) and nanopatterned (SQ) polycarbonate (PC) surfaces. 2 × 2 μm AFM 3D reconstruction images of (A) PL and (B) SQ demonstrate marked topographical differences between both surfaces. (C) and (D) correspond to AFM height scans for PL and SQ respectively, from which surface cross sections were obtained (E) (2 × 2 μm scans). From these cross sections, it is possible to observe nanopit depth and patterning on the SQ surface. (F) Although surface roughness was increased in SQ compared to PL surfaces, no differences were found in regards to surface wettability as both substrates were found to display a slightly hydrophilic behaviour.

Images of surface-bound S. aureus cells were successfully obtained with intermittent contact mode AFM imaging. More specifically, it was possible to observe the S. aureus cell surrounded by a microcapsule (Fig. 2a and b). This area appears to not only surround the bacterial cell but to also cover its surface partially, consistent with previous AFM observations which employed a similar S. aureus strain.³² Further phase contrast imaging, which has been previously used to differentiate cell from capsule in streptococcal species,³³ was used to demonstrate differences in physico-mechanical properties between the bacterial cell and adjacent area (Fig. 2c). Height images obtained with force–volume mapping in buffer are also consistent with this bacterial morphology, and although resolution is not as high as with intermittent contact imaging, the bacteria–capsule structure can still be clearly observed in the corresponding pixel map (Fig. 4a). Interestingly, previous research suggests that although S. aureus strain 8325-4 carries a serotype-5 capsule gene, it is defective in capsule expression.^34,35 However, Coldren and colleagues found very similar morphological characteristics in a serotype-8 capsule-positive S. aureus strain when imaged with AFM under comparable conditions.³⁶ Serotype-5 and serotype-8 bacterial capsules are considered to have similar characteristics,³⁴ and therefore it is possible that the strain is effectively expressing a capsule-like structure.

Fig. 2 AFM intermittent contact imaging of S. aureus 8325-4 adhered to PL and SQ surfaces. 3D reconstruction images of S. aureus attached to (A) PL and (B) SQ surfaces. It is possible to observe the S. aureus cell surrounded and partially covered by capsule (inset arrows) (C) phase contrast image obtained for the bacterium imaged in (A), which evidences distinct structural composition and physico-mechanical properties for both the S. aureus cell (cross) and capsule (stars). The capsule is seen to not only surround the cell but also cover it partially.

Although careful preparation was used, it was not possible to image the S. aureus capsule under the SEM-FIB, suggesting that it is destroyed or lost during sample dehydration and preparation (Fig. 3). Subsequently, upon exposing the S. aureus–PC interface with FIB milling, only minor contact between bacteria and surface could be observed and therefore it is believed that the bacteria–substrate interaction is mostly mediated by capsule rather than by the bacterial cell itself. Overall, imaging suggests that S. aureus capsule does not only account for a significant part of bacterial size observed in AFM imaging, but most importantly, it also increases the effective contact area between the bacterium and PC surface during surface attachment.

Fig. 3 SEM-FIB imaging and milling of the S. aureus–PC interface. Imaging of S. aureus cells attached to PL (A and B) and SQ (C and D) surfaces before and after FIB milling, respectively ((A and C) 50 000×; (B and D) 100 000×). S. aureus capsule is absent due to it being destroyed during sample preparation, nevertheless, a minor degree of interaction can still be observed between bacterial cells and PC surfaces after FIB milling.

3.2. Underlying substrate topography does not influence the nanomechanical properties of surface-bound S. aureus

The elastic properties of surface-bound S. aureus were obtained in force–volume mode by performing a number of force curves on the bacterial surface. Two distinct mechanical behaviours were observed for S. aureus, consistent with the presence of a stiffer cell body surrounded by capsule of decreased stiffness (Fig. 4b). This observation was consistent throughout measurements for S. aureus cells attached to both studied PC surfaces. YM for the bacterial cell was found to be 2.20 MPa for PL and 4.14 MPa for SQ, in the range of previously reported values for S. aureus 8325-4 elasticity.³⁷ However, surrounding the central bacterium, an area with significantly reduced YM of 116.58 kPa for PL and 92.89 kPa for SQ was observed (p < 0.05) (Fig. 4c and d).

Fig. 4 Nanomechanics of surface-bound S. aureus cells attached to PL and SQ in buffer. (A) 3D reconstruction image obtained during a S. aureus force–volume map, in which both the bacterial cell and capsule (arrow) can be observed. (B) 16 × 16 pixel stiffness map on the surface of a S. aureus cell attached to a PL surface. An area of decreased stiffness can be observed surrounding the bacterial cell (asterisk). Values for Young's modulus (YM) obtained with the Hertzian model for (C) the bacterial cell and (D) capsule are shown (n = 6 independent cells per group). A marked difference is observed between the two regions, with the bacterial cell showing values in the MPa range and capsule being in the kPa range. No significant differences in YM were found between PL and SQ, suggesting that surface nanotopography does not influence the mechanics of S. aureus cells (p > 0.05, Mann–Whitney).

Regarding the influence of the underlying surface on of S. aureus nanomechanics, no significant differences were found in YM between bacteria bound to PL and SQ substrates (p > 0.05) (Fig. 4c and d). As AFM imaging demonstrated that a typical S. aureus cell directly interacts with a number of nanopits on the SQ surface, any effect that nanotopography may have on bacterial cell elasticity should be clearly noticeable at the single-cell level. However, both S. aureus cell and capsule nanomechanics were not affected by the presence or absence of surface nanopatterning. It is also possible that nanopatterning may only exert a localised effect on elasticity in the vicinity of the bacteria–nanopattern interface, and therefore it cannot be explored by solely indenting the top region of attached S. aureus cells.

3.3. Adhesion forces between S. aureus–PC surfaces are increased by the presence of surface nanopatterning

To analyse the effect of surface nanopatterning on the early colonisation of S. aureus, functionalised AFM bacterial probes were constructed and probed against PC surfaces at 0 s and 1 s contact times (Fig. 5 and 6). Adhesion force and work between S. aureus and PL surfaces were found to be < 0.05 nN and <0.05 aJ at 0 s surface contact times delays. However, increasing the contact time to 1 s raised these values to 0.11 nN and 5.01 aJ respectively (p < 0.0001). Adhesion forces between S. aureus and SQ surfaces was increased at both time points compared to PL, with values of 0.10 nN at 0 s and 0.23 nN at 1 s surface delay times (p < 0.0001). A similar increase was observed for the work of adhesion, with values of 4.28 aJ and 18.75 aJ for 0 s and 1 s respectively (p < 0.0001). Altogether, these results suggest that both contact time and surface nanopatterning directly influence the early-adhesion of S. aureus to PC surfaces. In the literature, SCFS of S. aureus strain 8325-4 has been previously employed to measure its adhesion with Candida albicans hyphae and yeast cells and fibronectin-functionalised AFM cantilevers.^38,39 In both cases, reported adhesion forces were of higher magnitudes than the ones observed in the present study between S. aureus and PC. As these studies examined specific receptor–ligand interactions between S. aureus and biological substrates, it is possible to hypothesise that the reduced adhesion forces observed for the unbinding of S. aureus–PC, irrespective of patterning, is a reflection of a lack of specificity between the bacterial cell and surface. It also remains possible that increased loading forces were employed in these studies, which has been shown to promote bacterial adhesion to substrates.⁴⁰

Fig. 5 Adhesion forces and work between living S. aureus and PC surfaces at short contact times. Histogram of adhesion forces and work recorded between S. aureus-functionalised AFM probes and PL and SQ surfaces, with a surface delay of 0 s. For SQ, both parameters were significantly increased compared to PL surfaces (p < 0.05). The number of non-adhesive events per group is indicated in the upper left corner of each histogram.

Fig. 6 Adhesion forces and work between living S. aureus and PC surfaces at increased contact times. Histogram of adhesion forces and work recorded between S. aureus-functionalised AFM probes and PL and SQ surfaces, with a surface delay of 1 s. Increasing the contact time to 1 s increased adhesion forces and work in both studied surfaces (p < 0.05). Similar to 0 s contact times, both parameters were also found to be increased in SQ compared to PL surfaces (p < 0.05).

Representative force curves obtained for both PL and SQ surfaces can be observed in Fig. 7a. In both cases, ‘sawtooth-like’ unbinding peaks indicative of specific adhesion between the probe and surface were clearly observed throughout measurements. These unbinding events were fitted with the WLC model, yielding contour length values predominantly situated in the 50–400 nm range (Fig. 7b). The number of unbinding peaks found for SQ (n = 942) was slightly increased compared to PL (n = 889). Interestingly, it was possible to fit multiple curves to the contour length distributions of both PL and SQ. PL surfaces displayed peaks at 140 nm, 270 nm and 358 nm; while SQ surfaces were also found to have similar peaks at lengths of 147 nm, 253 nm, and 380 nm respectively (Fig. 7b). This data suggests that a range of surface and/or capsule receptors are involved in the adhesion between S. aureus and PC surfaces, with no important differences found regarding the presence or absence of nanopatterning. In a recent study, average contour lengths between S. aureus and titanium (Ti) implant surfaces were reported at 314.1 ± 9.3 nm.²⁶ Ti is more hydrophilic than PC and biomaterial surface hydrophilicity has been previously shown to favour bacterial adhesion.³¹ However, it has yet to be determined if contour lengths observed for S. aureus–PC unbinding correspond to the same Ti-binding receptors stretched to different lengths, or if they reflect another surface adhesion mechanism.

Fig. 7 Worm-like chain (WLC) modelling of force-extension peaks observed during S. aureus–PC unbinding. (A) Representative retraction curves observed during between S. aureus probes and PL and SQ surfaces. Single unbinding events can be observed in both cases, as indicated by the arrows. Insets represent AFM deflection images of each surface (2 × 2 μm scans). (B) WLC modelling yielded multiple peaks for contour lengths in both PL and SQ, as observed in the corresponding histograms.

Furthermore, Poisson analysis of S. aureus–PC unbinding was carried out to decouple overall adhesion forces into F_SR and F_LR. PL surfaces were found to have a F_SR of −0.08 ± 0.02 nN, while SQ surfaces showed an increased value of −1.42 ± 0.02 nN (Table 1). Interestingly, F_LR for PL surfaces was found to be a positive value of 0.38 ± 0.25 nN, and as such it indicates that the overall long-range forces acting between S. aureus and PL are repulsive in nature. Increased F_SR between S. aureus–SQ surfaces paired with a repulsive F_LR in S. aureus–PL may help explain the reduced adhesion force and work observed in the latter case.

Table 1 Poisson analysis of S. aureus unbinding from PL and SQ surfaces in buffer

Surface	Specific (FSR)		Non-specific (FLR)
	Mean (nN)	SE	Mean (nN)	SE
PL	−0.08	0.02	0.38	0.25
SQ	−1.42	0.02	−0.23	0.01

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Overall, the use of SCFS was an effective approach to study the early-colonisation of S. aureus onto PC surfaces at the nanoscale. In biomaterial infection, early bacterial colonisers are believed to come into contact with the surface with minimal to no external loading forces being applied. Therefore, in this PC-based model of biomaterial nanopatterning, loading forces for S. aureus probes were reduced (0.5 nN) to avoid deformation of the bacterial cell during probing. By utilising this ‘zero-force contact’ approach, it is believed that the influence of AFM probing on adhesion values can be kept to a minimum. High loading forces would therefore not effectively recreate the physiological process of bacterial adhesion, as they would be promoting the interaction between bacteria and surface. This effect was recently demonstrated by Chen et al., where the adhesion between S. aureus strain 8325-4 and a glass surface was found to be proportional to the loading force applied.⁴⁰

In the literature, the capsule has been considered to play an active role in bacterial attachment to biomaterial surfaces.^41,42 Considering the early contact times utilised in this study (0 and 1 s), it is believed that adhesion between S. aureus and PL and SQ surfaces at these time points is mostly mediated by the interaction between the capsule and substrate. Although nanopatterning theoretically reduces the contact area between cell and surface, adhesion was found to be increased for SQ surfaces compared to PL. It is hypothesised that this observation is a result of the physical interaction between the capsule and the roughness conferred by the nanopatterned surface. This mechanical retention might increase bacterial attachment to the surface during probe retraction, yielding increased adhesive forces probed with the AFM. Also, the interaction between capsule and/or surface receptors and the surface were observed, in the form of specific unbinding peaks. At this stage however, we do not believe that it is possible to pinpoint the exact receptors involved in the adhesion of S. aureus to PC surfaces; nevertheless, due to the predicted contour lengths observed with WLC modelling (∼150–400 nm), we believe that a range of receptors are involved in this interaction.²⁶

In the past, surfaces with nanoscale topographies were found to possess improved antibacterial properties against S. aureus, when bacteria are cultured for <1 h and macro-scale bacterial attachment assays such as fluorescence microscopy and spread plate methods were employed.^7,43–45 However, results from the present study suggest that nanopatterning increases the colonisation of biomaterial surfaces at early contact times (0 and 1 s). Although bacterial adhesion to surfaces is a crucial initial phase,⁴⁶ it does not account on its own for the entire process of biomaterial colonisation.⁴⁵ Therefore, it remains possible that although attachment of S. aureus to nanopatterned surfaces is initially increased at very short time points, bacteria may not be able to effectively colonise the surface due to reduced proliferation capabilities or decreased capsule secretion at increased contact times. Although contact times of ≤1 s may be short in relation to the lifetime of a biomaterial infection process, early-colonising bacteria could potentially become a ‘base-layer’ for the attachment of secondary bacteria at increased time points.⁴⁷ Future efforts should focus on further understanding the in vivo relevance of early biomaterial colonisation by S. aureus, and if promoting or inhibiting this initial bacterium–surface interaction can aid in the search for novel ways to control biofilm formation without compromising the increased biological properties of nanopatterned surfaces.

4. Conclusion

Both nanoindentation and AFM SCFS were found to be powerful tools to study the nanomechanics of living S. aureus cells in buffer conditions. Imaging of surface bound S. aureus showed the presence of adjacent capsule regardless of surface patterning, which was found to have reduced elasticity compared to the central bacterial cell. No surface-induced changes in bacterial nanomechanics were found in S. aureus attached to PL and SQ. However, SCFS with S. aureus functionalised probes demonstrated increased adhesion forces and work between bacteria and SQ surfaces. Poisson analysis suggests that this is due to higher short-range forces between S. aureus–SQ and repulsive long-range forces between S. aureus–PL. Overall, surface nanotopography was found to influence S. aureus attachment to PC surfaces at early time points, and further research should evaluate is this effect is observable at increased contact times and in other biomaterial surfaces of clinical relevance.

Acknowledgements

This work has been funded by the Becas-Chile PhD Scholarship Programme.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12504b

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