Influence of Fimbriae on Bacterial Adhesion and Viscoelasticity and

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Influence of Fimbriae on Bacterial Adhesion and Viscoelasticity, and Correlations of the Two Properties with Biofilm Formation Huabin Wang, Jonathan J. Wilksch, Ligang Chen, Jason W. H. Tan, Richard A. Strugnell, and Michelle L Gee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03764 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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Influence of Fimbriae on Bacterial Adhesion and Viscoelasticity, and Correlations of the Two Properties with Biofilm Formation Huabin Wang,*,†, ‡,‖ Jonathan J. Wilksch,§ Ligang Chen,† Jason W. H. Tan,§ Richard A. Strugnell,§ and Michelle L. Gee*,‡ †

Chongqing Key Laboratory of Multi-Scale Manufacturing Technology, Chongqing Institute

of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China ‡

§

School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia

Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty

Institute for Infection and Immunity, Parkville, Victoria 3010, Australia ‖

Key Laboratory of Interfacial Physics and Technology, Chinese Academy of Sciences,

Shanghai 201800, China

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ABSTRACT：The surface polymers of bacteria determine the ability of bacteria to adhere to a substrate for colonization, which is an essential step for a variety of microbial processes, such as biofilm formation and biofouling. Capsular polysaccharides and fimbriae are two major components on a bacterial surface, which are critical for mediating cell-surface interactions. Adhesion and viscoelasticity of bacteria are two major physical properties related to bacteria-surface interactions. In this study, we employed atomic force microscopy (AFM) to interrogate how the adhesion work and the viscoelasticity of a bacterial pathogen, Klebsiella pneumoniae, influence biofilm formation. To do this, the wild-type, type 3 fimbriae-deficient, and type 3 fimbriae-overexpressed K. pneumoniae strains have been investigated in an aqueous environment. The results show that the measured adhesion work is positively correlated to biofilm formation, however the viscoelasticity is not correlated to biofilm formation. This study indicates that AFM-based adhesion measurements of bacteria can be used to evaluate the function of bacterial surface polymers in biofilm formation and to predict the ability of bacterial biofilm formation.

KEYWORDS: bacteria, fimbriae, atomic force microscopy, adhesion, biofilm, viscoelasticity

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INTRODUCTION Understanding the fundamental mechanism of adhesion between bacteria and substrates is important for helping solve many bacteria-related issues such as food and water contamination, industrial and medical equipment biofouling and infectious diseases.1,

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Bacterial adhesion depends largely on the properties of bacterial surface components, such as the polysaccharide capsule and fimbriae.3, 4 The polysaccharide capsule is a pronounced layer of polysaccharides linked to the bacterial cell outer membrane via covalent attachments to either phospholipid or lipid-A molecules and has a thickness of several hundred nanometers.57

Fimbriae are proteinaceous, hair-like structures that radiate peritrichously from the cell

outer membrane to a distance of several hundred nanometers to several microns.4,

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The

influence of bacterial surface components on cellular adherence to surfaces has been studied using a variety of microbiology, immunology, and molecular biology techniques such as cell counting, bacterial labeling, light microscopic analysis, flow chamber or quantifying cells removed from the surface.8-11 However, these macro-level methods cannot be used to elucidate the fundamental biophysical mechanisms of various components contributing to bacterial adherence to a surface.8, 12 Atomic force microscopy (AFM) has emerged in recent years as a powerful technique in single-cell microbiology due to its capability for imaging live bacterial cells in physiologically-relevant environments and probing the physical properties of cell interactions at the nanoscale level.13, 14 AFM mechanical measurements revealed that bacterial cells are viscoelastic

materials

exhibiting

both

viscosity

and

elasticity

when

undergoing

deformation.15, 16 The viscosity of a material describes its resistance to strain and shear flow linearly with time due to the diffusion of molecules within the amorphous material when subjected to an applied stress while the elasticity describes the propensity of a material to completely recover to its original size and shape upon load removal.17, 18 The adhesion work 3


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and the viscoelasticity (deformation property) are two key physical properties of bacteria that occur between the bacterial cell and the surface, which is crucial for biofilm formation and can be measured using AFM.19-21 It has been demonstrated that AFM measurements of adhesion work at the nanoscale could be used to predict the macroscale adhesion behaviour and strength of bacterial cells,22 and that the viscoelasticity of a bacterium is closely related to properties of the bacterial envelope and plays important roles in biofilm formation and development.23 However, few studies have hitherto been conducted to investigate how the adhesion work and the viscoelasticity of bacteria influence biofilm formation, likely due to the highly interdisciplinary characteristics of such kind of research. Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic pathogen involved in a variety of hospital-acquired infections including pneumonia, urinary tract infections, surgical wound infections and bacteremia.4,

24, 25

Besides a dense capsule formed by

polysaccharides, most K. pneumoniae isolates produce two well-characterized types of fimbriae: type 3 fimbriae (~0.5-2 µm long) and type 1 fimbriae (~1 µm long).6, 26, 27 Type 3 fimbriae expression can strongly promote biofilm formation on abiotic surfaces.28-31 In our previous work, we investigated the biophysical mechanisms underlying how the capsular polysaccharides protect bacterial cells from external stresses and how the fimbriae influence the organization of the bacterial surface structure based on AFM nanomechanics measurements.7, 32, 33 In the present work, we expanded our work to examine the biophysical properties of bacteria including the adhesion (described by adhesion work) and viscoelasticity (described by the factor of viscosity) using AFM nanomechanics, and evaluated how these two physical properties influence biofilm formation. Our results show that the adhesion work is positively related to biofilm formation, however the factor of viscosity is not correlated to biofilm formation. The type 3 fimbriae can regulate these two physical properties, since both the adhesion work and the factor of viscosity can be

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changed by the amount of the type 3 fimbirae expressed on the bacteria surface. It was observed that reduced fimbriae expression could decrease the adhesion work, resulting in reduced biofilm formation ability. This study indicates that adhesion work can be an indicator used to predict the ability of biofilm formation and that regulating the amount of fimbriae could be a useful method for preventing bacterial fouling on a surface. The work presented here would be very helpful for designing effective strategies to prevent bacterial proliferation on medical device surfaces or in the body of patients.

MATERIALS AND METHODS Bacterial Strains, Culture Conditions, Harvesting and Static Biofilm Assays. K. pneumoniae AJ218 (capsule serotype K54) is a human urinary tract infection isolate.24 All strains were maintained on Luria-Bertani (LB) agar at 37°C. These cultures were used to inoculate LB broths which were grown for 16 hours at 37°C while shaking (180 rpm). Stationary-phase cells were subsequently harvested by centrifugation (10 min at 3500 × g) and washed twice with Milli-Q water (18.2 MΩ cm1). The final concentration of cells in Milli-Q water was approximately 2 × 108 CFU/mL. Kanamycin (50 µg/ml) was used to select for growth of type 3 fimbriae-deficient strains while chloramphenicol (60 µg/ml) was used to select for type 3 fimbriae-overexpressed strains. A type 3 fimbriae knockout mutant, in which the mrkA gene encoding the major fimbrial subunit was deleted and replaced with a kanamycin resistance cassette, was constructed in K. pneumoniae AJ218, as described previously.9 K. pneumoniae AJ218 which over-expressed type 3 fimbriae contained a plasmid vector (pMrk) carrying the type 3 fimbriae operon (mrkABCDF) operon, as described previously.9 Static biofilm assays were formed according to the method described in our previous work.9 Briefly, bacterial strains were grown overnight in LB at 37°C and then sub-cultured 5


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1:100 in 100 μL M63B1-GCAA minimal media (containing 1% glycerol and 0.3% casamino acids) in duplicate 96-well, flat bottom microtitre plates. Following 24 h static incubation at 37°C, non-adherent bacteria were removed and wells were washed twice with Milli-Q water. Biofilms were stained for 15 min with 125 μL of 0.1% (w/v) crystal violet solution (SigmaAldrich) and the wells were subsequently washed twice with Milli-Q water to remove unbound crystal violet. The bound dye was solubilised from adherent cells with 33% acetic acid and quantified by measuring OD595. Detection of type 3 fimbriae. Type 3 fimbriae expression was examined by mannoseresistant hemagglutination (MRHA) assays.34 Sheep erythrocytes were tanned by incubating equal volumes of 0.1% (w/v) tannic acid (Sigma-Aldrich) solution in saline and a 3% erythrocyte suspension in PBS for 15 min at 37°C. The erythrocytes were then washed twice in PBS. Bacteria strains were grown overnight in LB and subsequently washed and resuspended in PBS to approximately 1×1010 CFU/mL. A series of 2-fold dilutions of the bacterial suspension with or without 4% D-mannose was mixed with equal volumes of erythrocytes for 10 min. The minimum bacterial density (CFU/mL) needed to agglutinate erythrocytes was then measured. The experiments were performed in triplicate. Bacterial Sample Preparation for AFM Measurements. Polyethyleneimine (PEI)coated glass disks were prepared by depositing 0.5 ml PEI solution (0.005% w/v) on a freshly prepared glass disk for 4 h. After coating with PEI, disks were rigorously rinsed with Milli-Q water and air dried under sterile conditions. To immobilize bacteria, a drop of cell suspension was deposited onto the disk and left for 1.5 h at room temperature, then rinsed copiously with Milli-Q water and immediately immersed in Milli-Q water. All mechanical measurements were performed after 3-4 hours immersion. Atomic Force Microscopy and Force Measurement. All AFM experiments were performed with an MFP-3D instrument (Asylum Research, Santa Barbara, CA) in Milli-Q

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water. Silicon nitride cantilevers were purchased from Bruker Corporation (MLCT, Camarillo, CA) with a nominal spring constant of 0.01 N/m and a nominal probe curvature radius of 20 nm (according to product specifications), and their respective spring constants were calibrated through the thermal tune function included in the MFP 3D software. All cantilevers were cleaned in a BioForce UV/ozone cleaner (BioForce Nanosciences, Inc., Ames, IA) for 15 minutes before use. Silicon cantilevers are slightly negatively charged in Milli-Q water and widely used in bacterial force measurements to model nonspecific interactions between bacteria and negatively charged inert substrates such as soil and glass. 35, 36

The photodetector sensitivity was measured on a PEI-coated surface.37 The slope of the

linear upward region of the deflection-displacement curves obtained on the PEI-coated glass disk was used to convert the deflection, d, in millivolts to nanometers. No difference was observed on the slopes of deflection-displacement curves measured on PEI-coated glass disk and clean glass disk. The force (F) of cantilever deflection was calculated by F = k × d, where k is the spring constant of the cantilever.38 Force measurements were made by locating the tip at different points along the centreline of the apical surface of individual cells and acquiring a set of force curves at a loading rate of 2 μm s-1. This procedure can avoid artefacts due to the interaction between the side of the tip and the side of the cell to ensure highly reliable results.7, 39 The bacterial cells measured in our experimental conditions are alive, as demonstrated in our previous work.7 The same type of cantilever (MLCT, Santa Barbara, CA) from the same batch was used for all measurements to minimize experimental variations.38 It may be necessary to point out that using different types of cantilevers might lead to small different quantitative results, but the qualitative trend should not be influenced. Igor Pro (Version 6.04, Wavemetrics Inc., USA) and (Version R2010a, Mathworks) routines were written for the analysis of force curves to obtain adhesion work and viscous and elastic deformations. Origin 8.0 was used for the 7


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statistical analysis. Voigt model and ExpDec 1 model were used to fit the data of adhesion work and the factor of viscosity, respectively.

RESULTS AND DISCUSSION MRHA assays can be used to evaluate the amount of the type 3 fimbriae on bacterial surfaces.34 In this assay, the type 3 fimbriae can specifically bind to sheep erythrocytes, which causes clumping (agglutination) of the erythrocytes. The lowest titre of bacterial cells needed to cause a visible agglutination was diluted and cultured on agar to calculate the number of bacterial cells (expressed as CFU/mL). Compared to the wild-type bacteria (9.27×108 CFU/mL), much less type 3 fimbriae-overexpressed bacterial cells (3.33×108 CFU /mL) were needed to cause the agglutination, demonstrating that more type 3 fimbriae were expressed in the type 3 fimbriae-overexpressed strain than in the wild-type bacteria. At the 0.05 level, the difference between the two samples is significantly different, confirmed by the Student’s t-Test. The type 3 fimbriae-deficient strains could not cause an agglutination reaction. The bacterial sample was imaged in Milli-Q water using contact-mode AFM. The bacterial cells of all the three strains are rod-shaped and can be well-dispersed on the PEI substrate, as shown in Figure 1, of a typical height image of the wild-type K. pneumoniae cells. Prior to force measurements, the whole system was stabilized to minimize any possible drifts. The stability of the system was confirmed by comparing the position of a target bacterium in the images collected before and after force measurements. Following topographical imaging of the bacterium, the force curves were measured by positioning the AFM tip at varied points along the centre line of the apex of the bacterium.39 In our experiments, depending on the bacterial strains, the threshold force is slightly different, but all of them are around 2 nN. The threshold force should satisfy two conditions: (1) an overlaid region of the approach and

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retraction curves should appear at the high force end at the measured force curve, in order to calculate the elastic and viscous deformations of the bacteria completely. Otherwise, only non-complete parts of the viscous and elastic deformations can be measured which will lead to inaccurate results;40 and (2) the force applied to indent the bacteria should not be too high because bacterial cells could be punctured by the AFM tip if the loading force is too high.41

Figure 1. AFM 3D image of wild-type K. pneumoniae AJ218 attached to the surface of a PEI-coated glass disk in Milli-Q water. Scan size: 12 μm × 12 μm. Figure 2 is a force curve obtained from an individual bacterium as the AFM tip approaches to (black curve) and retracts (red) from the bacterium. The profile is similar to the force curves probed by AFM on other bacterial species.42-44 The distance 0 nm is set where the tip first registers a repulsive force. In the force measurement, the AFM tip pushes further into the cell surface until a threshold force is reached. Once the threshold force is reached, the tip retracts and moves away from the cell surface (red curve). During the withdrawal process, the overcoming of the force stored in the AFM cantilever to the adhesion force between the AFM tip and bacteria results in the snapping free of the cantilever at some points, indicating by a series of peaks. Once the tip retracts far enough, the tip jumps off from the bacterial surface and returns to its equilibrium positon and forces no longer exist between the tip and bacterial cell anymore. From the force profile, important information regarding the physical properties of bacteria can be extracted, including adhesion work and the factor of viscoelasticity.35, 44-47 9


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Bacterial adhesion work is an important parameter that can be used to assess the affinity of bacteria to interact with a surface.35, 48, 49 The adhesion work is the shaded area between the negative portion of the retraction curve and the zero force line. Besides showing the adhesion property, the force curve also shows the characteristics of the deformation of viscoelastic materials, as indicated by a hysteresis area between and a reasonable overlaid region of the approach and retraction curves before the AFM cantilever returns to its equilibrium position. 47, 50

This observation is consistent with previous reports that bacteria are viscoelastic

materials.15,

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The hysteresis between the approach and retraction curves indicate energy

dissipation during the indentation measurement.44, 51 Useful information can be drawn from such hysteresis by introducing the factor of viscosity, ψ, a parameter that characterizes the relative viscous/elastic deformation of materials under external stresses. ψ is defined as ψ = A1/(A1+A2), where A1 is the dissipated viscous energy due to viscous deformation, A2 is the elastic energy due to elastic deformation, and A1+A2 is the total work done on the sample in the indentation.52 The viscous energy (A1) can be calculated by integrating the area between the approach and retraction force curve above the zero force line, and is the irreversible work of indentation.47, 51 Accordingly, the elastic energy (A2) can be calculated by integrating the area of the retraction force curve above the zero force line, and is the reversible work of indentation, i.e., the deformation that can be recovered upon the withdrawal of the tip. Interested readers can refer to related references for more detailed information.47, 50-52

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Figure 2. An example a force curve for a wild-type K. pneumoniae cell, showing the approach (black) and retraction (red) curves. The cantilever bending has been corrected in the force curve. The zero point is the location where the cantilever starts to deflect upward. The yellow, green and blue areas represent viscous energy, elastic energy and adhesion work, respectively. Numbered peaks indicate adhesion events that contribute to adhesion work. Only peaks having absolute values larger than 0.025 nN were considered. In this case, three events were identified. Figure 3 shows typical force versus distance curves obtained on wild-type (Figure 3A), type 3 fimbriae-deficient (Figure 3B) and type 3 fimbriae-overexpressed (Figure 3C) K. pneumoniae strains in Milli-Q water. Regardless of the type of the bacteria probed, the general features of the force curves are very similar: there is a nonlinear part in the approach curves, a hysteresis between the retraction and approach curves and negative peaks in the retraction curves. All the force curves measured on the three bacterial strains used in this experiment have a viscous deformation region as well as an elastic deformation region. The bacterial surfaces are crowded with surface polymers, e.g., fimbriae and capsular polysaccharides, and are negatively charged as a whole in our experimental conditions.7, 32 The nonlinear part is a result from the interaction between the AFM tip and the bacterial surface charge, and then the bacterial surface polymers.7, 32 In our experiment conditions, the 11


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net charges on both the bacterial surface and the nitric AFM tip are negative, therefore there is a long range repulsive double layer force (electrostatic repulsive force) between the tip and bacterial surface before the tip contacts the bacterial cell.7, 32 Once the tip physically contacts the bacterial surface, cell surface polymers are compressed, with a nonlinear deformation character. Readers can refer to our recent work for more detailed information on this aspect. 7, 32, 44, 53

The hysteresis formed between the retraction and approach curves above the zero force line is due to energy dissipation in the compression process,47 which is very common when deforming soft materials like polymers and cells.46,

50

This phenomenon has also been

observed in the process of compressing bacteria in other studies.44 The peaks observed on the retraction force curves suggests that the bacterial surface polymers are peeling off the AFM tip when the tip moves away from the bacterial surface. It should be noted that the interactions between the AFM tip and bacterial surface represent intrinsically weak physical attachments (hydrogen bonding and van de Waals interactions) and no strong chemical bonds such as covalent or ionic bonds are expected.36 Abundant biomolecules grafted on the bacterial surface generally lead to the attachment of multi-molecules onto the AFM tip, leading to large adhesion forces which is larger than the electrostatic repulsive force in magnitude. The detachment of the molecules from the AFM tip results in multiple peaks in the retraction curve.54 As can be seen from Figure 3, multiple peaks were recorded in the retraction data for all three types of bacteria measured.

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Figure 3. Representative force versus distance curves for wild-type (A), type 3 fimbriaedeficient (B) and type 3 fimbriae-overexpressed (C) K. pneumoniae AJ218 in Milli-Q water. The viscous and elastic energies are indicated by the yellow and green areas, respectively. The blue area indicates the adhesion work. The approach and retraction force curves are indicated by black and red lines, respectively.

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As stated above, the adhesion work was obtained by integrating the negative areas of the retraction force curves. Bacterial surface polymers (e.g., the capsular polysaccharides) can have lengths from tens of nanometers to several hundreds of nanometers and composed of different chemical groups.54 Therefore, the adhesion forces between the AFM tip and the bacterial surface polymers are highly variable a wide range due to the heterogeneity of the distribution of the surface polymers. As a result, the retraction curves are generally not reproducible even on the same bacterium. Consequently, many force curves need to be measured to obtain useful information (about ten force curves from each bacterium) over multiple cells (at least five cells) from several independent samples (at least three independent samples). The statistical results of the extracted physical parameters from more than one hundred force curves for each type of bacteria are shown in Table 1.

Table 1 Summary of extracted parameters for the wild-type, type 3 fimbriae-deficient and type 3 fimbriae-overexpressed K. pneumoniae AJ218 cells. Wild-type

Type 3 fimbriae-

Type 3 fimbriae-

deficient

overexpressed

Adhesion work (nN·nm)

18.74

11.02

16.85

Factor of viscosity

0.42

0.26

0.51

As shown in Table 1, a general trend for the adhesion work is that the wild-type (18.74 nN·nm) bacteria show the strongest adhesion work, followed by the type 3 fimbriaeoverexpressed (16.85 nN·nm) bacteria, and finally the type 3 fimbriae-deficient bacteria (11.02 nN·nm). The Student’s t-Test results indicate that at the 0.05 level the differences of adhesion work between any two of the three bacterial strains are significant. The type 3

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fimbriae-overexpressed bacterial have less measured adhesion work than the wild-type bacteria, which can be ascribed to some steric hindrance applied to the capsular polysaccharides by the additional fimbriae.32, 55 The type 3 fimbriae-deficient bacteria have less adhesion than the wild-type bacteria, which could be attributed to two possible reasons: (1) the removal of fimbriae can lead to reduced numbers of polymers interacting with the AFM tip, which results in less adhesion work; and (2) the removal of fimbriae makes the bacterial surface more organized and crystalline, due to the easier forming of hydrogen networks between capsular polysaccharides, which can reduce the adhesion ability of bacteria.32 The factor of viscosities for these three types of bacteria are 0.51 (type 3 fimbriae over-expressed), 0.42 (wild-type) and 0.26 (type 3 fimbriae-deficient), which is understandable since more polymers grafted on the bacterial cell wall can dissipate more energy which means a higher factor of viscosity. The differences of the factor of viscosity between the three strains are also significant from each other, as confirmed by the Student’s tTest at the 0.05 level. From our previous studies, we know that bacterial capsular polysaccharides and fimbriae can act as a cushion surrounding the bacterial surface to dampen the mechanical force on the bacterial cytoplasm.7, 32 Since a higher factor of viscosity indicates more energy dissipated relatively under an external mechanical load, the results imply that under the same mechanical load (mechanical stressful conditions), in comparison with the wild-type bacteria, the type 3 fimbriae-overexpressed bacteria will experience the lowest mechanical impact (mechanical shock), while the type 3 fimbriae-deficient bacteria will experience the greatest mechanical impact. It has been reported that mechanical forces acting on bacteria can lead to a bacterial response at the gene level and that high shear forces can even kill bacteria.56-58 Therefore, alleviating the mechanical impact or stress is of great significance since in principle it can decrease adverse influences on bacterial survival.

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The trend of adhesion work of these types of bacteria is not in the same trend as that of the factor of viscosity. To interrogate how the adhesion work and the factor of viscosity influence biofilm formation, we performed static biofilm assays on wild-type, type 3 fimbriae-deficient and type 3 fimbriae-overexpressed K. pneumoniae AJ218 bacteria, as shown in Figure 4. It was found that the type 3 fimbriae-deficient bacteria formed the weakest biofilms while the wild-type bacteria formed the strongest biofilm, which is consistent with that of the adhesion work measured using AFM. These results indicate that the measured adhesion work from our AFM experiments can be used to predict the ability of bacterial biofilm formation. However, one may notice that the adhesion work is not proportional to the ability of biofilm formation. The normalized values of adhesion work for type 3 fimbriae-deficient and fimbriae overexpressed bacteria are 0.60 and 0.90, respectively, while that of biofilm formation are 0.06 and 0.61, correspondingly. This discrepancy is likely due to different time scales involved in AFM measurement and biofilm assays.

Figure 4. Comparison of the adhesion work (blue bars), the factor of viscosity (green bars) and biofilm formation (purple bars) of wild-type (WT), type 3 fimbriae deficient (T3FD) and type 3 fimbriae-overexpressed (T3FO) K. pneumoniae AJ218. All the data were normalized to the result of the wild-type bacteria, which is set to 1. The error bars indicate standard errors.

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Using a quartz crystal microbalance (QCM), Olsson et al. investigated the process of bacteria adhering to a gold surface and found that all bacterial strains used in their experiments need approximately 55 seconds to mature the adhesion after the bacteria initially contact with the QCM gold-coated sensor surface.2 In this period of time, the rearrangement of bacterial surface polymers occurred to position the bacteria in the energetically most favourable conformation and maximize the contact points and short range interactions, therefore increasing the adhesion strength between the bacterial cell surface and the substrate.2 This supports the observation that bacterial interfacial polymers can rearrange in the process of biofilm formation, which needs several hours.9 It is difficult to use AFM to investigate interactions for such long time scales, due to the intrinsic drift property of AFM systems in an aqueous environment. In our AFM experiments, the interaction time between bacterial surface polymers and the AFM tip is less than one second; therefore, the chance for the bacterial surface polymers to maximize and optimize the adhesion strength is unlikely to occur. Consequently, the measured adhesion work of the bacteria using AFM is not proportionally correlated to the ability of bacteria to form biofilms. Based on the above discussions, we suggest that the adhesive ability of bacteria at their initial contact with the substrate only partly determines their ability in biofilm formation, and the rearrangement of bacterial surface polymers occurs in the following stage is also a key process for determining biofilm formation.

CONCLUSIONS AFM measurements and biofilm growth experiments indicated that the measured adhesion work of bacteria is positively correlated to the ability of bacteria to form biofilms, although it was not observed to be proportional. This discrepancy is very likely due to the time scale for the measurement of adhesion work using AFM is much shorter than the actual time scale a bacterium needs to reach an equilibrium state in the process of adhering to a surface during 17


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biofilm formation. The measured factor of viscosity is not correlated with either the adhesion work or the biofilm formation. This study indicates that AFM-measured adhesion work can be used to predict the tendency of biofilm formation and that biofilm formation is not only influenced by the initial contact of bacteria to a surface but also influenced by the following rearrangement process of the bacterial surface polymers. In addition, the fimbriae number on the bacterial surface can influence the adhesion ability of bacteria, whereby less fimbriae leads to reduced adhesion ability. The findings in this study can provide useful insights into designing effective antifouling strategies.

AUTHOR INFORMATION Corresponding Authors *E-mails: [email protected] (H.W); [email protected] (M.L.Gee). Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the support of the Melbourne Materials Institute for interdisciplinary seed funding for this project and the Australian Government National Health and Medical Research Council (NHMRC) Project Grant 628770 for support of related project. H.W. acknowledges the support from the National Key Research and Development Program of China (2016YFC0101002), Chongqing Science and Technology Commission (cstc2015jcyjiA10057 and YJ500061LH1) and the Chinese Academy of Sciences (R52A500Z10).

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Influence of Fimbriae on Bacterial Adhesion and Viscoelasticity and

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