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Biological and Environmental Phenomena at the Interface

Effect of Substrate and Bacterial Zeta Potential on Adhesion of Mycobacterium smegmatis Diane Smith, Ali Dhinojwala, and Francisco Moore Langmuir, Just Accepted Manuscript • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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Effect of Substrate and Bacterial Zeta Potential on Adhesion of Mycobacterium smegmatis Diane E. Smith,∗,† Ali Dhinojwala,† and Francisco B.-G. Moore‡ †Department of Polymer Science, University of Akron, Akron, OH, USA ‡Department of Biology, University of Akron, Akron, OH, USA E-mail: [email protected] Phone: +1 (607) 351 5672

Abstract Bacterial adhesion is described as a multi-step process of interactions between microbes and substrate, beginning with reversible contact, followed by irreversible adhesion. We explore the influence of substrate zeta potential on adhesion of Mycobacterium smegmatis, a nonpathogenic bacterial model for tuberculosis-causing M. tuberculosis and common foulant of reverse osmosis filtration systems. Substrates having a range of zeta potentials were prepared by coating silica with the polycation, poly(diallyldimethyl ammonium chloride) (pDADMAC), by adjusting the pH of alumina, a pH-responsive material, and by coating silica with a hydrophobic self-assembled monolayer (SAM) coating of octadecyltrichlorosilane (OTS). Our observations using these surfaces demonstrated that adhesion of M. smegmatis increased significantly, by more than 200 % on the silica-pDADMAC system and more than 300 % on alumina substrates, as zeta potential became less negative, and that the variation of pH did not affect adhesion on

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alumina surfaces. Live and heat-killed bacteria were studied to investigate the contribution of biological response to adhesion with respect to zeta potential. While approximately 60 % fewer heat-killed M. smegmatis adhered to pDADMAC-coated silica substrates, the trend of significantly increasing adhesion with less negative zeta potential was still observed. These results show the influence of zeta potential on adhesion of M. smegmatis, which is a separate process from that of the biological response. Across the range of substrate surface chemistries, hydrophobicities, and zeta potentials tested, adhesion of M. smegmatis can primarily be controlled by zeta potential. The bacterial zeta potential was not changed by the various experimental conditions, and was -28.3 ± 2.4 mV.

Introduction Fouling of surfaces by mycobacteria poses major challenges in important industries including filtration, healthcare, and material transport. 1–4 Understanding the mechanism of mycobacterial adhesion is key to more effectively controlling fouling in these environments. In this investigation, we study the role of zeta potential in adhesion of Mycobacterium smegmatis. We focus on this genus of bacteria in particular because the cell wall of Mycobacteria is known to vary in its chemistry and structure from that of Gram-positive or negative bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, or Escherichia coli, which have been extensively studied by other investigators. 5,6 Such differences in cell wall are recognized to result in interactions with chemicals and substrates that are unique to Mycobacteria. 5,6 Therefore, the adhesive behavior of typical Gram-positive or negative bacteria is not necessarily predictive of that of Mycobacteria. Studying the influential factors in the adhesion of M. smegmatis adds to our understanding of how to control and mitigate mycobacterial fouling, which is relevant to healthcare, materials transport, and filtration industries. Bacterial adhesion is a progression of interactions between microbes and substrate that begins with reversible contact, followed by irreversible adhesion, often leading to biofilm 2


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formation. Non-specific interactions which are influenced by polar and non-polar electrostatic interactions dominate during reversible contact. During this stage, the bacteria are too far from or have not had sufficient time in contact with the substrate to respond biologically. 7 Irreversible adhesion is characterized by specific interactions between bacteria and substrate, caused by metabolic processes including receptor-ligand binding and production of extracellular polymeric material. 8–10 Mycobacterium smegmatis, the bacterial species used for the studies described here, is a nonpathogenic laboratory model for tuberculosis-causing M. tuberculosis and related to Mycobacterium species which foul reverse osmosis (RO) ultrafiltration membranes. 2,4 Driving factors involved in adhesion of M. smegmatis provide insights into methods of controlling the adhesion of these bacteria. 11 Elucidating the influence of non-specific factors on adhesion can inform material design, reducing the risk of exposure to bacteria adherent to common surfaces, and more effectively preventing fouling where it can be highly problematic. 11 Previous studies exploring non-specific factors of bacterial adhesion offer insights into the first stage of this process, which remains little-understood, especially in the case of M. smegmatis. 7,10,12–15 Computational approaches are commonly used to understand bacterial adhesion. These models include Derjanguin-Landau-Verwey-Overbeek (DLVO) and extended-DLVO (X-DLVO) theories, which have their origins as colloidal stability theories. DLVO theory takes into consideration the contribution of van der Waals and electrostatic interactions to predict the interaction free energy, as a function of distance, between surfaces across a given medium. X-DLVO additionally factors in acid-base interactions, which may be highly influential for anchoring bacteria to a substrate when the surfaces are in close proximity, aiding in the transition from reversible to irreversible attachment. 13,15 Both DLVO and X-DLVO have the potential to reveal conditions such as distance between bacteria and substrate, and substrate material that could result in an energy barrier that prevents adhesion. Additionally, these models indicate conditions under which secondary and primary energy minima occur, respectively corresponding to reversible and irreversible bacterial adhesion. 16 Though

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these theories were constructed with colloidal particles in mind, it is arguably reasonable to consider bacteria as such during the initial contact of bacteria with a substrate, before the influence of biological responses. 13 In spite of this, neither the DLVO nor the X-DLVO approaches to understanding bacterial adhesion are able to consistently predict adhesion. The link between adherent number of bacteria and interaction free energy has yet to be established. 13,15 Previous experimental work demonstrated the importance of a distribution of zeta potential in the capture of Staphylococcus aureus. Substrates with a variety of zeta potentials ranging from negative to positive were made by physically, irreversibly adsorbing polycation poly(dimethylamino ethyl methacrylate) (pDMAEMA) to albumin films, and used to capture the bacteria from flow. 12 Films with negative zeta potential resulting from low concentrations of adsorbed polycation were characterized as patchy surfaces, with surface-exposed areas of negative albumin and intermittent areas of polycation. Interestingly, bacterial capture increased significantly and reached a maximum in the presence of substrates with positive patches, but an overall negative zeta potential. 12 Enhanced capture of Mycobacteria on surfaces coated with polycation poly(diallyldimethyl ammonium chloride) (pDADMAC) was described in a recent patent. 17 Increased bacterial adhesion was attributed in part to the proximity of the two non-polar surfaces, the bacterial exterior and polymer backbone. In the aqueous environment, interstitial water between these surfaces leaves, forcing the non-polar surfaces together. This assertion was attributed to the observation of hydrophobic moieties of pDADMAC and the known hydrophobic character of M. smegmatis. 17 The contribution of zeta potential to adhesion in this environment remains unclear, but can be better understood using substrate systems having a range of zeta potentials, achieved using independent mechanisms. In this investigation, we examine the influence of zeta potential on adhesion of M. smegmatis. Silica (SiO2 ) surfaces were coated with polycation pDADMAC, and alumina (Al2 O3 ) substrates were exposed to phosphate buffered saline (PBS) of various pH values to alter

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their respective zeta potentials, onto which bacteria was allowed to adhere. Adhesion steps involving non-specific physicochemical and specific biological interactions were divided by heat killing the bacteria prior to adhesion on coated substrates. Heat killing isolated the bacteria from employing metabolic processes to contribute to their interaction with the substrate. Additionally, adhesion on these hydrophilic surfaces was compared to that of SiO2 coated with an octadecyltrichlorosilane self-assembled monolayer (OTS-SAM). The clear relationship between M. smegmatis adhesion and measured substrate zeta potential emerged, in spite of the differences in surface chemistry, hydrophobicity, and zeta potential. Particle and surface zeta potential measurements were used to investigate the zeta potentials of these surfaces under all experimental conditions.

Experimental Study Organism R Mycobacterium smegmatis mc(2)155 (ATCC 700084TM) were purchased from ATCC. M.

smegmatis was chosen as a non-pathogenic surrogate for M. tuberculosis and representative species for mycobacteria that foul RO ultrafiltration membranes, causing significant health and industrial challenges. 2,4 These bacteria were additionally chosen for their robust cell wall which allows for exploration of adhesion in PBS having a range of pH values and heat killing without disruption of the zeta potential or shape.

Materials Middlebrook 7H9 broth, 7H10 agar base, growth supplements (OADC and ADC), and pDADMAC (20 wt% in H2 O, 250,000 - 300,000 molecular weight) were purchased from Sigma and used as received. Carbol fuchsin powder, a hazardous material, (basic fuchsin and phenol, Sigma) was diluted to 1 wt% in H2 O for Ziehl-Neelsen (ZN) staining. 18,19 Acid alcohol solution for ZN 5


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staining was prepared by mixing 97 mL 95 % ethanol and 3 mL concentrated HCl. 19 Standard SiO2 microscope slides of dimensions 25 mm x 75 mm x 1 mm and coverslips of dimensions 18 mm x 18 mm x 0.15 mm were obtained from VWR. IR quality Al2 O3 windows 19.05 mm in diameter and 1.016 mm thick (MSW075/040) for use in bacterial adhesion experiments and IR quality Al2 O3 windows 6.35 mm in diameter and 5.08 mm thick (MSW025/020) for surface zeta potential measurements were purchased from Meller Optics. A 10 X concentration of PBS was made by mixing 80.0 g NaCl, 2.00 g KCl, 14.4 g Na2 HPO4 , and 2.40 g KH2 PO4 , and adjusting the final volume of 1 L with H2 O to pH 7.4. The ionic strength of PBS (1 X) is approximately 170 mM. Sterilization was achieved by autoclaving at 121

◦

C, 15 psi, for 15 min.

Mycobacterial Culture and Zeta Potential Characterization Mycobacterial growth and preparation. M. smegmatis were grown in Middlebrook 7H9 broth supplemented with ADC or Middlebrook 7H10 agar supplemented with OADC at 37

◦

C, according to the recommended ATCC protocol. Briefly, sterile, supplemented

broth was inoculated with M. smegmatis and incubated at static conditions until the desired cell density was reached. M. smegmatis was spread onto solid, sterile prepared agar plates to verify the viability and density of cells used in the experiments described below. These mycobacteria have a doubling time of approximately 3 to 4 h. 20

Heat killing of M. smegmatis. M. smegmatis were heat killed to isolate the bacteria in the first stage of adhesion, which is dominated by non-specific interactions between bacteria and substrate. By heat killing, bacteria were not allowed to progress to the second stage of adhesion, which is driven by interactions that depend on biological function. Experiments comparing live and dead bacterial adhesion were done only using SiO2 -supported substrates, as described below. M. smegmatis suspended in PBS were killed by heating to approximately

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100

◦

C for 1 minute, prior to studying adhesion.

Mycobacterial viability in PBS of pH 10, 8, 7.4, and 6, and after heat-killing. Bacterial viability was tested in solutions of various pH values, corresponding to all of those used in these studies. This analysis was meant to help determine whether or not the influence of cell lysis or death should be considered in bacterial adhesion results. NaOH was added to PBS to achieve pH values of 10 and 8, and HCl was used to yield pH 6 PBS. After incubating approximately 107 cfu/mL M. smegmatis in these solutions for 2 h at RT, mycobacteria were smeared and heat fixed on standard microscope slides, followed by Ziehl-Neelsen staining. Mycobacteria were visually inspected for cell fragmentation at a total magnification of 1,000 X using an Olympus DP70 camera attached to an Olympus BX51 optical microscope. Additionally, cells were grown on solid 7H10 media supplemented with OADC after 2 h of exposure to the various PBS solutions and examined for growth. Similarly, cell viability and morphology was tested before and after heat-killing to verify that 1 minute of boiling was sufficient to kill M. smegmatis, but preserve their shape. Cells were smeared and heat-fixed onto standard microscope slides before and after boiling, followed by Ziehl-Neelsen staining, and visual inspection at a total magnification of 1,000 X for changes in morphology. Before and after boiling M. smegmatis suspended in PBS, cells were plated on solid 7H10 media supplemented with OADC. Plates containing boiled cells were monitored over several weeks for growth.

Zeta potential of M. smegmatis. Zeta potential of M. smegmatis in PBS adjusted to pH values of 10, 8, 7.4, and 6 were analyzed using the Malvern ZetaSizer Nano-ZS90 at times of 0 and 2 h at 25 at 25

◦

◦

C. Zeta potential of heat-killed M. smegmatis was also measured

C before and after heat killing. Each measurement was repeated three times. The

Smoluchowski model was used to convert electrophoretic mobility data to zeta potential values. These experimental conditions are summarized in Table S1 of the Supporting Information. 7


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Al2 O3 and SiO2 Substrate Preparation Cleaning of Al2 O3 substrates. Al2 O3 windows were sonicated for 15 min in each of acetone, methanol, and toluene, and dried with N2 (g) before changing solvents. Substrates were plasma sterilized for 5 min at high radio frequency prior to experimental use (Harrick Scientific, PDC 32G). Clean Al2 O3 substrates were stored in clean SiO2 dishes until further use.

Manipulation of Al2 O3 zeta potential. Cleaned Al2 O3 substrates were allowed to equilibrate for at least 5 min in solutions of PBS adjusted to pH 10, 8, and 6 to achieve different zeta potentials immediately prior to bacterial adhesion experiments. The pH-responsive property of Al2 O3 has been previously reported. 21 Cleaning of SiO2 substrates. SiO2 substrates were thoroughly cleaned by submersion in base bath for 15 min followed by flushing with copious amounts of H2 O and drying. Substrates were plasma sterilized for 5 min at high radio frequency prior to coating as described below. Cleaning yielded SiO2 substrates with negative zeta potential. Cleaned SiO2 substrates were stored in clean SiO2 dishes until further use. Adsorption of pDADMAC on SiO2 substrates. Concentrations of 100 and 200,000 parts per million (ppm) pDADMAC in H2 O were physically adsorbed to cleaned SiO2 substrates by flooding slides or coverslips and rinsing off excess in H2 O after 5 min. Coated substrates were immediately used in mycobacterial adhesion experiments or surface zeta potential measurements.

Preparation of octadecyltrichlorosilane self-assembled monolayer coating on SiO2 . Cleaned SiO2 substrates were submerged in toluene in a clean SiO2 vessel capped with a rubber diaphragm with an inlet and outlet. For 15 min, N2 (g) was gently bubbled through the solvent via the inlet in the rubber diaphragm to remove O2 from the toluene that might 8


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quench the self-assembled monolayer (SAM) formation. Three wt % octadecyltrichlorosilane (OTS) was then added to the toluene and N2 (g) was continuously bubbled through the solution for the duration of the coating process, 4 h, at RT. The OTS-SAM-coated SiO2 substrate was removed from the toluene solution and rinsed with H2 O. Coated substrates were annealed overnight under vacuum at 150

◦

C and kept in clean SiO2 dishes until use.

Substrate Surface Characterization Contact Angle Measurements. Sessile drop contact angle measurements of OTS-SAMcoated SiO2 surfaces was done using the ramé-hart Contact Angle Goniometer. A 10 µL drop of MilliQ H2 O was placed, using a clean SiO2 syringe, on the OTS-SAM surface. DROPimage software was used to automatically take 10 sequential measurements of the contact angle of the drop on the surface. This procedure was repeated for three different locations on each prepared surface.

Surface Zeta Potential of Al2 O3 and SiO2 -Supported Substrates. Determination of surface zeta potential was generously facilitated by the Dr. Bi-Min Zhang Newby lab from the Department of Chemical and Biomolecular Engineering at the University of Akron. A cleaned Al2 O3 substrate of approximately 4 x 7 mm was mounted on the sample holder of the ZEN1020 Surface Zeta Potential Cell accessory using super glue. Solutions of 1 mM KCl were adjusted to pH 6, 8, and 10 using HCl or NaOH immediately prior to surface zeta potential measurements. KCl was used instead of PBS to yield the most accurate results. CML tracer particles (Invitrogen C37481), 0.5 µm in diameter, were suspended in prepared KCl solutions in a concentration of 109 particles/mL. These particles which have a negative zeta potential were used because we expected exposure of Al2 O3 to pH 6, 8, and 10 KCl to result in Al2 O3 having negative zeta potential, according to literature values. 21 A volume of 1.5 mL KCl solution containing CML tracer particles was added to the DTS0012 polystyrene cuvette (Malvern Instruments) into which the mounted Al2 O3

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flat was submerged. This apparatus was inserted into the Zetasizer Nano-ZS90 (Malvern Instruments) and the Zetasizer software was set to take 5 surface zeta measurements for each of 5 data points with 60 sec pauses between measurements. Five tracer particle zeta potential measurements were taken with 20 sec intervening pauses to calculate the final surface zeta potential using the Smoluchowski model. The same Al2 O3 substrate was used to take sequential surface zeta potential measurements with the prepared KCl solutions. The substrate was washed with copious amounts of H2 O and dried with N2 (g) between measurements. Cleaned SiO2 coverslips cut to dimensions of approximately 4 x 7 mm, and coated with pDADMAC or OTS as described above or left uncoated, were mounted to the sample holder of the ZEN1020 Surface Zeta Potential Cell accessory using super glue. PBS diluted to 0.1 X concentration was adjusted to pH 6, 8, and 10 using HCl or NaOH, or left at pH 7.4. CML tracer particles (Invitrogen C37481), 0.5 µm in diameter, were suspended in a concentration of 109 mL-1 in 0.1 X PBS solutions. The mounted samples were submerged in DTS0012 polystyrene cuvettes containing prepared 1.5 mL 0.1 X PBS solution. Zeta potentials of flats were measured using Zetasizer Nano-ZS90 (Malvern Instruments) using the Smoluchowski model, with the same software settings as those described for the Al2 O3 substrates, above. All surface zeta potential measurements were done at 25

◦

C.

These experimental conditions are summarized in Table S1 of the Supporting Information.

Mycobacterial Adhesion Mycobacterial adhesion on SiO2 -supported and Al2 O3 substrates. M. smegmatis, in a concentration of 107 cfu/mL, were added to SiO2 culture dishes containing 20 mL PBS and substrates, prepared as described above. In the case of Al2 O3 , PBS was adjusted to pH 10, 8, and 6 to change the Al2 O3 zeta potential, while SiO2 was either coated with various concentrations of pDADMAC or left uncoated and exposed to PBS of pH 10, 7.4, and 6. 10


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Bacterial solutions were incubated for 2 h at RT under static conditions. Substrates were then removed from solution, air dried, stained according to the Ziehl-Neelsen procedure, and imaged, as described below. All experimental conditions are summarized in Table S1 of the Supporting Information. For each adhesion experiment, several layers of replication were built into the experimental design which resulted in the data points shown in the corresponding figures. For instance, multiple bacterial cultures were used to test adhesion to each prepared substrate. Bacteria from every culture was exposed to three identical substrates, as described above. The use of multiple cultures was incorporated into our experiments to be able to account for any effects that culture conditions might have had on the final adhesion results. Ten images, each from different locations on a given substrate, were captured and analyzed to calculate the average area occupied by adherent bacteria per substrate. Thus, three values of average bacterial coverage were generated for each of the tested substrate preprations. These three averages were then used to calculate one average across the three identical substrates, resulting in one data point. A schematic in Figure S1 of the Supporting Information illustrates an example of this experimental design.

Ziehl-Neelsen staining, and mycobacterial imaging. Air-dried samples were heat fixed, flooded with 1 wt% carbol fuchsin and heated until steaming. Substrates were then allowed to sit for 10 min, during which time carbol fuchsin was periodically added, if needed, to prevent drying. Acid alcohol was washed over the substrates until runoff was colorless, followed by rinsing with H2 O. 18 Ten images were captured of each stained, air-dried substrate at a total magnification of 200 X using an Olympus DP70 camera attached to an Olympus BX51 or BX60 optical microscope. Percentage coverage of cells on substrates was then quantified using a custom program in ImageJ, which is detailed in Code S1 of Supporting Information.

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Results and Discussion Adhesion of M. smegmatis increases as substrate zeta potential becomes less negative, and is independent of pH. The influence of substrate zeta potential on adhesion of M. smegmatis was investigated by using Al2 O3 flats, which are pH-responsive, in contact with the bacteria. The zeta potential of Al2 O3 was adjusted to -81.0 ± 8.0, -69.8 ± 4.0, and -66.5 ± 6.8 mV by varying the pH of the surrounding PBS medium to pH 10, 8, and 6, respectively. In aqueous medium, the 0001 plane of Al2 O3 is hydroxylated and ideally exhibits an isoelectric point (IEP) at pH 4.1, according to previously recorded second harmonic generation and zeta potential measurements. 21 As the pH of the surrounding medium is changed, the associated singlyand doubly-coordinated hydroxyl groups on the surface of Al2 O3 become protonated or deprotonated, accounting for the change in zeta potential with respect to pH. 21 Singlycoordinated hydroxyl groups, according to the bond-valence model, have a high affinity for protonation, while the doubly-coordinated hydroxyl groups do not contribute to the pHresponse. Thus, by adjusting pH, zeta potential of Al2 O3 could be varied to study this influence on adhesion of M. smegmatis. Analysis of optical images of M. smegmatis on Al2 O3 flats, shown in Figure S2 of the Supporting Information, revealed that the quantity of adherent bacteria increases significantly, by more than 300 %, as the surfaces become less negative (Figure 1, ANOVA P = 0.0022). The results of statistical analysis on bacterial adhesion can be found in Table S2 of the Supporting Information. Because the pH of the surrounding aqueous medium, in this case PBS, was adjusted to change the zeta potential of Al2 O3 , the influence of pH on adhesion of M. smegmatis remains unclear from using Al2 O3 substrates alone.

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Figure 1: M. smegmatis adhesion to Al2 O3 substrates in PBS adjusted to pH 10, 8, and 6 to achieve zeta potentials of -81.0 ± 8.0, -69.8 ± 4.0, and -66.5 ± 6.8 mV, respectively (ANOVA, n = 3, P = 0.0022). Error bars represent standard error.

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Separating the contribution of pH from that of zeta potential within the Al2 O3 system was not possible because of the pH-responsive nature of Al2 O3 , which irrevocably intertwines pH and zeta potential. Therefore, we chose to investigate the influence of pH on adhesion of M. smegmatis using a system of SiO2 flats. This relationship was studied using substrates of uncoated SiO2 submerged in PBS of pH 10.00, 7.40, and 6.00 ± 0.02, containing M. smegmatis. The surface of SiO2 becomes hydroxylated upon contact with aqueous media and free counterions in solution associate with the surface, resulting in an IEP at approximately pH 2 and highly negative zeta potential that decreases slightly above pH 6, which has been previously reported. 22,23 In the SiO2 system, pH does not have the same effect on zeta potential as it does for the Al2 O3 system, allowing for independent testing of pH and zeta potential. No change in bacterial adhesion with respect to pH of PBS was observed on uncoated SiO2 substrates (Figure 2, ANOVA, P = 0.938), suggesting that the increase in bacterial adhesion observed on Al2 O3 substrates was driven by the change in zeta potential. The quantity of bacteria adherent to SiO2 substrates at pH 7.40 and 10.00 ± 0.02 in particular suggests that adhesion in this pH range is independent of pH. Though SiO2 exhibited different zeta potentials at pH 6.00 versus 7.40 and 10.00, no change in bacterial adhesion was observed. Representative images of M. smegmatis adherent to SiO2 flats can be seen in Figure S3, and the results of statistical analysis can be found in Table S3, of Supporting Information.

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Figure 2: Adhesion of M. smegmatis on SiO2 substrates submerged in PBS adjusted to pH 10, 7.4, and 6 to evaluate the influence of pH on bacterial adhesion (ANOVA, n = 3, P = 0.938). Error bars represent standard error.

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The previously discussed studies to investigate the role of Al2 O3 and SiO2 substrate zeta potential on adhesion of M. smegmatis centered around the zeta potential of the substrates in PBS of various pH. The pH-dependence of M. smegmatis zeta potential was evaluated to determine any possible contribution to adhesion. Bacteria were exposed to PBS of pH 10.00, 8.02, 7.42, and 5.99 ± 0.01 for up to two hours, equal to the duration and pH conditions of previous adhesion experiments, after which their viability and zeta potential was assessed. M. smegmatis were viable in this pH range and were able to grow on solid 7H10 media containing OADC growth supplement after exposure to a density of 107 cfu/mL. Particle zeta potential measurements showed that M. smegmatis has a zeta potential of -28.6 ± 1.5 mV after two hours in PBS at 25 ◦ C and physiological pH (Table 1). The zeta potential of the mycobacteria was negative and did not vary significantly between pH treatments (ANOVA, P = 0.2542). Interestingly, while the surfaces of both the bacteria and the prepared Al2 O3 and SiO2 -based substrates were negative, a significant increase in the quantity of adherent bacteria was nevertheless observed as substrates became less negative. This observation indicates that negative substrate zeta potential is not sufficient to resist adhesion by bacteria that exhibit negative zeta potential, though electrostatic repulsion would suggest otherwise. This phenomenon has been noted in a previous study, in which substrates of net-negative zeta potential with patches of adsorbed polycation captured significantly greater quantities of S. aureus than were captured on negative surfaces without positive patches. 12 The areas of positive zeta potential on the net-negative surfaces may be sufficient for significantly increasing bacterial capture.

Heat-killed and live M. smegmatis exhibit the same trend of increased adhesion with respect to substrate zeta potential. Adhesion of live and heat-killed bacteria onto prepared SiO2 -based substrates was assessed to investigate the contribution of physicochemical interactions and biological response during the adhesion process. Bacteria were boiled in PBS for one minute immediately prior to 16


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Table 1: Zeta potential of M. smegmatis in PBS solutions of various pH values for 0 and 2 hours. M. smegmatis zeta potential was measured at times of 0 and 2 hours in pH-adjusted PBS at 25 ◦ C using the Smoluchowski model (ANOVA, n = 3, P = 0.2542). Error values represent standard deviation. pH (± 0.01) 10.00 8.02 7.42 5.99

Zeta Potential (mV), 0 h -27.5 ± 0.5 -29.2 ± 1.4 -28.8 ± 1.5 -24.4 ± 0.8

Zeta Potential (mV), 2 h -28.1 ± 1.8 -25.8 ± 1.9 -28.6 ± 1.5 -25.6 ± 0.6

exposure to SiO2 coated with 0, 100, or 200,000 ppm pDADMAC to achieve zeta potentials of -59.9 ± 2.3, -14.5 ± 1.9, and -13.4 ± 1.9 mV, respectively. This method of varying zeta potential by coating SiO2 with the polycation pDADMAC, was chosen as a parallel to the pH-sensitive Al2 O3 system. That is, we selected systems within which a range of similar zeta potentials could be achieved by independent mechanisms of pH response and physical adsorption of a strong polyelectrolyte. Heat killing was done to prevent bacteria from progressing beyond the first stage of adhesion, in which non-specific interactions are dominant, to the second stage of adhesion, in which live bacteria undergo specific, biologically influenced interactions with the substrate. Bacteria boiled for one minute were unable to grow on solid 7H10 media containing OADC growth supplement. Figure 3 shows that the adhesion of both live and heat-killed bacteria increased significantly, by more than 200 %, as zeta potential became less negative, though adhesion of heat-killed bacteria to each substrate was consistently lower, by approximately 60 % for each surface, than that of live bacteria (ANOVA, P = 0.0011). These observations, representative images of which can be seen in Figure S5 in Supporting Information, suggest that while zeta potential is important for influencing the quantity of bacteria adherent to the substrate, the biological response of the bacteria increases the number of adherent bacteria beyond what can be achieved by physicochemical interactions alone. The results of statistical analysis on bacterial adhesion can be found in Table S4 of the Supporting Information. Previous studies note that bacteria can respond biologically to their environment in such a way that they more

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readily commit to a certain behavior, such as adhering to a given substrate, which may explain the difference shown in Figure 3 between number of adherent live and heat-killed bacteria. 24,25 These results suggest that in the two hours of the experiment, the live bacteria have progressed beyond the first stage of bacterial adhesion. This observation indicates that when M. smegmatis is exposed to a material, the antifouling quality of which is affected by its zeta potential, the interactions between bacteria and substrate quickly change from predominantly physichochemical, to a complex biological response, and the surface can no longer be considered for only its original properties. Also notable, is the difference in coverage on SiO2 -supported substrates shown in Figure 3, compared to Al2 O3 substrates shown in Figure 1. The quantity of adherent live bacteria is much greater on SiO2 -supported substrates than it is on Al2 O3 substrates, though the zeta potential of SiO2 is much lower than that of Al2 O3 . This observation may result from differences in surface chemistry of the substrates used. 26 Previous work acknowledges the importance of surface chemistry, particularly in specific interactions that generally affect bacterial adhesion. 8–10,27 Nevertheless, the statistically-supported trends remain, which indicate that bacterial coverage increases as zeta potential becomes less negative in each case.

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Figure 3: Adhesion of live (light grey bars) and heat-killed (dark grey bars) M. smegmatis on SiO2 -based substrates of various zeta potential. SiO2 substrates were coated with 0, 100, or 200,000 ppm pDADMAC to achieve zeta potentials of -59.9 ± 2.3, -14.5 ± 1.9, and -13.4 ± 1.9 mV, respectively (ANOVA, n = 3, P = 0.0011). Error bars represent standard error.

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The influence of heat treatment on zeta potential of M. smegamtis was tested using particle zeta potential measurements to evaluate any influence this factor might have on the results observed in Figure 3. Zeta potential of heat-killed M. smegmatis did not change significantly as compared with live bacteria used in the same experiment (Table 2; ANOVA, P = 0.7513). Table 2: Particle zeta potential of live and heat-killed M. smegmatis. M. smegmatis zeta potential was measured PBS at 25 ◦ C using the Smoluchowski model (ANOVA, n = 3, P = 0.7513). Error values represent standard deviation. Treatment Live Heat-killed

Zeta Potential (mV) -31.5 ± 0.6 -30.5 ± 2.5

Adhesion of M. smegmatis increases as zeta potential becomes less negative, regardless of substrate chemistry. Across the experiments we performed to understand the influence of substrate zeta potential on capture of M. smegmatis, an overall trend emerged. As previously described, these investigations were done on surfaces of which zeta potential could be changed by various mechanisms. In one instance, we studied adhesion of M. smegmatis on pH-responsive Al2 O3 . During experimentation, this surface was submerged in PBS of pH 10, 8, and 6, which yielded measured zeta potentials of -81.0 ± 8.0, -69.8 ± 4.0, and -66.5 ± 6.8 mV, respectively. We then measured bacterial adhesion on uncoated SiO2 in PBS of pH 10.00, 7.40, and 6.00 ± 0.02, having respective zeta potentials of -54.5 ± 7.6, -59.9 ± 2.3, and -38.0 ± 5.2 mV. Bacterial adhesion was then tested on SiO2 surfaces that were coated with 0, 100, and 200,000 ppm pDADMAC to achieve zeta potentials of -59.9 ± 2.3, -14.5 ± 1.9, and -13.4 ± 1.9 mV, respectively. Further, we tested adhesion on SiO2 coated with a self-assembled monolayer of octadecyl trichlorosilane (OTS). This surface was hydrophobic, having a water contact angle of 110◦ , and exhibited a zeta potential of -70.5 ± 1.0 mV in the aqueous environment. When submerged in aqueous solution, hydrophobic surfaces have a negative zeta potential, 20


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in spite of their net neutral chemistry, due to the association of -OH groups from the water. These hydroxyl groups make the contact of H2 O with the hydrophobic surface more thermodynamically favorable. 28 For all of these experiments, these substrates were exposed in static conditions to 107 cfu/mL M. smegmatis suspended in PBS for two hours prior to examination by light microscope and quantification of bacterial adhesion. These substrates vary in their hydrophobicity, surface chemistry, and zeta potential. Our process of experiments, as explained previously, sought to isolate as much as possible the effect of electrostatic interactions on bacterial adhesion. Nonetheless, Figure 4 shows that regardless of the surface chemistries tested for these studies, bacterial capture increases as zeta potential of the substrate becomes less negative. A linear fit line with a corresponding R2 value of 0.8211 in Figure 4 shows a strong relationship between capture of M. smegmatis and substrate zeta potential. The observations shown in this figure span a range of substrate surface zeta potential from -81.0 ± 8.0 to 13.4 ± 1.9 mV. Visually, it may appear that in the region between -50 and -10 mV, this relationship begins to weaken. If we were to consider the subset of data in this range, the trend of adhesion with respect to zeta potential would arguably not be as strong. The data points in this range, however, are part of a larger group of experimental results and therefore must be considered as such. In the context of the complete group of data shown in Figure 4, our statistical analysis supports that there is a significant relationship between the quantity of bacteria adherent to the substrate with respect to zeta potential, explaining 82 % of the variation in adherence, across widely varying surface chemistry (ANCOVA, P = 0.0003). The results of statistical analysis on bacterial adhesion can be found in Table S5 of the Supporting Information.This close of a fit is especially meaningful given the many biological factors that could contribute to adhesion of the bacteria under these conditions. The closeness of this linear fit is an indication of how important substrate zeta potential is for adhesion of M. smegmatis.

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Figure 4: The overall relationship of M. smegmatis capture and substrate zeta potential of across all live cell experiments. Treatment means were regressed on means for all combinations of substrate type (Al2 O3 , uncoated SiO2 , OTS-coated SiO2 , pDADMAC-coated SiO2 ), pH, and coating density treatments presented in this study. The linear fit explains explains over 82 % of the variation between the treatment and is highly significant (ANCOVA, P = 0.0003, Table S5). Surface zeta potential in parentheses was measured using the ZEN1020 Surface Zeta Potential Cell accessory with the Malvern ZetaSizer Nano-ZS90 using the Smoluchowski model.

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DLVO and X-DLVO theories are commonly employed to attempt to support and better understand experimental observations of bacterial adhesion. These models are quantitative in the sense that the change in free energy of adhesion with respect to the distance between surfaces coming into contact across a given medium can be calculated. This theoretical understanding cannot, however, be used to predict the number of bacteria found on a surface under given conditions, such as those presented in Figure 4. It is at times in qualitative agreement with others’ work, under certain experimental conditions. 29–31 While the use of DLVO and X-DLVO theories is abundantly common in related literature, we have not put forth these models to attempt to explain our findings for these reasons. It is important to note that generally, DLVO and X-DLVO theories tend to agree with the trend shown in Figure 4. That is, as zeta potential becomes less negative, electrostatic repulsion decreases, and so does bacterial adhesion, driven by van der Waals interactions. Further discussion on this topic is included in the Supporting Information in Discussion S1 on page S12.

Summary and Conclusions In this study, we investigated the influence of zeta potential on adhesion of M. smegmatis. The zeta potential of Al2 O3 substrates was changed by adjusting the pH of the surrounding medium, PBS, while that of SiO2 was changed by coating with polycation pDADMAC, conditions under which the zeta potential of M. smegmatis was statistically constant. Using these substrates, we observed that as zeta potential became less negative, a greater quantity of M. smegmatis adhered to the substrates, independent of the pH used for these studies. Additionally, adhesion to SiO2 -based substrates of live and heat-killed cells was compared to better understand the contribution of biological and physicochemical aspects of the bacterial adhesion process. We reported that live and heat-killed bacteria exhibit the same zeta potential dependence in their adhesion, though the heat-killed cells are found in lower quantity on each substrate as compared with live cells. In comparing adhesion on

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Al2 O3 , SiO2 , pDADMAC-coated SiO2 , and OTS-coated SiO2 , a strong relationship between M. smegmatis adhesion and zeta potential is apparent, in spite of the differences in surface chemistry, hydrophobicity, and zeta potential. These results may be used to better understand the contributing factors to fouling of surfaces and how to prevent problematic bacterial adhesion. Though our findings are not intended to be extrapolated to other bacterial species or substrate surfaces, the bacteria and surfaces tested in these studies are industrially and clinically relevant. This quantification of the importance of electrostatic interactions for M. smegmatis capture, together with the knowledge that these bacteria have a zeta potential of -28.6 ± 1.5 mV in physiological conditions, is suggestive of the possibility of leveraging electrokinetic methods to rapidly capture these bacteria.

Supporting Information Available The following files are available free of charge. • Supporting Information: ImageJ image analysis code, images of adherent M. smegmatis, and statistical analysis of M. smegmatis adherent to substrates. This material is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgement The authors thank Alex Nyarko and Dr. Bi-min Zhang Newby, who helped facilitate this work.

References (1) Statz, A. R.; Barron, A. E.; Messersmith, P. B. Protein, cell and bacterial fouling resistance of polypeptoid-modified surfaces: effect of side-chain chemistry. Soft Matter 24


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