Liquid-infused structured titanium surfaces: Antiadhesive mechanism

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Biological and Medical Applications of Materials and Interfaces

Liquid-infused structured titanium surfaces: Antiadhesive mechanism to repel Streptococcus oralis biofilms Katharina Doll, Ines Yang, Elena Fadeeva, Nadine Kommerein, Szymon P. Szafra#ski, Gesa Bei der Wieden, Andreas Greuling, Andreas Winkel, Boris N. Chichkov, Nico S. Stumpp, and Meike Stiesch ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b06817 • Publication Date (Web): 07 Jun 2019 Downloaded from http://pubs.acs.org on June 7, 2019

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ACS Applied Materials & Interfaces

Liquid-infused structured titanium surfaces: Antiadhesive mechanism to repel Streptococcus oralis biofilms

Katharina Doll1*, Ines Yang1, Elena Fadeeva2, Nadine Kommerein1, Szymon P. Szafrański1, Gesa Bei der Wieden1, Andreas Greuling1, Andreas Winkel1, Boris N. Chichkov2, Nico S. Stumpp1#, Meike Stiesch1#*

1Department

of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

2Institute

of Quantum Optics, Leibniz University of Hannover, Welfengarten 1, 30167 Hannover, Germany

ACS Paragon Plus Environment

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KEYWORDS

Slippery Liquid-Infused Porous Surface, bacterial adhesion force, oral multi-species biofilm, FluidFM, RNASeq, Streptococcus oralis genome sequencing

ABSTRACT

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the “slippery liquid-infused porous surfaces” (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer Streptococcus oralis and an oral multi-species biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces and planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC® 9811TM was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.


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INTRODUCTION

In Europe, more than 1.2 million dental implants are inserted annually, and this number is expected to double within the next 5 years1. However, despite continuous improvements in the materials used, dental implants still suffer from higher rates of implant-associated infections than other implantable medical devices, as they are constantly exposed to the oral microbiome2. Epidemiological surveys have found that the prevalence of peri-implantitis exceeds 20% after at least 5 years of implantation3. These infections are caused by bacterial biofilms in the oral cavity4,5. Biofilms are defined as attached multi-species microbial communities, embedded into a self-produced matrix of extracellular polymeric substances, with a reduced growth rate and altered gene expression when compared to planktonic bacteria5. Biofilm formation starts with bacterial adherence to a (implant) surface. This is mediated by bacterial surface structures, such as pili, fibrils and other adhesins6,7. The initial adhesion is influenced by a multitude of different factors, such as the presence of specific surface antigens, hydrophobicity and roughness of the surface, as well as other physical and chemical properties of the microenvironment6-9. When a bacterium comes into contact with an immobilized layer major changes in gene expression are induced, which e.g. initiate the production of extracellular polysaccharides involved in matrix formation, alter their metabolism and reduce growth rate5,6,9,10. The human mouth accommodates up to several hundred different bacterial species11,12. The dominant initial colonizers, which are able to adhere to solid surfaces such as teeth or implants, are streptococci (e.g. Streptococcus oralis) and actinomyces (e.g. Actinomyces naeslundii)13,14. Subsequently, bacteria like Veillonella dispar are attached to the biofilm. These coaggregate with the initial colonizers and are able to link them to late colonizing bacteria15. All of these bacteria are usually commensals and are not considered pathogenic for people of normal health


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status13,14,16. However, if the commensals’ biomass increases excessively, the local oral environment changes, which allows opportunistic key-stone pathogens, such as Porphyromonas gingivalis, to induce dysbiosis12,16,17. These pathogens can disarm host immune responses resulting in inflammatory lesions, cumulating in the symptoms of peri-implantitis such as gingival detachment and crestal bone loss18-20. Once a biofilm has developed, it exhibits inherent resistance mechanisms, making it up to 1000-fold less susceptible to common antibiotics than are planktonic cultures21. The extracellular matrix may serve as diffusion barrier, thus reducing the amount of locally delivered antibiotics and host phagocytosis21. Furthermore, as antibiotics often target bacterial gene expression, the reduced growth rate and altered gene expression patterns of bacteria living in biofilms result in additional protection21-23. In order to combat implant-associated infections, there is ongoing research on novel implant materials, which aim at inhibiting biofilm formation. A common approach is to equip the implant material with antibacterial chemicals that exhibit toxic effects on colonizing bacteria24-29. Besides other challenges, this may cause dead bacteria to remain on the surface, eventually masking the antibacterial coating and providing new attachment sites for further bacteria. Additionally, chemical surface functionalization, like antibiotics or silver ions, may lead to the development of bacterial resistance. To avoid these problems, other approaches focus on using surfaces that nonspecifically inhibit stable bacterial attachment. We have recently developed laser-structured liquid-infused titanium surfaces30, in accordance with the principle of “slippery liquid infused porous surfaces” (SLIPS) introduced by Aizenberg’s group31,32. These are based on superhydrophobic lotus leaf-like structures, composed of a combined micro- and nanopattern, generated by ultra-short pulsed laser-ablation on the common implant material titanium and are infused with a perfluoropolyether lubricant. This is immobilized as a thin liquid film on the structured surface, as a result of the specifically matched wetting characteristics, and then creates ACS Paragon Plus Environment

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a new slippery interface with the environment. In a multi-well plate assay, titanium SLIPS gave almost completely reduced initial bacterial adhesion and attached biofilm of the initial oral colonizer S. oralis30. The initial adhesion of eukaryotic cells – human fibroblasts and osteoblasts – was found to be almost completely inhibited as well30. Additional analyses revealed that this effect could not be attributed to toxic effects of the components used30. Even though SLIPS of various materials have demonstrated bacteria-resistant properties in other studies too30,31,33-37, little is known about their exact mechanism of action. The most prominent hypothesis is that bacteria are not able to stably adhere on the liquid layer and can therefore easily be removed by shear forces31,35,37,38. In a recent study, it was shown that several of these bacteria are more easily detached from the surfaces of immobilized liquid-layers, presumably due to weaker adhesion37. However, the details of the SLIPS mechanism have not yet been studied. The aim of the present study was to analyze the biofilm-repelling liquid-infused titanium in more detail, with a perspective on a future application as implant functionalization in the oral cavity and to shade more light on the mechanism of SLIPS. For this purpose, a recently introduced flow chamber system that simulates the hydrodynamic conditions of saliva flow was employed39. It more closely mimics the oral environment than do static experiments. Mono-species biofilms of the initial colonizer S. oralis and an oral four-species biofilm model, composed of S. oralis, A. naeslundii, V. dispar and P. gingivalis40,41, were used for the studies. To address the underlying mechanism, the adhesion forces of single bacterial cells of S. oralis on titanium SLIPS surfaces were directly measured using a combination of atomic force microscopy and a microfluidic pressure control system42,43. Furthermore, gene expression patterns of S. oralis cells forming biofilms on SLIPS surfaces were analyzed to reveal whether the lubricant layer influences biofilm formation. In order to facilitate this analysis, the genome of S. oralis strain ATCC® 9811TM was sequenced de-novo. ACS Paragon Plus Environment

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2 2.1

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MATERIALS & METHODS Titanium SLIPS fabrication and lubricant visualization

Disk-shaped titanium (grade 4) specimens of 12 mm in diameter were finished using 45 µm diamond abrasive polishing wheels. Specimens without further modification served as titanium control surfaces. For SLIPS fabrication, the respective specimen’s surface was entirely or partially structured with spikes using a commercially available amplified Ti-Sapphire femtosecond laser system (Femtopower Compact Pro, Femtolasers Produktions GmbH/Spectra Physics, Vienna, Austria). This delivers sub-30 fs pulses at 800 nm wavelength with energies of up to 1 mJ and a repetition rate of 1 kHz. An achromatic lens with a focal distance of 200 mm was used for focusing and the laser beam was scanned along its linear polarization direction (xdirection) with a speed of 800 µm/s. The translation step in the y-direction was 15 μm. Spike structures were generated by laser ablation at a fluence of 8 J/cm2. Circularly polarized laser light was used to obtain uniform isotropic structures. The spikes were silanized with a fluorinated polymer (Antispread E 2/30 FE 60, Dr. Tillwich GmbH Werner Stehr, Horb-Ahldorf, Germany) to increase hydrophobicity and coated with the perfluoropolyether lubricant Krytox GPL 104 (DuPont de Nemours, Neu-Isenburg, Germany). The SLIPS fabrication process is illustrated in Figure 1A. If the specimens were only partially spike structured, the unstructured part of the surface was protected by an adhesive foil from silanization and lubricant coating and served as the titanium control surface for biofilm volume quantification. Prior to all experiments, titanium SLIPS and control surfaces were sterilized by irradiation with UV light for 20 min. The lubricant was visualized on the surface by reflection microscopy using a confocal laser scanning microscope (CLSM, Leica SP8, Leica Microsystems, Mannheim, Germany). A 488 nm laser was used and emission was detected in the range of 485 – 490 nm. The lubricant thickness was


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calculated using the Imaris 6.2.1 software package (Bitplane AG, Zurich, Switzerland). Data visualization and statistical analysis were performed using GraphPad Prism software 6.01 (GraphPad Software Inc., La Jolla, USA). Gaussian distribution was assessed using D’Agostino & Pearson omnibus normality tests. To test for significant differences, repeated measures ANOVA with Tukey’s correction for multiple comparison (if more than one comparison) or unpaired t test (if only one comparison) were used at p ≤ 0.05. 2.2

Bacterial strains and culture conditions

Streptococcus oralis ATCC® 9811TM was obtained from the American Type Culture Collection (ATCC). Actinomyces naeslundii DSM 43013, Veillonella dispar DSM 20735 and Porphyromonas gingivalis DSM 20709 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). All bacteria were stored at -80 °C as glycerol stocks. For mono-species biofilms, S. oralis was precultured in Tryptone Soy Broth (Oxoid Limited, Hampshire, UK) supplemented with 10% yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37 °C for 18 h under aerobic conditions. For flow chamber biofilm experiments, a bioreactor containing Brain Heart Infusion growth medium (Oxoid Limited, Hampshire, UK) supplemented with 10 µl/ml vitamin K (Oxoid Limited, Hampshire, UK) (BHI/vitamin K) and 5% sucrose (Carl Roth GmbH + Co. KG) was inoculated to a final optical density of 0.026 at 600 nm. According to standard plate counting, this corresponds to 5x106 CFU/ml for S. oralis. Multi-species biofilms were grown according to the Hanoverian oral multi-species biofilm implant flow chamber (HOBIC) model described previously41. S. oralis, A. naeslundii, V. dispar and P. gingivalis were precultured individually in BHI/vitamin K under anaerobic conditions (80% N2, 10% H2, 10% CO2) at 37 °C for 18 hours. Precultures were adjusted to an optical density of 0.5 at 600 nm in BHI/vitamin K. This corresponds approximately


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to 2x1013 CFU/ml for S. oralis, 2x1010 CFU/ml for A. naeslundii, 5x108 CFU/ml for V. dispar and 1x109 CFU/ml for P. gingivalis. Equal volumes of the precultures were mixed and added to the bioreactor containing 1.8 l BHI/vitamin K at a dilution of 1:45. 2.3

Biofilm formation and volume quantification

Biofilms were grown in a flow chamber system developed by Rath et al.39, modified from a recirculating to a more realistic one-way system, in which bacteria were collected in a waste bottle instead of being pumped back into the bioreactor. To prevent an air/liquid interface crossing the SLIPS surface during the initial medium filling of the system, the flow chambers containing the titanium SLIPS or control specimens were filled and reassembled separately before being inserted into the system already filled with medium. Prior to bacterial inoculation, SLIPS and titanium control surfaces were equilibrated in sterile medium for 30 min at a flow rate of 100 µl/min to remove excess lubricant. For long term experiments, specimens were exposed to phosphate buffered saline for 13 days at a flow rate of 100 µl/min and 37 °C prior to bacterial inoculation. Biofilms were cultivated at 37 °C at a flow rate of 100 µl/min. The system was protected from light and, in the case of multi-species biofilm cultivation, kept anaerobic as described previously39. To quantify biofilm volume, partially structured SLIPS were used. After 24 h, the bioreactor was separated from the system and flow chambers were consecutively connected to reservoirs of washing, staining or fixation solution as described by Rath et al.39. Biofilms were washed by pumping phosphate buffered saline through the system to remove unbound bacteria and then stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, Darmstadt, Germany). Both fluorescent dyes, SYTO®9 and propidium iodide (PI), were applied simultaneously in a 1:2000 dilution in phosphate buffered saline pumped through the system. Biofilms were fixed with 2.5% glutardialdehyde in phosphate buffered saline. Time


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and flow rate of all steps are listed in Table 1. The flow chambers were subsequently separated from the system and examined by confocal laser-scanning microscopy (CLSM, Leica TCS SP2, Leica Microsystems, Mannheim, Germany) using a 488 nm laser and an emission range of 500 – 545 nm for SYTO®9 and 590 – 680 nm for PI. For each specimen, three image stacks with an area of 1200 x 1200 µm2 were acquired for SLIPS and control surfaces, respectively. The Imaris 6.2.1 software package (Bitplane AG) was used for 3D reconstruction, biofilm volume calculation and to quantify the viable (SYTO®9; green), dead (PI; red) and colocalized (SYTO®9 + PI; orange) proportions of the biofilms. Areas with colocalized fluorescence were considered dead, as PI was able to penetrate the membrane but had not completely removed SYTO®9. They were therefore subtracted from the SYTO®9 stained areas. Experiments were performed in 9 replicates for each condition. For data visualization and statistical analysis, GraphPad Prism software 6.01 (GraphPad Prism Software Inc.) was used. Gaussian distribution was assessed by D’Agostino & Pearson omnibus normality testing and differences were analyzed with Wilcoxon matched-pairs signed rank tests at p ≤ 0.05.


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Table 1. Conditions for biofilm washing, staining and fixation in the flow chamber system for biofilm volume quantification. Condition

Washing

Staining

Fixation

100 µl/min

Time [min] 30

Flow Rate [µl/min] 100

Time [min] 30


Time [min] 30


150 µl/min

20

150

20

150

20

150

250 µl/min

20

250

20

250

20

250

20

150

20

150

Pulsed

2.4

10

150

2 sec

1000

5

150

2 sec

1000

5

150

2 sec

1000

Fluorescence in situ hybridization (FISH)

Multi-species biofilms grown on titanium control specimens in the flow chamber system for 24 hours were fixed with 50% ethanol for 40 min at a flow rate of 150 µl/min. Specimens were removed under sterile conditions, air-dried and subjected to fluorescence in situ hybridization according to a previously published protocol40,41,44. In brief, cells were permeabilized with 1 µg/ml lysozyme before being hybridized with 8 µM of each 16S rRNA probe in urea-NaCl buffer for 30 min at 46 °C. The sequences and references of the applied probes are listed in Supporting Information Table S1. Stained biofilms were covered with PBS and analyzed by CLSM in a sequential process using PMT detectors (Leica TCS SP8, Leica Microsystems). The first sequence detected ALEXA Fluor®405 signals using a 405 nm laser and an emission range of 413 – 477 nm and ALEXA Fluor®568 signals using a 552 nm laser and an emission range of 576 – 648 nm. The second sequence detected ALEXA Fluor®488 signals using a 488 nm laser and an emission range of 509 – 576 nm together with ALEXA Fluor®647 signals using a 638 nm laser and an emission range of 648 – 777 nm. Image stacks with an area of 190 x 190 µm2 were acquired with a zstep size of 2 µm. The Imaris 6.2.1 software package (Bitplane AG) was used for image maximum projection and brightness/contrast processing.


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2.5

Single bacterial cell force spectroscopy

For bacterial adhesion force measurement, a FlexFPM atomic force microscope (Nanosurf AG, Liestal, Switzerland) connected to a FluidFM pressure control system (Cytosurge AG, Zürich, Switzerland) was mounted on an inverted microscope (Eclipse Ti-S, Nikon GmbH, Düsseldorf, Germany). The setup employs a hollow cantilever with a circular opening at the end, which enables the reversible immobilization of bacterial cells for spectroscopy by applying negative pressure45. Thus, it circumvents chemically-based cell fixation to cantilevers, which may impact bacterial surfaces, and allows measurement of more individual cells at higher throughput. Hollow silicon nitride cantilevers with a circular opening of 300 nm and a theoretical spring constant of 0.6 N/m (FluidFM Nanopipette, Cytosurge AG) were used. Prior to spectroscopy, the exact spring constant of each cantilever was measured based on the method described by Sader et al.46. It was always in the range of 0.6 ± 0.1 N/m. The cantilever was filled with degassed, filtered (0.22 mm pore size) phosphate buffered saline and its sensitivity was calibrated using the corresponding machine software scripts. 50 mm glass dishes (WillCo Wells B.V., Amsterdam, The Netherlands) were equipped with a glass ring of 1 mm thickness and a cavity for at-grade insertion of titanium SLIPS or control specimens. S. oralis precultures were suspended in filtered phosphate buffered saline to a final optical density of 0.005 at 600 nm (1x106 CFU/ml according to standard plate counting) and added to the glass dish. To capture a single bacterial cell on the glass ring, the cantilever approached with a set point of 10 nN and paused on the bacterium for 5 s to apply 400 mbar of negative pressure, before being retracted with a piezo velocity of 1 µm/s. The cantilever with the reversibly immobilized bacterial cell was transferred over the titanium SLIPS or control specimen to perform single bacterial cell force spectroscopy. The bacterium was approached to the surface with a setpoint of 0.75 nN, to prevent bacterial compression47 while maintaining a reliable approach. It was allowed to adhere to the surface for 0, 10 or 30 s ACS Paragon Plus Environment

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with force feedback enabled and retracted with a piezo velocity of 1 µm/s. For each surface and condition, 12 individual bacterial cells were measured 16 times, each at different positions on the titanium SLIPS and control specimens. After quality control (e.g. removal of curves without surface contact), N > 100 individual force/distance curves could be included in the analysis for each setting. To calculate maximum adhesion force, the program AtomicJ48 was used. The settings are listed in Supporting Information Table S2. The number of attachment points and the distance to detachment were quantified manually using the force/distance curves generated by AtomicJ. A custom Matlab (R2013b, The MathWorks GmbH, Aachen, Germany) script was used in order to calculate the area f between the baseline and the withdraw curves, which was interpreted as adhesion work. The area was computed according to 𝑛 ― 1 𝑦𝑖 + 𝑦𝑖 + 1 2

𝑓 = ∑𝑖 = 1

|

|

∙ (𝑥𝑖 + 1 ― 𝑥𝑖) ,

where n was the number of data points, 𝑦𝑖 the force (with the baseline set to zero) and 𝑥𝑖 the distance. GraphPad Prism software 6.01 (GraphPad Prism Software Inc.) was used for data visualization and statistical analysis. Gaussian distribution was assessed by D’Agostino & Pearson omnibus normality testing and significance was tested at p ≤ 0.05 with two-way ANOVA. 2.6

S. oralis ATCC® 9811TM genome sequencing, assembly and annotation

S. oralis ATCC 9811 was precultured in Tryptone Soy Broth supplemented with 10% yeast extract at 37 °C for 18 h under aerobic conditions. The bacterial suspension was centrifuged, the supernatant was removed, and the pellet was frozen at -20 °C. To isolate DNA, bacteria were lysed enzymatically in LM buffer (10 mM Tris; Carl Roth GmbH & Co. KG, 1mM EDTA, pH 8.0, 15 mg/ml lysozyme, 500 U/ml mutanolysin from Streptomyces globisporus ATCC 21553; all


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Sigma Aldrich Chemie GmbH, Munich, Germany) at 37 °C and 750 rpm for 2 h12. 10% SDS (final concentration 3%; Sigma Aldrich Chemie GmbH) and proteinase K (final concentration 200 µg/ml; PeqLab Biotechnologie GmbH, Erlangen, Germany) were added and incubated at 55 °C and 300 rpm for 30 min. DNA was extracted in phenol:chloroform:isoamylalcohol (mixing ratio 25:24:1; Carl Roth GmbH & Co. KG) in three repetitions. For purification, DNA was ethanol precipitated. Contaminating RNA was degraded by incubation with RNase A (Qiagen, Hilden, Germany) at a concentration of 0.1 mg/ml, 37 °C for 30 min. Magnetic beads (Agencourt® AMPure XP®; Beckman Coulter, Beverly, USA) were used for final DNA cleanup prior to DNA shearing. 4 µg of input DNA were applied to g-TUBE devices (Covaris, Woburn, USA) and centrifuged twice (11500 rpm, 60 s, Eppendorf Minispin® plus centrifuge) to acquire DNA fragments of approximately 6 kb in size. Shearing efficacy and fragment size distribution were checked on a 2100 Bioanalyzer instrument (Agilent Technologies, Palo Alto, CA, USA) and DNA concentration was determined on a Qubit 2.0 fluorometer (Life Technologies). Sequencing templates were prepared according to the manufacturer’s protocol for “Preparing SMRTbell™ Libraries using PacBio® Barcoded Adapters for Multiplex SMRT® Sequencing (PN 101-069-200-01)” using sequencing chemistry v2.0 and SMRT® 1M v2 sequencing cells, and the DNA library was subsequently sequenced on a PacBio Sequel instrument in a 10 h run (Software: SMRT® Link v5.0.1; Pacific Biosciences, Menlo Park, USA). Sequences were demultiplexed using SMRT Link v5.1.0 with a minimum barcode score of 30 and assembled with HGAP4 as implemented in SMRT Link v5.1.0 with the following non-default settings: Expected genome length 2,000,000 bp, minimum subread length 500 bp, aggressive assembly option, seed coverage 60, and PacBio-recommended FALCON cfg overrides pa_DBsplit_option = -x500 -s200; ovlp_HPCdaligner_option = -v -B24 -M16 -h35 -e.93 -l1000 -s100 -k25; pa_HPCdaligner_option = -v -B24 -M16 -h70 -e.75 -l1000 -s100 -k18. The 3 contigs of the


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resulting assembly were annotated using the RAST pipeline49. Completeness of the assembly was estimated using Benchmarking Universal Single-Copy Orthologs50. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SSNB00000000 (BioSample SAMN11401878). The version described in this paper is version SSNB01000000. Genome annotation can be found in Supporting Information Table S3. 2.7

RNA isolation, rRNA depletion, cDNA library preparation, sequencing and data analysis

RNASeq was applied to compare gene expression profiles of S. oralis biofilms on SLIPS surfaces, titanium control surfaces, and planktonic cells. Biofilms were grown in the flow chamber system for 20 h as described above. After cultivation, planktonic samples were taken from medium inside the bioreactor and mixed with RNAprotect Bacteria Reagent (Qiagen), incubated for 5 min, pelleted by centrifugation and stored at -20 °C until analysis. The remaining medium from the bioreactor was transferred to sterile tubes, centrifuged, and the bacteria-free supernatant was used to wash the system for 90 minutes at 100 µl/min to remove planktonic bacteria while maintaining cultivation conditions. To collect the biofilms, flow chambers were separated from the system, opened under sterile conditions, the biofilm covered specimens were removed and immediately transferred into RNAprotect Bacteria Reagent. Biofilms were removed from the surfaces by flushing with a micropipette and samples were processed similarly to planktonic cells. The experiment was performed in 4 replicates. RNA was isolated according to a previously described protocol12. Briefly, bacteria were lysed enzymatically in LM buffer at 25 °C and 250 rpm for 90 min. After adding RLT buffer (Qiagen) containing 1% β-mercaptoethanol, bacterial cells were mechanically disrupted by vortexing for 30 s in the presence of 50 µg sterile, acid-washed glass beads (diameter 106 µm; Sigma Aldrich Chemie GmbH), which was repeated 10 times with 1 min intervals on ice. Total RNA was isolated using the RNeasy Mini Kit


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(Qiagen) according to the manufacturer’s instructions. DNA was digested with DNase I (Qiagen), first on column and later in solution, followed by RNA purification according to the RNeasy cleanup procedure. Ribosomal RNA was depleted by capture method using the Ribo-Zero Kit for Gram-positive bacteria (Illumina, San Diego, CA, USA). The quality and quantity of total RNA, enriched mRNA and cDNA was assessed using the 2100 Bioanalyzer instrument and dedicated kits (Agilent Technologies). Due to poor RNA quality, one planktonic sample had to be excluded from further analysis. mRNA was converted to cDNA using ScriptSeq v2 RNA-Seq (Epicentre Biotechnologies, Madison, Wisconsin, USA). For sequencing, 15 ng of each library was used and 11 samples were multiplexed on a single lane. Cluster generation was performed with cBot (Illumina) using a TruSeq SR Cluster Kit v3–cBot-HS (Illumina). Samples were sequenced for 50 cycles on an Illumina HiSeq 2500 sequencer using the TruSeq SBS Kit v3 - HS (Illumina). Image analysis and base calling were performed using the Illumina pipeline v 1.8. Sequencing data are available at Gene Expression Omnibus under Accession Number GSE129981. RNA Sequencing data were analyzed on the Galaxy platform (https://usegalaxy.org/, last access: October, 2018). Where not stated otherwise, default settings were used. Data were trimmed using Trim Galore! (Galaxy Version 0.4.3.1; Adapter sequence to be trimmed: Illumina universal; Overlap with adapter sequence required to be trimmed: 5; Discard reads that became shorter than length N: 30) and quality controlled using FastQC (Galaxy Version 0.69; 51). Data were aligned to the genome of S. oralis ATCC 9811 using HISAT2 (Galaxy Version 2.1.0; 52). To count aligned reads, htseq-count (Galaxy Version 0.9.1; Mode: Intersection (nonempty); Stranded: No; Feature type: gene;53) and the genome annotation for S. oralis ATCC 9811 were used. Differently expressed genes were determined using DESeq2 (Galaxy Version 2.11.40.1; Output normalized count table: True;54) comparing each pair of datasets separately (biofilm on titanium vs. planktonic, biofilm on SLIPS vs. planktonic, biofilm on SLIPS vs. biofilm on titanium). The


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adjusted p-value threshold for statistical significance was set to 0.05. Expression values of the genes found to be significantly differently expressed in at least one of the comparisons were visualized as a heatmap depicting the arithmetic mean of the two normalized count values obtained for each condition. The heatmap was constructed using the heatmap.2 command of the R package gplots version 3.0.1 under R version 3.4.2 (The R Foundation, Vienna, Austria), with the row and column reordering options disabled. For optical reasons, the heatmap was first constructed using the log10 of the input values, and the color scale legend was manually adjusted to denote the original non-transformed values. Genes were assigned to different functional groups based on KEGG pathway annotation (http://www.genome.jp/kegg/annotation/enzyme.html, last access: November, 2018). 2.8

Statistical Analysis

The software and statistical tests used for data documentation and analysis are stated in the respective sections. Results are given as arithmetic mean (± standard deviation). Statistical significance was assessed at p ≤ 0.05, and is referred to “significant” in the results and discussion sections. 3 3.1

RESULTS Reduced biofilm volume on titanium SLIPS in the flow chamber system

Prior to biofilm examination, stability of SLIPS functionalization (Figure 1A) on the surfaces was verified by reflection microscopy in the flow chamber setup. Titanium SLIPS were analyzed after 30 min of equilibration in PBS with a flow rate of 100 µl/min, after 24 h in continuous PBS flow at 100 µl/min – conditions for mimicking biofilm formation – and after additional pulsed washing with PBS (the harshest washing condition). At all time points, a clear lubricant layer with an


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average thickness of 121 µm ± 68 µm was detected (Figure 1B). Despite some variation between different specimens, the lubricant layer on a single specimen did not change over time and no significant difference was observed between the lubricant layers at different time points. To analyze biofilm repulsion of titanium SLIPS, sessile communities of the initial oral colonizer S. oralis were grown for 24 hours on partially liquid-infused titanium in a flow chamber system. Biofilms were washed using different flow velocities to remove loosely attached or unbound bacteria prior to microscopic analysis of biofilm volume. As shown in Figure 1C and D, S. oralis biofilm volume was reduced on SLIPS surfaces compared to the titanium control, and this effect was significant for washing with 150 µl/min, 250 µl/min and pulsed washing. The mean biofilm volume on titanium control surfaces was 3.8 ∙ 107 µm3 (± 2.3 ∙ 107 µm3) per image, regardless of the washing condition. With washing with 100 µl/min flow rate, as used for biofilm cultivation, the biofilm volume on SLIPS surfaces slightly decreased by ~ 20% to 3.1 ∙ 107 µm3 (± 2.7 ∙ 107 µm3) per image. After washing with 150 µl/min and 250 µl/min, the decrease in biofilm volume on SLIPS surfaces was more pronounced (~ 65%) with 1.3 ∙ 107 µm3 (± 1.6 ∙ 107 µm3) and 1.4 ∙ 107 µm3 (± 1.4 ∙ 107 µm3) per image, respectively. The greatest reduction was observed after pulsed washing, which results in a biofilm volume of 2.9 ∙ 106 µm3 (± 1.9 ∙ 106 µm3) per image, a decrease of ~ 95% (as shown in Figure 1D). Live/dead staining revealed that all biofilms mainly consisted of viable cells (Figure 1E). The amount of viable bacteria in biofilms grown on SLIPS was slightly lower (on average 87.1% ± 6.5%) than on titanium control surfaces (on average 92.6% ± 4.2%), regardless of the washing velocity. These small differences were significant for washing with 100 µl/min, 250 µl/min and pulsed washing. To analyze the long term stability of SLIPS functionalization, the surface was exposed to a continuous flow of 100 µl/min PBS for 13 days prior to inoculation with bacteria. Reflection microscopy revealed that the SLIPS lubricant layer was maintained on the surface (Figure 2A). ACS Paragon Plus Environment

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No significant difference could be detected compared to the lubricant thickness directly after assembly of the flow chamber. After inoculation of bacteria and 24 hours of cultivation, biofilms were pulse-washed, stained and subjected to microscopic analysis. As shown in Figure 2B, biofilm volume on titanium control surfaces was 4.8 ∙ 107 µm3 (± 1.4 ∙ 107 µm3) per image. On SLIPS surfaces, the volume decreased significantly by ~ 55% to 2.2 ∙ 107 µm3 (± 1.6 ∙ 107 µm3) per image. The percentage of viable bacteria (Figure 2C) on SLIPS surfaces was 94.7% (± 3.0%), which was slightly but significantly lower than the amount of viable bacteria on titanium control surfaces (96.4% ± 3.3%). In the oral cavity, biofilms are multi-species communities. Therefore, titanium SLIPS were also subjected to in vitro colonization by an oral multi-species biofilm. The corresponding biofilm model has been established and characterized40,41. The incorporation in the biofilm of all four inoculated species was verified by fluorescence in situ hybridization staining (Figure 3A). S. oralis was the dominant species, followed by V. dispar, whereas A. naeslundii and especially P. gingivalis made up smaller fractions of the biofilm, as in line with previous results40,41. After 24 hours of cultivation followed by pulsed washing, the biofilm volume on SLIPS surfaces was significantly reduced (Figure 3B). On SLIPS surfaces, a biofilm volume of 6.2 ∙ 106 µm3 (± 4.3 ∙ 106 µm3) per image was calculated, which was ~ 60% less than the biofilm volume of 1.5 ∙ 107 µm3 (± 8.2 ∙ 106 µm3) per image on titanium control surfaces. The multi-species biofilm’s viable fraction was 92.0% (± 4.1%) on SLIPS surfaces and slightly higher with 94.6% (± 1.3%) on titanium control surfaces (Figure 3C).


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Figure 1. S. oralis biofilm on titanium SLIPS with different washing velocities. (A) Schematic illustration of SLIPS fabrication process. (B) Tukey box plots of SLIPS lubricant thickness directly after flow chamber assembly, after 30 min of equilibration in PBS with a flow velocity of 100 µl/min, after 24 h in continuous PBS flow at 100 µl/min - mimicking the conditions of biofilm formation - and after additional pulsed washing with PBS. (C) Representative 3D reconstruction of the biofilm at the SLIPS/titanium interface after pulsed washing. Viable


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bacteria are visualized in green and dead bacteria are visualized in red. Scale bars corresponding to 200 µm. (D) Tukey box plots of S. oralis biofilm volume per image and (E) mean ± standard deviation of biofilm live/dead distribution on SLIPS and titanium control surfaces after 24 hours growth in the flow chamber system, followed by washing with the different velocities stated in D. All graphs N = 9. (*) indicates statistical significance at p ≤ 0.05.

Figure 2. S. oralis biofilm on titanium SLIPS exposed to continuous flow for 13 days. (A) Tukey box plots of SLIPS lubricant thickness directly after flow chamber assembly and after 13 days exposure to continuous flow of PBS with 100 µl/min. Data that were not obtained in the same experiment designated by the dotted line. (B) Tukey box plots of S. oralis biofilm volume per image and (C) mean ± standard deviation of biofilm live/dead distribution on SLIPS and titanium control surfaces exposed to continuous flow of PBS with 100 µl/min for 13 days prior to biofilm formation for 24 h and pulsed washing. All graphs N = 9. (*) indicates statistical significance at p ≤ 0.05.


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Figure 3. Oral multi-species biofilm on titanium SLIPS. (A) Microscopic images of FISHstained oral multi-species biofilm grown for 24 hours on titanium in the flow chamber system. The individual bacteria were stained with 16S rRNA FISH probes: S. oralis – blue; A. naeslundii – green; V. dispar – yellow; P. gingivalis – red. The images on the left side show the single color channels for the four individual species; the image on the right side shows the overlay of the four color channels. Scale bars correspond to 30 µm. (B) Tukey box plots of multi-species biofilm volume per image and (C) mean ± standard deviation of multi-species biofilm live/dead distribution on SLIPS and titanium control surfaces after 24 h growth in the flow chamber system and pulsed washing. All graphs N = 9. (*) indicates statistical significance at p ≤ 0.05.


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Reduced bacterial adhesion forces on titanium SLIPS

In order to quantify bacterial adhesion forces on liquid-infused titanium surfaces, single bacterial cell force spectroscopy was performed. Representative force-distance curves for S. oralis spectroscopy on titanium control and SLIPS surfaces are shown in Figure 4A-C. The curves started with a positive deflection of 0.75 nN, linearly decreased to the maximum negative deflection and then gradually returned to the baseline of 0 nN deflection; the first major adhesion peak was followed by a number of minor adhesion peaks. As the steepness of the linear reduction in decreasing deflection corresponds to the stiffness and elasticity of the underlying material55, the steeper titanium withdrawal curves reflect its greater stiffness and lower elasticity compared to the lubricant-infused SLIPS. This result also confirmed that bacteria were not pushed through the lubricant layer to the underlying titanium, but were approached on the lubricant surface. From each force-distance curve, the maximum adhesion force, the number of attachment points, the detachment distance and the performed adhesion work were quantified (Figure 4A). All evaluated parameters increased with increasing adhesion time, regardless of the condition (Figure 4D-G). The mean values for each parameter are given in Table 2. After an adhesion time of 30 s, the maximum adhesion force (Figure 4D) and the number of attachment points (Figure 4E) on SLIPS surfaces were significantly lower than on titanium control surfaces. In contrast, after adhesion times of 10 s and 30 s, the distance to detachment of bacteria from the surface (Figure 4F) and the adhesion work needed to detach bacteria from the surface (Figure 4G) were significantly greater on SLIPS surfaces than on titanium control surfaces.


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Figure 4. S. oralis adhesion force on titanium SLIPS and control surfaces. (A) Schematic illustration of parameters analyzed from force-distance curves of single bacterial cell force spectroscopy. Representative force-distance curves of S. oralis single bacterial cell force spectroscopy on titanium control (B) and titanium SLIPS (C) surfaces obtained with a setpoint force of 0.75 nN and an adhesion time of 0, 10 or 30 s. Tukey box plots of maximum adhesion force (D), number of attachment points (E), detachment distance (F) and adhesion work (G) of S. oralis single bacterial cell force spectroscopy on titanium control and titanium SLIPS surfaces obtained with a setpoint force of 0.75 nN and an adhesion time of 0, 10 or 30 s. (*) indicates statistical significance at p ≤ 0.05.

Table 2. S. oralis single bacterial cell force spectroscopy. Mean ± standard deviation of parameters evaluated on titanium or SLIPS functionalized surfaces with increasing adhesion time. Adhesion time

Max adhesion force [nN]

Attachment points

Detachment distance [µm]

Adhesion work [aJ]

Titanium

SLIPS

Titanium

SLIPS

Titanium

SLIPS

Titanium

SLIPS

0s

0.82 ± 0.50

0.84 ± 0.56

4±4

3±3

0.22 ± 0.26

0.30 ± 0.24

102.9 ± 142.6

180.4 ± 211.6

10 s

1.44 ± 1.00

1.24 ± 0.63

6±3

6±4

0.37 ± 0.19

0.74 ± 0.57

194.2 ± 173.9

416.9 ± 367.9

30 s

3.67 ± 2.16

2.70 ± 1.34

8±6

7±4

0.47 ± 0.29

1.03 ± 0.63

718.8 ± 704.1

1252.7 ± 1253.7


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3.3

No differences in biofilm gene expression on titanium SLIPS and control surfaces

To detect potential changes in biofilm gene expression on titanium SLIPS compared to titanium control surfaces, mRNA isolated from bacterial biofilms and planktonic cultures was sequenced. As basis for this analysis, the genome of S. oralis ATCC® 9811TM was sequenced and annotated. The assembled genome consists of 1,881,450 bp in 3 contigs and has a GC content of 41.4%. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis indicated 99.8% completeness of the assembled genome50. RAST annotation predicted 1,795 protein encoding genes, 60 tRNA and 4 rRNA operons with 12 rRNA loci, which is comparable to characteristics of previously sequenced S. oralis genomes (https://www.ncbi.nlm.nih.gov/genome/2194, last access: December, 2018). Differential gene expression analysis of planktonic, biofilm on titanium and biofilm on SLIPS samples revealed 442 genes, with significantly different expression values between at least two of these conditions (Figure 5A). 409 genes were differently expressed in planktonic and biofilm on titanium samples and 337 genes were differently expressed in planktonic and biofilm on SLIPS samples. Among these, 304 genes were expressed in common in cells from both biofilm types when compared to planktonic samples (Figure 5B). While some variation between the biological replicates could be observed, no significant differences in gene expression between biofilm on titanium and biofilm on SLIPS samples could be detected. In comparison of gene expression in biofilms on both surfaces to planktonic samples, 186 genes were less expressed and 256 genes were more expressed in biofilms (Supporting Information Table S4). They can be assigned to different biological functions, such as sugar, amino acid or nucleotide metabolism, membrane transport or energy harvesting (Figure 5A). Exemplary genes are listed in Table 3. Genes that were expressed at significantly different levels only in either biofilms on the SLIPS or the titanium surface, compared to the planktonic control, did not show


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any specific pathway affiliation (Supporting Information Table S3). As mentioned above, none of these genes was expressed to a significantly different level in the two biofilm conditions.

Figure 5. S. oralis gene expression in biofilms on titanium SLIPS/control surfaces and planktonic samples. S. oralis biofilms were grown for 20 h in the flow chamber system on titanium SLIPS or


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titanium control samples at 100 µl/min and washed with the same velocity for 90 min with bacteria-free medium before samples were taken. Planktonic samples were taken from the bioreactor feeding the flow chamber system. (A) Heat map of mean normalized counts of genes differently expressed in at least two experimental conditions. Differently expressed genes were grouped on the basis of the KEGG pathway annotation. N = 4. (B) Venn diagram showing the distribution of differently expressed genes among the different comparisons of each two datasets.

Table 3. Exemplary genes with significantly different expression in biofilms on both surfaces vs. planktonic samples. BF – Biofilm

Gene/Operon Product Name and Function

Significant Log2 Fold Change BF Titanium vs. BF SLIPS vs. Planktonic Planktonic

Metabolism - Glycolysis NAD-dependent Glyceraldehyde-3-phosphate Dehydrogenase

- 1.11

- 0.99

Fructose-bisphosphate Aldolase Class II

- 1.01

- 1.04

Enolase

- 0.91

- 0.95

Glucose-6-phosphate Isomerase

- 0.74

- 0.78

Multimodular Transpeptidase-Transglycosylase

- 0.89

- 0.90

Oligoendopeptidase F

- 0.86

- 0.89

Cell Division Protein FtsX

- 0.67

- 0.73

L-Lactate Dehydrogenase

- 1.15

- 1.17

Acetate Kinase

1.03

1.24

Several Amino Acid ABC Transporter ATP-binding Protein

0.79 – 1.70

0.96 – 1.85

Several Amino Acid ABC Transporter Permease Protein

0.89 – 1.62

0.74 – 1.69

0.88 – 1.43

0.85 – 1.25

1.73

1.91

Cell Division

Fermentation

Membrane Transporters

Energy Harvesting Several ATP Synthase Components Gene Regulation Two-Component System Transcriptional Response Regulator, LuxR Family


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DISCUSSION

Since its introduction, the principle of slippery liquid-infused porous surfaces32 has been applied to numerous materials and has consistently led to reduced bacterial loads on the surface. Several different polymers coated with perfluoropolyethers or perfluorodecalin and SLIPS made from silicone showed reduced colonization with Escherichia coli, Pseudomonas pneumoniae and Staphylococcus aureus31,33-35. Different metal-based SLIPS were found to withstand colonization with P. aeruginosa, S. aureus and several Desulfovibrio species38,56. In a previous study, we developed laser-structured titanium SLIPS mimicking the structure of lotus leaves, which are coated with the liquid perflouropolyether Krytox GPL 104. We have shown that these repel biofilms of the oral initial colonizer S. oralis under static conditions30. As surfaces in the oral cavity are constantly exposed to the flow of saliva, a more sophisticated testing system was needed to obtain reliable results to support the prospective application of liquid-infused titanium in oral implantology. In the present study, the recently developed SLIPS were analyzed in a flow chamber system that more closely mimicked the situation in the oral cavity. Reflection microscopy revealed that the infused lubricant was stable up to flow rates of 1000 µl/min, as well as during long-term exposure to flow rates of 100 µl/min for 13 days. These flow rates are within the range of normal salivary flow, which ranges from 100 µl/min in the hibernation mode to 7000 µl/min when stimulated57. Previous studies also showed that SLIPS are generally stable under flow conditions lower than a design-driven critical shear stress58,59. Titanium SLIPS in the flow chamber system reduced attached biofilm volume by up to 95%. The remaining biofilm (Figure 1C) was anchored on the liquid layer, probably to denaturated proteins at the lubricant/liquid interfaces, and did not penetrate through the lubricant. The reduced biofilm ACS Paragon Plus Environment

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volume remained significant after long-term exposure to phosphate buffered saline. However, the magnitude of the biofilm-repulsion was somewhat decreased after 13 days of exposure to continuous flow conditions. This suggests that oral application of this SLIPS setup might benefit from replenishment of the lubricant. Significant reductions in biofilm volume were not only found in mono-species biofilms, but also in a multi-species assemblage mimicking an oral commensal biofilm40,41. The decrease in biofilm volume on SLIPS surfaces was slightly reduced with a multi-species biofilm, which may be attributed to additional adhesion capabilities60 and supportive interactions between the different bacterial species15. Nevertheless, the difference between mono-species and multi-species biofilms in the magnitude of the SLIPS effect emphasizes that it is important to employ complex in vitro models to achieve reliable results that are relevant to in vivo applications. In the present study, the rate of reduction in the biofilm volume increased with increasing shear forces, matching observations by MacCallum et al.35. Biofilm-repellent effects of SLIPS in flow systems are in the mean also smaller than in static experiments30,31,33-35,37,38. This apparent contradiction is most probably due to differences in selection pressure and the maximal shear forces applied: In static experiments, there is no selection pressure for firmly attached cells and the surrounding liquid is removed prior to most quantification methods. The resulting exposure of the biofilm to air creates high shear forces on less strongly attached cells. In flow systems, on the other hand, there is selective pressure for firmly attached cells and the liquid environment is also maintained during quantification processes, corresponding to a reduction in apparent shear forces. With increasing flow rates and shear forces in flow chamber systems, it can be predicted that the attached biofilm on SLIPS surfaces could be totally eliminated.


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Live/dead staining of biofilms revealed a slight but significant increase of ≤ 5% in the amount of dead cells on SLIPS surfaces when compared to titanium controls. This has already been shown in statically conducted experiments30. As this increase is much smaller than the biofilm repulsion, and the non-toxicity of the SLIPS components has been previously confirmed30,31,33, this may not serve as an explanation for the SLIPS mechanism. The most prominent hypothesis on the SLIPS mode of action is that bacteria are not able to stably adhere to the liquid slippery layer and can therefore easily be removed by shear forces31,35,37,38. This is supported by the observations of the present study: Biofilms can form on SLIPS surfaces under low flow conditions, but their volume is reduced by washing with elevated flow rates; this effect increases with increasing flow velocities. While we previously demonstrated reduced initial attachment of S. oralis on the liquid surface30, the contributing mechanisms have not been experimentally validated. The present study is the first to directly measure adhesion forces of bacterial cells to SLIPS surfaces, which is achieved by means of single bacterial cell force spectroscopy. The increase in adhesion strength over time, which was observed on both titanium SLIPS and control surfaces, is a known phenomenon45,61. This is mainly due to the removal of interfacial water and the stereochemical orientation of hydrogen bonding over time, which steadily increase the resulting adhesion forces45,61. Comparison between titanium and SLIPS surfaces shows that the maximum adhesion force and attachment points of S. oralis are significantly lower on the latter surface. The maximum adhesion force is driven by non-specific electrostatic Lifshitz-Van der Waals forces, whereas the attachment points represent specific hydrogen bonding61-63. On SLIPS surfaces, S. oralis exhibited lower non-specific interaction forces and less hydrogen bonding than on control surfaces. In contrast, detachment distance and adhesion work are significantly greater on SLIPS


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than on controls. The detachment distance is thought to reflect bacterial stretching upon withdrawal and the length of adhering molecules47,61. However, this relationship can only be established on a rigid surface such as titanium. A soft, deformable surface, such as the SLIPS lubricant layer, will stretch upon withdrawal of bacterial cells and thus lead to even longer detachment distances, which do not depend on bacterial adhesion forces. As the adhesion work depends on all these parameters, the drastically increased detachment distance also increases the adhesion work. Direct comparison of the detachment distance and adhesion work on titanium and SLIPS surface is therefore not possible, due to the different deformability of the two materials. Nevertheless, the significantly reduced non-specific physical adhesion forces and hydrogen bonding measured on a single cell level substantiate the hypothesis that S. oralis attachment on SLIPS surfaces is inefficient. As initial attachment is a key factor for initiating the biofilm phenotype, the reduced adhesion forces may have impaired this process6,9,10,22, which would additionally support the concept that the biofilm on the SLIPS surface is unstable. To address this possibility, S. oralis transcriptome of biofilms formed on SLIPS and titanium control surfaces was analyzed by means of RNAseq. With respect to gene expression patterns, no differences between biofilms on SLIPS and control surfaces could be established. However, this method does not account for spatial heterogeneity within biofilms. It is possible that bacterial cells in direct contact to the SLIPS surface might show alteration in gene expression patterns that could not be detected in our experimental setup. To address this problem, the transcriptional activity of cells in early single layer biofilms may be measured, reporter strains for genes of interest may be applied64, or alternatively in future, if technological advances allow, singe cell RNA sequencing could be performed for micromanipulated cells that had direct contact with the substratum65. In comparison to planktonic cultures, biofilms on either surface exhibited changes in approximately 22% of the annotated ACS Paragon Plus Environment

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genes. This may be attributed to common gene expression patterns associated with biofilm formation, including metabolic changes that are probably due to altered nutrient supply and pH or modifications in growth rate6,9,10,21-23,66. A detailed analysis of differentially expressed genes in biofilms and planktonic cultures has not been carried out and could be addressed in further studies. For the liquid-infused surfaces analyzed in this study, the overall observation suggests that S. oralis on SLIPS surfaces, even if less stably attached, form a biofilm largely identical to those on titanium surfaces. Taken together, the biofilm-repelling SLIPS surface reduces S. oralis bacterial adhesion forces without altering its biofilm transcriptome profile as judged by mRNA-level gene expression. As SLIPS coated with various lubricants exhibited the same biofilm-repellent effect31,33-35,38,67, the inefficient attachment is most probably due to the different underlying interface (solid-liquid vs. liquid-liquid), rather than the chemical composition of the lubricant. Therefore, the proposed hypothesis of unstable attachment can be confirmed for this bacterium. S. oralis biofilms are able to form on liquid-infused surfaces, but, due to insufficient attachment on the liquid-liquid interface, they can easily be removed by increased shear forces. Even if the SLIPS effect of reduced bacterial surface load is transferable to multiple other bacterial species31,33-35,38, the exact mechanism could be different. Streptococci adhere with surfaces molecules to a protein layer7. As proteins denaturate at the lubricant/liquid interface, they may serve as anchor for streptococci and enable (less stable) biofilm formation. For other bacteria exhibiting surface structures as flagella or pili, a direct contact to a solid surface is needed for the induction of a biofilm phenotype10,22. Thus, their behavior on the liquid SLIPS surface may differ. The results obtained for S. oralis in this study may assist in unraveling the interaction between SLIPS surfaces and other bacterial species.


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5

CONCLUSION

Biofilm-repellent surfaces based on the SLIPS mechanism possess enormous potential in developing implant materials that prevent infections. On the basis of this principle, we have recently developed liquid-infused titanium by combining a superhydrophobic micro- and nanopattern generated by ultra-short pulsed laser-ablation with a perfluoropolyether lubricant. This combination was strongly repellent to biofilms in multi-well plate assays and the SLIPS components had no inherent toxicity. In the present study, these surfaces were analyzed in a more sophisticated flow chamber system that simulates the flow of oral saliva. The titanium SLIPS surfaces were shown to be stable for 13 days of continuous flow and to steadily reduce the amount of attached biofilm with increasing flow rates - not only with S. oralis mono-species biofilms but also with an oral multi-species biofilm composed of S. oralis, A. naeslundii, V. dispar and P. gingivalis. This observation support the hypothesis that the SLIPS act by reducing bacterial adherence to the liquid layer, thus facilitating their removal by shear forces. To unravel the SLIPS mechanism in more detail, bacterial adhesion forces on the SLIPS surface were directly measured by single bacterial cell adhesion force spectroscopy of S. oralis. The results revealed that both non-specific physical forces and hydrogen bonding were reduced on SLIPS surfaces when compared to titanium controls. As this may have impaired biofilm formation, gene expression patterns of biofilms on SLIPS and control surfaces were analyzed. Both biofilms exhibited changes in approximately 22% of the coding genes when compared to planktonic cells, but no differences between the biofilms grown on the two different surfaces. According to the results of this study, the S. oralis biofilm-repellent properties of liquid-infused titanium are mainly based on reduced bacterial adhesion forces to the underlying liquid interface, without alterations in the ability to form biofilms.


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ASSOCIATED CONTENT Supporting Information Table S1. Species-specific 16S rRNA probes for FISH. Table S2. AtomicJ settings for single bacterial force spectroscopy data evaluation. Table S3. RAST annotation of Streptococcus oralis ATCC® 9811TM genome. Table S4. Significantly differently expressed genes between biofilms on SLIPS/titanium surfaces vs. planktonic samples with assigned functional groups. AUTHOR INFORMATION Corresponding Author *Corresponding authors: Lower Saxony Center for Biomedical Engineering, Implant Research and Development (NIFE), Stadtfelddamm 34, 30625 Hannover, Germany [email protected], Tel. +0049-511-532-1421 [email protected], Tel. +0049-511-532-4773 Author Contribution #These

authors contributed equally. The manuscript was written through contributions of all

authors. All authors have given approval to the final version of the manuscript. Funding Source


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The present study was carried out as an integral part of the BIOFABRICATION for NIFE initiative, which was financially supported by the State of Lower Saxony and the Volkswagen Foundation. ACKNOWLEDGEMENT The authors would like to thank: Marcus Stollhans and Costenoble GmbH & Co. KG for kindly providing Krytox lubricant; Patrick Lang and Henrik Peisker from Nanosurf AG and Dario Ossola from Cytosurge AG for their substantial support with single bacterial cell force spectroscopy; Marly Dalton and Rainer Schreeb for genome sequencing; Agnes Nielsen, Michael Jarek, Sabin Bhuju and Susanne Häußler from the Helmholtz Center for Infection Research for cDNA synthesis and RNASeq; and Lara Kühnle, Kerstin Elbert and Ronja Hagemeier for excellent technical assistance. REFERENCES 1. IData Research Inc. Europe Market Report Suite for Dental Implant Fixatures and Final Abutments. 2017. 2. Mombelli A, Müller N, Cionca N. The Epidemiology of Peri-Implantitis. Clin Oral Implants Res 2012;23:67-76. 3. Dreyer H, Grischke J, Tiede C, Eberhard J, Schweitzer A, Toikkanen SE, Glöckner S, Krause G, Stiesch M. Epidemiology and Risk Factors of Peri-Implantitis: A Systematic Review. Journal of periodontal research 2018;53:657-681. 4. Schaumann S, Staufenbiel I, Scherer R, Schilhabel M, Winkel A, Stumpp SN, Eberhard J, Stiesch M. Pyrosequencing of Supra- and Subgingival Biofilms from Inflamed Peri-Implant and Periodontal Sites. BMC Oral Health 2014;14:157. 5. Donlan RM, Costerton JW. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clinical microbiology reviews 2002;15:167-193. 6. Flemming HC, Wingender J. Biofilme - Die Bevorzugte Lebensform Der Bakterien. Biologie in unserer Zeit 2001;31:169-180.


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67. Sotiri I, Overton JC, Waterhouse A, Howell C. Immobilized Liquid Layers: A New Approach to AntiAdhesion Surfaces for Medical Applications. Exp Biol Med (Maywood) 2016;241:909-918.

ABSTRACT GRAPHIC


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Liquid-infused structured titanium surfaces: Antiadhesive mechanism

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