Carbon-Infiltrated Carbon Nanotubes Inhibit the Development of Staphylococcus aureus Biofilms

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Bowden, Jocelyn G. Wells, Katelyn M. Miller, Anton E. Bowden, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3283589/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2023 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Staphylococcus aureus forms biofilms that cause considerable morbidity and mortality in patients who receive implanted devices such as prosthetics or fixator pins. An ideal surface for such medical devices would inhibit biofilm growth. Recently, it was reported that surface modification of stainless steel materials with carbon-infiltrated carbon nanotubes (CICNT) inhibits the growth of S. aureus biofilms. The purpose of this study was to investigate this antimicrobial effect on titanium materials with CICNT coated surfaces in a variety of surface morphologies and across a broader spectrum of S. aureus isolates. Results: Study samples of CICNT-coated titanium, and control samples of bare titanium, a common implant material, were exposed to S. aureus. Viable bacteria were removed from adhered biofilms and quantified as colony forming units. Scanning electron microscopy was used to qualitatively analyze biofilms both before and after removal of cells. The CICNT surface was found to have significantly fewer adherent bacteria than bare titanium control surfaces, both via colony forming unit and microscopy analyses. This effect was most pronounced on CICNT surfaces with an average nanotube diameter of 150 nm, showing a 2.5-fold reduction in adherent bacteria. Since S. aureus forms different biofilm structures by isolate and by growth conditions, we tested 7 total isolates and found a significant reduction in the biofilm load in six out of seven S. aureus isolates tested. To examine whether the anti-biofilm effect was due to the structure of the nanotubes, we generated an unstructured carbon surface. Significantly more bacteria adhered to a nonstructured carbon surface than to the CICNT surface, suggesting that the topography of the nanotube structure itself has anti-biofilm properties. Conclusions: The CICNT surface possesses antimicrobial properties that result in fewer adherent S. aureus bacteria. These antimicrobial properties are consistent across multiple isolates of S. aureus and are affected by nanotube diameter. The experiments performed in this study suggest that this effect is due to the nanostructure of the CICNT surface. Physical sciences/Materials science Physical sciences/Materials science/Materials for devices Physical sciences/Materials science/Nanoscale materials Biological sciences/Microbiology Biological sciences/Microbiology/Antimicrobials Biological sciences/Microbiology/Bacteria Biological sciences/Microbiology/Biofilms Biofilm carbon nanotubes Staphylococcus aureus nanostructured surfaces antimicrobial surfaces surface modification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Staphylococcus aureus is a common gram-positive bacterium that leads to nearly 20,000 deaths each year in the US [ 1 ]. S. aureus forms biofilms, which are surface-associated assemblages of bacteria embedded in an extracellular matrix. Infections with established biofilms are very difficult to treat with traditional antibiotic regimens due to limited diffusion of antibacterial agents through the biofilm matrix. S. aureus biofilms cause severe infections in healthcare settings and are particularly problematic in the context of implanted hardware such as fixator pins or artificial joints. Orthopedic implants and prostheses are becoming more and more common—in 2018 over 1 million hip and knee implants were performed in the US alone [ 2 ]. Although these implants can relieve pain and restore freedom of movement, they are susceptible to post-operative periprosthetic joint infection (PJI), which is responsible for about 30% of implant failure [ 3 ]. Up to 70% of PJI cases are caused by S. aureus [ 4 – 7 ]. Infection rates are even worse for external fixator pins, where up to 80% of all patients experience a pin tract infection and most of these are caused by S. aureus [ 8 , 9 ]. Current efforts to reduce infection rates have proven insufficient as the yearly infection burden continues to rise [ 3 ]. Titanium (Ti) is often used in medical hardware for its high mechanical strength, but this material also offers a ready surface for bacterial colonization and biofilm formation. An ideal implant surface would exhibit innate physical resistance to the attachment and formation of bacterial biofilms. This physical resistance to biofilm formation is due to the texture of the surface at the nanoscale. Structural biofilm resistance is an attractive alternative to antibiotics for biofilm control because it could reduce the need for antibiotic use, which would help reduce the development of antibiotic-resistant bacteria. Many natural surfaces possess structural antimicrobial properties, including dragonfly [ 10 ] and cicada wings [ 11 ], shark skin [ 12 ], and lotus leaves [ 13 , 14 ]. Several synthetic analogs have been developed in an attempt to replicate the anti-biofilm effects of naturally biofilm-resistant surfaces [ 15 – 17 ]. While these materials have promising applications, they also suffer from limitations such as cytotoxicity to human cells and manufacturing difficulties, constraining their use in medical tools and implants. The search for a material that can overcome these limitations is ongoing, but one promising material is carbon-infiltrated carbon nanotubes (CICNT). The CICNT surface aims to replicate the structural biofilm resistance of naturally occurring antimicrobial surfaces. Carbon Nanotubes (CNT) are nanostructured cylindrical lattices of hybridized carbon atoms. CNT possess impressive structural and mechanical properties that make them of interest in a variety of applications in biotechnology [ 18 , 19 ]. They are synthesized using chemical vapor deposition techniques, flowing ethylene gas at high temperatures over a substrate such as silicon, Ti, or stainless steel [ 20 ]. Post-processing alteration of CNT is common for biological applications. When a post-processing carbon infiltration step is added, amorphous carbon is deposited on the nanotubes, resulting in carbon-infiltrated carbon nanotubes (CICNT). This infiltration step massively increases the diameter of the nanotube, resulting in a final volume ratio of around 99% bulk carbon to 1% CNT [ 21 ], as well as providing substantial structural reinforcement that changes the mechanical behavior of the CNT forest from individual tubes to a cohesive structural layer. The purpose of this study was to demonstrate that the CICNT surface has antimicrobial properties and to better understand these properties by determining whether changing nanotube size or the material at the nanotube/bacteria interface would affect bacterial adhesion. This was accomplished through the quantification of adhered S. aureus cells on CICNT as well as bare Ti by colony forming unit analysis. The CICNT surface offers a potential solution to the increasing burden of implant-associated infection. Results Optimizing CICNT diameter to reduce biofilm growth The size of individual nanotubes can be manipulated by altering the amount of carbon infiltration. CICNT samples of various diameters (50, 150, 250, and 350 nm, Fig. 1 A) were prepared in order to quantify the effects of CICNT diameter on surface biofilm growth. S. aureus strain JE2 was grown on each surface for 36 hours. JE2 was chosen because it is a USA300 strain, which is clinically relevant, and because it was found to have a biofilm matrix structure that was representative of many S. aureus isolates in quantities of polysaccharide, protein, and extracellular DNA [ 22 ]. After 36 hours, the biofilm was washed to remove unattached cells. Adherent cells were then removed from the sample surface and quantified by performing serial dilutions, then plating onto LB agar to count colony-forming units (CFU) of bacteria. Each diameter size of CICNT showed a significantly reduced number of bacteria as compared to a bare Ti control, ranging from a 1.4-fold reduction with 50 nm CICNT (p = 0.03) to a 2.5-fold reduction with 150 nm CICNT (p = 0.0005) after 36 hours (Fig. 1 B, Table 1 ). After determining that the 150 nm diameter was most effective, we used that size of CICNT for all future experiments. Table 1 P-values from statistical comparison of results from CICNT of various diameters 50 nm 150 nm 250 nm 350 nm 50 nm - 0.0011 0.33 0.031 150 nm - 0.0032 0.0048 250 nm - 0.14 350 nm - Scanning electron microscopy (SEM) images of biofilms grown on CICNT and bare Ti We considered the possibility that the differences in CFU counts could be due to a difference in the ability to retrieve cells from Ti vs CICNT surfaces, rather than their ability to adhere. Accordingly, we conducted an experiment where we performed SEM imaging of both surfaces after our typical process to remove cells. We found that only very rare cells were still adhered to either surface, suggesting that the cell retrieval process was similar and effective for both surface types (Fig. 2 ). In addition to colony forming unit enumeration, we used a scanning electron microscope to image the biofilms grown on both the 150 nm CICNT surface and bare Ti. More bacteria can be seen adhered to the Ti surface than to the CICNT surface, and the CICNT surface appears to have more bare spots lacking attached cells than the Ti (Fig. 3 ). Biofilm growth is slower on CICNT surfaces than on Ti surfaces To determine if the anti-biofilm effect was present over multiple time points, CICNT and Ti control samples were exposed to S. aureus for 12, 24, 36, or 48 hours and the adherent bacteria were quantified by performing serial dilutions onto agar plates. At 12 hours, no significant difference between the adherent bacterial load was found between the two groups. As time progressed, this difference became significant and the ratio between the number of bacteria on Ti and on CICNT was most substantial at 36 and 48 hours (Fig. 4 ). After determining that the 36-hour time point showed the most significant difference in CICNT versus the control, we used that time point for all future experiments Additionally, we performed a longitudinal analysis and found that the interaction effect of surface type and time was significant. A one-sided general linear hypothesis test on the hypothesis that the rate of biofilm growth was lower on the CICNT surface than on the Ti surface was significant (p = 0.0014). This indicates that the biofilm was growing more slowly on the CICNT surface than on the Ti surface. A variety of S. aureus isolates are inhibited from forming biofilms on CICNT We have previously shown that different S. aureus isolates produce biofilms that vary by overall biomass, as well as by polysaccharide, protein and extracellular DNA content. To determine if the reduction in adherent bacteria was consistent among different strains of S. aureus , we tested six additional isolates of S. aureus (JE2, used in previous experiments, is included as a reference). The clinical isolates chosen were found in a previous publication to represent a variety of relative biofilm strengths, with SH1000 forming the strongest biofilm and HA3 the weakest [ 22 ]. Additionally, both methicillin resistant and susceptible isolates were tested. We found that six of the seven isolates exhibited a significant reduction in the number of adherent bacteria on the CICNT surface as compared to bare Ti, while one isolate, HA2, showed a significant increase in the number of adherent bacteria on the CICNT surface (Fig. 5 ). Differentiating between chemical and structural biofilm resistance The reduction in adherent bacteria on the CICNT surface could be due to either an antibacterial chemical effect of the carbon used, a structural effect of the nanotubes, or both, on a developing biofilm. To differentiate between these two possibilities, we developed a carbon control consisting of nonstructured carbon deposited on the Ti surface. This carbon control, therefore, was chemically similar to the CICNT surface but possessed a different structure. The carbon control was found to have fewer adhered bacteria than the bare Ti surface (p = 0.003), but significantly more bacteria than the CICNT surface, which is also composed of carbon, (p = 4E-5, Fig. 6 ), indicating that the structure of the carbon affects the number of adhered bacteria. Discussion The purpose of this study was to investigate the antimicrobial properties of the CICNT surface. CFU analysis demonstrated that the CICNT surface effectively reduces the number of adherent bacteria as compared to bare Ti surfaces, with up to a 2.5-fold reduction in the number of adherent bacteria. SEM analysis confirmed this reduction in adherent bacteria. This reduction is consistent with the lower end of reported bacterial reduction values for the CICNT surface in the only previously published study of the material’s antibacterial properties to date [ 21 ], though in that study the CICNT surface modification was prepared on stainless steel rather than Ti. It should also be noted that the methods used by Morco et al. for that original study were different than ours both in their protocol for incubating bacteria on the CICNT surface and in their method for quantifying bacteria after incubation; Morco et al. incubated bacteria on CICNT in a bioreactor in 10% TSB for 48 hours and they counted bacteria remaining on the surfaces using SEM analysis. In this study the bacteria were grown in a still droplet on the sample surface in 66% TSB, and bacteria were quantified using CFU analysis. It is possible that differences in both biofilm growth conditions and bacterial enumeration played a role in the slight decrease in bacterial reduction found in this study compared to that of Morco et al. A reduction in adherent bacteria may indicate that a surface is chemically toxic to the cells, causing cell death, commonly referred to as bactericidal. Alternatively, a reduction in adherent bacteria could mean that the structure of the surface is preventing the bacteria from effectively attaching, known as anti-biofouling. SEM images can be used to suggest whether cell death is occurring [ 10 ]. SEM images of the S. aureus biofilm did not appear to show collapsed or deflated cells on the CICNT surface, suggesting that the reduction in adherent bacteria may be due to a prevention of attachment rather than cell death. This contrasts with the bactericidal properties of other surfaces such as dragonfly and cicada wings which demonstrate the greatest potency against gram-negative bacteria [ 23 ]. An anti-biofouling surface that prevents gram-positive bacteria from attaching and forming biofilms would play an important role in preventing infection, since antibiotics are much more effective against planktonic cells than against biofilms [ 24 , 25 ]. The results found in this study indicate that the surface nanostructure of the CICNT is important since different sizes of CICNT exhibit different levels of bacterial reduction. Furthermore, this implies that some material property such as nanotube diameter, curvature, stiffness, or the amount of space between nanotubes impacts the ability of bacteria to adhere to the surface. A diameter of 150 nm was found to exhibit the greatest degree of biofilm reduction. This size is within the range of nanostructure sizes found on dragonfly wings, which one study estimated to be between 83.3 and 195 nm [ 26 ]. Dragonfly wings also possess antimicrobial and bactericidal properties [ 27 ], though the topographies of dragonfly wings and the CICNT surface differ. The average size of carbon nanotubes tested in this study ranged from 50 to 350 nm in diameter, which is considerably smaller than that of S. aureus , which has a diameter of 0.5-1 µm. The fact that the CICNT surface features are smaller than S. aureus is in agreement with previously published literature which demonstrated that bacterial adhesion is reduced on surfaces with nanotopography smaller than the bacteria, possibly due to a reduction in bacteria-surface contact area [ 28 ]. However, the precise mechanism by which the CICNT surface reduces adherent bacteria remains to be understood. This study also tested biofilm growth at four different time points on both surfaces. At 12 hours, there was no significant difference in adherent bacteria between CICNT and Ti. However, at 24, 36, and 48 hours there were significantly fewer bacteria on CICNT surfaces than on Ti surfaces. The ratio between the number of bacteria found on Ti and CICNT surfaces was largest at 36 hours. The slight decrease at 48 hours may be due to our experimental conditions. We also found that the biofilm growth rates differed between surface types. The number of adherent bacteria grew more slowly on CICNT surfaces than on Ti surfaces. This could indicate that either fewer new bacteria were attaching to the surface or that the bacteria present on the surface were not replicating as quickly even though they remained viable. This supports the evidence collected thus far that the CICNT surface possesses anti-biofouling properties. We also tested the CICNT surface against seven different isolates of S. aureus . Most studies of materials suspected to possess anti-biofilm properties are only tested against one or two strains of bacteria [ 21 , 29 ]. However, the composition of the biofilm matrix in S. aureus is highly strain-, time-, and condition-dependent, so testing multiple strains is important to understand whether a material has broad anti-biofilm capabilities. The three main components of the biofilm matrix are proteins, polysaccharides, and extracellular DNA. These three components are important for attachment and structural components of the biofilm [ 30 ]. The isolates chosen represent both methicillin-resistant and methicillin-sensitive strains, as well as a variety of biofilm matrix compositions [ 22 ]. Six of the seven isolates tested showed significantly reduced adhesion on the CICNT surface compared to a bare Ti surface, and the CICNT surface reduced adhesion of both methicillin-resistant and methicillin-sensitive isolates. The HA2 isolate was the only isolate tested which appeared to adhere more strongly to the CICNT surface than to the bare Ti surface. This isolate was found by Ball et al. to have slightly elevated levels of protein in its matrix compared to other isolates tested, but was not significantly different from HA3, which was prevented from adhering to the CICNT surface. It is possible that some genetic difference between isolates is responsible for the difference in adherence to the CICNT surface, and that future work with additional strains may uncover such a genetic basis for why some isolates are susceptible and some are resistant to the effects of the CICNT surface. We also compared the growth of bacteria on a non-structured carbon surface to growth on the CICNT surface. This unstructured carbon control was found to have significantly more bacteria attached than the CICNT surface. This indicates that the specific structure of the surface affects the adhesion of the bacteria rather than the carbon itself having an antimicrobial effect and is in agreement with the results found by Morco et al. with CICNT grown on stainless steel [ 21 ]. It also echoes other work which describes how the antimicrobial effect of nanostructured surfaces is heavily affected by their precise arrangement and topographical structure [ 14 , 31 ]. Conclusions A Carbon-infiltrated carbon nanotube (CICNT) surface modification on Ti surfaces reduced biofilm formation of S. aureus and the magnitude of the effect was found to depend upon the CICNT diameter, the S. aureus isolate, and the topography of the CICNT surface itself. A CICNT diameter of 150 nm was found to provide optimal protection against adherent bacteria, with a 2.5-fold reduction. This effect was confirmed by CFU quantification and SEM analysis. Multiple isolates of S. aureus were investigated, and the antimicrobial effects of the CICNT surface varied by isolate but were shown to inhibit the growth of both methicillin-resistant and methicillin-sensitive isolates. Unstructured carbon did not exhibit the same antimicrobial effect, indicating that the CICNT nanostructure plays a role in biofilm reduction. Further investigation of the mechanism of the antimicrobial effect of the CICNT surface modification is warranted. Materials and Methods CICNT and Control Sample Preparation Carbon-infiltrated carbon nanotubes were grown as in previously published literature [ 21 ] with some adjustments. Briefly, 0.5 mm sheet stock of medical grade Ti6Al4V was cut into 9 mm squares. These squares were sonicated in isopropyl alcohol for 15 minutes, rinsed in deionized water, and dried. A 200 nm Al 2 O 3 thin film was deposited on the surface of each square using electron-beam deposition, which was followed by the deposition of 6 nm of iron using a thermal evaporator. The prepared samples were then placed into a furnace for CICNT growth. The furnace was heated to 750˚C with hydrogen gas flowing at 331 standard cubic centimeters per minute (sccm). Once this temperature was reached, ethylene gas flowing at 338 sccm was turned on for a one-minute growth step. The furnace was then heated to 900˚C and a carbon infiltration step was performed with hydrogen and ethylene gas flowing. The infiltration time determined the final average diameter of the nanotubes on a given sample. For 50 nm samples, this step lasted for 2 min, for 150 nm it was 8 min, for 250 nm it was 12 min, and for 350 nm it was 15.5 min. The samples were then cooled in argon flowing at 300 sccm to 200 ˚C before removal from the furnace. Carbon control samples were produced by depositing the Al 2 O 3 layer on Ti6Al4V and omitting the iron layer. Samples were then inserted into the furnace and heated to 900˚C with hydrogen and ethylene gas flowing for 10 minutes. This procedure produced samples with a layer of carbon but without nanotube topography (Fig. 5 ). All samples were sterilized before exposure to bacteria by the addition of 70% ethanol, followed by three washes with sterile water. Samples were allowed to dry completely before the addition of bacterial media. This method was tested for sterility, and no bacterial colonies were recovered. Bacterial Strains JE2 (BEI Resources NR-46543) is derived from the LAC strain, a well-characterized methicillin-resistant S. aureus strain isolated from the Los Angeles County jail in 2002 [ 32 ]. JE2 differs from the parent LAC strain by the removal of two plasmids [ 33 ]. The six other strains were chosen since they represent a variety of biofilm compositions. These strains have previously been tested for their relative composition of biofilm protein, polysaccharide, and extracellular DNA [ 22 ]. SH1000 (BEI Resources NR-55396) is a methicillin-sensitive human isolate with the addition of the rbs U gene for ribose uptake. TN112 (BEI Resources NR-46261) is a methicillin-resistant USA300 human isolate. SA29213 (ATCC, 29213) is a methicillin-sensitive clinical wound isolate. HA2, HA3, and HA4 are methicillin-resistant clinical isolates donated from a local hospital pathology lab. Bacterial Culture Cultures of MRSA SAUSA300_0794 (JE2) were grown up overnight in tryptic soy broth (TSB). They were then diluted to an optical density (OD) of 0.05 in broth consisting of TSB diluted to 66% in sterile water and with 0.5% glucose added. 25 µL of inoculated broth was pipetted as a droplet onto the surface of each sample. The purpose of this droplet method was to prevent confounding results from bacteria growing under the sample surface or on the plastic well. This method has been used before with good results [ 29 ]. The samples were then incubated at 37˚C for 36 hours unless otherwise indicated. In order to prevent premature evaporation of the droplet, CICNT and Ti samples were exclusively grown in the central wells of a 24-well plate, and the outer wells were filled with sterile water. CFU Analysis One of the most common methods for biofilm quantification is crystal violet. However, this method is inappropriate for use with the CICNT surface because the crystal violet dye is trapped by the porous nanotube surface, producing substantial background stain that confounds the results. Therefore, we used serial dilutions and CFU counts for quantification. After bacteria were cultured for the given amount of time, samples were washed once in sterile 1x phosphate buffered saline (PBS) and then removed to a well of a new, sterile plate with sterile forceps. 500 µL of PBS was added to the sample and pipetted vigorously to dislodge attached bacteria from the biofilms. The samples were then vortexed for 1 minute. 10 µL from each well was then removed and serially diluted in PBS before inoculation on Luria-Bertani (LB) agar plates. Plates were then incubated at 37˚C for 24 hours. For each sample, the countable plate with 20–200 colonies was selected and counted. This procedure was confirmed to effectively remove the bacteria by taking SEM images of the samples after the procedure (Fig. 2 ). SEM analysis Samples were prepared for SEM analysis by washing three times with sterile PBS, followed by fixation in 2.5% glutaraldehyde for 2 hours. They were then washed with PBS, followed by a wash in sterile water and a dehydration with a graded ethanol treatment for 30 minutes in 70% ethanol, followed by 30 minutes in 100% ethanol. Samples were then allowed to dry overnight. A 90-second timed sputter coat of an 80/20 gold/palladium mixture was then applied using a Quorum Q 150T ES sputter coater. Samples were imaged in a ThermoScientific Verios G4 UC SEM at multiple predetermined locations. Statistical Analysis For comparisons of CFU data, significant differences were determined by Student’s t -test. Statistically significant differences were attributed to variables with p ≤ 0.05. Analysis of data for bacteria grown at different time intervals was performed using a generalized linear model that included a first-order autoregressive correlation structure. An analysis of variance (ANOVA) was performed using this model to determine the significance of bacterial growth rate, and a general linear hypothesis test was performed to compare the CFU counts from different materials in individual time intervals. Abbreviations CICNT- Carbon infiltrated carbon nanotubes PJI- Periprosthetic joint infection Ti- Titanium CNT- Carbon nanotubes SEM- Scanning electron microscopy CFU- Colony forming unit PBS- Phosphate buffered saline LB- Luria-Bertani OD- Optical density Declarations Ethics approval and consent to participate Approval was granted by the Institutional Biosafety Committee of Brigham Young University to conduct this research (protocol IBC-2018-0046). No human subjects research was conducted in this study. Consent for publication All authors have read the final version of the manuscript, and consent to its publication. Availability of data and materials All primary data and materials contained in this manuscript are available by contacting the corresponding author (BKB), upon reasonable request. Competing interests The authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding No specific funding was used for this project. Authors' contributions - provide individual author contribution LB, AB, BJ, SH and BB conceived the original project. LB carried out most of the experiments, performed data analysis, created the figures, and wrote the first draft of the manuscript. JW carried out experiments under LB's supervision, with a focus on SEM analysis. BB mentored LB and JW, and assisted with project design, data analysis, and manuscript editing. KM performed statistical analyses. All authors reviewed the final draft of the manuscript. Acknowledgments The authors would like to acknowledge the BYU Electron Microscopy Facility for providing access to the equipment and expertise that allowed this project to be performed. Authors' information Lucy C. Bowden; [email protected] ; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA Jocelyn G. Wells; [email protected] ; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA Katelyn M. Miller; [email protected] ; Department of Statistics, Brigham Young University, Provo, UT 84602, USA Anton E. Bowden; [email protected] ; Department of Mechanical Engineering, Brigham Young University, Provo, UT 84602, USA Brian D. Jensen; [email protected] ; Department of Mechanical Engineering, Brigham Young University, Provo, UT 84602, USA Sandra Hope; [email protected] ; Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA Bradford K. 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Cite Share Download PDF Status: Published Journal Publication published 08 Nov, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 25 Sep, 2023 Reviews received at journal 21 Sep, 2023 Reviews received at journal 01 Sep, 2023 Reviewers agreed at journal 30 Aug, 2023 Reviewers agreed at journal 30 Aug, 2023 Reviewers invited by journal 27 Aug, 2023 Editor assigned by journal 27 Aug, 2023 Editor invited by journal 24 Aug, 2023 Submission checks completed at journal 24 Aug, 2023 First submitted to journal 21 Aug, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3283589","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":228588444,"identity":"d97368f6-6fec-430e-9f03-4f3d0e58f7a3","order_by":0,"name":"Lucy C. Bowden","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lucy","middleName":"C.","lastName":"Bowden","suffix":""},{"id":228588446,"identity":"a7172179-e424-47c6-abd8-91cff6afa12c","order_by":1,"name":"Jocelyn G. Wells","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jocelyn","middleName":"G.","lastName":"Wells","suffix":""},{"id":228588447,"identity":"0ab2eccf-d927-467e-b978-1f06283d7af2","order_by":2,"name":"Katelyn M. Miller","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katelyn","middleName":"M.","lastName":"Miller","suffix":""},{"id":228588448,"identity":"7dd74bc8-7002-4abc-91d1-f49534474b58","order_by":3,"name":"Anton E. Bowden","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anton","middleName":"E.","lastName":"Bowden","suffix":""},{"id":228588449,"identity":"d0fd7e8e-7fab-4425-b7e8-1f82d26d6acf","order_by":4,"name":"Brian D. Jensen","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Brian","middleName":"D.","lastName":"Jensen","suffix":""},{"id":228588450,"identity":"870798e4-5e57-4c73-ae41-2fcf1b40ca79","order_by":5,"name":"Sandra Hope","email":"","orcid":"","institution":"Brigham Young University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Hope","suffix":""},{"id":228588451,"identity":"abc9a7a3-81f5-4381-8c70-77cc587c2e8e","order_by":6,"name":"Bradford K. Berges","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACAyCWYGwAsxkfAAkePlK0MIM4PGykaGGTAJOEtJhLH354g3GHTb68e/uzyq85djJsDMwPH93Ao8WyL83YgvFMmuXGM2fMbstuSwY6jM3YOAefw84wmEkwth02MJyRw3ZbchszUAsPmzR+LezfgFr+GxjOf/6sWHJbPTFaeEC2HDCQl2AwY/y47TBhLZY9PMUWiWeSDQx4coylGbcd52FjJuAXcx72jTc+7rAzkG8//vDjz23V9vzszQ8f49MCBgkgFx4AxiUPiMdMSDkMyDcAU8wPYlWPglEwCkbBiAIArelChCu/8UYAAAAASUVORK5CYII=","orcid":"","institution":"Brigham Young University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bradford","middleName":"K.","lastName":"Berges","suffix":""}],"badges":[],"createdAt":"2023-08-21 20:29:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3283589/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3283589/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-46748-y","type":"published","date":"2023-11-08T15:01:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":42307702,"identity":"839738bc-b838-4fdd-8b9d-94a17ef3c23d","added_by":"auto","created_at":"2023-08-29 14:09:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":733292,"visible":true,"origin":"","legend":"\u003cp\u003eA) Scanning electron micrographs of CICNT of various diameters at 10,000x magnification. (B) Colony forming units (CFU)/mL of bacteria (strain JE2) for CICNT of various diameters and bare titanium (Ti) +/- standard error. Note that CICNT is denoted in blue and bare Ti in grey in this and all subsequent figures. Bars represent n=7 total samples and three independent experiments for each group. *p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/e866cf9b242dd28bc71c7448.png"},{"id":42307705,"identity":"aaa5a97a-5aa2-459e-bafb-df888502a8fb","added_by":"auto","created_at":"2023-08-29 14:09:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1020021,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of bare Ti or CICNT surfaces after performing our protocol for \u003cem\u003eS. aureus\u003c/em\u003e CFU analysis, indicating that the protocol is successful at removing the biofilm from both Ti and CICNT surfaces. Images were taken using a scanning electron microscope at 5000x. Red arrows indicate remaining bacteria.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/b6d5f0cffcf5de832c9e2d4b.png"},{"id":42309007,"identity":"18daeee6-15a2-4daa-a1f6-0bccf42d2441","added_by":"auto","created_at":"2023-08-29 14:17:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12762,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative scanning electron microscope images of JE2 biofilms on bare Ti or CICNT surfaces of diameter 150 nm at 10000x using immersion mode (for greater resolution) and 2500x in a nearby location using field free mode (for a wider view under lower magnification).\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/695aae6945072e1cba1a29c2.png"},{"id":42309008,"identity":"f0a04b54-f762-4002-a507-a35a1e9c1dd1","added_by":"auto","created_at":"2023-08-29 14:17:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1151901,"visible":true,"origin":"","legend":"\u003cp\u003eColony Forming Units/mL +/- standard error quantification of bacteria on CICNT and bare Ti at various time points. Bars represent n=7 total samples and three independent experiments. *p\u0026lt;0.01, ***p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/bac1ba69d2fc88f2d4d910fe.png"},{"id":42307701,"identity":"cb294be0-b566-4cc3-ae0b-2136bf3a9eb9","added_by":"auto","created_at":"2023-08-29 14:09:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26608,"visible":true,"origin":"","legend":"\u003cp\u003eCFU/plate for CICNT and bare Ti with different \u003cem\u003eS. aureus\u003c/em\u003e isolates grown in biofilms +/- standard error. Bars represent at least n=7 total samples from at least 3 independent experiments. *p\u0026lt;0.01, **p\u0026lt;0.001, ***p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Binder15.png","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/5bce67b714f0633f335b0aa2.png"},{"id":42307703,"identity":"369b1046-cfa0-4088-9ff2-f21a1061ab5b","added_by":"auto","created_at":"2023-08-29 14:09:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":478113,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of (A) bare Ti, (B) carbon control, and (C) CICNT at 10,000x. All images are prior to bacterial growth. (D) CFU/mL for bare Ti, carbon control, and\u003cem\u003e \u003c/em\u003eCICNT,\u003cem\u003e \u003c/em\u003e+/- standard error. Bars represent n=7 total samples and three independent experiments. *p\u0026lt;0.05, ***p\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Binder16.png","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/a30741f902e7a44f44d3f806.png"},{"id":46349231,"identity":"f968684a-c175-417f-aa19-873969b7a3ec","added_by":"auto","created_at":"2023-11-13 15:09:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3340414,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3283589/v1/55dbeff3-6f1c-405c-b809-ea3641bed040.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Carbon-Infiltrated Carbon Nanotubes Inhibit the Development of Staphylococcus aureus Biofilms","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a common gram-positive bacterium that leads to nearly 20,000 deaths each year in the US [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eS. aureus\u003c/em\u003e forms biofilms, which are surface-associated assemblages of bacteria embedded in an extracellular matrix. Infections with established biofilms are very difficult to treat with traditional antibiotic regimens due to limited diffusion of antibacterial agents through the biofilm matrix. \u003cem\u003eS. aureus\u003c/em\u003e biofilms cause severe infections in healthcare settings and are particularly problematic in the context of implanted hardware such as fixator pins or artificial joints.\u003c/p\u003e \u003cp\u003eOrthopedic implants and prostheses are becoming more and more common\u0026mdash;in 2018 over 1\u0026nbsp;million hip and knee implants were performed in the US alone [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although these implants can relieve pain and restore freedom of movement, they are susceptible to post-operative periprosthetic joint infection (PJI), which is responsible for about 30% of implant failure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Up to 70% of PJI cases are caused by \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Infection rates are even worse for external fixator pins, where up to 80% of all patients experience a pin tract infection and most of these are caused by \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Current efforts to reduce infection rates have proven insufficient as the yearly infection burden continues to rise [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTitanium (Ti) is often used in medical hardware for its high mechanical strength, but this material also offers a ready surface for bacterial colonization and biofilm formation. An ideal implant surface would exhibit innate physical resistance to the attachment and formation of bacterial biofilms. This physical resistance to biofilm formation is due to the texture of the surface at the nanoscale. Structural biofilm resistance is an attractive alternative to antibiotics for biofilm control because it could reduce the need for antibiotic use, which would help reduce the development of antibiotic-resistant bacteria. Many natural surfaces possess structural antimicrobial properties, including dragonfly [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and cicada wings [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], shark skin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and lotus leaves [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Several synthetic analogs have been developed in an attempt to replicate the anti-biofilm effects of naturally biofilm-resistant surfaces [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While these materials have promising applications, they also suffer from limitations such as cytotoxicity to human cells and manufacturing difficulties, constraining their use in medical tools and implants. The search for a material that can overcome these limitations is ongoing, but one promising material is carbon-infiltrated carbon nanotubes (CICNT). The CICNT surface aims to replicate the structural biofilm resistance of naturally occurring antimicrobial surfaces.\u003c/p\u003e \u003cp\u003eCarbon Nanotubes (CNT) are nanostructured cylindrical lattices of hybridized carbon atoms. CNT possess impressive structural and mechanical properties that make them of interest in a variety of applications in biotechnology [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. They are synthesized using chemical vapor deposition techniques, flowing ethylene gas at high temperatures over a substrate such as silicon, Ti, or stainless steel [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePost-processing alteration of CNT is common for biological applications. When a post-processing carbon infiltration step is added, amorphous carbon is deposited on the nanotubes, resulting in carbon-infiltrated carbon nanotubes (CICNT). This infiltration step massively increases the diameter of the nanotube, resulting in a final volume ratio of around 99% bulk carbon to 1% CNT [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], as well as providing substantial structural reinforcement that changes the mechanical behavior of the CNT forest from individual tubes to a cohesive structural layer.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to demonstrate that the CICNT surface has antimicrobial properties and to better understand these properties by determining whether changing nanotube size or the material at the nanotube/bacteria interface would affect bacterial adhesion. This was accomplished through the quantification of adhered \u003cem\u003eS. aureus\u003c/em\u003e cells on CICNT as well as bare Ti by colony forming unit analysis. The CICNT surface offers a potential solution to the increasing burden of implant-associated infection.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOptimizing CICNT diameter to reduce biofilm growth\u003c/h2\u003e \u003cp\u003eThe size of individual nanotubes can be manipulated by altering the amount of carbon infiltration. CICNT samples of various diameters (50, 150, 250, and 350 nm, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were prepared in order to quantify the effects of CICNT diameter on surface biofilm growth. \u003cem\u003eS. aureus\u003c/em\u003e strain JE2 was grown on each surface for 36 hours. JE2 was chosen because it is a USA300 strain, which is clinically relevant, and because it was found to have a biofilm matrix structure that was representative of many \u003cem\u003eS. aureus\u003c/em\u003e isolates in quantities of polysaccharide, protein, and extracellular DNA [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. After 36 hours, the biofilm was washed to remove unattached cells. Adherent cells were then removed from the sample surface and quantified by performing serial dilutions, then plating onto LB agar to count colony-forming units (CFU) of bacteria. Each diameter size of CICNT showed a significantly reduced number of bacteria as compared to a bare Ti control, ranging from a 1.4-fold reduction with 50 nm CICNT (p\u0026thinsp;=\u0026thinsp;0.03) to a 2.5-fold reduction with 150 nm CICNT (p\u0026thinsp;=\u0026thinsp;0.0005) after 36 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After determining that the 150 nm diameter was most effective, we used that size of CICNT for all future experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eP-values from statistical comparison of results from CICNT of various diameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150 nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e250 nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e350 nm\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0048\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e250 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e350 nm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM) images of biofilms grown on CICNT and bare Ti\u003c/h2\u003e \u003cp\u003eWe considered the possibility that the differences in CFU counts could be due to a difference in the ability to retrieve cells from Ti vs CICNT surfaces, rather than their ability to adhere. Accordingly, we conducted an experiment where we performed SEM imaging of both surfaces after our typical process to remove cells. We found that only very rare cells were still adhered to either surface, suggesting that the cell retrieval process was similar and effective for both surface types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to colony forming unit enumeration, we used a scanning electron microscope to image the biofilms grown on both the 150 nm CICNT surface and bare Ti. More bacteria can be seen adhered to the Ti surface than to the CICNT surface, and the CICNT surface appears to have more bare spots lacking attached cells than the Ti (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm growth is slower on CICNT surfaces than on Ti surfaces\u003c/h2\u003e \u003cp\u003eTo determine if the anti-biofilm effect was present over multiple time points, CICNT and Ti control samples were exposed to \u003cem\u003eS. aureus\u003c/em\u003e for 12, 24, 36, or 48 hours and the adherent bacteria were quantified by performing serial dilutions onto agar plates. At 12 hours, no significant difference between the adherent bacterial load was found between the two groups. As time progressed, this difference became significant and the ratio between the number of bacteria on Ti and on CICNT was most substantial at 36 and 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After determining that the 36-hour time point showed the most significant difference in CICNT versus the control, we used that time point for all future experiments Additionally, we performed a longitudinal analysis and found that the interaction effect of surface type and time was significant. A one-sided general linear hypothesis test on the hypothesis that the rate of biofilm growth was lower on the CICNT surface than on the Ti surface was significant (p\u0026thinsp;=\u0026thinsp;0.0014). This indicates that the biofilm was growing more slowly on the CICNT surface than on the Ti surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eA variety of S. aureus isolates are inhibited from forming biofilms on CICNT\u003c/h3\u003e\n\u003cp\u003eWe have previously shown that different \u003cem\u003eS. aureus\u003c/em\u003e isolates produce biofilms that vary by overall biomass, as well as by polysaccharide, protein and extracellular DNA content. To determine if the reduction in adherent bacteria was consistent among different strains of \u003cem\u003eS. aureus\u003c/em\u003e, we tested six additional isolates of \u003cem\u003eS. aureus\u003c/em\u003e (JE2, used in previous experiments, is included as a reference). The clinical isolates chosen were found in a previous publication to represent a variety of relative biofilm strengths, with SH1000 forming the strongest biofilm and HA3 the weakest [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, both methicillin resistant and susceptible isolates were tested. We found that six of the seven isolates exhibited a significant reduction in the number of adherent bacteria on the CICNT surface as compared to bare Ti, while one isolate, HA2, showed a significant increase in the number of adherent bacteria on the CICNT surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDifferentiating between chemical and structural biofilm resistance\u003c/h3\u003e\n\u003cp\u003eThe reduction in adherent bacteria on the CICNT surface could be due to either an antibacterial chemical effect of the carbon used, a structural effect of the nanotubes, or both, on a developing biofilm. To differentiate between these two possibilities, we developed a carbon control consisting of nonstructured carbon deposited on the Ti surface. This carbon control, therefore, was chemically similar to the CICNT surface but possessed a different structure. The carbon control was found to have fewer adhered bacteria than the bare Ti surface (p\u0026thinsp;=\u0026thinsp;0.003), but significantly more bacteria than the CICNT surface, which is also composed of carbon, (p\u0026thinsp;=\u0026thinsp;4E-5, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that the structure of the carbon affects the number of adhered bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe purpose of this study was to investigate the antimicrobial properties of the CICNT surface. CFU analysis demonstrated that the CICNT surface effectively reduces the number of adherent bacteria as compared to bare Ti surfaces, with up to a 2.5-fold reduction in the number of adherent bacteria. SEM analysis confirmed this reduction in adherent bacteria. This reduction is consistent with the lower end of reported bacterial reduction values for the CICNT surface in the only previously published study of the material\u0026rsquo;s antibacterial properties to date [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], though in that study the CICNT surface modification was prepared on stainless steel rather than Ti. It should also be noted that the methods used by Morco et al. for that original study were different than ours both in their protocol for incubating bacteria on the CICNT surface and in their method for quantifying bacteria after incubation; Morco et al. incubated bacteria on CICNT in a bioreactor in 10% TSB for 48 hours and they counted bacteria remaining on the surfaces using SEM analysis. In this study the bacteria were grown in a still droplet on the sample surface in 66% TSB, and bacteria were quantified using CFU analysis. It is possible that differences in both biofilm growth conditions and bacterial enumeration played a role in the slight decrease in bacterial reduction found in this study compared to that of Morco et al.\u003c/p\u003e \u003cp\u003eA reduction in adherent bacteria may indicate that a surface is chemically toxic to the cells, causing cell death, commonly referred to as bactericidal. Alternatively, a reduction in adherent bacteria could mean that the structure of the surface is preventing the bacteria from effectively attaching, known as anti-biofouling. SEM images can be used to suggest whether cell death is occurring [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. SEM images of the \u003cem\u003eS. aureus\u003c/em\u003e biofilm did not appear to show collapsed or deflated cells on the CICNT surface, suggesting that the reduction in adherent bacteria may be due to a prevention of attachment rather than cell death. This contrasts with the bactericidal properties of other surfaces such as dragonfly and cicada wings which demonstrate the greatest potency against gram-negative bacteria [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. An anti-biofouling surface that prevents gram-positive bacteria from attaching and forming biofilms would play an important role in preventing infection, since antibiotics are much more effective against planktonic cells than against biofilms [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results found in this study indicate that the surface nanostructure of the CICNT is important since different sizes of CICNT exhibit different levels of bacterial reduction. Furthermore, this implies that some material property such as nanotube diameter, curvature, stiffness, or the amount of space between nanotubes impacts the ability of bacteria to adhere to the surface. A diameter of 150 nm was found to exhibit the greatest degree of biofilm reduction. This size is within the range of nanostructure sizes found on dragonfly wings, which one study estimated to be between 83.3 and 195 nm [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Dragonfly wings also possess antimicrobial and bactericidal properties [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], though the topographies of dragonfly wings and the CICNT surface differ. The average size of carbon nanotubes tested in this study ranged from 50 to 350 nm in diameter, which is considerably smaller than that of \u003cem\u003eS. aureus\u003c/em\u003e, which has a diameter of 0.5-1 \u0026micro;m. The fact that the CICNT surface features are smaller than \u003cem\u003eS. aureus\u003c/em\u003e is in agreement with previously published literature which demonstrated that bacterial adhesion is reduced on surfaces with nanotopography smaller than the bacteria, possibly due to a reduction in bacteria-surface contact area [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the precise mechanism by which the CICNT surface reduces adherent bacteria remains to be understood.\u003c/p\u003e \u003cp\u003eThis study also tested biofilm growth at four different time points on both surfaces. At 12 hours, there was no significant difference in adherent bacteria between CICNT and Ti. However, at 24, 36, and 48 hours there were significantly fewer bacteria on CICNT surfaces than on Ti surfaces. The ratio between the number of bacteria found on Ti and CICNT surfaces was largest at 36 hours. The slight decrease at 48 hours may be due to our experimental conditions. We also found that the biofilm growth rates differed between surface types. The number of adherent bacteria grew more slowly on CICNT surfaces than on Ti surfaces. This could indicate that either fewer new bacteria were attaching to the surface or that the bacteria present on the surface were not replicating as quickly even though they remained viable. This supports the evidence collected thus far that the CICNT surface possesses anti-biofouling properties.\u003c/p\u003e \u003cp\u003eWe also tested the CICNT surface against seven different isolates of \u003cem\u003eS. aureus\u003c/em\u003e. Most studies of materials suspected to possess anti-biofilm properties are only tested against one or two strains of bacteria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the composition of the biofilm matrix in \u003cem\u003eS. aureus\u003c/em\u003e is highly strain-, time-, and condition-dependent, so testing multiple strains is important to understand whether a material has broad anti-biofilm capabilities. The three main components of the biofilm matrix are proteins, polysaccharides, and extracellular DNA. These three components are important for attachment and structural components of the biofilm [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The isolates chosen represent both methicillin-resistant and methicillin-sensitive strains, as well as a variety of biofilm matrix compositions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Six of the seven isolates tested showed significantly reduced adhesion on the CICNT surface compared to a bare Ti surface, and the CICNT surface reduced adhesion of both methicillin-resistant and methicillin-sensitive isolates. The HA2 isolate was the only isolate tested which appeared to adhere more strongly to the CICNT surface than to the bare Ti surface. This isolate was found by Ball et al. to have slightly elevated levels of protein in its matrix compared to other isolates tested, but was not significantly different from HA3, which was prevented from adhering to the CICNT surface. It is possible that some genetic difference between isolates is responsible for the difference in adherence to the CICNT surface, and that future work with additional strains may uncover such a genetic basis for why some isolates are susceptible and some are resistant to the effects of the CICNT surface.\u003c/p\u003e \u003cp\u003eWe also compared the growth of bacteria on a non-structured carbon surface to growth on the CICNT surface. This unstructured carbon control was found to have significantly more bacteria attached than the CICNT surface. This indicates that the specific structure of the surface affects the adhesion of the bacteria rather than the carbon itself having an antimicrobial effect and is in agreement with the results found by Morco et al. with CICNT grown on stainless steel [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It also echoes other work which describes how the antimicrobial effect of nanostructured surfaces is heavily affected by their precise arrangement and topographical structure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA Carbon-infiltrated carbon nanotube (CICNT) surface modification on Ti surfaces reduced biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e and the magnitude of the effect was found to depend upon the CICNT diameter, the \u003cem\u003eS. aureus\u003c/em\u003e isolate, and the topography of the CICNT surface itself. A CICNT diameter of 150 nm was found to provide optimal protection against adherent bacteria, with a 2.5-fold reduction. This effect was confirmed by CFU quantification and SEM analysis. Multiple isolates of \u003cem\u003eS. aureus\u003c/em\u003e were investigated, and the antimicrobial effects of the CICNT surface varied by isolate but were shown to inhibit the growth of both methicillin-resistant and methicillin-sensitive isolates. Unstructured carbon did not exhibit the same antimicrobial effect, indicating that the CICNT nanostructure plays a role in biofilm reduction. Further investigation of the mechanism of the antimicrobial effect of the CICNT surface modification is warranted.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCICNT and Control Sample Preparation\u003c/h2\u003e \u003cp\u003eCarbon-infiltrated carbon nanotubes were grown as in previously published literature [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] with some adjustments. Briefly, 0.5 mm sheet stock of medical grade Ti6Al4V was cut into 9 mm squares. These squares were sonicated in isopropyl alcohol for 15 minutes, rinsed in deionized water, and dried. A 200 nm Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e thin film was deposited on the surface of each square using electron-beam deposition, which was followed by the deposition of 6 nm of iron using a thermal evaporator.\u003c/p\u003e \u003cp\u003eThe prepared samples were then placed into a furnace for CICNT growth. The furnace was heated to 750˚C with hydrogen gas flowing at 331 standard cubic centimeters per minute (sccm). Once this temperature was reached, ethylene gas flowing at 338 sccm was turned on for a one-minute growth step. The furnace was then heated to 900˚C and a carbon infiltration step was performed with hydrogen and ethylene gas flowing. The infiltration time determined the final average diameter of the nanotubes on a given sample. For 50 nm samples, this step lasted for 2 min, for 150 nm it was 8 min, for 250 nm it was 12 min, and for 350 nm it was 15.5 min. The samples were then cooled in argon flowing at 300 sccm to 200 ˚C before removal from the furnace.\u003c/p\u003e \u003cp\u003eCarbon control samples were produced by depositing the Al\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e layer on Ti6Al4V and omitting the iron layer. Samples were then inserted into the furnace and heated to 900˚C with hydrogen and ethylene gas flowing for 10 minutes. This procedure produced samples with a layer of carbon but without nanotube topography (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll samples were sterilized before exposure to bacteria by the addition of 70% ethanol, followed by three washes with sterile water. Samples were allowed to dry completely before the addition of bacterial media. This method was tested for sterility, and no bacterial colonies were recovered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Strains\u003c/h2\u003e \u003cp\u003eJE2 (BEI Resources NR-46543) is derived from the LAC strain, a well-characterized methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e strain isolated from the Los Angeles County jail in 2002 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. JE2 differs from the parent LAC strain by the removal of two plasmids [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The six other strains were chosen since they represent a variety of biofilm compositions. These strains have previously been tested for their relative composition of biofilm protein, polysaccharide, and extracellular DNA [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. SH1000 (BEI Resources NR-55396) is a methicillin-sensitive human isolate with the addition of the \u003cem\u003erbs\u003c/em\u003eU gene for ribose uptake. TN112 (BEI Resources NR-46261) is a methicillin-resistant USA300 human isolate. SA29213 (ATCC, 29213) is a methicillin-sensitive clinical wound isolate. HA2, HA3, and HA4 are methicillin-resistant clinical isolates donated from a local hospital pathology lab.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Culture\u003c/h2\u003e \u003cp\u003eCultures of MRSA SAUSA300_0794 (JE2) were grown up overnight in tryptic soy broth (TSB). They were then diluted to an optical density (OD) of 0.05 in broth consisting of TSB diluted to 66% in sterile water and with 0.5% glucose added. 25 \u0026micro;L of inoculated broth was pipetted as a droplet onto the surface of each sample. The purpose of this droplet method was to prevent confounding results from bacteria growing under the sample surface or on the plastic well. This method has been used before with good results [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The samples were then incubated at 37˚C for 36 hours unless otherwise indicated. In order to prevent premature evaporation of the droplet, CICNT and Ti samples were exclusively grown in the central wells of a 24-well plate, and the outer wells were filled with sterile water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCFU Analysis\u003c/h2\u003e \u003cp\u003eOne of the most common methods for biofilm quantification is crystal violet. However, this method is inappropriate for use with the CICNT surface because the crystal violet dye is trapped by the porous nanotube surface, producing substantial background stain that confounds the results. Therefore, we used serial dilutions and CFU counts for quantification. After bacteria were cultured for the given amount of time, samples were washed once in sterile 1x phosphate buffered saline (PBS) and then removed to a well of a new, sterile plate with sterile forceps. 500 \u0026micro;L of PBS was added to the sample and pipetted vigorously to dislodge attached bacteria from the biofilms. The samples were then vortexed for 1 minute. 10 \u0026micro;L from each well was then removed and serially diluted in PBS before inoculation on Luria-Bertani (LB) agar plates. Plates were then incubated at 37˚C for 24 hours. For each sample, the countable plate with 20\u0026ndash;200 colonies was selected and counted. This procedure was confirmed to effectively remove the bacteria by taking SEM images of the samples after the procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSEM analysis\u003c/h2\u003e \u003cp\u003eSamples were prepared for SEM analysis by washing three times with sterile PBS, followed by fixation in 2.5% glutaraldehyde for 2 hours. They were then washed with PBS, followed by a wash in sterile water and a dehydration with a graded ethanol treatment for 30 minutes in 70% ethanol, followed by 30 minutes in 100% ethanol. Samples were then allowed to dry overnight. A 90-second timed sputter coat of an 80/20 gold/palladium mixture was then applied using a Quorum Q 150T ES sputter coater. Samples were imaged in a ThermoScientific Verios G4 UC SEM at multiple predetermined locations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eFor comparisons of CFU data, significant differences were determined by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Statistically significant differences were attributed to variables with p\u0026thinsp;\u0026le;\u0026thinsp;0.05. Analysis of data for bacteria grown at different time intervals was performed using a generalized linear model that included a first-order autoregressive correlation structure. An analysis of variance (ANOVA) was performed using this model to determine the significance of bacterial growth rate, and a general linear hypothesis test was performed to compare the CFU counts from different materials in individual time intervals.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCICNT- Carbon infiltrated carbon nanotubes\u003c/p\u003e\n\u003cp\u003ePJI- Periprosthetic joint infection\u003c/p\u003e\n\u003cp\u003eTi- Titanium\u003c/p\u003e\n\u003cp\u003eCNT- Carbon nanotubes\u003c/p\u003e\n\u003cp\u003eSEM- Scanning electron microscopy\u003c/p\u003e\n\u003cp\u003eCFU- Colony forming unit\u003c/p\u003e\n\u003cp\u003ePBS- Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eLB- Luria-Bertani\u003c/p\u003e\n\u003cp\u003eOD- Optical density\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval was granted by the Institutional Biosafety Committee of Brigham Young University to conduct this research (protocol IBC-2018-0046). \u0026nbsp;No human subjects research was conducted in this study. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read the final version of the manuscript, and consent to its publication. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll primary data and materials contained in this manuscript are available by contacting the corresponding author (BKB), upon reasonable request. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific funding was used for this project. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions -\u0026nbsp;provide individual author contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLB, AB, BJ, SH and BB conceived the original project. \u0026nbsp;LB carried out most of the experiments, performed data analysis, created the figures, and wrote the first draft of the manuscript. \u0026nbsp;JW carried out experiments under LB's supervision, with a focus on SEM analysis. \u0026nbsp;BB mentored LB and JW, and assisted with project design, data analysis, and manuscript editing. \u0026nbsp;KM performed statistical analyses. \u0026nbsp;All authors reviewed the final draft of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the BYU Electron Microscopy Facility for providing access to the equipment and expertise that allowed this project to be performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLucy C. Bowden; [email protected];\u0026nbsp;Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e\n\u003cp\u003eJocelyn G. Wells; [email protected];\u0026nbsp;Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e\n\u003cp\u003eKatelyn M. Miller; [email protected];\u0026nbsp;Department of Statistics, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e\n\u003cp\u003eAnton E. Bowden; [email protected];\u0026nbsp;Department of Mechanical Engineering, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e\n\u003cp\u003eBrian D. Jensen; [email protected];\u0026nbsp;Department of Mechanical Engineering, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e\n\u003cp\u003eSandra Hope; [email protected];\u0026nbsp;Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e\n\u003cp\u003eBradford K. Berges; [email protected];\u0026nbsp;Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eKavanagh KT: Control of MSSA and MRSA in the United States: protocols, policies, risk adjustment and excuses. Antimicrob Resist Infect Control 2019, 8:103.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003e2018 Hospital Inpatient National Statistics [ \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hcupnet.ahrq.gov/.]\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSpringer BD, Cahue S, Etkin CD, Lewallen DG, McGrory BJ: Infection burden in total hip and knee arthroplasties: an international registry-based perspective. Arthroplast Today 2017, 3:137\u0026ndash;140.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRaphel J, Holodniy M, Goodman SB, Heilshorn SC: Multifunctional coatings to simultaneously promote osseointegration and prevent infection of orthopaedic implants. Biomaterials 2016, 84:301\u0026ndash;314.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTuon FF, Cieslinski J, Ono AFM, Goto FL, Machinski JM, Mantovani LK, Kosop LR, Namba MS, Rocha JL: Microbiological profile and susceptibility pattern of surgical site infections related to orthopaedic trauma. Int Orthop 2019, 43:1309\u0026ndash;1313.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFernandes A, Dias M: The Microbiological Profiles of Infected Prosthetic Implants with an Emphasis on the Organisms which Form Biofilms. J Clin Diagn Res 2013, 7:219\u0026ndash;223.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTsai JC, Sheng WH, Lo WY, Jiang CC, Chang SC: Clinical characteristics, microbiology, and outcomes of prosthetic joint infection in Taiwan. J Microbiol Immunol Infect 2015, 48:198\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHamahashi K, Uchiyama Y, Kobayashi Y, Watanabe M: Delayed methicillin-resistant Staphylococcus aureus-induced osteomyelitis of the tibia after pin tract infection: two case reports. J Med Case Rep 2017, 11:23.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJauregui JJ, Bor N, Thakral R, Standard SC, Paley D, Herzenberg JE: Life- and limb-threatening infections following the use of an external fixator. Bone Joint J 2015, 97-B:1296\u0026ndash;1300.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBandara CD, Singh S, Afara IO, Wolff A, Tesfamichael T, Ostrikov K, Oloyede A: Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia coli. ACS Appl Mater Interfaces 2017, 9:6746\u0026ndash;6760.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePogodin S, Hasan J, Baulin VA, Webb HK, Truong VK, Phong Nguyen TH, Boshkovikj V, Fluke CJ, Watson GS, Watson JA, et al: Biophysical model of bacterial cell interactions with nanopatterned cicada wing surfaces. Biophys J 2013, 104:835\u0026ndash;840.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChung KK, Schumacher JF, Sampson EM, Burne RA, Antonelli PJ, Brennan AB: Impact of engineered surface microtopography on biofilm formation of Staphylococcus aureus. Biointerphases 2007, 2:89\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTripathy A, Sen P, Su B, Briscoe WH: Natural and bioinspired nanostructured bactericidal surfaces. Adv Colloid Interface Sci 2017, 248:85\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eElbourne A, Crawford RJ, Ivanova EP: Nano-structured antimicrobial surfaces: From nature to synthetic analogues. J Colloid Interface Sci 2017, 508:603\u0026ndash;616.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYa\u0026apos;ari S, Halperin-Sternfeld M, Rosin B, Adler-Abramovich L: Surface Modification by Nano-Structures Reduces Viable Bacterial Biofilm in Aerobic and Anaerobic Environments. Int J Mol Sci 2020, 21.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eQin H, Cao H, Zhao Y, Zhu C, Cheng T, Wang Q, Peng X, Cheng M, Wang J, Jin G, et al: In vitro and in vivo anti-biofilm effects of silver nanoparticles immobilized on titanium. Biomaterials 2014, 35:9114\u0026ndash;9125.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXu LC, Siedlecki CA: Submicron-textured biomaterial surface reduces staphylococcal bacterial adhesion and biofilm formation. Acta Biomater 2012, 8:72\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHe H, Pham-Huy LA, Dramou P, Xiao D, Zuo P, Pham-Huy C: Carbon nanotubes: applications in pharmacy and medicine. Biomed Res Int 2013, 2013:578290.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eVenkataraman A, Amadi EV, Chen Y, Papadopoulos C: Carbon Nanotube Assembly and Integration for Applications. Nanoscale Res Lett 2019, 14:220.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSperanza G: Carbon Nanomaterials: Synthesis, Functionalization and Sensing Applications. Nanomaterials (Basel) 2021, 11.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMorco SR, Williams DL, Jensen BD, Bowden AE: Structural biofilm resistance of carbon-infiltrated carbon nanotube coatings. J Orthop Res 2021, 40:1953\u0026ndash;1960.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBall AL, Augenstein ED, Wienclaw TM, Richmond BC, Freestone CA, Lewis JM, Thompson JS, Pickett BE, Berges BK: Characterization of Staphylococcus aureus biofilms via crystal violet binding and biochemical composition assays of isolates from hospitals, raw meat, and biofilm-associated gene mutants. Microb Pathog 2022, 167:105554.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHasan J, Webb HK, Truong VK, Pogodin S, Baulin VA, Watson GS, Watson JA, Crawford RJ, Ivanova EP: Selective bactericidal activity of nanopatterned superhydrophobic cicada Psaltoda claripennis wing surfaces. Appl Microbiol Biotechnol 2013, 97:9257\u0026ndash;9262.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShin H-J, Yang S, Lim Y: Antibiotic susceptibility of Staphylococcus aureus with different degrees of biofilm formation. Journal of Analytical Science and Technology 2021, 12:41.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBabushkina IV, Mamonova IA, Ulyanov VY, Gladkova EV, Shpinyak SP: Antibiotic Susceptibility of Staphylococcus aureus Plankton and Biofilm Forms Isolated in Implant-Associated Infection. 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Biotechnol J 2011, 6:1103\u0026ndash;1114.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJenkins J, Mantell J, Neal C, Gholinia A, Verkade P, Nobbs AH, Su B: Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress. Nat Commun 2020, 11:1626.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMoormeier DE, Bayles KW: Staphylococcus aureus biofilm: a complex developmental organism. Mol Microbiol 2017, 104:365\u0026ndash;376.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHasan J, Jain S, Padmarajan R, Purighalla S, Sambandamurthy VK, Chatterjee K: Multi-scale surface topography to minimize adherence and viability of nosocomial drug-resistant bacteria. Mater Des 2018, 140:332\u0026ndash;344.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, Mediavilla JR, Byrne KA, Parkins LD, Tenover FC, et al: Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proc Natl Acad Sci U S A 2008, 105:1327\u0026ndash;1332.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBose JL, Fey PD, Bayles KW: Genetic tools to enhance the study of gene function and regulation in Staphylococcus aureus. Appl Environ Microbiol 2013, 79:2218\u0026ndash;2224.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biofilm, carbon nanotubes, Staphylococcus aureus, nanostructured surfaces, antimicrobial surfaces, surface modification","lastPublishedDoi":"10.21203/rs.3.rs-3283589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3283589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e forms biofilms that cause considerable morbidity and mortality in patients who receive implanted devices such as prosthetics or fixator pins. An ideal surface for such medical devices would inhibit biofilm growth. Recently, it was reported that surface modification of stainless steel materials with carbon-infiltrated carbon nanotubes (CICNT) inhibits the growth of \u003cem\u003eS. aureus\u003c/em\u003e biofilms. The purpose of this study was to investigate this antimicrobial effect on titanium materials with CICNT coated surfaces in a variety of surface morphologies and across a broader spectrum of \u003cem\u003eS. aureus\u003c/em\u003e isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eStudy samples of\u003cstrong\u003e \u003c/strong\u003eCICNT-coated titanium, and control samples of bare titanium, a common implant material, were exposed to \u003cem\u003eS. aureus. \u003c/em\u003eViable bacteria were removed from adhered biofilms and quantified as colony forming units. Scanning electron microscopy was used to qualitatively analyze biofilms both before and after removal of cells.\u003cstrong\u003e \u003c/strong\u003eThe CICNT surface was found to have significantly fewer adherent bacteria than bare titanium control surfaces, both via colony forming unit and microscopy analyses. This effect was most pronounced on CICNT surfaces with an average nanotube diameter of 150 nm, showing a 2.5-fold reduction in adherent bacteria. Since \u003cem\u003eS. aureus\u003c/em\u003e forms different biofilm structures by isolate and by growth conditions, we tested 7 total isolates and found a significant reduction in the biofilm load in six out of seven \u003cem\u003eS. aureus \u003c/em\u003eisolates tested. To examine whether the anti-biofilm effect was due to the structure of the nanotubes, we generated an unstructured carbon surface. Significantly more bacteria adhered to a nonstructured carbon surface than to the CICNT surface, suggesting that the topography of the nanotube structure itself has anti-biofilm properties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe CICNT surface possesses antimicrobial properties that result in fewer adherent \u003cem\u003eS. aureus\u003c/em\u003e bacteria. These antimicrobial properties are consistent across multiple isolates of \u003cem\u003eS. aureus \u003c/em\u003eand are affected by nanotube diameter. The experiments performed in this study suggest that this effect is due to the nanostructure of the CICNT surface.\u003c/p\u003e","manuscriptTitle":"Carbon-Infiltrated Carbon Nanotubes Inhibit the Development of Staphylococcus aureus Biofilms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-29 14:09:22","doi":"10.21203/rs.3.rs-3283589/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-09-25T07:37:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-09-22T03:45:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-09-01T05:01:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89a73434-6ef6-4336-a4f8-08976bf715c8","date":"2023-08-31T00:58:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21b94aed-22d7-4df1-84c5-04f05c5f0bfd","date":"2023-08-31T00:37:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-08-27T20:34:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-08-27T20:23:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-08-24T09:00:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-24T08:55:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-08-21T20:18:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a394d3a6-4401-475c-9dc3-ba7b5cc6ed65","owner":[],"postedDate":"August 29th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":24248549,"name":"Physical sciences/Materials science"},{"id":24248550,"name":"Physical sciences/Materials science/Materials for devices"},{"id":24248551,"name":"Physical sciences/Materials science/Nanoscale materials"},{"id":24248552,"name":"Biological sciences/Microbiology"},{"id":24248553,"name":"Biological sciences/Microbiology/Antimicrobials"},{"id":24248554,"name":"Biological sciences/Microbiology/Bacteria"},{"id":24248555,"name":"Biological sciences/Microbiology/Biofilms"}],"tags":[],"updatedAt":"2023-11-13T15:06:32+00:00","versionOfRecord":{"articleIdentity":"rs-3283589","link":"https://doi.org/10.1038/s41598-023-46748-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-11-08 15:01:00","publishedOnDateReadable":"November 8th, 2023"},"versionCreatedAt":"2023-08-29 14:09:22","video":"","vorDoi":"10.1038/s41598-023-46748-y","vorDoiUrl":"https://doi.org/10.1038/s41598-023-46748-y","workflowStages":[]},"version":"v1","identity":"rs-3283589","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3283589","identity":"rs-3283589","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}