A Nanowire-Based Flexible Antibacterial Surface Reduces the

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A Nanowire-Based Flexible Antibacterial Surface Reduces the Viability of Drug-Resistant Nosocomial Pathogens Abinash Tripathy, Arvind Kumar, Atish Roy Chowdhury, Kapudeep Karmakar, Swathi Purighalla, Vasan K Sambandamurthy, Dipshikha Chakravortty, and Prosenjit Sen ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00397 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Applied Nano Materials

A Nanowire-Based Flexible Antibacterial Surface Reduces the Viability of Drug-Resistant Nosocomial Pathogens Abinash Tripathy‡, Arvind Kumar‡, Atish Roy Chowdhury¥, Kapudeep Karmakar¥, Swathi Purighallaѱ, Vasan Sambandamurthyѱ, Dipshikha Chakravortty*¥ and Prosenjit Sen*‡

‡-

Centre for Nano Science and Engineering, Indian Institute of Science, Bangalore, India

560012 ¥

- Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore,

India 560012 ѱ

- Mazumdar Shaw Centre for Translational Research, NH Health City, Bangalore, India

560099 *Corresponding Authors: [email protected], [email protected]

Abstract The global emergence of antimicrobial resistance poses a serious risk to patients by increasing the cost of healthcare with prolonged stay in hospitals, serious clinical complications and even death.

The ever-increasing challenges in discovering

antibacterial agents with novel mechanisms of action necessitates the development of smart antibacterial surfaces that have the potential to minimise colonisation of common hospital surfaces with bacterial pathogens. In this work, we report the antibacterial properties of flexible polydimethylsiloxane (PDMS) polymer decorated with copper hydroxide nanowires (PDMS_Cu) against a panel of drug resistant bacterial pathogens isolated from patients with bloodstream infection. The fabricated PDMS_Cu surface showed superior antimicrobial activity against both Gram negative (Escherichia coli and Klebsiella pneumoniae) and Gram positive (Staphylococcus. aureus) bacterial strains as compared to flat PDMS and glass coverslip which were used as controls. RAW 1 ACS Paragon Plus Environment

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macrophage and HeLa cells were seeded on the PDMS_Cu surface. Their viability was evaluated using confocal microscopy and MTT assay. PDMS_Cu surface supported the viability of both RAW macrophages and HeLa cells post 5 hours of incubation suggesting its potential application in a healthcare setting. Furthermore, we demonstrate the possibility of employing a thin film of PDMS_Cu surface as a protective covering over the microphone of a digital stethoscope to prevent the transmission of nosocomial pathogens between patients. In addition, this fabrication technique was used to coat commercially available gloves with a thin layer of PDMS_Cu which can be used in a hospital setting to curtail the spread of nosocomial infections while handling infectious instruments and surfaces. Key words - PDMS, superhydrophobic, antimicrobial, drug resistant, nosocomial

Introduction Antimicrobial resistance (AMR) is an emerging global epidemic resulting in increased mortality rates across various patient populations. AMR arises largely due to the ability of nosocomial pathogens to rapidly evolve resistance mechanisms against a wide variety of antibiotic classes

1,2,3.

This problem is further worsened by the widespread

indiscriminate use of antibiotics in developing countries. There is mounting evidence on the role of various surfaces as a major reservoir for the transmission of a variety of nosocomial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter spp., vancomycin-resistant enterococci (VRE), and Pseudomonas aeruginosa4,5. These nosocomial pathogens have evolved molecular mechanisms to acquire resistance against a large number of antibiotics, including carbapenems and third generation cephalosporins that are the mainstay in treating multi-drug resistant bacteria. 2 ACS Paragon Plus Environment

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In order to decrease the rates of nosocomial infection, numerous studies have highlighted the effectiveness of durable antibacterial surfaces towards preventing bacterial adhesion, colonisation and proliferation in healthcare settings6. The performance of antibacterial surfaces depends on several parameters such as surface morphology7–12, surface chemical composition13–18, physico-chemical properties19–21 of the surface etc. Metals such as silver22 and copper17,23–29 have also proven to be excellent in killing bacteria. However, the higher cost and rigidity of such surfaces limit their use for practical applications. Recently physico-mechanical killing of bacteria by sharp nanostructures has attracted attention of various researchers. This idea has been inspired from the wings of certain insects such as cicada and dragonfly. Their wings possess sharp nanostructures which kill bacteria by physically rupturing the cell wall. Following the same line of work, researchers have demonstrated antibacterial performance of natural and bioinspired nanostructured surfaces1,30–35. Despite various developments in this area, it is important to note that the fragile nature of antibacterial micro/nano structures limits their use in real world settings. Also, there are specific health care and household applications which require the antibacterial surfaces to be robust and flexible. Therefore, there is a compelling need for the development of flexible robust antibacterial surfaces. Additionally, the cost of manufacturing for these surfaces should be low so that they can be regularly substituted to address their durability issues. There are several reports on fabrication of antibacterial surfaces. Their efficacy against a wide variety of pathogenic bacterial strains have also been studied. However, fabrication of antibacterial surfaces for tackling multi drug resistant bacterial pathogens have received little attention and testing these surfaces in a real hospital environment has not been reported.

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In the present work, we have fabricated PDMS surface decorated with Cu(OH)2 nanowires (PDMS_Cu) by simple mechanical peeling method36. The strength of the technique is demonstrated by creating the nanostructure embedded PDMS surface on a commercial glove. Antibacterial efficacy of the fabricated surface was tested against multidrug resistant (MDR) nosocomial pathogens by enumerating bacterial cell viability using an agar plate method. The drug resistant bacterial strains used for testing the antibacterial efficacy of the PDMS_Cu surface in this work are different from the basic non-pathogenic laboratory strains as they possess different virulence factors37–40. Antibiofilm properties of the surface was tested using confocal microscopy by quantifying the amount of biomass present on the surface. Biocompatibility of these surfaces was evaluated by seeding RAW 246.7 macrophage and HeLa cells on the surface and testing their viability using confocal microscopy and MTT assay. Heartbeat recording has been carried out by placing the PDMS_Cu surface on the microphone of a digital stethoscope, thereby demonstrating its potential use on the stethoscope diaphragm to prevent patient to patient transmission of pathogenic flora in the hospital environment.

Materials and Methods Surface Fabrication Scheme 1 shows the process flow for the fabrication of the PDMS_Cu surface. Copper sheets were cleaned and etched to get the Cu(OH)2 nanowires as discussed in our previous work36. The Cu(OH)2 nanostructured surface exhibited superhydrophilic behaviour post etching. The surface wettability was tuned to superhydrophobic by dipping the susbtrates in 1H, 1H, 2H, 2H-Perfluorooctyltriethoxysilane. PDMS and the curing agent (ratio - 10:1) were mixed properly and then the mixture was poured on the 4 ACS Paragon Plus Environment

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superhydrophobic Cu(OH)2 nanostructured surface. To remove the air bubbles trapped inside the PDMS and curing agent mixture, the Cu(OH)2 nanostructured surface with PDMS on top was desiccated. Then the PDMS was allowed to cure at room temperature and after the PDMS became hard, it was peeled off from the Cu(OH)2 nanostructured surface gently36 (Scheme 1).

Bacterial growth conditions and sample preparation for hospital stains The bacterial sampling was carried out at Narayana Health City Hospital, Bangalore. Three clinical isolates cultured from blood cultures from sepsis patients were used: Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. The selected strains are typical representatives of multi drug-resistant (MDR) bacterial taxonomic lineages seen in the hospital. Isolates were identified and characterized using the MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) and liquid cultures of each isolate were stored at -70° C. Prior to each experiment to test the antibacterial efficacy of the test surfaces used in this work, bacterial cultures were refreshed on Luria Bertani agar (HiMedia) from stocks. In 5 of Luria Bertani broth (HiMedia) bacterial suspensions were allowed to grow overnight at 37° C. Bacterial cells were collected at the logarithmic stage of growth and the suspensions in phosphate buffered saline (PBS) were adjusted to O.D.600 0.1.

Antimicrobial susceptibility In vitro susceptibility testing was performed on all isolates and interpreted using the Phoenix™ (BD Diagnostic Systems) for the following antibiotics: ceftazidime, amikacin, tobramycin,

ciprofloxacin,

gentamicin,

ticarcillin-clavulanic

acid,

tazobactam-

piperacillin, colistin, cefepime, levofloxacin, meropenem, ceftriaxone, ampicillin, aztreonam,

ertapenem,

imipenem,

cefoxitin,

trimethoprim-sulfamethoxazole, 5

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amoxicillin-clavulanic acid and tigecycline. For S. aureus MIC was tested using the following antibiotics: amoxicillin, cefazolin, cefoxitin, chloramphenicol, daptomycin, gentamicin, linezolid, rifampicin, vancomycin. MIC values obtained by the above methods were categorized according to NCCLS breakpoints (Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. M100-S24. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.), as susceptible (S), intermediate (I), or resistant (R). Escherichia coli ATCC 39922 was used as quality control strains for the antimicrobial susceptibility test.

Bacteria Viability Assay for Hospital Stains The viability of the bacteria on the test substrates was evaluated using standard plate assay30,32,36. The substrates (glass coverslip, PDMS and PDMS_Cu) used in the experiments were dipped in PBS for 2 hours to remove the surface contaminants if present. Then the substrates were exposed to UV inside the laminar hood to ensure a sterile condition prior to the experiment. The test substrates (1 ) were kept inside a 24 well plate (BD Biosciences) in triplicates. 1 of bacterial culture from each strain was added on top of the substrates present inside the wells. The 24 well plates containing the bacterial culture on top of the test substrates were incubated for 0, 1, 2 and 4 hours at 37° C. At each incubation time point, 100 of bacterial culture was collected from each well and plated on LB agar plates followed by incubating the agar plates at 37° C for 24 hours. Then the number viable bacteria on the agar plates were calculated and the bacterial viability for each strain on different substrates was expressed in colony forming units (CFU)/. The antibacterial performance of the control surfaces (i.e. glass coverslip and PDMS) was compared with the PDMS_Cu surface based on the CFU/ data.


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Antibacterial Efficacy Testing by Exposing the Samples to Environment using Dry Swab Test To test the antibacterial efficacy of the PDMS_Cu surface in a real hospital setting, PDMS_Cu surface was kept in the hospital ward along with the control surfaces (glass coverslip and PDMS surface). Samples from all the three surfaces were taken (Figure S3) using sterile swabs after 2 hours and 4 hours of exposure and plated on chocolate agar plates. The plates were incubated at 37° C for 24 hours. Post incubating the bacterial culture plates for 24 hours, bacterial colonies on the plates were counted and compared.

Bacterial cells and culture conditions for biofilm formation Escherichia coli DH5α (E. coli), Klebsiella pneumoniae (K. pneumoniae) and Staphylococcus aureus (S. aureus) were plated on LB medium (Sigma) from glycerol stocks. The cultured were sub-cultured in LB broth at 37° C. Bacterial cells were suspended in sterile PBS to an optical density (O.D.) of 0.3, which approximates 107 / of each strain and used for further experiment.

Biofilm formation and sample preparation for confocal microscopy The submerged biofilm experiment was performed in a 24-well microtiter plates. Sterile glass slide and PDMS_Cu were placed in these wells. Two millilitres of LB media was dispensed in the wells and inoculated with around 107 cells (E. coli, K. pneumoniae and S. aureus) from an overnight culture. The microtiter plates were incubated under static condition for 5 days at 28o C. The glass slides and the PDMS_Cu blocks were washed with sterile PBS three times and dipped in 4% paraformaldehyde for 30 minutes. Samples were washed again with sterile PBS. The bacteria were stained with DAPI (0.1 /) and extracellular polysaccharide was stained with congo red (1 /) dye for 7 ACS Paragon Plus Environment


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30 minutes. The glass slide and PDMS_Cu substrates were subsequently washed with PBS and dried. Post drying samples were mounted over a glass cover slip and images were acquired using a confocal microscope.

HeLa and Macrophage cell preparation The murine macrophage cell line RAW264.7 and human epithelial cell line HeLa were maintained in DMEM media (Sigma- Aldrich) containing 10% FBS (Fetal Bovine Serum, Gibco) in 5% CO2 at 37° C.

MTT Assay for cell metabolism study 4 to 5 X 105 RAW264.7 & HeLa cells were seeded on sterile PDMS, PDMS_Cu surfaces in 24 well plates. Cells seeded in empty wells (without any test surface) were used as positive control (100% viability). PFA fixed cells were used as negative control. To enhance the attachment of the cells on to the test surface, the plates were spun at 700 rpm for 5 minutes & kept in incubator in presence of 5% CO2 at 37° C. After one hour of initial incubation, MTT dye (3-[4,5-Dimethylthiazol-2YI]-2,5-Diphenyltetrazolium Bromide) was added to each well at a concentration of 0.5 / from a stock of 5 / concentration and further incubated for 5 hours. The reaction was stopped using stop solution consisting of Dimethylformamide (1:1) and 10% sodium dodecyl sulfate (SDS). The O.D. of the culture supernatant was measured at 570 .

Durability Test The durability of the fabricated PDMS_Cu surface was tested under different conditions: dry heating, moist heating, exposure to UV ray, dust and algae solution. After exposure to all these conditions, wettability of the surfaces was quantified by contact angle and contact angle hysteresis measurements.


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Heartbeat sound monitoring For heartbeat sound monitoring mobile stethoscope app41 was installed on an Apple iPhone. The microphone of the mobile was directly placed on the chest and heartbeat sound was recorded using the mobile stethoscope app. After that PDMS_Cu was placed on the mobile’s microphone and heartbeat sound was recorded. The sound recording was carried out at a quiet place in order to avoid any external noise. This experiment was performed to ensure that heartbeat sound can be recorded by using a PDMS_Cu surface on the sound recording panel.

Scanning Electron Microscopy (SEM) Carl-Zeiss scanning electron microscopy instrument was used to capture the highresolution SEM images of the PDMS_Cu surface at different magnifications. For SEM imaging of the bacterial cells, substrates with bacterial cells on top were first fixed in 2.5 wt% of Glutaraldehyde in PBS for 5 minutes. The substrates were then desiccated in vacuum of for 48 hours. To avoid charging effect during imaging, a thin layer (~15 ) of gold was sputtered on the surface using Quorum Techport sputtering instrument. For RAW264.7 macrophage and HeLa cells imaging, cells were first fixed with paraformaldehyde (PFA). Post fixing serial dehydration was carried out in 30, 40, 50, 60, 70, 80, 90, 95 and 100% ethanol. Then the substrates were kept inside vacuum for 48 hours. SEM tool was operated at an operating voltage of 15 for carrying out the energy dispersive spectroscopy (EDS) of the PDMS_Cu surface. Scanning was performed at different locations on top of the PDMS_Cu surface to find the elements present.



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Confocal Microscopy for HeLa and Macrophage Cells 1.5 X 105 number of RAW264.7 & HeLa cells were seeded on sterile glass coverslips, PDMS & PDMS_Cu surfaces in 24 well plates. To enhance the attachment of the cells on to the test surface, the plates were spun at 700 for 5 minutes and kept in incubator in presence of 5% CO2 at 37° C. After 24 hours of incubation, the cells were washed once with PBS & fixed with 3.5% PFA for 30 minutes. Cells were permeabilized using 0.01% saponin dissolved in 3% BSA. Immunostaining was done using anti-Tubulin primary antibody (DSHB, University of Iowa) raised in mouse, followed by anti-mouse dylight 488 secondary antibody. Nucleus of the cells was visualized using DAPI staining. Image of the cells were taken in Zeiss Confocal Laser Scanning Microscope under 63X magnification. Cell morphology was analysed using Zen Blue edition software provided by Zeiss.

Confocal Microscopy and Image Analysis for Bacterial Biofilm Image acquisition of the biofilm was performed in Zeiss confocal microscope (LSM Meta 710). More than 50 random fields per sample were captured with a 40X objective. 3D reconstitution of biofilm was carried out using ZEN 2012 platform.

Atomic Force Microscopy (AFM) AFM (Bruker AFM instrument) was used for the roughness measurement of the PDMS_Cu surfaces. The scanning area was 20 20 for all the measurements. TESPA-V2 AFM tip was used (resonant frequency ∼320 and spring constant ∼42 /) and all the measurements were carried out in tapping mode.


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Contact Angle and Contact Angle Hysteresis Measurement Wettability of the PDMS_Cu surface was characterized by contact angle and contact angle hysteresis measurements. A custom-made Goniometer setup was used for the measurements. For contact angle measurement, 8 water droplet was placed on the PDMS_Cu surface and a CMOS camera was used for the image acquisition of the droplet. To measure the contact angles from the droplet images, ImageJ©3 software was used32,36 . To measure the contact angle hysteresis, the Goniometer set up was tilted and the droplet image was captured when the droplet first started to slide. After that the advancing contact angle and receding contact angle were measured using ImageJ software from the droplet image. Contact angle hysteresis was obtained by calculating the difference between advance and receding contact angles. All the measurements were performed three times to ascertain repeatability.

Results and Discussion The process flow for the fabrication of PDMS_Cu surface has been shown in Scheme 1(a). Cu(OH)2 nanowires were formed on the surface after etching the copper sheet in a solution containing ammonium persulphate and sodium hydroxide. The dimeter and length of the Cu(OH)2 nanowires were found to be ≈ 220 and ≈ 10 − 12 respectively (Scheme 1(b) and (c)). Figure 1(a) shows the photograph of the cured PDMS surface on top of a Cu(OH)2 nanostructured surface. The blue colour rectangular surface is the copper surface with Cu(OH)2 nanowires. Post curing, the PDMS surface was peeled from top of the Cu(OH)2 nanostructured surface (Scheme 1 and Figure 1(b)). The blue colour on top of the PDMS surface is due to the transferred Cu(OH)2 nanowires. Figure 1(c) shows the static water contact angle of water droplet on the PDMS_Cu surface. Water contact as high as 169° and contact angle hysteresis of less than 3° was 11 ACS Paragon Plus Environment


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obtained. SEM images of the PDMS_Cu confirmed the transfer of the Cu(OH)2 nanowires on to the PDMS surface. PDMS micro-bumps were seen across the PDMS_Cu surface. This micro-bump along with the Cu(OH)2 nanowires together form a hierarchical structure which provides the superhydrophobicity. EDS analysis was performed to determine the elemental composition of the PDMS_Cu surface (Figure 1(e)). Figure 1(f) shows the atomic force microscopy (AFM) image of the PDMS_Cu surface. Average roughness measured was ( ) 694 nm. Antibacterial efficacy of the PDMS_Cu surface was tested against three drug resistant bacteria isolated from a hospital environment, namely E. coli, methicillin resistant S. aureus (MRSA) and K. pneumoniae. These strains were isolated from patients with nosocomial bloodstream infections. Results from in vitro susceptibility testing of each isolate against a panel of antibiotics has been provided in Table S1 and the clinical significance of all the three bacterial isolates has been mentioned in Table S2. To test the bactericidal efficacy of each surface quantitatively, a standard agar plate assay method was used. The CFU/ in the suspension of E. coli, S. aureus (MRSA) and K. pneumoniae were quantified at 0, 1, 2 and 4 hours of incubation at 37o C (assay performed in triplicates). In brief, the bacterial cultures were incubated over the test substrates (coverslip, PDMS taken as controls and PDMS_Cu surface) without any mechanical agitation. The bacterial samples were collected aseptically from top of the substrates by touching the micropipette tip to the surface and plated onto LB agar plates using a sterile swab. The antibacterial efficacy of the nanostructured surfaces was determined based on the number of viable bacteria that could be cultured as CFU/. Figure S1 and Figure 2(a)-(c) shows the SEM images of the drug resistant bacteria on the fabricated surfaces. All the SEM images of the bacterial samples on all the surfaces (coverslip, PDMS and PDMS_Cu) were taken under the same condition at the same run. 12 ACS Paragon Plus Environment

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The bacterial cells were fixed in glutaraldehyde followed by dehydration in serially diluted ethanol solutions. The bacterial cells were dehydrated and empty after the fixing and dehydration steps and therefore they do not loose shape in vacuum. SEM tool was used in this work as it is one of the best techniques to study cell wall morphology under high vacuum42–44. Enhanced bacterial attachment was observed on the coverslip and PDMS surface as compared to the PDMS_Cu surface (Figure S2, scanning area 60 X 40 ). Also, the bacterial cells morphology looks healthy and intact on the coverslip and PDMS substrates (Figure S1), whereas, the cell wall morphology looks rough on the PDMS_Cu surface (Figure 2(a)-(c)). In our experiment initially the PDMS_Cu surface was superhydrophobic when the bacterial culture was poured. Hence the bacterial culture floats on the surface till trapped air in the micro/nano protrusions is escaped. After the air is escaped, the bacterial culture penetrates in to the micro/nano protrusions and react directly with them. This interaction of PBS with the Cu(OH)2 nanowires initiates the formation of the Cu2+ ions. The Cu2+ ions interact with the bacterial cell wall due to electrostatic interaction and the cell wall integrity is compromised leading the bacterial cell lysis. Even though the Cu(OH)2 nanowires are very sharp, their density on the PDMS surface is not sufficient enough to induce significant physical contact killing. Therefore, we have attributed the short term bacteriostatic action to superhydrophobicity which prohibits the initial attachment of the cells with the surface and the presence of copper hydroxide nanowires for the long term antibacterial activity in which the microbes are killed due to the copper ions45,46. Figure 2(d)-(f) show the CFU/mL data for clinical isolates of E. coli, S. aureus (MRSA) and K. pneumoniae on all the surfaces (coverslip, PDMS and PDMS_Cu) over the 4 hours



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of incubation period. Same inoculum of bacterial cells was added to all the three substrates in the first step. This is a standard procedure used to control the bacterial cell number in the initial load poured on the surfaces so that similar number of bacteria will interact with the surface30,32,47. Average values are plotted from the experiments that were carried out in triplicates. It is evident from the standard plate assay that bacterial viability on PDMS_Cu surface was much lower than the control surfaces. For all the bacterial strains i.e., E. coli, S. aureus and K. pneumoniae, there was clear statistical significance (Student’s t-test) for the number of live bacteria between the coverslip & PDMS_Cu and PDMS & PDMS_Cu pairs. Overall, both, superhydrophobicity and presence of copper hydroxide nanowires on the PDMS_Cu surface contributes to its enhanced antibacterial performance (Figure 3(a)). The maximum bacterial killing capacity of the PDMS_Cu surface against E. coli, S. aureus and K. pneumoniae was found to be !"

!"

!"

4 × 10 #$∗&#' ∗() , 1.5 × 10 #$∗&#' ∗() and 3.75 × 10 #$∗&#'∗() respectively.

A dry swab test was performed to test the antibacterial efficacy of the test substrates (coverslips, PDMS and PDMS_Cu) in real life scenario. The surfaces were exposed to circulating air by placing them near the air ducts in the patient ward (Figure S3(a)). After 2 and 4 hours of exposure, a swab was performed on the surfaces using a sterile swab and plated on chocolate agar plates (Figure S3(b)). The colonies were counted on the agar plates after 24 hours of incubation at 37° C. Figure 3(b) shows the bacterial colonies on the chocolate agar plates for 2 and 4 hours of exposure. PDMS_Cu surface was found to have the least number of bacterial colony post 4 hours of exposure (Figure 3(b2)) reassuring the fact that there was least viability on PDMS-Cu surfaces. Bacterial biofilm is a community of bacteria in which the microbes are embedded in an extracellular polymeric substances (EPS)48. Biofilms are formed on solid surfaces when bacteria adhere and produce extracellular polymeric substances. This provides the 14 ACS Paragon Plus Environment

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bacterial colony a firm attachment and protection from harsh environment. The main challenge in designing any antibacterial surface is to prevent/minimize the formation of bacterial biofilm. In this work, we have tested the antibiofilm efficacy of the PDMS_Cu surface (for experimental details see supporting information). Studied bacterial strains suspended in Luria Bertani broth (~ 10. /001/) were poured onto the test and control (coverslip and PDMS_Cu) surfaces. After 5 days of incubation at 28o C, the samples were processed for confocal microscopy. Figure 3(c) and Figure S4 shows the confocal microscopy images of the bacterial biofilms on the test surfaces for the different bacterial strains. The observed green colour is due to the staining of bacteria with DAPI (DAPI was pseudo coloured green in this case) and the red colour is the due to the staining of extracellular polysaccharide by the congo red dye. PDMS_Cu surface was found to have the least amount of bacterial biomass on its surface as shown in Figure 3(c) and Figure S4, thereby, demonstrating its superior antibiofilm efficacy against both Gram negative and Gram positive bacterial strains. Short term antibiofilm activity of the PDMS_Cu surface is due to the superhydrophobicity of the surface which prevents the initial attachment of bacteria. On longer term, the cytotoxicity of Cu ions reduces the number of live bacteria adhered onto the surface. Although the PDMS_Cu surface exhibited superior antibiofilm behaviour as compared to the glass slide, few bacterial colonies were still observed on the PDMS_Cu surface after 5 days exposure to bacterial culture in a nutrient rich Luria broth (LB) medium. Prolonged exposure of PDMS_Cu to the bacterial culture compromises the surface due to copper loss. Copper loss is due to ions leaching into the liquid medium. This loss of copper from the surface eventually reduces the amount of copper ions available on the surface. Hence, a few bacteria were able to survive on the surface after 5 days of continuous incubation.



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Biocompatibility is an essentially required property for any material to be used for healthcare applications. The biocompatibility of the PDMS_Cu surface was evaluated by testing the ability of the surface to support the viability of RAW264.7 and HeLa cells. RAW 264.7 is murine macrophage cell line which is phagocytic in nature. It is transformed with Abelson leukaemia virus and obtained from BALB/c mice. Macrophage is one of the most important white blood cells (WBCs), found in human blood. The major role of macrophage is to engulf any kind of pathogen, degrade the pathogen and represent the antigens of the pathogen towards T lymphocytes to activate host immune response. On the other hand, HeLa is human cervical cancer cell line which is epithelial in nature. It is obtained from Henrietta Lacks, a patient who passed away on 4th of October 1951. Both the cells are widely used in biological research. In this study we are proposing that PDMS_Cu surface can be used as antibacterial surface in hospital environment and will be routinely exposed to different body parts (skin, muscle, etc.) and body fluids (mainly blood etc.) of patients during surgical operation, as well as general treatment. So besides having antibacterial activity, this proposed test surface should also be host compatible and create less toxicity to the eukaryotic cells. In this preliminary study, we have decided to check the cytocompatibility of our proposed PDMS_Cu surface with one cell representing body fluid (RAW macrophage) and one cell representing human skin (outer most epidermis layer of human skin is composed of stratified squamous epithelial cells and hence, easily available HeLa cell has been used). The attachment of RAW264.7 and HeLa cells was found to be higher on the coverslip and PDMS substrates as compared to the attachment on the PDMS_Cu surface (see Figure S5). This can be attributed to the superhydrophobic property of the PDMS_Cu 16 ACS Paragon Plus Environment

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surface. Figure 4(a) shows the confocal microscopy images of both the cells on the coverslip, PDMS and PDMS_Cu surfaces. MTT assay was performed to assess the cell viability. ~51% of RAW264.7 and ~71% of HeLa cells were found to be viable after an incubation

period

of

5

hours

(Figure

4(b))

on

the

PDMS_Cu

surface.

Immunofluorescence microscopic data (Figure 4(a)) suggested that there was no significant change in the morphology of the cells on PDMS_Cu surface compared to the controls (glass coverslips, PDMS). The cytotoxicity of Cu ions on mouse L929 fibroblast cell line was reported to be 46 2 by Cao et al.49. This value (46 2) for fibroblast cell is greater than the concentration of (Cu) ion that was leached from our fabricated PDMS_Cu surface (i.e. 37.5 2) in PBS after 4 hours36. This copper ion leaching from the PDMS_Cu surface is expected to even decrease further over prolonged exposure as the source for copper ions will be exhausted due to continuous leaching. Though in our study we have not used L929 cells, but it can be apprehended from the reported data that the tolerance of heavy metal (Cu) ions by eukaryotic cell line is higher. There are previous published reports where growth of eukaryotic cells on copper surfaces have also been reported50. On the other hand the tolerance of heavy metal (Cu) by bacteria is comparatively lesser (e.g. 10-8 M or 0.6355 2 of copper ion concentration is cytotoxic for Klebsiella aerogenes, a Gram-negative bacteria51). Hence the PDMS_Cu surface can be used as an antibacterial surface for healthcare applications surface while supporting the survival of RAW macrophage and HeLa cells. The durability of the fabricated PDMS_Cu surface was tested under dry and moist heating conditions (Figure S6(a) and (b)). In case of dry heating, PDMS_Cu surface was placed on a hot plate at 120° C for 4 hours. To check the durability of the surface in moist heating condition, the surface was autoclaved at 121° C for 20 minutes. To test the



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effect of UV treatment on the hydrophobicity of the fabricated surface, PDMS_Cu substrate was placed in a laminar hood and kept under UV (76 3/ intensity) for 30 minutes (Figure S6(c)). The hydrophobicity was quantified using contact angle and contact angle hysteresis measurement as shown in Figure 5(a) following the three treatments. Post-exposure the change in surface hydrophobicity was not significant. The durability of the PDMS_Cu surface was also tested by exposing the surface to an extremely dusty environment (in close proximity to traffic) at 3 different locations for a period of 7 days (Figure S6(d)). After the test period, all the samples were brought to the laboratory and the hydrophobicity was checked under static and dynamic conditions. Figure 5(b) shows that the contact angle of the water droplet on PDMS_Cu surface before and after the exposure to the dusty environment. There was no significant decrease in the static contact angle post exposure (Figure 5(b)). Contact angle more than 150° was measured on all the surfaces as shown in Figure 5(b) (Unexposed: CA-169±2°, CAH-2.8±0.2°, Sample 1: CA-162±3.5°, CAH-5.5±0.3°; Sample 2: CA-166±3°, CAH-4.8±0.8°; Sample 3: CA-163±2.8°, CAH-5±1.2°). Drop impact study was carried out to test the hydrophobicity of the surface under dynamic condition. The contact time of the 10 droplet impact was obtained on the PDMS_Cu surface pre and post-exposure using video captured at 10000 frames per second (fps). Contact time of the water droplet did not change notably even after the (Unexposed: CT-17.3 ms; Sample 1: CT-19.4 ms; Sample 2: CT-18.7 ms; Sample 3: CT-19 ms) exposure. This study demonstrated the durability of the PDMS_Cu surface in a dusty environment. In hospitals, the transmission of nosocomial pathogens occurs via direct/indirect contact with infected patients or contaminated medical devices. Medical devices such as stethoscopes, otoscopes and monitoring devices are often contaminated with different bacterial species and are a potential source of infection transmission52. We recorded 18 ACS Paragon Plus Environment

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heart beat sound using a mobile stethoscope app41 (Figure 6(a), (b) and (c)) with and without the use of the PDMS_Cu surface (Figure 6(d) and (e)). There was a decrease of ~24 % in sound intensity in the heart beat sound when the heart beat was recorded via 1 mm thick PDMS_Cu surface using the mobile app (Video S1). Figure 6(f) and (g) show the histogram of the peak sound intensity occurring during the heart beat sound recording in the time series signal. To ensure the validity of our experiments, our recorded sounds were matched with the heart beat sounds library available53. The PDMS_Cu surface can be used as a thin covering on the stethoscope diaphragm in order to prevent the transmission of different pathogens in hospitals. Additionally, we fabricated hand gloves with a thin layer of PDMS_Cu surface on it. A thin layer of PDMS was poured on the commercially available nitrile gloves (Figure S7(b)) and then silanized copper hydroxide nanowires were pressed gently against the gloves as shown in Figure S7(c). Following this, the gloves along with the silanized copper foils were kept inside a vacuum chamber for 24 hours. Post desiccation, the copper foils were peeled off from the gloves (Figure S7(d)). The SEM images of the gloves after the peeling off process confirmed the transfer of the copper hydroxide nanowires on to the PDMS surface as shown in Figure S7(d). This highlights the potential value of these special gloves while handling contaminated equipment and surfaces in a hospital setting to minimise transmission of nosocomial pathogens between healthcare workers and patients. This will lead to fewer glove changes and hence reduced waste. Further, in contrast to other fabrication techniques where copper nanomaterial is uniformly dispersed inside the bulk, for our surfaces the copper nanomaterial is present only on the surface (top 10-20 ). This significantly reduces the amount of copper nanomaterial used. Further, gloves coated with PDMS_Cu can be collected after use. The used gloves with PDMS_Cu coating can be then recycled. As the 19 ACS Paragon Plus Environment


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nanomaterial is limited to the surface, the nanomaterial can be easily separated from the gloves and processed to prevent loss of nanomaterial to the environment. Collection of the used gloves in designated bins will stop its exposure to the environment. Also, recycling the used gloves will bring down the cost of manufacturing.

Conclusion In summary, we demonstrate the antibacterial properties of the PDMS_Cu surface to reduce the viability of a panel of multidrug resistant clinical pathogens (E. coli, S. aureus (MRSA) and K. pneumoniae). The PDMS_Cu surface exhibited superior antibiofilm activity as compared to a control glass coverslip. The antibacterial efficacy of the PDMS_Cu surface was also tested by exposing the surface in a patient ward along with controls (glass coverslip and PDMS substrates). PDMS_Cu was found to have the minimum number of bacterial colonies adhered on its surface as compared to controls showing its superior antibacterial property as compared to the controls in a real world setting inside a hospital environment. The biocompatibility of the PDMS_Cu surface was shown using confocal microscopy and MTT assay suggesting that PDMS_Cu supported the viability of RAW macrophage and HeLa cells. A potential application of using a thin film of PDMS_Cu surface as a thin covering on the stethoscope diaphragm to prevent the transmission of different pathogens from one patient to another in a hospital environment was demonstrated. The ability to coat a thin layer of PDMS_Cu onto a commercially available nitrile gloves provides compelling evidence and an attractive opportunity to introduce antibacterial gloves in a hospital setting to curtail the spread of nosocomial infections.


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Associated Content Supporting Information Sensitivity profile and clinical significance of drug-resistant bacteria used, FESEM images of bacteria, experimental procedure for dry swab test, confocal microscopic images of bacterial biofilms, SEM images of RAW macrophage and HeLa cells on silicon, PDMS and PDMS_Cu surfaces, images showing the procedure adopted for testing the reliability of the PDMS_Cu surface, photograph of antibacterial nitrile glove with a thin PDMS_Cu layer atop. (PDF) Video showing the heartbeat sound recording with and without the PDMS_Cu surface. (AVI)

Conflict of Interest The authors declare no competing financial interest.

Corresponding Authors Dipshikha Chakravortty - [email protected] Prosenjit Sen - [email protected]

Acknowledgement The authors would like to acknowledge the financial support from Unilever R&D, Bangalore. Authors would also like to thank Mazumdar Shaw Medical Foundation for providing the research facility. AT would like to thank Ministry of Electronics and Information Technology, Government of India for the financial support. AT and AK would like to thank Chandan and Shivani for their help during the experiments.



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Figures

Scheme 1: (a) Process flow for the fabrication of PDMS_Cu surface. SEM images of the Cu(OH)2 nanowires formed on the copper sheet after etching for 20 minutes: (b) Top view and (c) side view of the Cu(OH)2 nanowires formed (tip diameter ≈ 220 , nanowire length ≈ 10 − 12 ).



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Figure 1: (a) Photographs of the cured PDMS surface on top of the Cu(OH)2 nanostructured surface (b) PDMS_Cu after peeling (c) Static contact angle of 8 water droplet on top of the PDMS_Cu surface, (scale bar - 0.5 mm) (d) Representative FESEM image of the PDMS_Cu surface (e) EDS spectra showing the elemental composition of the PDMS_Cu surface (f) Atomic force microscopy of the PDMS_Cu surface (average roughness ~ 694 ).

Figure 2: SEM images of different multi drug resistant (MDR) bacterial strains on the (a)-(c) PDMS_Cu surfaces (Scale bar - 200 ). (d)-(f) CFU data of the drug resistant bacteria on all the test surfaces used in the experiments (Student’s t-test [∗∗ 5 6 0.01,∗∗ ∗ 5 6 0.001,∗∗∗∗ 5 6 0.0001]).


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Figure 3: (a) schematic explaining the antibacterial behaviour of the PDMS_Cu surface (b) Bacterial colonies on chocolate agar plates after (b1) 2 and (b2) 4 hours of exposure to the environment in a patient ward. (c) confocal microscopic images of the bacterial biofilm of E. coli on the coverslip and PDMS_Cu surfaces post 5 days of incubation in Luria broth (LB) medium. Scan area [70 (x-axis) X 70 (y-axis)].



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Figure 4: (a) Confocal Laser Scanning Microscopy images for inspecting the morphology of the RAW264.7 macrophage and epithelial HeLa cells on all the test surfaces (Green Tubulin, Blue - DAPI(Nucleus)) (b) Percentage viability evaluation of RAW264.7 macrophage and HeLA cells on all the test surfaces using MTT assay. [PDMS_CuPolydimethylsiloxane (PDMS) polymer decorated with copper hydroxide nanowires, PDMS- Polydimethylsiloxane, PFA- Paraformaldehyde fixed dead cells (Negative Control), MC- Media Control (Dulbecco’s Modified Eagle Medium (DMEM) media used as blank), Untreated- Untreated cells on glass coverslips (Positive Control- 100% viability).


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Figure 5: Contact angle and contact angle hysteresis of water droplet on the PDMS_Cu surface at post exposure to (a) dry and moist heating, UV radiation and (b) dust.

Figure 6: (a) Heart beat recording using mobile stethoscope app (b) 1 mm thick PDMS_Cu surface used for recording (c) The thin PDMS_Cu surface was placed on the mobile microphone and recording was carried out. Sound intensity of the recorded 33 ACS Paragon Plus Environment


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heart beat sound (d) without and (e) with the PDMS_Cu surface. Histogram of the heart beat sound intensity (f) without and (g) with the PDMS_Cu surface.


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27x17mm (600 x 600 DPI)

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A Nanowire-Based Flexible Antibacterial Surface Reduces the

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