TY - JOUR
AU - Heinemann, Jack A
AB - ABSTRACT Antimicrobial materials are tools used to reduce the transmission of infectious microorganisms. Photo-illuminated titania (TiO2) is a known antimicrobial material. Used as a coating on door handles and similar surfaces, it may reduce viability and colonization by pathogens and limit their spread. We tested the survival of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Saccharomyces cerevisiae on a nano-structured TiO2-based thin film, called ‘NsARC’, and on stainless steel under a variety of light wavelengths and intensities. There was significantly less survival (P <0.001) of all the organisms tested on NsARC compared to inert uncoated stainless steel under all conditions. NsARC was active in the dark and possible mechanisms for this are suggested. NsARC inhibited biofilm formation as confirmed by scanning electron microscopy. These results suggest that NsARC can be used as a self-cleaning and self-sterilizing antimicrobial surface coating for the prevention and reduction in the spread of potentially infectious microbes. nanostructured anatase rutile carbon, antimicrobial activity, antibiofilm activity, dark-active photocatalyst, scanning electron microscopy LIST OF ABBREVIATIONS LIST OF ABBREVIATIONS NsARC Nanostructured anatase rutile and carbon TiO2 Titanium (IV) Oxide, titania, titanium dioxide EPS Extracellular polymeric substances PP-MOCVD pulse-pressured metallorganic chemical vapour deposition SEM scanning electron microscopy AMA antimicrobial activity ABA antibiofilm activity EOP efficiency of plating Cfu/ml colony forming units per millilitre HMDS hexamethyldisilazane ANOVA analysis of variance BACKGROUND Nanomaterials exhibiting antimicrobial activity are generating attention because of their usefulness in pharmaceutical and biological applications (Fang et al. 2012; Leung et al. 2016). While the antimicrobial activity of nanomaterials such as titania (TiO2), silver (Ag), zinc oxide (ZnO) and copper is well known, the mechanism of antimicrobial action of these nanomaterials is not well understood (Fraud and Poole 2011; Chiang and Schellhorn 2012). Differences in the techniques used to characterise these nanomaterials, instability of some of the nanomaterials during testing (Guadagnini et al. 2015), variation in experimental methods (Djurišić et al. 2015; Guadagnini et al. 2015) and variation in the medium in which the testing is conducted can affect the results and contribute to the disagreement in the available literature explaining the mechanism of action of nanomaterials (Castellote and Bengtsson 2011; Fang et al. 2012). TiO2 is a photocatalyst (Castellote and Bengtsson 2011; Djurišić et al. 2015; Leung et al. 2016). Photocatalysis is the process whereby electronic carriers are generated under light excitation, and these react with surface species to produce reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), super oxides (O+) and other free radicals that are toxic to cell membranes of microorganisms (Yu et al. 2003; Verdier et al. 2014; Sadowski et al. 2015). Nanomaterials such as TiO2 could be of value because disease-causing microorganisms are gradually becoming resistant to the conventional methods that had been successful in inhibiting their growth and development (Cantón and Morosini 2011; Chowdhury et al. 2018). Conventional methods such as the use of chemotherapeutics and disinfectants are becoming less effective (Grass, Rensing and Solioz 2011; Tan et al. 2012; Abboud et al. 2014), as a result of the development of resistant phenotypes by these microorganisms due to indiscriminate use of antibiotics and other antimicrobial agents such as disinfectants and antiseptics (Dancer 2008; Cantón and Morosini 2011; Blair et al. 2015). The pathway by which these microorganisms develop resistance may be intrinsic, acquired or adaptive. Intrinsic resistance describes the species level of susceptibility to an agent. Adaptive resistance is a form of intrinsic resistance, but the phenotype is dependent on particular genes that are conditionally expressed. Exposure of bacteria to sublethal doses of antimicrobial agents can also induce an adaptive response that leads to an increase in tolerance of bacteria to antibiotics (Kohanski, DePristo and Collins 2010; Kurenbach et al. 2015). In acquired resistance, microorganisms acquire new genes or mutations in genes that change susceptibility, either through changes to the target or changes that affect the biologically relevant concentration of the agent. This form of resistance is not dependent on the environment. Microorganisms can also aggregate together and grow on solid surfaces, forming biofilms (Yang et al. 2016). Biofilms form through a series of maturation steps beginning with adherence to a surface. Intercellular binding within extracellular polymeric substance (EPS) provides mechanical stability, mediates adhesion to surfaces and protects the organisms from UV radiation, desiccation and toxic effect of antibiotics and other biocides (Jefferson 2004; Flemming and Wingender 2010; Koseki et al. 2014). Surgical implants, catheter and other materials used in hospitals have been shown to harbor biofilms (Arciola et al. 2012; Koseki et al. 2014). Biofilms could play a role in over 65% of microbial infections. The infections caused by resistant microorganisms are serious public health issues that increase hospital costs, morbidity and mortality (Cantón and Morosini 2011; Chowdhury et al. 2018). These infectious agents may be transferred through touching contaminated surfaces (Airey and Verran 2007; Dancer 2008; Chowdhury et al. 2018). Measures taken to reduce infections that can be acquired through contaminated surfaces are presently geared towards personal hygiene and cleaning of surfaces with disinfectants and antiseptics, but this has yielded limited success (Griffith et al. 2000; Page, Wilson and Parkin 2009; Leyland et al. 2016). Therefore, an alternative strategy is to reduce infections caused by microorganisms that persist on surfaces by making these surfaces antimicrobial (Yu et al. 2003; Verdier et al. 2014; Ganewatta et al. 2015). TiO2 is self-cleaning and self-disinfecting (Damodar, You and Chou 2009; de Niederhãusern, Bondi and Bondioli 2013) and is one of the materials of choice that can be used in making surfaces antimicrobial. Nanostructured anatase, rutile and carbon (NsARC) (Fig. 1) is a composite of titania and carbon (Krumdieck et al. 2019). NsARC has properties that make it promising as an antimicrobial surface coating: it is robust, super-hydrophilic and photoactive under UV and visible light (Krumdieck et al. 2017; Gardecka et al. 2019; Krumdieck et al. 2019). It is deposited using the direct liquid injection pulsed-pressure metal-organic chemical vapour deposition process (PP-MOCVD) technique and can be applied in conformal coatings onto complex geometries (Lee, Krumdieck and Talwar 2013). In previous work, we found that activation of NsARC by visible light reduced viable Escherichia coli populations by 3 orders of magnitude in 4 hours (Krumdieck et al. 2019). In addition, a 2 orders of magnitude reduction in viability was observed even in the dark (Krumdieck et al. 2019). Figure 1. Open in new tabDownload slide Images of the test samples (A), SEM image of the top view of the NsARC coating surface morphology. (B), SEM image of fractured cross-section of NsARC coating. (C) Typical photographs (left to right) of negative control (stainless steel), test material (NsARC) and positive control (copper). Figure 1. Open in new tabDownload slide Images of the test samples (A), SEM image of the top view of the NsARC coating surface morphology. (B), SEM image of fractured cross-section of NsARC coating. (C) Typical photographs (left to right) of negative control (stainless steel), test material (NsARC) and positive control (copper). In this study, we tested NsARC for antimicrobial (AMA) and antibiofilm activity (ABA). Here, we report on the antimicrobial properties of this material using a set of microbial species that serve as representatives of various kinds of pathogens and assess the ability of these microbial species to form biofilms on the material. The microorganisms chosen were from species that can cause infections in humans, or are model organisms used as surrogate indicators. They include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Saccharomyces cerevisiae. These microorganisms were chosen because they differ structurally and morphologically (Chao and Zhang 2011; Yao, Kahne and Kishony 2012). They are likely to respond differently to the killing effect of NsARC (Keren et al. 2013; Singla, Harjai and Chhibber 2014), and they represent varying responses of resistance to the killing effects of antibiotics and hospital disinfection (Foster et al. 2011; Koseki et al. 2014). METHODS Microbial strains and cultivation of test organism Bacterial strains used in this study are Escherichia coli ATCC8739 (Mills, Hill and Robertson 2012), Staphylococcus aureus 25923 (Mun et al. 2013) and Pseudomonas aeruginosa ATCC10145 (Kwiatek et al. 2017). Saccharomyces cerevisiae SY1229 (Bender and Sprague 1989) is the yeast strain used. The bacterial strains were stored in 15% (v/v) glycerol solution, while the yeast in 30% (v/v) glycerol solution all at −80°C. To recover bacteria, Lysogeny Broth (LB) (Lennox-L-Broth Base, Invitrogen (USA) broth and agar (Bacteriological Agar No.1, Oxoid (UK)) agar plates were inoculated using a loopful of the sample and incubated at 37°C for 18 hours. To recover the yeast, Yeast Peptone Dextrose (YPD) (Sigma-Aldrich (USA) broth and agar (Bacteriological Agar No.1, Oxoid (UK)) agar plates was inoculated with a loopful of the sample and incubated at 30°C for 24–48 hours. These plates were stored at 4°C for not longer than one week. To recover the microorganisms, LB or YPD broth was inoculated with a colony that was transferred from the plate. Liquid cultures were then aerated using a rotary shaker at 37°C (bacteria) and 30°C (yeast), respectively, and grown for 16–24 hours to saturation. The cells were then pelleted and then resuspended three times in phosphate buffer saline (PBS) to wash them. The cultures were then diluted to obtain a suspension of the organisms containing approximately 500 000 cells. Assessing the antimicrobial and antibiofilm activity of NsARC ISO 27447:2009, ‘test method for antimicrobial activity of semiconducting photocatalytic materials’ (Mills, Hill and Robertson 2012; Sadowski et al. 2015) formed the methodological basis of this assessment. Stainless steel (304) was used as a negative (inert) control material as recommended (Mills, Hill and Robertson 2012) and commercially pure copper, which is well-known for its antimicrobial activity (Grass, Rensing and Solioz 2011), was selected as a positive control material. Antimicrobial activity (AMA) testing The test organisms (50 μl of the culture containing ∼500 000 cells at stationary growth phase) were placed on the test and control surfaces (25 mm × 25 mm) under a sterile cover slip (24 mm × 24 mm) used to spread the culture on the surface. The samples were placed in petri dishes (60 mm × 15 mm) containing damp filter paper. Replicates were simultaneously exposed to high intensity visible light of 2100 lux (450-650 nm), UV light (365 nm), ambient light (650-750 nm) and also kept in the dark for a period of up to 8 hrs. The test and control samples were each placed in sterile cellophane bags and 1.95 ml tryptic soy broth with 0.05% (v/v) Tween 80 (TSB-Tween) (Sigma-Aldrich (USA)) was added. The samples were rubbed from outside the bags to wash off the cells. The samples were washed multiple times to ensure that all the cells were completely removed. Dilutions were made in PBS. 10 μl of samples of different dilutions of E. coli, S. aureus and P. aeruginosa were transferred onto the surface of tryptic soy agar (TSA) (Sigma-Aldrich (USA)), or for S. cerevisiae, onto the surface of YPD agar. The inoculated plates were incubated at 37°C for 18 hours for the bacteria, and at 30°C for 48 hrs for the yeast. All experiments were conducted three times to obtain biological replicates. Three samples of each (test and control) were used for each experiment to obtain technical replicates (Quick-R 2018). The colonies were counted and normalised to volume of solution to give colony forming units per ml (cfu/ml). This was compared for the various treatments (material and exposure conditions). There were variations in the values, thus, the cfu/ml counts were normalised to efficiency of plating (EOP) values, which is the ratio of live cells on the test samples to the initial cell counts on the negative control (Team RC). EOP was used in analysis with R and graphpad prism software (Prism - graphpad.com 2018). The morphology of cells was visualized by scanning electron microscopy (SEM) on the surface of NsARC and stainless steel for 8 hours before washing off . The cells were fixed with paraformaldehyde, dehydrated using various grades of ethanol (30%, 50%, 70%, 90% and 100%) and hexamethyldisilazane (HMDS) (Sigma-Aldrich (USA)) and coated with gold using a sputter coater (sputter current:120 mA, sputter pressure: 1 × 10−2 psi, Argon pressure: 12 600 psi and distance: 800 nm). They were mounted onto an SEM sample stub with a double-sided sticky tape for imaging in a JEOL JSM-7000F field emission scanning electron microscope. Twenty images from each sample replicate were capture and sixty images in total were saved. Antibiofilm activity (ABA) testing The procedure was similar to that used for AMA testing. Modifications included longer exposure times, up to 48 hours. The surface of the samples was washed with PBS to remove loosely bound cells, and the samples were placed into a sonication water bath (ultrasonic bath, Sigma-Aldrich, USA) and sonicated for up to 5 minutes. Then 100 μl of the recovered cells was serially diluted in PBS, and 10 μl transferred onto the surface of TSA agar. The inoculated plates were incubated at 37°C for 18 hours. All experiments were also conducted three times to obtain biological replicates. Three samples of each (test and control) was used for each experiment to obtain technical replicates. The colonies were counted and normalised to volume of solution to give colony forming units per ml (cfu/ml), that was used in analysis with R and graphpad prism software. SEM was used to visualize the cells that were still attached to the surface of NsARC and stainless steel samples after washing off with PBS to remove the loosely bound cells. The in situ biofilm samples were prepared in the same manner as described for assessing AMA testing (above). Samples with the fixed cells were mounted onto an SEM sample stub with a double-sided sticky tape for imaging in a JEOL JSM-7000F field emission SEM. About 20 images from each sample replicate were capture and sixty images in total were saved. Images were analysed using Fiji ImageJ. The total area covered by the biofilm was measured for all the images and the average surface area covered by biofilm for each organism was determined. Statistical analysis R was used for statistical analysis. In the experiment to determine AMA of NsARC, a multifactor analysis of variance (ANOVA) was performed on the EOP values to test for effect of the materials (NsARC and control) and exposure conditions (high intensity visible light, UV light, ambient light and dark). Residual plots were examined to determine if EOP values were normally distributed, which is an assumption for ANOVA. The plots were not normally distributed. So the EOP values were log transformed to meet the assumption. In each case, we tested for significant difference between materials. The null hypothesis was no difference between the EOP values from the materials under the various exposure conditions. We also tested for interaction between materials and exposure conditions. A Bonferroni's post hoc test was used to compare the EOP to determine if there is a difference between NsARC and stainless steel. The value for statistical significance was set as P <0.05. The ANOVA table and the results of each post hoc are available in the supplementary material. However, we were most interested in the difference in EOP between the following combinations: NsARC under ambient light and stainless steel under ambient light, NsARC under ambient light and NsARC in the dark, NsARC in the dark and Stainless steel in the dark, NsARC under visible light and stainless steel under visible light, NsARC under visible light and NsARC under UV light, NsARC under UV light and stainless steel under UV light. Contrast matrices listing these contrast of interest were drawn up and the test Interactions function in the phia package in R was used to evaluate the contrasts. For the experiment to determine biofilm formation, we were interested in comparing viable cells that were still attached to the surface of NsARC and stainless steel after 48 hours. So we tested to find out if viable cells recovered after sonication depended on the substrate material and exposure conditions. We also performed a multifactor ANOVA on the cfu/ml values. A Bonferroni's post hoc was also used to compare the cfu/ml values. Contrast matrices listing the contrast of interest as described earlier were drawn up. The value for statistical significance was also set as P<i < 0.>05 and ANOVA table and the results of each post hoc are also available in the supplementary material. RESULTS Antimicrobial activity NsARC in comparison with inert stainless steel (negative control) and a material with demonstrated antimicrobial activity (copper) demonstrated antimicrobial activity on all the microorganisms tested (Fig. 2). The viable population of all the organisms tested on NsARC was reduced by greater than 2-log under all the light exposure conditions, when compared with stainless steel. In contrast, copper reduced viability by greater than 4-log to below the detection limit (Fig. 2). Figure 2. Open in new tabDownload slide Survival of (A)E. coli, (B)S. aureus, (C)P. aeruginosa and (D)S. cerevisiae on stainless steel, NsARC and copper after 8 hours in the dark and exposure to UV, ambient and high intensity visible light. Error bars are standard error of mean. Asterisks indicate P values. * = P <0.05; ** = P <0.01; *** = P <0.001; ns = not significant. Figure 2. Open in new tabDownload slide Survival of (A)E. coli, (B)S. aureus, (C)P. aeruginosa and (D)S. cerevisiae on stainless steel, NsARC and copper after 8 hours in the dark and exposure to UV, ambient and high intensity visible light. Error bars are standard error of mean. Asterisks indicate P values. * = P <0.05; ** = P <0.01; *** = P <0.001; ns = not significant. A greater than 3-log reduction of viable E. coli was achieved on NsARC using UV and visible light, and a 2-log reduction was observed using ambient light or no light exposure, when compared to viability on stainless steel (Fig. 2A). These observations extend our earlier report of AMA from photoactivated (UV and high intensity visible light) and non-photoactivated NsARC surfaces to AMA under ambient light conditions. There was no significant difference in the effectiveness of NsARC in killing E. coli under visible and UV light (P = 0.966), But there was significantly greater killing on NsARC under ambient light compared to no light exposure (P <0.001). A greater than 4-log reduction in viability of S. aureus (Fig. 2B) on NsARC was achieved using UV and visible light, and a greater than 2-log reduction was observed using ambient light or no light exposure compared to stainless steel. There was no significant difference in the effectiveness of NsARC killing S. aureus under UV or visible light (P = 0.867), but there was significantly greater killing under ambient light compared to no light (P < 0.01). Furthermore, P. aeruginosa viability on NsARC was reduced by more than 3-log using either UV or visible light compared to stainless steel and 2-log reduction was observed using ambient light or no light exposure (Fig. 2C). There was significantly greater killing on NsARC under visible than UV light (P <0.003). There was also significantly greater killing under ambient light compared to no light (P <0.001). Viability of the fungus S. cerevisiae on NsARC was reduced by more than 3-log using either UV or visible light compared to the same light exposures on stainless steel (P < 0.01). About a 2-log reduction in viability was also achieved using ambient light or no light exposure (Fig. 2D). There was significantly greater killing on NsARC under UV light than under visible light (P <0.006). And as seen in all the other organisms tested, there was also significant killing under ambient light compared to no light (P <0.001). SEM was used to visualize the cells on stainless steel and NsARC. Images of cells on the same material under UV light, visible light, ambient light and no light were similar. Therefore, SEM of cells on stainless steel (Fig. 3A, B, C, D) and NsARC (Fig. 3E, F, G, H) under visible light for 8 hrs was presented. The cells appeared intact with little or no deformation after exposure to stainless steel, but most of the cells that were on NsARC appeared distorted (Table 1). Cell membrane damage and deformation have been observed in E. coli (Foster et al. 2010; Leung et al. 2016) and S. aureus (Cheng et al. 2009) after exposure to the surface of photoactivated TiO2 nanoparticles. Similar effects were also observed when Candida albicans and S. aureus were exposed to flame-synthesized nano-TiO2 coatings (De Falco et al. 2017). Figure 3. Open in new tabDownload slide SEM images of (A)E. coli, (B)S. aureus, (C)P. aeruginosa and (D)S. cerevisiae on stainless steel and (E)E. coli, (F)S. aureus, (G)P. aeruginosa and (H) S. cerevisiae on NsARC surfaces for 8 hours under high intensity visible light. (Each image represents 1 out of 60). Figure 3. Open in new tabDownload slide SEM images of (A)E. coli, (B)S. aureus, (C)P. aeruginosa and (D)S. cerevisiae on stainless steel and (E)E. coli, (F)S. aureus, (G)P. aeruginosa and (H) S. cerevisiae on NsARC surfaces for 8 hours under high intensity visible light. (Each image represents 1 out of 60). Table 1. Antimicrobial activity-associated damage. Organism . Average number of cells per field of view imaged . Average number of cells with distortions per field of view imaged (%) . Stainless steel NsARC Stainless steel NsARC E. coli 28 26 6 (21) 20 (77) S. aureus 23 21 8 (34) 19 (90) P. aeruginosa 31 26 10 (32) 22 (85) S. cerevisiae 33 29 9 (27) 25 (86) Organism . Average number of cells per field of view imaged . Average number of cells with distortions per field of view imaged (%) . Stainless steel NsARC Stainless steel NsARC E. coli 28 26 6 (21) 20 (77) S. aureus 23 21 8 (34) 19 (90) P. aeruginosa 31 26 10 (32) 22 (85) S. cerevisiae 33 29 9 (27) 25 (86) Open in new tab Table 1. Antimicrobial activity-associated damage. Organism . Average number of cells per field of view imaged . Average number of cells with distortions per field of view imaged (%) . Stainless steel NsARC Stainless steel NsARC E. coli 28 26 6 (21) 20 (77) S. aureus 23 21 8 (34) 19 (90) P. aeruginosa 31 26 10 (32) 22 (85) S. cerevisiae 33 29 9 (27) 25 (86) Organism . Average number of cells per field of view imaged . Average number of cells with distortions per field of view imaged (%) . Stainless steel NsARC Stainless steel NsARC E. coli 28 26 6 (21) 20 (77) S. aureus 23 21 8 (34) 19 (90) P. aeruginosa 31 26 10 (32) 22 (85) S. cerevisiae 33 29 9 (27) 25 (86) Open in new tab Antibiofilm activity Attachment of microorganisms onto the surfaces of stainless steel and NsARC was used as a model to determine biofilm formation. Biofilms on the surfaces was analyzed in two ways: firstly, by determining the number of viable cells that were detached from the surfaces after sonication and secondly, by using SEM to observe the surface area covered by the biofilm. Biofilms formed on NsARC compared to those formed on stainless steel are shown in (Fig. 4). There was a greater than 2-log reduction in viable E. coli recovered from NsARC compared to stainless steel under all light exposure conditions (Fig. 4A). There was significantly greater reduction on NsARC under UV light compared to on NsARC under visible light (P <0.001). There was also a significantly greater reduction on NsARC under ambient light than with no light. This suggested that NsARC was effective in preventing surface colonization by E. coli. Consistent with this, E. coli biofilms were adhered to a smaller proportion of the surface of NsARC (Fig. 5E and Fig. 6) compared to those formed on stainless steel (Fig. 5A and Fig. 6). Figure 4. Open in new tabDownload slide Number of viable (A)E. coli, (B)S. aureus, (C)P. aeruginosa and (D)S. cerevisiae cells recovered from biofilms on stainless steel and NsARC surfaces after 48 hours in the dark and exposure to UV, ambient and high intensity visible light. Error bars are standard error of means. Asterisks indicate P values. * = P <0.05; ** = P <0.01; *** = P <0.001; ns = not significant. Figure 4. Open in new tabDownload slide Number of viable (A)E. coli, (B)S. aureus, (C)P. aeruginosa and (D)S. cerevisiae cells recovered from biofilms on stainless steel and NsARC surfaces after 48 hours in the dark and exposure to UV, ambient and high intensity visible light. Error bars are standard error of means. Asterisks indicate P values. * = P <0.05; ** = P <0.01; *** = P <0.001; ns = not significant. Figure 5. Open in new tabDownload slide SEM images of (A) E. coli, (B) S. aureus, (C) P. aeruginosa and (D) S. cerevisiae biofilms that were formed on stainless steel and (E) E. coli, (F) S. aureus, (G) P. aeruginosa and (H) S. cerevisiae that formed on NsARC surface after 48 hours under high intensity visible light. (Each image represents one out of sixty). Figure 5. Open in new tabDownload slide SEM images of (A) E. coli, (B) S. aureus, (C) P. aeruginosa and (D) S. cerevisiae biofilms that were formed on stainless steel and (E) E. coli, (F) S. aureus, (G) P. aeruginosa and (H) S. cerevisiae that formed on NsARC surface after 48 hours under high intensity visible light. (Each image represents one out of sixty). Figure 6. Open in new tabDownload slide Proportion (%) of the surface area on stainless steel and NsARC that was covered by E. coli, S. aureus, P. aeruginosa or S. cerevisiae biofilms after 48 hours exposed to high intensity visible light. Figure 6. Open in new tabDownload slide Proportion (%) of the surface area on stainless steel and NsARC that was covered by E. coli, S. aureus, P. aeruginosa or S. cerevisiae biofilms after 48 hours exposed to high intensity visible light. Biofilms formed by S. aureus on NsARC also had fewer viable cells (about 2-log reduction) compared to those formed on stainless steel (Fig. 4B). In contrast to what was observed for E. coli, there was no significant difference in viable cells recovered from NsARC under UV compared to visible light (P<i = 1>) (Fig. 4B). But there were significantly greater viable S. aureus cells recovered from NsARC under Ambient light than no light (P >0.0098) (Fig. 4B). S. aureus biofilms occupied noticeably smaller areas of the surface on NsARC (Fig. 5F and Fig. 6) compared to stainless steel (Fig. 5B and Fig. 6). There was also a greater than 2-log reduction in P. aeruginosa biofilm formed on NsARC compared to stainless steel (Fig. 4C). There was no significant difference in the effectiveness of NsARC under UV or visible light (P <0.972). There was also no significant difference under ambient or no light (P = 0.067) . P. aeruginosa biofilms occupied noticeably smaller areas on the surface of NsARC (Fig. 5G and Fig. 6) compared to stainless steel (Fig. 5C and Fig. 6). S. cerevisiae biofilms had fewer viable organisms on NsARC than on stainless steel for all exposures (Figs. 4D). There was no significant difference in the effectiveness of NsARC under UV or visible light (P <0.375). There was also no significant difference under ambient or no light (P<i = 1>) . S. cerevisiae biofilms on NsARC also occupied a smaller surface area (Fig. 5H and Fig. 6) compared to biofilms on stainless steel (Fig. 5D and Fig. 6). DISCUSSION NsARC reduced the viability of all the microorganisms tested (Fig. 2). This confirms that NsARC is a broad-spectrum antimicrobial agent, similar to what has been observed for other TiO2-based materials (Pelaez et al. 2012; Sadowski et al. 2015; Xu et al. 2015). We observed cellular morphologies using SEM and found that those on NsARC were distorted compared to those that were on stainless steel. The observed cell morphologies are consistent with holes created following the photoexitation of electrons (Diebold 2003). Our observations are consistent with those made by others associating TiO2 with mortality through disruption of cell membranes. NsARC also reduced viability of microorganisms without light exposures and presumably without photoactivation. Light is necessary for photoactivation because undoped anatase and rutile phases of NsARC have a band gap that requires UV irradiation for photoexitation (Lee, Krumdieck and Talwar 2013; Krumdieck et al. 2017). This demonstrates that photoactivated ROS are not the only mechanism for cellular degradation. While the killing mechanism(s) in the dark is not known, there are several possibilities. This activity could arise from the carbon component of the NsARC. Carbon is also an antimicrobial agent that is inert as an element, but can be chemically active when combined with other compounds or elements (Cheng et al. 2009; Dizaj et al. 2015). Modification of the zeta potential of the cell membranes by direct contact with NsARC leading to increased permeability is another possible reason for the antimicrobial activity observed in the dark (Halder et al. 2015). The hydrophilic surface of NsARC, possibly linked to the very high specific surface area of the nanostructured coating (Gardecka et al. 2019; Krumdieck et al. 2019), may also desiccate cells over time (Yu et al. 2003). NsARC inhibited biofilm formation. Biofilms on NsARC were analyzed both by determining the number of surviving cells that were detached from the surface biofilm by sonication and by using SEM to show the surface area covered by the biofilm. Fewer viable biofilm-associated E. coli, S. aureus, P. aeruginosa and S. cerevisiae were measured on NsARC compared to stainless steel. For each species, biofilms covered less of the substrate surface area on NsARC compared to stainless steel. Contrary to what has been observed in other TiO2 formulations, where only photoexcitation can lead to prevention of biofilm formation (Cheng et al. 2009; Gomes Silva et al. 2011), NsARC inhibited biofilms even in the dark. In this study, we demonstrated the reduction in survival and morphological distortion of microbial cells caused by NsARC. However, we have not conclusively determined the mechanism of cell death. We hypothesize that damage to enzymes or membranes, disruption of metabolic pathways and leakage of the cytoplasmic content may lead to death (Foster et al. 2010; Foster et al. 2011). It is also not absolutely certain that the killing effect in the dark is fully explained by the carbon component of NsARC. Therefore we will be testing NsARC films that have been annealed in air to remove the carbon (Gardecka et al. 2018). This will enable us to monitor antimicrobial activity and see if there is a significant change when microorganisms are exposed to the annealed NsARC samples in the presence and absence of photoexcitation. The mode of action of some bactericidal antibiotics also involves the production of ROS that can induce cell death (Cantón and Morosini 2011; Verdier et al. 2014). However, low concentrations of ROS can also induce adaptive protective responses from the cell (Yu et al. 2003; Castellote and Bengtsson 2011). If the killing action of NsARC is due to the production of ROS, we hypothesize that it may be likely that sublethal exposure of bacteria to NsARC could induce some form of adaptive response. Further studies are therefore needed in order to determine if exposure of bacteria to NsARC could induce the development of unintended phenotypes like resistance to antibiotics. ACKNOWLEDGEMENTS The authors wish to thank Jack Aitkens and Hyunwoo Jun for helping to set up the experiments and reading through the manuscripts. Matt Walters for graphical design. We also thank the UC SEM technician, Shaun Mucalo. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Krumdieck's work has received funding from Koti Technologies Ltd. and she is a shareholder in the company, a university of Canterbury spin-out working to commercialize the pp-MOCVD coating technology. Author roles: Alibe Wasa: Original draft preparation, Investigation, Methodology, Writing, statistical analysis—Review and Editing; Johann Land: Production of test samples, Writing—Original Draft Preparation; Rukmini Gorthy: Characterisation of NsARC, SEM, Writing—Review & Editing; Susan Krumdieck: Funding Acquisition, Project Administration, Writing—Review & Editing; Catherine Bishop: Resources, Supervision, Writing—Review & Editing; Godsoe William: Statistical Analysis, Methodology, Supervision, Writing—Review & Editing; Jack A. Heinemann: Conceptualization, Methodology, Project Administration, Resources, Funding Acquisition, Supervision, Writing and Editing—Original Draft Preparation Grant information: This work was funded by grants from New Zealand Ministry of Business Innovation and Employment (MBIE) Contract UOCX1501. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Conflicts of Interest None declared. 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TI - Antimicrobial and biofilm-disrupting nanostructured TiO2 coating demonstrating photoactivity and dark activity
JF - FEMS Microbiology Letters
DO - 10.1093/femsle/fnab039
DA - 2021-05-03
UR - https://www.deepdyve.com/lp/oxford-university-press/antimicrobial-and-biofilm-disrupting-nanostructured-tio2-coating-alsYUfgkt0
VL - 368
IS - 7
DP - DeepDyve
ER -