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Biological and Environmental Phenomena at the Interface

Bactericidal Activity of Superhydrophobic and Superhydrophilic Copper in Bacterial Dispersions Ludmila B. Boinovich, Valery V. Kaminsky, Alexandr G. Domantovsky, Kirill A. Emelyanenko, Andrey V. Aleshkin, Eldar R. Zulkarneev, Irina A. Kiseleva, and Alexandre M. Emelyanenko Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03817 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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Bactericidal Activity of Superhydrophobic and Superhydrophilic Copper in Bacterial Dispersions Ludmila B. Boinovich,1,* Valery V. Kaminsky,2 Alexandr G. Domantovsky,1 Kirill A. Emelyanenko,1 Andrey V. Aleshkin,2 Eldar R. Zulkarneev,2 Irina A. Kiseleva,2 Alexandre M. Emelyanenko1

1A.N.

Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Leninsky prospect 31 bldg. 4, 119071 Moscow, Russia

2G.

N. Gabrichevsky Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., 125212, Moscow, Russia

KEYWORDS: extreme wettability, nosocomial infections, biocorrosion, primary adhesion mechanisms, laser surface modification, antibacterial surfaces, Escherichia coli, Klebsiella pneumoniae, contact killing, textured surfaces, nanoparticles

ACS Paragon Plus Environment

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ABSTRACT: A method based on nanosecond laser processing was used to design superhydrophilic and superhydrophobic copper substrates. Three different protocols were used to analyze the evolution of the bactericidal activity of the copper substrates with different wettability. Scanning electron microscopy was used to study the variation of cell morphology after attachment to superhydrophilic and superhydrophobic surfaces. The dispersions of Escherichia coli K12 C600 and Klebsiella pneumoniae 811 in Luria Bertani broth contacted with the superhydrophilic copper surface showed enhanced bacterial inactivation, associated with toxic action of both hierarchically textured copper surface and high content of Cu2+ ions in the dispersion medium. In contrast, the bacterial

dispersions

contacted

with

the

superhydrophobic

copper

substrates

demonstrated an increase in cell concentration with time until the development of corrosion processes. The resistance of bacterial cells to contact with the copper substrates is discussed on the basis of surface forces, determining the primary adhesion and of the protective action of a superhydrophobic state of the surface against electrochemical and biological corrosion.


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Introduction The antimicrobial activity of copper and copper alloys is now well documented in the literature1–7 and in clinical studies. In attempts to reduce the spread of nosocomial infections, copper has been evaluated for use on touch surfaces, such as toilet flush buttons, light switches, corridor handrails, door knobs and handles, bathroom fixtures, bed rails, etc. The use of copper in medicine as an antimicrobial agent became quite common beginning from the 19th century.8 Very recently, using copper vessels to render water drinkable has been discovered as a low-cost alternative of anti-microbial treatment for developing countries.9 At the same time, a copper intake, associated with accidental ingestion or exposure to high levels of copper, may cause adverse health effects,10–11 including neurological disorders like Alzheimer’s and prion diseases,11,12 and the inactivation of antibiotics.13 Since both copper deficiency and copper excess produce adverse health effects,11 and taking into account the antimicrobial copper activity, the precise amount and level of copper exposure requires further studies.


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One of the most prospective ways to manipulate the copper intake for antimicrobial purposes is based on the use of extreme wettability of the touch surfaces. Extreme wetting corresponds to either the superhydrophilic or superhydrophobic state of surfaces. During recent years, just a few successful attempts to combine the superhydrophobic state of copper containing materials with toxic action of copper have been made to enhance the self-bactericidal action of surfaces.14,15 The studies of anti-bacterial properties of superhydrophilic copper containing surfaces are scarce as well.16,17 However, to differentiate the different mechanisms of a combination of the extreme wetting and the toxicity of copper, the comparison of the anti-bacterial action of superhydrophilic and superhydrophobic copper surfaces having the similar surface morphology is required. To the best of our knowledge, such type of studies was not carried out previously. This work will shed light on different aspects of survival of bacteria, contacting with either a superhydrophilic or a superhydrophobic surface of mild copper alloy M1M, fabricated using nanosecond laser texturing and characterized by similar micro- and nanomorphology. We will consider two bacterial strains which are frequently considered in the laboratory studies targeted on protection


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against nosocomial infections. Namely, we will show the bactericidal action of our surfaces with respect to a nonpathogenic strain Escherichia coli (E. coli) K12 C600 and a pathogenic strain Klebsiella pneumoniae (K. pneumoniae) 811. The impact of extreme wettability on the bactericidal behavior of fabricated surfaces will be considered for bacterial cells contacted with the textured copper surfaces according to three different protocols.

Materials and Methods Chemicals and reagents. In this study, we fabricated and analyzed the antibacterial activity of superhydrophobic and superhydrophilic coatings on the surface of M1M copper alloy with the following chemical composition (in weight %): Cu 99.9, Fe 0.005, Ni 0.002, S 0.004, As 0.002, Pb 0.005, Zn 0.004, Ag 0.003, O 0.05, Sb 0.002, Bi 0.001, Sn 0.002. The samples fabricated for the studies of bactericidal properties according to protocols 1 and 2 had the size of 10×10×1 mm3. For studies of bactericidal properties according to protocol 3, the plates with dimensions of 25×25×1 mm3 were used. The wettability studies, as well as the analysis of the surface activity of bacterial cells in


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aqueous dispersion, were performed using flat sheets of copper alloy on samples with dimensions of 15×15×1 mm3. Luria Bertani broth and Mueller-Hinton agar were purchased

from

HiMedia

Laboratories

Pvt.

Ltd.

(India).

Methoxy-{3-

[(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl)oxy]propyl}-silane was synthesized in the laboratory of Prof. A.M. Muzafarov. Preparation of superhydrophilic and superhydrophobic coatings. The hierarchical surface morphology is a prerequisite for reaching both superhydrophilic and superhydrophobic state of materials.18,19 For a superhydrophilic surface, the value of the surface energy is high and aqueous droplet completely spreads along the sample. A superhydrophobic surface is characterized by low surface energy and aqueous phase repelling. Aqueous droplet wets such surface in heterogeneous wetting regime and forms with the substrate a high contact angle exceeding 150°. In our study, an Argent-M laser system (Russia) with an IR ytterbium fiber laser (wavelength 1.064 μm) equipped with a 2-axis beam deflection unit was used for surface processing. Prior to the laser treatment, the samples were ultrasonically washed


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in deionized water and air dried. Laser treatment was performed in a pure oxygen atmosphere at a temperature of 20-25 °C. The hierarchical roughness of samples SHPhil and SHPhob (superhydrophilic and superhydrophobic, respectively), was obtained by single pass laser beam raster scanning at linear speed of 100 mm/s with parallel line pitch of 0.0025 mm, pulse duration of 200 ns, repetition rate of 20 kHz and peak power of 0.95 mJ in TEM00 mode. Laser beam diameter was focused onto a sample surface into a 40 μm wide (the 1/e2 level) focal spot with peak fluence of ≈19 J/cm2. Laser ablation regime with subsequent deposition of nanoparticles, formed in the laser plum, onto the hot treated surface, leads to the formation of microgroove relief on the copper surface with nanoparticles sintered to the microtexture. As-prepared surface just after the laser treatment was superhydrophilic, which is verified by the rapid complete spreading and wicking of a water droplet by the surface. To prepare the superhydrophobic surfaces, the lasertreated samples were further exposed in the sealed vessel to saturated vapors of methoxy-{3-[(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl)oxy]-propyl}-silane for 1 h at T=95 °C. The subsequent drying for 1 h in an oven at 130 °C resulted in the


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formation of a low surface energy cross-linked layer of hydrophobic agent and in attaining the superhydrophobic state of the laser treated surface. Bare M1M plates without laser treatment were used as reference samples. Measurement Techniques. The sample morphology was analyzed with a fieldemission scanning electron microscope (FE-SEM) SUPRA 40 VP (Zeiss, Germany). An energy-dispersive X-ray spectroscopy (EDS) facility with an INCA PentaFETx3 detector (Oxford Instruments, UK) was used to characterize the chemical composition of studied samples. EDS spectra were registered at 5 kV acceleration voltage. The cross-sections of the treated samples were used to analyze the composition of the laser treated surface layers at different depths from the surface.

Wettability of the coatings was characterized by measuring the contact and roll-off angles. Digital video image processing of sessile droplets and Laplace fit optimization for determining the droplet shape parameters were used to measure the initial advancing contact angles. The roll-off angle for the sessile droplets was defined upon smooth substrate tilting until the droplet started to roll over the surface. Both the contact


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angles and roll-off angles were measured for 15 μL droplets at least at 10 different surface locations for each sample.

The electrochemical properties of the fabricated coatings were studied using an electrochemical workstation Elins P50x+FRA 24M (Elins, Russia). Measurements were carried out at 23 °C in a three-electrode cell PAR K0235 (Princeton Applied Research, USA) with a 0.5 M NaCl aqueous solution as an electrolyte. A silver/silver chloride electrode (Ag/AgCl) filled with saturated KCl solution served as a reference electrode and a Pt mesh as a counter electrode. For the electrochemical studies, the samples were immersed in the bacterial dispersion for a certain time from 30 min to 6 days. For the measurements, the samples were taken off the dispersion and placed into the threeelectrode electrochemical cell. The measurement of polarization curves was performed after 30 min equilibration of the sample to a sodium chloride solution. The potentiodynamic polarization curves were registered at a scan rate of 1 mV/s in the applied potential range from -100 to +400 mV (with respect to open circuit potential, OCP). Corrosion potential, Ecor, and current, Icor, were derived from the potentiodynamic polarization curves after Tafel extrapolation.


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For estimation of the electrical properties of the bacterial cells, their ζ-potential in a Luria Bertani broth as a dispersion medium was studied using a Zetasizer Nano (Malvern). Protocols used to study the bactericide activity of the substrates contaminated by E.

coli and K. pneumoniae. In this study, the bactericide activity of superhydrophilic, superhydrophobic and reference copper substrates was studied with respect to two strains of bacteria. A strain E. coli K12 C600 (“GСPM-Obolensk”, B-7158, Obolensk, Russia) was used as a gram-negative nonpathogenic bacterial culture. A pathogenic strain K. pneumoniae 811 was obtained from “GСPM-Obolensk”, В-7707, Obolensk, Russia. The tested surfaces were intentionally contaminated with one or another bacteria strain, and then the change in bacterial contamination level was monitored in time. For the contamination of the test samples, freshly prepared cultures were used, obtained by 18 h incubation at 37 °C and prepared on Luria Bertani broth with opacity standard corresponding to 109 CFU/mL (hereinafter CFU stands for colony forming units). The desired titer for contamination (107 CFU/mL) was obtained by sequential tenfold dilutions with Luria Bertani broth.


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Three different protocols were used to study the bactericide activity of the fabricated substrates with extreme wettability, to evaluate the role of dry and wet contact killing1 and to estimate the impact of the ratio of contact area to contact volume of bacterial dispersion. The schematic presentation of the protocols is given in the Supporting Information Figure S1. According to the first protocol, a 0.01 mL droplet of a bacterial suspension with a titer of 107 CFU/mL of either E. coli or K. pneumoniae was gently placed on the surface of each test sample for contamination and air dried during 20 min. Microbial purity of the substrates was tested for each type of samples 30 min, 1 h, and 2 h after drying of the contaminating droplets. For that, the sample with the residue of the dried droplet of dispersion was placed in a vial containing 1 mL sterile physiological solution and shaken on a shaker for 10 min at 250 rpm. Then a 0.1 mL sample of the resulting dispersion was applied onto a Petri dish containing sterile growth medium (Mueller-Hinton agar) and spread on the agar surface with a spreader. The Petri dish was placed in a thermostat for 24 h at 37 °C, and the number of colony-forming units was determined


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after that. Separate substrates were used to repeat the experiment 2 times for each surface and each test duration. In the frames of the second protocol, the test samples were placed inside the sealed sterile Petri dishes (one for each duration of the contact with the contaminant). Then a 0.01 mL droplet of a bacterial suspension with a titer of 107 CFU/mL of either E. coli or

K. pneumoniae was gently placed on the surface of each test sample for contamination. To suppress the evaporation of the contaminating droplet, the Petri dishes contained ash-free filter paper impregnated with sterile physiological solution. The Petri dishes were then stored for a prescribed time (30 min, 1 h, 2 h, 5 h, 24 h, 2 days, 4 days and 6 days). Microbial purity of the substrates was tested for each type of samples after the abovementioned time of contact with contaminating droplets, by the following procedure. The sample with the droplet of dispersion was placed in a vial containing 1 mL sterile physiological solution and shaken on a shaker for 10 min at 250 rpm; then a 0.1 mL sample of the resulting dispersion was applied onto a Petri dish containing sterile growth medium (Mueller-Hinton agar) and spread on the agar surface with a spreader. The Petri dish was placed in a thermostat for 24 h at 37 °C, and the number


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of colony-forming units was determined after that. Separate substrates were used to repeat the experiment 2 times for each surface and each test duration. In the third protocol, 15 mL of a bacterial suspension with a titer of 107 CFU/mL of either E. coli or K. pneumoniae was poured into sterile bottles. For each contaminating culture, three bottles were prepared. Then the superhydrophobic and superhydrophilic copper substrates were immersed in bacterial suspension, and the bottles were hermetically sealed. The third bottle with dispersion not contacting with the copper sample was used as a control sample for experiments, performed according to the third protocol. The bottles were stored at room temperature. To evaluate the anti-bacterial activity of the superhydrophobic and superhydrophilic copper substrates immersed in bacterial suspension, after the prescribed time (30 min, 1 h, 2 h, 5 h, 24 h, 2 days, 4 days and 6 days) the 0.5 mL of dispersion was collected from each bottle and the titer of survived bacterial cells was detected as described above for Protocol 2.


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Results Composition, morphology and wettability of fabricated samples. The morphology of laser-textured copper surface studied by the FE-SEM method is presented in Figure 1.

Figure 1. SEM images of the laser-textured copper surface at various magnifications. Low magnification top view (a) allows discerning ridges and trenches with an average period of around 100 μm. Higher magnification top view (b) shows that ridges are composed of globulelike aggregates of nanoparticles. Side view of partially scraped globule (c) well illustrates the porous structure of globules. The important features of the obtained surface texture, which affect the bactericidal activity of samples include surface ridges and trenches with an average period of around 100 μm (Figure 1a), decorated by globule-like aggregates of 5-10 μm composed of nanoparticles with an average size of around several tens of nanometers (Figure 1b,c). The bare sample of copper (reference sample) can be classified as a hydrophilic one since its advancing contact angle for water is 83.0±1.5°. The presence of texture


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elements belonging to different length scales indicates the multimodal roughness of the textured surface and serves as a basis for obtaining both the superhydrophilic and the superhydrophobic states of the textured samples. The achievement of extreme wettability was substantiated by the wettability studies of superhydrophobic and superhydrophilic samples using water droplets as testing liquid. Complete spreading and imbibition of water inside the grooves of the laser-processed sample indicated that just after laser treatment the samples were superhydrophilic. After chemisorption of fluorooxysilane, the significant decrease in surface energy in combination with the multimodal roughness resulted in establishing the heterogeneous wetting regime with water contact angles higher than 171° and roll-off angles less than 1.1°. To estimate the degradation of the coatings wettability under contact with the

E. coli bacterial dispersion in Luria Bertani broth with a titer of 107 CFU/ mL, we have measured the water contact angles on both superhydrophobic and superhydrophilic samples after 24 h and 6 days of immersion. It was found that initially superhydrophilic sample preserved its superhydrophilic state during the whole experiment and showed the water spreading. The superhydrophobic sample kept the superhydrophobicity after


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24 h of immersion, with a contact angle of 170.1±1.5° and roll-off angle of 4.7±1.8°. However, after 6 days of contact with bacterial dispersion, the water droplets ceased to roll-off the surface, although the contact angle for water droplet remained high enough (150±6°). Such degradation of the superhydrophobic state of fabricated coatings can be considered as an indication of the corrosion process, and this point will be discussed in more detail below. For further analysis and interpretation of data related to the bactericidal action of surfaces with extreme wettability, we would like to briefly recall the main peculiarities of the composition of laser fabricated textures on M1M alloy revealed in our recent study.20 X-ray diffraction studies indicated that after the laser treatment the surface is enriched with copper oxides. Both cupric and cuprous oxides are present on the surface, and the ratio of the oxides extracted from the diffraction pattern gives 71% Cu2O and 29% CuO for our samples. The detailed analysis of copper oxides distribution across the textured layer indicated that the cuprous oxide (Cu2O) is mainly occupying the inner layer, while cupric oxide (CuO) is mainly presented on the surface as the nanoparticles formed on the outer layer. The thickness of porous cupric oxide layer varied in the range of 1 to 10


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μm. Thus, the top functional layer of our coatings is constituted by cupric oxide nanoparticles whose bactericidal activity was confirmed in recent studies.21–23 For the superhydrophobic samples, the very top layer of nanoparticles is additionally covered by chemisorbed fluorooxysilanes. Antibacterial activity of copper alloy surfaces with extreme wettability. It was discussed in the literature1,2,5,21–25 that copper alloys, as well as copper and copper oxide nanoparticles, show the antibacterial activity against multidrug-resistant Gramnegative pathogens responsible for nosocomial infections, such as E. coli, K.

pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. The antibacterial effect increases with copper content in the substrate5 and with the area of contact between the copper sample and the bacterial medium. Thus, it is expected that the superhydrophilic substrates like that fabricated in our study will be very effective as an anti-bacterial tool due to developed high surface area. Besides, the higher is the interfacial area between the copper substrate and the liquid bacterial medium, the more intensively the corrosion processes develop. The latter due to the high concentration of copper ions in the dispersion medium facilitate the bacterial killing, whereas prolonged


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bacteria survival was shown for corrosion resistant copper substrates.24 Finally, the toxic action of nanotexture decorating the microrelief of a superhydrophilic surface contributes to bacterial killing efficiency as well.26–28 As for the superhydrophobic substrates, two important anti-bacterial mechanisms were mentioned in the literature in addition to the well documented cytotoxic action of surface texture and the copper ions present in the liquid medium. The first one is related to water repellence, which drastically reduces the contact area between the substrate and the bacterial dispersion.29–31 The second mechanism follows from the significant decrease in adhesion force between the bacterial cell and the superhydrophobic surface, as long as the liquid interlayer is present in between.32 The analysis of the titer for survived bacteria for both E. coli and K. pneumoniae cells after contaminating the bare alloy plate, superhydrophilic and superhydrophobic substrates according to the first protocol indicates the bacterial purity already 30 min after droplets of bacterial dispersions were dried. The increase in time of contact between the bacterial cells deposited onto the used three substrates upon drying to 2 h did not cause an increase in the number of survived bacterial cells atop of substrates.


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These data indicate that in conditions of “dry” contact method, all studied substrates demonstrate complete bacterial inactivation for two studied bacterial strains and the resistance to copper is not developed. Now let us consider the surviving of bacterial cells on our substrates in the experiments performed according to the second protocol, when the droplet with bacterial dispersion contacted to the studied surface in the conditions of saturated water vapor pressure for a prescribed time (30 min, 1 h, 2 h, 5 h, 24 h, 2 days, 4 days and 6 days). In these conditions, the evaporation is suppressed, and the droplet keeps constant volume during the time of contact with the substrate. In this type of experiment, the titer of survived bacterial cells, which were either sedimented to sample surface or remained dispersed in a sessile liquid droplet, was defined. The analysis of a number of bacterial cells inside the droplets deposited onto the bare copper and the superhydrophilic substrate, after 30 min of contact with the dispersion revealed the absence of survived cells for both E. coli and K. pneumoniae, similar to the result obtained in the first protocol experiments. Further increase in the contact time of the droplets with the substrates up to 6 days did not lead to the appearance of bacterial


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contamination. However, the surviving of bacterial cells on the superhydrophobic copper substrate was notably different. To better understand the reason for such difference, we have compared the morphology of bacterial cells precipitated onto the copper substrates with different wettability after 24 h of contact between the droplet of dispersion and the substrate (Figure 2).

Figure 2. SEM images of E. coli bacterial cells precipitated onto the bare copper (a), and onto textured superhydrophilic (b) and superhydrophobic (c) copper substrates after 24 h of contact between the droplet of dispersion and the substrate. False coloration was applied to images to highlight the bacteria. SEM analysis shows nearly uniform distribution of bacterial cells along the bare copper and the superhydrophilic copper substrates. The darker background under the adhered bacterial cells indicating partial release of cytosol, followed by some flattening of E. coli cell shape, was detected by the electron microscopy for the bare copper


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substrates (Figure 2a). The image of bacterial cell atop of superhydrophilic substrate (Figure 2b) shows that the cell adhered to the surface with hierarchical roughness underwent membrane piercing, deformation, the loss of intracellular fluid, and bacteriolysis. Although the partial destruction of cellular structures was detected for the superhydrophobic surfaces as well, the degradation of a shape of bacterial cells deposited onto this surface was less (Figure 2c). Besides, for the superhydrophobic samples very distinct distribution of bacterial cells was observed compared to the superhydrophilic and the reference ones. For the superhydrophobic sample, the bacteria mainly occupy the tops of ridges (Figure 3 a,b). This is explained by heterogeneous wetting mode of the superhydrophobic surface when the grooves capture the air and do not contact with the liquid medium.


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Figure 3. Various magnification SEM images of E. coli (a, c) and K. Pneumoniae (b, d) bacterial cells precipitated onto the textured superhydrophobic copper substrates after 24 h (a,b) and 48 h (c,d) of contact between the droplet of dispersion and the substrate. Survey images (a, b) evidence that the bacteria mainly sedimented on top of ridges (marked with circles). Higher magnification images (c, d) illustrate cell damage on the elements of the nanotexture. False coloration was applied to images (c) and (d) to highlight the bacteria. To quantitatively characterize the anti-bacterial activity of the superhydrophobic surfaces, we have analyzed the ratio of a titer for bacterial cells dispersed in a droplet at a prescribed time to the titer in a droplet before its deposition onto the superhydrophobic substrate. For E. coli K12 C600 cells, this ratio versus time is shown in Figure 4 in loglog coordinates by blue circles. Pink crosses are related to the corresponding ratio for K.

pneumoniae 811. For both strains, the number of survived cells at short contact time is


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nearly two orders of magnitude lower than that in the initial dispersion, indicating some bactericidal action of a superhydrophobic copper substrate. However, after 2 h of contact the opposite trend is developing. After 300 min of contact, the titer of K.

pneumoniae became two orders of magnitude larger, whereas for E. coli it only approaches the value characteristic of the droplet before its deposition onto the superhydrophobic substrate. After 2 days of contact, the stabilization of titer was observed for both bacterial strains. Besides, the absence of blue coloration of a droplet on a superhydrophobic substrate, which would be indicative of the enriching of dispersion medium with aqueous complexes of Cu2+ ions, allows concluding low copper ions concentration in dispersion and thus very high resistance of our superhydrophobic coating to corrosion processes and copper dissolution. The joint analysis of the evolution of the titer of survived bacteria and the corrosion processes allows concluding the development of resistance of bacterial cells dispersed in the broth to copper.


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1000 K. pneumoniae 100

Normalized bacteria titer

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10 E. coli 1

0.1

0.01

0.001

0.0001 10

100

Time, min

1000

10000

Figure 4. Variation of the titer of E. coli (blue circles) and K. Pneumoniae (pink crosses) in a droplet of bacterial dispersion with a time of contact between the droplet and the superhydrophobic copper substrate. The titer was normalized to the initial titer in the droplet before contact with the substrate. To understand the above phenomena in more detail, we have analyzed the titers of bacterial cells in dispersion medium according to the third protocol of studies. The data on titer of bacterial cells in the dispersion contacted with a substrate for prescribed times, normalized on the titer in the initial dispersion before bringing it into contact with large area superhydrophobic (red crosses) or superhydrophilic (blue circles) copper substrates are presented in Figure 5a for E. coli and in Figure 5b for K. pneumoniae. For the comparison, we present the normalized titer of the bacterial cells in the


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dispersion medium aged without contact with copper substrates (shown in Figure 5 by black diamonds).

Figure 5. Variation of the titer of E. coli (a) and K. Pneumoniae (b) in a vessel with bacterial dispersion with a time of contact between the dispersion and superhydrophilic (blue circles) or superhydrophobic (red crosses) copper substrates immersed in the dispersion. The reference line (black diamonds) represent the corresponding variation in the control vessel with bacterial dispersion without any substrate immersed in. The titer was normalized to the initial titer in the dispersion. The arrows near corresponding curves show the time of the beginning of blue coloration of a bacterial dispersion. As can be seen from the data obtained in the presence of a superhydrophilic copper substrates, the titer gradually decreases,


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correlating with an increase in the intensity of the blue color of the dispersion. For the E.

coli dispersion, the titer reaches zero value after 5 h of contact, while for the K. pneumoniae dispersion the zero titer was detected after 24 h. In the presence of a superhydrophobic copper substrate, the titer initially increased indicating the notable enhancement of bacterial contamination. Since the superhydrophobic coating was aimed to protect the metal substrate against its interaction with components of the dispersion and to enhance the resistance to corrosion, such coating leads to suppression of copper transition from the copper substrate to ionic form in the dispersion medium, thus minimizing the release of copper ions into the liquid medium. Also, the primary adhesion of bacterial cells to the superhydrophobic substrate is weakened due to surface forces action, which will be discussed below. The growth of titer was observed until an intensive corrosion process begins. Such process is accompanied by an increase in copper ions concentration in the dispersion and can be easily detected by dispersion coloration. After 2 days of contact of the superhydrophobic substrate with the bulk E. coli dispersion, and after 1 day of contact with bulk K.

pneumoniae dispersion, the number of living bacterial cells starts to decrease reaching


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the zero value for E. coli after 6 days of exposure to the superhydrophobic copper substrate. The resistance of K. pneumoniae cells to contact with copper ions is a bit higher. However, after 6 days the living cells titer becomes of the order of 1 percent of initial dispersion titer. We have studied the impact of corrosion processes on the morphology of the cells precipitated onto the superhydrophobic surface. SEM images of bacterial cells on superhydrophobic copper surfaces after 48 h of contact between the surface and bacterial dispersion show that for both E. coli cells (Figure 3c) and K.

pneumoniae cells (Figure 3d), the loss of cell integrity is observed, which results from the piercing of the cellular membrane on the elements of the nanotexture, cell stretching, and deformation. As for the evolution of bacterial titer upon dispersion aging in the control samples not contacting the copper, it follows from Figure 5a,b (black diamonds) that titer of both strains gradually increases until reaching the stationary value after 2 days of storage. Since the state of the superhydrophobic copper substrate plays a key role both for primary adhesion of bacterial cells to the substrate and for the concentration of copper ions in the dispersion, we have studied the electrochemical parameters and the


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corrosion resistance of the superhydrophobic samples in contact with the bacterial dispersion. Electrochemical behavior of fabricated superhydrophobic surfaces in contact with bulk dispersion of E. coli. In general, copper is characterized by appropriate stability against corrosion in aqueous environments due to the formation of a protective oxide film. However, in biological liquids, the pitting corrosion may occur on the copper surface in the presence of oxygen, some aggressive anions such as chloride ions, or some other species, appearing due to cell metabolism.33–36 Using the measurement of potentiodynamic polarization curves, we have measured the corrosion current for bare and textured copper samples immersed in bacterial dispersion of E. coli for a certain time. If the electrochemical and biochemical corrosion is taking place upon interaction of a copper surface with the bacterial dispersion, it will, as a rule, result in a deterioration of the barrier properties of samples and an increase in corrosion current. The polarization curves detected for bare, superhydrophilic and superhydrophobic samples immersed in the bacterial dispersion for 30 min, are shown in Figure 6a. The corrosion currents, Icor, derived from the potentiodynamic polarization


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curves after Tafel extrapolation, are as follows: 1.4×10-6 A for the bare sample; 1.5×10-5 A for the superhydrophilic and 5.5×10-9 A for the superhydrophobic sample. Thus, due to very developed surface area, the total current for the superhydrophilic sample appears to be one order of magnitude higher than that for the bare one, indicating the intense interaction of the sample with the dispersion medium and its rapid enriching with cupric ions. In contrast, for the superhydrophobic sample after 30 min of contact with the bacterial dispersion, the total current is nearly three orders of magnitude lower. As documented in the literature, the resistance of a superhydrophobic surface to corrosion, depending on the particular properties of the coating, might vary with time of contact with the corrosive medium.32,35 Therefore, we have measured the corrosion current for samples contacted with the bacterial dispersion for different prescribed times (30 min, 1 h, 2 h, 5 h, 24 h, 2 days, 4 days and 6 days) (Figure 6b). For the comparison, the value of corrosion current for the sample which was not in contact with the bacterial dispersion is shown by straight dashed line in Figure 6b. The evolution of the corrosion current versus time of contact with corrosive dispersion evidences the inhibition of physico-


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chemical processes at the interface, including charge and ions transfer. However, after two days of contact, the corrosion current exceeds the value of 2×10-8 A. 1E-7

b

Icor, A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1E-8

1E-9 10

100

1000

10000

Time, min

Figure 6. (a) The linear polarization curves for superhydrophobic (red), superhydrophilic (blue) and reference (black) copper substrates registered after 30 min of substrate contact with the bacterial dispersion of E. coli with a titer of 107 CFU/mL. (b) The dependence of corrosion current for the superhydrophobic copper substrate on time of contact with the E. coli dispersion. The values of corrosion current were determined by Tafel method for polarization curves registered in 0.5 M NaCl aqueous solution as an electrolyte. Dashed horizontal line corresponds to the value of corrosion current for the superhydrophobic copper sample which was not in contact with the bacterial dispersion.

Discussion


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Our experiments elucidated very drastic variation in behavior of the bacterial cell on copper surfaces with different surface treatment and at different experimental conditions. Thus, according to the first protocol of studies, corresponding to the socalled “dry” method1 of bacteria elimination, the 10 μL droplet of bacterial dispersion was rapidly evaporated from the substrate. Complete bacterial inactivation was characteristic of both studied bacterial strains, independently of the copper wettability, due to direct contact of bacterial cells with either flat metal surface or the cupric nanoparticles. These results are in a good agreement with the literature data on contact killing mechanism of dry copper and copper oxides,1,2,5,21-23 although some authors noted that CuO significantly inhibits contact killing, compared to pure copper.37 The mechanisms of contact killing were already discussed in the literature1,2 and include the cell damage associated with the capture of copper ions by the bacterial cell, the cell membrane deformation and piercing by the surface, leading to the loss of membrane integrity, the generation of reactive oxygen species, and finally, the genomic and plasmid DNA degradation induced by copper ions. And the key factor affecting the


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complete bacterial cells inactivation on all studied surfaces is related to direct contact between the cell and the metal or metal oxide surface. However, in the conditions of wet contact, which were realized in experiments according to the second and third protocols, the survival of the cells becomes sensitive to the wettability, which is a determining factor as well. It was discussed in our recent study,32 that the wettability of the surface affects three important features of the contact between the surface and the dispersion medium, containing the bacterial cells. The first specific feature is the area of contact between the surface and the dispersion. For nontreated bare copper, the droplet occupies although a limited but fairly large contact area, which is defined by the contact angle (=83.0±1.5°, see above). The distribution of bacterial cells, precipitated from dispersion under gravity, across this area allows for direct contact of cells with the copper surface. For the superhydrophilic surfaces, the complete spreading of a droplet of bacterial dispersion results in even distribution across the whole substrate with establishing of the direct contact between the individual bacterial cells and the texture elements on the surface. In contrast, for the superhydrophobic surface, the very limited area is wetted, and only tops of ridges


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contact the dispersion, and thus, the precipitation of bacterial cells leads to deposition of both individual cells and bacterial aggregates on tops of texture (Figure 3a,b). However, in that case, the cells establish the contact with the layer of long-chained hydrophobic agent – perfluorooxysilane, not with copper or copper oxide. The second peculiarity is the copper dissolution in the dispersion medium due to corrosion processes at the dispersion/copper interface. Bare copper is prone to both bio- and electrochemical corrosion. Relatively large contact area facilitates the enriching of dispersion with copper ions, contributing to bacterial cells contact killing. The superhydrophilic textured surface is characterized by the contact area with the bacterial medium, significantly higher than that for the bare copper substrate, which results in intensification of copper ions release into the dispersion medium. However, the thick surface textured layer of cupric oxide acts as a barrier to corrosion processes. Overall, as it was shown above, both the electrochemical data and the blue coloration of the droplet or bulk dispersion medium contacting the superhydrophilic surface during half an hour indicate the intensification of corrosion processes on the superhydrophilic surface in comparison to the bare one. This intensification enhances the contribution of


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bactericidal mechanism related to copper ion capture by the bacterial cells. For the superhydrophobic surface, as it follows from the presented electrochemical data, the corrosion is significantly suppressed leading to the inhibition of the copper ions release into the dispersion. The mechanisms of corrosion resistance of superhydrophobic coatings were discussed in detail recently.20,38 The electrochemical data presented above indicate a weak degradation of the barrier properties of the superhydrophobic copper surface to the transition of a charge and ions through the surface layer. At the same time, the electrochemical reactions accompanied by the transition of copper from the substrate to ionic form in the dispersion medium are evidenced by the value and the evolution of a corrosion current with a time of contact between the sample and the bacterial dispersion (Figure 6). However, the titer of survived cells inside the dispersion contacting with a superhydrophobic copper surface remains high indicating the development of the bacterial resistance to small concentrations of copper ions. And finally, the third specific feature deserving particular consideration is the primary adhesion of bacterial cells to the copper surface under the surface forces action.32,39 The most important types of surface forces are the van der Waals, electrostatic,


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structural and steric forces.40 The contribution of surface forces of different types is determined, in essence, by the structure of an outer membrane of gram-negative bacteria and the composition of the surface of the copper substrate. Lipopolysaccharide of the outer membrane, which contains glucosamine disaccharide acyl chains and a polysaccharide core, and an extended polysaccharide chain (the O-antigen)41 play a crucial role in the induction of steric interactions with the textured copper oxide layer, covered by chemisorbed long-chained fluorooxysilanes. For the analysis of the strength of primary adhesion, the concurrence of surface forces in the films of the order of a few water monolayers is important. For the Luria Bertani broth, the range of thicknesses of 2-4 water molecular diameters appears to be equal to or less than the inverse Debye length, indicating the very high contribution of electrostatic interactions to the primary adhesion of a cell to the surface. In contrast, the van der Waals interactions across the liquid film are expected to be weak. The main reasons for that are: the significant screening of the zero-frequency contribution into the energy of van der Waals interactions due to the presence of NaCl in broth, and the proximity of the dielectric properties of a bacterial cell and the broth film separating the cell and the sample surface. The steric interactions and structural forces for bare copper and the superhydrophilic copper/copper oxide substrates contacting the broth are weak.


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The ξ-potential of E. coli in 1 w% NaCl solution is around -20 mV.42 According to our measurements, the ξ-potential of K. pneumonia cell in Luria Bertani broth is equal to – 27 mV. The bare copper substrate or the superhydrophilic surface with oxide top layer are both positively charged in neutral solutions.43 Thus, the main physical force determining the primary adhesion of negatively charged bacterial cells to the positively charged substrates is the electrostatic attraction. For the superhydrophobic surface, the balance of forces is determined by the van der Waals, electrostatic, and steric forces. Since the differences in dielectric permittivities for broth interlayer/fluorooxysilane layer and the broth interlayer/lipopolysaccharide layer of the outer membrane of bacteria have the same sign, the van der Waals forces are attractive for both studied bacteria. Besides, due to the low dielectric contrast in the layered system under consideration, the van der Waals interaction is weak. The electrostatic interaction between negatively charged hydrophobized copper surface44–46 and the negatively charged bacterial cell is repulsive. Finally, the repulsive steric forces origin from the interaction between the well-ordered fluorooxysilane layer chemisorbed onto the superhydrophobic surface and the lipopolysaccharide layer.


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Thus, for a few nanometers thick broth interlayer between the cell and the substrate, the net surface forces should be repulsive, mainly defined by the electrostatic and steric repulsion. Such repulsion impedes the primary adhesion of bacterial cells to the superhydrophobic surface. However, the hydrolysis of fluorooxysilane molecules initiated by the presence of broth components and by the cell metabolism results in a desorption of the fluorooxysilane molecules from the surface and the development of the corrosion processes. In turn, the surface charge locally turns to a positive value, and the steric interaction weakens, overall allowing the direct contact between the cell and the surface texture. Thus, bacteria that are deposited on the surface, over time are subjected to direct contact with the surface, triggering the cell structure damage. One additional point worth brief discussing here is related to the development of bacterial resistance to copper. In the literature, several mechanisms were reported,47–53 such as relative impermeability of the outer and inner bacterial membranes to copper ions, intra- and extracellular sequestration of copper ions, based on scavenging of copper by specific cell components and proteins in the cytoplasm, periplasm and Slayers of outer membrane, and active extrusion of copper from the cell. Our experiments


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indicated the revealing of such resistance only for the case of bacterial dispersions contacted with the superhydrophobic copper substrate when the concentration of copper ions in the dispersion medium was low. The variation of the titer of survived bacteria in time of contact of the dispersion or the droplet containing K. pneumoniae cells with the superhydrophobic copper sample (Figure 4 and 5b) is well explained by the extracellular sequestration of copper ions, as a chief mechanism of bacterial tolerance. Exopolysaccharides bind copper ions by electrostatic forces and keep them trapped outside the cell.50,53 As long as the concentration of ions in the dispersion is small enough to be trapped by exopolysaccharides, both E. coli and K. pneumoniae cells dispersed in the broth survive, whereas the presence of high concentration of copper resulted in the notable decrease in a number of survived cells (Figure 5b). However, the inactivation of bacterial cells takes hours and days indicating the contribution of some other protection mechanisms against copper toxicity.

Conclusions


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A method based on nanosecond laser processing has been developed for designing superhydrophobic and superhydrophilic copper alloy substrates for bactericidal applications. Using Escherichia coli and Klebsiella pneumoniae as model bacterial cells, we studied the evolution of living cell concentration in dispersions contacted with the copper substrates with different wettability. Three different protocols used in our study allowed discriminating the role of the “dry” killing, the damage of cells directly contacted with the copper substrate in the presence of Luria Bertani broth, and the cell inactivation caused by copper ions in the dispersion. It was found that although the physical forces defining the primary adhesion are different for superhydrophilic and superhydrophobic substrates, the bacterial cells directly contacted with the hierarchically roughened substrates are subjected to piercing, deformation, and damage of the cellular membranes, leading to cell death. The balance of surface forces between the cell and the surface for the superhydrophilic surfaces results in the attractive total force, which promotes rapid direct contact, followed by cell damage. For the superhydrophobic surface, the surface forces acting in liquid interlayer which separates the cell and the surface, are initially repulsive. However, the processes such as perfluorooxysilane


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desorption, electrochemical and biological corrosion, trigger the inversion of primary adhesion from repulsion to attraction. The latter is easily detected by the variation in deposited cell morphology. The bactericidal action of the copper surfaces significantly varies with the variation of substrate wettability. The superhydrophilic surface due to very large contact area with the bacterial dispersion, accompanied by intense corrosion processes and the enrichment of dispersion medium with the copper ions, is characterized by the highest bactericidal effect. In contrast, the superhydrophobic surface demonstrates improved corrosion resistance. As long as the corrosion is not developed, the copper ion concentration in bacterial dispersion remains low, the primary adhesion of cells to the surface keeps weak, thus allowing enhancement in bacterial contamination. However, gradual transition from the superhydrophobic state of copper surface to the hydrophilic one triggers the scenario of cell killing mechanism, similar to that described for the superhydrophilic surfaces. AUTHOR INFORMATION

Corresponding Author


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*E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS The work was supported by the Russian Foundation for Basic Research (grants 17-0300142, 18-29-05008) and by the Program for fundamental studies P32 “Nanostructures: Physics, Chemistry, Biology, and Bases for Engineering” of the Presidium of the Russian Academy of Sciences.

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For Table of Contents Use Only

Bactericidal Activity of Superhydrophobic and Superhydrophilic Copper in Bacterial Dispersions Ludmila B. Boinovich,1,* Valery V. Kaminsky,2 Alexandr G. Domantovsky,1 Kirill A. Emelyanenko,1 Andrey V. Aleshkin,2 Eldar R. Zulkarneev,2 Irina A. Kiseleva,2 Alexandre M. Emelyanenko1


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Bactericidal Activity of Superhydrophobic and ... - ACS Publications

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