Article Cite This: ACS Appl. Nano Mater. 2018, 1, 1348−1359
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Effective Antibacterial Nanotextured Surfaces Based on Extreme Wettability and Bacteriophage Seeding Ludmila B. Boinovich,*,† Evgeny B. Modin,‡,§ Andrey V. Aleshkin,∥ Kirill A. Emelyanenko,† Eldar R. Zulkarneev,∥ Irina A. Kiseleva,∥ Alexander L. Vasiliev,‡ and Alexandre M. Emelyanenko† †
A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Leninsky prospect 31 building 4, 119071 Moscow, Russia ‡ National Research Centre “Kurchatov Institute”, Pl. Akad. Kurchatova 1, 123182 Moscow, Russia § Far Eastern Federal University, 8 Suhanova Street, 690090 Vladivostok, Russia ∥ G. N. Gabrichevsky Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov Street, 125212, Moscow, Russia S Supporting Information *
ABSTRACT: A method based on nanosecond laser treatment was used to design superhydrophobic and superhydrophilic aluminum alloy substrates showing enhanced cytotoxic activity with respect to Escherichia coli K12 C600 strain. It was shown that the survival of cells adhered to the superhydrophobic substrates was significantly affected by the presence of organic contaminants, which are ubiquitous in hospital practice and the food industry. The peculiarities of the texture also played a notable role in antibactericidal activity. It was found that the superhydrophilic surfaces had much higher toxicity than the superhydrophobic ones, which was explained by the mechanisms of adhesion of cells to the surface. Scanning electron microscopy and tomographic reconstruction of the adhered cells were used to study the variation of cell morphology after attachment to surfaces with different wettability. It was shown that the cytotoxicity of superhydrophobic surfaces could be significantly enhanced by using the combined antimicrobial action of bacteriophages and the superhydrophobicity of the objects. KEYWORDS: superhydrophobic surfaces, superhydrophilic surfaces, cell adhesion mechanism, cytotoxicity, antibacterial activity, deposited bacteria morphology
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INTRODUCTION Escherichia coli is a Gram-negative bacterium that is widely studied in the literature. This interest is motivated by the highly pathogenic character of certain strains of E. coli. Numerous recent reviews1−5 consider E. coli to be one of the most frequent causes of certain bacterial infections in humans and animals. For example, E. coli is associated with urinary tract infections and neonatal sepsis, enteritis, hemorrhagic colitis, septicaemia, and other clinical infections. Several strains, such as E. coli O157:H7 and O104:H4, are important foodborne pathogens,6,7 while others are mainly responsible for morbidity and mortality in hospitals through various medical deviceassociated infections from urethral and intravascular catheters, prosthetic joints and shunts, and prosthetic grafts.8,9 The danger of emergence of antimicrobial resistance complicates the therapeutic treatment of E. coli infections. In addition, E. colicaused infections are difficult to eradicate due to the formation of “biofilms”.10 The spread of multidrug-resistant E. coli strains is increasing worldwide and can be considered an increasing global public health concern. The restricted potential of modern medicine to effectively combat infections caused by E. coli strains was clearly shown during major outbreaks in Germany in 2011 and in Russia in 2013.11 Thus, the search for © 2018 American Chemical Society
new breakthrough approaches to treating infections is one of the main challenges facing the scientific community. Phage therapy is an alternative antimicrobial approach to antibiotics. Phages have many advantages, such as higher specificity for the intended bacteria and the ability to infect only one species, serotype, or strain without disturbing the normal bacterial flora in humans and animals. Lytic phages, which have been reassessed for their ability to prevent and treat bacterial infections, are currently receiving increased attention. The potential of phage therapy to reduce the distribution of E. coli infection and improve the antimicrobial effectiveness of phages for elimination of E. coli pathogens in vivo in humans and animals has recently been discussed in several reports.12−16 At the same time, there are still some disadvantages delaying the broad application of phages in clinical practice. The development of bacterial resistance to phages is among the most important. Another prospective antimicrobial approach is based on the use of extremal wetting of the surface of objects that may be in Received: January 17, 2018 Accepted: February 26, 2018 Published: February 26, 2018 1348
DOI: 10.1021/acsanm.8b00090 ACS Appl. Nano Mater. 2018, 1, 1348−1359
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ACS Applied Nano Materials
Figure 1. Scheme of treatment procedures used for sample preparation.
nants, which are ubiquitous in hospital practice and the food industry. We will analyze the cytotoxic action of the superhydrophilic and superhydrophobic substrates with respect to E. coli dispersed in a model hexane/physiological solution emulsion. We will also show that the cytotoxicity of superhydrophobic surfaces can be significantly enhanced by using the combined antimicrobial action of bacteriophages and the superhydrophobicity of objects that may come in contact with infected patients.
contact with infected patients and contribute to transmission of infection, such as hospital equipment and infrastructure, as well as various medical devices. Extremal wetting corresponds to either the superhydrophilic or superhydrophobic state of surfaces.17,18 Obtaining both superhydrophilic and superhydrophobic state of materials requires fabrication of hierarchical surface morphology.17,19 The main difference between the two wetting states is the value of the surface energy, which is high for the former surface and low for the latter. However, the mechanisms of antibacterial action for these two types of surfaces are very different. For superhydrophobic surfaces, the antibacterial effect resulting in the inhibition of bacterial cell colonization is provided by low cell adhesion to the superhydrophobic surface and by the reduced effective area for cell binding to the surface due to capture of air inside surface grooves.20−23 Another mechanism of antibacterial action of nanotextured superhydrophobic surfaces mentioned in the literature is related to piercing of the cellular membrane on nanotextured elements.24,25 However, note that surfaces with large contact angles do not always inhibit bacterial colonization, but may instead promote it.26,27 A detailed analysis of bacterial behavior on superhydrophobic surfaces is given in recent reviews.20,25,28 The antibacterial effect of superhydrophilic metallic or ceramic materials, where the surface texture of such materials is composed of micro- and nanoparticles, is based on oxidative stress-related cytotoxicity. 29−34 Recent reviews in the field25,28−32,35−37 have convincingly showed the antibacterial effect of metal and metal oxide micro- and nanoparticles. Since the nanoparticles themselves do not provide sharp selectivity of cytotoxic activity only to the desired bacteria strain, the application of nanoparticles in dispersions may cause wide dissemination of nanoparticles and damage to the normal bacterial flora. Using superhydrophilic substrates with texturizing nanoparticles that are rigidly bound to the substrate severely limits the overall toxicity of nanoparticles for the human body, which is an undisputed advantage. However, as stressed in the literature, the bactericidal efficiency of both superhydrophilic and superhydrophobic nanostructured surfaces depends on texture parameters, such as the dimensions and shape of texture elements, and on the type and size of bacteria.25 In this paper, we will describe a method for designing superhydrophobic and superhydrophilic aluminum alloy substrates for antibacterial applications. We will show that the survival of E. coli cells adhered to a superhydrophobic substrate is significantly affected by the presence of organic contami-
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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 AMG aluminum alloy with the following chemical composition (in weight %): Al 95.55, Mg 2.9, Mn 0.2, Cr 0.05, Cu 0.1, Fe 0.4, Si 0.4, Ti 0.1, Zn 0.2, and impurities 0.1. Flat sheets of AMG alloy on samples with dimensions 15 × 15 × 1 mm3 were used to perform the wettability studies, as well as the analysis of surface activity of E. coli and bacteriophage in aqueous dispersion and mixed aqueous-hexane dispersions. 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.38 Hexane (for high-performance liquid chromatography (HPLC), ≥97.0%) was purchased from Sigma-Aldrich. Preparation of Superhydrophilic and Superhydrophobic Coatings. As mentioned above, surface texturing leading to hierarchical (multimodal) surface morphology is a requisite stage for obtaining both the superhydrophobic and superhydrophilic state of the metallic surface. In our study, we used an Argent-M laser system (Russia) with an IR ytterbium fiber laser (wavelength 1.064 μm) equipped with a two-axis beam deflection unit for surface texturing. Prior to the laser treatment, the samples were degreased in a 1 M KOH solution, ultrasonically washed in deionized water, and air-dried. Laser treatment was performed in open atmosphere, at ambient conditions, with humidity of 40−50% and temperature of 20−25 °C. The treatment procedures used to fabricate superhydrophobic and superhydrophilic samples for studying antibacterial activity are sketched in Figure 1 and summarized in the Supporting Information (Table S1). Two types of textures with different laser treatment regimes were produced. The first type of texture (T1), characteristic of samples T1Phil and T1Phob (superhydrophilic and superhydrophobic, respectively), was obtained by onefold laser beam raster scanning at linear speed of 50 mm/s with parallel line pitch of 0.025 mm, pulse duration of 50 ns, repetition rate of 20 kHz, and peak power of 0.95 mJ in TEM00 mode. The second type of texture (T2), characteristic of samples T2Phil and T2Phob, was obtained by 10-fold laser beam raster scanning at linear speed of 50 mm/s with parallel line pitch of 0.1 mm, pulse duration of 50 ns, repetition rate of 20 kHz, and peak power of 0.95 mJ in TEM00 mode. 1349
DOI: 10.1021/acsanm.8b00090 ACS Appl. Nano Mater. 2018, 1, 1348−1359
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ACS Applied Nano Materials In both of these laser treatment regimes, the laser beam was focused into a 40 μm wide (the 1/e2 level) focal spot with peak fluence of ∼19 J/cm2 onto the sample surface. After laser treatment by either of the regimes, the metal surface became superhydrophilic, with quick complete spreading of an aqueous droplet touching the surface. To fabricate superhydrophobic surfaces on both types of laser-textured samples, their surface energy must be decreased.17,19 We used c h e m i s o r b e d m e t h o x y - { 3 - [ ( 2, 2 , 3, 3 , 4 , 4 , 5 ,5 , 6 , 6 , 7 , 7 , 8 , 8, 8 pentadecafluorooctyl)oxy]propyl}-silane for this purpose. According to the literature,39 long-chain fluorinated compounds have the lowest solid/air surface energy values of all known organic substances. To enhance the chemisorption of fluorooxysilane onto the textured aluminum surface, prior to fluorooxysilane deposition, the samples were exposed to UV−ozone treatment (Bioforce Laboratories) for 90 min, which resulted in grafting of surface hydroxyl groups serving as chemically active centers. Bare AMG plates without laser treatment were used as reference samples. Preparation of Escherichia Coli and Bacteriophage. In this study, we used two types of dispersion medium to prepare dispersions of both the bacteria and the bacteriophage. The first dispersion medium, commonly used in physical chemistry and biology, was a physiological solution (0.9% NaCl aqueous solution, pH of 7.4). The second was an emulsion of hexane in a physiological solution, which was obtained by stirring a 1:1 mixture of a physiological solution and n-hexane for 30 min at 170 rpm. This dispersion medium was used to mimic the influence of organic contaminants on the wettability and bactericidal effect of superhydrophobic surfaces. The source of such organic contaminants may be atmospheric pollutants, fats that contaminate the surface upon contact with the skin of an animal or human, etc. We used the latter composite dispersion medium to analyze the antibacterial activity of all studied substrates, to make more reliable comparison of the bactericidal activity of different superhydrophobic and superhydrophilic surfaces in conditions close to those in practical applications. Thermodynamic instability leading to quick emulsion coarsening and complete phase separation into oil and aqueous phases is an important feature of hexane−aqueous salt solution emulsions. Nevertheless, the aqueous phase after separation is saturated with hexane and therefore wets the superhydrophobic surface better than the parent physiological solution. This property makes it possible to deposit more phage/bacteria on the surface and mimics the influence of contaminants on surface wettability in natural conditions. Because of the low solubility of molecular hexane in water and high hexane volatility, this choice of model organic component results in quick spontaneous removal of hexane from the aqueous phase. Thus, the effect of an organic additive on the properties of the system being considered is restricted by the wetting stage and the stage of short-term contact between the liquid and the substrate. In our experiments, we used a nonpathogenic strain E. coli K12 C600 (“GCPM-Obolensk”, B-7158) and the original virulent polyvalent bacteriophage E. coli BPhEc1 (EcD7) (“DSMZ”, DSM 28572). The bacteriophage host range included shiga toxin-producing strains of E. coli O104:H4 and O157:H7, as well as the clinically relevant E. coli strains of other serogroups, such as O121, O145, and O103. A highly active sterile phage lysate purified from toxins was produced using our pilot technology40 with E. coli K12 C600 as a host strain for the bacteriophage EcD7. To evaluate the antibacterial activity of the superhydrophobic, superhydrophilic, and reference aluminum substrates, all surfaces were contaminated with a drop of E. coli K12 C600 dispersed at a titer of 1 × 107 CFU/mL (CFU = colony-forming units) in emulsion of hexane/sterile physiological solution prepared as described above. The desired titer was obtained by sequential 10-fold dilutions with physiological solution of fresh overnight cultures incubated at 37 °C and prepared on Luria−Bertani broth with opacity standard corresponding to 1 × 109 CFU/mL. A 0.1 mL drop for contamination was collected from the bottom water-rich part of the emulsion immediately after it was stirred. Bacterial titer was controlled at all stages of the study by parallel inoculation of 0.1 mL samples of the dispersion used in the experiment on Mueller−Hinton agar.
The extraction of bacterial cells and phage lysate by the physiological solution into the hexane in the physiological solution emulsion was proved in the following experiment. After the dispersion of E. coli K12 C600 was stirred at a titer of 1 × 109 CFU/mL in the emulsion of hexane in physiological solution, 0.1 mL samples of dispersion were taken from both “oil in water” and “water in oil” fractions and inoculated on Petri dishes with Mueller−Hinton agar. When a sample from the bottom (oil in water) phase was inoculated, the titer of the culture was analogous to the control bacterial dispersion before mixing with hexane, whereas when a top layer (water in oil) sample was inoculated, no bacterial colonies appeared in the Petri dish. We used preliminary substrate seeding by bacteriophage EcD7 to investigate the possibility of increasing the bactericidal activity of all studied substrates. A dispersion of bacteriophage in emulsion of hexane in sterile physiological solution was used for this seeding. A 0.1 mL droplet of phage dispersion (at a titer of 3 × 109 PFU/mL; PFU = plaque-forming unit) for substrate decontamination was taken from the bottom water-rich part of the dispersion and deposited onto the substrate immediately after the dispersion was stirred. Bacteriophage titer was controlled at all stages of the study by the Gratia method.41 The presence of bacteriophages on the surface of the contaminated substrates was confirmed as follows. After the test for bactericidal activity, the experimental plates with preliminarily seeded bacteriophage were immersed in a solution containing 0.1 mL of chloroform and 0.9 mL of physiological solution, stirred for 30 min at 170 rpm, and centrifuged for 30 min at 5000 rpm. Then supernatant was collected to define bacteriophage titer by the Gratia method on an 18 h (fresh overnight) E. coli K12 C600 culture. The experiments to define EcD7 phage content in the oil in water and water in oil phases by the Gratia method showed nearly complete extraction of EcD7 phage by the water-rich fraction of the hexane/ physiological solution emulsion, similar to the results described above for the extraction of bacterial culture. Analysis of Bactericidal Activity. Microbial purity of the five types of substrates, including two superhydrophilic (T1Phil and T2Phil), two superhydrophobic (T1Phob and T2Phob), and a reference substrate, was tested 24 h (1 d), 4 and 6 d after contamination with E. coli. Separate substrates were used to repeat the experiment three times for each test duration. To contaminate the substrates, we used the droplet evaporation method, where a 0.1 mL droplet of dispersion containing bacterial cells with a titer of 1 × 107 CFU/mL was deposited onto the test sample and stored until complete evaporation of the dispersion medium. In this method, all bacteria contained in the droplet were transferred to the substrate. A second route was additionally used to contaminate the superhydrophobic substrates from physiological solution dispersions (without hexane). Superhydrophobic substrates were placed on the bottom of a Petri dish with the E. coli K12 C600 dispersion at a titer of 1 × 107 CFU/mL for 1 h. Then the contaminated substrate was taken off and transferred to another Petri dish for storage. Contaminated substrates were stored at room temperature for the selected time in sealed sterile Petri dishes. To ensure the survival of the bacteria, the Petri dishes contained gauze impregnated with sterile physiological solution.42 Microbial purity of the substrates was evaluated by the following procedure: after the given storage time, the sample was placed in a vial containing 1 mL of sterile physiological solution and shaken on a shaker for 10 min at 150 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 surface of the dish with a spreader. After it was predried, the dish was placed in a thermostat for 24 h at 37 °C. The number of colony-forming units was determined after that. Two types of experiments were conducted simultaneously during the study: (1) definition of bactericidal activity of textured surfaces compared to a reference sample evaluated by artificial contamination of all 1350
DOI: 10.1021/acsanm.8b00090 ACS Appl. Nano Mater. 2018, 1, 1348−1359
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ACS Applied Nano Materials
expected and indeed found that the deposition of fluorooxysilane (at the hydrophobization stage) did not affect the surface relief, and thus the morphology of samples T1Phil and T1Phob was essentially the same. A similar situation occurred with samples T2Phil and T2Phob. Therefore, the images obtained just after different laser treatment regimes were sufficient to characterize the morphology of all textured samples. The textures characteristic of the T1 and T2 samples at various magnifications are presented in Figure 2.
types of plates with 0.1 mL of bacterial culture with subsequent definition of microbial purity. (2) definition of bactericidal activity of textured surfaces with applied bacteriophage compared to a reference sample also containing bacteriophages, in which case 0.1 mL of bacteriophage dispersion was applied onto all types of plates that were subsequently (after 24 h) contaminated with 0.1 mL of bacterial culture. Multiplicity of infection (MOI) for the second type of experiment was 100 bacteriophages per 1 bacterium. This choice of MOI was due to high-level decontamination of the plates and was based on experience gained from eradicating bacteria from nonmetal surfaces.43 Experimental Methods for Surface Morphology Characterization and Analysis of Bacterial Structure Modification after Its Adhesion to the Surface. The structures on the superhydrophobic and superhydrophilic surfaces of the AMG alloy were investigated by field-emission scanning electron microscopy (FESEM) and focused ion beam tomography (FIB-nanotomography) methods using an FEI Versa 3D double beam microscope. The FE-SEM images were recorded in secondary electron (SE) and backscattered electron (BSE) detection modes at accelerating voltages from 1 to 5 kV and currents up to 50 pA. The morphology of the asformed surface was also examined in cross sections prepared by FIB. The Slice&View (FEI G3 Slice&View software) technique was used for tomographic reconstruction of the desired area. A 20 nm thick Au layer was deposited onto the surface to avoid charging the Al2O3/ AlON surface layers during this reconstruction. To provide 3D reconstruction of the bacterial culture on top of the textured surface, the analyzed area was preliminarily covered with 0.5−1 μm of Pt using electron beam-induced deposition. The platinum layer preserved the bacterial structure on fragmentation by the focused ion beam into 10 nm thick slices. Bacteria SEM images were recorded in BSE detection mode with voxel size 2.8 × 2.8 × 10 nm and spatial drift correction. The number of slices in a bacterial reconstruction was typically greater than 300. The used parameters ensured a high level of detail in 3D reconstruction of the adsorbed bacteria. Measurement of Contact Angle and the Surface Activity of Bacteria and Bacteriophage. To characterize the wettability of the superhydrophobic and reference samples used in this study, the contact angles were measured for 15 μL of water or aqueous dispersion droplets at five different surface locations for each sample. We studied the wettability of samples with E. coli K12 C600 and bacteriophage EcD7 dispersions. As mentioned above (see Section Preparation of Escherichia Coli and Bacteriophage), two types of dispersion media were used in the experiments. The measurements were performed using a homemade experimental setup44 for recording optical images of sessile droplets and software for digital video image processing of the droplets and subsequent determination of droplet parameters. To measure the roll-off angle, 10 μL droplets of dispersion were deposited on the surface. After equilibration of the initial droplet shape, controlled substrate tilting was used to obtain the roll-off angle by averaging over 10 different droplets on the same substrate. To monitor droplet parameters, such as the contact angle, base diameter, droplet volume, and the droplet surface tension during long-term contact with the superhydrophobic substrates, a dispersion droplet was deposited onto the substrate inside the double-walled cell described in detail earlier.45 Continuous monitoring of the evolution of droplet surface tension for the dispersion droplet allowed us to study the surface activity of bacterial and bacteriophage culture. The time evolution of the droplet contact angle simultaneously with the surface tension behavior also provided information about the kinetics of biofilm formation on top of the superhydrophobic substrates and about the wetting regime (based on the method developed earlier46) for contact of the dispersion droplet with the superhydrophobic substrate.
Figure 2. FE-SEM top view (a, b, d, e) and cross-section (c, f) images of T1 (a−c) and T2 (d−f) samples.
The surface texture of the T1 samples (Figure 2a) was characterized by surface ridges and trenches with an average period of 40−45 μm, decorated by globulelike aggregates of 5− 10 μm composed of nanoparticles (Figure 2b). The size of the individual nanoparticles constituting the aggregates varied from a few nanometers to tens of nanometers. The texture of the T2 samples (Figure 2d) was characterized by well-ordered periodic surface ridges and trenches with an average period of 100 μm. The very top layer of ridges was decorated by raspberry-like aggregates with two characteristic scales of aggregates, the larger of order of 5−10 μm, and the smaller of ∼0.5−2 μm. These aggregates were composed of taper nanoparticles. The presence of several groups of characteristic sizes for both types of textures was responsible for the multimodal roughness of the textured surface and served as a basis for obtaining the superhydrophobic state of samples T1Phob and T2Phob. Wettability of Samples by Water, Bacterial, and Bacteriophage Dispersions. Wettability of the samples was assessed using deionized water droplets prior to analysis of the wetting behavior of biological liquids on our substrates. The data we obtained indicate that the T1Phil and T2Phil substrates fabricated without hydrophobic (fluorooxysilane) molecule deposition showed complete spreading and imbibition of water inside the grooves of laser-produced textures. Thus, according to modern classification,17,18 these substrates can be considered superhydrophilic. Contact angles higher than 170° were measured for the T1Phob and T2Phob substrates. At the
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RESULTS AND DISCUSSION Comparison of Sample Morphology. The morphologies of sample surfaces were studied by the FE-SEM method. We 1351
DOI: 10.1021/acsanm.8b00090 ACS Appl. Nano Mater. 2018, 1, 1348−1359
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Figure 3. Evolution of the surface tension (a) and the contact angle (b) of a dispersion droplet vs time of contact with the T1Phob substrate: (1)− for a droplet of E. coli dispersion in physiological solution; (2)−for a droplet of E. coli dispersion in emulsion of hexane/physiological solution; (3)− for a droplet of the same dispersion as (2), but on the T1Phob substrate preliminarily seeded by EcD7 bacteriophage. (insets) Optical images of corresponding droplets.
Figure 4. SEM images of E. coli cells on the superhydrophilic (a), superhydrophobic (b), and reference (c) surfaces. False color was applied to highlight the bacteria.
hydrophobic substrate for the bacterial dispersion in physiological solution was characterized by f = 0.015. An increase in the contact time between the droplet and the substrate was accompanied by an increase in the fraction of wetted area to f = 0.04. The latter value remained constant during continuous contact with water for more than 24 h (see Figure S1 in the Supporting Information). In contrast, the presence of hexane in the droplet resulted in notable growth of the fraction of wetted area to f = 0.05 even on short-time contact between the dispersion and the substrate, thus changing the contact area between the bacterial cells and the sample surface. Overall, the data presented in Table S2 (Supporting Information) indicate extreme water-repellent properties for the T1Phob and T2Phob samples with respect to both the aqueous salt solutions and the dispersions of organic objects and/or organic droplets in salt solutions. Bacterial Distribution over the Sample Surfaces. We observed surface morphologies by using scanning electron microscopy (SEM) to understand the interaction of bacterial cells with the studied substrates in more detail. SEM images obtained for different samples showed very distinct distributions of bacterial cells deposited from the dispersion droplets on the superhydrophilic samples compared to superhydrophobic and reference samples. To illustrate this difference, in Figure 4 we present SEM images of the T1Phil, T1Phob, and reference samples after deposition of bacteria from a droplet of E. coli dispersion (at a titer of 1 × 107 CFU/mL) in the hexane/ physiological solution emulsion. On the superhydrophilic surfaces, the adhered cells are visible both inside the grooves and on top of the texture (Figure 4a), whereas on the superhydrophobic surfaces, the bacteria mainly occupy the tops
same time, the roll-off angles were less than 1.5°, indicating low wetting hysteresis for both substrates. The combination of very high contact angle and low contact angle hysteresis indicated establishment of a heterogeneous (Cassie−Baxter) wetting regime upon contact of these substrates with an aqueous phase; that is, these substrates were superhydrophobic. Nevertheless, the nonwetting of these substrates with respect to E. coli K12 C600 and EcD7 bacteriophage dispersions should be tested separately. This is due to the presence of hydrophobic moieties both in outer protein shells in the bacteriophage and in the outer membrane and lipopolysaccharide layer of E. coli K12 C600.26 The results presented in Supporting Information (Table S2) for wettability of both types of superhydrophobic substrates with respect to dispersions of E. coli K12 C600 and EcD7 still show very high contact angles (>170°) and low rolloff angles (
