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Article Cite This: ACS Omega 2019, 4, 6891−6902
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Comparative Study of Candida albicans Inactivation by Nonthermal Plasma on Stainless Steel with and without Diamond-like Carbon Film Simone Maria Menegatti de Oliveira,†,‡ Newton Soares da Silva,§ Ana Sene,† Rinaldo Ferreira Gandra,‡ Daniele Schaab Boff Junges,‡ Marco Antonio Ramirez Ramos,† and Lucia Vieira*,† Laboratory of Nanotechnology and Plasma Processes and §Laboratory of Cell Biology and Tissue, University of Paraiba Valley, São José dos Campos, SP 12244-000 Brazil ‡ Laboratory of Mycology, Western Paraná State University, Cascavel, PR 85819-110, Brazil
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ABSTRACT: This paper reports the efficacy of nonthermal plasma (NTP) as a biocidal agent to treat 304 stainless steel (SS304) covered with a diamond-like carbon (DLC) film contaminated with Candida albicans biofilms. The association of two techniques using electric plasma was used. The first was plasma-enhanced chemical vapor deposition (PECVD) used to deposit a DLC film on the SS304. The second was NTP used on the surface of the SS304 with and without the DLC film. The combination of the SS304 surface with the DLC film was demonstrated to be effective when using the DLC film as corrosion protection. Comparing the proliferation of Candida species on the DLC film and SS304 surface, it was possible to conclude that Candida species stays longer in the DLC film than in SS304. The reduction of colony numbers was visible after 5 min using plasma on both surfaces; in addition, 99% of Candida species were eliminated after 15 min. Three C. albicans microorganisms were used. Two were from samples of urine and tracheal secretion, and one was from the American Type Culture Collection (ATCC #90028). Characterization of the plasma chemical species was performed using optical emission spectroscopy in order to understand the nature of the chemical species that inactivated the microorganisms. The DLC film was analyzed using profilometry, Raman spectroscopy, scanning electron microscopy, and tribocorrosion tests. The tribocorrosion tests were used to evaluate the effectiveness of the DLC film in protecting the SS304 surface against corrosion in simulated body fluid because corrosion species from the SS304 could interfere in biofilm growth and mask the effect of the plasma. The results of the factorial analysis of variance confirmed the statistical significance (p > 0.05) of the plasma as a biocidal agent, considering the reduction of colony-forming units of C. albicans. It was found that exposure of the samples to the plasma for only 5 min resulted in reductions ranging from 96.4 to 100.0% for all the microorganisms studied.
1. INTRODUCTION Stainless steel is a material commonly used in water supply systems, the food industry, medical/hospital materials, and the pharmaceutical industry. In health care, stainless steel has applications in fixation of fractures, stents, and surgical instruments, among others.1−5 However, contamination of the surfaces of equipment and utensils used in food processing, medicines, cosmetics, and medical and esthetic procedures is of concern to professionals in many different areas since the adherence and interaction of microorganisms on surfaces can be followed by cell growth and multiplication, which is highly undesirable.6 At the hospital level, it was found that 60% of infections are related to fungal infection, with 80% of these involving Candida albicans as the primary pathogen. This yeast study is clinically essential, due to its high virulence associated with rapid colonization and infection processes, since it is an opportunistic fungus.7 Recently, published surface disinfection processes have not shown great effectiveness. For example, the photodynamic © 2019 American Chemical Society
antimicrobial chemotherapy technique described by da Silva et al.,8 where the surface was exposed to toluidine blue and a light-emitting diode (LED) for 5 min, achieved elimination of only 20% of Candida krusei microorganisms. Disinfection of plasma health equipment has been implemented in hospitals.9 Some autoclaves were marketed using a low-temperature plasma process onboard.10−12 In addition, the U.S. Food and Drug Administration believes that chemical sterilization with liquids does not convey the same sterility assurance as sterilization using low temperature or thermal gas for surface sterilization methods.13 In the search for fast and effective surface disinfection processes, the combination of two technologies that have already been studied individually could provide a solution. Nonthermal plasma (NTP) technology has shown promising results in the elimination of various microorganisms,14−16 Received: December 27, 2018 Accepted: April 3, 2019 Published: April 16, 2019 6891
DOI: 10.1021/acsomega.8b03640 ACS Omega 2019, 4, 6891−6902
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Table 1. Time Used To Achieve Inactivation of Bacteria and Fungi by Plasma tested microorganisms
authors and ref
results
Aspergillus niger (fungi) and Candida lipolytica (yeast)
The areas of inhibition 30−40 mm in diameter were observed after 30−60 s of exposure. Trichophyton rubrum, Trichophyton interdigitale, Microsporum The in vitro plasma irradiation was able to kill over 90% of the fungal spores canis, and C. albicans within 30 s. The sensitivities of the vegetative forms of all bacteria were compared and were nine microbial species, including Gram-positive and Gramobserved that the inactivation of Candida and Geobacillus spores requires longer negative bacteria, Deinococcus radiodurans and G. exposure times. stearothermophilus spores, and the yeast C. albicans review of studies of various microorganisms: the efficiency of the Inactivation of bacteria was 2 to 4 min; those for yeast and fungi were 6 and 20− inactivation depends on the source of the plasma and the 30 min, respectively. operating conditions E. coli and Staphylococcus aureus, B. subtilis spores, and the yeast The inactivation of the vegetative bacteria occurred in approximately 1 min of Saccharomyces cerevisiae exposure, while several minutes were required for yeast, and decreased viability of bacterial spores only occurred after 1 h. M. canis and T. rubrum Plasma treatment sessions for 10 min daily (9 days) were more effective than single treatments. Aspergillus fumigatus Plasma with a dielectric barrier discharge by 45 s on a plate formed a halo. Fusarium graminearum, Fusarium oxysporum, and Neurospora crassa
Akishev et al.14 Daeschlein et al.15 Scholtz et al.16 Scholtz et al.22 Lee et al.26
Heinlin et al.27 Ghomi et al.45 Power and pulse duration time can control the production of oxygen radicals and Na et al.46 eventually control the efficiency of inactivation of fungal spores.
damage can also be caused to microorganisms during contact with DLC, causing membrane compromise and release of intracellular microbial metabolites.18 Biological materials can be exposed to plasma using two different methods: Direct exposure is when the sample being treated is in direct contact with the plasma, with all the plasmagenerated agents, including charged particles, achieving the sample. In the second method, the sample is placed at some distance from the plasma. In this configuration, the amount of heat transmitted to the sample is reduced, and charged particles do not play its role since they recombine before reaching the sample. While many neutral reactive species of short duration also do not reach the sample, just the ionized air and its species are in contact with the sample.35 1.1. Reactive Species. The species present in the plasma are subject to collisions; under collisions, molecules are dissociated into ions, at the same time that the ions are recombined and form molecules or reactive species. Thousands of collisions can occur until molecules, species, and ions achieve the sample. By controlling the working pressure inside the vacuum chamber, it is possible to control the collisions and the average free path of the species. The quasi-equilibrium plasma is usually called thermal plasma, and a nonthermal plasma is far from equilibrium. Examples of thermal and nonthermal plasma are the solar plasma and aurora borealis, respectively.36 Plasmas generated in atmospheric air, for example, are excellent sources of reactive oxygen and nitrogen species. The plasma plume with reactive species acts on a cell membrane. The most common reactive species in an air environment are O, O2, O3, OH, NO, NO2, and others.35 Among the reactive species, nitrogen monoxide (NO) is responsible for producing lipid peroxyl radicals (LOO·) as cited by Patel et al.37 Laroussi35 demonstrated that ozone (O3) is a well-known sterilization agent. Ozone has many uses in the health care due to its oxidizing potential in living tissues and its sterilization effectiveness against microorganisms. 38 It is useful in inactivation of pathogens including Escherichia coli,39 Bacillus subtilis, Clostridium sporogenes,40 and viruses,41 as well as spores resistant to other sterilization processes, such as those of Geobacillus stearothermophilus42 and Bacillus atrophaeus.43 Its mechanism of action involves attacking cell constituents such
while the application of a diamond-like carbon (DLC) coating can assist in reducing the interaction of microorganisms with the surfaces of materials.17−19 NTP is a partially ionized gas where energy is stored mainly in the free electrons and the total temperature remains low. NTP has been widely used for many years in applications such as low-temperature plasma chemistry and treatment of the feet of diabetics.20 However, over the past 10 years, the use of NTP has expanded to new biological applications such as the inactivation of microorganisms by plasma healing of skin wounds,21 the preparation of ready-to-eat foods, biofilm degradation, and health care.22 Mendis et al.23 and Laroussi et al.24 suggested that charged particles could play a very significant role in rupturing the outer membranes of bacterial cells. It was demonstrated that charge accumulation produced by electrostatic force on the outer surface of the cell membrane can overcome the tensile strength of the membrane and cause its rupture. Studies of the use of plasma in the inactivation of bacteria and fungi have shown promising results.16,25−27 Scholtz et al.22 concluded in their review that yeasts are inactivated after about 6 min of plasma exposure, with the inactivation efficiency being mainly dependent on the plasma source and its operating conditions. Plasma equipment assisted by high-frequency electrodes is widely used in esthetic clinics directly on human skin due to its fungicidal and bactericidal effects, stimulation of the circulation in the place where it is applied, and the vasodilating and hyperemia effects, which facilitate the penetration of systemic drugs.20 Such high-frequency systems are used to produce a plasma encapsulated in a glass electrode. Thin films, such as those composed of DLC, can improve surface properties, providing lubricant attributes and an adverse environment for infections.28 A DLC film is a metastable form of amorphous carbon with a significant fraction of sp3 binding. It has several desirable characteristics for technological applications, including high hardness, high wear resistance, chemical inertia, biocompatibility, and low friction coefficient.29,30 Also, the DLC film exhibits antimicrobial activity.31,32 This activity could be related to its chemical inertia due to the weakening of the chemical interface in the bacterial adhesion process,19,33 as well as to its surface energy, which includes a high dispersive energy component, and its hydrophobicity.34 Direct physical 6892
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as proteins, unsaturated lipids, respiratory enzymes in cell membranes, nucleic acids in the cytoplasm, proteins, and peptides of the protective layers of spores and viral capsules.44 1.2. Inactivation of Bacteria and Fungi by Plasma. Table 1 shows the state-of-the-art studies of inactivation of bacteria and fungi by plasma. A comparison of the references shown in Table 1 shows that C. albicans requires longer exposure times than bacteria, even with a different configuration for each plasma system. In the present work, high-frequency (HF) plasma was used, for the first time, to compare the efficiency of plasma applied using two different surfaces, one with a solid lubricant, the DLC film, and the other consisting of SS304; both surfaces were contaminated with C. albicans. 1.3. High-Frequency Plasma Equipment. A system using nonthermal plasma (NTP) was assisted by a power supply with high frequency employing plasma encapsulated in a glass electrode that has been used widely in esthetic clinics, because the ozone generated by air ionization has fungicidal and bactericidal effects, while the plasma stimulates the circulation in the location where it is applied. The energy is stored mainly in free electrons, and the total temperature remains low, avoiding burning of the skin. NTP has been widely used for many years in applications such as lowtemperature plasma chemistry, removal of gaseous pollutants, gas discharge lamps, and surface modification. However, over the past 10 years, the use of NTP has expanded to new biological applications as mentioned by Scholtz et al.22 Furthermore, the vasodilating and hyperemia effects caused by plasma facilitate the penetration of systemic drugs. The plasma also presents a local thermal effect that can stimulate metabolism and increase cellular oxygenation.27 1.4. DLC Associated with Plasma Action on Microorganisms. To the best of our knowledge, there have been no previously reported studies concerning the use of plasma with microorganisms on surfaces coated with DLC. It was hypothesized that the nonstick and lubricant characteristics of DLC would facilitate the action of plasma on microorganisms adhered to a surface with this type of coating. These properties would facilitate the disinfection or sterilization of materials and tools used in health procedures, beauty salons, and the food, pharmaceutical, and cosmetics industries. Use of the plasma technique for sterilization has the advantages of being fast and low cost, with the absence of high temperatures reducing risks to the operator and avoiding wear of materials, hence increasing the useful life of the item to be sterilized. Given the emerging importance of nonthermal plasma and DLC films, this work aimed to evaluate the efficacy of nonthermal plasma as a biocidal agent applied to SS304 contaminated with C. albicans in the absence and presence of the DLC film.
Figure 1. Optical emission spectrum of the IBRAMED plasma.
by direct dissociative electron excitation of water or by dissociative recombination of H2O+, where this radical is formed by metastable ions and subsequent recombination.47 In addition, the band of N2+ (B2Σu+, ν = 0 → X2Σg+, ν′ = 0) was observed from 380 to 440 nm and Hβ (487 nm) spectra.48 According to Ohno et al.,49 ionic N2+ at 391 nm and atomic oxygen lines could result from a Penning ionization model. When a molecule (M) meets with an excited atom (A*) with enough energy, an electron-transfer process can occur to yield an ionic state of the molecule (M+) and the ground state of the atom (A) together with an ejected electron (e−): A* + M → A + M+ + e− e− + N2+ → N2+* + e− direct electron impact excitation N2* + O2 → N2 + O + O Penning ionization
The active species in the plasma in contact with the cells were oxygen and nitrogen species with different levels of energy, which can cause degradation of the cell wall membrane, as reported by Patel et al.37 and also observed in this study, as shown in Figure 1. When ionized, nitrogen reacts with oxygen in the air to form various intermediates such as reactive oxygen species (ROS), nitrogen monoxide (NO), and nitrogen dioxide (NO2). In aqueous media, the rate of reaction between oxygen and NO is proportional to the two oxygen molecules forming NO2, being very slow for NO metabolism.50 However, in membranes and lipoproteins (hydrophobic media), there is an approximately 10-fold increase in oxygen-dependent NO consumption due to the 8- to 10-fold higher solubility of NO in the hydrophobic phase of lipid membranes. Taking into account the relative volumes in a cell with cytosol and membrane structures, the rate of NO reaction with oxygen is 300 times higher in the hydrophobic environment.51 This results in the formation of several intermediates, including dinitrogen trioxide (N2O3), dinitrogen tetroxide (N2O4), and the nitrogen dioxide free radical ·NO2.52 NO2 is a precursor of lipid peroxidation reactions that are propagated by lipid peroxyl radicals (LOO·). Oxidative damage to lipids mediated by NO2 or other oxidant species was studied by Patel et al.37 Higher concentrations of nitrogen and oxygen led to the formation of a variety of common species, including NO2 and NO, which are both able to initiate a cascade of oxidative reactions that cause damage to the lipid membrane. The equations for the formation of several reactive species are shown in Table 2. The oxidation of biological targets depends on how fast the oxidant in question reacts with its target, as well as the concentrations of the substrate and oxidants present. The lifetime of reactive species in a cellular environment is another critical factor in the study of redox processes. The lifetime and
2. RESULTS AND DISCUSSION 2.1. Optical Emission Spectroscopy (OES). OES was obtained from the IBRAMED plasma system assisted by a high-frequency electrode used in this work. Figure 1 shows the optical emission spectrum of the IBRAMED system using inlet and outlet slit widths of 13 μm and a Horiba i550 monochromator. The spectra from the plasma plume show the populations of the excited-state first-order ultraviolet OH band (A2Σ+, ν = 0 → X2∏, ν′ = 0) from 298 to 300 nm. The OH band was observed due to water vapor in the air environment, and the hydroxyl radical (HO·) can be generated 6893
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aromatic rings, olefin chains, and hydrogen-terminated bonds.28 Before and after plasma actuation under a specific time, the signature of the DLC film was kept the same. 2.3. DLC Film Morphology and Thickness. An optical profiler was used to obtain the average roughness of the samples with and without the DLC film using five measurements for each sample with a field of 301 × 229 μm2. Figure 3a,b shows a comparison of the surface morphology and arithmetic roughness (Ra) for the SS304 with and without a DLC film, from which it can be seen that deposition of the DLC film reduced the roughness of the surface by about 15%. Figure 3c shows an SEM image of the DLC film on SS304, revealing pores formed from clusters caused by the collision of the plasma species with the film surface. It is also possible to see marks from the sandpaper used in the polishing process. The presence of pores without a DLC film could increase the corrosion process and lead to film delamination. Therefore, tribocorrosion tests were performed in order to evaluate this possibility. The DLC thickness was measured using the attachment of Kapton tape to the SS304 substrate before film deposition. After the deposition process, the tape was removed, and the step was measured using optical microscopy. The average DLC thickness from five measurements was 1.00 ± 0.09 μm, as shown in Figure 4. 2.4. Tribocorrosion Assays. Figure 5 shows the open circuit potential (OCP) curves obtained during the tribocorrosion tests that were performed to evaluate the surface corrosion susceptibility of SS304 with and without DLC. The OCP was measured in three steps: static, dynamic, and static again. The static mode was measured to observe the evolution of the OCP before and after the reciprocating sliding friction mode, which can be useful for detection of a passive layer, as well as its recovery before and after a sliding movement. The black and red lines show the evolution of the OCP of the DLC-covered sample and the SS304 substrate, respectively. The OCP for the DLC-covered sample was approximately +0.025 V in static mode and 0.00 V in dynamic mode, indicating a very low tendency for corrosion and surface passivation promoted by the DLC film. On the other hand, the bare SS304 substrate (red line) presented high potential variation between the static and dynamic modes, decreasing from around −0.005 to −0.30 V. This decrease in potential was indicative of a higher corrosion tendency of the sample, as described previously.56 As soon as the sliding was started, the OCP of the uncovered sample underwent an abrupt decline due to the destruction of the fragile passive film at the surface, hence exposing the bare substrate to the corrosive effects of the simulated body fluid solution.
Table 2. Equations for Reactive Species Formation O2 + e− → O2−· O2−· + H2O → HO2−· + OH HO2−· + e− + H → H2O2 H2O2 + e− → ·OH + OH−
superoxide radical hydroperoxyl radical hydrogen peroxide hydroxyl radical
diffusion capacity of these species are directly related to the speed with which they react with the molecules present in the environment where they are generated. Reactive oxygen species include HO· (hydroxyl radical), O· (singlet molecular oxygen), O2−· (superoxide radical anion), and H2O2 (hydrogen peroxide), with lifetimes, diffusion, and speeds increasing in this order, while the reactivities of these species increase in the reverse order.53 In the case of highly reactive species such as · O2 and ·OH, it is believed that their reactivity is confined to regions close to their generation sites. Therefore, the plasma plume should be located close to the surface to be treated at least 10 mm. 2.2. DLC Chemical Structure Characterization. Figure 2 shows the Raman spectra. The standard signature of DLC
Figure 2. Raman spectra of the DLC film deposited on a silicon substrate.
was observed, consisting of two bands. A disorder band (D band) due to sp, sp2, and sp3 hybridization was centered at 1350 cm−1.54 The G band, centered at 1580 cm−1, represented the graphite phase.55 If the G band shows a shift to the left, this is indicative of the formation of a micro-graphite phase, while the shift of the G band to the right of the plot indicates the formation of open carbon chains. In the present case, the G band showed a shift of 33 cm−1 to the left, indicating that the DLC film contained a micro-graphite phase, graphite in
Figure 3. Sample surface images obtained using an optical profiler comparing the Ra values for SS304 with and without a DLC film (a,b). The SEM magnification shows details of the SS304 + DLC film surface (c). 6894
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1.0 = negative, >0.64 and
