Inactivation of Candida albicans by Cold Atmospheric ... - IEEE Xplore

770

IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 43, NO. 3, MARCH 2015

Inactivation of Candida albicans by Cold Atmospheric Pressure Plasma Jet Konstantin Georgiev Kostov, Aline Chiodi Borges, Cristiane Yumi Koga-Ito, Thalita Mayumi Castaldelli Nishime, Vadym Prysiazhnyi, and Roberto Yzumi Honda

Abstract— Nonthermal atmospheric pressure plasma jets (APPJs) are characterized by very reactive chemistry without the need of elevated temperatures. Also, plasma jets are capable of producing cold plasma plumes that are not spatially confined by electrodes, which makes them very attractive for biomedical applications. In this paper, we investigate the antimicrobial efficiency of a simple plasma jet device operating with pure He as working gas. The device was driven by an ac power supply operated at 31.0 kHz, 13.0 kV amplitude with mean power around 1.8 W. The jet was directed perpendicularly on a standard Petri dish (Ø90 mm × 15 mm) filled with agar. The jet fungicidal efficiency was tested against Candida albicans (reference strains SC 5314 and ATCC 18804) and five clinical isolates from previously obtained denture stomatitis lesions. In this paper, the effects of treatment time and distance to the target were evaluated. In most treatments the samples did not have direct contact with the plasma plume; therefore, the reactive oxygen species produced by interaction between the plasma jet and ambient air were the principal inactivate agent. Index Terms— Candida albicans (C. albicans), cold atmospheric plasma, decontamination, plasma jet.

I. I NTRODUCTION N THE last decades, nonthermal atmospheric pressure plasma sources, such as corona, dielectric barrier discharge (DBD), and cold plasma jet, have attracted significant interest, because they do not require expensive vacuum equipments and can be readily applied for in-line processing of industrial components. Atmospheric plasmas can be operated under ambient pressure and temperature conditions and have routinely been applied for surface treatment and functionalization of polymers, air pollution control, thin-film deposition, generation of nanotubes, flow control in aeronautic applications, improvement of combustion efficiency, and more recently for biomedical applications [1], [2]. Among other atmospheric plasma sources, the nonequilibrium plasma jets have drawn special attention because they are generated in

I

Manuscript received June 6, 2014; revised August 13, 2014; accepted September 4, 2014. Date of publication November 10, 2014; date of current version March 6, 2015. This work was supported in part by the National Council of Research and Development under Grant 470995/2013-0 and in part by the State of São Paulo Research Foundation under Grant 2014/098960. K. G. Kostov, T. M. C. Nishime, V. Prysiazhnyi, and R. Y. Honda are with the Faculdade de Engenharia de Guaratinguetá, São Paulo State University, São Paulo 12516-410, Brazil (e-mail: [email protected]; [email protected]; [email protected]; [email protected]). A. C. Borges and C. Y. Koga-Ito are with the Institute of Science and Technology, São Paulo State University, São Paulo 12247-004, Brazil (e-mail: [email protected]; [email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TPS.2014.2360645

open space rather than in confined volume and consequently can be used for treatment of irregular objects. Also, the plasma jets can be ignited inside small tubes, catheters, endoscopes, channels, which makes them appropriated for decontamination of hospital equipment [3]. There exists a large variety of atmospheric pressure plasma jets (APPJs) with different configurations that have been reviewed in [4]. The plasma jets are usually produced from a noble carrier gas and can be powered by pulsed or sinusoidal voltages at frequencies ranging from a few hundred Hz to tens of MHz [4], [5]. It has been revealed that the APPJs are actually trains of traveling supersonic plasma bunches called plasma bullets rather than a continuous plasma column. The fundamental physics of plasma bullets as well as the plasma bullets characteristics are discussed in [5]. Because of their highly nonequilibrium properties, the APPJs produce very reactive chemistry at room temperature, which makes them suitable for treatment of thermosensitive materials and biomedical applications in vivo and in vitro [6]–[9]. It has been shown that cold atmospheric plasmas are very effective for inactivating microorganisms even in liquid solution or in the form of pathogenic biofilm [8]. However, the exact mechanisms of microbial inactivation by plasma as well as the resulting microbial response are not fully understood yet [10]. Atmospheric plasmas are complex mixtures of charged particles, reactive atoms, and molecules (e.g., atomic oxygen, ozone, hydroxyl group, oxides of nitrogen, etc.), electric fields, and ultraviolet (UV) radiation. Each of one of these individual agents can cause bacterial deactivation. Charge accumulation on microorganisms can lead to electrostatic cell disruption [6]. Reactive oxygen compounds (O and O3 ) can physically etch the cell membrane and interfere with transport within the cell. Also, the reactive components can induce DNA breakage [10]. UV radiation (especially sub-260-nm wavelength photons) can induce damage to DNA and intracellular proteins [11]. In plasma, there is synergetic effect of all aforementioned inactivation mechanisms; therefore, plasma devices are very effective tools for bacterial decontamination. The most recent and important application of cold atmospheric plasma jets is in medicine, where they have been applied for wounds healing, disinfection, blood coagulation, inactivation of pathogens, microbial decontamination of medical equipment, and dental treatment [2], [6], [8]. In contrast to low-pressure plasmas, UV photons most likely are not the major inactivation factor in cold APPJs as the ambient air absorbs most high-energy photons and the intensity of UV radiation reaching the sample is too low [11], [12]. Usually in most medical devices the

0093-3813 © 2014 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.

KOSTOV et al.: INACTIVATION OF C. ALBICANS BY COLD APPJ

treated surface is not in direct contact with the plasma jet but with the plasma effluent alone. In this case, the charged plasma species are expected to recombine before reaching the substrate. However, in some cases as described in [12], negative O2 ions may be an important agent for the cells deactivation. Therefore, the role of charged particles in the process of microbial decontamination is not clear, yet. Most authors believe that under atmospheric conditions, reactive oxygen species (O, O3 , OH) produced by the plasma–air interaction, are most probably the key factor in microbial inactivation [11], [13]. For example, in [12] it was shown that admixture of small amount of O2 into He feed gas could greatly improve the plasma jet inactivation efficiency. The latter is also influenced by the kind of biological sample used (microbes plated in agar, in liquid solution or in biofilm) and especially for planktonic microorganisms by the solution pH [14]. In this paper, we report a simple atmospheric pressure plasma jet for fungal inactivation developed at FEG, São Paulo State University. In this preliminary paper, plasma jet was operated with He gas, rather than a gas mixture, because using pure helium was found to offer a lower breakdown voltage and higher discharge stability. In a forthcoming study, we intent to test a plasma jet operating with He/O2 mixture, which as reported in [12] is more efficient in microbial inactivation. In this paper for producing active radicals, we rely only on the natural penetration of air molecules into the jet effluent, which was applied on the microorganism Candida albicans (C. albicans) plated on agar. C. albicans is the most common pathogenic fungal species that is causal agent of oral and genital superficial infections. Also, C. albicans is associated to life-threatening systemic infections, in particular among immune suppressed patients. C. albicans is able to form biofilms on the surface of implantable medical devices that can be sources of infections. To deal with the problem different antimicrobial measures such as antibiotics, chemicals, heat, pressure, UV, and more recently plasma treatment have been applied. However, combating infections is still a challenge due to the growing microbial resistance to drugs. In addition, the reduced number of antifungal molecules and the similarity between fungal and human cells (both eucariotic) are additional issues. In this sense, the plasma treatment offers an interesting alternative for cells inactivation comparing to more traditional methods [2], [3]. Here we tested the fungicidal efficiency of a He APPJ against five different clinical C. albicans strains obtained from denture stomatitis lesions. II. E XPERIMENTAL S ETUP Fig. 1 shows a schematic diagram of the experimental apparatus. To generate plasma plume at atmospheric pressure a DBD system with one-electrode configuration was used. A 2.3-mm diameter, 10-cm long copper wire served as a highvoltage electrode. It was inserted into a 2.6-mm inner diameter, 1.7-mm thick quartz tube whose bottom end was closed. The quartz tube was installed coaxially in a cylindrical enclosure made from Delrin. It terminates with a 2-cm long, 1.5-mm inner diameter nozzle. The system was flushed with

771

Fig. 1.

Experimental setup.

He (99.5% purity) and gas flow rate was controlled by a rotameter within the range 0.1–10.0 L/min. The Cu wire was connected to a Minipuls4 ac power supply (GBS Elektronik GmbH, Germany), which was used to ignite plasma. The quartz tube tip ended 10 mm from the Delrin enclosure’s nozzle which a plasma jet was launched to air. The length of the plasma plume was about 2.5 cm at a gas flow rate of 5.0 L/min, which was kept fixed in all experiments. A grounded stage, which also served as a sample holder, was placed under the plasma plume. A low-inductance resistor of 100 in series to the grounded stage was used to measure discharge current. Alternatively, transferred charge was obtained by changing the resistor with a capacitor of 10 nF. The signals were monitored on a digital oscilloscope (Tektronix TDS 2024B, 200 MHz). A mean discharge power of the plasma jet was calculated using the Q–V Lissajous figure method. The tip of the plasma plume, produced in these conditions, is quite cold and can be touched by a bare hand. Optical emission spectroscopy (OES) in the wavelength range from 300 to 850 nm was used to identify the active species in the plasma plume. The operational parameters of the spectrometer (Andor Technology Shamrock 303i) were the following: grating of 150 g/mm, slit of 170 μm, and integration time of 0.06 s. The plasma jet was directed perpendicularly to a standard Petri dish filled with sabouraud dextrose (SD) agar on which microorganisms were plated. In this paper, the jet fungicidal efficiency was tested against C. albicans reference strains (SC 5314, ATCC 18804) and five clinical isolates from previously obtained denture stomatitis lesions (Ethical Board Committee approval, CONEP CAAE 31787814.1.0000.0077). Cultures were kept in SD broth supplemented with 20% glycerol at −6 °C. Fresh cultures were obtained by plating C. albicans cells in SD agar and incubating at 37 °C for 24 h. To obtain working suspensions, the fungal colonies were resuspended in a 0.9% NaCl solution (106 CFU/mL). These suspensions (100 μl) were distributed in SD agar plates with sterile swabs. Fifteen minutes later, the plates were exposed to plasma jet at 30, 60, 90, 120, 150, and 180 s, in triplicate.

772

Fig. 2.


Typical waveforms of applied voltage and discharge current. Fig. 4.

Fig. 3.

Q–V Lissajous figure.

As controls, plates were subjected to He gas flow only. After incubation (24 h, 37 °C), diameter of the circular inhibition zones of fungal growth was measured. III. R ESULTS AND D ISCUSSION Typical waveforms of the applied voltage and the discharge current are shown in Fig. 2. As can be seen the voltage signal is a slightly distorted sine wave with frequency of 31 kHz and 13 kV amplitude. The discharge current is composed by series of short current peaks superimposed on relatively large capacitive current. The current peaks always occur on rising or falling parts of the sinusoidal voltage. The amplitudes of current peaks in the positive half-cycle of the applied voltage is usually higher than ones in the negative half-cycle. This behavior of the current peaks is typical for the plasma jet devices and was reported by [15]. Fig. 3 shows a typical Q–V Lissajous figure, which in our case resembles a parallelogram. The area of this figure represents the electrical energy dissipated per one voltage cycle. Then the mean discharge power (1.8 W in this case) was

Emission spectrum at the plasma jet effluent.

obtained by multiplying the Lissajous figure area to the signal frequency. For all further experiments on fungal inactivation the same jet operation parameters: 31 kHz signal frequency, voltage amplitude 13 kV, and gas flow rate of 5 L/min were kept. When the He plasma jet is driven by the gas flow into air, ambient species (such as N2 , O2 , and humidity) penetrate into the plasma channel. The energetic plasma species (mostly He metastables) interact with air molecules forming reactive species (O, OH, O3 , NO2 , etc.). Therefore, even when pure He is used to ignite plasma the jet effluent (region where feeding gas and ambient air mix) is a highly reactive zone. In fact, in most plasma surface treatments the target is placed in this zone. Therefore, the composition of plasma-activated species in the jet effluent is very important for fungal decontamination efficiency. Fig. 4 shows an optical emission spectrum measured at the tip of the plasma plume. It is clearly seen that the spectrum is dominated by the excited N2 . The major emission lines come from the second positive nitrogen system (N2 C3 →B3 ). No emission from excited He atoms was detected in the jet effluent confirming the efficient energy transfer from He metastables to nitrogen molecules and formation of reactive nitrogen species as described in [16]. Because O2 molecules are also present in the air (tough in lesser quantity as N2 ), the formation of reactive oxygen species (ROS) in the jet effluent is also expected. Emissions from excited O atoms in He plasma jets effluent were observed in a previous work [13] but not in our experiments. We do believe that ROS were indeed produced in the jet effluent but characteristic O emission lines were not detected because of their weak emission intensity and also because of the low sensitivity of our spectrometer. It was shown that exactly the ROS (O, and O3 ) attack the cells membrane and are the main factor for microbial inactivation in atmospheric plasma jets [10], [12], [15]. First, the jet antifungal efficiency was tested against different C. albicans strains (two references and five clinical) keeping distance to the Petri dish fixed at 3.0 cm. As shown in Fig. 5(a), the plasma jet effluent was directed perpendicularly


773

Fig. 5. (a) Plasma jet directed on a Petri dish with agar. (b) Inhibition zones for time of treatment of (left) 30 s, (top) 60 s, (bottom) 90 s, and (right) 120 s.

Fig. 6.

Growth of the inhibition zones as function of the treatment time for all tested strains.

on a standard Petri dish (Ø90 and 15-mm height). In this way, the sample did not have direct contact with the plasma but the highly reactive species present in jet effluent still can reach the surface and interact with it. The jet effluent pointed on a desired spot on the inoculated Petri dish and at the end of desired exposure time we moved the plate to treat another spot. This process was repeated four times to treat total of four spots on the same dish as can be seen in Fig. 5(b). The treatment times used for the Petri dish shown in Fig. 5(b) were 30, 60, 90, and 120 s. The fungi that were not killed by the treatment grew during the incubation forming visible colonies with white color. Dark circles (inhibition zones) with the natural dark color of agar identified the places where microorganisms were inactivated by the plasma exposure. There were no any visual changes in the fungal growth of reference and clinical strains for 30 s of treatment. However, when fungi were exposed to jet effluent for 60 s inhibition zones with diameters of 4 ± 1 mm appear in all plates. The

diameters of the inhibition zones for all fugal strains were measured and they are presented in Fig. 6 as a bar-plot at different times of treatment. Upon increasing the treatment time, the fungal strains exhibited very similar behavior with inhibition zones whose diameter grew more or less linearly up to time of at 150 s after which a saturation value of around 10 ± 1 mm was achieved. Because the active species produced by the plasma jet have a limited lifetime, they cannot cover larger area of the sample even though the treatment time is further increased. Note that the size of inhibition zone was always larger than the plasma jet diameter (approximately equal to the nozzle diameter of 1.5 mm). As shown in [12] and [13], oxygen-reactive species generated by the plasma jet like O, and O3 , can diffuse radially outward and consequently deactivate cells. On the other hand, UV photons produced in atmospheric plasma jets propagate preferentially along the plasma column thus they interact only with a part of the target surface located just under

774

Fig. 7. Growth of the inhibition zones as function of the time of treatment for different distances.

the jet [17]. Therefore, judging by the size of the inhibition zone (much larger than the plasma column diameter) one can conclude that the reactive oxygen species produced by jet–air interaction are the major inactivation factor. As shown in [18] by model simulation of the gas flow dynamics of He/O2 plasma jet and oxygen reaction kinetics the O atoms formed in the discharge can propagate over a limited area with a maximum diameter of about several millimeters. However, ozone, which is well known for its fungicidal effect and is stable enough to survive the transport, can inactivate cells at large distances from the jet [10]. Therefore, it can be concluded that the fungi in our experiment were deactivated by the ROS (mostly O3 and may be O at short distances). The exact processes that lead to microbial deactivation are not fully understood yet but it is believed that in indirect plasma treatment the ROSs attack and compromise the integrity of cells membrane. A detailed discussion about the suggested mechanism for membrane destruction is presented in [11]. In the next set of experiments, we evaluated the effect of nozzle to Petri dish distance on the fungicidal efficiency. For these tests, Petri dishes were plated only with the reference strain ATCC 18804. Fig. 7 shows the growth of inhibitory zones on the Petri dishes as a function of the treatment time at three different distances of 2.0, 2.5, and 3.0 cm. As it was expected and also reported by [12] and [13], the plasma treatment at shorter distance resulted in larger inhibitory zones probably because larger amount of active oxygen species can reach the surface. However, the difference between the inactivation zones diameters for different distances was not so great as expected especially at long time of treatment. This finding can be explained by a so-called buoyancy effect reported in [18]. Ozone and atomic oxygen are efficiently produced in the jet effluent where He metastables and air molecules mix. At large distances (d > 2.5 cm), when the jet does not touch the surface, O3 molecules reach the surface and freely spread radially outward to inactivated cells.


However, as shown in [18] at short distances (d < 2.5 cm) the gas flow actually bounces back from the surface. In this case, the reflected He flow expands upward and drags up some active species that cannot reach the surface. Although this gas dynamics effect diminishes the efficiency of plasma treatments at shorter distances, still the inhibition zones are bigger. However, an undesirable effect of plasma jet treatment at short distances, such as the appearance of irregular (kind of ring-shaped) inhibitions zones, was reported in [13]. What exactly caused these patterns is not clear but most probably they were caused by turbulences of the reflected gas flow. In our experiments at 2 cm besides some shape irregularities of the inhibition zones, we also observed an indentation on the agar surface, which was caused by the gas flow. Therefore, treatments at very short distances have some shortcomings not to mention the danger of excessive sample heating. Thus, to achieve large inhibition zones with reproducible circular shapes the best operating conditions would be when the jet effluent almost touches the surface (∼2.5 cm in our case). IV. C ONCLUSION The fungicide efficiency of a homemade He plasma jet was proven against seven different strains of C. albicans. The microorganisms were plated on agar in Petri dishes and then exposed to the reactive species produced by the plasma jet. The size of inhibition zones on the Petri dishes was much larger than the jet diameter, which suggests that ozone, as a relatively stable byproduct of plasma jet–air interaction is the major inactivation factor. For all fungal strains, the size of inhibition zones scales with the time of treatment until a maximum diameter of the decontaminated area was reached. When the distance to the target is decreased, the inhibitions zones tend to increase but gas flow effects can lead to shape irregularities and agar indentations. In future work, the He plasma jet will be tested for inactivation of planktonic samples as well as C. albicans biofilms. R EFERENCES [1] M. Laroussi and T. Akan, “Arc free atmospheric pressure cold plasma jets: A review,” Plasma Process. Polym., vol. 4, no. 9, pp. 777–788, Nov. 2007. [2] T. von Woedtke, S. Reuter, K. Masur, and K.-D. Weltmann, “Plasmas for medicine,” Phys. Rep., vol. 530, no. 4, pp. 291–320, Sep. 2013. [3] K.-D. Weltmann and T. von Woedtke, “Basic requirements for plasma sources in medicine,” Eur. Phys. J. Appl. Phys., vol. 55, no. 1, pp. 13807–13817, Jul. 2011. [4] X. Lu, M. Laroussi, and V. Puech, “On atmospheric-pressure nonequilibrium plasma jets and plasma bullets,” Plasma Sour. Sci. Technol., vol. 21, no. 3, pp. 034005-1–034005-18, Apr. 2012. [5] X. Lu, G. V. Naidis, M. Laroussi, and K. Ostrikov, “Guided ionization waves: Theory and experiments,” Phys. Rep., vol. 540, no. 3, pp. 123–166, Jul. 2014. [6] E. Stoffels, “‘Tissue processing’ with atmospheric plasmas,” Contrib. Plasma Phys., vol. 47, nos. 1–2, pp. 40–48, Feb. 2007. [7] T. P. Ryan, K. R. Stalder, and J. Woloszko, “Overview of plasma technology used in medicine,” Proc. SPIE, vol. 8584, pp. 85840O-1–85840O-21, Feb. 2013. [8] M. Y. Alkawareek, Q. T. Algwari, S. P. Gorman, W. G. Graham, D. O’Connell, and B. F. Gilmore, “Application of atmospheric pressure nonthermal plasma for the in vitro eradication of bacterial biofilms,” FEMS Immunol Med Microbiol., vol. 65, no. 2, pp. 381–384, Jul. 2012.


[9] P. P. Sedghizadeh, M.-T. Chen, C. Schaudinn, A. Gorur, and C. Jiang, “Inactivation kinetics study of an atmospheric-pressure cold-plasma jet against pathogenic microorganisms,” IEEE Trans. Plasma Sci., vol. 40, no. 11, pp. 2879–2882, Nov. 2012. [10] A. Mai-Prochnow, A. B. Murphy, K. M. McLean, M. G. Kong, and K. Ostrikov, “Atmospheric pressure plasmas: Infection control and bacterial response,” Int. J. Antimicrobial Agents, vol. 43, no. 6, pp. 508–517, Jun. 2014. [11] J.-W. Lackmann and J. E. Bandow, “Inactivation of microbes and macromolecules by atmospheric-pressure plasma jets,” Appl. Microbiol. Biotechnol., vol. 98, no. 14, pp. 6205–6213, Jul. 2014. [12] X. Lu et al., “The roles of the various plasma agents in the inactivation of bacteria,” J. Appl. Phys., vol. 104, no. 5, pp. 053309-1–053309-6, Sep. 2008. [13] J. Goree, B. Liu, D. Drake, and E. Stoffels, “Killing of S. mutans bacteria using a plasma needle at atmospheric pressure,” IEEE Trans. Plasma Sci., vol. 34, no. 4, pp. 1317–1324, Aug. 2006. [14] H. Yamazaki, T. Ohshimira, Y. Tsubota, H. Yamaguchi, J. A. Jayawardena, and Y. Nishimura, “Microbicidal activities of low frequency atmospheric pressure plasma jets on oral pathogens,” Dental Mater. J., vol. 30, no. 3, pp. 384–391, May 2011. [15] J. L. Walsh, F. Iza, N. B. Janson, V. J. Law, and M. G. Kong, “Three distinct modes in a cold atmospheric pressure plasma jet,” J. Phys. D, Appl. Phys., vol. 43, no. 7, pp. 075201-1–1075301-15, Feb. 2010. [16] X. Pei et al., “Inactivation of a 25.5 μm Enterococcus faecalis biofilm by a room-temperature, battery-operated, handheld air plasma jet,” J. Phys. D, Appl. Phys., vol. 45, no. 16, pp. 165205-1–165205-6, Apr. 2012. [17] S. Schneider et al., “The role of VUV radiation in the inactivation of bacteria with an atmospheric pressure plasma jet,” Plasma Process. Polym., vol. 9, no. 6, pp. 561–568, Jun. 2012. [18] S. Schneider, J.-W. Lackmann, F. Narberhaus, J. E. Bandow, B. Denis, and J. Benedikt, “Separation of VUV/UV photons and reactive particles in the effluent of a He/O2 atmospheric pressure plasma jet,” J. Appl. Phys. D, Appl. Phys., vol. 44, no. 37, pp. 295201-1–295201-10, Jun. 2011.

Konstantin Georgiev Kostov received the B.S. degree in physics and the Ph.D. degree in plasma physics from Sofia University, Sofia, Bulgaria, in 1984 and 1994, respectively. He was a Post-Doctoral Fellow with McMaster University, Hamilton, ON, Canada, from 1995 to 1996. From 1998 to 1999, he held a post-doctoral position with the Institute for Space Research, São Paulo, Brazil, where he was a Visiting Researcher from 2001 to 2003. In 2004, he joined the Department of Physics and Chemistry with the Faculty of Engineering of Guaratingueta, São Paulo State University, São Paulo. His current research interests include gas discharges, plasma immersion ion implantation, and atmospheric pressure plasmas, such as dielectric barrier discharges and cold plasma jets for material surface modification and decontamination.

Aline Chiodi Borges was born in Piracicaba, Brazil, in 1984. She received the B.S. degree in pharmacy from the Universidade Ferderal de Ouro Preto, Ouro Preto, Brazil, in 2009, and the M.Sc. degree in oral biopathology from São Paulo State University, São Paulo, Brazil, in 2012, where she is currently pursuing the Ph.D. degree with the Oral Biopathology Graduate Program. Her current research interests include medical microbiology, environmental microbiology, innovative antimicrobial methods, and antimicrobial drugs from natural products.

775

Cristiane Yumi Koga-Ito was born in São Paulo, Brazil, in 1972. She received the D.D.S. degree in dentistry from São Paulo State University, São Paulo, in 1993, and the M.S. and Ph.D. degrees in oral biology and pathology from the State University of Campinas, Campinas, Brazil, in 1995 and 1997, respectively. She has held a post-doctoral position with the University of Torino, Turin, Italy, from 1997 to 1999, and Federal University of Belo Horizonte, Belo Horizonte, Brazil, from 2000 to 2001. She has been with the Instituto Carlos Malbran at Buenos Aires, Buenos Aires, Argentina, and the University of Hong Kong, Hong Kong, for research visits. She has authored over 115 articles and two chapters. She supervised one postdoctoral, four Ph.D., nine master’s, and 66 undergraduate research mentorship program students. She is currently supervising one post-doctoral, two Ph.D., and 10 undergraduate students. Her current research interests include medical microbiology, environmental microbiology, molecular biology, innovative antimicrobial methods, and antimicrobial drugs.

Thalita Mayumi Castaldelli Nishime was born in São Paulo, Brazil, in 1991. She received the B.S. degree in physics from São Paulo State University (UNESP), São Paulo, in 2013, where she is currently pursuing the M.S. degree in physics. She was an Undergraduate Research Assistant with the Faculty of Engineering of Guaratingueta/UNESP Plasma Physics Laboratory, UNESP, from 2010 to 2013. Her current research is focused on atmospheric pressure plasmas, polymer surface modification, and biological treatments using dielectric barrier discharges and cold plasma jets.

Vadym Prysiazhnyi was born in Kiev, Ukraine, in 1984. He received the B.S. and Magister degrees in applied physics from the Taras Shevchenko National University of Kiev, Kiev, in 2008, and the Ph.D. degree in plasma physics from Masaryk University, Brno, Czech Republic, in 2012. He was a Researcher with the Research and Development Centre for Low-Cost Plasma and Nanotechnology Surface Modifications, Masaryk University, from 2010 to 2013. Since 2013, he has been a Post-Doctoral Researcher with the Faculty of Engineering of Guaratingueta, São Paulo State University, São Paulo, Brazil. He has authored 14 articles in international peer-reviewed journals. His current research interests include cold atmospheric pressure plasmas (barrier discharges, corona discharges, and plasma jets), surface modifications of metalloids and oxides, and polymers and composites using those plasma discharges with special insight on surface analysis techniques.

Roberto Yzumi Honda received the B.S. degree in physics from the University of São Paulo, São Paulo, Brazil, in 1976, and the M.S. degree in science and the Ph.D. degree in electrical engineering from the State University of Campinas (UNICAMP), Campinas, Brazil, in 1980 and 1993, respectively. He was an Assistant Professor with the Federal University of Amazon, Manaus, Brazil, in 1981. In 1982, he joined the Tupa Project Team, which is a large theta-pinch device at UNICAMP. In 1985, he became an Assistant Professor with the Universidade Federal Fluminense, Niteroi, Brazil. In 1995, he joined the Faculty of Engineering of Guaratingueta with São Paulo State University, São Paulo, Brazil, as a faculty member. His current research interests include Q-machines, microwave plasma devices, plasma diagnostics, material processing, and teaching.