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Sep 9, 2015 - Xing-Min Shi, Wen-Long Liao, Zheng-Shi Chang, Guan-Jun Zhang, ... Cong-Wei Yao, Bing-Yu Ye, Ping Li, Gui-Min Xu, Si-Le Chen, and ...
IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 43, NO. 9, SEPTEMBER 2015

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Inactivation Effect of Low-Temperature Plasma on Pseudomonas aeruginosa for Nosocomial Anti-Infection Xing-Min Shi, Wen-Long Liao, Zheng-Shi Chang, Guan-Jun Zhang, Member, IEEE, Xi-Li Wu, Xiao-Feng Dong, Cong-Wei Yao, Bing-Yu Ye, Ping Li, Gui-Min Xu, Si-Le Chen, and Jing-Fen Cai

Abstract— Disinfection and sterilization in hospitals and other public places have always been the focus of attention. In our studies, dielectric barrier corona discharge (DBCD) was used to generate low-temperature plasma (LTP) to treat Pseudomonas aeruginosa (P. aeruginosa, one kind of bacteria responsible for nosocomial infections). The survival colonies of P. aeruginosa were counted with the standard plate-counting method after 30-, 60-, 90-, and 120-s exposures to DBCD. We discovered that LTP could lead to more than 5-log reduction of P. aeruginosa after 120-s treatment. Further experiments indicated that OH and excited N2 in LTP and lower pH value in bacterial suspension might synergistically inactivate P. aeruginosa by destroying its outer structure. Index Terms— Inactivation, low-temperature plasma (LTP), Pseudomonas aeruginosa (P. aeruginosa), reactive nitrogen species (RNS), reactive oxygen species (ROS).

I. I NTRODUCTION

T

HE application of atmospheric pressure low-temperature plasma (LTP) in the biomedical field has received increasing attention in recent years. Many researchers, including our group, have already proved that LTP can effectively inactivate a wide range of micro-organisms like vegetative forms, fungi, viruses, and spores [1]–[5]. LTP is regarded as one of the

Manuscript received November 24, 2014; revised March 23, 2015 and May 20, 2015; accepted August 3, 2015. Date of publication August 24, 2015; date of current version September 9, 2015. This work was supported in part by the China National Funds for Distinguished Young Scientists under Grant 51125029, in part by the National Natural Science Foundation of China under Grant 51221005, Grant 81372076, and Grant 51307133, in part by the Sci-tech Project of Shaanxi Province under Grant 2010K16-04, and in part by the Fundamental Research Funds for the Central Universities under Grant xkjc2013004. X.-M. Shi and J.-F. Cai are with the Environment and Genes Related to Diseases Key Laboratory of Education Ministry, School of Public Health, Xi’an Jiaotong University, Xi’an 710061, China (e-mail: shixingmin142@ 163.com; [email protected]). W.-L. Liao, Z.-S. Chang, G.-J. Zhang, C.-W. Yao, P. Li, G.-M. Xu, and S.-L. Chen are with the State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, Xi’an 710049, China (e-mail: [email protected]; [email protected]; [email protected]. edu.cn; [email protected]; [email protected]; [email protected]; [email protected]). X.-L. Wu and B.-Y. Ye are with the Second Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an 710004, China (e-mail: [email protected]; [email protected]). X.-F. Dong is with the Center for Disease Control and Prevention of Shaanxi Province, Xi’an 710054, China (e-mail: [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.2015.2465158

most promising approaches in the prevention of infectious diseases and nosocomial infections [6]. However, up to now, the inactivation mechanisms of LTP on micro-organisms have been unclear. In the field of electrical engineering, LTP can be generated in open air at atmospheric pressure and room temperature. So LTP is suitable for the treatment of heat-sensitive and/or vulnerable objects that cannot withstand vacuum, such as organic materials, foams, liquids, and living biological tissues [7]–[9]. Usually, dielectric barrier discharge (DBD) plasma and plasma jet are used to kill bacteria [10], [11]. Recently, a novel plasma reactor based on dielectric barrier corona discharge (DBCD), also called surface microdischarge plasma, has been invented by Morfill group and employed for hospital hygiene [12]–[14]. With advantages such as large scalable area, low cost, simple design, and convenient operation, it may be used to inactivate bacteria that can cause nosocomial infections. Pseudomonas aeruginosa (P. aeruginosa), a kind of Gram-negative bacteria, widely present in nature, is highly resistant to external environment. In hospital, P. aeruginosa can be found on water tap, bottled solution, disinfectant, liquid soaps, distilled water, and so on. It can also attach to biomaterials and catheters, as well as bronchia and urethra, forming a biofilm and causing nosocomial infections [15]. It has the highest detection rate in wound infections, especially infections after burn. If severe enough, such infections can cause septicemia, endocarditis, meningitis, visceral infections, and so on [15]. In recent years, several kinds of plasma devices have been developed, which can be used to inactivate microorganisms on the surface of endoscopic channels or to treat animal tumor cells at the single-cell level, such as plasma gun, capillary DBD, long and flexible tube DBD, and hollow core fiber plasma jets [16]–[20]. Such studies will for sure open up new opportunities for plasma technology in inactivating micro-organisms including P. aeruginosa on the surface of long channels of medical instruments and on the cavity surface in human body. In our research, DBCD was used to produce LTP in open air at atmospheric pressure, and P. aeruginosa American Type Culture Collection (ATCC) 15442 was chosen as the test model. The purpose was to investigate the inactivation effect and mechanism of LTP on P. aeruginosa.

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inoculated onto hexadecyl trimethyl ammonium bromide culture medium (hexadecyl trimethyl ammonium bromide 0.3 g/L, beef extract 3 g/L, tryptone 10 g/L, sodium chloride 5 g/L, and agar 20 g/L) and incubated for 24 h at 37 °C. Then the P. aeruginosa colonies were washed into a sterile test tube with 10-mL phosphate buffered saline (PBS). The test tube was fully shaken to obtain a homogeneous bacterial suspension. Finally, the suspension was diluted with PBS and the concentration of P. aeruginosa was determined to be approximately 7.12 × 107 colony-forming units (CFUs) per milliliters with standard plate-counting method. Fig. 1.

Experimental setup of DBCD plasma.

According to our results and other researchers’ reports, charged particles and reactive species (RS) in DBD plasma might play a dominant role in microbial inactivation [5], [21]. In this paper, we focused on the effect of RS during DBCD plasma treatment of P. aeruginosa. The components generated in LTP were detected through emission spectra. The concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in bacterial suspension were determined after P. aeruginosa was treated with LTP. Treated P. aeruginosa were observed with a transmission electron microscope (TEM). Thus, we explored plasma inactivation mechanism from the perspectives of plasma physics and bacterial morphology. II. E XPERIMENTAL A RRANGEMENTS A. Generation of LTP The DBCD plasma in ambient air was generated with a sandwich electrode configuration. The experimental system and the photo of discharge cell are shown in Fig. 1. The sandwich electrode configuration consisted of three parts: 1) a powered aluminum alloy electrode sheet of 1-mm thickness; 2) a 1-mm thick ceramic plate; and 3) a grounded stainless steel woven wire mesh (18 meshes/in). The wire diameter of the mesh was 0.25 mm. A high-voltage source was used to generate continuous sinusoidal wave with magnitude up to ∼30 kV and a frequency of ∼40 kHz. The voltage was detected with a high-voltage probe (Tektronix P6015A) and current was measured with a noninductance resistor Rm (100 ), and a capacitor Cm (0.1 μF) was used for discharge charge quantity measurement. A switch was employed for changing. Both waveforms of voltage and current were recorded through an oscilloscope (Tektronix DPO4034). Throughout our experiments, the applied voltage amplitude remained at 6 kV and 36 kHz. The discharge power density was 0.50 W/cm2 . The temperature of plasma generated by this supply was ∼26 °C, which was measured by a thermal infrared imaging viewer (Thermal CAM P30, FLIR, USA). A Mechelle spectrometer (Mechelle 5000, Andor) with an entrance slit was used to capture the emission spectra of plasma. B. Preparation of P. aeruginosa Suspension Fresh P. aeruginosa colony (ATCC 15442, supplied by the Academy of Military Medical Sciences of China) was

C. LTP Treating P. aeruginosa 50-μL suspension of P. aeruginosa was evenly spread on cover glass (2.4 cm × 5.0 cm, a thickness of 0.15 mm) for each experiment, which was operated in atmospheric air at a room temperature of ∼20 °C and a relative humidity of ∼60%. Cover glasses spread with bacterial suspension were placed under the grounded mesh electrode and treated with LTP. The distance between the grounded mesh electrode surface and the upper surface of bacterial suspension remained nearly 4 mm. The exposure times were set as 0 (meaning the control group), 30, 60, 90, and 120 s for each experimental group, respectively. After being treated with plasma, all samples remained liquid, and there was no obvious temperature change of the samples. Each cover glass was put into 5-mL PBS in a sterile centrifuge tube immediately, which was then shaken repeatedly to wash the test samples into PBS completely. After that, the centrifuge tube was centrifuged at 2000 r/min for 10 min, and the pellet was collected and suspended with 1-mL PBS. Then, P. aeruginosa was inoculated onto hexadecyl trimethyl ammonium bromide culture medium and incubated at 37 °C for 24 h. Finally, the survival colonies were counted with standard plate-counting method. In addition, the pH value of the treated bacterial sample was measured by a pH meter (Shanghai Leici Instrument Factory, Shanghai, China) to analyze possible causes of cellular rupture. D. Detection of Reactive Oxygen Species ROS is believed to be the essential element in plasma which is responsible for killing micro-organisms [6], [21]. Based on our previous plasma spectral analysis, there were many hydroxyl radicals (OH) in our LTP [22]. Therefore, OH concentration in the solution during LTP treatment was detected with the hydroxyl radical assay kit (KeyGEN BioTECH, Nanjing, China) at several time points. The measurement principles were listed as follows. Fenton reaction was commonly used to produce OH. The amount of H2 O2 was proportional to the amount of OH produced from Fenton reaction. When the electron acceptors were provided, H2 O2 reacted with Griess reagent to form a red substance. The assay was performed according to the manufacturer’s protocol with the supernatant of LTP-treated P. aeruginosa suspension. The optical density (OD) value was measured at 550 nm with a microplate reader (Tecan Spectra, Grodig, Austria). The concentration of OH in extracellular

SHI et al.: INACTIVATION EFFECT OF LOW-TEMPERATURE PLASMA ON P. AERUGINOSA FOR NOSOCOMIAL ANTI-INFECTION

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solution (Ct ) was calculated by the following formula: Ct (mM) =

ODt − ODc 1 mL × Cs × ODs − ODb Vt

(1)

where Ct represents the concentration of OH in extracellular solution; ODt , ODc , ODs, and ODb represent the absorbance of test, control, standard, and blank tubes, respectively; Cs denotes the concentration of standard tube, equal to 8.824 mmol/L (M); and Vt is the sample volume, being 0.2 mL in our experiments. There was only distilled water in the blank tube, distilled water and the acidic solution containing Fe2+ (catalyst of Fenton reaction) in the control tube, distilled water, the acidic solution containing Fe2+ , and 0.03% H2 O2 solution in the standard tube, and test sample and the acidic solution containing Fe2+ in the test tube. Standard tube was to verify the sensitivity of the test method. The blank tube and the control tube were to exclude the interference of the confounding factors (distilled water and the acidic solution containing Fe2+ ) on experimental results. E. Detection of Reactive Nitrogen Species Nitrates and nitrites are products when RNS diffuses into liquids [23]. The concentration of nitrates and nitrites in the solution during LTP treatment was detected with Griess-assay [24]. In short, the assay was performed with the supernatant of LTP-treated P. aeruginosa suspension. Nitrates in the solution were first converted into nitrites by the enzyme nitrate reductase, then nitrites reacted with Griess reagents and formed a colored azo dye product. The OD value was measured at 540 nm with a microplate reader (Tecan Spectra, Grodig, Austria). The concentration of measured nitrites (denoting the total nitrite and nitrate content) in extracellular solution (Ct ) was calculated by the following formula: Ct (μM) =

ODt − ODb × Cs ODs − ODb

(2)

where ODt , ODs , and ODb represent the absorbance of test, standard, and blank tubes, respectively; Cs denotes the concentration of standard tube, equal to 100 μM. The reason and signification of standard, blank, and test tubes were the same as those in Section II-D. F. Visualization by TEM After being treated with LTP, suspension of P. aeruginosa was washed into the centrifugal tube with PBS, which was then centrifuged at 3000 r/min for 10 min. After the supernatant was discarded, the deposit was washed three times with PBS and centrifuged at 3000 r/min for 5 min every time, then 2-mL glutaraldehyde (2.5%, Xi’an Runde Bio-Tech Co., Ltd., Xi’an, China) was dripped onto the deposit, which was then placed in refrigerator for 24 h at 4 °C. After that, the sample was fixed with osmium tetroxide (Xi’an Runde Bio-Tech Co., Ltd., Xi’an, China), gradient dehydrated with ethanol, embedded and polymerized with epoxy resin Epon812 (Shanghai Bioscience, Shanghai, China) to make a superthin slice-up. The slice-up was then double-dyed with uranyl acetate and lead citrate, and observed with a TEM

Fig. 2.

Spectrum of the DBCD plasma and its identification.

(H-600, Hitachi, Japan) in order to explore the ultrastructural change. The control group (suspension not exposed to plasma) was also visualized, and the results were compared and analyzed. G. Statistical Analysis The experiments were repeated three times for each treatment period. Values were expressed as mean ± standard deviation. All data were examined for statistical significance with the one-way analysis of variance. All analysis was conducted with Statistical Product and Service Solutions Version software (Version 13.0 for Windows). The difference with p < 0.05 was considered statistically significant. III. E XPERIMENTAL R ESULTS A. Emission Spectrum Distribution of LTP Fig. 2 shows the optical spectrum of the DBCD plasma and its identification. It was obtained in humid ambient air because LTP-treated model was bacterial suspension. The spectral lines of DBCD plasma distributed mainly over the range from 280 to 450 nm, which were identified and marked in corresponding positions. Fig. 2 demonstrates that the products of DBCD plasma in humid ambient air include ionized nitrogen molecules (N+ 2 ), excited hydroxyl radicals (OH), and lots of excited N2 , as indicated. The bands of N2 were marked with the vibration sequences v = i (i = −4, −3, −2, −1, 0, 1, 2), and their band head was 434.36 405.94, 380.49, 357.69, 337.10, 313.50, and 297.60 nm, respectively. It can be seen that excited N2 had strong intensity, while N+ 2 and OH had relatively weak intensity, indicating that excited N2 was the most common species. B. Inactivation Effect of LTP The experiments were repeated for three times. The original concentration of P. aeruginosa suspension was (7.12 ± 0.05)× 107 CFU/mL, with 50-μL bacterial suspension on each cover glass, and the original number of P. aeruginosa was approximately (3.56 ± 0.05)×106 CFU before experiment. The effect of LTP on P. aeruginosa is shown in Fig. 3.

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Fig. 3. Inactivation effect of LTP on P. aeruginosa. The numbers beside the dots indicate the standard deviation. The stars (∗ ) beside the dots indicate that P < 0.05 versus control (0 s).

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Fig. 5. Hydroxyl radical content in bacterial suspension after different plasma application times. The error bars show the standard deviation. The stars (∗ ) beside the dots indicate that P < 0.05 versus control (0 s).

Fig. 6. Total nitrite and nitrate content in bacterial suspension after different plasma application times. The error bars show the standard deviation. The stars (∗ ) beside the dots indicate that P < 0.05 versus control (0 s). Fig. 4. pH value of bacterial suspension versus exposure time. The error bars show the standard deviation. The stars (∗ ) beside the dots indicate that P < 0.05 versus control (0 s).

Fig. 3 shows that the number of survived colony of P. aeruginosa gradually decreased with longer exposure time. Compared with the untreated samples, the number of survived P. aeruginosa significantly reduced in each treatment group (P < 0.05). Furthermore, the survived P. aeruginosa decreased from (3.56 ± 0.05) × 106 to 18 ± 5 CFU within 120 s of exposure, indicating that more than 5 logs of bacteria were inactivated.

The OH content within 0-, 30-, 60-, 90-, and 120-s exposures was 48.20 ± 3.24, 804.54 ± 20.12, 467.15 ± 12.68, 378.17 ± 15.58, and 311.44 ± 12.54 mM, respectively. The results clearly show that the content of OH in the treated bacterial suspension increased sharply when the exposure time was less than 30 s. Then, the content decreased with longer exposure time. After the 60-s exposure, the decreasing rate slowed down. The OH content within 30-, 60-, 90-, and 120-s exposures was significantly higher than that of the control group (P < 0.05). E. Nitrates and Nitrites in Bacterial Suspension

The effect of LTP on the pH value of P. aeruginosa suspension is shown in Fig. 4. It can be seen that pH value rapidly decreased after the 30-s exposure. Then, the pH value gradually decreased with longer exposure. The pH value reduced from 7.03 ± 0.03 to 3.52 ± 0.12 within the 120-s exposure. The pH value within 30-, 60-, 90-, and 120-s exposures significantly decreased compared with that of the control group (P < 0.05).

The total nitrite and nitrate content in 100-μL bacterial suspension after different LTP treatment times was measured as described in methods section (Fig. 6). The results indicated that the change of nitrite and nitrate content was similar to that of hydroxyl radicals. The nitrite and nitrate content drastically increased from 27.65 ± 6.58 μM to 2003.62 ± 41.20 μM during the inception period of 30 s. Then, the content decreased with as the exposure went on. The nitrite and nitrate content within 30-, 60-, 90-, and 120-s exposures was significantly higher than that of the control group (P < 0.05).

D. Hydroxyl Radicals in Bacterial Suspension

F. TEM Images

The concentration of OH in 200-μL bacterial suspension after different LTP treatment times was assessed (Fig. 5).

Fig. 7 shows the TEM images (×60 000) of P. aeruginosa treated with LTP. Normal P. aeruginosa had a smooth surface

C. pH Value of Bacterial Suspension

SHI et al.: INACTIVATION EFFECT OF LOW-TEMPERATURE PLASMA ON P. AERUGINOSA FOR NOSOCOMIAL ANTI-INFECTION

Fig. 7. TEM photographs (×60 000) of P. aeruginosa. (a) Normal P. aeruginosa. (b) 30-s plasma treatment P. aeruginosa. (c) 60-s plasma treatment P. aeruginosa. (d) 120-s plasma treatment P. aeruginosa.

and intact cell wall and cell membrane, as shown in Fig. 7(a). Cytoplasm uniformly distributed in the intracellular space. After being treated with LTP for 30 s, morphological characteristics of bacteria had no significant difference compared with that of control, with intact cell wall and cell membrane [Fig. 7(b)]. However, when the exposure time was 60 s, most of the bacteria were damaged [Fig. 7(c)]. Cell wall and cell membrane of some bacteria were broken, and cytoplasmic contents were leaked. Edema or cytoplasmic dissolution might occur in some bacteria, resulting in a few blank areas in the cytoplasma. Fig. 7(d) displays P. aeruginosa treated with LTP for 120 s. The surface of most bacteria turned significantly rough, and their cell wall and cell membrane were broken. In the cytoplasma, nuclear material (DNA) condensed, dissolved, or disappeared. Here, the TEM picture with LTP for 90 s is not presented, and it is similar to that of 120 s. IV. D ISCUSSION Since nosocomial transmitted infections induced by P. aeruginosa are transmitted by hand contact [15], inactivation of P. aeruginosa in hospitals is very important. Now ultraviolet (UV) ray and chemical disinfectants are commonly used to disinfect air, medical instrument, water, food, and daily necessities in hospitals to kill P. aeruginosa. However, there exist shortcomings such as high costs, possible environmental pollution, adverse health effects, and so on. Fig. 3 shows that LTP induced by DBCD could reduce P. aeruginosa by more than 5-log after 120-s treatment, indicating that LTP could rapidly and effectively inactivate P. aeruginosa. Therefore, LTP will be a promising alternative disinfection technique available to inactivate micro-organisms of nosocomial infections.

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In recent years, much attention has been paid to the inactivation mechanism of LTP on micro-organisms, but it is still largely unknown. In plasma physics, UV ray, electric field, charged particles, and RS (including ROS and RNS) of the plasma are all considered bactericidal agents [25]–[28]. In our experiment, Fig. 2 shows the optical spectrum of DBCD plasma ranged from 280 to 450 nm. As UV of 200–275-nm wavelength had bactericidal effect [29], effects of UV could be neglected in this paper. Our previous study proved that effects of electric field in atmospheric LTP inactivating micro-organisms could also be neglected [5]. Considering that discharge was restrained upon the surface of grounded mesh electrode and there was no discharge channel reaching the bacterial suspension, the role of charged particles could be neglected reasonably. Therefore, RS in plasma might play a dominant role in the process of inactivating P. aeruginosa. It is known that P. aeruginosa has abundant lipid, protein, and peptidoglycan in cell wall and cell membrane [30]. The outer membrane of the cell wall and the cell membrane of P. aeruginosa are made of lipid bilayers, whose important component is unsaturated fatty acids [30], which are susceptible to attacks by OH [31], [32]. It is reported that the oxidizing nitrites can also react with unsaturated fatty acids and initiate lipid peroxidation directly [33], [34]. Embedded in the lipid bilayer, protein molecules are essentially linear chains of amino acids and are also susceptible to oxidation when placed in the radical-rich environment of the plasma [31]. Four peptide side chain (including L-alanine, D-glutamate, L -lysine, and D -alanine) is an important component part of peptidoglycan [30], which can be oxidized and broken. As shown in Fig. 2, our LTP in this paper contained ROS (OH) and RNS (excited N2 ), which were converted into nitrates and nitrites in solution. Moreover, the plasma jet supplies gas-phase electrons to the liquid surface, and the electrons react with H2 O is taken place at the plasma/liquid interface [35], [36]. For this reason, H2 O in the bacterial suspension acted as the other source of OH radicals through the following reaction: H2 O + e → OH + H + e [31]. Therefore, OH and excited N2 in LTP were the main compositions to greatly compromise the integrity of the cells of P. aeruginosa, leading to the eventual destruction. This was verified with the cell rupture in TEM observation (Fig. 7). In addition, it has been reported that RS can oxidize various biomacromolecules such as protein, lipid, nucleic acid, and inside and outside bacterial cells [5], [10], [31], [37], [38]. Figs. 5 and 6 show that, with longer exposure time, the content of OH and nitrates/nitrites in bacterial suspension initially increased, reached the peak value at 30 s of treatment, and then decreased. The possible reason might be explained as follows. During the inception period of 30 s, the outer structures (cell wall and cell membrane) of the majority of P. aeruginosa were not destroyed, RS could not enter the bacterial cells to react with various biomacromolecules described above. When the exposure time was prolonged (more than 30 s), the outer structures of most P. aeruginosa were broken and cytoplasmic contents leaked to extracellular solution. RS could not only react with various macromolecules on the outer structure and inside bacterial cells but also reacted with them in

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extracellular solution. In this way, the consumption of RS was gradually increased, leading to the results shown in Figs. 5 and 6, which was consistent with cell morphology evolution as exposure time extension (Fig. 7). The effect of pH value alteration in sample medium on the process of LTP inactivating micro-organisms has attracted attention from many researchers. Yu et al. [39] reported that LTP induced by DBD could lead to a decrease in the pH value of the medium, which might contribute to a remarkable rupture of the inactivated yeast cells. Tang et al. [40] considered that a decreased pH value was the mechanism whereby plasma exerted deleterious effects on microalgae in aqueous environments. The pH value of bacterial suspension versus exposure time of DBCD plasma is shown in Fig. 4. The pH value in bacterial suspension gradually decreased from 7.03 ± 0.03 to 3.52 ± 0.12. The optimum pH for P. aeruginosa growth was 7.2 [30]. So a pH value of about 3–4 in an extracellular environment was effective in killing P. aeruginosa. Aqueous solutions of nitrite ions at pH below 4.0–5.0 were known to be antimicrobial [41]. Therefore, we considered that reduction in pH value in bacterial suspension also played a significant role during LTP inactivating P. aeruginosa. V. C ONCLUSION In summary, DBCD plasma can effectively inactivate P. aeruginosa. OH and excited N2 in LTP and lower pH value in bacterial suspension might play a crucial role during the process of LTP inactivating P. aeruginosa. DBCD plasma has potential to inactivate micro-organisms that can cause nosocomial infections. The most promising application of this kind of plasma device in hospital hygiene is antimicrobial sanitation of disinfectants and biofilms on the surface of biomaterials and catheters. ACKNOWLEDGMENT The authors would like to thank Dr. C. Huang at the School of Medicine, Xi’an Jiaotong University, for his excellent technical help. R EFERENCES [1] G. Daeschlein et al., “Skin decontamination by low-temperature atmospheric pressure plasma jet and dielectric barrier discharge plasma,” J. Hospital Infection, vol. 81, no. 3, pp. 177–183, Jul. 2012. [2] K. Lee, K.-H. Paek, W.-T. Ju, and Y. Lee, “Sterilization of bacteria, yeast, and bacterial endospores by atmospheric-pressure cold plasma using helium and oxygen,” J. Microbiol., vol. 44, no. 3, pp. 269–275, Jun. 2006. [3] G. Fridman, G. Friedman, A. Gutsol, A. B. Shekhter, V. N. Vasilets, and A. Fridman, “Applied plasma medicine,” Plasma Process. Polym., vol. 5, no. 6, pp. 503–533, Aug. 2008. [4] X.-M. Shi, G.-J. Zhang, Y.-K. Yuan, Y. Ma, G.-M. Xu, and Y. Yang, “Research on the inactivation effect of low-temperature plasma on Candida albicans,” IEEE Trans. Plasma Sci., vol. 36, no. 2, pp. 498–503, Apr. 2008. [5] X.-M. Shi et al., “Effect of low-temperature plasma on deactivation of hepatitis B virus,” IEEE Trans. Plasma Sci., vol. 40, no. 10, pp. 2711–2716, Oct. 2012. [6] V. Boxhammer et al., “Bactericidal action of cold atmospheric plasma in solution,” New J. Phys., vol. 14, no. 11, p. 113042, Nov. 2012. [7] M. Marschewski et al., “Electron spectroscopic analysis of the human lipid skin barrier: Cold atmospheric plasma-induced changes in lipid composition,” Experim. Dermatol., vol. 21, no. 12, pp. 921–925, Dec. 2012.

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[32] T. C. Montie, K. Kelly-Wintenberg, and J. Reece Roth, “An overview of research using the one atmosphere uniform glow discharge plasma (OAUGDP) for sterilization of surfaces and materials,” IEEE Trans. Plasma Sci., vol. 28, no. 1, pp. 41–50, Feb. 2000. [33] N. Hogg and B. Kalyanaraman, “Nitric oxide and lipid peroxidation,” Biochim. Biophys. Acta, vol. 1411, Vol. 2–3, pp. 378–384, May 1999. [34] H. Rubbo et al., “Nitric oxide regulation of superoxide and peroxynitritedependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives,” J. Biol. Chem., vol. 269, no. 42, pp. 26066–26075, Oct. 1994. [35] M. Witzke, P. Rumbach, D. B. Go, and R. M. Sankaran, “Evidence for the electrolysis of water by atmospheric-pressure plasmas formed at the surface of aqueous solutions,” J. Phys. D, Appl. Phys., vol. 45, no. 44, p. 442001, Nov. 2012. [36] M. G. Kong and D. X. Liu, “Researches on the interaction between gas plasmas and aqueous solutions: Significance, challenges and new progresses,” High Volt. Eng., vol. 40, no. 10, pp. 2956–2965, Oct. 2014. [37] K. G. Kostov et al., “Bacterial sterilization by a dielectric barrier discharge (DBD) in air,” Surf. Coatings Technol., vol. 204, nos. 18–19, pp. 2954–2959, Jun. 2010. [38] X.-M. Shi, G.-J. Zhang, X.-L. Wu, Y.-X. Li, Y. Ma, and X.-J. Shao, “Effect of low-temperature plasma on microorganism inactivation and quality of freshly squeezed orange juice,” IEEE Trans. Plasma Sci., vol. 39, no. 7, pp. 1591–1597, Jul. 2011. [39] H. Yu et al., “Inactivation of yeast by dielectric barrier discharge (DBD) plasma in helium at atmospheric pressure,” IEEE Trans. Plasma Sci., vol. 33, no. 4, pp. 1405–1409, Aug. 2005. [40] Y. Z. Tang, X. P. Lu, M. Laroussi, and F. C. Dobbs, “Sublethal and killing effects of atmospheric-pressure, nonthermal plasma on eukaryotic microalgae in aqueous media,” Plasma Process. Polym., vol. 5, no. 6, pp. 552–558, Aug. 2008. [41] M. J. Traylor et al., “Long-term antibacterial efficacy of air plasmaactivated water,” J. Phys. D, Appl. Phys., vol. 44, no. 47, p. 472001, Nov. 2011.

Xing-Min Shi was born in Shaanxi, China, in 1974. He received the bachelor’s degree in public health and the M.S. and Ph.D. degrees from the School of Medicine, Xi’an Jiaotong University (XJTU), Xi’an, China, in 1999, 2004, and 2008, respectively. He has been a Teaching Assistant with the School of Medicine, XJTU, since 1999, where he is currently an Associate Professor. He has authored over 40 journal articles and conference papers. His current research interests include the mechanism of low-temperature plasma on medicine.

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Guan-Jun Zhang (M’02) was born in Shandong, China, in 1970. He received the B.S., M.S., and Ph.D. degrees in electrical engineering from Xi’an Jiaotong University (XJTU), Xi’an, China, in 1991, 1994, and 2001, respectively. He was a Visiting Researcher with the Tokyo Institute of Technology, Tokyo, Japan, from 1998 to 1999, where he was involved in electroluminescence and discharge phenomena of solid insulating materials. From 2006 to 2007, he was a Visiting Scientist with the Princeton Plasma Physics Laboratory, Princeton University, Princeton, NJ, USA, where he was involved in secondary electron emission characteristic and plasma simulation. In 2008, he was a fellow of the Japan Society for the Promotion of Science with Saitama University, Saitama, Japan, where he was involved in flashover physics in vacuum. He has been a Teaching Assistant with the State Key Laboratory of Electrical Insulation and Power Equipment and the School of Electrical Engineering, XJTU, since 1994, where he has also been a Full Professor since 2004. He has authored over 150 journal articles and conference papers, and holds 12 Chinese patents. His current research interests include high voltage insulation and discharge phenomena, in particular, dielectric response phenomena and condition maintenance of oil–paper insulation system, surface discharge and flashover across solid insulation, and low-temperature plasma and biomedical applications. Dr. Zhang was a recipient of the 2003 China National Top 100 Excellent Doctoral Dissertation Award, the 2004 China New Century Excellent Talents in University, the 2006 China Fok Ying Tong Research Award for University Young Teachers, the 2008 IEEE Chatterton Young Investigator Award of the 23rd International Symposium on Discharges and Electrical Insulation in Vacuum, the 2011 Distinguished Young Scholar of the National Natural Science Foundation of China, and other awards and prizes from the Chinese Government.

Xi-Li Wu was born in Shandong, China, in 1975. He received the bachelor’s degree in Chinese traditional medicine, the master’s degree in clinical integrated traditional and Western medicine, and the Ph.D. degree in internal medicine from Zhangjiakou Medical College, Zhangjiakou, China, in 1999, 2002, and 2010, respectively. He has been a Physician with the Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China, since 2002. He has authored over 50 journal articles and conference papers. His current research interests include the mechanism of kidney disease and traditional Chinese drug.

Wen-Long Liao received the B.S. degree from Xi’an Jiaotong University, Xi’an, China, in 2012, where he is currently pursuing the Ph.D. degree in electrical engineering. His current research interests include lowtemperature plasma technology and applications.

Xiao-Feng Dong was born in Shandong, China, in 1974. He received the M.S. degree in public health from the School of Medicine, Xi’an Jiaotong University, Xi’an, China, in 2007. He has been an Associate Chief Physician with the Center for Disease Control and Prevention of Shaanxi Province, Xi’an, since 1999. His current research interests include the inactivating mechanism of disinfectants on microorganism.

Zheng-Shi Chang received the M.S. degree in physics from Northwest Normal University, Lanzhou, China, in 2010. He is currently pursuing the Ph.D. degree in electrical engineering with Xi’an Jiaotong University, Xi’an, China. His current research interests include microplasma and its applications.

Cong-Wei Yao was born in China in 1990. He received the bachelor’s degree in electrical engineering from Xi’an Jiaotong University, Xi’an, China, in 2013, where he is currently pursuing the Ph.D. degree with the State Key Laboratory of Electrical Insulation and Power Equipment. His current research interests include modeling and application of low temperature plasma.

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IEEE TRANSACTIONS ON PLASMA SCIENCE, VOL. 43, NO. 9, SEPTEMBER 2015

Bing-Yu Ye received the bachelor’s degree in integrated Western and Chinese medicine from the Shannxi University of Chinese Medicine, Xi’an, China, in 2012. She is currently pursuing the master’s degree in integrated Western and Chinese medicine with Medical College, Xi’an Jiaotong University, Xi’an. Her current research interests include adriamycininduced nephropathy, calcium–ion channel protein, and the effects of microplasma on cell proliferation.

Si-Le Chen was born in Shaoyang, China, in 1992. He is currently pursuing the master’s degree with the School of Electrical Engineering, Xi’an Jiaotong University, Xi’an, China. His current research interests include atmospheric pressure nonthermal plasmas and its applications.

Ping Li was born in Yingshang, China, in 1981. He received the M.S. degree in electrical engineering from Guangxi University, Nanning, China, in 2007. He is currently pursuing the Ph.D. degree with the School of Electrical Engineering, Xi’an Jiaotong University, Xi’an, China. He is a Teacher with the College of Electrical and Information Engineering, Anhui University of Science and Technology, Huainan, China. His current research interests include atmospheric pressure cold plasmas and its applications.

Gui-Min Xu was born in Shandong, China, in 1984. He received the B.Sc. degree in electrical engineering from Chang’an University, Xi’an, China, in 2006, and the M.Sc. degree in electrical engineering from Xi’an Jiaotong University, Xi’an, in 2009, where he is currently pursuing the Ph.D. degree with the School of Electrical Engineering. His current research interests include gas discharge and plasma technology.

Jing-Fen Cai received the Bachelor of Medicine degree from Shanxi Medical University, Taiyuan, China, in 2014. She is currently pursuing the master’s degree in public health with Xi’an Jiaotong University, Xi’an, China. Her current research interests include the applications of low-temperature plasma in medical field.