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Sterilization efficiency of a novel electrochemical disinfectant against Staphylococcus aureus Qian Zhang, Ruonan Ma, Ying Tian, Bo Su, Kaile Wang, Shuang Yu, Jue Zhang, and Jing Fang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05108 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 17, 2016
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Environmental Science & Technology
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Sterilization efficiency of a novel electrochemical disinfectant against
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Staphylococcus aureus
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Qian Zhang1, #, Ruonan Ma 1, #, Ying Tian1, Bo Su1, Kaile Wang 1, Shuang Yu1, Jue Zhang1, 2, *
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and Jing Fang 1, 2
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1
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, P. R. China
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2
College of Engineering, Peking University, Beijing, P. R. China
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#
Qian Zhang and Ruonan Ma contributed equally to this work.
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*
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[email protected] (Dr. J. Z.).
Corresponding
Author ： Tel:
+8610-62755036 ； Fax:
+8610-62753562;
E-mail:
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Abstract
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Disinfection of hazardous microorganisms which may bring challenge to the environmental safety
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is a crucial issue for the economic and public health. Here, we explored the potential of a novel
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electrochemical disinfectant named plasma activated water (PAW), which was generated by
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non-thermal plasma, for inactivating Staphylococcus aureus (S. aureus). Meanwhile, the influence
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of bovine serum albumin (BSA) on the PAW disinfection efficacy was investigated. In the presence
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of BSA, PAW treatments achieved a reduction of S. aureus ranging from 2.1 to 5.5 Log, and when
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without BSA it reached 7 Log. The sterilization efficacy depended on the PAW treatment time of S.
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aureus and plasma activation time for PAW generation. The results of electron spin resonance
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spectra showed the concentration of hydroxyl radical (OH•) and nitric oxide radical (NO•) in water
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activated by plasma for 10 min (10-PAW) was higher than water activated by plasma for 5 min
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(5-PAW). Additionally, the physiological analysis of S. aureus demonstrated that the integrity of cell 1
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membrane, membrane potential, intracellular pH homeostasis as well as DNA structure were
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damaged by PAW, and the molecule structure and chemical bonds of S. aureus were also altered due
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to PAW. Thus, PAW can be a promising chemical-free, and environmentally friendly
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electrochemical disinfectant applied in the medical and food industry.
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INTRODUCTION
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The environment can act as a reservoir and disseminator for infection due to the extensively
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persisted pathogens in the environment. For example, meticillin-resistant Staphylococcus aureus (S.
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aureus) can persist on objects for up to seven months.1 As described in the literature, the infectious
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disease can transmit from the environment through various ways, such as person-to-person and via
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inanimate surfaces, water, hands, food, and household surfaces.2 In the last decade, we have
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witnessed a dramatic increase in the absolute number and economic cost of healthcare-associated
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and community-onset bacterial pathogens infections. 3 , 4 For instance, the World Health
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Organization estimates that approximately 4.5 million and 1.7 million patients per annum in Europe
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and the USA, respectively, are affected by Healthcare-associated infections, accounting for 0.1
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million and 0.037 million deaths per annum.5 Guidelines by the Centers for Disease Control and
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Prevention, the Food and Drug Administration, the Environmental Protection Agency, and the
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International Scientific Forum on Home Hygiene figured out the high incidence of disease due to
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the insufficient disinfection. One of the means for prevention of disease is through proper 2
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disinfection2. S. aureus is a frequent cause of infections in both the community and hospital.
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Unfortunately, the increasing resistance of this pathogen to various antibiotics complicates
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treatment of S. aureus infections. Effective measures to prevent S. aureus infections are therefore
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urgently needed.6
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Traditionally, there are a variety of disinfection methods involving radiation, thermal and
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chemical means. However, most of the methods have their own application limitations. Irradiation
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is an effective sterilization method, but the radiation can alter the physical properties and
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appearance of materials and ultimately affecting the functionality of a device utilizing these
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materials.7 For thermal method, steam autoclave is the oldest, safest and most cost effective method
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of sterilization in the medical equipment industry. However, many surgical instruments are not
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designed to withstand prolonged heat and moisture of the steam sterilization process. 8 The
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chemical methods, such as chlorine dioxide, chloramines, and ozone have now become clear that
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some of these chemicals or associated inorganic by-products bring several physical and health
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hazards to personnel and patients.9,
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With the ever increasing standard of drinking water supply and the stringent environmental
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regulations, electrochemical technologies, which are not only comparable with other technologies in
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terms of cost but also are more efficient and more compact, have regained their importance during
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the past two decades.12,
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technology, has also been extensively investigated and have shown promising results in various
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biomedical applications, thus leading to the emergence of “plasma medicine”.14,15,16
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Nowadays, non-thermal (cold) plasma, which is a novel electrochemical
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Low temperature plasma is a mixture of electrons, ions, free radicals, excided and neutral
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molecules, which has proved that can react with the water so that the plasma activated water (PAW) 3
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possesses the ability to inactivate microbial cells. 17 ,
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bactericidal activity of PAW derives from the synergistic effects of a high positive oxidation
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reduction potential (ORP) and low pH. Among various reactive chemical species in PAW, hydroxyl
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radical (OH•), atomic oxygen (O), ozone (O3) and hydrogen peroxide (H2O2) are the main reactive
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oxygen species (ROS) significantly contributing to the high ORP. Moreover, reactive nitrogen
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species (RNS) have also be considered to result in the high ORP, such as nitric oxide (NO•) and its
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derivatives formed with water, including nitrites (NO2 − ), nitrates (NO3 − ) and peroxynitrites
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(ONOOH).21,
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acidification of PAW. Recently, it was reported that PAW could also inactivate food-borne pathogen
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on strawberries and maintain postharvest quality of button mushrooms. 27 ,
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highlights that PAW is a potential alternatives to conventional sterilization methods in numerous
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settings (the food industry, hospitals and public places). To our knowledge, most of these studies
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were conducted in a clean condition which did not mimic a disinfection process in the real situation.
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However, medical sterilization often involves both bacterial inactivation and protein destruction. It
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is therefore essential to study the influence of protein on the inactivation ability of PAW before they
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can be widely accepted as a viable hospital sterilization disinfectant. Meanwhile, the
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physicochemical properties and disinfection mechanisms of PAW were not well understood. More
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importantly, to our knowledge, no evidences were provided whether reactive radicals, which might
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be responsible for the chemical and biological effects, were existed in the PAW.
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It is generally agreed that the
Meanwhile, production of RNS also plays a dominant role in the
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These studies
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In our work, a novel electrochemical disinfectant named plasma activated water PAW, which
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was generated by non-thermal plasma, for inactivating Staphylococcus aureus (S. aureus) was
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investigated. The plasma microjet (PMJ) with air as the working gas was used to generate PAW and 4
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then S. aureus was used as a model bacterium to verify its disinfection efficacy. In order to mimic a
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disinfection process in the real dirty situation and to study the influence of protein on the
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inactivation ability, disinfection tests in the presence of bovine serum albumin (BSA) were
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performed. The concentration of BSA is set according to the European Standard EN-1276.29
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Furthermore, the physicochemical properties of PAW were assessed by ORP/pH/conductivity
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meters, and electron spin resonance (ESR) spectroscopy was used to detect the short lived radicals in
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PAW. In addition, the disinfection mechanisms were further studied through assessing the cell
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membrane integrity, membrane potential (∆ψ), intracellular pH (pHi), DNA fragment as well as the
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chemical bonds of cell surface.
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MATERIALS AND METHODS
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Microorganism and Culture Conditions
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S. aureus (China General Microbiological Culture Collection Center, CGMCC number 1.2465) was
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the microorganism tested throughout this study. Loopfuls of cells from the bacterial strain was
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aseptically transferred into agarslantculture-medium. After incubation at 37 °C for 24 h, the bacteria
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were washed with saline phosphate buffer, followed by centrifugation for 5 min at 4500 rpm. The
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bacterial cells were resuspended in sterile saline to a final concentration of approximately 109
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CFU/ml. The bacterial concentration was estimated by optical density (OD) measurements at 600
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nm (OD600), considering that an OD600 = 1.5 corresponded to 109 CFU/ml of bacterial cells.
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Non-thermal Plasma Device and PAW Generation
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The air plasma generator is schematically illustrated in Figure 1, which was designed based on
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Dielectric Barrier Structure with Hollow Electrodes (HEDBS) structure. A detailed description of 5
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how HEDBS works was presented
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the supporting information (SI).
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by Yu et. al..30 The detailed description of the device was in
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As shown in Figure 1, PAW solution was produced by PMJ under the water surface, and the
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distance between the end of the plasma jet and the water surface was about 2 cm. Sterile distilled
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water of 15 mL was activated by plasma for 5 min and 10 min to obtain PAW, respectively defined
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as 5-PAW and 10-PAW. A thermal imaging camera (FLIR E50, USA) was employed to measure the
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temperature values of PAW during its generation. The mean temperature of the non-thermal plasma
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near the water surface is 34.2 °C (Figure S1).
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Disinfection procedure
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In order to mimic disinfection process in the real situation under dirty conditions, and to investigate
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the influence of proteins on the antimicrobial efficacy, bovine serum albumin (BSA) were added
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into the PAW after its generation to a final concentration of 3 g/L (according to the European
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Standard EN-1276). For the disinfection experiment, 10 mL of S. aureus cell suspension (a
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concentration of approximately 109 CFU/mL) were harvested by centrifugation for 10 min at 5000
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rpm, and then resuspended in 1 mL sterile distilled water to obtain cells final concentration of 1010
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CFU/mL. Subsequently, 100 µL of S. aureus cell suspension (concentration of approximately 1010
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CFU/ml) were added into the 9.9 ml fresh PAW with or without BSA immediately for 3, 5, and 10
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min treatment. The S. aureus suspension treated by sterile distilled water was set as the
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non-PAW-treated control. The initial bacteria concentrations for the control and experiment group
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during experiment were both around 108 CFU/ml. Disinfection procedures were performed at least
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three times for each conditions tested.
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Figure 1. (a) The photo of PAW generation, and (b)A schematic diagram of plasma jet and the
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experimental arrangement, including PAW generation and PAW treatment of S. aureus with or
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without BSA.
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Analysis of Sterilization Ability of PAW
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Colony count assays were used to demonstrate kinetic killing curves and the antimicrobial effects of
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PAW.19 In brief, after PAW treatment, ten-fold serial dilutions of 100 µL treated suspension were
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plated on Luria-Bertani (LB) agar culture medium and incubated at 37 °C for 21 h for a subsequent
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colony forming unit (CFU) count. The total number of viable bacterial cells in 10 mL PAW was
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estimated by the standard semi-log plot method. The detection limit was 100 CFU, which means if
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the number of microorganisms fell below the detection limit, i.e., no viable microorganisms were
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found, the max bacteria number in 10 mL PAW was considered to be 100 CFU. The sterilization
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ability of PAW was evaluated via the Log Reduction, which was further calculated by the formula
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below:
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Log Reduction = Log (CFUcontrol/CFUtreated)
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Evaluation of the Physicochemical Properties of PAW
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Electron Spin Resonance Spectroscopy
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The hydroxyl radical (OH•) and nitric oxide radical (NO•), as the important reactive oxygen 7
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nitrogen species (RONS), were measured immediately after PAW generation by electron spin
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resonance (ESR) spin-trapping spectroscopy, which is a magnetic resonance technique based on the
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interaction of unpaired electron spins with an external magnetic field.31 Due to the short life time of
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OH• (around 10-9 s)32 and NO• (around 5 s)33, 20 µL 5, 5-dimethyl-1-pyrroline N-oxide (DMPO)
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(0.8 M) (Sigma-Aldrich, USA) was added into the 20 µL PAW and mixed thoroughly to spin trap
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OH•, and a mixture of 0.5 mL Diethyldithiocarbamate (DETC) and 0.5 mL Fe2+ was added into 1
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mL PAW to spin trap NO• via forming a longer-lived spin adduct mononitrosyl-iron-DETC complex
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((DETC)2-Fe2+-NO). A detailed description of the ESR technique and spin-trapping approach can be
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found in the literature.34
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ORP, pH and Electrical Conductivity Measurement
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ORP, pH and electrical conductivity of PAW were immediately measured after PAW generation.
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ORP is an indicator of the general level of reactive oxygen species (ROS).35 ORP, pH were
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measured by a multimeter pH & Redox, (Mettler-Toledo LE501, Switzerland). Electrical
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conductivity was measured with electric conductivity meter (DDB-303A).
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Evaluation of the Physiological Changes of S. aureus
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The physiological changes of S. aureus after PAW treatment were evaluated in a separate
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experiment. The parameters examined after PAW were as follows: the leakage of intracellular
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nucleic acid and protein, cell membrane potential, intracellular pH (pHi), structural changes of
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DNA, and the changes of chemical bonds of molecule. The detail of the protocols is described in
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the SI.
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Statistical Analysis
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All Data were obtained from at least three replicate experiments (n ≥ 3). Values from all 8
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experiments were expresses as the mean ± standard deviation (SD). Statistical analysis was
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performed using statistical product and service solutions (SPSS) statistical package 17.0 (SPSS Inc.,
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USA). An analysis of variance (ANOVA) was conducted to evaluate the sterilization efficiency and
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intracellular pH of different PAW treatments, as well as the effects of different plasma activation
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time on ORP, pH value and electrical conductivity, and significant differences were identified by the
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Student-Newman-Keul’s multiple range tests with a confidence level at P ≤ 0.05. Moreover, the
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paired-sample t-test was applied to compare the effects of different PAW treatments on the release
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of intracellular materials. The significant difference is expressed as *P < 0.05, **P < 0.01, and ***P
