Cold, or non-thermal, atmospheric-pressure plasma jets (CAPJ) have gained attention in biomedical applications [1
] due to unique characteristics, comprising of a complex plasma chemistry without the need for elevated gas temperatures as required for traditional thermal plasma [7
]. In addition, the high energy electrons of CAPJ can produce reactive oxygen species (ROS) with slightly higher temperature than ambient environment in an open air. The ROS produced by CAPJ plays a significant role in promising inactivation of bacteria [9
], which can be used in developing antimicrobial treatments for infectious diseases or in sterilization of reusable thermal sensitive medical devices to prevent the outbreak of antibiotic-resistant bacteria [11
]. Although these properties have led to extensive use of CAPJ in material processing and biomedical applications, consistent use of CAPJ without risks remains difficult. As the performance of CAPJ is dependent on the operational parameters, understanding how each parameter impacts the plasma’s properties is necessary and can allow for adjustment of the plasma for use in different applications.
is often used as an indicator of hygiene and safety in food products [12
], and it is commonly found in community and hospital-acquired infections. E. coli
is one of the most common and deadly pathogens causing pediatric urinary tract infection, intra-abdominal infection, or acute lung injury [13
] and can lead to life-threatening intestinal infections in immunocompromised patients [17
]. In addition, empiric antibiotic therapy may be ineffective with multidrug-resistant E. coli
which will delay care and may cause further harm to the patient [18
]. Therefore, we selected E. coli
to investigate the antimicrobial efficiency of CAPJ treatment. The aim of this work is to develop the interventions by CAPJ treatment to prevent E. coli
The influence of operational parameters of CAPJ, such as operation power, CAPJ-sample distance, gas mixtures composition, and treatment periods need to be determined to generate consistent results when using CAPJ. In general, a large number of experiments are required to optimize the processing parameters to improve the antimicrobial efficiency by changing one parameter value at a time. Such approach is time consuming, costly, and labor intensive. In this study, a systematic and efficient approach using the Taguchi experimental method design [20
] is applied to the CAPJ system in determining the optimal process parameters for the highest antimicrobial efficiency. The optimal condition of CAPJ was determined experimentally and the antimicrobial mechanism of CAPJ was explored using DNA damage assay and microbial microstructure analysis.
2. Experimental Section
2.1. Plasma Experimental Setup
depicts the setup of the CAPJ used. A quartz tube with 4 mm inner diameter was covered with two parallel cylindrical pure copper electrodes, which were connected with a high voltage pulsed direct current (DC) power supply for generating the plasma jet [24
]. The distance between two Cu electrode was 15 mm. The frequency and the duty cycle of the power supply were fixed at 10,000 Hz and 50%, respectively. In this work, the He gas flow rate was kept at 5 slm (standard liter per min), and the argon gas flow rate was changed from 0 to 500 sccm (standard cubic centimeter per min). Taguchi method with L9 experiment was applied to discover the optimal sterilization effect. The CAPJs were generated under four different processing parameters in three various conditions as listed in Table 1
, including the applying voltage of 6.5, 7.5, and 8.5 kV; the distance between CAPJ downstream and sample surface of 10, 20, and 30 mm; the CAPJ treating time of 60, 180, and 300 s; and the Ar gas flow rates of 0, 200, and 500 sccm. The radical compositions at the CAPJ downstream of each experiment was detected by an optical emission spectrometer (OES, AvaSpec ULS2048L, Avantes, Louisville, CO, USA) in the wavelength ranging from 200 to 1100 nm. The integration time for the OES detection was 80 ms.
2.2. Bacterial Strain and Culture
) was used as a reference strain, which was described elsewhere [25
]. The bacteria were routinely cultured in Luria–Bertani (LB) broth (Becton Dickinson, Franklin Lakes, NJ, USA) at 37 °C for 24 h to reach the logarithmic phase and performed in the following experiments.
2.3. In Vitro Growth Inhibition Assay
was cultured in LB broth at 37 °C for 24 h. The bacterial suspension was adjusted to an optical density of 1.0 at 600 nm (OD600
), which corresponded to 1 × 105
colony-forming units (CFUs)/mL. The bacteria were untreated by CAPJ under various combinations of experimental parameters assigned as S1–S9 revealed in Table 2
. The untreated E. coli
was assigned as S0 (control). The bacteria were serial diluted in phosphate buffer saline (PBS) and plated onto LB agar plates. After incubation at 37 °C for 24 h, the viable CFUs were counted. The bactericidal activity was represented as a percentage of CAPJ treated one divided by the untreated control group (S0). The results were expressed as the means of three independent experiments performed in duplicate.
The bactericidal activity = (Nx/N0) × 100%
are the number of the viable bacteria on an untreated control S0 and CAPJ treated Sx
(x = 1–9) experiment after incubation at 37 °C for 24 h, respectively.
2.4. Taguchi Method
Four control factors of the Taguchi experiment including (A) application voltage (kV), (B) CAPJ downstream-sample distance (mm), (C) Ar gas flow rate (sccm), and (D) CAPJ treatment time (s) were applied to analyze the influence of the sterilization parameters on E coli.
Three levels were considered for each factor and shown in Table 1
. Table 2
represented the Taguchi design and structure of L9 orthogonal array reflecting the parameters and levels in Table 1
. The higher the better concept was applied to estimate the significant parameters of CAPJ sterilization treatment by data analysis and signal-to-noise (S/N) ratios. The contribution percentages of individual parameters were determined by analysis of variance (ANOVA) [26
]. The S/N ratio is defined as follows:
is the number of experiments and y
is the observed data.
A confirmation test was carried out to validate the Taguchi’s optimization approach. The summary statistic S/N at optimal conditions was calculated after the sanative parameters, which can be used to determine the optimal condition.
(S/N)opt = m + (mx − m) + (my − m)
where m is the overall mean, and mx
are the mean effect sensitive parameters at the optimal level [19
]. The L9 orthogonal array for sample designation and detailed CAPJ treated parameters was tabulated in Table 3
, which was the designed L9 orthogonal array in Table 2
filled with the corresponding parameters assigned in Table 1
2.5. Bacterial Viability Assay
The LIVE/DEAD Bacterial Viability Kit (Thermo Fisher Scientific, Camarillo, CA, USA) was subjected to analyze the viability of bacterial populations based on the membrane integrity [27
]. Two nucleic acid fluorescent dyes, SYTO9 and propidium iodide (PI), were used to determine the bacterial viability. The E. coli
bacteria suspensions, which were untreated (S0) and CAPJ treated under S10, were collected and washed with PBS. The prepared samples were stained with SYTO9 and PI, according to the manufacturer’s instructions. The stained bacteria were then analyzed by a confocal laser scanning microscope (LSM 780; Carl Zeiss, Göttingen, Germany). The quantification of fluorescence intensity was performed by using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
2.6. DNA Damage Assay
A plasmid DNA, pGL3 (2 μg/μL) (Promega, Madison, WI, USA) was untreated (S0) or S10 CAPJ treated. Both the S0 and S10 CAPJ treated plasmids were then dissolved in DNA suspension buffer. DNA solution was loaded on 1.0% agarose gel for electrophoresis. Ethidium bromide-stained DNA was visualized under UV light. Photograph was taken by using a UV transilluminator (Azure Biosystems c400; Dublin, CA, USA).
2.7. Field Emission Scanning Electron Microscope Analysis
The morphologies of untreated S0 and S10 CAPJ treated bacteria were analyzed using a field-emission scanning electron microscope (FE-SEM, JSM 6701F, JEOL, Akishima, Japan). The bacteria samples were fixed in slides with 2% glutaraldehyde for 2 h, followed by washing with saline solution, and then exposed to 25%, 50%, and 75% of ethanol for 20 min, respectively, and finally immersed in 100% of ethanol for one hour. The slides were dried using a critical point dryer for 2 h afterwards. The bacteria samples were coated with a thin platinum layer around 5 nm thick by a sputter system (JFC 1600, JEOL, Akishima, Japan).
2.8. In Vivo Evaluation
Animal experiments were performed in accordance with the ethical standard approved by the Institutional Animal Care and Use Committee, Chang Gung University (Approval No. CGU105-032). Three male Sprague-Dawley (SD) rats with body weight range of 550 ± 30 g were study. The animals were housed under controlled conditions of temperature of 21–22 °C, relative humidity of 55% to 65%, and a 12 h light/dark cycle with artificial lighting. The animals received a standard feed and water ad libitum and were acclimatized under the aforementioned conditions before wounding experiment to create one full-thickness wounds with 17 mm in diameter on each side of rat’s shoulder as described in literature [28
]. The right-side wounds were treated with S10 CAPJ, and the left side wounds were kept untreated as control (S0). Two bacterial swabs were immediately taken from each superficial wound site right after CAPJ treatment on day 0 and day 4. Swabs were immediately immersed in 3 mL PBS and inoculated 200 μL sample on LB agar plates. After incubation at 37 °C for 24 h, the CFUs were counted.
2.9. Statistical Analysis
During the ANOVA calculation, the relation of between-group comparisons was performed using the chi-square with Fisher exact test by SPSS program (version 18.0, SPSS, Inc., Chicago, IL, USA). A p-value less than 0.05 was considered statistically significant.
Infectious disease remains a great challenge in the field of public health as it is among the top 10 causes of death and also leads the cause of disability [32
]. Inadequate antibiotic therapy worsens the control of pathogenic microbes and leads to drug resistance. The discovery of new and effective antibacterial agents or methods is quite a challenge because of its time and financially consuming nature. In addition, poor sanitation and increasing international travel have made transmission of infectious diseases easier.
This study focused on the development and optimizations of a sterilization technique using CAPJ utilizing the Taguchi method to shorten to experimental time spent on trial and error. In general, a design of orthogonal arrays of Taguchi analysis provides a maximum number of main factors to be estimated to minimize experimental trials. S/N ratios are transferred from the responses in the bactericidal activity by CAPJ treatment. ANOVA can further determine the contribution of CAPJ operating parameters on the preferable efficiency of antimicrobial condition. The effectiveness of Taguchi’s optimization approach is evaluated by antimicrobial effect, where 100% was obtained with the CAPJ using parameters obtained from the Taguchi method. As determined by the Taguchi method, the optimal antimicrobial activity is achieved with CAPJ parameters of application voltage 8.5 kV, APJ-sample distance 10 mm, Ar gas flow rate 500 sccm, and treatment time 300 s. We found CAPJ treatment time affected the plasma’s efficacy the most, and it required 5 min of treatment to achieve 100% bactericidal activity. By extension, the principles and concepts of Taguchi approach can also benefit industry reducing experimental trials of the performance, quality, and cost [22
A number of studies represent very promising results for infection control by atmospheric pressure plasma [25
]. In this work, we demonstrated that CAPJ built in our lab inhibited bacterial growth. The beneficial effects of plasma in sterilization are not yet fully understood, so we used our CAPJ to explore proposed mechanisms described in the literature. The observed temperatures of this CAPJ clearly indicated that consecutive plasma treatment by CAPJ remains below human body temperature for 30 min without causing thermal injury by utilizing the application voltage lower than 8.5 kV (Figure 2
), which is an important requirement for biological applications. Previous studies reported that reactive oxygen, hydrogen peroxide, and UV photons produced by cold plasmas might target cell membrane and cell wall for antibacterial activity [34
]. From the plasma diagnostics by OES (Figure 3
), there is nearly no emission in the germicidal UV-C region around 254 nm [36
]. The effect of heat and UV radiation as the main antimicrobial mechanism for plasma component, therefore, is unlikely.
On the other hand, The Ar mixed into He-based plasma described in this study contains OH and NO radicals, nitrogen and oxygen species, and metastable species of He and Ar (Figure 3
), which probably interact with biological organisms to generate further reactive species [37
]. Previous reports demonstrated ROS from different technologies might target different components of bacterial cells [38
], which subsequently leads to the destruction of bacterial cell wall to achieve antimicrobial strategies. For example, ROS attacks the polyunsaturated fatty acids of the fatty acid membrane to initiate a self-propagating chain reaction [39
], induces lipid peroxidation in Gram-negative bacteria [40
], decomposes macromolecules such as DNA and protein [41
], and breaks important C–O, C–N and C–C bonds of the peptidoglycan structure [42
]. The distinguished emission at 309 nm in Figure 3
is a measurement for substantial amount of OH radicals, which are produced by plasma chemical reactions of dissociation and excitation of water molecules present in the air and are likely the part of plasma that is lethal toward living bacterial cells [43
]. The intensity of OH produced by CAPJ is influenced by the feed gas mixture and increases with increasing Ar flow rate similar to a previous report [45
]. The amount of OH radicals positively correlates to the antimicrobial efficiency as well. Deleterious OH radicals are the dominant reactive species and play a significant role in cell death [46
It has been known that bacterial colonization of wounds exacerbated inflammation around the injury sites and slowed the skin healing. A previous study in bacteria infected skin diseases shows that E. coli
is one of the four main Gram-negative bacteria among 90 isolated bacteria cultured from skin ulcers [48
]. In the present study, we first assessed whether CAPJ possessed bactericidal effects on pathogenic E. coli
and validated the bactericidal activity by using the animal experiment. We showed that S10 CAPJ treatment dramatically decreased bacterial load on day 4 as compared to untreated rat. However, we did not identify the bacterial species that were presented on the wounds. Our results are in line with previous in vitro studies, which showed that cold atmospheric plasma can decrease bacterial load independent of the strains [49
]. Although CAPJ can effectively reduce bacterial loads in the animal study, further identification of bacterial species is worth studying in the future research.
According to the observation of both the DNA damage assay (Figure 7
) and the morphology of bacteria after CAPJ treatment by FE-SEM (Figure 8
), CAPJ disrupts bacterial cell walls and induces DNA damage to exercise its antimicrobial effect in plasma mediated reactions. Figure 10
is a schematic that suggests the antimicrobial mechanism of CAPJ treatment noted in this study.
In this work, experimental design using the Taguchi method with a L9 orthogonal array was confirmed by S/N ratios and ANOVA, to optimize the operating parameters of CAPJ, achieving the best antimicrobial efficacy against E. coli. Parameters obtained via the Taguchi method were confirmed by 100% antimicrobial activity, with the final parameters of 8.5 kV CAPJ application voltage, 10 mm CAPJ-sample distance, 500 sccm Ar gas flow rate, and 300 s CAPJ treatment time. These parameters were further applied to wounds created on a rat model and showed a marked decrease in microbial load compared to an untreated wound, suggesting CPAJ have safe and effective application in vivo. As the intensity of hydroxyl radical produced by CAPJ is positively correlated to its antimicrobial efficiency, reactive species likely play a significant role for the plasma sterilization in this study. According to the observation of both the DNA damage assay and the bacterial cell wall integrity test after CAPJ treatment, the antimicrobial mechanism of CAPJ works through cell wall destruction and further DNA damage, thus ensuring antimicrobial activity. This makes CAPJ a promising and effective antimicrobial technique.