Sterilization efficiency of a novel electrochemical disinfectant against. Staphylococcus aureus

Transcription

1 Sterilization efficiency of a novel electrochemical disinfectant against Staphylococcus aureus Qian Zhang 1, #, Ruonan Ma 1, #, Ying Tian 1, Bo Su 1, Kaile Wang 1, Shuang Yu 1, Jue Zhang 1, 2, * and Jing Fang 1, 2 1 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing , P. R. China 2 College of Engineering, Peking University, Beijing, P. R. China # Qian Zhang and Ruonan Ma contributed equally to this work. *Corresponding Author :Tel: ;Fax: ; zhangjue@pku.edu.cn (Dr. J. Z.). Pages: 7 Figures: 1 Figure S1: The thermographic measurement of plasma near the water surface during PAW generation. S1

2 Supplemental Data 1. Non-thermal Plasma Device and PAW Generation The air plasma generator is schematically illustrated in Figure 1, which was designed based on HEDBS structure. A detailed description of the working principle of HEDBS has been introduced by Yu et. al.. 1 The detailed description of device was in the SI. The whole system, mainly consisting of copper electrodes and quartz dielectric, is set at the end of a quartz tube with the inlet diameter of 1.5 mm. Air with 260 L/h gas flow rate is injected into the quartz tube, and the high voltage electrode is connected to a power source with 20 khz. A homogeneous plasma is generated in the discharge gap of 0.5 mm and a plasma jet reaching lengths of up to 7 mm long is ejected through the end outlet of 0.5 mm. All the parts of the device are fixed to each other to prevent accident displacement. A thermal imaging camera (FLIR E50, USA) was employed to measure the temperature values of PAW during its generation. The mean temperature of the non-thermal plasma near the water surface is 34.2 C (Figure S1). S2

3 Figure S1. The thermographic measurement of plasma near the water surface during PAW generation. 2. Evaluation of the Physiological Changes of S. aureus The objective of this experiment was to evaluate the physiological changes of S. aureus after PAW treatment to explore the mechanisms of PAW sterilization. The parameters examined after PAW were as follows: the leakage of intracellular nucleic acid and protein, cell membrane potential, intracellular ph, structural changes of DNA, and the changes of chemical bonds of molecule. Experimental protocol Detection of the Leakage of Intracellular Nucleic Acid and Protein For the evaluation of cell membrane integrity, 1ml of the S. aureus suspension was added into the 9 ml PAW generated as above to harvest enough cells for the following detection. After 5-PAW treatment for 3, 5, 10 min, the bacterial suspensions were centrifuged immediately for 10 min at 5000 rpm, 4 C. The released DNA/RNA and proteins in supernatant were detected through detecting the absorbance of 3µL S3

4 samples at 260nm and 280nm on a SPECTROstar Omega absorbance plate reader with a Rapid UV/Vis spectrometer (BMG LABTECH, Germany). Cell Membrane Potential Measurement The BacLight Bacterial Membrane Potential Kit (Invitrogen, B34950) was used to determine whether PAW treatment induced a drop in the membrane potential by flow cytometry following the manufacturer s (Becton Dickinson, USA). Non-PAW-treated cells treated with Carbocyanine dye 3, 3 -diethyloxa-carbocyanine iodide (DiOC 2 (3)) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were set as the negative and positive control, respectively. For each measurement, at least cells were analyzed by flow cytometry. A detailed description of this approach can be found in our previous paper. 2 Intracellular ph Detection The ph standard buffer A consisted of 133 mm KCl, 7 mm Choline Chloride, 1 mm CaCl 2, 2 mm KH 2 PO 4, 5 mm glucose, and 6 mm HEPES, and its ph was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. The buffer B contained 135 mm NaCl, 5 mm KCl, 1.8 mm CaCl 2, 0.8 mm MgSO 4, 5 mm glucose, and 10 mm HEPES, and the ph of buffer B was adjusted to 7.4. These two buffers were stored at 4. A 5 mm Nigericin (Sigma, USA) stock solution was stored at -20. The ph sensitive fluorescent probe 2, 7 -bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (BCECF-AM) (Beyotime, China) was dissolved in DMSO (5 S4

5 mm) and stored at -20. The ph i was measured by the BCECF-AM probe as previously described. 3 The ph standard curve needs to be established by following procedure. The stationary cultures of S. aureus were grown overnight (OD600, approximately 1.3), and exponential-phase cultures were harvested from mid-exponential growth (OD600, approximately 0.4). The exponential-phase bacterial cells were washed twice (5 min each time) with the buffer B; then each group of the cells were incubated in buffer B containing 5 mm BCECF-AM for 1 h and washed twice (5 min each time) with the buffer B; then each group of the cells were incubated in buffer B containing 5 mm BCECF-AM for 1 h and washed twice with each given-ph buffer A. Thereafter, each group of cells was treated with 5 mm Nigericin dissolving in each given ph buffer A for 15 min. After above described procedure, the cells in each group were resuspended in 1 ml buffer A with graded ph. The BCECF fluorescent intensity was measured by fluorimeter (Synergy H4 Hybrid Reader, BioTek, USA). Set the fluorimeter to alternately measure at excitation wavelengths 435 nm and 495 nm, both with an emission wavelength of 525 nm. Set the sample chamber temperature to 30. Obtain a calibration curve by calculating the ratio of fluorescence at 495 nm to 435 nm for each ph value of solution A. The fluorescence ratio is then plotted vs. ph to get a calibration curve. After 5-PAW treatment for 3, 5 and 10 min, the ph i value of S. aureus were measured as above-described, except that the solution A was replaced by the solution B without Nigericin. The fluorescence ratio was recorded and the ph i of PAW-treated bacterial cells was calculated using the ph i calibration curve. S5

6 Analysis of Structural Changes of DNA To measure the structural changes of intercellular DNA after PAW treatment, the genomic DNA of bacteria were extracted by Genomic DNA Mini Preparation Kit (D0063, Beyotime, China) with spin column following the standard molecular biology protocol and resuspended in 50 µl water. The genomic DNA extracted from cells treated by 5-PAW for 3, 5, 10 min was loaded on a 1% agarose gel and run for 0.5 h. And then, the DNA bonds was visualized on a long wave UV light box and photographed with a Polaroid camera. Assessment of the changes of Chemical Bonds of Molecule in S. aureus Conformational changes in proteins and internal vibrational modes of lipid acyl chains of the membrane of S. aureus were measured by Fourier transform infrared spectroscopy (FTIR). S. aureus were washed and harvest after treatment, and next were dried in a drying oven at 37. Then, the cells were mixed with bromate kalium (KBr)powder. The sample and KBr powder is transferred carefully to a Specac pellet die. The pellet die is evacuated for three minutes and placed inside a Specac hydraulic pellet press. The axial load is increased slowly to a maximum value of 15 tons, held at pressure for 3 minutes and very slowly released to atmospheric. The solid KBr specimen is immediately placed in a sample holder and placed in the purged infrared spectrometer. The resulting solid pellet of sample suspended in KBr is crystal clear. KBr is hydroscopic and exposed to water vapor clouds very quickly. S6

7 Since the KBr is precisely weighed, the resulting pellet is always the same thickness. An absorption spectrum is collected over wave numbers ranging from 400 to REFERENCES: 1. Yu, S.; Chen, Q.; Liu, J.; Wang, K.; Sun, S.; Jiang, Z.; Zhang, J.; Fang, J. Dielectric Barrier Structure with Hollow Electrodes and Its Recoil Effect. Appl. Phys. Lett. 2015, 106 (244101), Tian, Y.; Ma, R.; Zhang, Q.; Feng, H.; Liang, Y.; Zhang, J.; Fang, J. Assessment of the Physicochemical Properties and Biological Effects of Water Activated by Non-Thermal Plasma Above and Beneath the Water Surface. Plasma Process. Polym. 2015, 12, Chen, M., Huang, S., Zhang, X., Zhang, B., Zhu, H., Yang, V., and Zou, X., J. Cell. Biochem. 2012, 113, S7