Atmospheric-Pressure Plasma Jet Irradiation onto Main Components of the Cell Wall and Membrane of Escherichia Coli

To clarify the mechanism underlying the inactivation of Escherichia coli (E. coli) cells subjected to atmosphericpressure argon (Ar) plasma jet irradiation, we focused on phospholipids, the main components of the cell wall and membrane of E. coli. Commercially available standard samples of phospholipids, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were irradiated with an atmospheric-pressure Ar plasma jet in air for 5 s. The phospholipids irradiated with the plasma jet were separated by thin-layer chromatography (TLC), and the results showed that PE was unchanged even after plasma jet irradiation, whereas PG was degraded into several substances. One of them was identified as lysophosphatidylglycerol (LPG), a lysophospholipid of PG, by liquid chromatography-mass spectrometry (LC-MS). From these results, we inferred that PG in the cell wall and membrane was degraded into LPG and a fatty acid by plasma jet irradiation, and that the cell wall and membrane were disrupted, inactivating E. coli. [DOI: 10.1380/ejssnt.2014.400]


INTRODUCTION
Currently, medical instruments are sterilized by various techniques, such as autoclaving (high-pressure vapor sterilization), the use of ethylene oxide gas (gas sterilization), and irradiation with gamma rays, ultraviolet rays, or electron beams (irradiation sterilization).However, all these techniques have both merits and demerits [1].As an alternative to these methods, plasma sterilization has been studied [2][3][4][5].Recently, sterilization using an atmospheric-pressure plasma jet has attracted attention [6][7][8][9][10].Our atmospheric-pressure argon (Ar) plasma jet has a low gas temperature of approximately 30 • C [11] and can achieve pinpoint irradiation as a result of the shape of the ejected plasma.
Previously, we reported that Escherichia coli (E.coli) AB1157 on an agar medium was killed upon irradiation with an atmospheric-pressure Ar plasma jet in air for 2 s, on the basis of the proliferation of cultured E. coli and the scanning electron microscopy (SEM) observation results [12,13].Because an SEM image of E. coli cells irradiated with a plasma jet showed that the cells were punctured but retained their original shape to a certain degree, we speculated that the structures of the cell wall and membrane were changed by Ar plasma jet irradiation.
In this study, we hypothesize that the changes in the components of the cell wall and membrane of E. coli upon plasma jet irradiation kill E. coli.The cell wall of E. coli is composed of a peptidoglycan layer and an outer membrane consisting of a lipopolysaccharide (LPS) outer layer and a phospholipid inner layer [14,15].The cell membrane is composed of phospholipids [16].Commercially available standard samples of phospholipids, the main components of the cell wall and membrane, were irradiated with an Ar plasma jet to investigate its effect.
The phospholipids in the cell wall and membrane of E. coli are phosphatidylethanolamine (PE, 70-80%), phosphatidylglycerol (PG, 15-25%), and cardiolipin (CL, a dimer of PG, 1-10%) [17].In this study, we focused on PE and PG.Phospholipids have a polar hydrophilic head and nonpolar hydrophobic tail [16].The head consists of a basal glycerin backbone, the C3 of which is bound to a phosphoric acid.The phosphoric acid is bound to ethanol amine in PE and glycerin in PG.In the tail, the C1 and C2 of the glycerin backbone are ester-bound to fatty acids (hydrocarbon chains).
The phospholipids were separated by thin-layer chromatography (TLC).Liquid chromatography-mass spectrometry (LC-MS) was performed to identify the degradation products of the phospholipids irradiated with a plasma jet.

II. EXPERIMENTAL
Figure 1 shows a schematic of our experimental setup.In this setup, a copper tube (inner diameter, 4 mm; outer diameter, 6 mm) used as a discharge electrode is inserted into a dielectric quartz tube (length, 50 mm; inner diameter, 6 mm; outer diameter, 8 mm), around which copper foil (thickness, 0.05 mm; width, 10 mm) is wrapped as a grounding electrode [18].When a high AC voltage is applied, dielectric barrier discharge is induced in the quartz tube between these electrodes, and the inflowing Ar gas is excited to form a plasma that is then released into the atmosphere.
The Ar plasma jet was generated using a high-voltage power source (LHV-10AC, Logy Electric Co., Ltd.) with a frequency of 9 kHz and an applied voltage of 10 kV at an Ar gas flow rate of 10 L/min.
As phospholipids, the following standard samples from Wako Pure Chemical Industries, Ltd., were used: unsaturated yolk-derived PE, saturated dipalmitoyl-PE (DPPE), and saturated dipalmitoyl-PG (DPPG).First, 100 µg of phospholipid was dissolved in a solvent (chloroform : methanol = 1 : 1) and a few droplets of the solution were dropped onto the center of a glass Petri dish and dried for solidification.Subsequently, the phospholipid was irradiated with an Ar plasma jet.The irradiation duration was 5 s and the irradiation distance (from the tip of the quartz tube to the phospholipid) was 15 mm.The plasma-irradiated phospholipid was extracted using the above solvent, and 10 µg of the phospholipid was spotted onto the lower end of a TLC plate (silica gel 60, Merck KGaA), which was then developed in a chamber.The solvent used for the development was composed of chloroform, methanol, and water (65 : 25 : 4).After the development, the TLC plate was sprayed with primulin reagent and observed under 312 nm ultraviolet light.
Negative electrospray ionization mass spectra of plasma-irradiated samples were measured with an LCMS-IT-TOF instrument (Shimadzu, Japan) combined with a Prominence high-performance liquid chromatography system (Shimadzu, Japan).A reverse-phase column (Devosil C30, 50 × 1.0 mm i.d.; Nomura Chemical, Japan) was eluted with elution solvents, solvent A (12.5% water, 10 mM ammonium formate, and 0.1% formic acid in methanol) and solvent B (2.5% water, 10 mM ammonium formate, 0.1% formic acid, and 50% 2-propanol in methanol), using a gradient elution of 20% B in A for 5 min, from 20% to 100% B in A for 20 min, and 100% B for 5 min at a flow rate of 0.05 ml/min.

III. RESULTS AND DISCUSSION
Figure 2 shows an Ar plasma jet irradiated onto a phospholipid.The entire phospholipid was completely irradiated with the Ar plasma jet.
Figure 3 shows the results of TLC for the phospholipids irradiated with an Ar plasma jet.As shown in Figs.3(a)-3(d), no changes were observed in PE, suggesting that PE may not be degraded by Ar plasma jet irradiation.For DPPG irradiated with an Ar plasma jet, in contrast, several bands other than its original band were observed, as shown in Figs.3(e) and 3(f).This result indicates that DPPG was degraded by Ar plasma jet irradiation.The degradation of DPPG was not considered to be at- tributable to the heat of the plasma because the gas temperature of the plasma used in our experiment was as low as approximately 30 • C. The factors that may have contributed to the degradation of DPPG include (1) charged particles, such as Ar + ions and electrons, and electroneutral excited Ar atoms in the Ar plasma, (2) excited nitrogen molecules (N 2 ), oxygen (O) radicals, and hydroxyl (OH) radicals generated during the reaction between the plasma and the surrounding air and water, and (3) the electric field.However, which of these factors contributed to the degradation remains unclear.The above substances were almost simultaneously generated and their actions are difficult to distinguish.In Fig. 3(f), the band below the DPPG original band is denoted as band A. The new band on the upper end of the TLC plate is denoted as band B.
In normal phase chromatography, as in this experiment, bands on the lower side of the TLC plate correspond to higher polarity hydrophilic substances, whereas bands on the upper side of the TLC plate correspond to lower polarity hydrophobic substances.Therefore, DPPG was found to be degraded into substances with high and low polarities by Ar plasma jet irradiation.
There are reports on the separation of phospholipids by TLC [19,20].The positional relationship between PG and PE on the TLC plate in this experiment was in good agreement with that in those previous reports.
Flieger et al. performed TLC to separate DPPG and reported that the band of lysophosphatidylglycerol (LPG), a lysophospholipid of DPPG, was observed below the DPPG original band [21].Considering that report, we speculated that band A in Fig. 3(f) corresponds to LPG.
Since PG was degraded by Ar plasma jet irradiation but PE was not, there may be a difference in the effect of Ar plasma jet irradiation between phospholipids.In the TLC plate, PG was located lower than PE, indicating that the polarity of PG is higher than that of PE.The difference between PE and PG lies only in the structure of their head.Therefore, the effect of Ar plasma jet irradiation on phospholipids is considered to depend on the polarity of the head of the phospholipids, and a substance with higher polarity may be more affected by the plasma.
For unsaturated phospholipids with double bonds in the fatty acids (hydrocarbon chains) in their tails, the double bonds are broken by ozone, in a process called ozone oxidation [22,23].As shown in Figs.3(a) and 3(b), no degradation products were observed for unsaturated PE, and ozone oxidation did not occur.Therefore, ozone was not considered to be present in the plasma jet.
Figure 4 shows a fragmentation pattern of DPPG in negative-ion-mode mass spectrometry.The mass-tocharge ratios (m/z) are 721 for [M-H] − , 483 for [M-H-FA] − , and 255 for [FA-H] − .Here, M is DPPG, H is a hydrogen atom, and FA is a fatty acid.
LC-MS was performed for the plasma-irradiated DPPG to identify band A corresponding to the degradation product obtained by Ar plasma jet irradiation.Figure 5 shows the LC-MS results for DPPG.For DPPG before plasma jet irradiation [Fig.5(a)], a peak is observed at approximately 22.5 min.For DPPG after plasma jet irradiation [Fig.5(b)], another low peak is additionally observed at approximately 3.4 min.Because of the reversed-phase chromatography carried out in this experiment, the sub- stance attributable to the low peak has a higher polarity and is more hydrophilic than DPPG.Therefore, the degradation product from plasma-irradiated DPPG was found to have a high polarity.Mass spectra were analyzed to identify the degradation product.As shown in Fig. 5(c), m/z for the peak observed at approximately 22.5 min was 721.According to Fig. 4, this peak was found to correspond to [M-H] − , the ion produced when a hydrogen atom is desorbed from DPPG.In contrast, m/z for the low peak observed at approximately 3.4 min was 483, as shown in Fig. 5(d).According to Fig. 4, the low peak was found to correspond to [M-H-FA] − , the ion produced when a hydrogen atom and a fatty acid are desorbed from DPPG.These results indicate that the degradation product from plasma-irradiated DPPG is a substance produced when a fatty acid is desorbed from DPPG, that is, a lysophospholipid of DPPG called LPG.Therefore, the substance corresponding to band A in Fig. 3(f) was identified as LPG.Moreover, band B in the same figure was considered to correspond to the fatty acid (simple lipid) desorbed from DPPG.
The desorption of a fatty acid from the plasmairradiated phospholipid indicates that the ester bond between the glycerin backbone and the fatty acid in the phospholipid was broken.
Lysophospholipids exert a surface active action that destroys the cell membrane and dissolves cells.Therefore, the inactivation of E. coli upon Ar plasma jet irradiation, as observed in our previous studies, is considered to be caused by the degradation of PG in the cell wall and membrane of E. coli into LPG and a fatty acid and the resulting disruption of the cell wall and membrane.
Although lysophospholipids are produced when phospholipids are degraded by phospholipase [24], the production conditions are complicated.In this study, lysophospholipids were produced simply by Ar plasma jet irradiation, demonstrating that Ar plasma jet irradiation is very effective for producing lysophospholipids.

IV. CONCLUSIONS
We focused on phospholipids, the main components of the cell wall and membrane of E. coli, and carried out experiments in which commercially available standard sam-ples of phospholipids, PE, and PG, were irradiated with an atmospheric-pressure Ar plasma jet in air for 5 s to clarify the mechanism underlying E. coli cell inactivation.
The results of TLC and LC-MS indicated that PG was degraded into LPG and a fatty acid upon Ar plasma jet irradiation for 5 s, whereas PE was not.Therefore, the effect of Ar plasma jet irradiation on phospholipids was considered to depend on the polarity of their head; phospholipids with a higher polarity are more affected by Ar plasma.The degradation of phospholipids into lysophospholipids indicated that the ester bond between glycerin and a fatty acid in the phospholipids was broken upon Ar plasma jet irradiation.
From the above discussion, PG in the cell wall and membrane of E. coli was concluded to be degraded into LPG and a fatty acid by Ar plasma jet irradiation and the cell wall and membrane were disrupted, causing E. coli cell inactivation, as observed in our previous studies using Ar plasma jet irradiation.