22G03
Molecular Biological Mechanism of Plasma Sterilization in Liquidwith the Reduced pH Method
低pH法によるプラズマ液中殺菌の分子生物学的メカニズム
Satoshi Ikawa and Katsuhisa Kitano井川 聡1), 北野 勝久2)
1)Technology Research Institution of Osaka Prefecture
2-7-1, Ayumino, Izumi, Osaka 594-1157, Japan
大阪府立産業技術総合研究所 〒594-1157 大阪府和泉市あゆみ野2-7-1
2)CAMT, Graduate School of Engineering, Osaka University
2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
大阪大学工学研究科 原子分子イオン制御理工学センター〒565-0871 大阪府吹田市山田丘2-1
Atmospheric pressure plasma jet with low gas temperature has been applied to bacterial suspension tosterilize bacteria in liquid. Successful method of bacterial inactivation in liquid was developed by theacidification of bacterial suspension (called "Reduced pH method"). Plasma treated Escherichia coli
maintained some physiological activity such as aerobic glucose metabolism, whereas proliferation function
was completely inactivated. Quantitative PCR analysis revealed plasma treatment in liquid causes
extremely little or no DNA damages as seen by UV irradiation. On the other hand, some protein moleculein plasma treated E. coli showed the increase in its molecular weight. These results suggests that themechanism of plasma sterilization in liquid is due to the chemical modification of bacterial proteins, whichleads to physiological inactivation of the proteins.

1. Introduction
Although many studies of plasma sterilization
against dried target of bacteria were carried out, notmany studies against wet target, like liquid or
animal tissue. For medical application of cold
atmosphericpressure
plasma,however,development of the method for bacterial
inactivation in liquid is necessary. Typically,
bacterial inactivation in whole of the liquid ishardly achieved by plasma application to thesurface of the liquid, because components ofplasma, electron and excited ions can not penetrate
into the liquid. Successful method of bacterial
inactivation in liquid was developed byacidification of bacterial suspension (called "Low
pH method") [1]. It is considered that strong
bactericidal activity of "Reduced pH method" isbrought by hydroperoxy radical (HOO•) generated
from the association of hydrogen ion (H+) andsuperoxide anion radical (O2–•), as shown in Fig 1.
Oxygen molecules included in atmospheric gas areexcited to produce O2–• at the interface of plasma
and atmospheric gas, then O2–• diffuses into an
acidic liquid that allows association of O2–• with H+
to generate HOO• [2]. As the nature without electriccharge, HOO• can diffuse into bacterial cytosol via
hydrophobic cell membrane, therefore HOO• shows
extremely stronger bactericidal activity thannegative charged O2–• [3].
As well as the investigations of active species

generated with plasma discharge, to elucidate themechanism to affect target bacterial composition
such as proteins or nucleic acids, that is very
important ensuring safety in plasma medicine. In
the most of studies in plasma sterilization, damagesof plasma exposed bacteria are estimated by only
electron microscopic observation. However, surface
observation presents almost no information aboutmolecular mechanism of bactericidal activity. In
this study, we examined the damages of plasma
exposed bacteria by using molecular biologicalmethods to elucidate the molecular mechanism ofbacterial inactivation in liquid by plasma exposure.

–Fig. 1 Scheme for the model of O2 • permeationthrough the cytoplasmic membrane.

2. Result and discussion
To examine bacterial inactivation in liquid,
atmospheric pressure cold plasma jet called LF(low-frequency) jet was used [4]. When
Escherichia coli suspended in acidic buffer (pH3.7) was exposed to LF jet for 10 min, no colony
was obtained on LB agar plate. However,microscopic observation of this suspension proved
no morphological change in the bacterial cells,suggesting bacterial inactivation was resulted fromphysiological damages on cytoplasmic membraneor in cytosol. To examine the physiological activity
on cytoplasmic membrane of plasma expoesd E.
coli, WST assay, a method for detection of aerobic
metabolism of glucose, was carried out. Results
show that plasma exposed E. coli, completely
inactivated colony formation ability, maintain themetabolic activity for about 40% comparing with
intact cells. This fact suggests that plasma treatedbacteria were inactivated in proliferative function
while maintaining other physiological functions. A
similar state of impaired cells is found atsterilization by ultraviolet (UV) irradiation causing
DNA damage. Thus, a quantitative PCR wasperformed to compare the effect on DNA moleculeof plasma exposure with that of UV irradiation. As
shown in Fig. 2, UV-irradiation to E. coli resulted
in decrement of DNA amplification, depending on
the irradiation period, it was confirmed that theaccumulation of DNA damage. On the other hand,plasma exposure to E. coli had no effect on DNA
amplification. These results indicate that amolecular mechanism of bacterial inactivation byplasma exposure is not due to the damage on DNA
brought by UV light.

Fig. 2 Quantitative PCR of plasma exposed andUV irradiated E. coli.

Finally, the effect of plasma exposure to proteinsin a bacterial cell was examined. Total proteins ofplasma treated E. coli were analyzed by SDS polyacrylamide gel electrophoresis, visualizing

each protein to individual band separated in its
molecular weight. When E. coli was exposed toplasma at lower pH, distinct band shift indicating
increase in molecular weight was found at some
protein molecule, whereas no shift was observed inplasma exposure at neutral pH, at which nobactericidal activity is seen. This suggests that
distinct band shift is correlated with bactericidal
activity. Increase in the molecular weight of protein
is considered to be due to chemical modification inamino acid residues, such as oxidation or nitration.
It is considered that excessive or critical chemical
modification of amino acid residues leads tophysiological inactivation of bacterial protein,
thereby the proliferation function is inactivated byplasma exposure. Additional investigation ofproteins from plasma treated bacteria is expected tobe elucidated the molecular mechanism of plasma
sterilization in liquid.
In addition to the measurement of the generated
active species and the physicochemical reactions inliquid, the affects of plasma exposure to humanbody for hemostasis, disinfection, wound healing
and so on in plasma medicine should be achievedby a similar technique.
References[1] S. Ikawa, K. Kitano, S. Hamaguchi, Plasma
Process. Polym., 7, 1, pp.33, (2010).
[2] A. Tani K. Kitano, K. Mizotani, S. Ikawa, S.
Hamaguchi, Proc. of Int. Interdisciplinary-Symposium on Gaseous and Liquid Plasmas,(ISGLP, Sep. 5-6, Sendai, Japan), p263, (2008).
[3] S. S. Korshunov and J. A. Imlay, Mol. Microbiol.,
43, pp. 95, (2002).
[4] K. Kitano, H. Furusho, Y. Nagasaki, S. Ikawa, S.
Hamaguchi,
ProceedingsofInternational
Conference on Phenomena in Ionized Gases
XXVIII,(Prague, July 2007), p1131-1134,(2007).