Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addtion

Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition Wei Chen, Jun Huang, Ning Du, Xiao-Di Liu, Xing-Quan Wang et al.. Treatment of

Treatment of enterococcus faecalis bacteria by a helium atmospheric cold plasma brush with oxygen addition

Wei Chen, Jun Huang, Ning Du, Xiao-Di Liu, Xing-Quan Wang et al

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Treatment of enterococcus faecalis bacteria by a helium atmospheric

cold plasma brush with oxygen addition

Wei Chen,1,a),b)Jun Huang,1,a)Ning Du,2Xiao-Di Liu,2Xing-Quan Wang,1Guo-Hua Lv,1

Guo-Ping Zhang,1Li-Hong Guo,2and Si-Ze Yang1,3

1

Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics,

Institute of Physics, Chinese Academy of Science, 100190 Beijing, China

2

Department of Oral Biology, Peking University School and Hospital of Stomatology, 100080 Beijing, China

3

Fujian Key Laboratory for Plasma and Magnetic Resonance, Department of Aeronautics, School of Physics

and Mechanical and Electrical Engineering, Xiamen University, Xiamen 361005, China

(Received 3 April 2012; accepted 30 May 2012; published online 6 July 2012)

An atmospheric cold plasma brush suitable for large area and low-temperature plasma-based

sterilization is designed Results demonstrate that the He/O2 plasma more effectively kills

Enterococcus faecalis than the pure He plasma In addition, the sterilization efficiency values of

the He/O2plasma depend on the oxygen fraction in Helium gas The atmospheric cold plasma

brush using a proper ratio of He/O2 (2.5%) reaches the optimum sterilization efficiency After

plasma treatment, the cell structure and morphology changes can be observed by the scanning

electron microscopy Optical emission measurements indicate that reactive species such as O

and OH play a significant role in the sterilization process.V C 2012 American Institute of Physics

[http://dx.doi.org/10.1063/1.4732135]

I INTRODUCTION

In recent years, nonthermal atmospheric pressure

plas-mas have been widely studied for several novel applications

in biomedicine116 and nanotechnology.17,18 Among the

novel applications, sterilization by low temperature

atmos-pheric pressure plasmas, which are partially ionized gases, is

attracting significant attention.19–22 Very recently, a

room-temperature, battery-operated, handheld air plasma jet was

designed and applied to effectively inactivate multilayered

Enterococcus faecalis biofilms by Lu and co-workers.23

Enterococcus faecalis is a Gram-positive facultative

anaero-bic bacterium, which has a strong adaptability to temperature

and resistance to multiple antibiotics The conventional

steri-lization methods such as heat and chemical agents are not

suitable to treat these notorious heat- and drug-resistant

pathogens By contrast, low temperature atmospheric

pres-sure plasma is a more effective technology, which can

pro-vide many chemically and biologically active species,

including various atoms, ions, energetic electrons,

metasta-ble particles, and UV irradiation It has been shown that

when these plasmas generated at atmospheric pressure are

used for sterilization, they do not cause bulk destruction of

living tissue, do not damage heat-sensitive materials, and

may be touched by humans without any harm.1However, the

available atmospheric plasma sterilization processes in

cur-rent medical uses have some drawbacks that the treatment

sizes from plasma jets,24plumes,25or plasma needles26with

a diameter of a few millimeters or less are rather small

In this paper, we report an experimental study on

inacti-vation of Enterococcus faecalis bacteria by means of an

atmospheric cold plasma brush driven by an ac power

supplier The influences of oxygen addition into the helium atmospheric plasma brush on its sterilization capability are studied After the plasma treatment, scanning electron mi-croscopy (SEM) is used to inspect the bacterial cell structure changes According to the optical emission spectra of the he-lium atmospheric plasma brush with and without oxygen addition, the roles of the various plasma agents in the inacti-vation of bacteria are investigated in detail

II EXPERIMENT

A Atmospheric cold plasma brush

Figure1(a) is a schematic diagram of the experimental setup Our atmospheric cold plasma brush comprises of two parts: a discharge chamber and two electrodes placed outside the discharge chamber The discharge chamber with a vol-ume of 80 35  1 mm3is made of quartz Both electrodes are made of copper foil of 10 mm wide wrapping the dis-charge chamber The gap between the inner edges of the cop-per foil is 15 mm The ground electrode is on the upstream side; the active electrode is on the downstream side and

10 mm apart from the chamber nozzle A sinusoidal ac high-voltage (11 kHz, 22 kV peak to peak) is applied between two electrodes for the excitation and sustaining of the discharges High-purity helium and oxygen are used as the working gas, and the flow rate is controlled by the flow meter Throughout the experimental procedure, the applied power (P) is fixed at

24 W, and the He flow rate always maintains 4500 SCCM (SCCM denotes standard cubic centimeter per minute) The separation between the nozzle and the sample is 5 mm Fig-ure1(b)shows the discharge photograph of the atmospheric cold plasma brush with 4500 SCCM helium (P¼ 24 W) The visually uniform brush-shaped plasma is formed and extended out of the discharge chamber through the narrow slit at outlet

a)

Wei Chen and Jun Huang contributed equally to this work.

b) Author to whom correspondence should be addressed Electronic mail:

chwbetter@163.com.

B Sample preparation

An overnight culture containing approximately 107

CFUs/ml is prepared (CFUs denotes colony-forming units)

Then, with filter papers as the supporting media, 5 lL of

sus-pension containing Enterococcus faecalis bacteria are

dropped onto sterilized filter papers (5 mm diameter), and

the papers are allowed to dry in a moderate vacuum

incuba-tor at 37C for 1 h Afterward, the filter papers containing

Enterococcus faecalis bacteria are exposed to the

atmos-pheric cold plasma brush sustained with He/O2 gas After

plasma treatment, the plasma treated filter papers are

trans-ferred into a 1.5 ml centrifuge tube containing 1 ml

physio-logical saline and mixed using a vortex mixer for 1 min The

bacterial strains are grown into the sterile Brian Heart

Infu-sion agar, and the number of the living bacteria cells is

counted after incubation at 37C for 48 h

III RESULTS AND DISCUSSION

A I–V curve

Voltage is measured using a 1000:1 high voltage probe

(Tektronix P6015A, maximum input voltage: DC 20 kV,

bandwidth: 75 MHz) For current measurements, a magnetic

core current probe (Tektronix P6021, maximum discharge

current: 15 A, bandwidth: 60 MHz) is utilized Signals from

the current and voltage probes are acquired and recorded by

a digital Tektronix TDS 210 oscilloscope It can be seen

from Fig 2 that the breakdown of working gas in

atmos-pheric cold plasma brush, resulting in a large number of

cur-rent filaments, the so-called micro-discharges, which are

randomly distributed both in time and space In this

filamen-tary mode,27–29the discharge starts with local gas breakdown

at many points in the discharge volume This mode is

charac-terized by a periodic current constituted by many discharge

pulses in each half cycle An inverse current peak is also

observed when the polarity of the applied voltage changes

B Sterilization effect of atmospheric cold plasma brush

Figure 3 shows the change of the survival curves of Enterococcus faecalis bacteria in the He with addition of dif-ferent amounts of O2(1%, 2.5%, 5%, 10%) into atmospheric cold plasma brush (P¼ 24 W) As can be seen in Fig.3, a 5 log reduction of the cells required only 30 s exposure time with about 2.5% oxygen addition into the helium plasma, while 90 s were required in the pure helium plasma It was also noted that 2.5% O2addition provided the faster killing speed for a complete kill of the bacteria within 60 s exposure time, while it needed 120 s to completely kill the bacteria using pure helium plasma, as shown in Fig 3 The experi-mental data shows that sterilization efficacy of He/O2plasma

is better than that of pure He plasma

C Scanning electron microscopy

Scanning electron microscopy (SEM) (Model S-5200, Hitachi, Japan) was used to examine the morphology and structural changes ofEnterococcus faecalis after plasma ex-posure The controlled and the plasma treated samples were placed in the fixation and then coated with a thin layer of plasma sputtered platinum Figure 4 shows the SEM

FIG 2 Typical oscillograms of applied voltage and discharge current of a filamentary discharge in He/O 2

FIG 3 Survival curves of Enterococcus faecalis bacteria in the He with dif-ferent O 2 additions into atmospheric cold plasma brush (P ¼ 24 W).

FIG 1 (a) Schematic diagram of the experimental setup (b) Photograph of

the atmospheric cold plasma brush with 4500 SCCM He (P¼ 24 W).

photographs of untreated control and plasma treated

Entero-coccus faecalis with pure helium plasma brush and with

oxy-gen addition at an amount of 1% and 2.5%, respectively It

can be seen from Fig.4that the plasma treatment of

Entero-coccus faecalis resulted in a significant alteration in cell

structure and morphologies when compared with the

untreated controls As seen from images in Figs.4(b)–4(d),

more structure damages were observed onEnterococcus

fae-calis with increasing the oxygen addition amount with

vol-ume percent of 0, 1%, and 2.5% Consistent with the cell

surviving curve shown in Fig.3, He plasma with 2.5% O2

addition provided a much faster killing of the bacteria From

Fig.4, it was also noted that the oxygen-based plasma

spe-cies were mainly responsible in improving the plasma

inacti-vation efficiency and oxidation capacity of Enterococcus

faecalis

D Optical emission spectra

To identify the various reactive species generated by the

atmospheric cold plasma brush, the optical emission

spec-trum (Stellarnet, EPP-2000 C) of He/O2plasma is measured

in the 200–850 nm wavelength range at atmospheric pres-sure Figure 5 shows the optical emission spectra of the plasma with 4500 SCCM He/O2(2.5%) taken at 5 mm bot-tom the nozzle along axis It is well known that UV radiation

in the 200–300 nm region with doses of several milliwatt per square centimeter may cause lethal damage to cells How-ever, the UV radiation intensity in 200–300 nm wavelength range is below 50 lW/cm2 In addition, the inactivation effect on bacteria by ultraviolet radiation is mostly related to the DNA/RNA damage in UV-C (200–280 nm).30Therefore, the UV emission plays a minor role in the inactivation of the bacteria It clearly shows that excited O lines (285 and

777 nm), OH line (309 nm), molecular nitrogen lines (C3Gu— B3Gg) (316, 337, 357, 380, and 391 nm), Oþline (427 nm), Haline (656 nm) and excited He atom lines (501,

587, 640, 669, 707, and 729 nm) are presented in the plasma brush Atomic oxygen is probably due to the direct electron impact dissociations of oxygen molecule (eþ O2

! O þ O þ e).31 O may also be formed through Penning ionization (N2þ O2! N2þ O þ O).32 , 33We assign the fea-ture at 309 nm to the OH (A2Rþ— X2G) transition;34,35the formation of which is attributed to the reaction of excited O with water vapor (H2O þ O* ! 2OH) and the electron impact dissociation (H2Oþ e! H þ OH þ e) The pres-ence of N2(C!B) lines clearly reveals the air entrainment

in the plasma brush The Ha emission line at 656 nm is formed by the collision between water vapor molecules and electrons (H2Oþ e! H þ OH þ e) The formation of

O2þ is probably due to Penning ionization (He*þ O2

! He þ O2 þþ e).36Furthermore, the addition of oxygen to the He plasma decreases He molecules’ emission,37 but enhances intensity level of reactive oxygen radicals This can be accounted for from that some electrons are probably consumed to produce O radicals Others then collided with air, water vapor, and He to produce N, OH and He radicals

In fact, reactive oxygen species (ROS) play a major role in

FIG 4 Scanning electron micrographs of Enterococ-cus faecalis bacteria of (a) untreated control, (b) helium plasma treatment, (c) He þ 1% O 2 plasma treatment, (d) He þ 2.5% O 2 plasma treatment Plasma conditions were 4500 SCCM helium flow rate and 60 s exposure time (P ¼ 24 W).

FIG 5 Emission spectra of the plasma with 4500 SCCM He/O 2 (2.5%)

taken at 5 mm bottom the nozzle (P ¼ 24 W).

bacterial inactivation38,39 and lead to various biological

effects in the intracellular space.40 These reactive species

could directly act on microorganisms, especially their outer

membranes, and damage them because of a strong oxidation

mechanism To increase the content of OH and O radicals,

increasing O2concentration is a useful method

IV CONCLUSION

In this study, an atmospheric cold plasma brush is

designed and fabricated The effect of oxygen on the

deacti-vation of Enterococcus faecalis by an atmospheric cold

plasma brush is presented Experimental results show that

oxygen addition into the plasma brush can improve the

deac-tivation efficiency of Enterococcus Faecalis The

atmos-pheric cold plasma brush using a proper ratio of He/O2

(2.5%) reaches the maximum sterilization efficiency After

plasma treatment, SEM images show obvious cell structure

damages in morphology of the organisms In addition,

opti-cal emission spectroscopy clearly indicates that there are

excited OH, O, N2, and He in the atmospheric cold plasma

brush, and OH and O radicals are mainly responsible for the

bacteria death

ACKNOWLEDGMENTS

This work was supported by the Young Scientists Fund

of the National Natural Science Foundation of China under

Grant No 11005151

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