BLP-TES produces nitrogen gas plasma by means of a fast high-voltage pulse applied using a SI thyristor power supply as described in our previous studies (5–7). Cathode electrodes (earth electrodes) were placed between the anode electrodes (high voltage electrodes). All bacterial spore samples and chemical indicators were placed on a plastic net on the earth electrodes during nitrogen gas plasma treatment. First, the chamber box containing the sample was decompressed and degassed, and then nitrogen gas (99.9995%, Okano, Co., Ltd., Kadena, Japan) was introduced. The pressure in the box was maintained at ~0.5 atmospheres during the discharge at 1.5 kilo pulse/sec (kpps).

A filter paper strip and SUS disk coated with G. stearothermophilus ATCC7359 (SGM Biotech Inc., Bozeman, MT, USA) were used for treatment. In certain experiments, a tablet containing G. stearothermophilus EZ-SPORE (Microbiologics, Inc., St. Cloud, MN, USA) was used. Each tablet was suspended in 1 ml of hydration buffer (Microbiologics, Inc.) in accordance with the manufacturer's protocols and then spotted and dried onto a cover glass. The SUS disk, filter paper, or cover glass were treated with nitrogen gas plasma. In all cases, the treated and untreated G. stearothermophilus was mixed with tryptic soy broth (TSB) pH 7.3±0.2 (Raven Japan Co., Ltd., Koshigaya, Japan) and pH indicator bromocresol purple (BCP) prior to incubation at 56°C. A color change in the medium as a result of proliferation of the bacteria served as an index of viability.

Plasma-treated or untreated G. stearothermophilus samples on paper filters or SUS disks were resuspended in distilled water and genomic DNA was then eluted by heat treatment at 100°C for 15 min. The liberated DNA samples were subjected to PCR amplification comprising 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a final incubation at 72°C for 10 min. GEOBAC primers (GEOBAC-F and GEOBAC-R) were used in the amplification and based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences. GEOBAC-F (5′-TAAGCGTGAGGTCGGTGGTTC-3′) targeted the gene of tRNA^Ile, and GEOBAC-R (5′-GCGCTCTCGGCTTCTTCCTT-3′) targeted the 3′ end region of Geobacillus ITS (13). Gstearo-16S primers were targeted to 16S rRNA of G. stearothermophilus and designed to the DNA sequence with Genbank Accession no. EU484358 (www.ncbi.nlm.nih.gov/genbank). The primer sequences were as follows: Forward, 5′-CTTCGGGTCGTAAAGCTCTG-3′ termed F1′ and reverse, 5′-CCTTTGAGTTTCAGCCTTGC-3′ termed R1 and 5′-GAATTCCGCTCTCCTCTCCT-3′ termed R2. All the amplifications were performed using a PC320 model thermocycler (Astec Co. Ltd., Kasuya, Japan). The PCR products were initially analyzed by agarose gel electrophoresis and then subsequently verified by DNA sequencing. The PCR products obtained by standard PCR and qPCR were subcloned into pT7Blue T-vector (EMD Millipore, Billerica, MA, USA) and subjected to DNA sequencing (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems; Thermo Fisher Scientific, Inc.) to verify the identity of the amplified product.

The extracted genomic DNA samples were subjected to quantitative PCR using SYBR Premix Ex TaqII (Tli RNase H Plus; Takara Bio Inc., Otsu, Japan) and the following primers for 16S rRNA of G. stearothermophilus (Genbank Accession no. EU484358): Forward, 5′-CACACTGGGACTGAGACACG-3′ and reverse, 5′-CATTGCGGATTCCCTAC-3′ for region 1; forward, 5′-ACGGTACCTCACGAGAAAGC-3′ and reverse, 5′-TCGCCCCCTACGTATTACC-3′ for region 2; and forward, 5′-CATTCGGTTGGGCACTCTA-3′ and reverse, 5′-AAGGGGCATGATGATTTGAC-3′ for region 3. The thermocycling conditions used for the quantitative PCR were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 1 min. Relative quantification was performed using the 2^−∆∆Cq method (14).

The G. stearothermophilus-contaminated paper strips were treated with nitrogen gas plasma (1.5 kpps) for 0 and 15 min, and then fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) overnight at 4°C. The cover glasses were subsequently treated with 2% osmium tetroxide at 4°C for 3 h. Samples were dehydrated through a graded ethanol series (50–100% ethanol) at room temperature. Finally, the samples were subjected to critical point drying and evaporation coating by osmium plasma ions. SEM was performed using a JSM-6320F (JEOL Ltd., Tokyo, Japan) instrument at 5 kV using a magnification of ×20,000.

The temperature in the box during operation of the instrument was measured using THERMO LABEL 5E (NiGK Corporation, Kawagoe, Japan), which was placed on the earth electrode. The ambient temperature in the nitrogen gas plasma instrument box was measured using a fiber optic thermometer (FT1420A; Takaoka Electric MFG. Co. Ltd., Tokyo, Japan) during nitrogen gas plasma treatment.

Indicator label-H (NiGK Corporation) was placed on the earth electrode and treated with nitrogen gas plasma (1.5 kpps) for 0–30 min. Analysis of emission during operation of the nitrogen gas plasma instrument was performed using a USB multichannel spectrophotometer (S-2431; Soma Optics, Ltd., Tokyo, Japan).

Chemical indicators were used to estimate the respective concentration of 2–80 mg/l NO₂⁻ (nitrite; Kyoritsu Chemical-Check Lab., Corporation, Tokyo, Japan), 10–500 mg/l NO₃⁻ (nitrate; Kyoritsu Chemical-Check Lab. Corporation) and 0.5–25 mg/l H₂O₂ (Merck Ltd. Tokyo, Japan). The following indicators were used: Quantofix Nitrite (Macherey-Nagel, GmbH & Co. KG, Düren, Germany), Quantofix Nitrite 3000, Quantofix Peroxide 25, Quantofix Peroxide 100, Nitrite, Quantofix Nitrate and Quantofix Active oxygen, all obtained from Macherey-Nagel GmbH, Duren, Germany), which were placed on a plastic net on the earth electrodes prior to treatment with nitrogen gas plasma using a BLP-TES device at 1.5 kpps for 0–30 min.

For heat treatment, a suspension of G. stearothermophilus at 3.1×10⁴ colony-forming unit (CFU)/ml was incubated at temperatures ranging from 40–100°C for 30 min using a block incubator (BI-516S; Astec Co. Ltd). For UV treatment, filter papers contaminated with G. stearothermophilus (2.1×10⁶ CFU) were exposed to long wavelength UV-A and short wavelength UV-C radiation from a handheld UV transilluminator (UVGL-58; UVP, Inc., Upland, CA) for 30 min on each side of the paper strip (distance from lamp to paper strip, 1.3 cm). The energy (mJ/cm²) of UV-A and UV-C was estimated on the basis of a color change of UV indicator (UV label-H). Samples of G. stearothermophilus suspension (3.1×10⁴ CFU/ml) were subjected to oxidative stress by incubation in the presence of hydrogen peroxide (0–3%; Wako Pure Chemical Industries, Ltd., Osaka, Japan), peroxynitrite (0–4.92 mM; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) or 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1; 1.0 mM-1.0×10⁻⁵ mM; Dojindo Molecular Technologies, Inc.), which produces superoxide anions and nitric oxide resulting in the generation of peroxynitrite.

G. stearothermophilus samples on filter papers were frozen at −80°C overnight and dipped into 500 µl of High Molecular Weight (HMW) buffer containing 10 mM Tris-HCl (pH 8.0), 0.1 M ethylenediaminetetraacetic acid (pH 8.0), and 0.5% sodium dodecyl sulfate prior to boiling for 10 min. Following centrifugation at 10,000 × g for 15 min, an aliquot (50 µl) of the supernatant was subjected to the ELISA. To detect 8-hydroxy-2′-deoxyguanosine (8-OHdG), Highly Sensitive 8-OHdG Check ELISA kit (Japan Institute for the Control of Aging, NIKKEN SEIL Co., Ltd., Fukuroi, Japan) and 8-OHdG Assay Preparation reagent set (Wako Pure Chemical Industries, Ltd.) was used in accordance with the manufacturer's protocols. In this kit, a competitive ELISA utilizing monoclonal antibody (clone N45.1), which is highly specific for 8-OHdG, was employed. The concentrations of 8-OHdG was quantified by comparison of absorbance at a wavelength of 450 nm with standards of 8-OHdG diluted with HMW buffer.

The results are presented as the mean ± standard deviation of replicate experiments (n=6). The statistical analysis of significant difference between plasma-treated and untreated samples was performed by Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.

The inactivation efficiency of the nitrogen gas plasma was investigated (Fig. 1). The bacterial spores of G. stearothermophilus were spotted and dried onto a SUS disk or filter paper and treated with nitrogen gas plasma at 1.5 kpps for 0, 5, 15 or 30 min. The spores were then incubated in TSB medium containing BCP at 56°C for 6, 24 and 48 h and the change in pH of the culture broth was used as the index of bacterial proliferation. The results after 6 h incubation of the spores taken from the SUS disk (initial population, 1.9×10⁶ CFU) and filter paper (initial population, 1.4×10⁶ CFU) indicated that nitrogen gas plasma treatment decreased the number of viable bacterial spores on the surface of the two materials. However, longer incubation of the samples (24 and 48 h) demonstrated that the inactivation efficiency was greater for the spores recovered from the filter paper compared with those from the SUS disk.

The fraction negative method, calculated using the Halvorson-Ziegler formula (15) and Stumbo-Murphy-Cochran procedure (16), indicated the decimal reduction time (D-value) for the filter paper samples was 2.48 min at 15 min of treatment, while the D value for the SUS disk could not be calculated, suggesting lower inactivation efficiency in the SUS disk.

SEM analysis was performed to investigate the effect of nitrogen gas plasma treatment on the structure of the bacterial spore surface (Fig. 2). Careful examination indicated no evidence of shrunken or aggregated spores after nitrogen gas plasma treatment. However, spores subjected to nitrogen gas plasma treatment for 15 min exhibited a slight increase in surface roughness compared with untreated spores.

Potential changes to the bacterial genomic DNA induced by nitrogen gas plasma treatment were observed (Figs. 3 and 4). SUS disks (disk) and filter papers (strip) were treated with nitrogen gas plasma for 0 and 15 min and then subjected to PCR analysis using the three pairs of primers designed against the sequence of G. stearothermophilus 16S rRNA (F1+R1, F1+R2, and GEOBAC-F+GEOBAC-R; Fig. 3). Agarose gel electrophoresis indicated that no PCR products were amplified from genomic DNA samples of G. stearothermophilus after 15 min treatement with nitrogen gas plasma on a SUS disk or filter paper. By contrast, PCR products of the anticipated size were generated from the untreated samples. Quantitative analysis using qPCR, which employed three pairs of primers designed against three different regions of 16S rRNA (region 1–3), demonstrated a decrease of intact genomic DNA in the nitrogen gas plasma treated bacterial spores of G. stearothermophilus compared with untreated samples (Fig. 4). The quantity of intact DNA detected in the nitrogen gas plasma treated samples was ~1/10 that of the untreated samples. DNA sequence analysis demonstrated that in each case the anticipated product had been amplified. Specifically, 99–87% (F1+R1), 99–96% (F1+R2) for Genbank Accession no. EU484358, and 100% (GEOBAC-F+GEOBAC-R) for Genbank Accession no. EU157949, 99% (region 1), 100% (region 2) and 100% (region 3) for Genbank Accession no. KJ722527. Furthermore, the ELISA demonstrated a significant increase in the level of 8-OHdG in G. stearothermophilus containing filter paper and SUS disk following nitrogen gas plasma treatment (P<0.01; Fig. 5). 8-OHdG is a product of oxidative damage to DNA formed by hydroxyl radicals, singlet oxygen and direct photodynamic action (17). Thus, the results indicate nitrogen gas plasma treatment of G. stearothermophilus spores induces oxidation of the DNA.

Previous studies have confirmed that at least three variables are generated during operation of the nitrogen gas plasma instrument BLP-TES that may be responsible for its sterilizing action, namely heat, UV-A and oxidative stress (10). This is similar to other gas plasma instruments (18,19). Temperature measurement of the sample box in the BLP-TES instrument using a fiber thermometer gave readings of 65°C at 7.5 min, 75°C at 15 min, and 85°C at 30 min during operation of the device. UV measurements using a paper indicator indicated a color change in a time-dependent manner (equivalent to 0 mJ/cm² at 0 min, 25 mJ/cm² at 7.5 min, and 50 mJ/cm² at 30 min). Consistent with these findings, UV-A was detected by analysis of the emission using a spectrophotometer (S-2431; data not shown). In addition, the levels of chemicals were further estimated by the index of color change. The increase of hydrogen peroxide depended on treatment time and was estimated to be 7.5±0.9 mg/l at 30 min. Production of nitrite was also observed during operation of the instrument and was estimated to be 5.5±0.7 mg/l at 30 min, while that of nitrate was 92.7±19.0 mg/l at 30 min (data not shown).

The individual contribution of these three variables (heat, UV, and oxidative stress) to inactivation efficiency was examined. Following exposure to each of these variables, at a level equivalent to that observed during operation of the gas plasma device, spores of G. stearothermophilus (3.1×10⁴ CFU) were incubated for 48 h. The change in color of the medium was then checked against an index to determine the bacterial proliferation. The results indicated that heating at 40–100°C for 30 min did not affect the viability of the bacterial spores (Table I). UV-C treatment for 30 min (>300 mJ/cm²) using a transilluminator (UVGL-58) completely inactivated bacterial spores (100%), while UV-A treatment (75–142 mJ/cm²) for the same length of time did not (Table II). In the case of oxidative stress, treatment with peroxynitrite (4.92–4.92×10⁻⁵ mM), SIN-1 (1.0–1.0×10⁻⁵ mM) or hydrogen peroxide (3×2⁻⁵-3×2⁻⁹%) for 30 min did not inactivate the bacterial spores (Tables III–V). However, exposure of the spores to an elevated level of hydrogen peroxide (3–3×2⁻⁴%) resulted in their complete inactivation (Table III).