Introduction

The antimicrobial effect of direct non-thermal plasma treatment is has been widelyinvestigated and is becoming a well- known phenomenon1,2,3,4,5,6.There are different factors and mechanisms which influence the antimicrobial effect ofnon-thermal plasmas. The most common routes of microbial inactivation are reported as:UV photons generated during plasma discharge, plasma generated reactive oxygenspecies2 and reactive nitrogen species1 in the gasphase and diffusion of these species through the bacterial cell wall and membrane,physical effect of electron discharge, electrical field, UV and reactive species thatcause damages to the cell surface and localized heating effects to cell surface. Allthese routes are capable of inactivating microorganisms synergistically5,7,8. Recently, plasma treated liquids have shown an increasing interestdue to their stable antimicrobial activity even up to two years9.Different groups have reported antimicrobial properties of liquids including water, 0.9%saline solution and phosphate-buffered saline (PBS) that are treated with differentnon-thermal plasma sources. The acidification of liquids following plasma treatment isone of the most commonly reported chemical modifications9,10,11,12,13,14,15,16,17,18,19,20. In previous publication, ourgroup defined fluid-mediated plasma treatment, where a particular fluid is treated witha plasma discharge and then exposed to microorganisms in order to achieve microbialinactivation9. In fluid mediated plasma treatment, bacteriadon’t come in contact with UV and electron discharge. Therefore theinteraction of plasma generated electrons and UV radiation with the fluid being treated,and the subsequent diffusion of plasma generated ROS and RNS into treated liquid arethought to be the main cause for the antimicrobial effect9. The decreasedpH of plasma treated liquid is attributed to generation of HNO2,HNO3 and, H3O+ 9,12,16. The acidification of plasma treated liquid is critical for amicrobiocidal effect; however, it is reported that acidic pH is not the main source ofantimicrobial effect10,20,21. Ikawa et al. have reported thatacidic pH is essential for the antimicrobial effect of plasma treated liquids. They havedemonstrated that when the pH is below 4.7, the antimicrobial effect of plasma-treatedliquid is significantly higher compared to liquid with a pH greater than 4.7. Theyclaimed that pH 4.7 is the critical value for microbial inactivation and it is almostuniversal for different types of bacteria including Gram-positive, Gram-negative,aerobic and anaerobic bacteria14. In addition to decreased pH, alsonitrate (NO3−), nitrite(NO2−) and hydrogen peroxide(H2O2) have been detected in plasma treated liquids16,22,23. Plasma also generates other ROS and RNS such as OH radical,superoxide and nitric oxide3,7,11. A direct interaction of thesespecies in liquid with the electron discharge and the plasma generated ROS and RNS inthe gas phase and their diffusion into liquid may be responsible for the presence ofNO3−,NO2− and H2O2 in plasmatreated liquids. Reactions of ROS and RNS lead to the formation of other species thatmight contribute to antimicrobial effect. It is reported that plasma generates NO andsuperoxide (O2−) and their interaction yields toperoxynitrite (ONOO−) formation3,7,11,24,25,26. Another possible route for peroxynitrite formation inplasma treated liquids is the formation of nitrosooxidanium(H2NO2+) via the reaction of H+cation with NO2− anion in a highly acidicenvironment. Nitrosooxidanium (H2NO2+) is brokendown into nitrosonium (NO+) a cation that is highly reactive and canattack biomolecules in the cell and form peroxynitrous acid (ONOOH) in the presence ofhydrogen peroxide. Finally peroxynitrous acid is dissociated to peroxynitrite in aqueousmedium24,25. Lukes et al. have shown the formation ofNO2, NO. and OH. radicals and NO+ ions by plasma operatedin ambient air at the gas-liquid interface and peroxynitrite generated in plasmatreated water which played a significant role in the antimicrobial activity of plasmatreated water26. Peroxynitrite is highly reactive and can easily diffusethrough the cell membrane due to its high permeability. It attacks various biomoleculesin the cell and causes protein and lipid nitrosylation and also intracellular oxidation.Intracellular damage induced by peroxynitrite usually can’t be restored bycellular repair mechanisms and cells die27,28,29. Acidified nitrite andnitrate have been known for decades for their antimicrobial effect both in vitroand as a part of natural protection mechanism of the body30,31,32.Salivary nitrite comes in contact with the acidic content of the stomach when swallowedand acts as a natural host defense mechanism through the formation of biocidalspecies33. The antimicrobial effect of acidified nitrite is closelyrelated to RNS involving mechanisms. For example, NO release from skin has beenreported. Also natural flora of the skin reduces nitrate to nitrite. In the acidicmilieu of the skin RNS, including nitrous acid (HNO2), dinitrogen trioxide(N2O3) and peroxynitrite (ONOO−)are produced via NO and nitrite and nitrate that act as non-specific protection againstpathogens on the skin34. Various groups have demonstrated that acidifiednitrite has antimicrobial effect on various skin and oral pathogens33,34,35. The composition of the liquid being treated should beconsidered as an important factor in order to correlate the mechanism of antimicrobialeffect. As opposed to plasma-treated water and PBS, Oehmigen et al. have reportedthe antimicrobial effect of plasma-treated 0.85% saline solution (NaCl). However theyconcluded that effect of chlorine species that arises fromCl− ion could be neglected15.

Previously our group has reported that N-Acetylcysteine (NAC) solution gainsantimicrobial effect when treated with non-thermal, atmospheric dielectric barrierdischarge (DBD) plasma (operated in ambient air, without using technical gases). NACsolution was capable of inactivating a broad range of multi drug resistant (MDR)bacteria and fungi in their planktonic and biofilm forms. It was also observed thatalthough the pH of the solution dropped to the acidic side (pH 2.35), it was not themajor reason for microbial inactivation9. During the same preliminarystudy, generation of hydrogen peroxide was detected (0.42 mM,1.67 mM and 0.93 mM respectively, after 1-minute, 2-minutes and3-minutes of non-thermal atmospheric DBD plasma treatment). The interesting observationwas that the hydrogen peroxide concentration reached to saturation after2 minutes of plasma treatment and then dropped after 3 minutesof plasma treatment9.

We hypothesized that plasma treatments of NAC solution generate RNS and ROS and theirinteractions further give rise reactive product which in the presence of plasma-inducedacidic pH gets stabilized and exhibit strong antimicrobial properties. Therefore we setout to detect the major reactive species and to characterize their intermediate chemicalspecies and correlate these interactive species with antimicrobial efficacy. In thepresent study the techniques such as nitrite and nitrate detection, UV-vis spectrumanalysis, FT-IR analysis, NMR analysis, were performed in order to evaluate chemicalmodifications in NAC solution following non-thermal atmospheric DBD plasma treatment.Also, NAC solution was separated to its constituents by evaporating liquid phase.Chemical characterization was also performed for remaining NAC powder and evaporatedliquid phase following separation of plasma treated NAC solution in order to understandchemical modifications in the constituents of plasma treated NAC solution. Thus, througha combination of physico-chemical analysis we predicted that during plasma treatment ofNAC solution, ROS and RNS are formed in both gas phase and liquid phase. Low pH is thecommon result of the liquid-mediated plasma treatment. Even though high aciditydoesn’t contribute to the antimicrobial effect, it is an essential componentfor the microbial inactivation. The antimicrobial effect of this solution originatesfrom diffused ROS and RNS in liquid as opposed to direct plasma treatment, wherephysical impacts such as UV, electrical field and electron bombardment are majorcontributors to the biocidal effect.

Materials and Methods

Nonthermal Plasma settings and NAC Treatments

Plasma treatment of NAC solution was carried out as previously explained9. In brief, a custom-made glass liquid container was built whichcan hold 1 mL of liquid and can maintain 1 mm of liquidcolumn. Plasma treatment was performed for 1, 2 and 3 minutes with aDBD electrode(38 mm × 64 mm insize) that was placed above fluid holder with 2 mm discharge gap.Plasma treatment parameters were fixed as 31.4 kV and15 kHz, which yield 0.29 W/cm2 powerdistributions.

NAC solution was prepared from 100 mM stock solution. Stock solutionwas prepared by dissolving NAC powder in 1X sterile PBS and sterilized through a0.2 uM sterile membrane filter and aliquots of stock solution werekept at −20 °C. The 5 mM ofworking solution of NAC was prepared by diluting stock solution in 1X sterilePBS.

Nitrite and Nitrate Detection

A Nitrite-Nitrate Test Kit from HACH (Loveland, CO, USA) was used for thedetection of nitrite and nitrate concentrations in plasma-treated NAC solution.Plasma treated NAC samples were diluted when required. The results werevalidated by comparing with standard solutions of nitrite and nitrate followingmanufacturer’s instructions.

UV-Visible Spectrum Analysis

UV-visible spectra of solutions were collected. These solutions include the NACsolution that was treated for different time points, the peroxynitrite standardsolution, the supernatant (after centrifugation) of bacterial cell suspensionafter treatment with plasma-treated NAC solution, the peroxynitrite solution andrehydrated (with PBS) liquid of dried remnant of plasma-treated NAC solution.The UV-visible spectra were generated with a UV-visible spectrophotometer(Thermo Scientific, Hudson, NH, USA) between 190 nm and1100 nm wavelengths with 1 nm interval.

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis of thePlasma-Treated NAC Solution

Untreated and plasma-treated NAC solutions were evaporated with a rotaryevaporator. Infrared spectra of the remaining powders from the evaporation ofuntreated and 3-minute plasma-treated NAC solutions were obtained with anOlympus BX51 microscopy system (Olympus, Japan) and modified with aFourier-Transform infrared spectrometer IlluminatOR that is equipped withan ATR lens (Smith Detection, USA).

Nuclear Magnetic Resonance (NMR) of Plasma-Treated NAC Solution

NMR analysis of the NAC solutions was conducted using deuterated water(D2O) (Sigma Aldrich, St. Louise, MO, USA). Proton NMR spectrawere collected using a Varian INOVA 500 MHz FT-NMR device (PaloAlto, CA, USA). Antimicrobial effect of plasma-treated NAC solutions prepared inD2O was also tested in order to assure that its antimicrobialproperty was same as NAC solution that was prepared in H2O.

Antimicrobial Effect of Acidity and Plasma-Generated Species

In order to determine whether the reduction in pH after plasma treatment isresponsible for the disinfection effect, acetic acid (CH3COOH),nitric acid (HNO3) (Fischer Scientific, Pittsburgh, PA, USA),sulfuric acid (H2SO4), hydrochloric acid (HCl) andphosphoric acid (H3PO4) (Sigma Aldrich, St. Louise, MO,USA) solutions were prepared and adjusted pH to ~2.3 (equivalent to3 min of plasma treatment). The prepared acid solutions were exposedto the same volume of 107 CFU/ml E. coli andheld for 15 minutes. Then colony-counting assay was performed forthe quantification of surviving bacteria as described previously9. Also, the antimicrobial effect of plasma-generated species was tested bypreparing the determined concentrations of each species in deionized water. Forspecies combination experiments, final concentrations of each species wereadjusted accordingly. Hydrogen peroxide solution (30%), nitric acid (1N),glacial acetic acid (Fisher Scientific, Pittsburgh, PA, USA), sodium nitrite andcysteic acid (Sigma Aldrich, St. Louise, MO, USA) were used to prepare desiredconcentrations of tested substances. Superoxide thermal source (SOTS-1) (Cayman,Ann Arbor, MI, USA) was used to prepare superoxide solution. SOTS-1 is providedas crystalline powder and its stock solution was prepared by dissolving it indimethyl sulfoxide (DMSO) and stored at−80 °C. The working solution was prepared bydissolving stock solution in PBS. Peroxynitrite(ONOO−) (Cayman, Ann Arbor, MI, USA) solution wasdiluted in deionized water just before exposure to bacteria. Peroxynitritesolution was provided in 0.3 M sodium hydroxide (NaOH). Thereforeantimicrobial effects of corresponding concentrations of NaOH were tested inorder to make sure that it doesn’t interfere with antimicrobialeffect. All species and all combinations of them were exposed to equal volume of107 CFU/ml E. coli and held for15 minutes. Then colony-counting assay was performed for thequantification of surviving bacteria as described previously9.

Antimicrobial Effect of Components of Plasma-Treated NACSolution

The NAC solution was prepared by dissolving NAC powder (Sigma) in PBS solution(Sigma). To demonstrate the contribution of two components of plasma-treated NACsolution [NAC molecule (solute) and PBS (solution)], study chemicalmodifications and correlate with antimicrobial activity, an experiment wascarried out to separate the components of treated NAC solution. In brief, after3-minute of plasma treatment, the NAC solution was immediately transferred to aglass beaker, the glass beaker was covered with a glass petri dish and heated ona hot plate to 50 °C until all the solvent wasevaporated. The evaporated and condensed solvent was collected separately in amicrotube. Dried, plasma-treated NAC powder (solute) was reconstituted in threedifferent ways: 1) with untreated PBS, same volume (equivalent toevaporated liquid) to yield 5 mM final concentration, 2) withuntreated PBS, to half of the volume of evaporated liquid to yield10 mM final concentration and 3) in the same volume ofactual evaporated liquid (to give final concentration of 5 mM). Alsothe condensed liquid part of plasma treated NAC solution was exposed to bacteriain 1:1 (50 μl bacteria: 50 μlsolution) and 1:2 (50 μl bacteria:100 μl solution) ratios. The analyses were carried outfor the measurement of pH, antimicrobial activity and UV-vis spectra. Forantimicrobial assays, each liquid was exposed to the equal volume(50 μl:50 μl) of cell suspensioncontaining 107 CFU/ml E. coli, held for15 minutes and colony counting assay was performed as previouslydescribed. Also NAC powder was treated with DBD plasma for 3 minutesand then NAC solution was prepared by dissolving the treated NAC powder inuntreated PBS to create a solution of 5 or 10 mM (finalconcentration). These solutions were exposed to equal volume of107 CFU/ ml E. coli, held for15 min of holding or contact time and colony counting assay and pHmeasurements were performed The final concentrations do not represent how muchactive species are generated by plasma treatment, but refer to the initialconcentration of untreated NAC (5 mM).

Antimicrobial activity of plasma-treated NAC solution and the effect ofdiluent

A colony count assay was carried out in order to evaluate persistence ofantimicrobial activity of plasma treated NAC solution following dilution withdiluent (untreated PBS). In colony count assay experiments plasma-treated NACsolution was mixed with bacteria suspension, held for 15 minutes inorder to let the bacteria and NAC solution to come in contact. After15 minutes of holding time, plasma-treated NAC solution– bacteria suspension mixture was further serially diluted to obtaincountable number of bacteria. The possibility of persisted antimicrobialactivity of plasma-treated NAC solution following dilution was considered.Therefore, an experiment in which antimicrobial activity of first plasma-treatedand then diluted NAC solution was carried out; and the findings are shown inFigure S1.

Data Analysis

All experiments had built-in negative and positive controls as stated. Theinitial concentrations(1 × 107 CFU/ml)of bacteria (untreated samples or 0 time treatment samples) were taken as 100%surviving cells to calculate relative percent inactivation (unless specificallystated). All of the experiments were carried out thrice in triplicate. Whereverneeded, Prism software v4.03 for Windows (Graphpad, San Diego, CA) was used foranalysis. A P-value was derived using pair comparisons between twobacterial groups with the Student t test and one-way analysis of variancefor multiple comparisons. The P-value of <0.05 was consideredstatistically significant.

Results

Nitrite and Nitrate Detection

Both nitrite and nitrate concentrations in plasma treated NAC solution increasedwith plasma treatment time. The nitrite concentrations in plasma treated NACsolution were determined as 0.03 mM, 0.16 mM, and0.33 mM after 1-minute, 2-minute and 3-minute of plasma treatment,respectively (Fig. 1A). Nitrate concentrations in plasmatreated NAC solution were measured as 1 order magnitude more than nitrite, and2.07 mM, 5.46 mM and 9.35 mM nitrate weregenerated after 1, 2 and 3 minutes of plasma treatment, respectively(Fig. 1B).

Figure 1
figure 1

Generation of Nitrite and Nitrate in NAC Solution during PlasmaTreatment.

Nitrite (A) and nitrate concentration (B) in plasma-treated NACsolution increases in a plasma treatment time-dependent manner. The datashows relative concentrations to 0 min plasma treatment time(untreated NAC solution). In 3 minute of plasma treatments, thesignificantly high concentrations of nitrite (0.33 mM) andnitrate (9.35 mM) were detected in plasma treated NAC. (Bar, SD;*p < 0.05;n = 3).

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UV-Visible Spectrum Analysis of Plasma Treated NAC Solution

UV-visible spectra of plasma treated NAC solutions were obtained after 1, 2, and3 minute of plasma treatments. In the spectrum of 1-minute plasmatreated NAC solution, a specific peak appears at 332 nm, whichbelongs to S-nitroso-N-acetyl cysteine (SNAC) that is a type of S-nitrosothiol.This peak disappears in the spectra of 2 and 3-minute plasma treated NACsolutions. In the spectrum of 2-minute plasma treated NAC solution a new peakstarts to appear at 302 nm and the absorbance value of this peakincreases after 3 minutes of plasma treatment (Fig.2A). The specific peak (B), molecular structure (C) and color (D) ofS-nitroso-N-acetyl-Cysteine developed during plasma treatment is shown here(Fig. 2).

Figure 2
figure 2

UV-Visible Spectra of Plasma Treated NAC Solution.

(A) Following 1-minute plasma treatment of NAC solution, a peak at332 nm (representing formation of S-nitroso N-acetyl cysteine(S-NAC); a type of S-nitrosothiol molecule) was observed. Following 2-minuteof plasma treatment, the peak at 332 nm was shifted to302 nm and the intensity of the peak at 302 nmincreased in the plasma treatment time dependent manner. (B) Specificsecondary peak for S-nitroso N-acetyl cysteine (S-NAC) in 1-minute plasmatreated NAC solution. (C) S-nitroso N-acetyl cysteine (S-NAC)molecule, H atom is abstracted from thiol group and NO group is bonded after1-minute of plasma treatment. (D) A change in color was observed in1-minute plasma treated NAC solution, which is characteristic fors-nitrosothiols. Our observation on plasma treated NAC solution along withUV-vis results suggests the formation of S-NAC in 1-minute plasma treatedNAC solution.

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FT-IR Analysis of Plasma Treated NAC

The graphical presentation of FT-IR data on untreated and 3 minplasma treated NAC is shown (Fig. 3A). The IR peakinterpretation of untreated NAC molecule at3375 cm−1,2547 cm−1,1718 cm−1, and1535 cm−1 correspond to thestretching motion of N–H in CONH group, S–H,C = O and CONH group, respectively are shown (Fig. 3B). The absorptions at these peaks disappear afterplasma treatment of NAC solution. New peaks after plasma treatment appear at3600–3000 cm−1 (broad),and 1344 cm−1 (narrow), which correspondsto the stretching motion of −NH2 and−SO3H, respectively are shown in Fig.3(C). FT-IR spectra of untreated and 3 minutes of plasmatreated NAC solution suggest that the SH group was converted to−SO3H. Moreover the cleavage of N-acetyl group leadsto formation of acetic acid (CH3COOH) and –NH2formation due to the cleavage of the of the CONH group. These results suggestthat NAC molecule was converted to acetic acid (CH3COOH) and a−SO3H-containing compound, most likely cysteic acid(C3H7NO5S), due to oxidation of thiol groupas a result of plasma treatment. Peaks corresponding to acetic acid cannot beseen on the spectrum because of the evaporation of acetic acid during theevaporation of water before FT-IR analysis.

Figure 3
figure 3

FT-IR and NMR analysis of plasma treated NAC.

(A) A graphical and schematic diagram showing specific peaksrepresenting NAC molecule in untreated NAC solution and cysteic acidmolecule as a product of NAC in consequence of 3 minute plasmatreatment of NAC solution. (B) Peaks of untreated NAC molecule at3375 cm−1,2547 cm−1,1718 cm−1, and1535 cm−1 (from Fig. 3A) correspond to the stretching motion of N–Hin CONH group (shown with number 4 in spectra and on molecule),S–H (number 3), C = O (number 2) andCONH group, respectively. (C) New peaks after plasma treatment appearat 3600–3000 cm−1 (Fig. 3A; number 2), and1344 cm−1 (Fig.3A; number 1), which correspond to the stretching motion of−NH2 and −SO3H,respectively, which suggest the formation of cysteic acid. (D) NMRspectrum of untreated and 3-minute plasma treated NAC solution which showsspin coupling patterns of untreated N-acetyl cysteine (numbered on spectrapeaks and molecule), which is being chemically converted to cysteic acid asa result of plasma treatment. Arrows indicate increase in intensity ofmultiplets found in 3.54–3.61 and3.24–3.42 ppm. Also, decrease in intensity of themultiplet located at 2.95–3.01 ppm was observed.Proton shifts suggest that about 90% of N-acetyl cysteine is converted tocysteic acid via cleavage of the thiol group.

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Nuclear Magnetic Resonance (NMR) Analysis of Plasma Treated NACSolution

The NMR spectra of untreated and 3 min plasma treated NAC solutionare shown (Fig. 3D). The NMR spectra of the plasma-treatedN-acetyl cysteine solution showed that NAC was first oxidized to cysteic acid bycleavage of the N-acetyl group from the NAC molecule and oxidation of the thiol.Increasing duration of plasma treatment of N-acetylcysteine resulted in increasein intensity of multiplets found in 3.54–3.61 and3.24–3.42 ppm, as well as decrease in intensity of themultiplet located at 2.95–3.01 ppm. The proton shiftssuggest that majority of thiol groups of NAC may have been cleaved.

Antimicrobial Effect of Acidic pH and Plasma-Generated Species

Figure 4 shows the findings on E. coli responsesagainst various conditions that simulate the chemical species/products generatedafter 3 min plasma treatment. The data in Fig.4(A) demonstrates that various acids at pH ~2.3 (exceptacetic acid) alone do not have significant inactivation on107 CFU/ml of E. coli. Acetic acid exhibited~3.5 log reduction. Acetic acid is a weaker acidcompared to other acids that are used in this experiment. Therefore, higheramount of acetic acid needed to obtain pH 2.3 solution of it led to higherconcentration of acetate ion(CH3CO2−). Excepthydrochloric acid (HCl), when all tested acids were combined with0.93 mM H2O2 (the detected concentration ofH2O2 in NAC solution after 3-minute of plasmatreatment), did not show significant improvement in antimicrobial effect. Theincreased antimicrobial effect of HCl when it is combined withH2O2 may be attributed to the formation ofhypochlorous acid, the halogenating agent34. In addition, aceticacid loses its antimicrobial activity when it is combined withH2O2. Similarly, the combinations of nitrate ornitrite with hydrogen peroxide were also tested for their bacterial inactivationproperty and the findings suggested that a combination of nitrite andsuperoxide has excellent bactericidal effect (Fig.4B).

Figure 4
figure 4

Colony assays showing surviving bacterial cells in response to acids,peroxynitrite and combinations of ROS and RNS, simulating 3-minute plasmatreated NAC solution.

(A) Different acids having equivalent pH (set to 2.3) do not showsignificant antimicrobial effect, except acetic acid. However, the effect ofacetic acid diminishes when hydrogen peroxide is added. With addition ofhydrogen peroxide to acids also do not show a significant antimicrobialeffect, except hydrochloric acid (which is due to formation of hypochlorousacid). (B) Combination of superoxide with nitrite resulted in 7 logreduction of E. coli. However, this effect diminishes when hydrogenperoxide and nitrate are added. These results suggest RNS could be the mostdominant source for antimicrobial effect in present plasma-treated NACsolution. (C) The findings of concentration-dependent antimicrobialeffect of peroxynitrite are shown. Since peroxynitrite solution was providedin sodium hydroxide, antimicrobial effect of corresponding sodium peroxideconcentration for each tested concentration of peroxynitrite was alsoincluded to make sure that presence of sodium hydroxide does not interferewith antimicrobial effect. Peroxynitrite interval(0.18 mM–0.36 mM) showed very highinactivation of E. coli suggesting that peroxynitrite might be amajor source for antimicrobial effect of plasma treated NAC solution. Theconditions of PBS and 3% H2O2 are shown as positiveand negative controls for growth, respectively; and Fig. 4(B) testconditions contain 0.93 mM H2O2 (theamount detected in 3 min plasma-treated NAC solution).

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Table 1(A) summarizes the concentrations of plasmagenerated products via the interaction of the NAC molecule with plasma dischargein the present study. The concentrations of each given species were tested fortheir antimicrobial activity in order to reveal an effective contribution ofspecies generated in NAC solution during plasma treatment. Thus, theantimicrobial effects of detected concentrations of nitrite(0.33 mM), nitrate (9.35 mM), hydrogen peroxide(0.93 mM), acetic acid (~5 mM) and cysteicacid (~5 mM) in 3-minute plasma treated NAC solutionwere tested on 107 CFU/ ml E. coli by itself,and in combination with each other. The pH of all tested mixtures was set to~2.3–2.5. Table 1(B) shows alltested species and their combinations.

Table 1 Chemistry of plasma treated NAC solution.
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None of the tested species or their combinations was not able to show asignificant antimicrobial effect and most showed <1 log reduction in CFU.In further experiments, the determined concentrations ofH2O2, NO3−, andNO2− were combined with arbitrarilyselected concentrations (0.1, 0.5 and 1 mM) of superoxide that wasgenerated by SOTS-1 (superoxide thermal source) compound. Since the SOTS-1 isdissolved in DMSO, antimicrobial effects of corresponding concentrations of DMSOwere also tested in order to make sure that DMSO doesn’t interferewith results. Superoxide in given concentrations and in combination withdetected concentration of H2O2 did not show anysignificant antimicrobial effect (<1 log reduction in CFU). Similarly,0.1 mM or 0.5 mM of superoxide with nitrate(9.35 mM) combinations have shown less than 1-log reduction in CFU.Combination of 1 mM superoxide with 9.35 mM nitrateinactivated close to 1 log bacteria. Finally, when the given concentrations ofsuperoxide were combined with 0.33 mM nitrite the results revealedthat only combination of 1 mM superoxide with 0.33 mMnitrite was able to achieve more than 6.5-log reduction of CFU. This effect wasdisappeared when hydrogen peroxide and nitrate were added to superoxide-nitritemixture (Fig. 4B). These results led us to believe thatRNS might be primary reason for the antimicrobial effect of plasma treated NACsolution. Therefore, we tested the antimicrobial effect of determinedconcentration of peroxynitrite (ONOO−) on107 CFU/ml of E. coli. Since theperoxynitrite solution was provided in NaOH solution, the antimicrobial effectof corresponding concentrations of NaOH alone were also tested to ensure thatNaOH does not contribute or interfere with antimicrobial effect. As demonstratedin Fig. 4(C), 0.36 mM and 0.18 mMof peroxynitrite showed more than 5.5 and 3.5 log reduction, respectively.

Antimicrobial Effect of Separated Components of Plasma-Treated NACSolution

As explained in the materials and methods section, the schema of separation ofcomponents of plasma-treated NAC solution is depicted in Fig.5(A). The different methods were used for reconstitution of plasmatreated NAC solutions. In the first case no antimicrobial effect was observed.In the second and third cases 7-log reduction was observed. When condensedliquid portion of plasma treated NAC solution alone was used, no inactivationwas observed. The pH of reconstituted solutions was found to be slightlyincreased and measured as 4.2, 3.5 and 3 for the 1st,2nd and 3rd cases respectively. Also, thecondensed liquid retained its acidity, where the pH was measured as 3. Figure 5(B) summarizes the inactivation and pH measurementresults of the reconstitution experiments. In case of DBD plasma treatment ofNAC powder by itself and preparation of NAC solution with plasma-treated NACpowder (dried crust), no inactivation effect was achieved, with both5 mM and 10 mM solutions. The pH of prepared solutionswere measured as ~6 (Fig. 5C). The UV-visiblespectra obtained from the reconstitution experiments revealed that the intensityof the peak at 302 nm is directly related to the concentration ofperoxynitrite. In other words, after heating, the peak at 302 nmdecreases and is therefore attributable to liquid that was removed by theevaporation process. When the remaining powder is reconstituted with PBS in a1:1 ratio (same as the evaporated liquid), the absorbance of the peak at302 nm is measured as 0.31 and when the powder is reconstitutedwith PBS in a 2:1 ratio (half volume of the evaporated liquid), the peak at302 nm is measured as 0.62. With a basic dilution calculation wecalculated that if the dried powder is reconstituted with PBS in a 1.5:1 ratio(0.75 times of evaporated volume), the peak intensity should be same as the3-minute plasma treated NAC solution. Therefore, the dried NAC powder wasreconstituted with PBS in a 1.5:1 ratio, its antimicrobial effect was testedusing colony counting assay and nitrite, nitrate and hydrogen peroxideconcentrations were measured using HACH nitrite/nitrate detection kit andhydrogen peroxide assay kit and the UV-visible spectrum was obtained. Figure 5(D) demonstrates that all tested solutions showintensity of the peak at 302 nm and at this peak, a similarabsorbance (0.47 AU) is observed by 3-minute plasma treated NACsolution and the dried powder reconstituted solution of 1.5:1 ratio. Inaddition, the dried powder that was reconstituted in 1.5:1 ratio had achieved7-log reduction and nitrate, nitrite and hydrogen peroxide concentrations weremeasured as 0.8 mM, 0 mM and 0 mMrespectively. Through these results we speculate that the peak at302 nm represents peroxynitrite and products of the NAC or unreactedNAC molecule somehow act similar to a spin trap and play a role in thestabilization of plasma generated reactive species. However the stabilizationprocess is unclear and further studies are required in order to understand themechanism of stabilization of plasma-generated species.

Figure 5
figure 5

Separation schema and features of plasma-treated NAC solutioncomponents.

(A) Separation schema of plasma-treated NAC solution to liquid portionand solute portion. Plasma-treated NAC solution was heated; evaporatedliquid portion was condensed and collected separately. Remaining solid(powder) portion was reconstituted using untreated PBS solution in differentratios or in condensed liquid portion. (B) Colony assays wereperformed using reconstituted samples following separation of 3-minueplasma-treated NAC solution. When separated dried NAC portion wasreconstituted in 1:1 ratio (to achieve final [5 mM] NAC) usinguntreated PBS, no significant microbial inactivation was observed; butsolution prepared using condensed liquid to untreated PBS in 2:1 ratio(means remaining powder concentration was doubled) resulted as 7 logreduction (complete inactivation). The pH values of each condition are alsoshown. (C) Colony assays showing fresh NAC powder, treated withplasma-discharge for 3 minutes by itself, then dissolved inuntreated PBS to obtain 5 mM and 10 mM finalconcentration of NAC, the solution did not show significant antimicrobialeffect. (D) UV-visible spectrum of 3-minute plasma-treated NACsolution, 1:1 and 2:1 ratio reconstituted samples and 1.5:1 ratioreconstituted sample to obtain same intensity of the peak at302 nm that was obtained from 3-minute plasma treated NACsolution using Beer-Lambert Law.). Following reconstitution (1:1 ratio ofplasma-treated NAC solution; after heating and evaporation of liquid part),intensity of the peak at 302 nm relatively decreased (explainedin manuscript as 1st condition). When remaining powder wasreconstituted in 2:1 ratio (to double the concentration of remaining powder/dried portion), the intensity of peak was doubled (explained in manuscriptas 2nd condition), suggesting the products of NAC moleculeprobably stabilized peroxynitrite. When remaining powder (dried portion) wasreconstituted in 1.5:1 ratio the same intensity of the peak at302 nm (obtained from 3-minute plasma treated NAC solution) wasobserved; and no hydrogen peroxide, no nitrite and 0.8 mMnitrate (<10% what was detected in 3-minute plasma-treated NACsolution) was detected and 7 log reduction of cells was achieved. Thesefindings suggest that peroxynitrite is a major source for microbialinactivation.

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Since we postulate the presence of peroxynitrite in plasma treated NAC solutionfrom our findings, the concentration of peroxynitrite in plasma treated NACsolution was calculated by using the Beer-Lambert Law. The extinctioncoefficient of peroxynitrite (εONOO) is given as1670 M−1.cm−11,27,36,37,38,39 and concentration of peroxynitrite in3-minute plasma treated NAC solution was calculated as 0.28 mM.UV-visible spectra of reconstituted solutions showed a specific peak at302 nm similar to plasma treated liquid. Peaks at 302 nmrepresent ONOO− and concentrations ofONOO− calculated as 0.18 and 0.36 mMfor the above-mentioned 1st and 2nd reconstitutioncases respectively (Fig. 5D).

Discussion

According to our findings, the antimicrobial effect of plasma treated NAC solutionseems to be from the chemical modifications in NAC solution during plasma treatment.The previous literature which have reported a moderate reduction in pH of PBS orwater always had a plasma discharge gap of more than 2 mm through45 mm and the amount of liquid more than 1.5 ml through10 ml10,16,17,20. This drastically changes thechemical properties and pH equation of these solutions. Our findings show thatacidic pH has no direct significant contribution to bacterial inactivation. Similarobservations are also reported by other groups10,20,21. However,acidic pH seems to be a crucial for the antimicrobial effect of the solution, andusually can be referred to antimicrobial effect of acidified nitrites30,31,32.

During analysis, we did not see significant antimicrobial activity at the detectedconcentrations of plasma-generated species alone. In partly, this might be due tolack of sufficient concentration of individual plasma-generated species. Plasmatreated liquids are complex mixtures of ROS and RNS and carry various charged anduncharged molecules. Further detailed chemical analyses and chemical kineticexperiments are required to better understand the exact roles of plasma-generatedspecies on microbial inactivation. Oehmigen et al. have reported that themixture of 0.074 mM H2O2 and 0.032 mMNO2− at pH 3 is able to inactivate 3.5logs E. coli15. However this effect is mostly related toexposure conditions of bacteria to hydrogen peroxide-nitrite mixture. In theirstudy, bacteria were exposed 50 times more volume of mixture(50 μl bacteria: 2.45 ml mixture) as opposed toour 1:1 (50 μl bacteria: 50 μlmixture or plasma treated NAC) ratio and held for 60 minutes as opposedto our 15 minutes of holding time. Therefore our findings on thenitrite-superoxide mixture, which demonstrates 7 log reductions under the similarexposure conditions with plasma treated NAC, seems to be more significant, andrelevant to our plasma treatment method. In addition, the inactivation rate ofperoxynitrite in the detected range supports our hypothesis of prominentcontribution of RNS in E. coli inactivation. However precise quantificationof superoxide is required for better understanding of the interactions betweenoxygen and nitrogen species.

An interesting chemical modification was observed in 1-minute plasma treated NACsolution. In the UV-visible spectrum of 1-minute plasma treated NAC solution, we sawa peak at 332 nm, specific for S-nitrosothiols (in our caseS-nitroso-N-acetyl cysteine). Also, S-nitrosothiols have another specific peak at545 nm with 16 M−1cm−1 molar extinction coefficient, which is moresuitable for concentration calculation due to lack of possible interference by othernitrogen species40. According to the absorbance of the peak at545 nm and the molar extinction coefficient, the concentration ofS-nitroso-N-acetyl-cysteine is calculated as 0.75 mM by usingBeer-Lambert equation (Fig. 2B). S-nitrosothiols, with thegeneral structure of RSNO (R is an organic group) are the S-nitrosylated products ofthiols and are known NO (nitric oxide) donors and carriers, with a characteristicpink color2,41,42. Our findings and observations on1 minute plasma treated NAC solution regarding the formation ofS-nitroso-N-acetyl cysteine (Fig. 2C) is consistent, andsupported by previous literature. We observed a pinkish color development in1 minute plasma treated NAC solution (Fig. 2D).Formation of S-nitrosothiols (RSNO) involves the reaction of a thiol (RSH) with NOand NO derivatives such as NO2,NO2− and N2O3. Byitself NO reaction with a thiol leads to disulfide formation rather than RSNO.However, in the presence of oxygen or other oxygen species such as hydrogenperoxide, NO oxidation leads to formation of S-nitrosothiols43.Oxidation of thiols with plasma generated ROS involves the production of sulfonylradical that later react with NO to form S-nitrosothiol as shown in the equationsbelow44.

Thus, plasma generated ROS and RNS cause the modification of NAC to S-NAC by the endof 1-minute plasma treatment. The interaction ofO2− and NO leads to peroxynitrite(ONOO−). S-nitroso-N-acetyl cysteine acts as NO donorvia decomposition for peroxynitrite formation in plasma treated NAC solution. Notonly plasma generated ROS and RNS but also UV light that is generated during plasmatreatment and acidic pH as a result of plasma treatment of liquids, have influenceon the production and the decomposition of SNAC. UV light and the presence of thiols(unreacted NAC) in acidic environment induce decomposition of S-nitrosothiols (inour case S-NAC)42. Nitric oxide5 is released viadecomposition of S-NAC and reacts with plasma-generated superoxide(O2−) for peroxynitrite formation. Anothermechanism for the S-nitrosothiol mediated peroxynitrite formation which is relevantto plasma treated liquids, involves the reaction of hydrogen peroxide with S-NAC.This mechanism involves the dissociation of hydrogen peroxide and the reaction ofhydroperoxyl (HO2−) with S-NAC as shown in thefollowing equations45.

In addition to plasma-generated superoxide, by itself the S-nitrosothiol formationmechanism can lead to superoxide production via reduction of O2 byRSNO-H, a radical intermediate, as shown in following equations46.

As stated above, in the plasma treated NAC sample that was dried and reconstituted in1.5:1 ratio, hydrogen peroxide and nitrite was totally diminished and nitrateconcentration was measured as 0.8 mM, which in contrast, is less than10% of the nitrate concentration found in 3-minute plasma treated NAC solution. Thissample was capable of 7 log reduction, supporting our speculation on the presence ofperoxynitrite and supports its relation with the peak at 302 nm.

Our FT-IR and NMR data suggests that about ~90% NAC is converted tocysteic acid. This mechanism involves the oxidation of NAC as given in equations1, 2, 3.Oxidation of thiols results in formation of disulfides that later are likely to beoxidized to sulfonic acid (in our case cysteic acid)44,47. In suchreaction, where NAC is converted to cysteic acid, acetic acid is also formed due tocleavage of acetyl group. The overall reaction is shown in the equation below:

It can be observed from equation 8 that in order to fulfill theatomic balance in the reaction, NAC should react withHOO− which is present in the plasma treated NACsolution, probably due to the dissociation of H2O2 as shown inequation 4 or protonation of superoxide. This possible reactionmechanism explains the decreased hydrogen peroxide concentration in3 minute plasma treated of NAC solution compared to 2-minute plasmatreated NAC solution. The formation of cysteic acid and acetic acid contributes tothe rapid and drastic pH drop in NAC solution following plasma treatment.

Conclusion

The findings suggest that plasma treatment turns NAC solution into an acidic mixtureof ROS and RNS where both contribute to bacterial inactivation. NAC seems to be inthe center of all reactions and interactions between ROS and RNS. NAC by itselfserves as a source of RNS by releasing NO and also the source of ROS through theintermediates upon its reaction with other ROS. Although the antimicrobial effectcan be attributed to both ROS and RNS, based on our results we speculate that ROSplay an important role in the modification of NAC molecule, while RNS seems tocontribute to the antimicrobial effect more dominantly. This is the first report toour knowledge of the generation of peroxynitrite during nonthermal plasma treatedNAC solution which is capable of inactivating7 log of CFU of E. coli. Furtherstudies on the mechanism of action ROS and RNS on microbial inactivation andresponse of bacteria to those species are underway.

Additional Information

How to cite this article: Ercan, U. K. et al. Chemical Changes inNonthermal Plasma-Treated N-Acetylcysteine (NAC) Solution and Their Contribution inBacterial Inactivation. Sci. Rep.6, 20365; doi: 10.1038/srep20365 (2016).