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# Degradation kinetics of cold plasma-treated antibiotics and their antimicrobial activity

## Abstract

Antibiotics, such as ofloxacin (OFX) and ciprofloxacin (CFX), are often detected in considerable concentrations in both wastewater effluents and surface water. This poses a risk to non-target organisms and to human health. The aim of this work was to study atmospheric cold plasma (ACP) degradation of antibiotics in water and meat effluent and to explore any residual antimicrobial activity of samples submitted to the plasma process. The results revealed that ACP successfully degraded the studied antibiotics and that the reaction mechanism is principally related to attack by hydroxyl radicals and ozone. According to the disk diffusion assay, the activity of both antibiotics was considerably reduced by the plasma treatment. However, a microdilution method demonstrated that CFX exhibited higher antimicrobial activity after ACP treatment than the corresponding control revealing a potentially new platform for future research to improve the efficiency of conventional antibiotic treatments. Importantly, short-term exposures to sub-lethal concentrations of the antibiotic equally reduced bacterial susceptibility to both ACP treated and untreated CFX. As a remediation process, ACP removal of antibiotics in complex wastewater effluents is possible. However, it is recommended that plasma encompass degradant structure activity relationships to ensure that biological activity is eliminated against non-target organisms and that life cycle safety of antibiotic compounds is achieved.

## Introduction

The objective of this work was to study the efficacy of atmospheric air plasma for the degradation of antibiotics in different liquid matrices, such as water and a model meat effluent. We aimed to elucidate the intermediate or transformed products in order to propose a reaction pathway. Furthermore, the antimicrobial property of the treated antibiotic solutions was also assessed with the potential for adaptive responses to any breakdown products in addition to exploring the possibility for enhanced biological safety.

## Results and Discussion

### Evaluation of degradation efficiency and kinetics

Ofloxacin (OFX) and ciprofloxacin (CFX) are among the most frequently detected antibiotics in the environment5,6,7,8. Although of the same chemical class, these antibiotics differ slightly in their molecular structure, which in the current research allowed reporting on the intrinsic factors influencing the efficacy of ACP for the removal of antibiotics from the environment.

Atmospheric air plasmas can generate many chemical reactions, which are responsible for generating various active chemical species at ambient conditions, such as negative and positive ions, neutral molecules (H2, O2, O3, and H2O2), and free radicals (•OH, •O, •H, and HO2). Among these species, ozone (O3), hydrogen peroxides (H2O2), hydroxyl radicals (•OH), as well as RNS (peroxynitrites, nitrogen oxides) are generally identified as the main active species responsible for the degradation of antibiotics40,41,42, although the role of other reactive species may also be important in the degradation process through other chain reactions. In the current work, the concentration profiles revealed that the degradation depends on the applied voltage (Fig. 1). Increasing the voltage from 70 to 80 kV increased the plasma degradation efficacy of antibiotics in water from 75% to 89% for CFX and from 88 to 92% for OFX, respectively. This can be attributed to generation of more reactive species. It was also observed that the degradation of antibiotics was dependent on the treatment time with higher degradation rates achieved after longer plasma exposure. The degradation kinetics of antibiotics can be explained by a first-order kinetic model (model parameters can be found in Supplementary Table S1). The rates of reaction were increased from k = 0.039 to k = 0.054 min−1 with an increase in applied voltage from 70 kV to 80 kV.

The degradation efficacies and the reactive species generated are influenced by many factors, such as input energy, the nature of the gaseous atmosphere as well as pH, conductivity, temperature of the solution, chemical structure of the contaminant and the solution matrix43. In the current work, the influence of the solution matrix was studied to reflect more realistic challenges. The degradation efficacy of ACP was influenced by the presence of meat organic matter, which decreased the efficacy by 10% for all applied voltages as compared to the water samples. This is due to the competition between antibiotics and the organics present in the model meat effluent, where the plasma generated active species are consumed not only in reactions with antibiotics, but also in reactions with the organic matter and antibiotic degradants. During plasma treatment, the active species, such as ozone, hydrogen peroxide and hydroxyl radical, react with unsaturated bonds and aromatic rings of the organic compounds (proteins, carbohydrates, fat) leading to the splitting of bonds and the dissociation of the rings, following the Criegee mechanism44. The removal efficiency may be characterized by the amount of contaminant degraded per unit of energy (yield). The yield can be influenced by many factors, including system design, the type and the concentration of the compound. For the antibiotic solutions studied, a decrease in energy yield values was observed with increase in time and voltage. With increase in voltage, the energy yield has reduced from 328 × 10−6 to 221 × 10−6 g/kW h and from 204 × 10−6 to 103 × 10−6 g/kW h for CFX and OFX in water, respectively.

Hydrogen peroxide (Eo = 1.77 V/SHE) is one of the most reactive species formed in gas-liquid plasma system. It is generally formed by the recombination of generated hydroxyl radicals in oxygen free water45,47. Hydrogen peroxide under ambient conditions is relatively stable in aqueous phosphate buffer solution with the reported stability of several weeks48. This can also be used as an efficient indicator of the •OH generation in the DBD contained plasma reactor. In order to evaluate the efficacy of the DBD reactor, the formation of H2O2 in antibiotic solutions was quantified (Fig. 3). It can be observed that the concentration of H2O2 in the liquid phase generated by the DBD reactor is dependent on the treatment time and applied voltage. The concentrations of H2O2 ranged from 0.174 to 2.7 mM for antibiotics solutions in water with slightly higher values (0.21 to 3.6 mM) observed for antibiotics in the meat effluent. Similar results have been reported by previous authors49,50. Focusing on the degradation of antibiotics by DBD plasma, Hama Aziz et al.43 reported that the presence of pharmaceuticals or organic matter can enhance the formation of H2O2.

The presence of oxygen in the gas atmosphere induces the production of reactive species like oxygen radicals and ozone. Ozone is one the most stable active species generated in DBDs with a high oxidation potential of 2.02V51. The interaction between ozone and organic compounds can be either direct or indirect where decomposition occurs through a series of reactions. It was believed that ozone formed in the gas phase during the discharge is dissolved in deionized water until saturation. In this study, the gas phase ozone concentrations measured after 10 min of plasma treatment were found to be 2100 and 3200 ppm (within ± 10% errors) for applied voltages of 70 and 80 kV, respectively. In our study, very low concentrations of dissolved ozone were achieved (data not shown). This is probably due to the rapid consumption of generated ozone during the degradation process of the antibiotics. Several authors have explained the formation of •OH radicals during the discharge in a chain reaction with ozone in water52. The lower dissolved ozone values can also be attributed to the consumption of ozone by the antibiotic itself and their intermediate products.

The variation of pH values for the solution after plasma treatment process is shown in Fig. 4. The pH value was lowered with increasing treatment time for all antibiotic solutions. After the first 5 min of treatment the pH values dropped to less than 4.5 and subsequent increases in treatment time resulted in a slow decline. This variation of pH values in the antibiotic solutions is caused by the formation of several specific acidic substances, such as nitric acid and nitrous acid during plasma treatment in air27. Also, a certain contribution to the pH variation during the plasma treatment may come from carboxylic intermediates, produced from the degradation of antibiotics. To confirm the acidification of the antibiotic solutions caused by air plasma the treated solutions were analyzed for the presence of nitrates, nitrites and several carboxylic acids. The concentrations of nitrate in the antibiotic-water solutions after 25 min of plasma treatment were found to be 0.98 mM and 2.38 mM for applied voltages of 70 and 80 kV, respectively (Fig. 5). An increase in nitrate values was observed with increases in treatment time and applied voltage. Similarly, the formation of high nitrate concentrations in the solution were reported for gliding arc plasma treatments53,54. Such increases in nitrates cause a drop in pH. Moreover, no nitrites were detected in the present study, which is due to the easy conversion of nitrite to HNO2 under acidic conditions. Nitrates formed under acidic conditions undergo several reactions to form peroxynitrite acid55. Oxidative ability of reactive nitrogen species is lower than reactive oxygen species, as they are formed by consuming the stronger oxidant •OH, which in turn converts them to HNO356.

Several authors have reported that the ultimate oxidation by-products of pharmaceuticals are low-molecular weight carboxylic acids, such as oxalic acid, acetic acid, formic acid57,58. However, the toxicity of the degradation by-products may still be a matter of concern. It is known that ACP treatment of aqueous organic pollutant results in the formation of several intermediate products, such as carboxylic acids, formic acid and oxalic acid. The concentrations of formic and oxalic acids were determined using ion chromatograph and are presented in Fig. 6(A,B), respectively. It can be observed that the concentration of both organic acids increased with treatment time and all applied voltages. Similar results in the formation of organic acids as by-products have been reported by Vasquez et al.59 in plasma-treated aqueous antibiotic solutions. The formation of carboxylic acids resulted in slow mineralization and low total organic carbon (TOC) removal values after plasma treatment.

The plasma degradation of organic contaminants takes place through several reactions. In the presence of air/oxygen the mechanism is not only based on the hydroxyl radical or ozone formation but also based on photo-oxygenation, photo-isomerisation or photo hydrolysis28,51. The carbon-centered radicals may yield peroxy radicals, which can be further decomposed to form corresponding oxidation products. Other mechanisms include the formation of superoxide radicals, which could further recombine, rearrange or hydrolyze to final products. Like photolytic or photocatalytic processes, the organic contaminant during plasma treatment undergoes reactions through excitation or ionization60. These excited contaminants may also quench molecular oxygen with formation of singlet oxygen. It is reported that oxygen in its singlet oxygen state (E0 = 1.77 V) is more reactive compared to the molecular oxygen. Consequently, singlet oxygen is significantly more electrophilic61. This singlet oxygen can react rapidly with unsaturated carbon-carbon bonds neutral nucleophiles, such as sulfides62 and amines60, as well as with anions, and form an hydroperoxide as an intermediate compound.

In order to understand the antibiotic degradants present post plasma treatment, the changes of the LC–MS/MS chromatograms in full scan mode were examined. Samples taken at the time when the greatest range of by-products appeared were further analyzed by comparison to the literature to identify their molecular structures. The proposed pathways of these intermediate products are expected to include multiple routes due to the presence of several reactive sites in the parent compound but in the present study the occurrence of two oxidation mechanisms by both molecular ozone and hydroxyl radicals was considered. Based on the experimental data and literature survey the degradation of the quinolone group of antibiotics occurs as follows: the plasma generated active species, such as •OH and O3, attack the carboxyl group of the quinolone moiety at the first place, followed by subsequent attack of the piperazinyl substituent and oxazinyl substituent63. In our study, no transformation products were found corresponding to the degradation of the oxazinyl group. These pathways are in line with work published by Carbajo et al.12.

### Antibacterial activity

The degradation of antibiotics should be accompanied with the loss of biological function. The residual antibacterial activity of CFX and OFX dissolved in either water or meat effluent was examined using a disk diffusion assay and measuring the inhibition zone diameter (mm) for E. coli, B. atrophaeus and P. aeruginosa (Figs S6 and S7). It should be noted that all controls resulted in no inhibition zone formed around the disks and that the disk assay involving P. aeruginosa was not valid due to the resistance of this microorganism to studied antibiotics at the concentration obtained on disks (~0.2 μg disk−1). In both cases the inhibition zone diameter was recorded as 6 mm, which was equal to the diameter of the disk. In general, the efficacy of ACP treatment for the degradation of antibiotics was influenced by the duration of the treatment and was affected by the type of antibiotic and the sample matrix. The antibacterial activity of OFX and CFX is linked to the presence of carboxylic and carbonyl groups in the quinolone molecule. Thus, the antimicrobial activity of both antibiotics dissolved in water was significantly reduced (p < 0.05) after either 15 or 25 min of treatment as was recorded for the microorganisms tested (Fig. 7A). A lower degradation ability of ACP was observed when the antibiotics were dissolved in the meat effluent (Fig. 7B). Moreover, in this case the effect of the type of antibiotic on ACP degradation ability was more apparent, with CFX exhibiting higher resistance to ACP than OFX. The CFX mechanism of degradation also supports the results from the observed antibacterial activity. It was found that the quinolone moiety of OFX was completely transformed. However, the CFX transformation was reserved only for piperazine, fluorine substituents, with the core quinolone structure remaining intact. Therefore, the efficacy of plasma treatment for antibiotic degradation depends on the susceptibility of the core structure of the chemical compound to oxidation pathways. Plasma treatment may not sufficiently oxidize or mineralize the quinolone ring of CFX, but can cause significant transformations to the auxiliary functional groups, which could also decrease the antibacterial activity. A relationship between degradation efficiency and antimicrobial activity reduction was observed.

To confirm the degradation efficacy of plasma, the effect of the longest treatment time (25 min) on the antimicrobial activity of the antibiotics suspended in a simple sample matrix (water) was quantitatively assessed using the broth microdilution method. This estimated the MIC values for the three microorganisms selected (Supplementary Table S7). However, a relatively low agreement between the two tests was found in the case of the remaining antimicrobial activity of CFX. According to the MIC values for E. coli, B. atrophaeus and P. aeruginosa, the CFX samples exhibited a higher antimicrobial activity after ACP treatment than the corresponding untreated control. In contrast, higher MICs were recorded for ACP treated OFX as compared with MICs of the untreated control for all microorganisms tested. This indicates that the reduction of the antimicrobial activity of this antibiotic (OFX) is due to the plasma treatment, which is in agreement with the disk diffusion assay. Therefore, for OFX, the degradation processes did not generate by-products with a greater antimicrobial activity than the parent compound. These observations were also demonstrated by De Witte et al.69 for ozonation and by Paul et al.70 for UVA photo catalysis.

The increased activity of CFX highlights the generation of compounds more toxic to cells due to the plasma exposure, which was not reflected with the disk diffusion assay. Such discrepancy between the two techniques could be due to the decreased stability of the generated by-products that exhibit antimicrobial properties in the disk diffusion assay or impaired diffusion capacity of these compounds into the agar. The antimicrobial potential of the water (antibiotic diluent) subjected to plasma treatment was tested using a two-fold microdilution method to examine its potential microbial toxicity. Plasma treated water was not toxic to cells at concentration below 12.5% for P. aeruginosa and 6.2% for E. coli and B. atrophaeus that do not correspond to the dilutions at which MICs for the two antibiotics were reached (Supplementary Table S7). These results demonstrated that the retention of antimicrobial potential of treated antibiotics could not be attributed to the antimicrobial properties of the plasma treated water.

Unintentional exposure of microorganisms to sub-inhibitory concentrations of biocides in the environment can lead to the development of antimicrobial resistance, which is a global healthcare problem71. Because of the demonstrated increase in the antimicrobial potency of ACP treated CFX, a study on bacterial adaptation to CFX was conducted. For E. coli, the tendency of bacterial adaptation after repeated exposure to untreated CFX was observed, with a significant, 8-fold increase in MIC recorded (p < 0.05). Importantly, there was only 1.5-fold increase in MIC value noted in the case of ACP treated antibiotic (Fig. 8A), which is a useful observation indicating synergistic effects of antibiotics and plasma treatment. Such an effect could improve the efficiency of conventional antimicrobials and/or antibiotic treatments. A significant reduction (p < 0.05) in susceptibility to both ACP treated and untreated CFX was observed for B. atrophaeus and P. aeruginosa, with a 4-fold increase in the MIC values found (Fig. 8B,C, respectively). This study revealed that short-term exposure to sub-lethal concentrations of antibiotics present in effluents, whether they were treated with cold plasma or not, equally reduced antibiotic susceptibility, which could potentially select for bacterial populations with stable genetic mutations. Therefore, it is recommended that cold plasma as an AOP for industrial effluents that may contain antibiotics or residues is optimized for complete loss of biological activity or is combined with a separate process to ensure life cycle safety.

## Methods

### Materials

Analytical grade standards of ofloxacin (OFX) and ciprofloxacin (CFX) of purity (>98%), HPLC grade methanol, acetonitrile, ethyl acetate, ammonium hydroxide solution (32%) puriss p.a. (NH4OH), acetic acid (AcOH), formic acid (HCOOH), sodium acetate (NaOAc), ammonium formate, tertiary butanol alcohol (TBA), carbon tetrachloride (CCl4) and LC-MS grade water were obtained from Sigma-Aldrich (Ireland).

### Sample Preparation

A fresh model effluent was prepared before each experimental run using procedure of Barrera et al.72. The raw synthetic meat effluent consisted of distilled water, commercial meat extract powder 1950 mg l−1; glycerol (C3H8O3), 200 mg l−1; ammonium chloride (NH4Cl), 360 mg/l and sodium chloride (NaCl) 50 mg l−1. The model effluent was used to overcome the inherent variability found in commercial effluent composition. Large particulate matter was removed by filtering the model effluent through a Whatman (UK) filter paper and a 0.45 μm membrane (Millipore). Because of the structural characteristics and persistence in the environment, ofloxacin (OFX) and ciprofloxacin (CFX) were selected in this work. Each antibiotic (OFX and CFX) was dissolved in methanol to obtain a standard stock solution with the concentration 1000 mg l−1. The prepared stock solution was either diluted in water or spiked with the model effluent to obtain a minimum concentration of antibiotic 10 mg l−1.

### Atmospheric air cold plasma treatment

The high voltage in package ACP DBD system employed for this work is described in Sarangapani et al.73 and was fully characterized in Moiseev et al.74 and Patil et al.75. The schematics of the system is presented in Supplementary Fig. S8. For each experiment 25 ml of water or meat effluent spiked with antibiotics at initial concentration of 10 mg l−1 was subjected to ACP treatment. Atmospheric air was used as the working gas. Plasma treatment was performed at variable voltage (70–80 kV) and treatment duration (5–25 min). Treatment was carried out in duplicate at ambient temperature (~18 °C). The temperature increase inside the container and at the surface of the samples due to plasma treatment was <5 °C. After processing, containers were stored at room temperature of ~18 °C for 24 h in line with our previous findings that post treatment retention time is useful for biocontrol. This treatment approach allows extended contact time of the generated and contained chemical reactive species with the samples. Control samples were not plasma treated. Ozone concentrations were measured using short-term ozone detection tubes obtained from Gastec (Product No. 18 M, Gastec, Japan), while dissolved ozone measurements were carried out according to the procedure of Bader76.

### Analytical Methods

Standard curves for the antibiotics were established using standard solutions ranging between 0.01 mg l−1 and 10 mg l−1. The linear correlation coefficients (r2) were 0.997 and 0.998 for OFX and CFX, respectively. The plasma treated effluents were firstly extracted by solid-phase extraction (SPE) according procedure of Sarangapani et al.49. Antibiotics quantification was performed using a HPLC system (Waters, Ireland). The mobile phase consisted of 70% acetonitrile and of 30% 0.03 M phosphate buffer solution at pH 2.7, and the flow rate was set at 1 ml min−1. The detector wavelength was set at 277 nm. Chromatographic data was collected and processed using Empower2 software (Waters, Ireland).

### Degradation kinetic modeling study and data analysis

The removal efficiencies (η) of antibiotics were calculated according to the following equation: η = (C0C)/C0 × 100, where η is removal efficiency of each antibiotic, $$C$$ is the concentration of antibiotic at time ‘t’ and $${C}_{0}$$ the initial concentration of the antibiotic. Plasma degradation of antibiotics in aqueous media can be described by the following equation: C = C0 exp (−kt), where ‘k’ is the degradation rate constant (min−1) of the reaction and ‘t’ is the treatment time (min).

### Antimicrobial activity

#### Disk diffusion assay

To determine the remaining antimicrobial activity of tested antibiotics (OFX and CFX), standard disk diffusion assay was utilized involving three single species test microorganisms, namely Escherichia coli ATCC 25922, Bacillus atrophaeus var. niger (Sportrol®/Namsa®, VWR International, ATCC 9372) and Pseudomonas aeruginosa ATCC 27853, obtained from School of Food Science and Environmental Health of the Dublin Institute of Technology. Each bacterium was inoculated in tryptic soy broth (TSB, Scharlau Chemie, Spain) and incubated overnight at 37 °C. The density of each overnight culture was adjusted to McFarland 0.5. Resulting cell suspensions were further used for inoculation of tryptic soy agar (TSA, Biokar Diagnostics, France). Either ACP treated (15 and 25 min) or non-treated antibiotic solutions were filtered (0.2 μm pore size) and 20 μl aliquots were deposited on sterile discs (diameter 6 mm, Whatman®), while either ACP treated or untreated sterile deionized water and meat effluent served as a control. The disks were dried inside the laminar flow biosafety cabinet and placed on inoculated TSA plates. Following incubation for 24 h at 37 °C the zone of inhibition (diameter, mm) produced around the disks was measured at the point where no growth was observed. Experiments were performed in triplicate and replicated twice (n = 6).

#### Minimum inhibitory concentration (MIC)

MIC of either treated (ACP treatment − 25 min) or untreated antibiotic solutions in water for E. coli, B. atrophaeus and P. aeruginosa were determined using broth microdilution method78. Controls included each antibiotics concentration without bacterial cells (blank), TSB and bacterial cells (positive control) and two-fold diluted plasma treated water in TSB with final concentration of plasma treated water ranging from 50–0.01%. Plates were incubated for 24 h at 37 °C. MICs of either treated or untreated antibiotic for three microorganisms were determined as the lowest concentration showing no turbidity as comparing to corresponding blank controls. Plasma treatment experiments were replicated three times and two biological replicates were included in each plate (n = 6). The lowest antibiotic concentration that inhibited the growth of the microorganism was detected by the lack of visual turbidity (matching corresponding blank control) and was designated as MIC79.

To study the possible development of resistance to ACP treated CFX, three biological replicates of each microorganism (E. coli, B. atrophaeus and P. aeruginosa) and three independently treated antibiotic in water (ACP treatment − 25 min) were used (n = 9). Overnight cultures were adjusted to ~6 log10 colony forming units (CFU) ml−1 in TSB. Either treated or untreated antibiotics were filter sterilized (pore size 0.2 μm) and twofold dilution series were prepared in 96 well plates with highest concentration corresponding to the double of the lowest MIC value established and the lowest concentration corresponding to 1/8 MIC. Bacterial cell suspensions (50 μl) were added to resulting concentrations of each antibiotic (50 μl). TSB without antibiotics was used as a control. Plates were incubated for 24 h at 37 °C. After incubation, MICs for each antibiotic were recorded and bacterial samples from the well containing sub-inhibitory concentration corresponding to ¼MIC were streaked on TSA80. The TSA plates were incubated for 24 h at 37 °C. Using isolated colonies the concentration of cells was visually adjusted with saline (0.85% NaCl) to a turbidity equivalent to the McFarland 0.5 standard. Bacterial suspensions were further diluted in TSB to a final cell concentration of ~6 log10 CFU ml−1. Again, aliquots of bacterial inoculum (50 μl) were dispensed into the wells containing doubling dilutions of each antibiotic with final absorbance measured at 600 nm corresponding to ~0.05. Viable count of the inoculum suspension using TSB without antibiotics was performed each time to ensure that final concentration of the inocula in the wells corresponds to ~5 log10 CFU ml−1. This procedure was repeated for ten consecutive cycles. Bacterial cells recovered after 1st, 5th and 10th exposures to sub-lethal doses of antibiotics were used to determine the MIC values, which were compared with MICs determined for cells exposed to TSB.

### Statistical analysis

Statistical analysis was performed using IBM SPSS statistics 21 Software (SPSS Inc., Chicago, USA). The values of the inhibition zone diameter measured for either untreated control or ACP treated for 15 and 25 min antibiotic samples for E. coli, B. atrophaeus and P. aeruginosa and MIC of CFX for each bacteria in bacterial adaptation study were subjected to Analysis Of Variance (ANOVA). Means of the diameters and MICs were compared according to the method of Fisher’s Least Significant Difference-LSD at the 0.05 level for each type of bacteria and antibiotic group.

## Data Availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files).

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## Acknowledgements

The authors would like to acknowledge funding from the Food Institutional Research Measure administered by the Department of Agriculture, Food & the Marine, Ireland (Grant number: 13F442), Science Foundation Ireland (Grant Numbers 14/IA/2626), the Irish Research Council New Foundations Strand 3a project PlasmaAPPS and the RCUK-BBSRC under Grant Reference BB/P008496/1.

## Author information

Authors

### Contributions

C.S., D.Z. and P.Be. conceived and conducted experiments; D.B., B.F.G., P.J.C. and P.Bo. conceived and designed the experiments. All authors reviewed manuscript.

### Corresponding author

Correspondence to Dana Ziuzina.

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The authors declare no competing interests.

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Sarangapani, C., Ziuzina, D., Behan, P. et al. Degradation kinetics of cold plasma-treated antibiotics and their antimicrobial activity. Sci Rep 9, 3955 (2019). https://doi.org/10.1038/s41598-019-40352-9

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