According to the available literature, the planktonic form of Enterococcus spp. was applied on diverse types of solid surfaces or carriers and exposed to NTP in a variety of experimental setups. Daeschlein et al. (2010) tested the action of argon NTP toward suspension of typical wound infection-associated pathogens, including E. faecium, inoculated on agar surface imitating wound colonization. The monitored microorganisms on agar media were exposed to NTP three times for up to 2 min (with this experimental setup, a contaminated area of 55 cm² was treated completely within 6 min). The NTP efficacy was generally almost as strong as the power of antiseptics; however, the lowest reduction factor (RF = 1.9) was obtained only for E. faecium (initial concentrations of 10²–10³ log CFU/plate). In a subsequent study, Daeschlein et al. (2014) compared 194 tested clinical wound pathogens from 13 varied species including E. faecium and E. faecalis. After 3 s treatment, they observed the inhibition zones, which differed from species to species and showed the higher susceptibility of Gram-positive bacteria. The largest zones were observed for enterococci, with inhibition areas of 480 and 20 mm² for DBD and plasma jet, respectively. Ermolaeva et al. (2011) inactivated the clinical isolates of seven pathogenic bacterial species, including E. faecium, inoculated on agar plates in a concentration range of 10³–10⁵ CFU/plate. Overall, the Gram-negative bacteria appeared to be more susceptible to argon NTP treatment than did the Gram-positive bacteria. E. faecium displayed medium resistance. A 10-min treatment completely suppressed bacterial survival on agar plates. Also, Scholtz et al. (2010) compared the effects of NTP on various microorganisms grown on agar plates. In accordance with the previously mentioned publication, E. faecium proved to be moderately sensitive: inoculated on agar in the 10⁶-CFU/cm² concentration and exposed to NTP, it provided a 38-mm² inhibition zone of a circular shape free of any surviving bacteria. Nishime et al. (2017) also observed an inhibition zone of 100 mm² after several minutes of exposure. Lis et al. (2018) treated E. faecium, K. pneumoniae, A. baumannii, and other bacterial suspensions at an initial concentration of 10⁶ CFU/cm² dried on stainless steel surfaces (3.14 cm²) with NTP for 5, 10, and 20 min. A 3.66 log reduction was achieved for E. faecium after 20 min of exposure. Comparable results were shown for the other ESKAPE pathogens tested without any significant differences between the representatives of Gram-positive and Gram-negative bacteria. In general, bacterial cells covered with bovine serum albumin (BSA) for the simulation of natural organic material were significantly less inactivated in comparison with the cells in phosphate-buffered saline (PBS) suspension. In the study of Klämpfl et al. (2014), an even higher sensitivity (a 3 log reduction) of bacterial endospores (Clostridium difficile) on a stainless steel carrier exposed to NTP for 10 min was observed in comparison with vegetative bacteria including E. faecium (only a 2 log reduction). Finally, Chen et al. (2012) designed an NTP source for large area sterilization. Helium and oxygen were used as the working gas. The helium/oxygen NTP killed E. faecalis more effectively than did the pure helium NTP. As determined by optical emission spectroscopy, reactive species such as O and OH radicals were probably responsible for the sterilization effect.

Jiang et al. (2012) verified the hypothesis that E. faecalis biofilm may be inactivated by NTP in vitro. An E. faecalis monoculture biofilm was cultivated on the surface of hydroxyapatite disks for 6 days and exposed to NTP and/or 5.25% sodium hypochlorite (NaOCl) solution. The CFU counts and scanning electron microscopy (SEM) showed that treatment of the E. faecalis biofilm with NTP and NaOCl for 5 min resulted in 93 and 90% reductions, respectively. Nearly no intact bacteria were discernible after NTP exposure. Similarly, Theinkom et al. (2019) studied the effect of NTP on E. faecalis inoculated on agar plates or on its biofilm form. The 5- and 10-min exposures of 24-h-old biofilms resulted in more than 3 and more than 5 log reductions in CFU/plate, respectively. In biofilm experiments, chlorhexidine (CHX) and UV-C radiation were only slightly more effective than NTP. Moreover, there was no damage of the cytoplasmic membranes upon NTP treatment; thus, in this case, it seems that membrane damage is not the primary mechanism of NTP action. In another study, Cao et al. (2011) evaluated the efficacy of NTP against E. faecalis and S. aureus suspensions on agar plates and E. faecalis biofilm on nitrate membrane filters. In agar cultures, inhibition zones were observed after NTP treatment; the diameter of the zones for both bacteria increased with prolongation of the treatment duration. However, ultrastructural changes in the E. faecalis biofilm were observed by electron microscopy after 2 min treatment. It was concluded that NTP could serve as an effective supplement to standard endodontic microbial treatments.

Enterococcus faecalis is one of the most important bacteria causing failure of root canal treatments; thus, many studies have been devoted to this problem as well. As a fundamental review in this field, the one by Hoffmann et al. (2013) should be mentioned. The important methods of NTP production are listed and described therein, namely, DBD, atmospheric pressure plasma jets, radio frequency plasma jets, pulsed direct current-driven plasma jets, plasma needle, and plasma pencil. Among the working gases, the use of helium, argon, nitrogen, a helium and oxygen mixture, and air is recommended. State-of-the-art lab-scale research and clinical trials of NTP in stomatology were reviewed by Li et al. (2017).

Du et al. (2012) tested the efficiency of NTP and antiseptic CHX against E. faecalis biofilm formed on coverslips and in root canals simulating the conditions during endodontic therapy. NTP treatment was comparably effective to CHX for bacteria inactivation in infected root canals after 5 and 15 min exposures (both agents with 6 log decreases in CFU/ml). The results were confirmed using confocal laser scanning microscopy (CLSM) and micro-computed tomography. In their follow-up study, Du et al. (2013) focused on the investigation of the actions of NTP and CHX on E. faecalis and multispecies (mixture of bacteria from human dental root canal infections) biofilms grown on bovine dentin disks. Biofilms (1 and 3 weeks old) were exposed to NTP, modified non-equilibrium NTP with CHX, and CHX for 2 and 5 min. The exposed samples were examined with CLSM and 3D reconstruction analysis. No significant difference was detected between the actions of NTP and CHX. Modified NTP with CHX was the most effective in killing bacteria; significantly more cells were killed in 1-week-old biofilms than in 3-week-old biofilms. Simoncelli et al. (2015) described the dental applications of NTP against E. faecalis on Tryptone Soya Agar plates, quantitatively evaluated on bacterial suspensions and tested in root canals of extracted teeth. The direct treatment of root canals in a dry environment was shown to cause the highest level of bacterial inactivation, but a relevant bacterial load reduction was also obtained when the root canal system was irrigated with PAW. Hüfner et al. (2017) aimed to compare the effect of NTP with NaOCl and to use their combination on E. faecalis biofilm formed in root canals of extracted human teeth. It was shown that the mutual treatment with NaOCl and NTP has an additive effect (but not statistically significant) in the log CFU reduction of E. faecalis biofilm as compared to NaOCl monotherapy. The results were also confirmed using SEM. Zhou et al. (2016) used NTP with helium working gas bubbled through 3% hydrogen peroxide to treat the root canal infected for 1 week with E. faecalis. This experimental setup was also compared with ones without hydrogen peroxide. The greatest reductions in CFU/ml (a 7 log decrease) were obtained after 4 min exposure of NTP with helium bubbled through hydrogen peroxide. Regarding the disinfection mechanism, atomic oxygen and hydroxyl radicals are considered responsible for the improved antibacterial effects. Li et al. (2015) treated endodontic E. faecalis biofilm (formed for 3 weeks in root canals) with argon/oxygen NTP (12-min exposure) and compared it with those treated with calcium hydroxide, 2% CHX gel, and calcium hydroxide/CHX for 1 week. The most effective treatment was the NTP exposure; there were no detectable bacteria alive after 12 min of NTP exposure, as confirmed by CFU counting, SEM, and CLSM. The microhardness and roughness of root canal dentin showed no significant difference after NTP treatment. Pan et al. (2013) treated a set of single-root teeth infected with E. faecalis biofilm. NTP treatment for 8 and 10 min had a significant anti-biofilm efficacy with a total reduction in CFU/sample. SEM and CLSM showed that the bacterial membrane was ruptured and the structure of the biofilm was fully destroyed. Finally, the study by Üreyen Kaya et al. (2014) compared the antimicrobial efficacy of NTP with a gaseous ozone delivery system and NaOCl solution rinsing on E. faecalis-contaminated root canal walls and dentine tubules performed on extracted human mandibular premolars with straight root canals. A superior efficacy of NTP compared with NaOCl was demonstrated. Both NaOCl and NTP were better than ozone at the coronal and middle parts of the root canals.

The idea of PAW treatment was improved by Ercan et al. (2013), who tested the antibacterial effect of 100 mM PAW solution of N-acetylcysteine (NAC) against planktonic cells and biofilm of various bacteria. The total inactivation of 10⁷ CFU/ml of E. faecalis, S. aureus, A. baumannii, and P. aeruginosa in both planktonic and biofilm forms was observed after 15 min of application. In a follow-up work, Ercan et al. (2014) demonstrated a low but significant inhibitory activity of plasma-activated solutions of methionine, threonine, glucose, cysteine, proline, glycine, glutamine, heparin, and arginine on E. faecalis, A. baumannii, S. aureus, and K. pneumoniae.

In planktonic form, Burts et al. (2009) observed a 4 log reduction in CFU/ml after exposure of S. aureus inoculated on agar plate to NTP for 10 min. On the other hand, Yong et al. (2019) tried to disinfect S. aureus inoculated on pork jerky pieces exposed to NTP for 30 min, but achieved only a 1 log reduction in the CFU counts. Usta et al. (2019) exposed a cell suspension of S. aureus for 15 min, which resulted in only a 0.25 log reduction in CFU/ml. The increment of the treatment time to 30 min resulted in total inhibition, indicating a frequent leap effect, where NTP acts very mildly for long time and then suddenly kills all cells with the increment of exposure time. Liao et al. (2018b) experimented with the adaptation of cells to various physical stresses such as increased acidity, osmotic pressure, and temperature. In this case, non-adapted cells of S. aureus were reduced by 7.1 log CFU/ml after 30 s exposure to NTP. The acidity-adapted cells were even more susceptible toward NTP treatment, with the same exposure time resulting in a 7.5 log reduction in survival counts. On the other hand, when the cells were adapted to osmotic pressure, heat or cold, their susceptibility dropped significantly, and the same NTP treatment resulted in a lower reduction of only 3 log CFU/ml. Such study indicates how environmental properties affect the cell persistence of S. aureus, which is relevant especially in the food industry for the proper packaging and storing of food. Its importance also lies in humidity of the surrounding atmosphere, as described by Ki et al. (2019). They examined the effects of 20–50% and 70–90% humid air on the efficacy of NTP exposure in S. aureus cell suspension inhibition. The low humidity of air resulted in only a 1.5 log reduction in CFU/ml after 60 min of NTP exposure, while the high humidity of air completely inhibited cell growth. The increased efficiency of such NTP exposure is obviously caused by the different proportions of reactive species formed, with water-originated radicals as the most prominent. Such finding would benefit the NTP decontamination of wet food samples, although its effect on the sensory properties of treated food must be thoroughly studied. Food contamination by S. aureus was also studied by Choi et al. (2018), who focused on paprika powder loaded with drops of S. aureus suspension. The authors used multiple-cycle treatments for only 2 min with various power settings of NTP. After a five-cycle treatment with 1.5 kW power, there was only a 2 log reduction in CFU/ml. They then combined NTP treatment (2 min, 1 kW) with radio frequency heating (2 min, 1.5 kW) for two cycles, which resulted in the complete inhibition of S. aureus on paprika powder. The combination of NTP exposure with other physical treatments was also proven to be effective by Liao et al. (2018a), who treated the S. aureus cell suspension with NTP for 5 min and subsequently with ultrasound for 10 and 20 min. The subsequent ultrasound treatment for 10 min resulted in a 3.8 log reduction in CFU/ml, which was greater than that with NTP acting alone (2.5 log reduction). The combination of 5 min NTP exposure with 20 min of ultrasound treatment resulted in complete inhibition.

Zhang et al. (2017) exposed a cell suspension of S. aureus to 7 min of NTP and achieved complete inhibition of growth. In this study, they also tried to decrease the exposure times; the 5-min treatment resulted in only a 3.4 log reduction. Comparable results were also achieved by other researchers, with treatment times ranging from 2 to 4 min needed for prevalent and complete inhibition (Guimin et al., 2009; Alkawareek et al., 2014; Flynn et al., 2015; Parkey et al., 2015; Yoo et al., 2015; Lunov et al., 2016a).

An interesting study by Vaze et al. (2017) evaluated the effect of NTP on bioaerosol of cell suspension created by nebulizer and achieved total suppression in the CFU/ml counts, indicating that treatment of very few nebulized drops of liquid might be a very effective form of therapy.

The pathogenicity of S. aureus is mostly facilitated by its ability to form compact biofilms on both biotic and abiotic surfaces. The occurrence of biofilm-related S. aureus infection is very common, so it is not surprising that NTP is primarily used to eradicate such contamination or to prevent surface biofilm formation. Miletić et al. (2014) focused on the treatment of cell suspension before the biofilm formation in polystyrene microtiter plates using NTP discharge working at two power settings. Total inhibition of biofilm formation (a 5 log decrease in CFU/ml) was achieved either by 2-min treatment with NTP produced at 0.9 W or only a 1-min exposure to NTP formed at 1.6 W. Different power regimes for NTP formation were also used in Modic et al. (2017). The NTP used in this study achieved total inhibition (6.5 log decrease) of biofilm formation on coupons after 2 min at both working powers (8 and 34.5 W), similarly to Miletić et al. (2014). Cotter et al. (2011) studied the effect of NTP on S. aureus biofilms formed on coverslips. NTP exposure for 1.5 h resulted in 90% inhibition in biofilm cell viability as quantified by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) viability assay. A similar result was also achieved in the study of Usta et al. (2019). NTP exposure caused an 86% inhibition of the viability of S. aureus biofilm cells after 2 h of treatment. Interestingly, treatment of only 30 min resulted in the total suppression of cell survival as determined by CFU/ml counting (5 log decrease), which only highlighted the problematic evaluation of biofilms and the effects of various antimicrobial agents on them. Alkawareek et al. (2014), also mentioned for P. aeruginosa, eradicated the S. aureus biofilm pre-formed on the peg lid of the Calgary Biofilm Device after several minutes.

Since biofilms are complex structures consisting of both cells and the extracellular matrix in between them, Xu et al. (2015) studied the efficacy of NTP exposure in killing S. aureus biofilm with LIVE/DEAD staining on CLSM. They found that the first to the fourth layer (0–5 μm) of biofilm cells were indeed killed, but the deeper layers (6.2–10 μm) showed only partial cell killing. The layers close to base (11–18.5 μm) of the biofilm were completely unharmed. Ferrell et al. (2013) also studied the effect of NTP on the properties of S. aureus biofilm. Using COMSAT analysis (a routinely used image analysis of CLSM z-stack pictures), they evaluated the biofilm porosity, thickness, biovolume, and roughness, among other properties. After all exposure times used, the biofilm volume decreased and the porosity and roughness increased, indicating damage of the upper biofilm layers, which correlates with the previously mentioned study. Czapka et al. (2018) also studied the effect of NTP on biofilm morphology and found that NTP exposure resulted in the increased porosity and roughness of the formed biofilm. Interestingly, they also found that, in comparison to S. aureus, Escherichia coli was less susceptible toward NTP, and its biofilm formation was not inhibited or even slightly affected.

Xiang et al. (2019) prepared PAW for only 1 min and mixed it with the cell suspension of S. aureus for 6 min. The treatment resulted in a 2.3 log reduction in CFU/ml. Laurita et al. (2015) prepared PAW for longer times (5 and 10 min). PAW exposed to NTP for 5 min resulted, after 25 min treatment of S. aureus cells, in a 6.5 log reduction in CFU/ml. When the prolonged time of PAW preparation (10 min) was used, the same reduction was already achieved at 15 min treatment of suspension cells. Ma et al. (2015) used PAW exposed for 10 and 20 min for the decontamination of S. aureus-loaded strawberries. PAW prepared for 10 min resulted, after 15 min treatment, in a 1.6 log reduction of CFU/ml, while PAW prepared with 20 min NTP exposure caused a 2.3 log reduction after the same amount of time. Schmidt et al. (2019) used an even longer NTP exposure time (30 min) in the preparation of PAW, which, after a 30-min treatment of S. aureus cells, completely suppressed cell survival. Chen et al. (2018) used the same exposure time for the preparation of PAW; after 3 h of cell treatment, this resulted in only a 2.3 log reduction in CFU/ml. Interestingly, such findings were also complemented with information on the metabolic activity of the treated cells reflecting the actual viability. Although the CFU/ml reduction was not complete, most cells were metabolically inactive (99% in comparison with the control samples) according to the MTT viability assay. Such lack of correlation between the cell viability and culturability was also found in other studies, which, however, used the direct NTP effect (Cotter et al., 2011; Flynn et al., 2015; Lee et al., 2015; Seo et al., 2017; Czapka et al., 2018). This reflects that, although most cells were metabolically inactivated after relatively short NTP exposure times, the complete suppression in culturability afterward was achieved in approximately twice the time in most of the mentioned studies. The endurance of cells (especially in biofilm form) toward antimicrobials, including NTP exposure, should not be underestimated, and the fact that their viability decreased due to treatment does not guarantee the complete suppression of cell survival. Zhang et al. (2013) used PAW exposed to NTP for 20 min, which resulted in the total suppression of cell survival after 10 min treatment of cells (a 6 log decrease in CFU/ml). In addition, they observed the effect of storage on the efficacy of PAW and found that, after 24 h storage, the same PAW needed 40 min to achieve complete suppression of cell growth. Similarly, Shen et al. (2016) evaluated the effect of temperature and storage time on the effectivity of PAW on S. aureus cells. They studied storage temperatures ranging from −80 to 25°C and storage times from 0 to 30 days. For the storage temperature, −80°C proved to be the most efficient in the conservation of the effect of PAW. Without storage, the stated PAW caused a 5 log reduction in the survival counts, whereas after 30 days of storage at −80°C, such reduction dropped to only 3.7 log. In comparison, analogically stored PAW at 25°C lost its antimicrobial activity almost completely, resulting in a 0.8 log reduction. Although storage of PAW at −80°C was proven to be effective, its immediate use is better for achieving greater antimicrobial effects.

The effectivity of PAW against S. aureus growth is also affected by experimental arrangements of NTP exposure. Tian et al. (2015) studied the effect of a plasma microjet used for PAW preparation placed above or beneath the surface of the liquid. PAW prepared by exposure to NTP placed above the liquid surface showed a decreased antimicrobial activity in comparison to PAW prepared by placing the electrode beneath the liquid surface. They also found distinct differences in the electrical conductivity, hydrogen peroxide content (reflecting the creation of ROS), and the pH of both types of PAW. PAW created with electrode under the surface of the liquid showed an increase in almost all parameters in comparison to PAW created with the electrode placed above the surface, indicating the greater effectivity of placement beneath the liquid surface for preparing effective PAW.

Lastly, there are also several studies on the use of plasma-activated solutions such as saline, PBS, and even alginate gels. Oehmigen et al. (2010) studied saline and PBS activated by NTP and found the plasma-activated PBS to be more effective than saline, resulting in total inhibition after 15 min treatment of cells. As mentioned in the section on E. faecalis, Ercan et al. (2013, 2014) reported on the antibacterial effect of PAW solution of NAC, methionine, threonine, glucose, cysteine, proline, glycine, glutamine, heparin, and arginine against S. aureus. Interesting results were also presented by Poor et al. (2014), reporting plasma-activated alginate wound dressing as a strong antimicrobial agent. Alginate gel is a common emulsifier or gelling agent, which is non-toxic but has no meaningful antimicrobial property. On the other hand, alginate gels exposed for 15 s to NTP completely inhibited 10⁷ CFU/ml of S. aureus and A. baumannii in planktonic forms within 15 and 30 min, respectively. The 80% inhibition of the viability of biofilm cells was observed after 15 min treatment.

The sensitivity of planktonic cells to NTP is similar to that of other species. Treatment of suspension cells was described in Gabriel et al. (2016), where a 5 log decrease was observed after 25 min of exposure. The efficiency of decontamination of agar plates was studied in several papers (Justan et al., 2014; Metelmann et al., 2018; Gorbunova, 2019; Lee et al., 2019), where inhibition zones were observed from several seconds to 10 min of exposure depending on the NTP source and arrangement. In addition, a more than 3 log decrease of viable cells on inoculated steel plates was observed after 20 min NTP exposure in the study by Lis et al. (2018).

Gilmore et al. (2018) reviewed the fundamental descriptions of biofilms, their specific characteristics, and the mechanisms of their interactions with NTP. In this general overview, K. pneumoniae was only briefly mentioned. Furthermore, Gupta and Ayan (2019) reviewed recent advances in NTP application on biofilms. The decontamination and sterilization of numerous bacterial biofilms are summarized here. For K. pneumoniae, the use for the sterilization of venous and urinary catheters is proposed. O’Connor et al. (2014) reported on the possibility of using NTP to prevent nosocomial bacterial and viral infections. Methodically, it was proposed to use DBD and plasma jet for the decontamination of skin, wounds, tissue cultures, and medical and dental instruments, including biofilm removal. Good efficacy was reported for several bacteria and viruses. K. pneumoniae was mentioned here as an important target, but without specific results. Lee et al. (2019) investigated the antibacterial properties of titanium used for dental implants treated with NTP. Klebsiella oxytoca and K. pneumoniae were used as the tested species, together with Streptococcus mutans and S. aureus. The adhesion and the biofilm formation rate of bacteria were significantly reduced on the plasma-treated titanium surfaces compared with those of the untreated samples. Both adhesion and the biofilm formation rate were significantly lowered for Gram-negative bacteria than for the Gram-positive ones. Thus, NTP treatment could be useful for preventing bacterial adhesion and biofilm formation on titanium dental implants.

Plasma-activated water was used for the decontamination of tiger nut samples in Muhammad et al. (2019), where a 4 log decrease (from an initial 7 log) was observed after 15 min of PAW application. The activation of other various solutions (glucose, heparin, and amino acids) by NTP was described in Ercan et al. (2014). The authors declared that the plasma-activated methionine solution exhibited a strong inhibitory activity against not only K. pneumoniae but also other tested bacterial species. In addition, this solution prevented the formation of biofilms by about 70% compared to untreated controls and inhibited the biofilm formation in less than 30 min exposure to the plasma-activated methionine solution. This suggests that plasma-activated solutions have the potential to prevent biofilm formation.

As described in detail by El-Sayed et al. (2015), NTP may be used for the treatment of wastewater. This application has already been mentioned in the historical work on plasma production (Siemens, 1857). The design of the ozone generator, known today as an ozonizer, was described there. Wastewater samples were collected from a food processing and a leather processing plant. Twenty-two bacterial species were identified using 16S rDNA sequences. The samples were exposed to NTP for 30–90 s, and extensive results were summarized. Klebsiella spp. showed good sensitivity to NTP treatment. From the complex results, the following might be mentioned as typical examples: dominant bacterial groups in leather processing wastewater changed greatly upon exposure to NTP for 30 and 60 s, with Klebsiella spp., Enterobacter aerogenes, and Acidithiobacillus ferrooxidans. Extension of the exposure time to 90 s resulted in an 80% reduction in bacterial populations and the elimination of all bacterial groups, except for the resistant Pseudomonas spp. and Citrobacter freundii.

Gorbunova (2019) described the possibilities of using NTP in the food industry for processing and preserving meat products. A collection of bacterial strains was exposed in model experiments, where K. pneumoniae was inhibited after a 10-min exposure by 36%. Among the other results of this work are the interesting attempts to decontaminate real meat samples, reducing almost half the incidence of contaminating bacteria while maintaining the sensory properties of the exposed meat. Another work (Kan, 2014) described the use of NTP treatment in the textile industry. Besides various aspects of fiber modifications or coloring, the bacterial inactivation and the antimicrobial functionality of textiles were also mentioned. For K. pneumoniae inhibition in wool, the combination of enzyme, peroxide, and argon plasma pretreatment is recommended as the most effective.

Dong et al. (2010) described the use of NTP to generate a stable strain of K. pneumoniae with improved 1,3-propanediol production. This compound is important due to its large potential in commercial applications, particularly as a monomer of polyesters, polyethers, or polyurethanes. It may be prepared from glycerol by the specific activities of glycerol dehydrogenase, glycerol dehydratase, and 1,3-propanediol oxidoreductase of K. pneumoniae. The change of their activities gave rise to the improved 1,3-propanediol production, which could be considered as a mutation to a new strain designed as Kp-M2.

The initial series of references is devoted to the ability of NTP to inactivate A. baumannii inoculated on the surface. The following papers focused on wet surfaces. Svarnas et al. (2019) reported that 1 ml of PBS suspension of 3 × 10⁶ CFU/ml was inactivated after 20 min of treatment. On agar surface inoculated with 10⁴ CFU/cm², NTP exposure of 240 s created a circular inhibition zone of 15 mm in diameter. Atta (2019) exposed cultures on agar surfaces and obtained circular inhibition zones of 4 cm in diameter after 60 s of NTP exposure. Moreover, damage of the DNA, protein structure, and bacterial morphology caused by the NTP exposure was also mentioned. In Kolb et al. (2012), A. baumannii was inactivated on agar surface at 10⁵ CFU/cm² concentration. Complete inhibition on a 2 × 2 cm² area was observed within 120 s. Heller et al. (2012) used NTP to inactivate A. baumannii on the surface of both agar plates and porcine skin, which mimics clinical application. The 6 log decrease in bacterial count meant an almost complete inactivation on agar plates, which was observed after 215 s of NTP exposure. On porcine skin, a reduction of only 2–3 log was observed. This lower effect may probably be explained by the scabrous surface serving as protection for the bacteria.

Cahill et al. (2014) inactivated MRSA, VRE, and A. baumannii on dry surfaces. Contaminated surfaces of marmoleum, mattress, polypropylene, powder-coated mild steel, and stainless steel were exposed to NTP for up to 90 s. The exposure successfully reduced the bacterial load by 1.7 log for A. baumannii. A similar multi-jet arrangement applied on the same bacteria was presented in Cahill et al. (2017). NTP was applied to 5-cm² sections of stainless steel and mattress for up to 45 s, where at least 3–4 log and 3–6 log reductions were achieved on the mattress and stainless steel, respectively. Lis et al. (2018) inoculated A. baumannii, E. faecium, S. aureus, and K. pneumoniae on a stainless steel surface. They reported a 3 log decrease after 20 min of NTP exposure. However, after the addition of BSA serving as protection for the bacteria, the inactivation efficacy fell to only a 2 log decrease.

The last two papers described NTP decontamination of various types of suspensions. Parkey et al. (2015) used helium and helium/air mixture (97:3) gas NTP to inactivate A. baumannii and S. aureus in 150 μl of PBS suspension. While the pure helium gas NTP was only less effective, the helium/air mixture gas NTP led to the full inactivation of A. baumannii (7 log decrease). A complete inactivation of A. baumannii was also reported by Ruan et al. (2018), who used the NTP in open air. A rapid decrease of the surviving bacteria was observed in 2 ml suspension of 10⁶ CFU/ml concentration after 5 min of NTP exposure; a total inactivation occurred after 10 min.

Flynn et al. (2019) used the helium/oxygen plasma jet for up to 540 s to inactivate the A. baumannii biofilm grown for up to 72 h. As expected, the younger 24-h-grown biofilm was more sensitive to the NTP treatment than were the biofilms grown for 48 and 72 h. While the 24-h biofilm was inactivated from the initial 10⁶ CFU/peg (i.e., peg lid of the Calgary Biofilm Device) to approx. 10 CFU/peg in 540 s, the others were inactivated to only 10²–10³ CFU/peg. The other differences presented in this paper were not statistically significant. Lis et al. (2018) reported a 4 log reduction of the biofilm of carbapenem-resistant A. baumannii isolate formed on stainless steel after a 20-min exposure to NTP.

As also mentioned under E. faecalis, Ercan et al. (2013, 2014) reported the antibacterial effect of plasma-activated solutions of NAC, methionine, threonine, glucose, cysteine, proline, glycine, glutamine, heparin, and arginine against A. baumannii. Also, as mentioned under S. aureus, Poor et al. (2014) reported that a plasma-activated alginate wound dressing completely inhibited within 15 min 10⁷ CFU/ml of A. baumannii in planktonic form.

The efficiency of NTP against P. aeruginosa in planktonic form was examined in many relevant studies (Gadri et al., 2000; Ermolaeva et al., 2011; Alkawareek et al., 2012a; Lunov et al., 2016b; Mai-Prochnow et al., 2016; Mohd Nasir et al., 2016; Abbas et al., 2017; Navon-Venezia et al., 2017; Kondeti et al., 2018; Mohammed and Abbas, 2018; Humud, 2019). For details, see Supplementary Table 5 (also with other studies referred to in this section). Alkawareek et al. (2014) tested a selected panel of clinically significant bacterial species that included Bacillus cereus, MRSA, E. coli, and P. aeruginosa exposed in planktonic form directly to NTP. All these bacteria were completely inactivated within 2 min of NTP exposure. The reduction of the cell population was 10⁷ CFU/ml in the case of P. aeruginosa. In addition, the damaging effects of NTP on the cellular components, including DNA, a model protein enzyme, and lipid membrane integrity and permeability, were observed in this study. The 10-min NTP exposure was used for the investigation of its impact on the lipopolysaccharide toxicity of P. aeruginosa in Barakat et al. (2019), where a complete reduction in endotoxin concentration was achieved.

In Puač et al. (2014), bacterial suspensions of P. aeruginosa and E. faecalis were treated for 60–180 s. Complete inhibition of bacteria was achieved after 180 s of NTP exposure (10³ CFU/ml decrease). A more efficient sterilization was achieved in the case of P. aeruginosa. Lunov et al. (2016a) treated E. coli, P. aeruginosa, S. aureus, and Bacillus subtilis inoculated on agar plates. After 60 s of NTP exposure, no survival was detected. The SEM images confirmed damage of the cells. Sohbatzadeh et al. (2010) also treated bacterial suspension of P. aeruginosa (tested in comparison with B. cereus and E. coli) with 10 min of NTP exposure and observed total inactivation of cells determined as a decrease in optical density. In the study of Yang et al. (2009), P. aeruginosa on contaminated polyethylene terephthalate (PET) sheets was treated using NTP. The results, confirmed by SEM, showed total inactivation in a short time (60 s). In Nishime et al. (2017), NTP was applied directly on agar plates inoculated by E. faecalis, P. aeruginosa, and Candida albicans. For P. aeruginosa, an inhibition zone of 100 mm² was detected after 180 s of NTP exposure.

Liu et al. (2021) studied the action of NTP on A. baumannii exposed on the surface of Petri dishes. NTP was produced by corona discharge on an array of pin electrodes. The positive discharge appeared to be more effective than the negative discharge. A 99.99% sterilization efficiency was achieved within 9 min.

Pseudomonas aeruginosa was examined by Alkawareek et al. (2012a) alongside other microorganisms (B. cereus, S. aureus, and E. coli) for the eradication of its biofilm pre-formed on the peg lid of the Calgary Biofilm Device. An exposure time lower than 240 s was necessary for the complete eradication of Gram-positive bacterial biofilms, while the Gram-negative biofilms required a longer treatment time (6 log decrease after 600 s). The following research by the same laboratory group (Alkawareek et al., 2012b) confirmed the eradication of P. aeruginosa biofilm pre-formed on the peg lid of the Calgary Biofilm Device and polycarbonate coupons after direct exposure to NTP. The authors demonstrated that the parameters of NTP generation (particularly the frequency) had a significant effect on the bacterial inactivation rate. There was total decrease in the number of surviving cells (6.2 log CFU/ml). The anti-biofilm activity of NTP against P. aeruginosa was also confirmed using the XTT {sodium 3′-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate} metabolic assay and CLSM. A similar experimental setup was used by Patenall et al. (2018), who investigated the effect of NTP against P. aeruginosa biofilm grown on polycarbonate membranes exposed to NTP at different stages of its development. The results showed that the stage of biofilm formation exposed to NTP has a crucial role in its ability to overcome the NTP effect and regrow. A reduction of the culturability of cells of 10⁴–10⁵ CFU/ml was achieved for the early stage biofilm (0–8 h) exposed to NTP, while only a 10²-CFU/ml decrease was shown for the developed and mature biofilms (12–24 h). These findings were confirmed using SEM and CLSM. The study of Mai-Prochnow et al. (2016) described the effect of NTP against different single-species (B. subtilis, Staphylococcus epidermidis, Pseudomonas libanensis, and P. aeruginosa) and mixed-species bacterial biofilms formed on glass and stainless steel coupons. The authors reported that the efficacy of NTP is directly correlated with the bacterial cell wall thickness. While the biofilm of B. subtilis (55.4-nm cell wall) showed less susceptibility to NTP exposure (<10 CFU/ml reduction after 600 s treatment), P. aeruginosa (2.4-nm cell wall) was almost completely eradicated (3.5 log CFU/ml decrease) at the same conditions. In addition, the cell membranes and the biofilm matrix also played significant roles. Mixed-species biofilms corresponded to the properties of the least sensitive species in the culture. In Gupta et al. (2017), the effectivity of NTP exposure on the biofilm of P. aeruginosa pre-formed on titanium coupons was also examined in combination with the biocide CHX digluconate, which was used for 5–15 min. The authors found that complete elimination of the biofilm (10⁸ CFU/ml reduction) can be achieved after this combinatorial treatment. When using NTP alone, only a 10³-CFU/ml decrease was achieved. Soler-Arango et al. (2019) studied the eradication of P. aeruginosa biofilm pre-formed on stainless steel 316L, which showed 6.5 log CFU/ml decrease after 30 min of NTP exposure. Moreover, significant damage on the biofilm matrix was described, where mostly the carbohydrates and environmental DNA (eDNA) succumbed to chemical and structural changes.

In two works from one research group (Zelaya et al., 2010; Vandervoort and Brelles-Mariño, 2014), P. aeruginosa biofilm was inactivated. Firstly, a significant reduction of the adhesiveness of P. aeruginosa biofilm to borosilicate coupons in batch cultures and a reduction of the thickness of the grown biofilms were reported. In addition, after 5 min of NTP exposure, no culturable cells of P. aeruginosa were detected. The group’s further research in a continuous culture system showed total decrease (8 log CFU/ml) of culturable cells after 30 min of NTP exposure.

Triandafillu (2003) investigated P. aeruginosa adhesion on PVC surfaces previously exposed to oxygen–plasma for 120 s. This treatment made a hydrophilic surface of PVC, therefore reducing the number of adhering bacteria by as much as 70% (adhered cells counted using a light microscope). However, it was considered as insufficient for the prevention of P. aeruginosa colonization of PVC endotracheal intubation devices. Gabriel et al. (2016) applied a cocktail of four P. aeruginosa strains on the surface of stainless steel 316 and 304 for cell attachment. NTP was used for the early stage biofilm treatment for 90 s, with a 99.9% reduction in the cell population (a decrease of 5 log CFU/ml was achieved). In our previous study (Paldrychová et al., 2019), we examined the effect of NTP on the redevelopment of pre-formed biofilms of different P. aeruginosa strains on Ti-6Al-4V titanium alloy. There was a significant difference in the sensitivity of non-hospital and clinical strains to NTP action. While the biofilms of the clinical isolates were not eradicated after 60 min of NTP exposure, the non-hospital strains were almost eliminated. In addition, quorum sensing signaling molecules [acyl-homoserine lactones (AHL)] were detected and the lower level of AHL production was determined in non-hospital strains. In Flynn et al. (2016), commercially available AHL molecules (produced usually by P. aeruginosa) were exposed to NTP for 240 s. This treatment caused the degradation of the AHL molecules and their conversion into a series of by-products. In Ziuzina et al. (2014), the biofilm of P. aeruginosa grown on 96-well microplates and glass coverslips was exposed to NTP for 300 s. This treatment reduced the metabolic activity of biofilm cells by 70%, determined using the XTT assay. This research was expanded in Ziuzina et al. (2015) by monitoring the efficiency of the discharge burns under the same conditions on the reduction of quorum sensing-regulated virulence factors of P. aeruginosa. The production of pyocyanin was significantly inhibited after a short treatment time (60 s), but the reduction of elastase LasB was notable only after 300 s and no reduction in the actual biofilm formation was achieved according to the CFU counts.

Hübner et al. (2010) compared the impact of NTP exposure for 30 s with the action of CHX and polyhexanide (PHMB) on the biofilm of P. aeruginosa grown on polystyrene microplates and silicone swatches. A 10³-CFU/ml decrease was achieved in P. aeruginosa biofilm exposed to NTP, while PHMB showed a reduction of 10–100 CFU/ml. The most effective was CHX applied on the biofilm formed on silicone swatches (10⁴ CFU/ml decrease). In Matthes et al. (2013b), the same research group investigated the anti-biofilm activity of NTP and CHX against P. aeruginosa and S. epidermidis biofilms grown on 96-well microplates. The effectiveness of both agents was comparable, but NTP treatment led to a higher reduction effect (5.5 log CFU/ml decrease) depending on the exposure time and the gas used. Moreover, Matthes et al. (2013a, 2014) revealed very similar results on polycarbonate disks. NTP pretreatment was used for the enhancement of the action of antibiotics (gentamicin, ceftazidime, and polymyxin B) against P. aeruginosa biofilm in our recent study (Paldrychová et al., 2020). The strongest anti-biofilm effect was observed in the case of the combined action of NTP and gentamicin. The P. aeruginosa biofilm was completely eradicated by these agents, which was also confirmed using fluorescent microscopy and SEM.

As mentioned under E. faecalis, Ercan et al. (2013) reported that a total inactivation of 10⁷ CFU/ml of P. aeruginosa in both planktonic and biofilm forms was observed after 15 min with the plasma-activated solution of NAC.

A special application of NTP was examined by Scally et al. (2018), who described the possibility of treating low-density polyethylene for the acceleration of the biodegradation of this material with P. aeruginosa. NTP was applied on the surface of polyethylene for 300 s. The biodegradation of this material was increased, probably due to an increase in oxidative species causing better cell adhesion and acceptance on the polymer sample surface. Another special application of NTP was investigated by Hammann et al. (2010) when testing eyeballs extracted from commercially slaughtered pigs artificially contaminated with S. aureus and P. aeruginosa. The efficacy of NTP exposure for 600 s resulted in a higher effectivity (RF = 2.4–2.9) compared with that of povidone iodine and polyhexanide. An in vivo study was also described by Yu et al. (2011), who investigated the effect of NTP on P. aeruginosa colonization on skin wound in mice. The counts of the bacterial colonies decreased from an initial 9 log to 3 log CFU/ml; therefore, it was suggested that NTP facilitates wound healing by suppressing bacterial colonization.

The possibility of the inactivation of microorganisms on the surface of polyethylene and polyvinyl chloride substrates using NTP is mentioned in Tiwari and Chaturvedi (2018). For Enterobacter spp., a short exposure time of 1 min was required for their inactivation. For other bacteria, e.g., Listeria or Klebsiella spp., an exposure time of at least 3 min was required (Agnihotri et al., 2018). On the other hand, in the small number of studies on such problems, 3 min was proven to be a decent treatment time, as shown, for example, in Parkey et al. (2015). They achieved complete suppression of the planktonic growth of E. cloacae on polystyrene microtiter plates after NTP treatment for 3 min. A high antimicrobial effect of NTP was also proven in the case of E. agglomerans in Ren et al. (2012). They achieved a 90% decrease of the viability of planktonic cells with a 4-min treatment. Similarly, total inhibition of E. aerogenes growth on stainless steel (7 log CFU/ml decrease) after a 3-min exposure to NTP was achieved in Lu et al. (2011).

Although this species is not often linked to its biofilm formation and to its further virulent potential, there are a handful of studies on the ability of NTP to inhibit the biofilm formation of the genus Enterobacter. Mai-Prochnow et al. (2016) tested the efficacy of NTP exposure on E. cloacae biofilms formed on stainless steel and found that treatment for 10 min caused a 3.5 log CFU/ml reduction in survival counts (approximately by half, original inoculum of 6 log CFU/ml). The anti-biofilm effect of NTP on E. cloacae was further observed only by the previously mentioned study of Flynn et al. (2015), who investigated NTP treatment of all the ESKAPE pathogens. In this study, total inhibition of biofilm formation on microtiter plates with NTP treatment for only 2 min was achieved. It is interesting to compare the small amount of data on the susceptibility of E. cloacae planktonic cells to NTP with those of biofilm cells, as the average effective treatment time for NTP to achieve complete inhibition of planktonic growth is 3 min, but Flynn et al. (2015) completely suppressed the biofilm formation of this species after only 2 min. This was highly dependent on the arrangement of the device for NTP generation, which also generally results from the parameters described in Supplementary Table 6.

In clinical settings, a multiple-cycle NTP exposure was proven effective in the treatment of heart assist device-related infections. Urayama and McGovern (2018) described the NTP treatment of patients with infected left ventricular assist device (LVAD) (applied to cure heart failure). The infection was located in the pump pocket or on the driveline. Out of six treated patients, Enterobacter spp. were found in three, usually accompanied by Pseudomonas spp., Klebsiella spp., or Staphylococcus spp. Treatment for 2–9 weeks caused complete or nearly complete healing, which strongly highlights the great medical potential of NTP.

El-Sayed et al. (2015) described the efficacy of NTP in the decontamination of wastewater containing E. aerogenes. The presence of the species, among others, was completely suppressed in wastewater samples treated with NTP for 30 s.

Finally, NTP treatment may be a useful tool for mutation. In addition to the information mentioned above, Ren et al. (2012) carried out an efficient agar plate mutagenesis and screening technique for improving the mutation frequency. E. agglomerans is well known as a phosphate-solubilizing plant-associated bacterium, and NTP was applied to its mutation in order to improve the phosphate-solubilizing activity. The results showed that the phosphate-solubilizing activity of mutants increased compared with that of the original strain, and the phosphate-solubilizing activity of the best mutants was 1.49-fold that of the original strain. It demonstrated that NTP treatment has high efficiency and that it will be a useful method for mutation. Comparable results were presented by Lu et al. (2011): the positive mutant of E. aerogenes obtained after 3 min of NTP exposure showed a 26% increase of the total hydrogen yield per mole of glucose and was genetically stable after more than 25 subcultures. The increase of the hydrogen production by the mutant strain makes it promising to produce hydrogen as an alternative clean energy source.