3.1. Bactericidal Efficacy of Tb-PAW on Different MicroorganismsThe antibacterial effect of Tb-PAW compared to the untreated TRIS-buffer control for the respective pathogens is shown in Figure 3. The Tb-PAW was able to achieve significant reductions of the CFU of Gram-negative bacteria species E. coli (5.12 ± 0.46 log), F. necrophorum (5.04 ± 0.03 log) and P. levii (4.98 ± 0.61 log) after a 1 min exposure time (Figure 3a). Whereas the CFU of E. coli, F. necrophorum and P. levii were decreased below the detection limit, only low but not significant reduction rates of the bacteria numbers were obtained for Gram-positive bacteria C. sporogenes (0.20 ± 0.75 log) and S. aureus (0.36 ± 0.40 log) within 1 min compared to the control samples. After increasing the Tb-PAW exposure time of C. sporogenes and S. aureus to 5 min (Figure 3b) S. aureus was significantly reduced by about 1.76 ± 0.27 log. C. sporogenes showed insignificant decreases of the CFU (0.36 ± 0.40 log). For both bacterial species, an additional exposure time of 15 min was applied, functioning as highest possible practice-oriented treatment duration. The prolonging contact time did enhance the antimicrobial efficacy, leading to significant reductions of 3.06 ± 0.38 log for C. sporogenes and 3.66 ± 0.16 log for S. aureus (Figure 3b).We observed that the Tb-PAW used in this study could reduce both Gram-negative and Gram-positive bacteria up to at least 3.0 log. However, S. aureus and C. sporogenes were more resistant to the Tb-PAW treatment, which reflected in elevated treatment times compared to E. coli, F. necrophorum and P. levii. A comparison of the present results with previously published studies [28,29] is difficult as antimicrobial effects of different PALs are quite variable. This high variability might be due to the application of different parameters such as voltage, working gases and gas flow rate, the treatment regime (used liquid and plasma treatment time), the content of reactive species, the acidity of the PAL or differences of the bacterial strains [24,30,31,32]. For example, in the present study the inactivation for S. aureus within 15 min was higher compared to the study of Tsoukou et al. [28]. Rothwell et al. [29] presented an increased efficacy for E. coli (6 log) within 1 min compared to our results. An effect which might be attributed to a lower initial CFU number in the present study. Some studies showed that the utilized bacterial species (E. coli, S. aureus) were only inactivated after high exposure times. Furthermore, other authors also reported a higher sensitivity of Gram-negative bacteria against PALs [24,28,33,34]. For example, Zhao et al. [24] observed that S. aureus, treated with PAW, needed 5 h to be significantly reduced by 3 log steps, whereas E. coli reached this reduction within 30 min. Li et al. [34] compared the reduction of the Gram-negative bacterium Porphyromonas (P.) gingivalis and the Gram-positive bacterium Actinomyces viscosus after PAW treatment and described a higher inactivation rate for P. gingivalis within a shorter treatment time.The higher effect on the Gram-negative compared to the Gram-positive bacteria may be due to their differences in the cell wall structure. Gram-positive bacteria show a reduced susceptibility to reactive species due to their significantly thicker cell wall (20–80 nm vs. 10–15 nm) [33]. Furthermore, we have shown that the treatment of anaerobes with Tb-PAW also leads to different inactivations. Our findings depicting an increased impact of Tb-PAW on anaerobic bacteria is supported by the study of Li et al. [34]. They also found that aerobic bacteria, such as Streptococcus mutans, are less sensitive than anaerobic bacteria when exposed to comparable concentrations of different ROS species. A strong interdependency of antibacterial efficacy of the applied Tb-PAW’s and bacteria species is apparent.To date, the exact mode of action of PAW has not been fully elucidated. As described by Laroussi et al. [35] the effect of other ROS and RNS produced in PTW besides NO3− is not well understood. According to Li et al. [36], plasma-activated chemical solutions (PACS) containing H+, NO2− and H2O2 and especially their conversion to peroxynitrite, play a key role in microbial decontamination. Zouh et al. [25], who emphasized the role of peroxynitrite in their work, also supported this statement. However, other authors have noted that H2O2, hydroxyl radicals (•OH), and ozone (O3) for example, play important roles (Zhang et al. [37]). Thus, further research is necessary to understand the mode of action of PAW.The TRIS-buffer used in the present study caused the ph-value of the Tb-PAW to be neutral, giving the opportunity for their in situ application on open wounds and skin of living animals. Some studies compared the results between PAW and plasma-activated buffer in relation to their acidity and figured a lower pH-value would increase the inactivation rate [16,17,38]. Consequently, we decided on the one hand to buffer the PAL, on the other hand to adjust the control solution to the pH-value of the Tb-PAW, which was also associated with high inactivations. 3.2. Tb-PAW Inactivation Ability of E. coli, F. necrophorum and P. levii under the Influence of Bovine Serum AlbuminIn the present study the impact of bovine serum albumin (BSA) on the antimicrobial capacities of Tb-PAW against E. coli, F. necrophorum and P. levii was tested evaluating if proteins, which are present for example, on the skin of the cattle with DD might influence the overall Tb-PAW efficacy. The reduction rates of Tb-PAW applications containing 5% BSA and without BSA are shown in Figure 4. As described above, the Tb-PAW could significantly (p ≤ 0.05) reduce all bacterial species below the detection limit. Under the influence of 5% BSA E. coli was significantly reduced by 4.48 ± 0.42 log values. The reductions for F. necrophorum and P. levii using 5% BSA were 4.39 ± 0.28 and 4.55 ± 0.37 log values, respectively. The addition of 5% BSA to the Tb-PAWs revealed no significant impact on occurring reduction rates.The 30% BSA solution contained NaN3 as an antimicrobial agent, thus control samples with the existing concentrations of NaN3 (0.1 mg/L) were additionally analyzed during the BSA-impact study. No significant differences between all groups could be obtained. The results are reported in the Supplementary Material (see Table S1).The presented results disagree with other studies revealing a buffer effect of organic materials, which can considerably reduce antimicrobial activities of PALs [39,40]. BSA is described as a protein that can not only form a physical barrier between microorganisms and reactive species, but also actively interacts with free radicals leading to a decrease in the antimicrobial properties of PALs [39,41]. For example, Zhang et al. [39] demonstrated that BSA reduced the antimicrobial efficacy of PAW against S. aureus after a treatment time of 10 min. In the study by Xiang et al. [40], bacterial suspensions were added after combining beef extract or peptone with PAW (different protein concentrations) and after a waiting time of 15 min resulting in decreasing bacterial reduction properties with increasing protein concentrations. In contrast to the previous studies in the present experimental setup the bacteria were exposed to Tb-PAW and BSA at the same time which might explain the different effects between the present and other studies. However, the present setup seems more logical. For example, if a PAL is applied to the skin of living animals, the PAL is likely to come into contact with the microorganisms as well as with proteins and other (probably disturbing) substances simultaneously.Other studies indirectly support the present results. If liquids containing bacteria were treated with plasma to obtain a PAL, the proteins and other components in the media had no impact on the bacterial inactivation capacity [42,43]. For example, Rowan et al. [42] inactivated microbial pathogens, such as E. coli and Campylobacter jejuni in chilled poultry wash water, achieving an increased reduction compared to samples in distilled water. Gurol et al. [43] showed in their study that after treating different types of milk (whole, semi skimmed and skimmed milk) with low pressure plasma, the inactivation of E. coli decreased by 54% after 3 min.

In conclusion, the protective effect of proteins against microbial inactivation by PAL seems to depend on several parameters, including the protein source, the amount of protein, the experimental setup (treatment time and treatment procedure), as well as the plasma source and its settings, as evidenced by these various study results.

3.3. Tb-PAW Inactivation of E. coli after Different Storage Times and TemperaturesIn a third study, Tb-PAW was stored at three different temperatures (7 °C, 21 °C and 30 °C) and monitored at four time points (4 h, 8 h, 12 h and 24 h). The bactericidal efficacy of those Tb-PAWs against E. coli was tested (Table 1). The comparisons between untreated TRIS-buffer control samples and the corresponding Tb-PAW samples showed significant reductions of the CFU in Tb-PAW at all temperatures and storage times. These ranged from 5.08 ± 0.52 log steps (30 °C, 8 h) to 0.73 ± 0.23 log steps (30 °C, 24 h). The residual microbial content of the Tb-PAW sample at 30 °C and 24 h was significantly higher compared to the 7 °C and 21 °C samples, whereas the other time points revealed no differences between the temperature groups. In addition, we found significantly higher bacterial counts in the 30 °C treatment group at 24 h compared to the other three time points. We therefore verified that Tb-PAWs stored at 7 °C and 21 °C can be used for a period of 24 h and for at least 12 h when stored at 30 °C.These results are consistent with findings of the study by Shen et al. [44], who also reported prolonged preservation of bactericidal activity when PAW was stored at low temperatures. In this study, the bactericidal properties of a PAW used against S. aureus also increased with decreasing temperatures (25 °C Traylor et al. [17] investigated the bactericidal activity of PAW and plasma-activated PBS (PAPBS). They found that storage of PAW resulted in a decreasing reduction effect with time. The PALs were stored for seven days after preparation. Each day, E. coli was incubated with the PALs for 15 min as well as for 3 h. Over two days, the 3 h exposure resulted in a 5.0 log reduction before it began to decline, while the 15 min exposure dropped to 2.4 log on the first day (30 min after production) and remained at about 1.0 log until the end of the second day. In contrast to the PAW, the PAPBS did not reach a high reduction at any of the time points and remained below 1.0 log.The effect of the PALs in these studies appears to decrease with time and at elevated temperature, which is in good agreement with our own findings. However, the decrease varies greatly depending on the study. In the researches described, much longer exposure times (4 min to 3 h) were used compared to our study. In addition, we were able to achieve high reductions rates with Tb-PAW compared to the PAPBS of Traylor et al. [17] using only 1 min exposure time resulting in reduction rates of up to 2.61 logs (7 °C) and 3.18 logs (21 °C) after 24 h.