## 3 Results and Discussion  
### Bactericidal Efficacy of Tb-PAW on Different Microorganisms  
The 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 \(\pm\) 0.46 log), _F. necrophorum_ (5.04 \(\pm\) 0.03 log) and _P. levii_ (4.98 \(\pm\) 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 \(\pm\) 0.75 log) and _S. aureus_(0.36 \(\pm\) 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 \(\pm\) 0.27 log. _C. sporogenes_ showed insignificant decreases of the CFU (0.36 \(\pm\) 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 \(\pm\) 0.38 log for _C. sporogenes_ and 3.66 \(\pm\) 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. necrorophorum_ 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 [PERSON] et al. [28]. [PERSON] 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, [PERSON] 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. [PERSON] et al. [34] compared the reduction of the Gram-negative bacterium _Porphyomonas (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  
Figure 3: (**a**) Inactivation of _Escherichia (E. coli, Staphylococcus (S. aureus, Fusobacterium (F.) necrorophorum, Porphyomonas (P.) levii_ and _Clostridium (C.) sporogenes_ after 1 min of Tb-PAW treatment (**b**) Inactivation of _C. sporogenes_ and _S. aureus_ after 1 min, 5 min and 15 min of Tb-PAW treatment. Results represent the mean \(\pm\) standard deviation. Significant differences are defined as * _p_\(\leq\) 0.05. Crtrl = Control (TRIS-buffer); Tb-PAW = TRIS-buffered plasma-activated water. a reduced susceptibility to reactive species due to their significantly thicker cell wall (20-80 nm vs. 10-15 nm) [33].