Plasma-mediated inactivation of E. coli: Influence of protein on wet surface and in liquid medium

Application of cold atmospheric-pressure plasma (CAP) for wound treatment but also for decontamination of food or water often includes the presence of proteins. These proteins contain amino acids with functional groups such as thiol (SH) groups. Plasma-mediated effects in biological systems involve reactive oxygen and nitrogen species (RONS). In this regard, the redox signaling is often mediated via thiol groups. The aim of this study was to investigate the influence of protein and SH group containing amino acid on the antimicrobial properties of plasma-treated saline solution (0.85% NaCl, w/v) and on the direct CAP efficacy on solid wet agar plates. Plasma treatment of saline solution was realized using an AC-driven pin-to-liquid discharge. After 10min plasma treatment time, the amino acids L-cysteine (contains SH group) or L-alanine (no SH group) or bovine serum albumin (BSA; with approximately 6% cysteine content) was added together with the test microorganism Escherichia coli K-12 (DSM 11250/NCTC 10538) for an exposure time of up to 60min. The total viable cell count was determined in appropriate time intervals. A concentration-dependent repeal of the antimicrobial efficacy was determined. Thus, 0.0025% of BSA did not have any influence whereas 0.25% of BSA (w/v) as well as the tested amino acids resulted in no inactivation of E. coli. The difference between L-alanine and L-cysteine was negligible, suggesting only a minor effect of the presence of SH groups. Dimerization of L-cysteine was shown by LC/MS analyses whereas no derivatization of L-alanine was detected. To test the influence of proteins in direct plasma treatment on wet surfaces, E. coli was plated together with BSA on soybean casein digest agar surface. Another setup based on agar plates which contained different concentrations of peptone (mixture of peptides and amino acids). The agar plates were punctually treated by the argon-driven CAP jet kINPen((R)) Med (neoplas tools GmbH, Greifswald, Germany). After overnight incubation, inhibition zones were analyzed. The bacterial growth was independent of protein or peptone content for this direct plasma treatment. Summarizing all, the antibacterial effect of plasma-treated solution is strictly limited in the presence of proteins. This inhibiting effect is not dependent on chemical structures containing SH groups. In direct plasma application, none of the tested protein or peptide/amino acid content resulted in a diminished antimicrobial effect of the kINPen Med((R)) when applied in a protein-containing environment.