Non-thermal (low-temperature) physical plasma is under intensive study as an alternative approach to control superficial wound and skin infections when the effectiveness of chemical agents is weak due to natural pathogen or biofilm resistance. The purpose of this study was to test the individual susceptibility of pathogenic bacteria to non-thermal argon plasma and to measure the effectiveness of plasma treatments against bacteria in biofilms and on wound surfaces. Overall, Gram-negative bacteria were more susceptible to plasma treatment than Gram-positive bacteria. For the Gramnegative bacteria Pseudomonas aeruginosa, Burkholderia cenocepacia and Escherichia coli, there were no survivors among the initial 10 5 c.f.u. after a 5 min plasma treatment. The susceptibility of Gram-positive bacteria was species-and strain-specific. Streptococcus pyogenes was the most resistant with 17 % survival of the initial 10 5 c.f.u. after a 5 min plasma treatment. Staphylococcus aureus had a strain-dependent resistance with 0 and 10 % survival from 10 5 c.f.u. of the Sa 78 and ATCC 6538 strains, respectively. Staphylococcus epidermidis and Enterococcus faecium had medium resistance. Non-ionized argon gas was not bactericidal. Biofilms partly protected bacteria, with the efficiency of protection dependent on biofilm thickness. Bacteria in deeper biofilm layers survived better after the plasma treatment. A rat model of a superficial slash wound infected with P. aeruginosa and the plasma-sensitive Staphylococcus aureus strain Sa 78 was used to assess the efficiency of argon plasma treatment. A 10 min treatment significantly reduced bacterial loads on the wound surface. A 5-day course of daily plasma treatments eliminated P. aeruginosa from the plasma-treated animals 2 days earlier than from the control ones. A statistically significant increase in the rate of wound closure was observed in plasma-treated animals after the third day of the course. Wound healing in plasma-treated animals slowed down after the course had been completed. Overall, the results show considerable potential for non-thermal argon plasma in eliminating pathogenic bacteria from biofilms and wound surfaces.Abbreviation: CI, confidence interval.

SummaryPrfA, a transcription factor structurally related to Crp/ Fnr, activates Listeria monocytogenes virulence genes during intracellular infection. We report two new PrfA* mutations causing the constitutive overexpression of the PrfA regulon. Leu-140Phe lies in a a a a D adjacent to the DNA-binding motif in the C-terminal domain, like a previously characterized PrfA* mutation (Gly-145Ser). Ile-45Ser, in contrast, maps to the N-terminal b b b b -roll, a structure similar to that of the Crp cAMP binding site. The in vitro transcriptional properties of recombinant PrfA* I45S and PrfA* G145S were compared to those of PrfA WT at two differentially regulated PrfA-dependent promoters, P plcA and P actA . The two PrfA* mutations increased the affinity for the target DNA to a different extent, and the differences in DNA binding (PrfA* G145S > PrfA* I45S >>> PrfA WT ) correlated with proportional differences in transcriptional activity. The use of the PrfA* proteins revealed that P plcA had a greater affinity for, and was more sensitive to, PrfA than P actA . RNA polymerase (RNAP) initiated transcription independently of PrfA at P plcA , but not at P actA , consistent with bandshift experiments suggesting that P plcA has a greater affinity for RNAP than P actA . Thus, differences in affinity for both PrfA and RNAP appear to determine the different expression pattern of PrfA-regulated promoters. Modelling of the PrfA* mutations in the crystal structure of PrfA and comparison with structure-function analyses of Crp, in which similar mutations lead to constitutively active (cAMPindependent) Crp* proteins, suggested that PrfA shares with Crp an analogous mechanism of cofactormediated allosteric shift. Our data support a regulatory model in which changes in PrfA-dependent gene expression are primarily accounted for by changes in PrfA activity.

SummaryVirulence genes from the facultative intracellular pathogen Listeria mono cytogenes are controlled by the transcriptional regulator PrfA. Although PrfA synthesis is activated at 37 ∞ ∞ ∞ ∞ C, PrfA-dependent expression remains low in rich medium. However, a strong induction of the PrfA regulon is observed when L. monocytogenes is cultured in the presence of activated charcoal. Here, we show that the 'charcoal effect' results from the adsorption of a diffusible autorepressor substance released by L. monocytogenes during exponential growth. Analyses using an L. monocytogenes strain in which the prfA gene is expressed constitutively at 37 ∞ ∞ ∞ ∞ C from a plasmid indicate that the autoregulatory substance represses PrfA-dependent expression by inhibiting PrfA activity. PrfA presumably functions via an allosteric activation mechanism. The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA. Our data indicate that the listerial autorepressor is a low-molecular-weight hydrophobic substance. We suggest that this diffusible substance mediates a quorum-sensing mechanism by which L. monocytogenes restricts the expression of its PrfA virulence regulon. This autoregulatory pathway could serve L. monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37 ∞ ∞ ∞ ∞ C. It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA-dependent virulence factors in the PrfA-activating cytosolic compartment.