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.

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Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N(omega)-nitro-L-arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160-240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.

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Using scanning electron microscopy techniques we measured the diameter of adhesive tubular appendages of Salmonella enterica serovar S. Typhimurium. The appendages interconnected bacteria in biofilms grown on gallstones or coverslips, or attached bacteria to host cells (human neutrophils). The tubular appendage diameter of bacteria of virulent flagellated C53 strain varied between 60 and 70 nm, thus considerably exceeding in size of flagella or pili. Nonflagellated bacteria of mutant SJW 880 strain in biofilms grown on gallstones or coverslips were also interconnected by 60-90-nm tubular appendages. Transmission electron microscopy studies of thin sections of S. Typhimurium biofilms grown on agar or coverslips revealed numerous fragments of membrane tubular and vesicular structures between bacteria of both flagellated and nonflagellated strains. The membrane structures had the same diameter as tubular appendages observed by scanning electron microscopy, indicating that tubular appendages might represent membrane tubules (tethers). Previously, we have shown that neutrophils can contact cells and bacteria over distance via membrane tubulovesicular extensions (TVE) (cytonemes). The present electron microscopy study revealed the similarities in size and behavior of bacterial tubular appendages and neutrophil TVE. Our data support the hypothesis that bacteria establish long-range adhesive interactions via membrane tubules.